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was then isolated with the Tri-Reagent. Strong-stop DNA, the first product of HIV RT, was analyzed by the qPCR with primers specific for strong-stop DNA. The DNA concentrations of the each sample were normalized by equal DNA loading confirmed with primers for GAPDH. Data analysis Data were presented as the mean ± SD from at least three independent experiments, and statistical significance was measured by Student's t-test or one-way analysis of variance followed by the Newman-Keul's test where appropriate. Statistical significance was defined as P < 0.05 or P < 0.01. Tlr3 signaling of iecs induces iFns Activation of TLRs 3, 7, and 9 could trigger intracellular IFN-mediated innate immunity against virus infections (37). Therefore, we first examined the expression of TLRs in IECs. As shown in Figure S1A in Supplementary Material, IECs expressed mRNAs for all known human TLRs except TLR5. To investigate whether the antiviral TLRs (TLR3, 7,9) are biologically functional in IECs, we transfected the cells with the ligands to TLR3 (Poly I:C), TLR7 (Imiquimod), TLR8 (ssRNA40), and TLR9 (ODN2006). As shown in Figure S1B in Supplementary Material, the IECs expressed functional TLR3 and TLR8, as the ligands to these TLRs could induce the expression IFN-β and IFN-λ. In contrast, the ligands of TLR7 and TLR9 had little effect on IFN induction. TCI, a TLR3 complex inhibitor, could significantly block the effect of Poly I:C ( Figure S2 in Supplementary Material). We thus focused on the impact of TLR3 signaling on IFN induction in IECs in the subsequent experiments. As shown in Figure

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1, TLR3 activation of IECs induced IFN-β and IFN-λ at both mRNA ( Figure 1B) and protein ( Figure 1C) levels. These effects of Poly I:C stimulation on IFN-β and IFN-λ expression in IECs were dose-and time-dependent ( Figures 1A,B). We next examined whether IRF3 and IRF7, key regulators of the IFN signaling pathway, are involved in the TLR3 action on IFN induction by IECs. As shown in Figure 2, TLR3 signaling of IECs induced the phosphorylation of both IRF3 and IRF7, which were positively associated with the dose of Poly I:C transfected into IECs. iecs-Derived exosomes can Be Taken up by Macrophages Exosomes released from donor cells could deliver their cargo to recipient cells and subsequently modulate host cell function (21). We thus isolated and characterized the exosomes from activated IECs cultures by detecting the common exosome-carried proteins (Alix, CD63, and LAMP2) (Figure 3A). To determine whether macrophages (recipient cells) can take up the exosomes released from IECs, we incubated macrophages with exosomes labeled with green fluorescent dye PKH67. As shown in Figure 3B, PKH67-labeled exosomes were observed within macrophages treated with SN from activated IECs cultures. Tlr3 signaling of iecs inhibits hiV infection of Macrophages As shown in Figure 5A, macrophages treated with SN from Poly I:C-stimulated IECs cultures had less HIV infection-induced (Figures 5B-E). This IECs SN-mediated HIV inhibition was positively associated with the concentrations of Poly I:C used to activate IECs (Figures 5B,D) and the percentage of IECs SN added to macrophage cultures (Figures 5C,E). To decipher the roles of each subtype

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of IFNs in IECs-mediated anti-HIV activity, we used the neutralization antibody against IFN-β to pretreat the IECs SN or antibody against IFN-λ receptor to pretreat macrophages, respectively. As shown in Figure 5F, antibody to IFN-β significantly reduced the anti-HIV activity of SN from activated IECs cultures. In addition, anti-IL10 receptor β (IL-10Rβ a subunit of IFN-λ receptor) antibody pretreatment of macrophages could also block the anti-HIV activity of the IECs SN. We then investigated whether ISGs could be induced in macrophages treated by IECs SN. As shown in Figure 5F, TLR3 signaling of IECs induced the expression of ISG (ISG15, ISG56, OAS-1, OAS-2, MxA, MxB, GBP5, and Viperin) and several known HIV restriction factors, including Tetherin and APOBEC3G/3F in macrophages. To ensure the IECs cultures are polarized (38), we used the transwell system to determine whether the polarized stimulation IECs could mediate HIV inhibition in macrophages. As shown in Figure 6, HIV GAG gene expression was suppressed in macrophages treated with SN from either upper (apical side) or lower (basolateral side) chambers of the transwell cultures. No significant difference in HIV inhibition was observed between SN from the upper level chambers and those from the lower level chambers. iecs-Derived exosomes contribute to hiV inhibition in Macrophages To evaluate the role of the exosomes in IECs-mediated anti-HIV activity in macrophages, we added the activated IECs-derived exosomes to macrophage cultures. As shown in Figures 7A-D, macrophages treated with the exosomes showed less expression of cell-associated as well as extracellular HIV GAG gene as compared with untreated macrophages. We

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then examined the anti-HIV potency of IECs SN with or without exosome depletion. As indicated in Figure 7E, SN from Poly I:C-stimulated IECs significantly suppressed HIV, while the depletion of exosomes from IECs SN diminished IECs-mediated anti-HIV activity in macrophages. Tlr3 signaling of iecs induces cc chemokines CC chemokines (MIP-1α, MIP-1β, RANTES) are the ligands of the HIV entry co-receptor, CCR5. We examined whether IECs upon the TLR3 activation can produce these CC chemokines. As shown in Figure 8, Poly I:C treatment of IECs dose-dependently induced the CC chemokines at both mRNA Figure 8A) and protein (Figure 8B) levels. We then examined the ability of IECs SN to block HIV entry into macrophages. As shown in Figure 8C, the pretreatment of macrophages with the IECs SN resulted in a marked decrease in strong-stop DNA of HIV. DiscUssiOn HIV infection provides ample pathogen-associated molecular patterns that can be detected by a variety of PRRs of the innate immune system (39). Among the PRRs, TLR3 is implicated in sensing dsRNA structures during viral infections, including HIV (40). While it has been reported that intestinal epithelial cell lines Caco-2 and HT-29 express functional TLR3 (41), there is little information about TLR3 activation of IECs and its role in antiviral activity against HIV infections of macrophages. We demonstrated that human IECs expressed functional TLR3, the activation of which resulted in the production of multiple antiviral factors, including the type I and III IFNs (Figure 1), ISGs, HIV restriction miRNAs (Figure 4), and CC chemokines (Figure 8). Importantly, we found that

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when added to primary human macrophage cultures, SN from the activated IECs cultures could potently suppress HIV infection and replication. In our early work of studying factors that influence the activation efficiency of TLR3 by Poly I:C (15), we found that the direct addition of Poly I:C to the cultures of primary macrophages or a neuroplastoma cell line could effectively activate TLR3. However, the transfection was necessary and needed in order to have efficient TLR3 activation by Poly I:C in the human hepatic cell line (Huh7) and brain microvascular endothelial cell line (hCMEC/ D3). In addition, we demonstrated that the efficiency of TLR3 activation by high molecular mass Poly I:C was significantly higher than that by low molecular mass Poly I:C. These findings indicated that cell types and the size of Poly I:C are the crucial factors in Poly I:C-mediated TLR3 activation. As demonstrated in Figure S3 in Supplementary Material, we examined difference in the TLR3 activation efficiency between the direct addition and transfection of Poly I:C in IECs, showing that the levels of induced IFNs were significantly higher in IECs transfected with Poly I:C as compared to direct Poly I:C treatment. Therefore, we used the transfection technique for Poly I:C stimulation of IECs in this study to conceptually prove that as non-immune cells in GI tract, IECs can produce antiviral factors that can be transported through exosomes to macrophages, inhibiting HIV replication. The HIV inhibition in macrophages was also seen in macrophages treated with SN from either apical side or basolateral side of the polarized/

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activated IEC cultures (Figure 6). It was reported that there were little differences in TLR3 expression at different sites or between non-inflamed and inflamed mucosae in tissues from ulcerative colitis patients (42). Also, the polarized IECs responded to the TLR ligands, including TLR3, secreting IL-8 into the basolateral chamber, either exclusively on basolateral stimulation, or on apical stimulation. In non-polarized IECs, as expected, there was no difference in the response to all of these ligands (33). Although IECs are non-immune cells, they are able to produce IFN-driven antiviral factors, including ISGs. Studies have shown that the ISGs, including ISG15, ISG56, MxA, MxB, OAS-1, OAS-2, and GBP5 have anti-HIV activities (43)(44)(45). ISG15 plays a crucial role in the IFN-mediated inhibition of late stages of HIV assembly and release (46); MxB inhibits HIV infection by inhibiting the capsid-dependent nuclear import of subviral complexes (47); GBP5 reduces HIV infectivity by interfering with Env processing and incorporation (48). In addition to the ISGs, Poly I:C-stimulated IECs expressed HIV restriction miRNAs (Figure 4), including miRNA-17, miRNA-20, miRNA-28, miRNA-29 family members (miR-29a, 29b, and 29c), and miRNA-125b. It is known that miRNA-28 and miRNA-125b can target the 3'UTR of HIV transcripts (49). miRNA-29 family members interfere with virus replication, as they can target a highly conserved site in various HIV subtypes (50). Studies have shown that miRNA-17 and miRNA-20 target p300/CBP associated factor (PCAF), a cellular cofactor of the HIV Tat protein (51). Furthermore, we found that CC chemokines (MIP-1α, MIP-1β, RANTES), ligands of HIV entry co-receptor CCR5, were induced in activated

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IECs (Figure 8). The observation evidenced the role of CC chemokines in IECsmediated HIV inhibition that SN from TLR3-activated IEC cultures could block HIV entry into macrophages. IFN-β and IFN-λ in IECs SN appeared to be responsible for the induction of these anti-HIV factors, as the antibodies to IFN-β and IFN-λ receptors could block the inhibitory effect of IECs SN on HIV (Figure 5). The investigation on the mechanisms for the induction of IFNs showed that there was upregulation of IRF3 and IRF7 in activated IECs (Figure 2). IRF3 and IRF7 are the key regulators of type I and III IFNs during viral infections (52). IRF3 and IRF7 phosphorylation is a crucial step in activating type I and III IFNs-mediated antiviral response (53). Both IRF3 and IRF7 require phosphorylation-induced activation in order to translocate to the nucleus to activate IFNs (54). Specifically, during viral infections, IRF3 is important in the early phase of inducing the transcription of IFN-α and IFN-β, which then can IECs SN was preincubated with anti-IFN-β (10 µg/ml) for 1 h and then used to treat macrophages 24 h prior to HIV Bal infection (p24, 20 ng/ml). For IFN-λ receptor pretreatment, the anti-IL10Rβ neutralization antibody (10 µg/ ml) was added to macrophage cultures for 1 h prior to the addition of SN. HIV GAG expression was then measured by qRT-PCR for 8 days postinfection. (g) Effect of Poly I:C-stimulated IECs culture SN on ISG expression of macrophages. IECs were stimulated with Poly I:C for 48 h and culture SN was collected for treatment

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of macrophages (10% v/v) for 12 h. RNA was extracted, and the expression of ISGs was measured by qRT-PCR. Representative data were the mean ± SD of three independent experiments using macrophages of three donors. Asterisks indicate that the differences between the indicated groups are statistically significant (*P < 0.05, **P < 0.01). (55). IRF7 not only induces IFNs, but also actives many ISGs, among which PKR, OAS, and the Mx protein have been well characterized for their antiviral activities (56). As one of the primary targets for HIV infection and persistence, macrophages have been implicated as an important HIV reservoir. Our early investigations (26,57) showed that TLR3 activation of macrophages potently suppressed HIV infection and replication through multiple antiviral mechanisms at both the cellular and molecular levels. Despite being a major producer of type I IFNs, the biological functions of macrophages are significantly compromised in IFN induction upon HIV infection (17,18). In contrast to macrophages, IECs are not the target of HIV. Therefore, it is unlikely that HIV has a direct and negative impact on IECs. As the first line of cells in the GI system, the IECs have to encounter a number of stimuli and immune cells, including HIV-infected macrophages (58). Thus, the activation of these nonimmune cells in the GI tract is inevitable. We found that activated IECs SN could induce the expression of several key HIV restriction factors in macrophages, including Tetherin and APOBEC3G/3F (Figure 5). Tetherin is a transmembrane protein that specifically inhibits HIV release from infected cells (59), APOBEC3G/3F are

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single-stranded DNA deaminases that inhibit HIV replication through deaminating cytidine to uracil on the minus strand of the HIV proviral DNA (60). Thus, the activation of IFNmediated antiviral responses by IECs should be beneficial for GI protection. As a non-HIV target cell in the GI tract, it is unlikely that the ability of IECs to mount an IFN-mediated anti-HIV response would be compromised by HIV infection. We as well as others have shown that IFNs were produced not only by the immune cells but also by the nonimmune cells in the CNS, such as neurons and astrocytes (34,61). In contrast to Poly I:C induction of both IFN-α and IFN-β in the immune cells, TLR3 signaling of IECs induced only IFN-β expression. This finding is consistent with the report by Starace et al. showing that Poly I:C induced IFN-β but not IFN-α in mouse Sertoli cells (62). These observations along with the findings of this study support the notion that IECs and other nonimmune cells in the GI tract could be important bystanders in mounting effective antiviral responses, which may have a key role in restricting HIV infection/replication in the GI system. To understand how IECs could transport the antiviral factors to macrophages, we examined whether IECs can produce and release exosomes which are known to have the ability to shuttle biologically active molecules. Exosomes have a vital role in a variety of biologic processes, such as cell proliferation, apoptosis, and immune responses (63,64). A major recent study in the intestinal mucosa field unveiled the capacity of

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exosomes to mediate the functional transfer of genetic materials (mRNAs and miRNAs) between immune cells (65). We found that IECs-derived exosomes could be taken up by infected macrophages, inhibiting HIV replication (Figure 7). We also observed that exosomes from Poly I:C-stimulated IECs were enriched with antiviral cellular ISGs and miRNAs (Figure 4), including miRNA-17, miRNA-20, miRNA-28, miRNA-29 family members (miR-29a, 29b, and 29c) and miRNA-125b. miRNA-28 and miRNA-125b are known to target 3'UTR of HIV transcripts (66). miRNA-29 family members interfere with virus replication, as they can target a highly conserved site in various HIV subtypes (50). Studies by several groups The inhibition of HIV replication by IECs culture SN with or without exosome depletion. To deplete exosomes, the SN from Poly I:C-stimulated IECs were incubated with anti-CD63 antibody-conjugated Dynabeads overnight at 4°C and then separated in a magnetic field. Representative data were the mean ± SD of three independent experiments using macrophages of three donors. Asterisks indicate that the differences between the indicated groups are statistically significant (*P < 0.05, **P < 0.01). showed that miRNA-17 and miRNA-20 target p300/CBP associated factor (PCAF), a cellular cofactor of the HIV Tat protein (67). Collectively, we have provided the experimental evidence that TLR3 activation-induced antiviral factors in IECs could be transported to macrophages through exosomes released by IECs and internalized by macrophages (Figure 9). Because HIV has evolved several mechanisms to evade TLR3 mediated intracellular innate immunity in target cells, such as macrophages (68,69), anti-HIV support from non-immune bystander cells is helpful in restoring the HIV-suppressed system

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in infected cells. Given that macrophage is an important cellular reservoir for HIV infection/persistence, to control and eradicate HIV in macrophages is clinically significant. Although the precise cellular and molecular mechanisms by which activated IECs could inhibit HIV replication in macrophages remain to be determined, the induction of IFNs, antiviral ISGs, HIV restriction miRNAs, and CC chemokines should account for much of IECs-mediated anti-HIV activity. However, further in vitro and in vivo investigations are necessary in order to determine whether the TLR3 signaling of IECs is indeed beneficial in protecting GI macrophages from HIV infection. Currently, the therapeutic TLR agonists are being developed for the treatment of cancer, allergies and viral infections. A number of TLR agonists are now in clinical or preclinical trails such as the anti-HIV TLR3 agonist (Poly I:C 12U) (70)(71)(72). These studies support the notion for further developing a TLR3 agonist-based therapy for HIV disease in which host cell innate eThics sTaTeMenT In this in vitro study, we obtained primary human monocytes from the Immunology Core at the University of Pennsylvania School of Medicine. The Core has the Institutional Review Board approval for blood collection from healthy donors. Anyone who obtains human cells from the Core is considered as secondary use of de-identified human specimens, which does not subject to human subject review by both NIH and IRB. aUThOr cOnTriBUTiOns LG, LZ, XW, J-LL, and W-ZH designed the study. LG, X-QX, R-HZ, J-BL, BZ, and HL performed the experiments. W-ZH supplied reagents needed for this study. LG analyzed and interpreted the data

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and wrote the manuscript. LG and W-ZH reviewed and revised the manuscript. All the authors have read, reviewed, and edited the manuscript and agreed for submission to this journal.

