Type 3 secretion system induced leukotriene B4 synthesis by leukocytes is actively inhibited by Yersinia pestis to evade early immune recognition.

Subverting the host immune response to inhibit inflammation is a key virulence strategy of Yersinia pestis. The inflammatory cascade is tightly controlled via the sequential action of lipid and protein mediators of inflammation. Because delayed inflammation is essential for Y. pestis to cause lethal infection, defining the Y. pestis mechanisms to manipulate the inflammatory cascade is necessary to understand this pathogen's virulence. While previous studies have established that Y. pestis actively inhibits the expression of host proteins that mediate inflammation, there is currently a gap in our understanding of the inflammatory lipid mediator response during plague. Here we used the murine model to define the kinetics of the synthesis of leukotriene B4 (LTB4), a pro-inflammatory lipid chemoattractant and immune cell activator, within the lungs during pneumonic plague. Furthermore, we demonstrated that exogenous administration of LTB4 prior to infection limited bacterial proliferation, suggesting that the absence of LTB4 synthesis during plague contributes to Y. pestis immune evasion. Using primary leukocytes from mice and humans further revealed that Y. pestis actively inhibits the synthesis of LTB4. Finally, using Y. pestis mutants in the Ysc type 3 secretion system (T3SS) and Yersinia outer protein (Yop) effectors, we demonstrate that leukocytes recognize the T3SS to initiate the rapid synthesis of LTB4. However, several Yop effectors secreted through the T3SS effectively inhibit this host response. Together, these data demonstrate that Y. pestis actively inhibits the synthesis of the inflammatory lipid LTB4 contributing to the delay in the inflammatory cascade required for rapid recruitment of leukocytes to sites of infection.

Risk perceptions of drinking bottled vs. tap water in a low-income community on the US-Mexico Border.

BACKGROUND: Previous studies have shown that low-income Latinos generally drink bottled water over tap water and might be at increased risks for cavities from unfluoridated bottled water. In order to better design interventions, it is important to understand the risk perceptions of this unique high-risk yet historically marginalized group. METHODS: We interviewed low-income Latino households (n = 90) from Nogales, Arizona who primarily drink bottled water and asked them to evaluate potential health risks of drinking tap water compared to 16 other voluntary activities. Unpaired t-tests were used to determine if statistically significant (α = 0.05) differences occurred in perceived risk by drinking-water source and differences among demographic groups in their level of (dis)agreement with statements regarding tap or bottled water safety. To assess significant differences (α = 0.05) in perceived risks and voluntariness to engage in a number of activities, including drinking local tap water and drinking water in different geographic regions, a one-way analysis of variance (ANOVA) followed by Scheffe's post-hoc test (a conservative post-hoc test) with adjustment for the number of pairwise comparisons was used. RESULTS: Participants viewed bottled water to be significantly safer to consume than tap water (p < 0.001). On a Likert scale from 1 (low risk) to 5 (high risk), "drinking tap water in Nogales, Arizona" received an average score of 4.7, which was significantly higher than the average perceived risk of drinking San Francisco, California tap water (µ = 3.4, p < 0.001), and as risky as drinking and driving (µ = 4.8, p = 1.00) and drinking Nogales, Sonora, Mexico tap water (µ = 4.8, p = 1.00). Ninety-eight percent of participants feared that drinking local tap water could result in illness, 79% did not drink their water because of fear of microbial and chemical contamination and 73% would drink their water if they knew it was safe regardless of taste. CONCLUSIONS: These results suggest that fear of illness from tap-water consumption is an important contributing factor to increased bottled water use. Future efforts should focus on the development of educational and outreach efforts to assess the safety and risks associated with tap-water consumption.

Proactive multi-modality treatment of Pancreatic Neuroendocrine Tumours (PNETs): Potential survival benefits.

BACKGROUND/OBJECTIVES: Primary and metastatic pancreatic neuroendocrine tumours (PNET) can be treated with combination of surgery, locoregional and systemic therapy. Survival benefits from individual treatments have been well reported, however, the combined outcome from multimodal treatments are not well described in the literature. We report outcomes in a cohort of PNET patients treated with proactive, multimodality therapy. METHODS: 106 patients were identified from a single tertiary referral centre prospective database. Outcomes of treatment were studied, with the primary end point being death from any cause. RESULTS: Median follow-up was 71 months and overall 5-year survival of 62%. In patients with stage I-III disease (51 patients) estimated 5-year survival was 90%. Median survival in patients with stage IV disease was 51 months with an estimated 5-year survival of 40% in this group. A total of 80 patients (75%) had surgery of which 16% suffered complications requiring intervention. There was no perioperative mortality. CONCLUSIONS: This study demonstrates that proactive multimodal treatment is safe and may confer a survival benefit to patients in this cohort compared to historical data.

