The current preclinical pipeline for drug discovery can be cumbersome and costly, which limits the number of compounds that can effectively be transitioned to use as therapies. Chemical screens in zebrafish have uncovered new uses for existing drugs and identified promising new compounds from large libraries. Xenotransplantation of human cancer cells into zebrafish embryos builds on this work and enables direct evaluation of patient-derived tumor specimens in vivo in a rapid and cost-effective manner. The short time frame needed for xenotransplantation studies means that the zebrafish can serve as an early preclinical drug screening tool and can also help personalize cancer therapy by providing real-time data on the response of the human cells to treatment. In this Review, we summarize the use of zebrafish embryos in drug screening and highlight the potential for xenotransplantation approaches to be adopted as a preclinical tool to identify and prioritize therapies for further clinical evaluation. We also discuss some of the limitations of using zebrafish xenografts and the benefits of using them in concert with murine xenografts in drug optimization.
The zebrafish is a powerful model system for studying human cancer. The ease with which these organisms can be genetically manipulated, the opportunity for direct observation in transparent embryos, and the capacity for forward genetic and chemical screens has enabled zebrafish to be employed in the investigation of many gain-of-function and loss-of-function mutations implicated in cancer (Amatruda et al., 2002; Beckman, 2007; Beckwith et al., 2000; Berghmans et al., 2005; Dovey et al., 2009; Gutierrez et al., 2011; Yang et al., 2004). Many of the oncogenes and tumor suppressor genes that have been identified as major players in human cancers have zebrafish homologs, and important signaling pathways regulating cell proliferation, migration, apoptosis and differentiation are well conserved (Feitsma and Cuppen, 2008; Payne and Look, 2009; Stoletov and Klemke, 2008). In parallel with the basic characterization of important cancer-related genes and pathways in the zebrafish, a toolkit of experimental approaches has also been developed that has enabled cancer modeling in this species. This toolkit includes many reverse genetic approaches to generate loss-of-function phenotypes, such as TILLING (targeting induced local lesions in genomes) (Wienholds et al., 2003), morpholino oligonucleotides (Nasevicius and Ekker, 2000), and more recently, zinc-finger nucleases (Doyon et al., 2008), TALENs (transcription activator-like effector nucleases) (Huang et al., 2011) and the CRISPR (clustered regularly interspaced short palindromic repeats) system (Hwang et al., 2013). Gain-of-function approaches have also been developed, including transgenic techniques to introduce foreign oncogenes into the genome, which can incorporate meganucleases (Soroldoni et al., 2009), Tol2 transposons (Kwan et al., 2007) or bacterial artificial chromosomes (BACs) (Suster et al., 2011). Through these approaches, numerous mutant and transgenic zebrafish have been generated to model human cancers, and these have also been used in chemical screens. In addition to these cancer models, a number of mutant and transgenic zebrafish lines have been generated that facilitate the observation of oncogenic pathways in living fish, such as the casper mutant (White et al., 2008), a transparent zebrafish strain that maintains embryonic transparency through to adulthood because it lacks melanocyte and iridophore cell populations, and the vascular fluorescent reporter line tg(fli1a:eGFP) (Lawson and Weinstein, 2002). Together with advances in imaging technology, these lines have provided new opportunities to develop the zebrafish as a xenograft model with potential for in vivo drug screening.
Xenotransplantation is the transfer of living cells or tissue from one species to another (Cariati et al., 2011). The appeal of zebrafish xenotransplantation of human cancers for drug discovery and evaluation lies in the ability to directly observe the drug response of human tumor material, particularly primary patient-derived biopsy specimens that are often hard to maintain in vitro, in a cost-effective and time-efficient manner. The approach circumvents some of the current hurdles with rodent xenografts, such as those related to availability of sufficient patient material or challenges in optimizing murine hosts to enhance engraftment. Moreover, zebrafish xenotransplantation approaches have the potential to serve as a first step in a preclinical pipeline to screen for drugs before moving on to the more costly and time-consuming mouse and ultimately human trials. The zebrafish xenograft platform could also, for the first time, provide a bona fide real-time in vivo platform for personalizing cancer therapeutics.
Small-molecule drug screens in zebrafish
Pioneering work undertaken by several laboratories has demonstrated the efficiency of large-scale drug screens in zebrafish embryos. Arguably, these studies and the therapeutic revelations that have emerged from them represent some of the most clinically relevant contributions the zebrafish model has made in the last decade (Tamplin et al., 2012; Zon and Peterson, 2005). One of the first chemical screens using zebrafish embryos was piloted by Randall Peterson’s group (Peterson et al., 2000). They conducted a screen using a panel of structurally diverse chemical compounds and evaluated toxicity in embryonic development, including on the central nervous and cardiovascular systems. The authors identified several compounds that influenced the development of the central nervous system, altering its general morphology by significantly increasing the size of the hindbrain ventricle and producing tissue artifacts such as ‘sawtooth-like’ projections within the organ (Peterson et al., 2000). Subsequently a number of studies have demonstrated the utility of chemical screening in zebrafish embryos for identifying and evaluating the efficacy of potential anti-cancer agents (Murphey et al., 2006; Ridges et al., 2012; Yeh et al., 2009).
Zebrafish are ideally suited to high- and medium-throughput screens because their small size enables them to be arrayed in a variety of isolated well plates (12-well, 24-well, 96-well and larger) and bathed in water that contains the compound(s) of interest (reviewed in Peterson and Macrae, 2012). This approach provides a high-throughput platform that is substantially more rapid than injecting mice. Furthermore, embryo screens have the potential to reveal important information on absorption, distribution, metabolism and excretion when whole organisms are exposed to a drug (Makky et al., 2008; Peterson and Macrae, 2012). However, investigating these properties in embryo screens is still in its infancy. Zebrafish embryos are transparent and develop externally, which can aid in understanding drug absorption from the surrounding medium specifically when chemicals possess inherent fluorescence to facilitate direct visualization of drug absorption into the embryo. Subsequent drug excretion following treatment can also be observed and measured by exploiting the transparency of zebrafish embryos. However, this approach requires that compounds are water-soluble. Thus, drug solubility characteristics need to be established at the outset, including maximum water solubility or drug solubility in other common delivery solvents, such as dimethyl sulfoxide (DMSO) or methanol, before undertaking these types of screens. If compounds are not water-soluble, direct injection into the body of the embryo can be performed to ensure drug uptake. Both diffusion and injection approaches can be used for toxicity screens and to evaluate specific efficacy signals.
Evaluating drug toxicity
Systemic drug toxicity can be investigated using overall embryonic mortality as a metric from which the working range of a particular drug can be determined. For example, embryos (one or more per well) can be arrayed in multi-well plates and exposed to a series of drug concentrations to enable the maximum tolerated dose of a compound to be determined (Parng et al., 2002; Taylor et al., 2010). These studies can uncover toxic side effects within the context of a living animal that are not discernible in tissue culture. In this way, appropriate dosing can be determined prior to performing a chemical screen to evaluate drug efficacy in order to ensure that observations are not confounded by non-specific drug toxicity.
Candidate compounds from in vitro tissue culture screens can also be further evaluated using the zebrafish. Precise phenotypic observations can be made to determine drug-induced effects on specific aspects of development and tissue function. For example, the development of pericardial edema, changes in specific tissue or cell types made fluorescent through versatile transgenic strains, or altered growth of different organ systems (Barros et al., 2008; Eimon and Rubinstein, 2009; Sipes et al., 2011). For instance, a compound that selectively kills cancer cells in the tissue culture dish but that also causes severe central nervous system toxicity or renal damage in vivo might not be a good candidate drug for further development.
Applications of drug screening in blood diseases and angiogenesis
Zebrafish screens have made important contributions to the study of both normal and leukemic hematopoiesis. A chemical screen in wild-type embryos conducted by North, Zon and colleagues in 2007 (Durand and Zon, 2010; North et al., 2007) revealed a previously unrecognized role for prostaglandin E2 (PGE2) in the development of hematopoietic stem cells (HSCs). Using whole-mount in situ hybridization for increased expression of the HSC markers runx1 and c-myb, ten of the effective compounds were found to modulate the prostaglandin pathway. This discovery was subsequently validated in mice and led to a recent clinical trial (Cutler et al., 2013), suggesting that the use of PGE2 can enhance HSC engraftment following cord blood transplantation. This finding represents the first time a chemical screen in zebrafish has led to a clinical trial in patients and highlights the tremendous potential of embryonic screens for drug discovery. In another study (Yeh et al., 2009), the transgenic zebrafish strain tg(hsp:AML-ETO) was used to screen for chemical modifiers of AML1-ETO, an oncogenic fusion protein that is prevalent in acute myeloid leukemia (AML) that results in hematopoietic dysregulation in fish and elicits a malignant phenotype similar to human AML. An inhibitor of cyclo-oxygenase 2 (COX-2), nimesulide, was identified as an antagonist to AML1-ETO in hematopoietic differentiation. Furthermore, this screen exposed a role for COX-2 and PGE2 in the subsequent phenotypes associated with AML1-ETO expression (Zhang et al., 2013).