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Reproduction and production in a buffaloes farm of the Marche region: a ten-year study Riassunto Aspetti riproduttivi e produttivi in un allevamento bufalino della regione Marche: un decennio di attività. Sono stati valutati i parametri riproduttivi e produttivi di un’azienda bufalina locata nell’area Marchigiana dell’Appennino Centrale, per un periodo di attività di 13 anni. L’età media al primo parto di 35,9 mesi, sottolinea una notevole precocità riproduttiva degli animali e buone performance riproduttive della mandria (intervallo interparto medio 443 d e durata dell’asciutta 156 d). La produzione di latte è aumentata nel periodo di osservazione fino al massimo produttivo di 2321 kg di latte nell’anno 2002. La non eccellente qualità del latte, prodotto in una zona d’allevamento non vocata, suggerisce la necessità di una stima dei fabbisogni reali delle bufale per raggiungere un titolo lipidico più idoneo alla trasformazione casearia del latte bufalino, che in Italia è destinato unicamente alla produzione di mozzarella. INTRODUCTION -Water buffalo milk reproductive and productive traits are fundamental to evaluate farm efficiency and dairy economy. Due to the buffalo cows' long life these aspects are also important to assess the improvement of genetic animal potential and to reach optimal productive performance. Currently this knowledge is critical since the Italian cattle population is decreasing whereas the national buffalo sector is expanding; this trend is partially depending on the reconversion of some dairy cattle' farms into buffalo cows' farms. The present investigation was undertaken to study non-genetic factors affecting reproductive parameters and milk production traits in a farm located in a not traditional

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area for buffalo farming. MATERIAL AND METHODS -589 complete records regarding the productive careers of 111 buffalo cows from 1 st up to 11 th lactation, reared in a farm of the Marche region, were analysed to outline the productive performance over a period of 13 years: from 1992 up to 2004. The buffalo farm is located in the Marche Apennine area (689 m asl), where climatic and environmental conditions are deeply different from those characterising the Italian buffalo traditional farming areas (Campania, Lazio and Puglia). Reproductive parameters were analysed: age at calving, length of calving interval and length of dry period. The quali-quantitative parameters of buffalo milk produced throughout 1992-2004 were effective lactation length and related milk production, protein and fat percentage and fat/protein ratio. Moreover an estimation of milk energy content was computed according to the NRC prediction equation (1989) based on milk fat content (kcal/kg = 96.2 Fat + 351.2). A three-way ANOVA was performed (Statistica, 1989) to estimate the effects of the non genetic variables year, parity and calving season, known to affect reproductive and productive milk buffaloes traits. For this analysis parities greater than the 7 th were grouped in the same class. RESULTS AND CONCLUSIONS -ANOVA showed a high significant effect of year on all parameters but the length of dry period; parity, as expected, affected age at parturition, length of period between two subsequent deliveries, milk yield and lactation length; the season of calving, which is known to influence buffalo cows careers, affected all parameters but lactation length and

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fat/protein ratio (Table 1). Regarding the reproductive parameters, tightly related to an adequate feeding of heifers in growing phase and to the age at first oestrus, the primiparae showed an average age of 35.92 mo at first calving (Table 2), that was lower than those reported in literature for dairy buffaloes breeds reared in the tropics (www.ssdairy.org) and for Italian buffaloes cows (39.5 mo) controlled by A.I.A. (Matassino and Rossi, 1998). For age at following parturitions a regular increase, depending on the calving interval, was found. Length of period between calvings was in the range of 421-467 days according to results reported by Tekerli et al. (2001) for Anatolian buffaloes; due to its low heritability this trait could be lowered through a better nutrition and an early breeding. Dry period estimated as days between the end of lactation and the beginning of the next one, ranged from 142 up to 165 days. Table 1. Results of ANOVA for the evaluated dependent variables. According to calving season, deliveries where mainly distributed in Summer and Autumn (28.6 and 36.8% respectively); calving interval was shorter for delivery occurring in Autumn (416 d) and higher for the ones occurring in Spring (483 d) certainly because of the buffaloes' seasonal nature and bull presence in the herd (Borghese et al., 1994). The dry period by calving season was shorter for Autumn calves and higher for deliveries occurring in Spring as also observed by Zicarelli et al. (1977). For 607 deliveries occurring in 13 year period, the rate of twinning was very

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low (0.66%) but higher then values reported in literature for swamp buffaloes (Buffalo Production, 1992); for 606 records regarding calf 's sex, the sex ratio was 49.84 % males vs. 50.16 % females. The results of productive parameters are listed in Table 3.

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Meat Me Halfway: Sydney Meat-Loving Men’s Restaurant Experience with Alternative Plant-Based Proteins : Within the theoretical framework of psychological reactance and impression management, this study conducted in Sydney, Australia, in 2020–2021, explores the acceptance by men of alternatives to animal-based foods. Qualitative in-depth interviews were conducted with 36 men who have visited a vegan restaurant and have eaten a plant-based burger. The findings from the study show that, despite the increasing popularity of these novel food options, men are unlikely to include the plant-based alternatives as a permanent feature of their diets as explained by the theory of psychological reactance. However, the study’s male participants acknowledged the importance of women for their visit to the vegan restaurant which can be explained by impression management theory. Using excerpts from the interviews, men’s experience is highlighted, particularly as it relates to concerns linked to masculinity, dietary identity and social perception by others. The analysis reveals the complexity of transitioning to more sustainable food choices within a gender-constructed social environment. Whether the new plant-based alternatives to meat are going to be a short-lived trend or a more lasting option in the men’s diets is also discussed. Practical implications for social marketing as a tool to influence collective behaviour are drawn. They emphasise the role of women, changing social perceptions and transparency about the new plant-based products. Introduction Considered a personal choice in western countries, food options are part of the psychological freedom of consumers who decide their own preferences. By doing so, people exhibit particular behaviours and express individual

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identity. When food choices are perceived to be somehow restricted or non-voluntary, they are cast by consumers as potentially freedom-threatening [1]. The psychological theory of reactance details consumers' response to such perceived reduction in everyday life's personal freedoms [2]. Reactance arises when people feel pressured to make certain choices or are deprived of some options and consequently tend to move in the opposite direction to restore their freedom [1]. On the other hand, when dietary choices are voluntary, they often become part of a dramaturgical act of expressing a person's identity [3] with well-calculated and judged motives to manage a positive impression upon others. Goffman's dramaturgical framework [3], based on theatrical metaphors of acting on a stage, explains the socially constructed self-identity related to food, made even easier nowadays with the use of social media. Human diets and food create a social problem. While in the West there is an increasing number of flexitarians-people who conscientiously decrease their consumption of meat and other animal-based products, there is also reluctance by many to include more plantbased options [4]. Despite the scientific evidence about the many environmental, health, Compared to other cities around the world, such as Barcelona in Spain or Didim on the Aegean coast of Turkey [19], Sydney, with a population of close to 6 million, is not renowned for its vegan options. This, however, may be changing as currently there are 75 entirely vegan restaurants in Sydney and the city is aspiring to become one of the most vegan-friendly places in the world [20]. Many

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of the meals and ingredients used in these restaurants are part of the burgeoning plant-based alternatives sector. We approached some of the male visitors in these vegan restaurants to understand their experience and whether, according to them, there is a future for such plant-based protein options, particularly the plant-based burger. Although many of our findings are not surprising, as men's preference for meat has been widely known, we believe this is the first study to explore male attitudes towards plant-based alternatives. The men interviewed in this study have "crossed the line" by trying such new products and it is of interest to see that women played a big part in this process. Despite this, many challenges remain in order to mainstream such dietary behaviour. The article first presents a brief synopsis of the two theories used in the analysis and the role of social marketing. This is followed by sections describing the methodology of the study and the male sample researched. All in-depth interview questions were openended, and the findings are discussed around five main themes, namely: experience in a vegan restaurant, how important is the perception about masculinity, acceptability of plant-based alternatives, taste experience compared with real meat, and whether these options have a long-term future. The insights from these interviews indicate that many men who have been exposed to such plant-sourced food options are reluctant to embrace them as mainstream preferences. Their behaviour can be explained to a certain degree by the theories of psychological reactance and impression management. This has implications for social

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marketing [21] that can better influence more sustainable food behaviour within society. The contributions of the study and concluding remarks are presented in the final sections of the paper. Psychological Reactance, Impression Management and Social Marketing In the domain of food, it is common for a person to have a range of alternatives and to freely express preferences. In the theory of psychological reactance [2], this is described as freedom, forming the basis for individuals' free behaviours [22]. However, this freedom of choice may be threatened to be diminished or fully eliminated [22] when there is outside influence. Such external influence may come from social pressure to make a particular choice or simply from the availability, or lack thereof, of particular foods. In most cases, this leads to the individual's motivation to re-establish the threatened or eliminated freedom. When people recognise a threat and start experiencing difficulties in exercising their freedom due to external pressure, they experience reactance [22] or unpleasant motivational arousal resulting in behavioural efforts to restore liberty of choice accompanied by negative emotions, such as feeling uncomfortable, hostility, being angry, or aggressiveness [23,24]. According to psychological reactance theory, when a person feels that someone or something is taking away their choices or limiting the range of alternatives, four components of response are effected to resist the social influence of others and restore the freedom. These components are: perceived freedom, threat to freedom, reactance and restoration of freedom [25]. In relation to food choices, the theory of psychological reactance was used to test behavioural

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freedom related to emotional eating [26], prevention of anorexia [27], health education campaign messages [28], excessive drinking and alcohol consumption [29,30], antismoking and tobacco use [31][32][33][34]. It has also been used in other health-related areas, such as condom use for safe sex [35], teeth flossing [36], sun protection [37], skin-cancer protection [38] and anti-inhalant appeals [39]. We use this theory to explain the reactions and behaviours of men in relation to new food choices based on plant-sourced proteins, particularly men's reluctance to maintain these choices as part of their regular diets. With eating being an important social process, people's behaviour can also be seen as acting, and impression management in the eyes of their companions is particularly important [40]. This could be a sufficient trigger for someone to modify their eating behaviour, consciously or subconsciously, to create a particular impression of themselves [41,42], an explanation at the heart of impression management theory [43]. According to Sinha [44], impression management is an active self-presentation of a person who wants to enhance their image in the eyes of others. Newman [45] (p. 184) further explains that impression management is an "act presenting a favourable public image of oneself so that others will form positive judgments" while Scheff [46] stresses that the acting is often aimed at avoiding embarrassment. Impression management involves two motives in response to social norm expectations or restrictions, namely instrumental and expressive. The instrumental motive is guided by the desire for self-esteem and seeks the gaining of rewards, acceptance and respect, while the expressive motive

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pertains to being in control of one's own behaviour and identity, often to present to others something different [47]. Eating in the company of others, particularly in a public place, such as a restaurant, easily becomes a type of stage acting and impression management, as described by Goffman. Neither of these two theories has been previously applied for explaining genderbased food-related behaviour, and particularly men's attitudes toward novel plant-based alternatives such as the plant-based burger (also described as vegan or veggie burger). However, as this study shows, they offer a powerful way to understand men's behaviour and identity when it comes to consuming plant-based alternatives. In a time when dietary changes are urgently needed to respond to the challenges of climate change and planetary boundaries [5], understanding male behaviour when offered plant-based alternatives is very important. Men's diet tends to have a higher environmental footprint, mainly because of the larger intake of red meat [48], and the emissions associated with non-vegetarian, compared with plant-based, diets are also significantly higher [48]. If tackling climate change is to be successful, in addition to finding theoretical explanations about consumer behaviour (as offered by the theories of psychological reactance and impression management), we need to also investigate possible ways for changing people's practices. Men in wealthier countries, such as Australia, need to make a dietary shift towards more plant-based options which are tasty, nutritious and better from a human health perspective. Social marketing can be an avenue for encouraging behavioural changes for improved individual well-being and for the common good

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[49]. The internationally adopted consensus definition that guides approaches to developing research-and theorybased social change programs, is as follows: "Social Marketing seeks to develop and integrate marketing concepts with other approaches to influence behaviours that benefit individuals and communities for the greater social good" [50] (p. 1). Science-supported social marketing campaigns work and Australian examples include sunscreen protection and reduction in the use of tobacco [51]. Knowing how men perceive their experience with new plant-based food options can inform the possible messages of social marketing to make it more effective. Men's reactions need to be properly understood and the theories of psychological reactance and impression management offer tools for explaining male behaviour. Possible ways of framing any social marketing messages are put forward in order for them to be able to influence broader public behaviour. Methodology This qualitative study was based on 36 semi-structured in-depth interviews with male participants who usually consume meat but who had visited vegan restaurants in Sydney, Australia in 2020 or 2021. We wanted to understand the experience of relatively young male Australians who have been exposed to quality professionally prepared vegan food, mainly vegan burgers. In addition to having visited a vegan restaurant, the interviewees were largely selected based on the following criterion: being male representatives of Generation Z (Gen Z) or the Millennials, that is, aged between 18 and 40 years, as these are the population groups most likely to embrace flexitarian or meat reductionist practices and incorporate them as part of their diets [52]. This population are more likely

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than older generations to replace animal-sourced meat with plant-based alternatives. In addition to demographic information related to age, education, employment status and frequency of meat consumption, we used six open-ended questions (see Table 1) and included prompts to direct the flow of the conversation. It was made clear to the participants that the aim of the study was to understand their experience-there were no right or wrong answers. We believe that we managed to establish a relationship of trust with each participant, and that this allowed for open and honest expression of opinion. A snowballing technique was used to recruit the participants starting with a couple of men who have visited a vegan restaurant and agreed to be interviewed. They further suggested other men who would normally eat meat but had experienced the vegan options. We were not interested in men who are usually vegetarian or vegan as they represent a very small fraction of Sydney's population, estimated at less than 6% [53]. We continued the recruiting of participants through snowballing until we achieved repetition of the issues raised during the interviews indicating data saturation or exhaustion of the number of unique opinions offered [54]. The interviews were carried out during the COVID-19 pandemic and precaution was taken to abide by any restrictions. They were conducted at locations convenient for the respondents and in line with any existing requirements at the time. These locations included public open spaces, such as parks, coffee shops and, on a few occasions, the vegan restaurants themselves. There was an equal