A novel in vitro model system for smooth muscle differentiation from human embryonic stem cell-derived mesenchymal cells.

The objective of this study was to develop a novel in vitro model for smooth muscle cell (SMC) differentiation from human embryonic stem cell-derived mesenchymal cells (hES-MCs). We found that hES-MCs were differentiated to SMCs by transforming growth factor-ß (TGF-ß) in a dose- and time-dependent manner as demonstrated by the expression of SMC-specific genes smooth muscle α-actin, calponin, and smooth muscle myosin heavy chain. Under normal growth conditions, however, the differentiation capacity of hES-MCs was very limited. hES-MC-derived SMCs had an elongated and spindle-shaped morphology and contracted in response to the induction of carbachol and KCl. KCl-induced calcium transient was also evident in these cells. Compared with the parental cells, TGF-ß-treated hES-MCs sustained the endothelial tube formation for a longer time due to the sustained SMC phenotype. Mechanistically, TGF-ß-induced differentiation was both Smad- and serum response factor/myocardin dependent. TGF-ß regulated myocardin expression via multiple signaling pathways including Smad2/3, p38 MAPK, and PI3K. Importantly, we found that a low level of myocardin was present in mesoderm prior to SMC lineage determination, and a high level of myocardin was not induced until the differentiation process was initiated. Taken together, our study characterized a novel SMC differentiation model that can be used for studying human SMC differentiation from mesoderm during vascular development.

Evaporation kinetics of aqueous acetic acid droplets: effects of soluble organic aerosol components on the mechanism of water evaporation.

The presence of organic surfactants in atmospheric aerosol may lead to a depression of cloud droplet growth and evaporation rates affecting the radiative properties and lifetime of clouds. Both the magnitude and mechanism of this effect, however, remain poorly constrained. We have used Raman thermometry measurements of freely evaporating micro-droplets to determine evaporation coefficients for several concentrations of acetic acid, which is ubiquitous in atmospheric aerosol and has been shown to adsorb strongly to the air-water interface. We find no suppression of the evaporation kinetics over the concentration range studied (1-5 M). The evaporation coefficient determined for 2 M acetic acid is 0.53 ± 0.12, indistinguishable from that of pure water (0.62 ± 0.09).

2021 AAPM Equity, Diversity, and Inclusion Climate Survey Executive Summary.

PURPOSE: The American Association of Physicists in Medicine (AAPM) shares the results, conclusions, and recommendations from the initial Equity, Diversity, and Inclusion Climate Survey conducted in 2021. METHODS AND MATERIALS: The climate survey targeted medical physicists who are full members of the AAPM and included demographic inquiries and questions intended to assess the working environmental climate in terms of a sense of belonging and inclusion, experiences of discrimination and harassment, and obstacles to participation within the AAPM. The survey invitation was sent to 5,500 members. Responses were collected from 1385 members (response rate of 25%) between January and February 2021. RESULTS: Overall, the medical physics workplace climate was positive. However, some demographic and professional subgroups reported lower levels of agreement with positive characteristics of their workplace climates. Compared with men, women ranked lower 7 of 8 categories that characterized the workplace climate. Other subgroups that also ranked the workplace climate descriptors lower included individuals not originally from the United States and Canada (3/8). Most respondents strongly agreed/agreed that the climate within the AAPM was welcoming. However, 17% of respondents reported personally experiencing or witnessing microaggressions within the AAPM. Overall, medical physicists reported low levels of agreement that opportunities within the AAPM were available to them, from 34% to 60% among 8 categories, including opportunities to volunteer, join committees, and compete for leadership positions within the AAPM. Several subgroups reported even lower levels of agreement that these opportunities are available. Asian and Asian American respondents (3/8) and physicists with origins in countries outside the United States and Canada (7/8) reported fewer opportunities to participate in the AAPM. Medical physicists reported their experiences of discrimination and sexual harassment in their workplaces and within the AAPM. For those who reported personal experiences of sexual harassment, only 24% (15/63) felt comfortable reporting when it occurred within their workplaces, and 35% (9/26) felt comfortable reporting when it occurred within the AAPM. CONCLUSIONS: The report concludes with several recommendations for action.