Other zebrafish drug screens have been designed to identify agents targeting tumor angiogenesis. Angiogenesis is a physiological process in which normal blood vessels are formed during embryonic development to deliver oxygen and nutrients to the required tissues of the body. Tumor cells have been shown to co-opt this process and use angiogenic recruitment for cell growth and subsequent metastasis (Camus et al., 2012; Chan et al., 2002; Cross et al., 2003; Radi et al., 2012; Tran et al., 2007). Many of these studies have used a non-cancer model, the tg(fli1a:eGFP) zebrafish strain, in which the fli1a promoter drives the expression of green fluorescent protein (GFP) in embryonic blood vessel endothelial cells, as a surrogate assay (Lawson and Weinstein, 2002). Common readouts in zebrafish drug screens have included changes to local blood vessel and hematopoietic cell organization and formation, alterations of organ development such as the heart and kidneys, and behavior changes such as swimming and reaction responses, following exposure to a chemical agent (Burns et al., 2005; Cao et al., 2009; Kokel et al., 2010; Murphey et al., 2006; North et al., 2007; Paik et al., 2010; Peterson et al., 2004; Ridges et al., 2012; Stern et al., 2005; Yeh et al., 2009).
As highlighted above, some of the most successful zebrafish screens have been in the identification of a new application for an existing drug, so-called drug repurposing. This is advantageous when familiarity and available human safety data can permit rapid transition to the clinical setting, as illustrated by the prostaglandin example discussed above (Durand and Zon, 2010). However, new drugs are also being identified in this way. For example, Ridges et al. (Ridges et al., 2012) recently applied a chemical screening strategy to a panel of T-cell acute lymphoblastic leukemia (T-ALL) mutant embryos and identified lenaldekar, a new compound, as a selective inhibitor of lymphocyte proliferation. Over 26,000 compounds were screened for activity that could reduce overall fluorescent-tagged lymphoblast populations in mutant embryos. Lenaldekar was shown to induce remission in a zebrafish T-ALL model without causing substantial toxicity. Similar observations and drug efficacy was confirmed using murine xenograft models (Ridges et al., 2012). These studies underline the versatility and robustness of using the zebrafish in small-molecule chemical screens with similar experimental design but diverse goals. As pharmaceutical companies increasingly make available their substantial chemical library resources to academia, there is no doubt more such discoveries will be made, and the zebrafish will be prominent in their screening.
In summary, drug screens in zebrafish have been used for and have the potential to validate existing compounds for use in many human disorders. In drug discovery studies, promising new compounds can be identified from panels of thousands in smaller quantities than in rodent studies, which require large volumes of reagents. Zebrafish embryos can be used to identify general drug toxicity and gross developmental abnormalities caused by drugs and to prioritize agents that should be further evaluated in additional preclinical model systems.
Human tumor xenografts in zebrafish are an innovative approach that can take full advantage of the inherent opportunities afforded by the zebrafish but with the added benefit of addressing any lack of conservation in drug response or molecular oncogenic pathways between humans and fish, and zebrafish xenografts are gaining traction for advancing cancer drug development.
Transgenesis has been a traditional approach to modeling human malignancies in zebrafish (Carradice and Lieschke, 2008; Chen and Langenau, 2011; Forrester et al., 2011; Jessen et al., 1999; Le et al., 2007; Leacock et al., 2012; Li et al., 2013; Project et al., 2005) but, more recently, the xenotransplantation of human cancer cell lines and primary human tumor samples has been successfully used to study human cancers in vivo (Corkery et al., 2011; Eguiara et al., 2011; He et al., 2012; Jung et al., 2012; Konantz et al., 2012; Nicoli et al., 2007; Taylor and Zon, 2009).
Xenotransplantation involves the transfer of one species-specific tissue to another animal species and has been used as a tool for many years to study human cancer (Cariati et al., 2011; Johnson et al., 1995; Merk and Adams, 1972). In particular, xenograft experiments have been extensively performed in mouse models of human disease, enabling the analysis of cancer cell proliferation, invasion, migration and induction of growth advantages such as increased angiogenic recruitment (Cheon and Orsulic, 2011), as well as enabling drug screening and evaluation (Huynh et al., 2011; Kelland, 2004; Sharpless and Depinho, 2006). Although murine models remain the ‘gold standard’ for xenotransplantation studies and drug evaluation, the time required, high cost and complexity of these systems mean that it is not always feasible to use mice. This is particularly relevant when funds to conduct such large-scale drug screens are limited. Moreover, the use of highly immunecompromised mice calls into question the actual relevance of findings in these models back to the bedside. By contrast, the ease and relative cost-effectiveness of performing these screens in zebrafish can be adapted to embryos transplanted with human cancer cells without the need for immunosuppression because, for the first month of life, the larvae have not developed an adaptive immune response (Lam et al., 2004; Traver et al., 2003).
The zebrafish xenotransplantation model can also be applied to drug screening and rivals transgenic approaches by allowing the direct evaluation of the most clinically relevant tissue, namely human cell lines or primary samples. This circumvents the limitations imposed by the lack of zebrafish-specific tools, such as antibodies for flow cytometry or for western blot confirmation, and the apparent absence of certain key genes that regulate human oncogenic pathways, such as the BRCA1 and INK4a/ARF tumor suppressors (Howe et al., 2013). The absence of certain genes in the zebrafish can also be exploited as a means of identifying human xenografted cells from the zebrafish embryo in ex vivo analyses and immunohistochemistry. For example, we have recently used the promyelocytic leukemia (PML) protein, for which the gene is absent in the zebrafish, as a marker of human primary T-cell leukemia cells during ex vivo proliferation analyses (Bentley et al., 2013). Finally, it should be noted that the human tumor xenografts interact with, and in many instances remodel, the host tissue following engraftment; for example, host vasculature can be recruited by tumor xenografts (Zhao et al., 2011). This is a useful property of the xenotransplantation system in zebrafish, allowing analysis of tissue remodeling, innate immune cell interactions with the human tumor and neoangiogenesis (tumor-associated angiogenesis) induced by the human tumor. These changes to the host might lead to developmental toxicities, precluding analysis of tumor progression. For this reason, all zebrafish xenotransplantation experiments begin with pilot experiments to ensure that the given tumor being studied can successfully engraft and that the zebrafish embryo can survive engraftment long enough to allow meaningful observations to be made.
Technical advances in zebrafish xenotransplantation
Lee et al. (Lee et al., 2005) first pioneered human tumor xenotransplantation in zebrafish by engrafting human melanoma cell lines, demonstrating that human cells could survive and migrate in zebrafish. Melanoma cells were injected into the blastula stage of zebrafish embryos. These cells were tolerated within the zebrafish for the first 8 days after injection and could home to the epidermis. Human fibroblasts and normal healthy melanocytes were also injected into zebrafish and displayed similar survivability to that of the melanoma cells, but lacked the mobility of the latter. Further studies have developed and optimized the parameters for transplanting human cancer cell lines, including breast cancer, leukemia and sarcomas, into zebrafish embryos and analyzing the transplanted recipients for cellular behavior and response to drugs (Corkery et al., 2011; Pruvot et al., 2011; Haldi et al., 2006; Marques et al., 2009). Human cancer cell lines survived, formed tumors and exhibited cell migration in zebrafish embryos.
Haldi et al. optimized transplantation parameters such as: site of injection, age of transplant recipients, injection cell quantity and post-injection incubation considerations (Haldi et al., 2006). Although numerous sites of injection were trialed, including the hindbrain ventricle and intravenous routes, the yolk sac of 2-day-old embryos was demonstrated to be an ideal site of injection because it provides a nutrient-rich environment for injected cells and is acellular, allowing cell growth and migration (achievable only through active cell motility) to be monitored. This group also showed that 35°C is the optimal temperature at which to incubate embryos following xenotransplantation. This temperature permits both natural embryonic development of the zebrafish transplant recipients and satisfactory human cell survival and proliferation. Marques et al. expanded on the field of zebrafish xenotransplantation by qualitatively capturing hallmark oncogenic events of human cancer cells post-embryonic injection (Marques et al., 2009). Patient-derived gastrointestinal tumor cells displayed cell proliferation, migration through the circulation and micrometastases within 24 hours following transplantation – events that were not observed using non-tumor cell lines (Marques et al., 2009). Injected pancreatic tumor cells transplanted into the cloche zebrafish mutant, which lacks a complete functional vasculature, displayed no cell migration (Herpers et al., 2008). These observations demonstrate that migration observed post-xenotransplantation of human cell lines into zebrafish embryos occurs via active transport mechanisms such as a circulatory route, and not by diffusion or other passive means.