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representation of participants from both generations, namely 18 Millennials and 18 Generation Z, with two people per year. Informed consent was obtained from all participants. The interviews lasted between 30 min and 1 h and were conducted face-to-face and recorded. Handwritten notes were not taken during the conversations, in order to help the researcher focus on the interview content and the verbal prompts. Although it took longer to recruit participants during COVID-19 and conduct the interviews due to several lockdowns, we do not consider that the pandemic has had a major influence on the responses we collected. This statement cannot be confirmed and there were many conflating factors around the time of the interviews, such as the 2019-2020 bushfires in Australia, coronavirus outbreaks in meat-processing facilities in the Australian state of Victoria, a quarter more than the average rainfall in Sydney in 2020, followed by a heatwave at the start of 2021. Nevertheless, as the findings from the interviews reveal, the interviewed men were not convinced that, despite the increasing popularity, plant-based meat alternatives would stay as an enduring feature in their diets. The interviews were initially analysed manually and then with NVivo11 to identify the main themes in the respondents' answers. Overall, there was a high level of consistency between the various opinions expressed. Given that this is a qualitative study, any generalisation should be treated with care. The aim was not to generate evidence for statistical generalisation, but "to provide a rich, contextualised understanding of human experience through the intensive study of particular cases"

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[55], (p. 1452). It allowed us to apply theoretical insights that are not exclusive to the analysed sample of young men and their particular circumstances but convey higher-level concepts [55]. This is described as analytical generalisation based on the transferability of the results on a case-by-case basis when similarities exist [53]. Therefore, it is essential to present thick description [56] of the cases that forms the basis for analysis and from which conclusions are drawn. We achieved this by using extensive quotes which outline the circumstances and line of thinking of the participants. Furthermore, to enhance the potential for replication of the findings, we used multi-site sampling of participants across different vegan restaurants in Sydney's metropolitan area, including in the Central Business Area (CBD), Bondi, Enmore, Glebe, Manly, Marrickville, Newtown, Parramatta, Potts Point, Randwick and Surry Hills. Table 2 presents the main demographic characteristics of the interviewed sample. Millennials and Generation Z were equally represented (n = 18 each). All 36 participants were relatively well-educated with at least high school completed-in addition, 12 had a master's degree, 13 held a bachelor's degree and another 6 were pursuing further university studies. In this respect, the selected interviewees were well above the average educational level of Sydney's population, only 14% of whom have a university degree according to the most recent 2016 Australian population census [57] compared with 69% in our case. A higher level of education implies better knowledge and awareness about environmental, human health and social problems. Description of the Interview Sample Furthermore, 23 of the

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participants were in full-time, and 7 in part-time, employment, and 6 were studying at university full-time. Overall, the sample represents men of a variety of career paths who all had the same experience of eating out at a vegan restaurant. This was out-of-the-norm behaviour for many of the participants, given that 24 (66.6%) of the participant men consumed meat daily and the remaining 12 (33.3%) consumed meat 4 to 5 times per week. Again, the sample was not representative of the Sydney population, as our 2018 research indicated that 38% of men consume meat daily and 41% 4 to 6 times per week, with a further 14% 2 to 3 times per week, and 7% once per week or not at all [58]. The relatively higher frequency of meat consumption in the study sample was of interest to us as it represents the section of the population that most needs to reduce its intake of animal proteins if we are to transition to better and more sustainable food choices. Results and Discussion Current food trends show that some of the main reasons why people choose to switch to plant-based options are the smaller ecological footprint, ethical concerns about animal cruelty, and the health benefits of a vegetable-rich diet [12]. With the rise of flexitarianism, people do not have to be vegan to reduce meat in their diets and opt for more plantbased foods. Our male sample represented men who, at least for that meal, have opted to exclude animal-based proteins and were doing this openly in

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a public environment. The latter fact is of importance as a lot of prejudice and misconception exists in Australia against vegetarian men [53]. It is also interesting in relation to the impression management theory [43] we use in this study looking at whether modifying one's food choices and eating behaviour would be beneficial to creating a particular impression in other people's eyes [40]. Both instrumental (seeking self-esteem and rewards) and expressive (being in control) motives were evident during the interviews outlining the men's experience. Furthermore, psychological reactance theory helps to shed light on whether such a behaviour is likely to persist. All four components (perceived freedom, threat to freedom, reactance and restoration) were present and communicated by the interviewees. We present below wordfor-word quotes and the overall thoughts of the participants, following the identified themes from their interviews. This structure is suitable for a thick description as the identified themes emerged directly from the empirical material and were not constrained by the theoretical constructs. The two theories allowed us to understand how meat-loving men felt and what are the possible levers to influence them in the long run. To the best of our knowledge, this is a new approach and a contribution to knowledge. Experience in a Vegan Restaurant Australian men who normally consume meat are rarely confronted with the hostile social attitudes experienced by vegetarians or vegans [59]. On the contrary, for our sample of men, dining at a vegan restaurant was an interesting novelty. They wanted to have first-hand experience with new alternative proteins,

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namely a plant-based burger, which they referred to as a "burger experience". Consciously or subconsciously, this experience was targeted at creating a positive image while downplaying any negative connotations and, as explained in social psychology [41], the impression management was with the purpose of controlling the attributions formed by others about these men. Visiting a vegan restaurant is a suitable "situation" in Goffman's sense [43] where meat-eating men can control their self-presentation and guide the impressions others may form of them. The popularity of new vegan burgers had attracted many of the men who shared how they felt: You don't need to be a vegan to go and try a veggie burger. I am not a vegan, but everyone is talking about it (the burger). I am not even kidding, they are so popular. When we went there, . . . .there were so many people. I was happy to line up with them. It was real fun to chat with the others while meekly waiting for my turn to come. There was . . . kind of fun atmosphere for everyone. I had the feeling that the people waiting around somehow united with me. It's incredible how you could make laugh with other people in the queue. This indicates strong social pressure to try the plant-based burger and for men in particular, to be part of such an experience. They do not want to be perceived wrongly by others in relation to this new trend. This reflects the impression management of the men who want

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to be in control of their own self-esteem and the way they are seen by others. Impression motivation and impression construction [42] are interwoven in their behaviour. In some cases, men are motivated to go to a vegan restaurant because they want to please their partners who may not consume animal-based foods. Although being meateaters themselves, they respect their female partners' choice and occasionally want to join in. Being in a visible conformity with their partners is not only flattering for their loved ones, but is also a good motivator as it enhances their relationship. The interviewee below talks about this while distancing himself from "the propaganda" about meat: The importance of impression construction is signified by the men's desire to control the way others see them. Creating the right impression is not only a conscious act, but also an intentionally embodied desired identity as a person who cares about the values shared by his partner. Vegan burgers are seen as a new trend for which people are prepared to stay in a queue to have that novel experience. With this comes the social pressure and men find themselves being part of this new wave. In a situation like this, men are not convinced they fully possess the freedom of choice, but are happy to construct an image of being open-minded. This may also unite them or just offer a different experience: I didn't know these burgers are becoming too trendy among my friends. We used to go out and eat steaks and burgers in pubs

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and steakhouses, but instead, now we are mingling with the veggie burger eaters. Strange world! ( This man's reaction to reduction in freedom is expressed with the imposition of "mingling with the veggie burger eaters" in a place where conventional meat is not available. However, he also accepts the attractiveness of the new food products. Impression management has been shown to be particularly important in romantic relationships [40]. A big factor in the decision to eat out at a vegan restaurant is the men's feelings and love for their female friend or partner, indicating the social importance of eating. It seems that in such a setting they are less interested to target other men to demonstrate their manliness [17] but instead they want to enjoy the company of the loved ones and an atmosphere free of machoness, where you can connect to your feelings: The defensive position this man takes seeks to protect his image of a meat-eater. This impression management a man strategically makes requires a special effort [43] and can explain the reason behind the decision to eat plant-based alternatives. Modifying eating behaviour in order to create a positive impact on the woman and appear more attractive in a romantic relationship [40] is impression management in action. When a carnivorous man is in love or wants to make a good impression on the opposite sex, he immerses himself in the atmosphere of a vegan restaurant. His negative reactions to consuming only plant-based food are suppressed and he is accepting to give up meat for

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once. To be fully accepted by the woman, he wants to make a positive impression on her: When you want to make your girl happy, you just do whatever it takes to make her happy and this included eating plant-based burgers. (Interviewer: Is she an influencer for the food you eat?) Not really. We eat lots of meat and she cooks some of it when around. But I try to please her when I can. (Male, 19 years old, daily meat eater) Not all men enjoyed the taste of the plant-based burger but pleasing and making their partner happy seemed to be a significant motivator in the vegan restaurant experience. There was, however, an immediate attempt to save the man's freedom with the response "Not really" to whether the female next to him was influencing his food choices. In line with psychological reactance theory, when men's food choices are being threatened, freedom and control need to be re-asserted [2,22]. Impression management and the desire to support the woman in her choice of food seemed to take precedence over the real food experience, despite the impossibility of exercising behavioural freedom: Vegan meal is a totally irritating experience, but when you love someone, you don't have much choice, but just to be there for her. (Interviewer: Was it that difficult for you?) It was not difficult to accompany her at all, but then when I had to eat the burger I ordered, I experienced a certain difficulty. Why? It didn't have that meaty consistency I'm used to eating.

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It was very soft, somehow it didn't smell very good to impress my taste. (Interviewer: Did you share what you just described about your meal with her?) No way. She was going to be so much upset and unhappy . . . There is no way she could hear it. (Interviewer: Do you think this is a good thing to do, especially if you love her?) Not really good, but it's saving the family peace. It's more important than to admit next to her how much I hated this burger. (Male, 40 years old, daily meat eater) Maintaining the "family peace" and the happiness of the romantic partner win over the expressed resistance toward the plant-based alternatives. Experiencing the difficult situation by consciously adopting a behaviour opposite to their inner desire, the men ate the vegan burger recommended by their loved ones. It is important to keep in mind this reaction and worth considering the part women play in men's food choices. Eating out for a plant-based meal often happens in the context of romantic dating, which offers an intriguing behavioural situation where gendered conceptions of impression management are common [40]. Without the need to display that "real men eat meat", a latent positive attitude towards the plant-based burger may persist in a softer gentler environment and with the right impression management [40,43]: At the beginning I was a bit afraid to say that everything on the menu was vegan/vegetarian, but then something clicked, and I haven't put too many thoughts on the meal, but on

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the atmosphere and the woman next to me. This makes me still feel a real macho man despite the plant burger I needed to eat. (Male, 27 years old, daily meat-eater) A negativity towards vegetarian and vegan men was not expressed in the atmosphere of public places serving only plant-based foods. In a social environment where animal flesh is excluded, the men did not need to negotiate their manliness and, in fact, could focus on other aspects of their lives and relationships. The norm is not to eat animal-based foods and this seemed acceptable to most of them. Some, however, still resented the fact that they were not eating animal-based meat, reacting to the loss of freedom and adopting an opinion contrary to the one expected by their partner [23]. In the context of persuasion from their romantic partners, this threat to their freedom was seen by men as attempts at social influence: I felt kind of trapped when at the veggie burger place. My girl enjoyed the many "tasty" options, and I didn't want to make her unhappy, but actually I felt miserable and even I did not enjoy my burger. There wasn't any meat in it to enjoy. (Male, 32 years old, daily meat eater) It seems that irrespective of whether men really liked the plant-based burger or not, there were other important social aspects that they valued more. Excluding the meat-based dishes from the menu seemed to reduce social tensions and could potentially create a better milieu where food dilemmas are seen differently.

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Meat avoidance by men might signal to women many things, including righteousness, self-control, non-violence and agreeability, creating a particular positive impression. It's All about Masculinity Masculinity remained essential for the Sydney men we interviewed, but most importantly, it was seen as restoration of the freedom linked with eating meat. As one of the participants explained: A burger, even plant-based, is simply a burger. I don't think it is a big deal to consume it here and there. The problem arises after that. (Interviewer: Why? What do you mean by "the problem"?) Because I prefer real meat. (Male, 37 years old, daily meat eater) Eating has a physical function to sustain the living body. Essentially, it is about intake of calories that provide energy and essential proteins which the human organism cannot produce. For men in a western society such as Australia, eating has a much broader social function which is about the experience but also how they are perceived by others. It is important for men to demonstrate their masculinity gender traits related to virility, power and hegemony. Although presumably consumed voluntarily, psychological reactance theory suggests that the plant-burger is a symbol of eliminated freedom. The coercive avoidance of meat makes a man feel and believe his perception of masculinity and freedom are sacrificed. By explaining his action was forced, the man tries to restore his manliness and to regain a masculine image. The threat was clearly visible from the words of the interviewees, who felt obliged to counter-react. Such a loss of freedom is further

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exacerbated by the general perception that individuals who eat a meat-based diet are more masculine than people who follow a vegetarian diet [40,59]. Ironically, high consumption of meat and dairy products has been associated with many negative consequences, including increased rates of erectile dysfunction [60] and unpleasant scent [61]. Previous research has shown that "men can use impression management through food intake to bolster their masculine identity" [40], (p. 76). This is expressed by selecting what are perceived to be masculine food options, particularly when their identity is threatened [40]. Visiting a vegan restaurant is a perfect situation which requires impression management. The image that men portray is extremely important, more so for other males. Being ridiculed for not eating meat is a big concern: I did it only because my girlfriend asked me to do this for her. Male needs to be strong and play by the rules to make their girl happy. When she is happy, I am happy. This is how things work . . . This man's words clearly show the application of the two theories-he is divided between the need to defend his threatened freedom and maintain self-presentation. Changing his consumption behaviour is not simple because the needed compliance with the romantic partner's desires generates psychological reactance [2], as well as fear of potential damage to his social self-presentation. The above interviewee touches on being ridiculed by others for going vegan. This is seen as a betrayal of masculinity, even among the younger generations of Millennials and Generation Z. They feel

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compelled to remain as macho as possible and food choices are part of building this image [40]. The interview excerpt below shows that, although the man liked the burger, which he describes in the same terms he would use for animal-based meat, he still wants to be seen as being "a carnivore": My partner is trying very hard to sway a carnivore like me and I can tell you that some of the attempts are quite spot on, like with the vegan burger I ate the other week. It was juicier, less bleeding than contemporary meat, but in her company, I am easy to get swayed. (Male, 33 years old, daily meat eater) Being exposed to the broader public with the image of a non-meat eater is another fear that the interviewed men have. Food selfies in the social media are similarly seen at the crossroads between the two theories applied. On the one hand, they symbolise positive impression management in the eyes of others, and in particular the women men love. They provide documentation of the personal satisfaction from the eating experience. On the other hand, publishing a vegan selfie can destroy a man's masculine self-actualisation and reputation among his peers. Sharing food selfies on social media from the vegan restaurant is seen as a formula for trouble and irreversible image destruction. One of the participants explained his feeling of being trapped when his girlfriend takes a photo of him eating at a vegan restaurant and makes it available on Instagram. Although the feelings are