Use of human embryonic stem cell derived-mesenchymal cells for cardiac repair.

Human mesenchymal stem cells (hMSC) have proven beneficial in the repair and preservation of infarcted myocardium. Unfortunately, MSCs represent a small portion of the bone marrow and require ex vivo expansion. To further advance the clinical usefulness of cellular cardiomyoplasty, derivation of "MSC-like" cells that can be made available "off-the-shelf" are desirable. Recently, human embryonic stem cell-derived mesenchymal cells (hESC-MC) were described. We investigated the efficacy of hESC-MC for cardiac repair after myocardial infarction (MI) compared to hMSC. Because of increased efficacy of cell delivery, cells were embedded into collagen patches and delivered to infarcted myocardium. Culture of hMSC and hESC-MCs in collagen patches did not induce differentiation or significant loss in viability. Transplantation of hMSC and hES-MC patches onto infarcted myocardium of athymic nude rats prevented adverse changes in infarct wall thickness and fractional area change compared to a non-viable patch control. Hemodynamic assessment showed that hMSCs and hES-MC patch application improved end diastolic pressure equivalently. There were no changes in systolic function. hES-MC and hMSC construct application enhanced neovessel formation compared to a non-viable control, and each cell type had similar efficacy in stimulating endothelial cell growth in vitro. In summary, the use of hES-MC provides similar efficacy for cellular cardiomyoplasty as compared to hMSC and may be considered a suitable alternative for cell therapy.

Radiobiology for eye plaque brachytherapy and evaluation of implant duration and radionuclide choice using an objective function.

PURPOSE: Clinical optimization of Collaborative Ocular Melanoma Study (COMS) eye plaque brachytherapy is currently limited to tumor coverage, consensus prescription dosage, and dose calculations to ocular structures. The biologically effective dose (BED) of temporary brachytherapy treatments is a function of both chosen radionuclide R and implant duration T. This study endeavored to evaluate BED delivered to the tumor volume and surrounding ocular structures as a function of plaque position P, prescription dose, R, and T. METHODS: Plaque-heterogeneity-corrected dose distributions were generated with MCNP5 for the range of currently available COMS plaques loaded with sources using three available low-energy radionuclides. These physical dose distributions were imported into the PINNACLE(3) treatment planning system using the TG-43 hybrid technique and used to generate dose volume histograms for a T = 7 day implant within a reference eye geometry including the ciliary body, cornea, eyelid, foveola, lacrimal gland, lens, optic disc, optic nerve, retina, and tumor at eight standard treatment positions. The equation of Dale and Jones was employed to create biologically effective dose volume histograms (BEDVHs), allowing for BED volumetric analysis of all ROIs. Isobiologically effective prescription doses were calculated for T = 5 days down to 0.01 days, with BEDVHs subsequently generated for all ROIs using correspondingly reduced prescription doses. Objective functions were created to evaluate the BEDVHs as a function of R and T. These objective functions are mathematically accessible and sufficiently general to be applied to temporary or permanent brachytherapy implants for a variety of disease sites. RESULTS: Reducing T from 7 to 0.01 days for a 10 mm plaque produced an average BED benefit of 26%, 20%, and 17% for (103)Pd, (125)I, and (131)Cs, respectively, for all P; 16 and 22 mm plaque results were more position-dependent. (103)Pd produced a 16%-35% BED benefit over (125)I, whereas (131)Cs produced a 3%-7% BED detriment, independent of P, T, and plaque size. Additionally, corresponding organ at risk physical doses were lowest using (103)Pd in all circumstances. CONCLUSIONS: The results suggest that shorter implant durations may correlate with more favorable outcomes compared to 7 day implants when treating small or medium intraocular lesions. The data also indicate that implant duration may be safely reduced if the prescription physical dose is likewise diminished and that (103)Pd offers a substantial radiobiological benefit over (125)I and (131)Cs irrespective of plaque position, implant duration, and tumor size.

BEDVH-A method for evaluating biologically effective dose volume histograms: application to eye plaque brachytherapy implants.