With the establishment of these foundational parameters, xenotransplantation methods have continued to improve, with the incorporation of single-cell-based assays to quantitatively interpret in vivo human cell behavior. We have established approaches to reproducibly quantify cell proliferation rates and cell migration in zebrafish xenotransplantation, a necessity for applying xenotransplantation to determining drug responses (Corkery et al., 2011). Cancer cells are fluorescently labeled and injected into the embryos, and cell proliferation is monitored by live-cell microscopy. Embryos are then enzymatically dissociated into cells, which are stained to confirm that they are intact and then imaged to determine average cell counts per embryo (Corkery et al., 2011). Proliferation rates of several cell lines, including leukemia, sarcoma and breast cancer lines, have been measured using this assay, and proliferation rates of cells injected in zebrafish embryos demonstrate similar kinetics to those observed in vitro. When fluorescence cannot be used to mark human cells either because the cells are dividing too rapidly or the drugs being tested are inherently fluorescent (e.g. doxorubin), an immunocytochemical technique can be applied that uses a fluorescent antibody directed against a human protein not conserved in zebrafish (Bentley et al., 2013) (V. L. Bentley, C.J.V., D. P. Corkery, J. B. Pinder, M. A. LeBlanc, K. Bedard et al., unpublished results). Using these approaches, we have demonstrated the utility of zebrafish xenotransplantation for the study of specific drug-tumor interactions for both chronic myelogenous leukemia and acute promyelocytic leukemia by specifically inhibiting their proliferation in xenotransplanted zebrafish using imatinib mesylate or all-trans retinoic acid, respectively (Corkery et al., 2011). We have also utilized the zebrafish xenotransplantation assay to evaluate transcription factors that may be critical for migration and metastasis in childhood sarcomas (Veinotte et al., 2012) (A. M. El-Naggar, C.J.V., C. E. Tognon, D. P. Corkery, C. Hongwei, F. Tirode et al., unpublished results).
Many zebrafish xenotransplantation studies performed to date have used embryos at 48 hours post-fertilization (hpf) as transplant recipients. This developmental time point is considered the optimal stage for injection because it offers a relatively large transplant site (yolk sac) and absence of adaptive immune responses. Immunosuppression is required for xenotransplantation in zebrafish older than 30 days post-fertilization (dpf), when adaptive immunity is well-established (Lam et al., 2004). Other advantages that are apparent in transplanting human cancer cells into 2- to 5-day-old (48–120 hpf) embryos include the large number of animals that can be produced for experimentation, injected and housed simultaneously.
The yolk sac has been considered the optimal site of transplantation because it provides a favorable acellular site of injection that can nurture a variety of human cancer cell lines prior to and/or during tumor growth and cell migration. Other anatomical locations have also been used (Fig. 1), although the yolk sac remains the most widely used site of embryonic xenograft injection to date. The site selected can influence how a tissue sample engrafts, the efficiency of drug administration and delivery to tumor-engrafted materials, and, subsequently, response to therapy. Many of the sites used for injection are not native to the tumor tissue, so heterotopic engraftment is necessary. Although the zebrafish shares homology with many of the mammalian pathways and organ systems, it does lack several tissues found exclusively in mammals, which affects the possibility of orthotopic injection. Despite the lack of orthotopic sites for tissues such as the lung, breast and prostate, it might be possible to ‘add-back’ the required cells or growth signals that would allow normal growth cues associated with orthotopic injection. For example, embryo water could be supplemented with tissue-specific growth factors (e.g. androgens or estrogens), human cells expressing growth factors could be co-injected with the cells of interest, or transgenic fish expressing human growth factors, receptors and/or cytokines could be generated to enhance engraftment and tumor growth.
Zebrafish xenotransplantation as a tool to study tumor-microenvironment interactions
Another opportunity afforded by zebrafish xenotransplantation is that it enables direct observation of the impact of a particular treatment on interactions between tumor cells and the tumor microenvironment. A number of studies have highlighted the impact of the tumor microenvironment on the proliferation and migration of a variety of human cancers (Gupta and Massagué, 2006; Hanahan et al., 2000; Yu and Rak, 2003). Many normal cell and/or tissue behaviors are often co-opted by and for malignant cells. Solid tumor cells harness the oxygen and nutrients needed for their survival from oncogenic-induced blood vessels sprouting from healthy normal vasculature (Kaya et al., 2009; Singh et al., 2007; Zetter, 1998). Immune cells that normally combat infection might function in either pro-tumorigenic or anti-tumorigenic ways depending on cytokines and chemokines released by tumor cells and other inflammatory mediators generated in the local microenvironment (Kees and Egeblad, 2011; Spano et al., 2012). Studying and uncovering the interactions that take place between tumor and normal cell populations is essential for understanding cancer progression and for determining new ways to therapeutically inhibit pro-tumorigenic responses and enhance anti-tumorigenic behavior.
Fluorescent transgenic zebrafish reporter lines represent a key tool that can be paired with xenotransplantation to study cancer cell progression in a living microenvironment (Fig. 2). Currently available fluorescent reporter lines include tg(fli1a-eGFP) (Lawson and Weinstein, 2002), which expresses enhanced GFP (eGFP) in endothelial cells; tg(mpx:eGFP), which expresses GFP in neutrophils (He et al., 2012; Renshaw et al., 2006); and tg(mpeg1:eGFP) (Ellett et al., 2011), which expresses GFP in monocytes/macrophages. Combining such transgenic strains of zebrafish with transparent strains, such as the casper mutant, allows for the observation and analysis of tumor cell interactions with key players in the tumor microenvironment.
As mentioned above, there is a large time lapse of a number of weeks between the development of innate and adaptive immune systems in the zebrafish embryo. This temporal separation in the development of neutrophils, macrophages/monocytes and mast cells, and subsequently B and T cells, provides a unique opportunity to evaluate the specific contribution of each of these lineages to cancer progression and therapeutic response (He et al., 2012). He et al. showed that neutrophil migration could control tumor cell mobility within zebrafish embryos through pre-conditioning of extracellular matrix, resulting in the formation of metastatic niches. The group also showed that vascular endothelial growth factor receptor inhibitors reduce local tumor growth of xenografted embryos and blood vessel recruitment to the tumor site. These compounds also enhanced the rate of neutrophil migration, which in turn increased the migratory potentially of the injected tumor cells, resulting in micrometastases. These observations demonstrate how zebrafish xenotransplantation can be used to study tumor cell and tumor microenvironment interactions, validate drug efficacy of compounds that directly target elements of the microenvironment to prevent cancer progression, and uncover any unwanted or harmful bystander cell effects prior to moving a compound further along the preclinical pipeline.
Limitations of the zebrafish xenotransplantation platform for drug screening
Although all of the characteristics described above enable the completion of multiple statistically robust experiments simultaneously, the zebrafish xenotransplantation platform is not without its limitations. Although the lack of an adaptive immune response is beneficial for initial transplantation and injection, it might become a limitation to translation of findings, because adaptive immune cells can play vital roles in promoting or inhibiting the progression of human cancers and the effects of certain cancer treatments (Gupta and Massagué, 2006; Kees and Egeblad, 2011; Spano et al., 2012).
Human and zebrafish genomes are 70% similar based on conservation of individual genes (Table 1), with several cancer-associated genes found in mammals but not in the zebrafish, including BRCA1, p16 (CDKN2A), BRCA1, LIF, OSM, IL6 (Howe et al., 2013) and PML (G.D. and J.N.B., unpublished observation). This presents several challenges when studying the functions of these ‘missing’ genes or the pathways in which they play a role; for example, senescence studies in the fish might prove to be challenging given the lack of a p16 homolog, a major marker of senescence in mammals (Salama et al., 2014). Moreover, when foreign tissue and cells are introduced into fish, there is no guarantee that all of the molecular mechanisms linking the recipient and the xenograft tissue are completely conserved, which might impact interactions between host cells and the cancer xenograft. For example, human growth factors cannot support zebrafish hematopoiesis (Stachura et al., 2009). This issue is particularly relevant for tissues such as the lung, breast and prostate for which orthotopic sites do not exist in the fish. However, as discussed above, it might be possible to ‘add-back’ the required cells or growth signals to mitigate this problem during xenotransplantation, or to ‘humanize’ the fish by creating transgenic animals that express appropriate human growth factors, receptors and/or cytokines, as has been done in mice (Brehm et al., 2013).