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not as strong to be seen as anger and aggression, often associated with psychological reactance [23,24], the man expresses psychological arousal [24] and refers to the need to search for explanations to justify his true identity: If the expectation is that after going to a vegan restaurant and experiencing plantbased food men would change, this did not seem to be the case with the people we interviewed. They were particularly worried about the judgments by others based on their, albeit temporary, plant-based food choice. Plant-based burgers are not the right option for me. It's destroying my manly image. (Male, 39 years old, daily meat-eater) For them, eating meat is a masculinity fortress that needs to be protected. The social perception of masculinity which disassociates from eating plants, was dominant even when seen by others who also ate plant-based foods. A judgment based on a choice away from the established norms for men causes stress: I was under constant stress how to get out of the situation, especially if some acquaintance of mine accidentally shows up. And this is entirely possible, because now everyone is crazy and wants to try vegetarian burgers. (Male, 38 years old, daily meat-eater) This is an expression of reactance measures intended to explain greater variance in attitude toward plant-based burgers. There were negative concerns voiced by some of our male participants around the impact plant-based alternatives may have on them physically: It's not a quality thing to eat these plant-based burgers. Such food effeminates you, you could end up growing some soya

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enhanced breast like the same thing you get when you eat chicken fed with hormones. (Male 37 years old, daily meat eater) Similar concerns were raised in other parts of the world that plant-based meat could possibility make men more feminine and "grow boobs" [63]. If men share such concerns in relation to plants, it would be difficult to justify why they would drink cow's milk. Consumption of new meat alternatives by people who usually are not keen on these dietary options may increase their understanding of those who regularly consume plantbased foods. They may even begin to show empathy for others who make such a choice. However, for this to happen, many social barriers must be overcome, especially perceptions related to reduced masculinity. Eating plant-based meals needs to become the new normal, as is the case for their female friends and partners, without whom they would not have had the vegan restaurant experience. However, as explained by the psychological reactance theory, the public has shown resistance to many cogent health messages [28] and is likely to equally reject points related to environmental persuasion. Instead of trying to restore the threatened or eliminated freedoms, men could accept the plant-based options as the new normal and use impression management in their new role. This would require changes in the subsequent perceptions about vegetarian and vegan men by making them similarly normal. A lot of the social fear seems to exist about labelling somebody as a "vegan", which is an important observation for any marketing, including social marketing,

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that promotes the consumption of more plant-based foods. What is considered normal is likely to shift in our lifetimes and people, particularly men, can choose to be one of the first to change, or alternatively be one of the laggards. This particular notion could be related, not to threatened freedoms to food choices and expression of masculinity, but to masculine notions of leadership, taking responsibility and of being a trendsetter. Plant-Based Alternatives Some of the nuances revealed during the in-depth interviews indicated a degree of acceptance of the plant-based options. The acceptance of their presence as an option was expressed with specific attention to maintaining the desired impression and reaffirming the possible discrepancy between how they wanted to be perceived and how other people may perceive them [64]. As one of the participants explained: I am pretty much a carnivore, but I have gladly given one of the plant-burger a go. (Male, 35 years old, daily meat eater) This also advances knowledge toward understanding the complexity of the factors influencing food intake in relation to the stereotype of "real men eat meat" [16] and impression management as an important influence on people's eating behaviour [40]. Some of the beliefs expressed by the participants can be also seen as self-awareness and self-deprecating jokes. In a similar vein, another participant explained his acceptance of occasionally eating plant-based options: I think consuming meat analogues in the form of burgers is fine here and there as long as it doesn't screw my reputation. (Male, 24 years old, consumes meat 4-5

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times per week) Interestingly, with Australia being a multicultural society, there was a broad acceptance if men who ate plant-based burgers because of religious beliefs. This somehow protected them from being labelled "vegan" or unmanly: Many people are eating these analogues as part of their religious practices, like India, Bangladesh and other countries or just to merge with the surroundings and the people around them. (Male, 33 years old, consumes meat 4-5 times per week) Some participants however questioned whether putting forward religious or health justifications was not a way to hide the real intention to give up meat-another reasoning for potential reactance. This was seen as a plot or mass betrayal by people who are giving up the present status quo: Religious, animal welfare . . . people are having heaps of excuses for not consuming one thing or another. Many people are following them to justify their own vegetarian consumption. (Male, 22 years old, daily meat-eater) The novel plant-based analogues are also seen as a new undesired trend penetrating Australian society, including the supermarkets: Once I bought some, I think Danish burgers, by mistake. I didn't know there are selling fake meat next to real meat. (Male, 21 years old, daily meat-eater) While women seem to play the main part in this trendsetting, men are prepared to follow to a certain degree, just to maintain an impression in the eyes of their romantic partners: My wife is fanatic about new food and always wants to try things. There were so many experiments with some

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plant-based options I never understood from where she was bringing them home. Another acknowledgement about women setting the trend is present in the words of the following interviewee who sought to balance the benefits of plant-based and animal meat options: I don't think there is some sort of belief and structure that guide my wife's daily eating decisions. Even she eats less meat, she continues eating meat because it is a good source of iron she is lacking. Also, vitamins, minerals, B12, zinc, everything precious and much needed for our body to function is in meat. The desire to visit vegan restaurants in my opinion is not because she is that much in love with the veggies, but purely trend-based. She likes these kinds of things and obviously she guides my dietary practices and choices too, ha-ha-ha. (Male, 40 years old, daily meat-eater) Some saw the plant-based alternatives as a non-Australian thing, particularly as Australia prides itself on producing quality meat. Such options were perceived as foreign to the Australian industry and another justification for creating a feeling of limitation and reactance: I believe there are not many plant-based products in Australia. I've heard about some American brands "Beyond" and "Impossible", but not other Aussie brands. Maybe Sanitarium and Nestle do something. I am sure I saw something there . . . but apart from these I don't know others. (Male, 33 years old, consumes meat 4-5 times per week) This expressed opinion highlights potential opportunities for domestic producers in Australia, as national brands for meat

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alternatives are likely to be important and respected amongst consumers. The emphasis on the foreign origin of the plant-based alternatives mimicking meat may well be because people do not know how to properly use plant-based ingredients in food preparation at home or feel that the taste is simply inferior: Till recently I didn't know these things existed. I thought they have them only at restaurants. Maybe if you buy them and don't know how to cook it, you may not have a good experience with it. A friend of mine bought some plant-based mince and tried to use it for the Bolognese sauce. She said that the final result was absolutely yuck . . . inedible. Maybe we will need some practice to familiarise ourselves with these products. (Male, 26 years old, daily meat-eater) Others are simply not prepared to explore such new foods in their normal buying routine: I never buy any of these animal-free products. I am not familiar with them. I heard they sell them in Coles and Woolies [major Australian supermarket chains], but I never paid any attention to them. (Male, 34 years old, daily meat-eater) Some of the interviewees perceive them as something that is interfering with their usual choice and creates unwanted limitations to what they want to consume: Not sure why we need these alternatives when there are varieties of already existing cuts of meat, salami, sausages, bacon, all good stuff we all enjoy on a daily basis. Sometimes I feel suppressed by their existence and the desire of

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my girlfriend to consume plant-based imitations, it's honestly giving me a headache. (Male, 25 years old, daily meat-eater) There needs to be a lot of effort to break down the conservatism associated with food. Messages which are constructed in a freedom-threatening language or are understood as direct persuasion, are likely to provoke even stronger reactance [25,28]. Instead, the use of choice-enhancing language and arguments which emphasize the availability of different options, is more appropriate [28]. It is important to focus on adding another option to the list of foods rather than limiting the freedoms of Australian men and denying them the right of access to "real meat". Taste Experience The sensory experience is important for any type of food, and this applies equally to plant-based alternatives. While temporarily modifying their eating behaviour to create the desired impression management in the eyes of others, eleven of the interviewees (40% of the sample) expressed concerns about the taste of these new foods: It's like consuming fried or minced bun between buns. (Interviewer: Interesting description. Why do you think that?) Tasteless for me . . . not even close to real meat. You could have it once but that's it . . . You can't repeat it again. It's a waste of time. Many properties of the plant-based alternatives, which directly impact on people's taste buds, such as taste, texture, flavour and juiciness, were seen as a barrier to future consumption and essentially a loss of freedom of choice, unless they are improved: I can't say they (plant-based burgers)

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were anything special. Rather a little greasy and not meaty. (Male, 38 years old, daily meat eater) Sensory properties constitute crucial parameters whether the plant-based meat "looks, cooks and satisfies like beef" [65] and delivers "the juicy, delicious taste you know and love, while being better for you and the planet" [66], especially for meat-loving men. While food companies claim that these new "impossible" products made from plants use 87% less water, generate 89% less greenhouse gas emissions and require 96% less land [67], the meat-eating consumer ultimately cares most about how they taste. Good sensory properties are likely to shift men's perceptions about alternative proteins from dull to desirable, with a 2018 survey by Mintel showing that taste was the top reason why US adults ate such foods [68]. Both the sensory properties of plant-based alternatives and consumer perceptions of such products are improving over time [69]. The unfamiliarity and uncertainty about the taste qualities of the new food products are a major barrier for Australian consumers as well: I was sceptic about plant-based burgers as everywhere they are advertised with the promises of the same taste as meat, same appearance as meat etc. At the end it is unclear whether I was going to have the same eating experience as before when I was chewing on a real steak. (Male, 39 years old, daily meat eater) Even concerns about possible COVID-19 outbreaks or infections with other diseases are not seen as threats to consumer freedom. They take backstage compared to the taste experience: Bleeds,

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taste, maybe will be able to avoid some food worries around contracting diseases, but it still needs a big room for improvement. (Male, 33 years old, consumes meat 4-5 times per week) There is a strong message from this group of consumers that the taste experience needs to be comparable to animal meat for them to accept the new alternatives and not be seen as resistant or reactant to the social influence of others in favour of consuming plant-based burgers. In addition to the issues related to taste, the complexity surrounding consumption stereotypes and impression management, including the viewpoints about meat and masculinity, is not yet well-understood [40]. This is also directly linked to how the meat-eating men see the future of plant-based proteins. Future of This Trend Plant-based alternatives are projected to reach substantial growth [10,12]. Although most of the interviewees agreed that these products were likely to remain a constant feature on the food menu, some (namely, 13 or 36% of the sample) were of the view that this was only a temporary trend. They highlighted a range of concerns that will impede the long-term adoption of these products and their penetration as a widely accepted choice. Below, we discuss three main reasons expressed as to why the current trend may lose momentum, namely: this is just a curiosity which will wear off, the new products are unhealthy, and there is a lack of transparency about how they are made. Curiosity Many people try plant-based meat alternatives out of curiosity. Being a driver of

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progress, curiosity relates to inquisitive thinking, which makes people enter the unknown, unexplored new areas of knowledge, search for novel experiences, face and be attracted to unfamiliar situations. Many people feel the same way about food, although some may experience neophobia [70]. Among the participant sample, there was a shared opinion that men wanted to have this new experience but not much support that this would lead to a more permanent inclusion of such plant-based options in their diet: I was glad I had the chance to try the plant-based burger as I have heard so many things about it and was wondering whether I will like it or not. (Interviewer: How was it?) I can't say I was too impressed; it was a bit greasy for my taste, but I tried it and I kind of ticked the box. (Male, 30 years old, consumes meat 4-5 times per week) "Pure curiosity" was driving many of the men who also wanted to reaffirm their current food choices, leaving little, if not no, space for adding these options on their menu: Now they are popular as they are something pretty new. I was trying of pure curiosity. I don't think we will be eating these (plant-based) and abandon our meat, but I was just curious to try and reassure myself that this is the case. (Male, 29 years old, eats meat 4-5 times per week) Many of the interviewees were aware of the multitude of problems associated with current livestock production, including industrial farming, overuse of environmental

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resources, pollution and contribution to antimicrobial resistance, but that was not convincing enough to see the plant-based alternative proteins as a long-term solution, despite admitting their current popularity: I can say that plant-based meat is truly having its moment, right now, but not forever. It's despite the livestock industry induced problems with factory farming, antibiotic resistance and environmental problems. Plant-based is having a momentum, so do their producers . . . . I don't think that it will replace consumers' meat purchases. (Male, 25 years old, daily meat-eater) Health concerns also featured as part of plant-based alternatives being only something to try but not to adhere to in the long-run: I was listening to a podcast last week and they were discussing the projections of plantbased alternatives to go to the roof. Consumers' demand and sentiments linked to animal welfare, health and the environment were pointed to as reasons for the growing trend, but these are all gimmicks. Everyone is keen to try as it's something new because people are curious to know what is out there, but people will be quickly fed up with it . . . (Male, 18 years old, consumes meat 4-5 times per week) In line with the prevalent opinion among our research participants, the 2020 survey by the International Food Information Council Foundation [71] also placed curiosity as the primary reason for consuming plant-based alternatives and ahead of benefits for the environment and human health [72]. Curiosity was also linked to the availability of choices and the understanding of the

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freedoms a consumer has. If plant-based protein options are perceived as enhancing people's image and self-presentation, then curiosity may deliver longer-term effects as indicated by the impression management theory. Concerns about Unhealthiness Eight of the interviewed men (22% of the sample) believed that the current trend of plant-based alternatives will discontinue because these new options are unhealthy to consume. This opinion was expressed predominantly by Generation Z consumers who were also well-informed about the benefits of meat reduction: Why should we eat plant-based meat if we have real meat? Plant-based are unhealthy. They contain high sodium contents and saturated fat, basically equivalent fat and caloric contents to meat. Maybe it is far less saturated fat than animal meat on average, but it is still saturated fat. If you reduce your meat intake, you will have to digest less saturated fat than you will with plant-based alternatives. I don't think the whole madness with the plant-based is for real. (Male, 20 years old, consumes meat 4-5 times per week) Generation Z, which is the largest age cohort in Australia, as well as in the world, wanted reassurance about the health benefits of the new plant-based proteins before they committed to eating this food on a regular basis: Plant-based is nothing better for our health compared to meat. They are ultra-processed imitations, and I think no matter how hard the industry tries to replicate the taste and the cooking, sizzling and whatever experience of meat, I am not convinced that I want to consume it without a clear

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indication of plant-based analogues' health benefits (Interviewer: If you are given this reassurance are you going to react differently?) Mmmm, honestly, I am not sure. Even if they want to make them sound healthier, they can't. I read they are too processed . . . It's difficult to decide. I am more inclining toward not eating them. (Male, 21 years old, daily meat-eater) Scepticism about the health credentials of plant-based meat analogues seems to be common among consumers despite evidence that they are lower in cholesterol and have less salt than many animal-based foods. This was also confirmed by the 2020 survey of the International Food Information Council Foundation [73], in which a sizeable minority of 25% of Americans believed plant alternatives to be unhealthier. Products being highly processed was a common concern as explained by the participant below: The ways the industry uses soybeans, peas and wheat to create ingredients for plant-based alternatives are not good as the product is highly processed. Then we are offered to eat it without being aware of the price. (Male, 24 years old, daily meat-eater) Concerns about the novel plant-based meat alternatives being unhealthy were confirmed with the expressed desire to eat vegetables and fruits directly. This was seen as restoration of the freedom to consume better and healthier alternatives: Eating vegetables is good for you but being plant-based does not automatically makes it healthy. Plant-based meats are just using ingredients that come from plants, and they are the food chemist pride. We don't know what people like about

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the taste of meat. Believe it or not, even considered not so healthy to eat, meat is meat, and it will remain the preferred food for the majority of people in Australia. (Male, 22 years old, daily meat-eater) There were also some who saw plant-based alternatives as something that may be consumed by others, but not themselves, because of concerns related to healthiness and taste: These products are marketed as containing protein from plants and as long as I am aware, they are formulated to provide an option for non-meat consumers. Not me by the way, as I love eating meat. But made from plants does not necessarily mean they are providing a healthy protein. Who knows what is in there? . . . . Also, by default, plants contain less protein than real meat. (Interviewer: Do you think plant-based alternatives have a chance with meat-eaters like you?) Ha-ha-ha. Maybe if they make it similar to at least processed meat like salami, sausages, plant-based options will have the best chances to replace meat. But they . . . I mean the producers, the industry is not yet there. (Male, 37 years old, daily meat-eater) The survey of the International Food Information Council [69] found that consumers perceive plant-based alternatives as being healthier when it comes to vitamins and minerals content, including specific amounts but were concerned about the presence of sodium. This makes ingredient disclosure very important and is again related to the freedom of choices for the consumer. Lack of Transparency The Generation Z participants