PURPOSE: A method is introduced to examine the influence of implant duration T, radionuclide, and radiobiological parameters on the biologically effective dose (BED) throughout the entire volume of regions of interest for episcleral brachytherapy using available radionuclides. This method is employed to evaluate a particular eye plaque brachytherapy implant in a radiobiological context. METHODS: A reference eye geometry and 16 mm COMS eye plaque loaded with (103)Pd, (125)I, or (131)Cs sources were examined with dose distributions accounting for plaque heterogeneities. For a standardized 7 day implant, doses to 90% of the tumor volume ( (TUMOR)D(90)) and 10% of the organ at risk volumes ( (OAR)D(10)) were calculated. The BED equation from Dale and Jones and published α/ß and µ parameters were incorporated with dose volume histograms (DVHs) for various T values such as T = 7 days (i.e., (TUMOR) (7)BED(10) and (OAR) (7)BED(10)). By calculating BED throughout the volumes, biologically effective dose volume histograms (BEDVHs) were developed for tumor and OARs. Influence of T, radionuclide choice, and radiobiological parameters on (TUMOR)BEDVH and (OAR)BEDVH were examined. The nominal dose was scaled for shorter implants to achieve biological equivalence. RESULTS: (TUMOR)D(90) values were 102, 112, and 110 Gy for (103)Pd, (125)I, and (131)Cs, respectively. Corresponding (TUMOR) (7)BED(10) values were 124, 140, and 138 Gy, respectively. As T decreased from 7 to 0.01 days, the isobiologically effective prescription dose decreased by a factor of three. As expected, (TUMOR) (7)BEDVH did not significantly change as a function of radionuclide half-life but varied by 10% due to radionuclide dose distribution. Variations in reported radiobiological parameters caused (TUMOR) (7)BED(10) to deviate by up to 46%. Over the range of (OAR)α/ß values, (OAR) (7)BED(10) varied by up to 41%, 3.1%, and 1.4% for the lens, optic nerve, and lacrimal gland, respectively. CONCLUSIONS: BEDVH permits evaluation of the relative biological effectiveness for brachytherapy implants. For eye plaques, (TUMOR)BEDVH and (OAR)BEDVH were sensitive to implant duration, which may be manipulated to affect outcomes.

Exercise-induced specialized proresolving mediators stimulate AMPK phosphorylation to promote mitochondrial respiration in macrophages.

OBJECTIVE: Physical activity has been shown to reduce the risk of CVD mortality in large-cohort longitudinal studies; however, the mechanisms underpinning the beneficial effects of exercise remain incompletely understood. Emerging data suggest that the risk reducing effect of exercise extends beyond changes in traditional CVD risk factors alone and involves alterations in immunity and reductions in inflammatory mediator production. Our study aimed to determine whether exercise-enhanced production of proresolving lipid mediators contribute to alterations in macrophage intermediary metabolism, which may contribute to the anti-inflammatory effects of exercise. METHODS: Changes in lipid mediators and macrophage metabolism were assessed in C57Bl/6 mice following 4 weeks of voluntary exercise training. To investigate whether exercise-stimulated upregulation of specialized proresolving lipid mediators (SPMs) was sufficient to enhance mitochondrial respiration, both macrophages from control mice and human donors were incubated in vitro with SPMs and mitochondrial respiratory parameters were measured using extracellular flux analysis. Compound-C, an ATP-competitive inhibitor of AMPK kinase activity, was used to investigate the role of AMPK activity in SPM-induced mitochondrial metabolism. To assess the in vivo contribution of 5-lipoxygenase in AMPK activation and exercise-induced mitochondrial metabolism in macrophages, Alox5-/- mice were also subjected to exercise training. RESULTS: Four weeks of exercise training enhanced proresolving lipid mediator production, while also stimulating the catabolism of inflammatory lipid mediators (e.g., leukotrienes and prostaglandins). This shift in lipid mediator balance following exercise was associated with increased macrophage mitochondrial metabolism. We also find that treating human and murine macrophages in vitro with proresolving lipid mediators enhances mitochondrial respiratory parameters. The proresolving lipid mediators RvD1, RvE1, and MaR1, but not RvD2, stimulated mitochondrial respiration through an AMPK-dependent signaling mechanism. Additionally, in a subset of macrophages, exercise-induced mitochondrial activity in vivo was dependent upon 5-lipoxygenase activity. CONCLUSION: Collectively, these results suggest that exercise stimulates proresolving lipid mediator biosynthesis and mitochondrial metabolism in macrophages via AMPK, which might contribute to the anti-inflammatory and CVD risk reducing effect of exercise.

Measured and Modeled Comparisons of Chemical and Microbial Contaminants in Tap and Bottled Water in a US-Mexico Border Community.