Clinical applications of the zebrafish xenotransplantation platform
Xenotransplantation approaches can be used for small-molecule screens using similar procedures to those employed by traditional zebrafish transgenic models, including arraying injected embryos into multi-well plates, with the added benefit of being able to measure cell proliferation and metastasis in the presence or absence of a drug.
A recent area of growth for zebrafish xenotransplantation in chemical screens is the progression towards enhanced experimental automation and rapid high-resolution imaging (Ghotra et al., 2012). Most xenotransplantation studies require manual injection of the human cells, which can be a time-consuming technical hurdle. Although a skilled researcher can inject a few hundred embryos in an hour, the use of automated methods would greatly expedite this process.
Efforts are now underway to apply this approach to patient-derived specimens. Primary human tumors, such as gastric cancers, prostate and primary leukemia samples, can be injected into embryos, where they proliferate and disseminate (Bansal et al., 2014; Marques et al., 2009; Pruvot et al., 2011) (V. L. Bentley, C.J.V., D. P. Corkery, J. B. Pinder, M. A. LeBlanc, K. Bedard et al., unpublished results). Murine xenotransplantation studies using primary patient samples can be very complex and relatively expensive. The total sample needed is several times greater in mice than in zebrafish xenografts (typically 100–200 cells are needed per zebrafish embryo), so zebrafish provide a key advantage when working with precious limited biopsy samples. Zebrafish engraftment can also take as little as 2–3 days, whereas, in a murine model, primary tissue grafts can take weeks to months, if engraftment occurs at all.
Given the speed of zebrafish xenotransplantation, it could be used as a predictive tool for patient responses to drug therapies. Transplanting malignant patient tissue or cells into hundreds of zebrafish embryos and monitoring the response to planned investigational drug treatments could yield valuable information on the most suitable drug to be administered for that particular patient. Evidence of a response to a small-molecule inhibitor might indicate effective targeting of an actionable genetic driver lesion, as we observed for a child with T-ALL who expressed a novel γ-secretase-responsive Notch mutation (Bentley et al., 2013) (V. L. Bentley, C.J.V., D. P. Corkery, J. B. Pinder, M. A. LeBlanc, K. Bedard et al., unpublished results). The ability of zebrafish xenotransplantation to provide such actionable personalized clinical information is promising and, with further validation, has the potential to find its way into future clinical trials.
The approach could be most readily adapted in leukemia, where the incorporation of targeted therapeutic strategies is becoming commonplace and diagnostic material is easily accessible. In contrast to current strategies where a targeted therapy could be randomly assigned to a patient who might or might not be screened to confirm the presence of a particular actionable genetic lesion, incorporation of the zebrafish xenograft platform could provide a more practical functional genomics readout with real time in vivo response data, which could be followed by genomic validation. Applicability to solid tumors is also feasible; however, here there is a need to overcome the challenges of limited availability of biopsy material and tumor heterogeneity (which could mean that the sample being engrafted is not representative of tumor genetics or behavior).
Diagnostic leukemia or solid tumor samples can also be used to predict responses to experimental therapies that might be considered in the event of refractory disease or relapse. Anecdotally, we have undertaken these studies, which could inform the selection of a particular Phase I/II trial. In most cases the eligibility for these studies is refractory or relapsed disease without specific evidence for the presence of a particular target or activity of a given pathway. Evidence of an efficacy signal from pre-screening available Phase I/II drug options using a zebrafish xenograft model could provide timely guidance in choosing the most promising therapy. Although encouraging in its ability to more specifically tailor therapy at relapse, this strategy could be limited by clonal evolution, which has the potential to significantly alter the genetics and response of a tumor from the time of diagnosis.
As is the case for other embryonic zebrafish chemical screens, in addition to evaluating drug efficacy, zebrafish xenotransplantation can help determine drug toxicity. By using xenografted embryos, the value of co-administration of a toxicity-mitigating agent can be studied in the context of ongoing primary drug efficacy. Using this approach, we confirmed that two novel compounds could prevent cardiotoxicity from anthracycline antibiotics without compromising cytotoxicity in an engrafted T-ALL cell line (Y. Liu, A. Asnani, L. Zou, V. L. Bentley, M. Yu, Y. Wang et al., unpublished results). Similarly, drug efficacy and neurotoxicity could be evaluated. The optical clarity of the zebrafish embryo enables clear visualization of neural structures and there is a battery of probes that label each distinct component of the central nervous system, facilitating the evaluation of drug-induced anatomic changes. Drugs of lower molecular weights more easily cross the embryonic blood-brain barrier, a factor to be taken into consideration when evaluating the impact on brain development of a compound given systemically. Finally, myelotoxicity is inherently evaluable in the zebrafish xenograft context. By conducting xenograft drug response studies that incorporate transgenic reporter lines that label neutrophils (Hsu et al., 2004; Renshaw et al., 2006), erythrocytes (Long et al., 1997) or thrombocytes (Lin et al., 2005), the impact of a drug on cell numbers can be visually determined and subsequently quantified using fluorescence-activated cell sorting.
Transgenic and xenotransplantation approaches have long been used in a complementary manner in mice to evaluate responses of tumors to drugs. Transgenic approaches in zebrafish have become well established, with a growing acceptance and appreciation for the tremendous efficiency of high-throughput chemical screens in genetically modified embryos to provide new indications for the repurposing of available agents and reveal the potential efficacy of novel compounds. Although still in its infancy, the zebrafish xenotransplantation platform capitalizes on many of the advantages inherent in the zebrafish system, with additional opportunities in the evaluation of patient-derived samples, tumor-microenvironment interactions and the potential ability to inform treatment decisions in an actionable time frame. Like all techniques, zebrafish xenotransplantation is not without its limitations, and additional proof-of-principle studies are needed to conclusively show that features that have made the mouse xenotransplantation model the ‘gold standard’ for preclinical studies, such as the ability for orthotopic engraftment and serial transplantation, are equally robust in the fish (Table 1). Given current pressure on the pharmaceutical industry to use more cost-effective pre-screening tools for drug discovery, and the clinical demand for more targeted and personalized therapy, we predict that the popularity of the zebrafish xenotransplantation model will continue to increase and will provide a uniquely powerful tool to improve the outcome for patients with cancer.
This article is part of a Special Issue, Spotlight on Zebrafish: Translational Impact. See all the articles in the issue at http://dmm.biologists.org/content/7/7.toc.
The authors declare no competing financial interests.
G.D. and J.N.B. are funded by a Collaborative Health Research Project from the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council CPG-12-10-29/CHRP-41-38-59. C.J.V. and J.N.B. are funded by a C17 Canadian Pediatric Hematology/Oncology Network/Ewing’s Cancer Foundation of Canada Grant and the Cancer Care Nova Scotia Peggy Davison Clinician Scientist Award.
- © 2014. Published by The Company of Biologists Ltd
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.


Whenever I’m back home in the US, I’m always grabbing books about the nation to help the kids learn more about their American side. One of my favorite books is Laurie Keller’s The Scrambled States of America, which I just read to them again today.
In it, Kansas decides he’d like to meet some other states, so all the states have a party to meet each other. That leads to the idea of swapping places. It’s a great, great read, with all the states deciding they want to go back home in the end. My kids are entranced by it and love parts like Nevada and Mississippi falling in love, plus some of the trivia the book has.
Even I learned things, such as Michigan having a thumb region. That got me to thinking about the various ways I learned to remember some of the states, which I’ve been passing on to the kids.
California is easy, in that they know it’s Daddy’s state. But I did initially remind them that if you look at it, it sort of forms the shape of a C for California. Similarly, Texas always looked like a lower case T to me, while Louisiana looks like an L.
Then there are parts of states that have names based on what they look like, such as the Oklahoma panhandle and the aforementioned Michigan thumb. I’d love to know what some other areas of states are referred to by those who live there. It’s one of those things you don’t know if you’re from out of state. If I eventually enable comments on the blog, please post some you know of below.
At the end of the book, all the states are listed alphabetically. I made the mistake of singing these for the kids to the tune of Fifty Nifty United States, or what I can remember of it from learning it in elementary school. I always lose the tune near the middle.
Turns out, the song was written by Ray Charles, which you can listen to and buy here.


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We’ve all heard of amino acids, but what exactly are they, and why are they essential to our diets?
Amino acids are the building blocks of protein. They are organic compounds containing an amino group (-NH2) and a carboxy group (-COOH). As about twenty percent of the human body is made up of protein, amino acids make up a large proportion of our cells, muscles, and tissue.
Amino acids are integral to to the biological processes that happen within our bodies, such as giving cells their structure, transporting and storing nutrients, as well as forming our organs, glands, arteries and muscles. They’re also essential for healing wounds and repairing tissue, especially in the muscles, skin, bones, and hair.