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in our study expressed demand for clear labelling and production transparency for these products. This can be seen as a beneficial step for the industry to build consumer confidence and avoid future undesired surprises. Reactance should be seen not just as an opposition to availability of choices but also as a strategy of empowerment [28] where the consumer has the right to know: You know, producers need to let us know what they are putting into these plant-based options. There is not much transparency about the hidden ingredients. I know they are including the general stuff in the label, but there are many small doses of chemicals that are not disclosed. I read about this recently in one news article. It's quite disturbing. When people find out what is in it, this will be the end of it. (Male, 19 years old, daily meat eater) Reactance triggers cognitive processes and acts as motivation forcing individuals to do something to change the circumstances associated with the threat [22,24]. Parallels can be drawn with Generation Z's attitude towards cultured meat where they strongly question the motivation and quality of the food products [74]. Better transparency could help plantbased alternatives to be perceived more favourably by consumers. This is quite important for Generation Z who are already feeling the burden of inheriting the problems created by preceding generations, including climate change, biodiversity loss, reduced soil fertility, contaminated land and waterbodies, plastics pollution and many other environmental problems. Unknowns and reluctance related to accepting genetically modified organisms (GMOs) were also

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stated as part of the need for transparency, particularly when linked to the freedom of choice and the ability to make informed decisions: Not sure if they use GMOs and other ingredients that are against my health-conscious lifestyle. We need to know and this needs to be visibly included in the labels. (Male, 26 years old, consumes meat 4-5 times per week) As can be seen from the questions posed to the Sydney men (see Table 1), we did not specifically target issues related to perceptions about masculinity. However, this theme emerged strongly during the interviews. The association between meat and masculinity may be the result of toxic masculinity [75] or hegemonic masculinity [76] within Australian society. We also did not attempt to measure the degree of meat-loving men's psychological reactance, an area that is increasingly gaining momentum [23,24]. These aspects require further investigation. The role of friends and social media in influencing what men think was also not investigated. Most importantly, there were no questions soliciting answers related to Sydney men's environmental attitudes, animal welfare considerations, or concerns about climate change. These themes were expressed in some of the interviews but, overall, they were brushed over as issues of minor importance. This was unexpected for us as we assumed that the main reason for people to eat out at a vegan restaurant would be environmental and animal welfare concerns. However, this was not the case with the meat-loving Sydney men whose experience and intentions we present here. Although concerns were raised about the potential unhealthiness

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of the new plant-based alternative proteins, there was no discussion of the problems associated with livestock production. In fact, the perception was that animal-based foods are healthy, nutritious and should be a preferred option. This came as a surprise to us given the fact that the sample consisted of men who were relatively highly educated (see Table 2) compared to the average Australian population. All the above issues can be investigated further. Meet Me Halfway: Social Marketing Implications We interviewed the participant men about their behaviour in a public space, namely at a vegan restaurant, but have no indication whether this experience has affected their food choices in the private realm. We hope this to have been the case, but there were no signs that these meat-eaters have embraced plant-based alternatives in one way or another. Our study, however, confirmed the power of the two theories to explain men's behaviour in the public sphere in the company of girlfriends, partners, friends and when they can be seen by others. There were strong voices of disagreement and suspicion raised by the men in our study who, on the surface, can be considered as part of the shift we need to see to ensure the availability of healthy and nutritional food across the globe. Previously, we have argued for social marketing to be used to facilitate a transition towards better and more sustainable food choices [51]. The analysis in this study, however, presents a new perspective and potential pitfalls. We were able to identify two complementary reactions supported

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by the theories of psychological reactance and impression management. Currently, Australian men who are used to regularly consuming meat are unlikely to include plant-based alternatives as an enduring feature of their diets as predicted by the theory of psychological reactance which explains that persuasion poses a threat to the person's free behaviour and induces resistance to change [24,25]. However, the study's male participants acknowledged the importance of women for their visit to the vegan restaurant which can be accounted for by impression management theory. If social marketing is pushed too much, it can be perceived as limiting people's freedoms. On the other hand, building on positive impressions can help steer better food choices. These Australian men expressed a broader unwillingness to consume plant-based alternatives despite any good intentions behind the development and introduction of the new products. What the analysis was able to suggest is that certain levers can be used in social advertising for the common good. Below are several points that can inform social marketing initiatives: • Women have a very important role to play in any food transitions, not only as mothers but also as partners to the men who want to maintain good relationships. Eating is a daily necessity and the power of women, be it subtle or by expressing explicit preferences, is important. • The link between meat and masculinity is mainly targeted at other men rather than women. Men should think about how their dietary choices are seen by women and about the overall food-related perceptions that exist in society.

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Industry marketing has contributed much to creating messages linking meat and machoness. Social marketing needs to break these mental connections and create images that are better suited for our time of an environment and climate emergency. Manliness has also a caring side and this should be emphasised in relation to climate change and environmental deterioration. • It is important to stop labelling people as "vegan", "flexitarian" or "meat-eaters" as this causes divisions and disagreements among all stakeholders in this complex problem area. Plant-based meat alternatives should aim to become just another, better food option for people, particularly in western countries. • By being transparent and delivering genuinely healthier options, the new food industry can gain credibility and broader acceptance. Its claims, including through marketing, will be monitored closely by Generation Z, Millennials and any other people who are hesitant to change their diets. Social marketing, informed in this case by the theories of psychological reactance and impression management, has a role to play in providing a space free of misinformation and false claims while offering possible solutions to the complexity of the food problem. The theories of psychological reactance and image management explain human behaviour; in this case we used them for a situation when men were challenged in a new food environment. Even with good intentions, men are at the crossroads, raising concerns related to what is considered masculine behaviour, and about mistrust of the new plant-based alternative proteins. The mere fact that they have dined out at a vegan restaurant shows that they are

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willing to explore and be challenged. They need to be met halfway on the road of transformation without fear of being ridiculed, misled, or physically or emotionally compromised with their sense of freedom threatened. Using women as influencers, men's attachment to meat needs to be overcome for the broader personal, social and environmental benefits. Social marketing has been used to address other complex social problems, such as alcohol and tobacco consumption, and, in isolated cases, for meat consumption [77]. It is time to start using the power of "social marketing theories, concepts, discourses and practice, to generate critique . . . and change that facilitates social good" [21] (p. 86) for transitioning to environmentally and nutritionally better diets. Contributions of the Study In a time when there is a growing body of convincing scientific evidence that dietary shifts in high-income countries, such as Australia, can generate substantial climate dividends [78], behaviour changes are essential. This study is responding to these challenges by extending the application of existing theories to a new area which positions it in the middle of the contribution continuum ranging from straight replication to new theory development [79]. Its main contributions are as follows. Firstly, the theories of psychological reactance and impression management have not been previously used individually or in combination to explain male attitudes towards plant-based alternative proteins. This aspect is particularly important as men tend to consume higher amounts of meat. In the latest Australian dietary survey, men were found to consume 32% more meat-based products than women [80]. Moreover,

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Australians have one of the highest per capita consumptions of meat in the world [78]. The two social behaviour theories explain the importance of perceptions about freedom of choice and the construction of male identities within society. They confirm that eating is much more than satisfying hunger and is a social process where men want to be in control of the choices they make and how they are perceived. Without working on these two aspects, including through social marketing, a dietary change among Australian male population is unlikely to occur within a foreseeable future. This means that there will be missed opportunities for acting on climate change through reducing the greenhouse gas emissions of food, on increasing the potential for carbon sequestration by freeing up land currently used for livestock grazing and feed as well as improving public health. Backed-up by empirical evidence and informed by theory, the study was able to identify the nature of social marketing messages that need to be communicated to Australian men. Secondly, although the study focused on men only, it confirmed that women play a major role in the dietary choices of their partners. This continues to be the case with new plant-based alternative proteins. Both theories explained different aspects of the role of women. The impression management theory is particularly strong in highlighting the desire of men to construct an image that is pleasing for their romantic partners. Psychological reactance on the other hand showed the importance of women not limiting the food choices men have and emphasising the

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availability of choice. This is in line with previous research in flexitarianism [4] which aims at reduction in meat consumption rather than elimination of animal-based food options. Thirdly, this study was able to identify very strong attitudes of mistrust towards the industry players in the field of new plant-based alternative proteins. Psychological reactance acts as a tool of empowerment where individuals emphasise their right to know and the need for transparency about the nutritional and health values of the new products on the market. Impression management on the other hand can be used by such empowered men to create new male identities built around making good and environmentally responsible behaviour. Fourthly, this is the first analysis which links psychological reactance, impression management and social marketing in relation to a dietary shift towards more sustainable food options. Social marketing aims to educate people so that they can make informed healthier choices regarding their diet, health and lifestyle [81]. It is being supported by governments, industry, charity organisations and broadly within society [81]. Message relevance and message framing are essential to avoid persuasion resistance, as explained by psychological reactance, and counteract existing trends within society often associated with particular negative images or labelling of people, as expounded by impression management. The study was able to identify possible social marketing messages and ways to frame the most-needed food transition. Conclusions The two theories that we used to describe men's behaviour following their experience in a vegan restaurant, namely psychological reactance and impression management, have been around for a long

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time. They have been used for more than half a century in many other situations to explain social behaviour. We were able to show that they equally apply in the case of novel alternative plant-based proteins. Good theories are indeed expected to possess explanatory power. Understanding how the meat-eating men react to what they perceive as their right of choice and how they manage the impressions constructed by others, is also essential in making the transition from theory to practice. We expect social marketing with research-backed messages (for example, as identified in this study) to influence this food transition. New plant-based alternatives, including the vegan burger, may have a role in the future but, for now, Australian men need to be convinced. Those of them who love meat demonstrate strong reactance to the deprivation of choice and fervent attempts for impression management that disassociates them from plant-based food options. There are however other contributions that this study was able to make that relate to the framing of food choices and the male identities: • Plant-based foods, including the veggie burger, need to be communicated as options which empower consumer choices with supporting evidence that they are nutritionally healthier and ecologically better -this is likely to suppress reactance towards them; • From food being at the core of the male identity as represented by meat-based options, it needs to become the centre of care about the future -masculinity needs to be communicated as being defined not by what men eat but what men do about other people

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and the planet. Given the heavy environmental footprint of our current meat consumption, plantsourced foods will need to have a prominent presence in the future [5,78]. Such a scenario may change some of the current explanations provided by the theories of psychological reactance and impression management; however, for now, they provide important insights about men's behaviour. Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Curtin University Human Research Ethics Committee. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: All data is contained within the manuscript.

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System-wide identification and prioritization of enzyme substrates by thermal analysis Despite the immense importance of enzyme–substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change their thermal stability. In our proof-of-concept studies, SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, opening opportunities to investigate the effect of post-translational modifications on signal transduction and facilitate drug discovery. 'differential' approach. This is certainly an interesting adaptation of the TPP strategy that may address an existing need in screening for enzyme substrates, for instance when studying these enzymes as potential drug targets. Unfortunately, the presentation of the manuscript is far from optimal and a number of points listed below have to be addressed. Major 1. The authors use a lysate (mild conditions) and add recombinant enzyme + substrate to study in vitro substrates of this enzyme. Although their approach undoubtedly generates interesting data, this strategy may be prone to false positive identifications. PTMs are mainly regulated spatially by bringing substrate and enzyme in close proximity (or not). Now, in a lysate this level of regulation is

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completely gone. I do not necessarily see a problem in using such a strategy, but I definitely miss a clear statement on this approach in order to make the reader aware of potential shortcomings. For instance, protein/substrate interactions may be specific to a certain organelle. I was first also wondering about secondary effects, which the authors exclude based on the fact that the lysate is 77x fold diluted compared to the cell, but that also means that a huge excess of enzyme is added to drive the reaction which can lead to unspecific reactions. The authors validate some of their findings using recombinant proteins, some of which being expressed in E coli, which may also be problematic due to different PTM patterns compared to human expression. The incubation of recombinant potential substrate with the enzyme and cosubstrate can again lead to artificial modifications that may not occur under physiological conditions and, unfortunately, I cannot see any kind of negative control for any of the three validation experiments, which is essential to ensure that not any protein will be modified in this setting. 2. In the same context, the authors used a method to "track phosphate release" which is not wellexplained, stating that they preferred this over p-proteomics, as the phosphate release represents all p-sites, whereas MS will only show individual sites (and I guess may miss some based on sequence inaccessibility). Indeed, MS would be an important additional validation to ensure that there is not too many different p-sites on the recombinant proteins under the given

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conditions, as the identification of too many sites would support the note of low specificity in such experiments. Also the sites should be compared with Phosphositeplus. 3. Line 164: The authors speculate that secondary reactions would occur when using NADPH alone, as well, but this is not completely obvious to me ---do the authors refer to the same reactions? 4. Line 330: the authors discuss how the cutoffs for considering a protein as shifted can be changed and how this impacts the list of potential substrates. Using a suitable cutoff is of course a common problem in quantitative proteomics, but the authors have to discuss this in the context of the robustness of the method ---how is the variation in melting temperature between replicates? Indeed, the cutoff being used in the end should be based on this variation in order to produce more robust data, than by applying kind of arbitrary cutoffs. 5. Line 365-372. The authors speculate on the effects of PTMs on protein stability and that this will be hard to generalize. The beginning and end of this 8-line section is somewhat redundant. The authors should mention the concept of PTM crosstalk and the PTM code. Indeed it would be really surprising if there was a general trend, as it seems that the entirety of PTMs dictate a proteins structure and function rather than individual PTMs, which may be one reason why the authors fail to verify some known substrates, they may simply be to heterogeneous within the sample which may even have functional/biological

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implications. 6. In general the writing has to be revised, as many statements throughout the manuscript are somewhat confusing or not ideal. a. For instance the section between lines 98 and 101 should be rephrased as it is hard to read. b. Lines 148-149: What do the authors mean with top 100-400 SIESTA proteins, this is not very specific. c. What exactly is meant with "variable influence on projection" VIP values? It would be helpful for the reader to get a short statement on that, as it may not be common knowledge. d. Lines 178-183: Please rephrase this part, it is confusing. e. Line 232: Please summarize the assay to track phosphatase release in one sentence. f. Line 252: Please mention that NAD is a cosubstrate, this is not obvious from the text. g. Line 270 (and elsewhere): The term targeted MS is used in a wrong way. This typically refers to SRM/MRM and PRM assays, but what the authors used here is a triggered second MS/MS scan based on a decision-tree. Also the second MS/MS used EThcD ( the term is not used though) while the corresponding figure 4c reads "targeted ETD MS/MS", which is not correct in two ways. h. Line 288: "coverage with trypsin digestion was not complete" -it was also not complete for the previously mentioned examples, and a 100% sequence coverage might be extremely rare. i. line 307: although the hot spot TPP was briefly mentioned in the introduction, it would be helpful to have a short statement how it actually

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works, particularly as the authors refer to this repeatedly, e.g. line 318 where the statement is hard to access without actually knowing how it works. j. line 402: I don't think a protein concentration can be distributed in aliquots. k. line 343 (and was also somewhere else): please exchange 'protein molecules' which could refer to different proteins by 'copies of the same protein' or similar to clarify what is meant here. Minor 1. Line 55: the authors should add work from the Krogan lab (PMID 22817900) to their introduction. 2. Line 59: Please add some references to the statement on using modified substrates as readout. 3. From the main text (e.g. figure 2) it is not obvious that Cys-levels were indeed checked using iodo-TMT. This was confusing and only made sense when reading the method section later. 4. The terms substrate and co-substrate in connection to enzymes are widely used. The authors should rephrase their statement on page 4, lines 88-90, which sounds as if this was specific for the manuscript at hand. 5. Line 322: The authors mention that different PTMs induce different thermal shifts. Please summarize the average/median shifts observed in the 3 different examples given in this study. 6. Line 347: The authors mention sub-cellular fractionation as a way to increase the depth of SIESTA. This implies that the lysis must be so mild, that organelles are still intact. The authors should comment on strengths and limitations of this. 7. The supplemental tables should have more explanations on what is actually presented. It

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would be good to have a better connection between the "raw" data and the lists of substrates, maybe by combining them as different sheets into a single table rather than having so many individual tables per experiment. Reviewer #3 (Remarks to the Author): "System-wide identification and prioritization of enzyme substrate by thermal analysis" submitted for publication in Nature Communication. In this manuscript Amir Ata Saei et al. propose a workflow based on the mass spectrometry CEllular Thermal Shift Assay (MS-CETSA) to discover post-translational modifications produced by enzymes. Their hypothesis is that enzyme-induced post-translational modifications of substrate proteins may change the substrate thermal stability. This assumption is supported by multiple kinase-substrate pairs studied in vitro. Very recently this principle was generalized on a proteome-wide scale, as numerous shifts in protein melting temperatures were observed in response to site-specific phosphorylation events. Building on this concept, the authors aimed to detect post-translational modifications produced by, in principle, any enzymatic activity on its substrates with MS-CETSA. They found candidate protein substrates of three enzymes (TXNRD oxidoreductase, AKT1 kinase and poly-ADP ribose polymerase) by incubating human cell lysates with the enzymes purified in vitro and their co-factors. Their method named SIESTA is an interesting idea that could fill a gap in the field, as the (de)-stabilization events measured in MS-CETSA experiments are often difficult to interpret mechanistically. Although the screening of all potential substrates comes at the cost of producing recombinant enzymes, SIESTA would be very useful to reveal new enzyme-substrate associations. The method described in this manuscript could have an

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important impact in an area of biology where large scale studies are still scarce. However, I would suggest some conceptual and methodological clarifications of the content of this manuscript. MAJOR POINTS: CONCEPTUAL 1) The authors stated that the biological relevance of a protein modification correlates to the extent of the Tm shift that is observed in the thermal proteome profiling, and use this principle to prioritize substrates accordingly to their altered stability in their SIESTA assays. This idea follows the phosphorylation sites 'hotspots' theory proposed by Huang et al in a recent manuscript cited by the authors. However, two independent research groups questioned the conclusions of this work arguing that the experimental design was flawed. By reanalyzing the published data and performing independent experiments they showed that the extent of stability-altering phosphorylation is much less prevalent than what Huang et al proposed originally. At present, considering the open debate, I would consider the 'hotspot' theory at least controversial, therefore I would recommend to minimize references to the concepts originating from this paper and mitigate the statements regarding 'biological importance' and its association with 'larger Tm shifts'. 2) The criteria used for the definition of an enzyme substrate hit in SIESTA are not well described. What are the thresholds applied? How does the ranking of the hits work? I understand that the specificity of the measured responses is calculated from the orthogonal partial least-square discriminant analysis (OPLS-DA) method referenced in the text. However, the authors should guide the readers summarizing what OPLS-DA does in a couple of

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sentences in the main text, as the audience will likely not know OPLS-DA. The discrimination of the candidate hits seems to be based on the so called 'VIP value'. This concept is unclear for people who have not done an OPLS-DA analysis but it is the key discriminant for the discovery of the highest stabilized proteins. This should be made more accessible for a non-specialist audience. For instance, with the current format it is not trivial to understand how to interpret the data shown in the figures. For instance, what do the acronyms 'pq[1]' and poso[1] mean in the axes of the plot of figures 2d, S2c and S3c? What is a "negative reference context"? Since these three plots are showing the key results of the paper for the three enzymes under analysis, it is essential that sufficient elements are provided for the evaluation of this data. 3) There is a reference to the interaction databases GeneCards in line 147 of the main text and it is mentioned that GeneCard scores were also used to select putative substrates for validation. How the combination of the VIP and GeneCard scores were objectively used to prioritize or/and rank the candidate substrate? 4) A possible internal quality control for SIESTA could be to use the experiments with vehicle and cosubstrate only. The Nordlund group and others have essentially done the same experiments before in these publications (http://dx.plos.org/10.1371/journal.pone.0208273, http://www.nature.com/articles/s41467-019-09107-y). What is the overlap among the ATP, NADPH and NAD datasets? 5) It is meaningfully pointed out that MS-CETSA experiments with

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vehicle and co-substrate find cosubstrate binding proteins, as is has been extensively shown before. Can the authors comment whether they also observe significant Tm shifts between vehicle treated and enzyme treated samples? Is there any case in which substrate detection was achievable in absence of the cosubstrate by adding recombinant enzymes to the lysates, as they do? Was any interaction between substrate and enzyme in absence of a post-translational modification observed in this dataset? 6) The duplicates of the 'vehicle only' melting curves figures 2e, 3b, 4b and in the supplementary figures should also be reported for completeness. They would provide a better prospective of how the different components of the binding reaction affect the substrate stability. 7) Adding the enzyme of interest in large quantities to protein lysates could potentially lead to artifact post-translational modification of proteins that are not substrates of the enzymes in physiological conditions. This important issue is not discussed. Was this considered at all when designing the experiments? 8) The text is at times hard to follow due to excessive use of abbreviations (ex lines 166-183). This is also dangerous because it leads to misinterpretation. For instance, line 200, the authors write that "ACTB and MAP2K4 are new ATP binding proteins". I disagree. ACTB and MAPK4 are not novel ATP binding proteins. ACTB is cytoplasmic actin, and it is well known that ATP participates in the polymerization cycle of actin filaments. MAPK4 is a kinase component of the MAP kinase signal transduction pathway, and being a kinase binds ATP by definition.

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9) Expressions like: "one could estimate the false positive rate to be not higher than 30%" (line 178) should be avoided if the sample size is 7. Similar issue at lines 300-301. The discussion about "positive and -negative rates" should be toned down, if clear and objective criteria to define substrate hits are not provided, as discussed above (point 2 and 3). EXPERIMENTAL: 10) The authors used separate multiplexed TMT10 samples to process and analyze experimental replicates in SIESTA experiments. Since this choice could introduce a certain experimental bias, can the authors show if this was taken into account for instance by examining the consistency between experimental duplicates? Can the authors also explain why the AKT1 experiment require much more extensive peptide fractionation than the TXNRDq and PARP10 experiments (24 final fractions instead of 8)? 11) The validation of the monomer -dimer transition of GULP1 of supplementary figure 1d lacks a control. The total amount of GULP1 loaded in the two conditions should be shown in a SDS-PAGE gel run in denaturing conditions. 12) It would be interesting to comment about advantages/disadvantages of SIESTA versus REDOX proteomics for analyzing oxidoreductases substrates. The corresponding author's lab has a record of publications in the field of REDOX proteomics, so should be in a good position to briefly revise this in the discussion. MINOR POINTS: 13) The scatter plot of Figure 2a does not show stabilization of known NADPH interacting proteins, rather consistency between replicates. 14) Some sentences are too vague. For instance, lines 135-138: "The analysis of specific

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∆Tm shifts in the TXNRD1+NADPH treatment revealed that in the presence of NADPH, TXNRD1 destabilized both known and novel candidate substrate proteins (Supplementary Data 3). In general, the expected asymmetry in Tm shifts in favor of 138 destabilization was well pronounced (Fig. 2c)." Please report the exact number of destabilized and destabilized proteins in the main text to illustrate the content of figure 2c in the main text. 15) Lines 270-274: References to figure 4c and figure S3D appear to be missing. Comment 4. Line 218: "31% (123/396) of the proteins annotated in Uniprot as ATP binders were also verified in our experiment." The authors could also compare their results to previous TPP experiments with ATP, since even in lysate proteins that are stabilized are not necessarily binding ATP directly. Response: Unfortunately, the processed data has not been made available in the previous studies. However, we present a comparison with UniProt, which has a compiled list of validated ATP, NADPH and NAD binding proteins. We have intentionally used 500 µM of ATP, at which concentration it mainly shows substrate activity, according to PMID: 30858367. Comment 5. In Fig 2-4, when showing melting curves for potential substrates, I think the controls should also be included. Response: We have now included the control melting curves for all the enzyme systems. Andre Mateus Reviewer #2 (Remarks to the Author): Saei et al. describe an adaptation of the Thermal Proteome Profiling (TPP) methodology, they term SIESTA and which can be used to particularly screen for enzyme substrates. As the authors

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point out, proteome-wide screening for substrates of a particular enzyme is not necessarily straightforward. The authors demonstrate the utility and versatility of SIESTA on three model enzymes, namely the thioredoxin reductase TXNRD1, the protein kinase AKT1, and poly-ADP-ribosyltransferase PARP-10 and show that known as well as novel substrates can be identified for these enzymes using a 'differential' approach. This is certainly an interesting adaptation of the TPP strategy that may address an existing need in screening for enzyme substrates, for instance when studying these enzymes as potential drug targets. Unfortunately, the presentation of the manuscript is far from optimal and a number of points listed below have to be addressed. Response: We thank the reviewer for the positive appraisal of our work and the valuable comments. Comment 1. [A] The authors use a lysate (mild conditions) and add recombinant enzyme + substrate to study in vitro substrates of this enzyme. Although their approach undoubtedly generates interesting data, this strategy may be prone to false positive identifications. PTMs are mainly regulated spatially by bringing substrate and enzyme in close proximity (or not). Now, in a lysate this level of regulation is completely gone. I do not necessarily see a problem in using such a strategy, but I definitely miss a clear statement on this approach in order to make the reader aware of potential shortcomings. For instance, protein/substrate interactions may be specific to a certain organelle. [B] I was first also wondering about secondary effects, which the authors exclude based on the fact that the lysate is

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77x fold diluted compared to the cell, but that also means that a huge excess of enzyme is added to drive the reaction which can lead to unspecific reactions. [C] The authors validate some of their findings using recombinant proteins, some of which being expressed in E coli, which may also be problematic due to different PTM patterns compared to human expression. [D] The incubation of recombinant potential substrate with the enzyme and cosubstrate can again lead to artificial modifications that may not occur under physiological conditions and, unfortunately, I cannot see any kind of negative control for any of the three validation experiments, which is essential to ensure that not any protein will be modified in this setting. Response: We thank the reviewer for these critical comments. These issues are now mentioned in the discussion. [A, B] It reads "Furthermore, using lysate might distort the spatial regulation of enzyme substrate interaction and yield substrates that are not active in the biological context. The excess of the enzyme may also lead to unspecific reactions". We tried to keep the conditions as physiological as possible. For example, in the case of ATP, we used 500 µM concentration where it mainly has cosubstrate activity (PMID: 30858367). The amount of added enzymes was not that high; we were actually limited by the amount of available recombinant enzymes. We now present the amount of added enzyme compared to the total amount of enzyme found in untreated cell lysate. Within the current study, for the TXNRD1, AKT1 and PARP10 systems, the

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ratio of added enzyme was ~10, ~20 and ~ 1.5 fold compared to untreated lysate, respectively. Given the dilution of lysate by ≈77 fold, these ratios are well within the physiological concentrations. The Supplementary Figure 7 was added to illustrate the results. [C] We added "Moreover, recombinant proteins expressed in E. coli may lack some important PTMs necessary for their activity in human cells". [D] We did not include an unrelated protein as a negative control during the incubation of recombinant potential substrate with the enzyme and cosubstrate (as it might create a bias). However, we have done other validation experiments on the same day and under the same exact conditions. For example, 7 redox proteomics experiments were performed in parallel for validating TXNRD1 substrates, with only 5/7 validated. For PARP10, we could not validate Caspase 6, meaning that our validation experiments did rule out false positives. For AKT1, we now complement our study with phosphoproteomics validation experiments where we use AKT1 inhibitors in cells. The results are presented in Fig. 3. In Figure 1 we included validation as a crucial step in the SIESTA workflow. Comment 2. In the same context, the authors used a method to "track phosphate release" which is not wellexplained, stating that they preferred this over p-proteomics, as the phosphate release represents all p-sites, whereas MS will only show individual sites (and I guess may miss some based on sequence inaccessibility). Indeed, MS would be an important additional validation to ensure that there is not too many different p-sites on the

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recombinant proteins under the given conditions, as the identification of too many sites would support the note of low specificity in such experiments. Also the sites should be compared with Phosphositeplus. Response: In response to the reviewer comment, we performed phosphoproteomics experiments with AKT1 inhibitors added to living cells and now provide in several cases the potential sites of modifications changing protein stability. The validated proteins included some of the genuine substrates of AKT1 such as BCL3, TRIP12 and MEF2D, confirming our findings. This statement was also added: "For example, in two direct microarray screenings of human cells, 165 and 51 AKT1 substrates have been found, respectively, with no overlap between them. In the PhosphoSitePlus database containing an accumulated list of 206 human protein substrates for AKT1, there are 4 and 7 overlapping proteins with the two mentioned studies, respectively, while the 72 SIESTA substrates obtained with a 0.5°C cutoff, similarly gave 4 overlaps." Comment 3. Line 164: The authors speculate that secondary reactions would occur when using NADPH alone, as well, but this is not completely obvious to me ---do the authors refer to the same reactions? Response: For better clarity, we rephrased this sentence that now reads: "Furthermore, if secondary reactions were present, they would also occur in lysates treated with NADPH alone (as the basal levels of cellular TXNRD1 was also present there), and thus would be filtered away in our analysis". Comment 4. Line 330: the authors discuss how the cutoffs for considering a protein as shifted can be changed and how

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this impacts the list of potential substrates. Using a suitable cutoff is of course a common problem in quantitative proteomics, but the authors have to discuss this in the context of the robustness of the method --how is the variation in melting temperature between replicates? Indeed, the cutoff being used in the end should be based on this variation in order to produce more robust data, than by applying kind of arbitrary cutoffs. Response: In choosing the final cutoff, we had also calculated the median variation between the replicates for different treatments for each enzyme system. This statement is now added: "The 1°C cutoff was chosen by analysis of the variation in Tm between replicates. In TXNRD1, AKT1 and PARP10 systems, the median Tm variation for different treatments was 0.50, 0.45 and 0.59 °C, respectively." Furthermore, as now detailed in the text, our final cutoffs allow for a minimum false discovery rate, and are thus robust. Comment 5. Line 365-372. The authors speculate on the effects of PTMs on protein stability and that this will be hard to generalize. The beginning and end of this 8-line section is somewhat redundant. The authors should mention the concept of PTM crosstalk and the PTM code. Indeed it would be really surprising if there was a general trend, as it seems that the entirety of PTMs dictate a proteins structure and function rather than individual PTMs, which may be one reason why the authors fail to verify some known substrates, they may simply be to heterogeneous within the sample

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which may even have functional/biological implications. 7 Comment 7. The supplemental tables should have more explanations on what is actually presented. It would be good to have a better connection between the "raw" data and the lists of substrates, maybe by combining them as different sheets into a single table rather than having so many individual tables per experiment. Response: We merged the tables in the revised version of the manuscript. Reviewer #3 (Remarks to the Author): "System-wide identification and prioritization of enzyme substrate by thermal analysis" submitted for publication in Nature Communication. In this manuscript Amir Ata Saei et al. propose a workflow based on the mass spectrometry CEllular Thermal Shift Assay (MS-CETSA) to discover post-translational modifications produced by enzymes. Their hypothesis is that enzyme-induced post-translational modifications of substrate proteins may change the substrate thermal stability. This assumption is supported by multiple kinase-substrate pairs studied in vitro. Very recently this principle was generalized on a proteome-wide scale, as numerous shifts in protein melting temperatures were observed in response to site-specific phosphorylation events. Building on this concept, the authors aimed to detect post-translational modifications produced by, in principle, any enzymatic activity on its substrates with MS-CETSA. They found candidate protein substrates of three enzymes (TXNRD oxidoreductase, AKT1 kinase and poly-ADP ribose polymerase) by incubating human cell lysates with the enzymes purified in vitro and their co-factors. Their method named SIESTA is an interesting idea that could fill a gap in the field, as the (de)stabilization events measured in MS-CETSA experiments are often difficult to interpret mechanistically.