There are 23 proteinogenic (protein building) amino acids in total, and over 100 natural amino acids, which are non-proteinogenic. Of the proteinogenic amino acids, 9 are essential, 11 which are nonessential, and 3 of which are not found in the human body.
Essential amino acids are not produced naturally by the body, so they have to be obtained from the foods we eat. The 9 essential amino acids are: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. Each of these amino acids have unique properties and play crucial roles in our working bodies.
Non-essential amino acids are produced in the human body, so they are not essential to our diets. There are also three amino acids (selenocysteine, pyrrolysine, and N-formylmethionine) which are not found in humans, but are non-standard protein-building amino acids found in plants and other organisms.
Branched-Chain Amino Acids (BCAAs) refers to three essential amino acids: leucine, isoleucine, and valine. These are amino acids that have aliphatic side-chains with a branch in their atomic structure. Branched-Chain Amino Acids account for 35% of the essential amino acids in our muscles.
So how do we get the amino acids that we need, and what exactly do they do? Here’s a quick insight into each of these powerful little molecules.
The 9 Essential Amino Acids
Leucine helps to stimulate muscle strength and growth, and helps to retain lean muscle when dieting. Leucine is the main amino acid directly responsible for activating an essential compound in muscle called mTOR(mammalian target of rapamycin), which is directly responsible for up-regulating protein synthesis. Leucine provides the basic building blocks for muscle and helps to synthesise more. Leucine also helps to regulate blood sugar levels by moderating insulin into the body during and after exercise, and has a positive impact on our brain and neurotransmitters.
Isoleucine is an isolated form of leucine that helps the body produce haemoglobin. Haemoglobin carries iron in the blood and regulates blood sugar which is burned for energy in the muscles during exercise. Whey protein isolate is naturally high in Isoleucine.
Isoleucine also assists nitrogen growth within the muscle cells, which is a large part of our structural and DNA makeup.
Sources of Isoleucine: soy, meat and fish, dairy and eggs, cashews, almonds, oats, lentils, beans, brown rice, legumes, chia seeds.
Lysine is one of the main amino acids that is responsible for muscle repair and growth, and has also been shown to boost the body's immune system. Lysine also helps the absorption of other minerals in the body and is needed for the synthesis of collagen which is the main element needed for the formation of connective tissue and bones in the body.
Sources of Lysine: eggs, meat, poultry, beans, peas, cheese, chia seeds, spirulina, parsley, avocados, almonds, cashews, whey protein.
Methionine is important for the growth of new blood vessels and muscle growth, and it contains sulphur, which is integral to tissue and muscle health. Without enough sulphur in the body, people can be susceptible to arthritis, damaged tissue, and have trouble healing. Methionine also aids in the production of muscle growth and the formation of creatine, which is needed for energy. Methionine can also dissolve fat within the body and reduces fat deposits in the liver.
Sources of Methionine: meat, fish, cheese, dairy, beans, seeds, chia seeds, brazil nuts, oats, wheat, figs, whole grain rice, beans, legumes, onions, and cacao.
Phenylalanine is turned into the amino acid tyrosine within the body, which is needed to make proteins and brain chemicals such as epinephrine, L-dopa, norepinephrine, and thyroid hormones. Phenylalanine therefore has a large impact on our mood and mental health.
Sources of Phenylalanine: milk and dairy, meat, fish, chicken, eggs, spirulina, seaweed, pumpkin, beans, rice, avocado, almonds, peanuts, quinoa, figs, raisins, leafy greens, most berries, olives, and seeds.
Threonine supports health function of the immune system, liver, heart, and the central nervous system. It is also needed to create glycine and serine, amino acids that are necessary to produce elastin, collagen, and muscle tissue. It is essential for the healthy working of the muscles, and help to keep them strong and elastic. Threonine also helps to build strong bones, and can help to accelerate the healing of wounds and tissue injuries.
Sources of threonine: lean meat, cheese, nuts, seeds, lentils, watercress and spirulina, pumpkin, leafy greens, hemp seeds, chia seeds, soybeans, almonds, avocados, figs, raisins, and quinoa.
When tryptophan is absorbed by the body, it is eventually turned into serotonin - the chemical responsible for making us feel happy, is a neurotransmitter, and helps to lower stress levels and depression. Tryptophan is also known for inducing a relaxing effect on the body, and promotes healthy sleep patterns, as well as supporting brain function and nervous system function.
Sources of tryptophan: chocolate, milk, cheese, turkey, red meat, yogurt, eggs, fish, poultry, chickpeas, almonds, sunflower seed, pepitas, spirulina, bananas, and peanuts.
Valine is essential for optimal muscle growth and repair. It helps to supply the muscles with extra glucose responsible for energy production during physical activity, making it essential for endurance and overall muscle health. It also helps to smooth working of the nervous system and cognitive function, as well as curing metabolic and liver diseases.
Sources of valine include: cheese, red meat, chicken, pork, nuts, beans, spinach, legumes, broccoli, seeds, chia seeds, whole grains, figs, avocado, apples, blueberries, cranberries, oranges, and apricots.
Histidine supports brain health and neurotransmitters (in particular, the neurotransmitter histamine). It also helps to detoxify the body by producing red and white blood cells, which are needed for overall health and immunity. Histidine can even help protect tissues from damage caused by radiation or heavy metals.
Sources of Histidine: red meat, cheese, white meat and poultry, seafood, soybeans, beans, legumes, chia seeds, buckwheat, potatoes.
So what is the best source of amino acids?
There are many amino acids on the market that are manufactured chemically. This can be done with chemical synthesis or extraction from protein sources. Synthesized amino acids differ in effect, depending on the way that they were genetically engineered. We recommend obtaining amino acids from a natural protein source, rather than a synthesized substitute.
Whey protein is one of the few sources that naturally contain all 20 amino acids, making it a complete protein.
Bare Blends’ whey protein isolate blends have a superior amino acid profile, and are especially undenatured. They provide our bodies with the most functional protein to recover, repair and build muscles, and also boosting our immunity.
The convenience factor of our vegan protein blends, or whey protein blends is an important one - as with after exercise it is important to fuel our bodies with amino acids straight away so they can begin recovering our muscles immediately.
These blends are also extremely handy for quick, nutrient-dense breakfast smoothies when you don't have time for anything else. Blending up a serve of our WPI with dairy/nut milk or your liquid of choice, with some frozen fruit is a delicious healthy breakfast which will keep you sustained, and contain the protein and amino acids which your body needs to recover and perform optimally.


A key performance indicator refers to a measurable value which indicates how a firm is achieving its primary business objectives (Marr, 2012). In most cases, organizations use KPIs to evaluate if they have achieved their targets. Some of the financial key performance indicators include gross profit margin, net profit margin, current ratio, aging accounts receivable, and net profit. The indicators are also used to show the financial performance of a company.
Profit is considered the most important financial performance indicator as it entails analyzing both the net and gross profit of the company to see how it is performing (Parmenter, 2007). Increased profit is an indication that the company is performing well financially. Cost is another important financial indicator to consider. The cost metric measures the cost effectiveness of the company. It also shows that the company should find the best ways of managing and reducing costs for it to make profits. The metric of cost goods sold enhances the company to get a rough idea of how the profit margin and product markup is supposed to be to ensure that the company is performing well (Marr, 2012). Furthermore, it is an important key that is used to knowing how the firm can outsell its competition. If the company can compete effectively with the competitors, then it is an indication that it is performing well. The day sales outstanding considers the accounts receivables of a company then divide them with the credit sales. The number is then multiplied by the total number of days in the timeframe of the firm. In case the number achieved after computation is low then it indicates that the company is performing well.
Target versus LOB revenue helps in comparing the projected revenue and the total revenue of the company. Minimizing the costs is not the only way that can be used to know if the company is performing well. The customer lifetime value should also be put into considerations when measuring the financial performance of a company as it shows if the company has a long-term relationship with the customers. According to the information provided, it is evident that 75% of the customers usually continue with the project after designing. Using this metric helps the company to get the best customers at reasonable prices. The customers retention and satisfaction are another important issues to consider when analyzing the performance of a company. According to this concept, the customers should be motivated for them to continue yearning for a firms services (Parmenter, 2007). Motivated customers usually put more efforts in ensuring that the products produced are of high quality and the customers are served well. Different performance indicators can be used to measure this metric including the number of consumers making purchases frequently.
The net promoter is another important performance indicator although it is used in measuring the long-term growth of the firm (Marr, 2012). Through this metric, the customers will get the chance of recommending the company to their friends, colleague, and relatives which will result to increase the sales and profits of the company. The number of customers a company is gaining or losing helps it to know if the needs of the consumers are met (Parmenter, 2007). When the company is gaining more customers, then this is an indication that it is performing well. However, when the customers are reducing then it means that the company is not performing well. Thus it should find ways of evaluating the needs of its consumers.
The current ratio describes the capability of the company to pay its bills. It is calculated by diving the current ratio with the current liabilities (Parmenter, 2007). Through this, the creditors of the company will easily know if their debts will be settled. Furthermore, aging accounts receivable involves sending bills to the consumers. In case some of the customers pay their bills in time while other pay their bill late, the company can find the reason for its cash flows problem. The company can resolve this issue by commencing to write off the debts or commence charging interest on the accounts that are overdue.
The following are the main financial indicators that determine the financial health of Royal Design Inc. the gross profit is the most important financial performance indicator used in determining the financial health of the company.
Gross profit= (revenue-the cost of goods sold)
$10506 9245 = $1261.
In comparing the gross profit for the financial year 2016 and 2015, the one for the financial year 2016 is low which indicates that the company is not performing well. In 2016, both the cost of sales and revenue have decreased. All the same, the gross profit of the business is large enough to cover its fixed operating expenses. Therefore, although the profitability of the company is decreasing it has the ability to cover its fixed operating expenses. The net profit is the amount the company is left with after paying all the bills. It is computed by subtracting the total expenses from the total revenue. In 2015 and 2016, the company made a net profit of $339 and $(67) respectively. The company made losses in 2016 which is an indication that it is not performing well financially. The loss made by the firm means that it does not have enough capital to keep its operations during the slow periods as it made a loss.
Net profit financial indicator helps in showing the company the percentage of revenue that was profit. It is computed by dividing the net profit by the total revenue. 67/10, 506 = 0.006377. The value of the profit margin is very low. Thus, it cannot assist the company to set goals and project the future profits. Most of the firms use the aging accounts receivable as an eye opener. It indicates the duration of time the consumers take to pay their debt. According to the information provided, it is evident that only 15% of your consumers proceed with their projects at a later date. Therefore, the firm does not have a large number of debtors. However, it can use the method of writing off debts or placing interest for the consumers who take a long period before repaying their debts.
The current ration financial key performance indicator is computed by dividing the current assets by the current liabilities. In 2015, the current ratio of the company is 2677/2168 = 1.2 while in 2016, 2487/ 1942 = 1.3. Therefore, it is evident that the current ratio of the company of the company reduced from the previous year. However, ideally, current ratio should be between 1.5 and 3. The current ratio indicates that the company has a small capability of paying its bills.
Non-financial key performance indicators
The number of customers that a company has is an indication of how the company is performing. A rise in the number of customers purchasing the products of the firm is an indication that it is performing well (Parmenter, 2007). Furthermore, if the number of new customers increases then it is an indication that the needs and preferences of the existing consumers are met. Responsible investment is another non-financial key performance indicator. A company should have a good investment capability to show that it is growing. Having a high percentage of investment capabilities is an indication that the firm is performing well (Marr, 2012). The cultural entropy is also used in measuring the amount of limiting or negative values that exist in a company which can lead to unproductive work. If the cultural entropy is low, then this indicates that the firm has a healthy culture.
Donating to the community is another important factor in determining is the company is performing well. It indicates that the company is concerned about its community and it is doing something important to give back to the community (Parmenter, 2007). Employee satisfaction and engagement allows a company to create a workforce which is engaged. Increasing the morale of the employees makes them put more effort into their activities. Therefore, when a company gives its employees incentives, and they put more effort in their different activities, then it is an indication that it is performing well.
The quality of work produced by a firm is a non-financial key performance indicator. It is evident that RAD is performing well in quality because 75% of the customers usually proceed with the projects after they are designed. Offering quality products may result in an increase in sales and profits of the organization. Offering good customer service to the customers helps in providing good experience as they are satisfied with the products of the firm (Marr, 2012). Making the consumers feel valued will give the firm more business in the future. Additionally, when the employees have good interactions with customers, then it is an indication that it is performing well.
Informal conversations and surveys with the employees help the firm to assess its level of consumers service that the firm is currently providing. Public image is also used when measuring the non-financial key performance of accompanying (Parmenter, 2007). When a company improves the way the general public views it, then it is an indication that it is performing well. When the general company views a company well, then it means it can make stronger relationships with the community. The aim of every company should be to maintain its professional image (Marr, 2012). Donating funds and time to the charitable organizations helps in establishing the firm as a fixture in the society.
Reasons for decline in profitability
The increase in costs incurred by a company may have resulted to decrease in profit. The cost of labor per hour paid to the employees are high (Pwc, 2013). Additionally, the cost the company uses in purchasing the raw materials is high which shows that the company uses much of its capital in paying the labor costs. Additionally, the fixed costs of the company are high. Thus, the firm should produce a lot of its product for it to benefit from the economies of scale. The high fixed costs of the company are the reason why the profitability is decreasing. The decrease in the sales is another reason why the profitability of the company has declined.
In comparing the sales of the company in the financial year 2015 and 2016, the sales declined in 2016. The decrease in the sales of the company may be as a result of pricing issues. The prices charged by the firm on its products are low. The prices are almost similar to the cost of producing the products. On the other hand, the competitors might have dropped their prices which cause the firm to shift. In the case of this company, the competitors are a non-issue because 75% of the customers continue with their projects after the company had designed them. Creativity changes is another strategy that can be used to determine the reason for the decrease in sales (Osburg & Schmidpeter, 2013). The company might have designed its products without testing.
Although the financial costs of the company declined from the previous year, the costs are still high because the company is small. The high financial costs might have need caused by increased interest rates or even increased capital expenditure (Osburg & Schmidpeter, 2013). Another reason why profitability of the company decreased may be increased competition. The market of the products the company is selling is very competitive. Thus, the consumers might have resolved to purchase products from the companies that are charging cheaply. Furthermore, in considering the type of business the company is doing, it is easy for new firms to enter the market as compared to the other industries.
The decline in demand for the products sold by the company resulted in declining in profitability. The decline in the demand is evident through the cost...
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|Understanding and Enhancing the Value of Female Corrections Professionals|
|By Robert Winters, JD, Professor, School of Criminal Justice, Purdue Global University|
The first female warden in the United States was Mary Weed, who after the passing of her husband in 1793 became caretaker of the Philadelphia Walnut Street Jail (and who enjoyed, it should be noted, an administration remarkably free of corruption). It was another quarter-century before the first female correctional officer was appointed, at New York’s Sing Sing prison in 1822. Thereafter, the growth of women in corrections slowed drastically, not improving until the late twentieth century.
In 1969, women made up 12 percent of the correctional workforce, and that only in facilities housing female inmates. Although a 1972 amendment to the Civil Rights Act of 1964 prohibited gender discrimination in hiring by state and local governments, and the National Advisory Commission on Criminal Justice Standards and Goals promulgated guidelines for hiring women in all correctional facilities in 1973, as of 1978 four state agencies still did not hire females for male correctional facilities. (One of those states, Texas, had no female correctional officers in 1978 but had 8,528 by 1998.) While various researchers and surveys have used different methodologies over the years, we can nevertheless obtain snapshots of the progress of women entering the field of corrections.
By 1995 the overall general correctional staff in the U.S. was 41 percent female. In 1999, 69,199 of 207,600 jail employees (33 percent) were female. In 2001 24.5 percent of correctional officers in male facilities were women. As of 2005 69,299 of 419,637 or 16.5 percent of federal and state officers were female. By 2007 women represented 37 percent of the adult correctional workforce and 51 percent of the juvenile workforce. The Bureau of Justice Statistics study Women in Law Enforcement, 1987-2008, published in June 2010, shows that in 1998 12 percent of Bureau of Prisons officers were female, up to 14 percent in 2008.
While these numbers represent significant improvement in just over three and a half decades, the percentages seem anemic when one considers that the total U.S. labor force is about 47 percent female. Moreover, as of 2007 over one million women were under the supervision of the U.S. criminal justice system, with over 200,000 of those incarcerated, and the number of female inmates increased by 404 percent from 1985 to 2007 compared to 209 percent for males. Clearly, there will be a significant female offender population requiring oversight.
Research has demonstrated repeatedly that there are very real differences between the genders (although it is important to recognize that those differences represent strengths that can be complementary in a mixed-gender environment, not weaknesses of one gender or the other). Specifically, women overall tend to be more communicative, collaborative, inclusive, and skilled with people, particularly in terms of being patient, empathetic, and nurturing. They are also usually desirous of information and interested in intricate relationships, whether those involve people, processes, or data.
It should not be surprising, then, that research has shown that female correctional staff have a positive effect on the environment even of all-male facilities. A 1980 study reported that male offenders did not feel that female officers invaded their privacy and did not resent taking orders from females. A San Quentin warden found female officers to be more observant and more attentive—important considerations for both security and improved inmate relations. Research in a prison in the southern U.S. revealed that male offenders were less likely to engage in violence in the presence of female officers and considered those officers to be just as competent as their male counterparts. In fact, another study found that male inmates viewed female correctional officers more favorably than did male staff members.