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Although the screening of all potential substrates comes at the cost of producing recombinant enzymes, SIESTA would be very useful to reveal new enzyme-substrate associations. The method described in this manuscript could have an important impact in an area of biology where large scale studies are still scarce. However, I would suggest some conceptual and methodological clarifications of the content of this manuscript. MAJOR POINTS: CONCEPTUAL Comment 1. The authors stated that the biological relevance of a protein modification correlates to the extent of the Tm shift that is observed in the thermal proteome profiling, and use this principle to prioritize substrates accordingly to their altered stability in their SIESTA assays. This idea follows the phosphorylation sites 'hotspots' theory proposed by Huang et al in a recent manuscript cited by the authors. However, two independent research groups questioned the conclusions of this work arguing that the experimental design was flawed. By reanalyzing the published data and performing independent experiments they showed that the extent of stabilityaltering phosphorylation is much less prevalent than what Huang et al proposed originally. At present, considering the open debate, I would consider the 'hotspot' theory at least controversial, therefore I would recommend to minimize references to the concepts originating from this paper and mitigate the statements regarding 'biological importance' and its association with 'larger Tm shifts'. Response: Thanks for the comment. We also noticed the debates over the "hotspot" theory after we submitted the manuscript and have mitigated our citations to this paper in this version. Thus we added to the

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discussions: "In should be noted that recent findings from two independent groups raised doubts in the extent of stability-altering phosphorylation postulated by Huang et al.". However, we still believe that substrates can be prioritized based on the size of the shift they exhibit in SIESTA. Comment 2. The criteria used for the definition of an enzyme substrate hit in SIESTA are not well described. What are the thresholds applied? How does the ranking of the hits work? Response: The selection criteria are now clearly stated in materials and methods: "For selection of putative substrates, the following criteria were used: 1) R2 > 0.7 between the measurement and the fitted curve, 2) the standard deviation between the replicates was <2.5°C, 3) p values between the Enzyme-Cosubstrate treatment against Enzyme and Cosubstrate treatments < 0.05 for one condition and <0.1 for the other; 4) the absolute mean ΔTm was larger than 1°C for both conditions (a similar approach was used for selection of cosubstrate binding proteins). We also now added: "The 1°C cutoff was chosen by analysis of the variation in Tm between replicates. In TXNRD1, AKT1 and PARP10 systems, the median Tm variation for different treatments was 0.50, 0.45 and 0.59 °C, respectively." We also added "The proteins passing the significance thresholds were ranked by absolute ΔTm or VIP values obtained from OPLS-DA analysis". I understand that the specificity of the measured responses is calculated from the orthogonal partial least-square discriminant analysis (OPLS-DA) method referenced in the text. However, the authors should guide the readers summarizing what

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OPLS-DA does in a couple of sentences in the main text, as the audience will likely not know OPLS-DA. The discrimination of the candidate hits seems to be based on the so called 'VIP value'. This concept is unclear for people who have not done an OPLS-DA analysis but it is the key discriminant for the discovery of the highest stabilized proteins. This should be made more accessible for a non-specialist audience. For instance, with the current format it is not trivial to understand how to interpret the data shown in the figures. For instance, what do the acronyms 'pq[1]' and poso[1] mean in the axes of the plot of figures 2d, S2c and S3c? What is a "negative reference context"? Since these three plots are showing the key results of the paper for the three enzymes under analysis, it is essential that sufficient elements are provided for the evaluation of this data. Response: We have now described the OPLS-DA approach and the attributed parameters in the TXNRD1 results section (on the first use) and included the Supplementary Figure 1 to explain the approach and the way data must be interpreted. "pq[1]" and "poso[1]" were components 1 and 2 of the OPLS-DA model, and were changed accordingly in the new figures for clarity. The following paragraph was also added: "OPLS-DA is a multivariate supervised modeling tool for pinpointing the variables (here proteins) that have the largest discriminatory power between the two or more statistical groups (samples) 26. In the "loading plot" or "score scatter plot" for

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two-group comparison models, the predictive component is the x-axis, while y-axis is related to the orthogonal components that is irrelevant in this study ( Supplementary Fig. 1a). In the loading plot ( Supplementary Fig. 1b), each protein is represented by a dot. In SIESTA, the protein Tm for single treatments is contrasted with those from the combination treatment. The large dots on either side of the plot are the reference points for the treatments. Therefore, proteins specifically stabilized by the modification will move close to the reference point of the combination treatment and the destabilized proteins will be further away on the opposite side. The proximity of a protein to the reference point on either side of the x-axis is a measure of the magnitude of the thermal stability change upon modification and its reproducibility among the replicates. Each protein can also be characterized by the variable influence on projection (VIP-value). The VIP-values quantify the impact each variable (i.e., protein) has on the OPLS-DA model, with a higher value corresponding to a greater contribution. Thus the proteins with the highest VIP values are suitable as candidates for validation. For more detailed explanation, see Umetrics documentation." Comment 3. There is a reference to the interaction databases GeneCards in line 147 of the main text and it is mentioned that GeneCard scores were also used to select putative substrates for validation. How the combination of the VIP and GeneCard scores were objectively used to prioritize or/and rank the candidate substrate? Response: We deleted this analysis from the manuscript

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due to the collective comments from reviewers. Comment 4. A possible internal quality control for SIESTA could be to use the experiments with vehicle and co-substrate only. The Nordlund group and others have essentially done the same experiments before in these publications (http://dx.plos.org/10.1371/journal.pone.0208273, http://www.nature.com/articles/s41467-019-09107-y). What is the overlap among the ATP, NADPH and NAD datasets? Response: This is a valuable suggestion, but unfortunately, the processed data has not been made available in these studies. However, we present a comparison with UniProt, which has a compiled list of validated ATP, NADPH and NAD binding proteins. Comment 5. It is meaningfully pointed out that MS-CETSA experiments with vehicle and co-substrate find cosubstrate binding proteins, as is has been extensively shown before. [A] Can the authors comment whether they also observe significant Tm shifts between vehicle treated and enzyme treated samples? [B] Is there any case in which substrate detection was achievable in absence of the co-substrate by adding recombinant enzymes to the lysates, as they do? [C] Was any interaction between substrate and enzyme in absence of a post-translational modification observed in this dataset? Response: [A] Yes, we have. Please see our response to the Comment 3 Reviewer 1. [B] Yes, there is. For instance, four proteins were known to interact with AKT1 in the literature. We performed a pulldown experiment using PARP10 and analyzed the overlap in PARP10 interactors with SIESTA results. These results are now fully described in the paper and Fig. 5 and supplementary Fig. 6 are added. Some of the identified hits were indeed

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known substrates of the enzymes under study, for example MAZ for AKT1 system. [C] Yes, there might be a few known substrates such as MAZ that interact with an enzyme, but do not show the additional stability change upon modification, but we have noticed that the reverse is more likely; i.e. a protein identified as a substrate be known as an interacting protein. The latter is discussed for AKT1 system in the text. Comment 6. The duplicates of the 'vehicle only' melting curves figures 2e, 3b, 4b and in the supplementary figures should also be reported for completeness. They would provide a better prospective of how the different components of the binding reaction affect the substrate stability. Response: We have now included the control melting curves for all the enzyme systems in all the figures. Comment 7. Adding the enzyme of interest in large quantities to protein lysates could potentially lead to artifact post-translational modification of proteins that are not substrates of the enzymes in physiological conditions. This important issue is not discussed. Was this considered at all when designing the experiments?

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Ultrasonic-Assisted Synthesis of Fe–BTC–PEG Metal–Organic Complex: An Effective and Safety Nanocarrier for Anticancer Drug Delivery The porous metal–organic complexes are emerging as novel carriers for effective and safe delivery of drugs for cancer treatment, minimizing the side effect of drug overuse during cancer treatment. This study fabricated the Fe–BTC–PEG metal–organic complex from Fe ions, trimesic acid, and poly(ethylene glycol) as precursors using an ultrasonic-assisted method. The morphology and crystallinity of the resultant complex were observed by scanning electron microscopy (SEM) and X-ray diffraction (XRD), respectively. FTIR spectroscopy was employed to investigate the functional groups on the surface of the Fe–BTC–PEG complex. The result showed that the prepared Fe–BTC–PEG complex was in particle form with low crystallinity and diameter ranging from 100 to 200 nm. The obtained Fe–BTC–PEG complex exhibited a high loading capacity for the 5-fluorouracil (5-FU) anticancer drug with a maximal capacity of 364 mg/g. The releasing behavior of 5-fluorouracil from the 5-FU-loaded Fe–BTC–PEG complex was studied. Notably, the acute oral toxicity of the Fe–BTC–PEG metal–organic complex was also carried out to evaluate the safety of the material in practical application. INTRODUCTION One of humankind's most well-known "death penalty" is cancer, which is increasing in the modern world. 1 Several methods could be employed to cure cancer, including surgery, chemotherapy, hormonal therapy, radiation therapy, adjuvant therapy, and immunotherapy; however, their effectiveness is still questionable. 2 Of these cancer treatment techniques, chemotherapy is considered to be the most common. 3 Even though proven to be an effective cancer treatment technique, chemotherapy lacks selectivity toward cells and

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drug molecules, which could damage the normal cells, causing side effects of significantly weakening other organs. 4 Furthermore, the cancer treatment efficiency of the chemotherapy is also hindered by the low solubility, stability, and bioavailability. 5 Thus, we need to find practical pathways to deliver drug molecules to the targeted tumor and cancer cells. Many strategies have been developed to accurately deliver the anticancer drug to the tumor, such as the direct introduction of the anticancer drug into the tumor, routes of drug delivery, systemic delivery targeted to the tumor, drug delivery targeted to blood vessels of the tumor, special formulation and carriers of anticancer drugs, transmembrane drug delivery to intracellular targets, and biological therapies. 6 Nanoparticles as carriers for drug delivery belong to the unique formulation, and carriers of the anticancer drugs category have been extensively studied and employed for this purpose. The anticancer drugs are encapsulated into nanocarriers and delivered to the targeted tumors before release into the tumor space, resulting in the effective uptake of the drug molecules by tumor cells. 7,8 The major disadvantage of drug delivery using nanocarrier systems is to actively release the drug molecules to the suitable tumors at the right time. Many factors such as heat sensitivity, pH controls, employment of proteases or phospholipases, and controlling the glutathione levels have been considered to design the delivery systems for the anticancer drugs using nanoparticles. 9,10 Controlling these factors enables the release rate of the anticancer drug molecules when they are in the cancer cells while remaining stable in the

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environment of the normal cells, consequently minimizing the side effects and enhancing the therapeutic efficiency. The recent emergence of porous metal−organic complexes (MOCs) has attracted significant research interest from scientists. MOCs are porous crystalline materials selfassembled by metal ions or clusters and organic ligands to form frameworks. 11,12 MOCs are known for their extraordinarily high specific surface areas, tunable pore size, and adjustable internal surface properties. These inherent features render MOCs promising in various applications, such as gas adsorption/separation, 13−15 catalysis, 16,17 and drug deliv-ery. 18−20 The MOCs possess several outstanding features, including the mesoporous architecture with mesoporous cages and microporous windows, a giant cell volume, and a high surface area. The unique combination of properties highlighted above makes MOCs an excellent candidate for industrial applications, including drug delivery systems (DDSs). 21 Because of facile synthesis, high loading capacity, biocompatibility, and degradability, MOCs have been extensively employed as carriers for stimuli-sensitive drug delivery. 22−24 5-Fluorouracil (5-FU) is a chemotherapeutic agent employed to treat several deadly cancers, such as breast, colorectal, and head and neck cancers. It has been demonstrated to be effective in treating various types of cancers as it has a mode of action based on interfering with thymidylate synthesis, which controls the development of cancerous cells. 25 The main challenge of using 5-FU is its short biological half-life, low selectivity, and toxic side effects on the bone, marrow, and gastrointestinal tract. 26,27 To minimize these limitations, drug delivery systems have been considered for the controlled release of 5-FU drugs to targeted tumors. 28,29 Many

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nanoparticles have been employed for the 5-FU drug delivery, such as mesoporous silica nanoparticles, 30 nanogels, 31 magnetic nanoparticles, 32 metal−organic frameworks (MOFs), or metal−organic complex. 20,33 To the best of our knowledge, the use of the Fe−BTC−PEG complex obtained from the green synthesis method of ultrasonication has not been investigated for the delivery of the 5-FU drug. In this work, the new Fe−BTC−PEG metal−organic complex is synthesized using a facile approach with the assistance of an ultrasonicator. The detailed characterizations of the prepared metal−organic complex will be thoroughly investigated. The drug-loading capacity and release for the 5fluorouracil will also be studied. Significantly, the acute oral toxicity and repeated dose 7-day oral toxicity of the 5-FU- Figure 1. It can be seen from Figure 1a,b that the Fe−BTC−PEG complex in the shape of the small particles tends to aggregate to form large clusters. The morphology of the Fe−BTC−PEG complex is similar to the Fe−BTC fabricated from the ultrasonication approach; however, the particle size of the Fe−BTC−PEG is significantly smaller than that of Fe−BTC ( Figure S1). This indicates that the addition of the PEG modifier could slow down the growing process of the Fe−BTC, reducing the particle size. The TEM images (Figure 1c) exhibit the round shape of the Fe−BTC− PEG complex with diameters ranging from 100 to 200 nm. The high-resolution TEM image in Figure 1d indicates that the Fe−BTC−PEG was well prepared and identical in the complex structure. The thin outer layer is ascribed to the coverage of PEG polymer around the Fe−BTC metal−organic

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particles. This morphology is in good agreement with the Fe−BTC shape of the previous study. 34 The crystallinity of the prepared Fe−BTC−PEG metal− organic complex was investigated using a powder X-ray diffraction pattern, as shown in Figure 2a. The low and relatively broad peaks (or maybe overlapping) observed in the XRD pattern indicate the Fe−BTC−PEG materials' semiamorphous nature. 34,35 This may be due to the disordered structure (SEM and TEM images) or the effect of the PEG organic coating. Because of the low crystallinity of the Fe− BTC, the XRD characterization for this material was limited, with no reported crystal structural data in the literature. However, the position of the broad peaks in the XRD pattern are well-matched with the ones reported previously. 36,37 Figure 2b exhibits the FTIR spectrum of the Fe−BTC−PEG complex to investigate the chemical structure and functional groups on the material surface. The broad adsorption from 3300 to 3700 cm −1 is assigned to the O−H bending vibration in adsorbed moisture and the O−H stretching vibrations in the BTC and PEG molecules. The characteristic peaks that appeared at 1632, 1581, 1451, and 1380 cm −1 are ascribed to the asymmetric and symmetric carboxylate groups' vibrations in BTC molecule ( Figure S2), respectively, which demonstrates the coordination between carbonyl group in BTC ligand with the iron sites. 38 The materials' surface area and functional groups are among the decisive factors to evaluate the loading capacity for drug delivery. The BET surface area plot of the Fe−BTC−PEG metal−organic complex was determined using and nitrogen

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adsorption−desorption plot, as shown in Figure 3. The results show that the Fe−BTC−PEG metal−organic complex has a surface area of 21.4718 m 2 /g. This relatively low surface area is probably due to the coverage of the PEG molecule inside and outside of the Fe−BTC micropores. Even with low surface area, the drug adsorption capacity by Fe−BTC−PEG metal− organic complex might be significantly enhanced by functional groups induced by the PEG modification, which will be demonstrated by the drug-loading study in the following sections. Furthermore, the Fe−BTC−PEG metal−organic complex's pore volume and pore diameters are 0.078 cm 3 /g and 24.86 nm, respectively, making it suitable for the 5-FU drug loading. 41 2.2. 5-FU Loading and Releasing by the Fe−BTC−PEG Complex. Many drugs have been effectively utilized for cancer treatment. Among them, 5-fluorouracil (5-FU) could be employed for treating many types of cancers such as skin, stomach, breast, anal, and colorectal. As mentioned above, the Fe−BTC−PEG metal−organic complex is suitable for functional groups, particles size, pore diameter, and pore volume to deliver 5-FU to the targeted cell with minimum side effects. In this work, the prepared complex was loaded with 5-FU via immersing approach. The presence of 5-FU in the complex structure after encapsulation was confirmed by FTIR spectroscopy, as shown in Figure 4. The FTIR spectrum of the 5-FU-loaded Fe−BTC−PEG reveals all characteristics peaks of the Fe−BTC−PEG complex discussed above. Additionally, the appearance of the vibration band at around 750 cm −1 is attributed to the C−H stretching group in the CFCH plane of the

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5-FU; this characteristic peak is also observed in the FTIR spectrum of the 5-FU as shown in Figure S4. The surface morphology of the Fe−BTC−PEG metal−organic complex was also observed, showing a negligible change compared to the morphology of the sample before drug loading ( Figure S5). The BET surface area, pore volume, and pore diameter of the Fe−BTC−PEG metal−organic complex after 5-FU loading were determined to be 17.4 m 2 /g, 0.034 cm 3 /g, and 8.27 nm, respectively, which decreased in comparison with those of the initial Fe−BTC−PEG sample, further demonstrating the success of the 5-FU loading on the sample ( Figure S6). The drug-loading capacity is one of the critical properties to evaluate the applicability of the materials for drug delivery. To determine the maximal 5-FU-loading capacity of the Fe− BTC−PEG complex, the complex was immersed into the 5-FU solution for 14 days to reach an equilibrium adsorption state. The result showed that the prepared complex has a high loading capacity for 5-FU with a maximal capacity of 364 mg/g material, accounting for 36.4% weight of the carrier material, which was higher than the drug-loading capacity of the zincbased metal−organic framework. 42 It is noteworthy that the slow release behavior was one of the crucial properties when using advanced materials for drug delivery. Figure 5 illustrates the releasing profile of the 5-FUloaded Fe−BTC−PEG metal−organic complex. Two evident releasing stages could be clearly observed during the release process of 5-FU from the loaded complex. The first stage is the release of the 5-FU in

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free form, which forms no or a weak bond with the complex. This stage is expected to occur very quickly (Figure 5a). The second stage is a slow-releasing process of the 5-FU drug because of intense interaction with the functional groups on the surface or in the micropores of the BTC−PEG metal−organic complex. For the free-standing 5-FU drugs, more than 90% of the 5-FU could be released in vitro media after 30 min. 43 When loaded with the Fe−BTC− PEG metal−organic complex, only 35% of 5-FU is released after 2 h of dissolution in the first releasing stage (Figure 5a). In the second stage, the releasing rate is significantly reduced, with only a few percentages of 5-FU released after each immersion (Figure 5b). The slow release of 5-FU is ascribed to solid host−guest bonding between 5-FU and the Fe−BTC− PEG metal−organic complex. The complete release of 5-FU from the loaded complex in the PBS solution was only evident after 14 days of the experiment, which is considered to be highly stable in the body-simulated media. The drug-releasing time of the Fe−BTC−PEG/5-FU system is much slower than that of the Fe−BTC−PEG/5-FU system, which is advantageous in cancer treatment. 25 This slow 5-FU release minimizes the side effect of the cancer drug to the healthy cells and improve the treatment effectiveness of the drug. 2.3. Acute Oral Toxicity. Evaluating the toxic properties of the Fe−BTC−PEG metal−organic complex is crucial when this material is employed as a carrier for drug delivery in the body. Figure 6 shows the optical

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images of the Albino BALB/c mice administered Fe−BTC−PEG orally in different doses of 2, 4, 6, 8, and 10 g material/kg mice using a stomach tube. Results reveal that the Fe−BTC−PEG complex does not cause any toxicity in mice even at the highest dose of 10 g/kg bodyweight. All mice treated with Fe−BTC−PEG survived and did not show any toxic symptoms or abnormal behavior at the tested doses over the 7 days of observation. The testing mice treated with Fe−BTC−PEG showed little change in bodyweight, food intake, or water consumption compared to the control group. Acute treatment with Fe−BTC−PEG showed that the healthy white mice (six males and six females) moved and ate usually and responded well to light and sound. The change in the bodyweight was also investigated to assess the oral toxicity of the prepared metal−organic complex toward the mice after the oral uptake of materials for 7 days. The result is shown in Figures 6 and 7. It is evident from the figure that the mice normally developed with the bodyweight of tested mice almost the same as that of the control sample when the treatment dose was 2−6 g/kg. After 7 days of treatment with a 2−6 g/kg dose, the bodyweight of mice is approximately 24 g. When the Fe−BTC−PEG increases to 8 g/kg, the bodyweight of the mice slightly decreases compared to that of the controlled sample after 7 days of the experiment. The bodyweight of the mice treated with an 8 g/kg dose is around 22.4 g after 7 days

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of treatment. Especially at the treatment dose of 10 g/kg, the bodyweight of mice decreases compared to those of the mice before the administration of the Fe−BTC−PEG complex with the bodyweight of only 21.9 g after 7 days of treatment. However, the mice treated with this dose were still healthy with no sign of abnormal behaviors. No change was observed in the food and water consumption of the mice at all groups, including the controlled group and treated groups with various doses of the prepared complex throughout the testing period. The results indicate that the Fe−BTC−PEG metal−organic complex can be a safe carrier for drug delivery for cancer treatment in humans. The effectiveness of the 5-FU-loaded Fe−BTC−PEG metal−organic complex compared to the free-standing 5-FU for the inhibition of the human gastric cancer cells in various treatment times was evaluated. For the free-standing 5-FU drug, the cells inhibiting percentage is determined to be around 74% after 24 h of incubation time and tends to decrease along with the increase of the incubation time. When Fe−BTC−PEG/5-FU was employed to treat the human gastric cancer cells, the inhibiting percentage increased along with the incubation time and retained the inhibiting effect of higher than 80% even after 120 h of incubation time ( Figure 8). This long-lasting effectiveness of the Fe−BTC−PEG/5-FU system is ascribed to the slow release of the 5-FU drug in the Fe−BTC−PEG metal−organic complex, which effectively provides the 5-FU drug for killing cancer cells. CONCLUSIONS In summary, the Fe−BTC−PEG metal−organic complex has been successfully synthesized using a

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facile ultrasonic-assisted approach. The resultant Fe−BTC−PEG complex has diameters ranging from 100 to 200 nm with functional groups on the surface, which is suitable to be utilized as an effective carrier for drug delivery. The Fe−BTC−PEG complex exhibits a high loading capacity for the 5-fluorouracil drug with the maximal capacity of 364 mg/g complex, accounting for 36.4% weight of the carrier material. Significantly, the 5-FU-loaded Fe−BTC− PEG revealed a prolonged release of 5-FU in the in vivo media. The acute oral toxicity also showed no death or signs of toxicity in mice administrated with the Fe−BTC−PEG complex at doses ranging from 2 to 10 g/kg. No change was also observed in the food and water consumption of the mice during the testing period, which indicates that the Fe−BTC− PEG complex could be safely used in the human body. With high drug-loading capacity and safety of use, the Fe−BTC− PEG metal−organic complex is considered a promising carrier for drug delivery for the effective and safe treatment of various cancers. The functional groups of the complex before and after loading with 5-FU were studied by Fourier transform infrared spectroscopy (FTIR, TENSOR II, Bruker). The crystallinity of the samples was investigated by the XRD pattern obtained on the A X'Pert PRO PANalytical instrument with a radiation source of 0.154 nm Cu Kα. The BET TriStar II Plus instrument was used to obtain an N 2 adsorption isotherm to measure the surface area of the Fe−BTC−PEG metal−organic complex. EXPERIMENTAL SECTION 4.3. Drug-Loading and Release Batch Experiment. The 5-FU solution with

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a concentration of 10 g/L was prepared by diluting the 5-FU powder in distilled water. The Fe−BTC−PEG metal−organic complex was then immersed into the 5-FU solution for 10 days before taking out, drying, and determining the loading capacity. The 5-FU drug delivery experiment was carried out as follows: seven samples of the 5-FU-loaded Fe−BTC−PEG metal−organic complex immersed in the body-simulated media (seven vials containing 5 mL of PBS solution) at the temperature of 37°C. After each interval, one vial was taken out and the vacuum filter removed the material. The 5-FU release was calculated by measuring the absorbance of the remaining filtrate at the wavelength of 265 nm. All experiments were repeated three times to obtain the average value with standard derivation. 44 Forty-two mice were randomly divided into seven groups with doses of 0, 2, 4, 6, 8, and 10 g material/kg bodyweight, with six mice in each group. The Fe−BTC−PEG suspension was orally administered to mice once a day for seven consecutive days. The controlled mice received water only. The general appearance of all mice was observed daily. The animals were weighed and food consumption was monitored first, fourth, and seventh day of the experiments. 4.5. In Vitro Toxicity Study of Fe−BTC−PEG/5-FU. The in vitro toxicity testing of the Fe−BTC−PEG/5-FU was carried out following the Monks' protocol. 45 The Fe−BTC− PEG/5-FU system and the free-standing 5-FU drug were diluted at the concentration of 10 μg/mL 5-FU. The diluted reagents were added into 96 wells and incubated at various testing times of 24, 48, 72,

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96, and 120 h. After a certain time, the cells were fixed using trichloracetic acid and coloring by sulforhodamine B dye in 30 min at 37°C, then washed with acetic acid, and dried at room temperature. The optical density of dyed cells in the wells was read on the ELISA Plate reader at the wavelength of 515−540 nm. The data were used to calculate the cell-inhibiting percentages.

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Prevalence of Musculoskeletal Disorders among Self-Employed Female Tailors in Selected Places of Bengaluru Prevalence Introduction Musculoskeletal disorders (MSD) include a wide range of inflammatory and degenerative conditions affecting the muscles, tendons, ligaments, joints, Corresponding Author: Pavana, HOD & Associate professor, Community Health Physiotherapy Department, RV College of Physiotherapy, No. CA 2/83-3, 9th Main Road, 4th Block, Jayanagar, Bengaluru.E-mail: pavanaholla.rvcp@rvei.edu.inperipheral nerves and supporting blood vessels.Work related MSDs (WRMSDs) usually occur when there is a mismatch between the requirements of the job and the physical capacity of the human body, depending upon the physical movement characteristics, ergonomics, and mechanical design of work tasks. 1 Work-related musculoskeletal disorders have been known to affect workers in a wide variety of occupations and are major causes of lost times from work, worker's disability, increase in compensation claims and health care costs. 2 The pattern of workrelated musculoskeletal disorders has been reported to vary among different occupational groups. Tailoring involves monotonous, highly repetitive tasks like cutting, assemble, pressing and finishing performed in sitting working posture with upper back curved and head bent over the sewing machine.Working in this awkward posture for a long duration increases the chance of developing work-related musculoskeletal disorders in them. 3The tailoring industry of India is an unorganized sector, mostly run by private establishments provides employment for both men and women majority from the lower economic classes.The employees of this industry hardly ever benefit from occupational health-andsafety provisions.They lack any type of social security, so their ill-health and poverty go hand-byhand and create a stupendous pressure from which

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they can hardly come out. The present study highlights the burden of musculoskeletal disorder among the females engaged in tailoring occupation as well as their discomfort related to ergonomically deranged workstation.Although majority of musculoskeletal disorders are benign and self-limiting in nature, the consequences of its persistent presence led to chronic (and persistent) disabling pain, sickness absence, work disability and increasing health care costs.If not corrected early, this health problem can seriously undermine their physical capacity hence leading to permanent physical disability.All these can adversely affect their earning power, thus setting a vicious cycle of no work, no money, malnutrition, and weakness.Intervention at work site like providing the workers with improvised workstation combined with ergonomic education and training, frequent short breaks to stretch and/or change position in between their working hours and jobs/task rotation through tasks that do not require constant leg pressure and awkward leg posture would go a long way in reducing the incidence of musculoskeletal disorders among them.Concerted efforts by the government and nongovernmental agencies as well as the employers to bring them under the umbrella of an organized sector with provision of social security measures and quality health care coverage would be helpful to mitigate their suffering. Every occupation has its own adverse effects on health.Low back pain is considered to be one of the most common occupational health problems.It is defined by Last JM et al as "a non-specific condition that refers to complaints of acute or chronic pain and discomfort in or near the lumbosacral spine". 4raemer et al1985 stated prolonged

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sitting leads to isometric contraptions of back muscles leading to endow muscular pressure restricted blood flow, resulting in ischemia which reduces energy requirements of the muscles causing fatigue. 5Prolong flexion of the spine leads to increase intervertebral joint laxity and fluid loss in the intervertebral discs. 6nd the intra-dismal pressure at the lumbar region is high both in sitting and trunk-bent-forward posture.7 Insufficient recovery time is the contributing factor to developing low back pain.8 Over two decades ago,Kuorinka and colleagues presented the general Standardized Nordic Questionnaire as a screening instrument that comprised just three questions regarding musculoskeletal pain that is been widely utilized in the absence of any other rigorously reliable assessment tool.The Nordic Musculoskeletal Disorder Questionnairetool used in this study was adapted from Dawson et al 9 which collects reliable information regarding the point,12month, and lifetime prevalence and consequences of musculoskeletal symptoms in nine body regions. This study aims to estimate the prevalence of work-related musculoskeletal disorders among female tailors and the severity of the disability due to low back pain among female self-employed tailors populations. Objectives of the Study • To estimate the prevalence of musculoskeletal disorders among self-employed female tailors. • To assess the prevalence of severity of disability due to low back pain among selfemployed female tailors. Descriptive statistics: All the categorical variables were presented in the form of frequency tables and graph wherever necessary.The quantitative variables were summarized using. Mean ± standard deviation with 95% confidence interval. Inferential statistics: The prevalence of work-related musculoskeletal disorder among self-employed female tailors from selected

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