With the shift toward reentry preparation, the particular strengths of female correctional staffers have significant value. For example, one study found that offenders were more likely to be open with female officers regarding medical and educational concerns. The typical female approach to interpersonal communication is less confrontational, and women are socialized to view physical aggression as a last resort; these two traits produce lower levels of violence and fewer assaults. An experiment in the late 1990s in the Israeli Prison Service found that “the presence of women helped prevent trouble; they had a sharper sense for impending problems....” Also, because women tend to have a more collaborative decision-making process, as leaders they tend to produce subordinates who are better at problem-solving and in general produce more creative solutions.
How, then, do we incorporate more women into corrections? One of the first areas to consider is training. Opinions do differ on whether female officers should receive gender-specific training. Many authorities, including the South Carolina Department of Corrections and the National Institute of Corrections (NIC), feel that such training is appropriate and vital. Others, such as the female warden of a 1,000-bed male facility that is manned by nearly 80 percent female staffers, insist there is no such need. It is perhaps worth considering that in promoting gender-specific training, both South Carolina and NIC refer in part to the challenges of working in a male-dominated environment. It stands to reason, then, that once the correctional workforce more nearly represents the gender composition of the general population, the need for such training might well fade away. On the other hand, training that helps both male and female staffers to understand the aforementioned differences in the genders (and more not discussed here) could be a real benefit to all concerned.
While women have entered the correctional workforce, their promotion into supervisory and other leadership roles has been slower. Good mentorship is key to developing leaders, and with few female role models, women correctional professionals have suffered a dearth of mentorship. As women gradually enter the leadership ranks in greater numbers, a “critical mass” of potential mentors will develop. However, given their relative scarcity it is vital that female leaders act as mentors at every opportunity.
Along with the military, corrections is one of the last remaining male-dominated fields. But correctional institutions can benefit tremendously from what women have to offer, and it is important that women have the opportunity to fulfill their potential.
Corrections.com author, Robert Winters, holds a Juris Doctorate degree and is a Professor with Kaplan University. He is also a member of the National Criminal Justice Association and serves as a Western Regional Representative, a member of the National Advisory Board and their National Elections Committee.
Other articles by Winters
IN CASE YOU MISSED IT


Oatstraw: Medicine from the hills
The hills surrounding Yolo County are covered with oatstraw. In the coming months this annual green grass will turn to gold. As summer rolls in, the green and gold of the hills give way to a darker brown. The “golden rolling hills of California” are honored in paintings, songs and poems. Have you ever wondered which plants give us this wonderful display of changing colors? Well, one of them is oatstraw.
Oatstraw (Avena sativa) is a plant that has colonized millions of acres. It escaped the farmer’s oat fields and has naturalized throughout California. It chokes out native plants and can literally take over entire hillsides. The good thing about it is that almost everyone can benefit from drinking oatstraw tea. It grows in such abundance that unlike many other medicinal plants, it does not matter how much of it you harvest. You can pick as much oatstraw as you want without having to worry about the environmental impact of your harvest.
Oatstraw is usually ready to harvest from mid February through mid April depending on its exposure to sun and access to water. As with harvesting any herb, do not pick any plants unless you are absolutely sure that you have identified the correct plant. It is best to attend an herb walk or have a knowledgeable person introduce you to a plant. When harvesting any herb you want to make sure that you pick them in a place that is as clean as possible. Never harvest near a road, from an area that may have been sprayed with herbicides or from a place where dogs or cats frequent.
Oatstraw needs to be harvested while it is still green. If the plant is beginning to turn gold, it is past the harvesting stage and you need to wait until next year. It is important to harvest oatstraw while there is still a milk-like substance in the seed pods. Squeeze the seed pods, if they are milky, then harvest the top seven inches of the plant including the milky seed pods. Let the plant dry in a cool, dark place and then store it in a jar or a paper bag in a dark cabinet. If you are not able to harvest the herb yourself, dried oatstraw is readily available at the Davis Food Co-op.
Why would you want to go through all of this trouble to have a bunch of dried oat grass around your house? Oatstraw is a wonderful nourishing herb that is one of the best remedies that I know of for stress, emotional challenges, and a burned out nervous system.
Oatstraw helps with insomnia, a chattering mind, anxiety, nervous exhaustion, nerve pain, recovery from substance abuse, emotional trauma, tension, depression, P.M.S., and nervous headaches. When someone seems overwrought, on the edge, overdone, frazzled, and generally worn out, I suggest oatstraw. Oatstraw helps to regenerate and nourish the nervous system. It is gentle, calming and soothing. Oatstraw is not a quick fix herb; it is an herb that can be quite effective if used on a regular basis over time. Every person and every situation is different, but a general guideline could be to drink two to three cups of infusion, three or four times a week for several weeks.
Oatstraw is one of my favorite herbal remedies for emotional challenges and stress and it actually has a very nice taste! Hardly a week goes by without use this gentle healing medicine from my surrounding hills.
Add four tablespoons of dried oatstraw to one quart of water. Bring it to a boil, turn off the heat, let it sit in the pot with the lid on for four hours, then strain the herb from the infusion. Drink at room temperature throughout the day or re-heat your infusion as desired.
Kami McBride is the author of The Herbal Kitchen and has helped thousands of people learn to use herbs in their daily lives in ways that are healthy, safe and fun. She is the director of Cultivating the Herbal Medicine Woman Within, an experiential training in using herbs in the home for everyday health. An intuitive and inspiring teacher, Kami works to revive the cultural art of home herbal care and teaches herbology as a relationship with the Earth and a way of life. For a schedule of classes or herbal consultations Kami can be reached at (707) 446-1290 or sign up for her free herbal e-newsletter at: www.livingawareness.com


I believe that the term “governor” and “corruption” have been synonymous since at least 70 B.C. when Cicero made the legal case against Caius Verres, the Roman governor of Sicily. Amongst a host of other allegations, Cicero charged that Verres had famously stripped the interior of that contented island of everything of value, and then forced the city of Syracuse to build and crew a new ship each year to transport Verres’ plunder back to Rome, where he kept the plunder and sold the ship - and kept the money. Before Cicero had even finished presenting his case, on the advice of his own lawyer, Verres fled Italy with a fair part of his wealth still intact. We know this because years later Mark Anthony had Verres executed, in order to steal what Verres had stolen from Sicily. The murder of corrupt Roman officials by other corrupt Roman officials had, by then, become part of the circle of life.
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Availability of CCS was found by the IPCC  to lower overall climate change mitigation cost to society, based on the results of integrated assessment models. In this presentation, drawing on recent studies [2, 3], we use the integrated assessment model GCAM to explore the ways in which CCS could be used in a future carbon-constrained world, focusing on its possible roles across different sectors ─ electricity, liquid fuels (predominantly, biofuels for transportation), and industry ─ and coupled to different primary fuels (oil, gas, coal, and biomass). To generate scenarios, we assume an increasing economy-wide global price on GHG emissions high enough to match radiative forcing targets, approximating a mitigation pathway that is least cost to society for a given target.
The total scale of CCS deployment largely depends on the stringency of the target, with more CO2 stored with more ambitious mitigation. For example, in a RCP 2.6 scenario, GCAM estimates that about 350 Gt of CO2 would be cumulatively stored by 2100, compared to approximately 190 Gt in a RCP 4.5 scenario.
Results show that the deployment of CCS technologies is not limited to fossil fuels, nor to power plants. There is potential for significant long-term climate change mitigation from application of CCS in the use of biomass to produce both electricity and liquid fuels [2, 3]. Moreover, in the more stringent climate change mitigation scenarios examined with GCAM (e.g. RCP 2.6 and 4.5), most biofuels and bio-electricity over the century use CCS to reduce their emissions.
Our results (see Figure) show a near-term role for some industrial process (such as natural gas processing, and cement and fertilizer production), because of the lower cost of CCS in some instances. This could drive CCS development and technology cost reduction. However, these industrial processes currently account for only a small fraction of global GHG emissions. Thus, deployment of CCS technologies at a scale that contributes significantly to long-term climate change mitigation entails large-scale deployment in the power sector and/or coupling to production of biofuels (if biofuel production were to grow substantially in the future), simply based on the projected scale of these sectors .
Our results indicate that the use of biomass coupled to CCS in the electricity sector could become significant (reaches 5% of the total CCS applications) around 2050 in the RCP 4.5 scenario. In the more stringent RCP 2.6 scenarios the carbon price is significantly higher, and bio-electricity with CCS is deployed as early as 2030, accounting for about 10% of the total CCS applications . In both scenarios, there is greater deployment of CCS coupled to biomass than to coal, when considering cumulative use over this century .
The generation of energy systems transformation pathways over this century is subject to deep uncertainty and entails numerous assumptions. The deployment of CCS technologies competes with other mitigation options on a relative cost basis in GCAM―with climate policy, fuel costs, and technology costs as key factors. The inter-sectoral deployment of CCS technologies in GCAM is found, for example, to depend on assumptions about how future CCS and biofuel technology costs evolve . In the electricity sector, CCS cost adders (i.e. the additional cost implementing CCS at a plant) are key technology parameters, whereas for biofuel production the non-energy costs (i.e. not including the cost of biomass feedstock production and transport) for producing biofuels are more relevant. CCS cost adders for biofuels represent a smaller portion of the cost increase when competing with petroleum-based fuels. By testing the sensitivity of CCS cost adders and biofuel non-energy costs we show that the share of CCS used in electricity generation and in biofuel production (for use primarily in transportation) can vary significantly, as can the share of CCS coupled with fossil fuels versus bioenergy (bio-electricity and biofuels).
1. IPCC 2014, Climate Change 2014: Synthesis Report.
2. Muratori, M., Kheshgi, H., Mignone, B., Clarke, L., McJeon, H., and Edmonds, J, 2017. Carbon capture and storage across fuels and sectors in energy system transformation pathways, International Journal of Greenhouse Gas Control, 56, pp 34–41, DOI: 10.1016/j.ijggc.2016.11.026.
3. Muratori, M., Kheshgi, H., Mignone, B., McJeon, H., and Clarke, L., 2017: The future role of CCS in electricity and liquid fuel supply, Energy Procedia, in press.
Figure: Rate of CO2 stored via CCS technologies in a RCP 4.5 scenario, from .


Baroque refers to a style of art, architecture, music, literature and drama popular in the 17th and 18th Centuries. It began in Italy and spread throughout Catholic Europe. The Spanish and Portuguese also exported the baroque style to their colonies in Latin America. Baroque art is sumptuous and spectacular, characterized by curved lines and surfaces. Light and shadow create strong contrasts and dramatic effects. The style is characterized by its intent to surprise and overwhelm, whether in painting, architecture, literature, theater or poetry. The origins of the word “baroque” are uncertain. It seems to come from the Portuguese words aljofre barroco, Spanish "barrueco," or French "baroque," all terms used to designate an irregularly shaped pearl. In the 18th Century, critics considered the baroque style strange and eccentric, and used the term pejoratively. However, baroque art was reevaluated during the 19th and 20th Centuries, and is now considered an authentic expression of 17th-century culture.
In the 17th Century, scientific discoveries undid the balance of the Renaissance. Man was removed from his position at the center of the universe. As a result, reality was represented in a markedly complex and articulated way. The overloaded shapes of baroque art were a testament to this new development. In baroque art, the prevailing central perspective was replaced by the use of multiple viewpoints, rendering vision more complex. One of the foremost practitioners of Baroque art was Gian Lorenzo Bernini. A sculptor and architect, Bernini mostly worked in Rome. He designed the monumental colonnade in St. Peter’s square, whose elliptical shape symbolizes the church’s embrace of its believers. Bernini’s sculpture, with their elaborate draping and dramatic effects, fully express baroque sensibility.
During the same period, Pietro da Cortona decorated churches and palaces with spectacular frescos that seemed to make ceilings and walls disappear, and the interiors expand. Peter Paul Rubens’s rich colors and elaborate shapes also indicate a baroque sensibility. The famous Flemish artist spent a long time in Italy and Spain, where his extravagant style influenced local artists. Although baroque visual art was centered in Rome, Spain produced the movement’s most important literary artists. Luis de Gongóra’s poems, full of metaphors and allegories, show the same taste for wonder that characterized visual arts. Symbolic and fantastic elements recur in the comedies of Pedro Calderón de la Barca, who passionately addressed religious topics as well. The baroque style spread throughout Europe. Its powerful displays were widely supported by the church and nobility. Numerous baroque artworks can be found in churches, palaces and museums in Rome. Examples of baroque architecture can also be found in Lecce, Puglia and Noto, in Sicily.


I hope you’re enjoying this piece in print. If not, then there’s a good chance you’re reading this on a cell phone.
Take a moment to think about that. Bytes of data are sitting in a server somewhere, getting sent through “a series of tubes,” and finally appearing as crisp text within 10 seconds. All of this is done with what we consider conventional computing, where computers use transistors as switches to step through one computation at a time.
Through what’s called Moore’s law, every 18 months the number of transistors we can fit on the same microchip doubles, while, as though through radioactive decay, the price continually halves. Although the law started as only an observation, it’s become a trend that industry professionals treat as a quasi-fact when they make new products. And it’s allowed us to do amazing things. You can video chat with a significant other on the other side of the world; compute the trajectory a rover takes to land on Mars; or watch all of “House of Cards” without having to leave the couch. All of this is done with what is, fundamentally, a beefed up calculator.
But conventional computers have their drawbacks. For one, we’re quickly approaching the limit of Moore’s law: As transistors get smaller and smaller, they’ll cease to be reliable carriers of information and generate too much heat to function properly. Computational boosts won’t be possible by simply squeezing more transistors onto a plate. Secondly, as my 10 percent battery warning reminds me, conventional computers use a lot of power. And lastly, they’re sometimes just plain bad at solving what often seems like simple problems. Compute pi to a million digits: easy as pie; have Siri identify a type of pie based solely on a photo: a scary computational problem.
All the while, humans have long known about and used a different type of computer. This one performs tasks such as face recognition and natural language recognition “much better and more accurately” than anything we’ve been able to make a computer do so far, according to John Harris, Ph.D, chair of the Electrical and Computer Engineering (ECE) Department at the University of Florida.
Compute pi to a million digits: easy as pie; have Siri identify a type of pie based solely on a photo: a scary computational problem.
If you haven’t guessed by now, this computer is the brain.
With just 20 watts of power — about how much your laptop consumes — your brain can navigate you through Turlington without hitting a pedestrian on your bike. With just a tiny electrical nudge, you can find your friend’s voice from across a loud room and immediately gather from his tone that he wants to leave.
Still, despite everyone having one, the brain remains a mystery, Harris said. In fact, we still haven’t been able to construct computers that work as well as the brain.
“It is embarrassing,” Harris said, “that we can’t do as well as a rat running around.”
But a field coming into its own, neuromorphic engineering, seeks to use the brain as a biological inspiration for new computers. In his lab, Harris used neuron-like circuits to design a camera that works with less power and provides a higher dynamic range than even high-end models.
Another approach to this field, taken by Karim Oweiss, PhD, a professor in the ECE department, is to understand the brain not by modeling it, but by working on it directly.
Oweiss’ team focuses on developing Brain-Machine Interfaces (BMI). Prior to the development of modern neurobiology, we could only learn about the brain by observing neural input and the resulting action. For example: Touch a hot pan, and your hand snaps back instantly. In Oweiss’ lab, BMIs are used as a window through which we can read the individual firings of neurons in a brain, showing us what the brain does between touching the pan and snatching your hand away.
This has allowed the Oweiss lab to see how neurons adapt themselves when they’re damaged or needed for other tasks — a property known as “neural plasticity.”
In a current project, Oweiss places a sensor into mice brains that detects when neurons fire. He then trains the rats to move robotic arms simply by thinking about which direction they would like to move their bodies.
Oweiss said this work could provide relief to paralyzed patients who are unable to move. By applying similar BMI to those patients’ brains, it would be possible to train them to independently move a robotic arm.
In Oweiss’ lab, BMIs are used as a window through which we can read the individual firings of neurons in a brain, showing us what the brain does between touching the pan and snatching your hand away.
But training requires mental gymnastics, with patients learning to think through very specific movements. Try it out yourself: To work the arm, you’d need to walk through every tiny motion required to, say, flip this page (or your phone, I’m still not going to assume). It’s not easy.
For this reason, Oweiss’ former Ph.D. student has attempted to train mice to move the robotic arm with higher level, ‘“goal-oriented” signals. Instead of the mouse thinking through all the necessary in-between steps — arm up, arm down, squeeze — Oweiss’ team believes it has been able to detect and act on the signal. Now they believe the robotic arm is simply acting on the urge to get food.
In other words: Oweiss’ team may be reading the mices’ thoughts.
This is why neuromorphic computing is so exciting. By trying to answer engineering questions, we are building up the necessary tools to answer what were once considered philosophical questions. And as scientists and engineers continue to explore neurobiology, we see more and more that the mind-melting innovation of science fiction could become an everyday reality.
And Harris pointed out the profound possibilities best: “There is seemingly no roadblock to getting consciousness into a computer.”