Tap water quality concerns and advertisements often drive increased bottled water consumption, especially in communities with historical tap water quality problems (e.g., Nogales, Arizona). The study objective was to assess contamination of municipal tap and bottled water in Nogales, Arizona. Bottled (sealed, open/partially consumed bottles, and reusable containers for vended water) and tap water samples were collected from 30 homes and analyzed for chemical and microbial contaminants. Fisher exact tests and Wilcoxon rank sum tests were used to compare proportions of positive samples and contaminant concentrations between tap and bottled water samples. While none of the chemical contaminants were above MCLs, there were statistically significantly greater concentrations and proportions of positive samples for some contaminants, including arsenic, in tap vs. bottled water. E. coli concentrations were >0 CFU/100mL in some unsealed bottled water samples but not for sealed bottles. This study demonstrates that 1) the measured concentrations in tap and bottled water likely pose low risks, as they are below the MCLs, 2) more education in this community on hygiene maintenance of refillable or opened bottled water containers is needed, and 3) using tap water over bottled water is advantageous due to likely lower E. coli risk and lower cost.

Gamma spectrometry analysis of radioiodine in charcoal from high volume aerosol samples.

Following accidental releases, gamma spectrometry of impregnated charcoal filters is used to measure gaseous 131I contamination, but is subject to sampling inhomogeneity. In this study two germanium detectors are calibrated using a charcoal multi-gamma standard. Activities in samples spiked with a matrix of 131I aliquots are compared based on measurement, spike known activity, and monte-carlo simulation, and used to test a simple mixing method. Measurement efficiency, and removal of 11% inhomogeneity effect by mixing, was successfully reproduced in GESPECOR calculations.

Cohort Generation and Characterization of Patient-Specific Familial Hypercholesterolemia Induced Pluripotent Stem Cells.

Homozygous familial hypercholesterolemia (hoFH) is a rare disorder caused primarily by pathological mutations in the low-density lipoprotein receptor (LDLR), which disrupts LDL-cholesterol (LDL-C) metabolism homeostasis. hoFH patients are at extremely high risk for cardiovascular disease and are resistant to standard therapies. LDLR knockout animals and in vitro cell models overexpressing different mutations have proved useful, but may not fully recapitulate human LDLR mutation biology. We and others have generated induced pluripotent stem cells (iPSC) from hoFH patient's fibroblasts and T cells and demonstrated their ability to recapitulate hoFH biology. In this study, we present the generation and characterization of a cohort of seven hoFH-iPSC lines derived from peripheral blood mononuclear cells (PBMC) collected from four homozygous and three compound heterozygous patients. The hoFH-iPSC cohort demonstrated a wide range of LDLR expression and LDL-C internalization in response to rosuvastatin that correlated with the predicted pathogenicity of the mutation. We were able to confirm that hoFH-iPSC cohort were pluripotent by differentiation toward all three germ layers and specifically to hepatocyte-like cells (HLC), the cell with primary LDL-C metabolic regulatory control, by expression of hepatocyte markers. hoFH patient PBMC-derived iPSC recapitulate the LDLR dysfunction of their specific mutation. They were capable of differentiating to HLC and could be useful for early developmental studies, pharmacology/toxicology, and potentially autologous cell therapy.

Childhood adversity, mental health and suicide (CHASE): a methods protocol for a longitudinal case-control linked data study.

INTRODUCTION: Suicide is a tragic outcome with devastating consequences. In 2018, Scotland experienced a 15% increase in suicide from 680 to 784 deaths. This was marked among young people, with an increase of 53% in those aged 15-24, the highest since 2007. Early intervention in those most at risk is key, but identification of individuals at risk is complex, and efforts remain largely targeted towards universal suicide prevention strategies with little evidence of effectiveness.Recent evidence suggests childhood adversity is a predictor of subsequent poor social and health outcomes, including suicide. This protocol reports on methodology for harmonising lifespan hospital contacts for childhood adversity, mental health, and suicidal behaviour. This will inform where to 1) focus interventions, 2) prioritise trauma-informed approaches, and 3) adapt support avenues earlier in life for those most at risk. METHODS: This study will follow a case-control design. Scottish hospital data (physical health SMR01; mental health SMR04; maternity/birth record SMR02; mother's linked data SMR01, SMR04, death records) from 1981 to as recent as available will be extracted for people who died by suicide aged 10-34, and linked on Community Health Index unique identifier. A randomly selected control population matched on age and geography at death will be extracted in a 1:10 ratio. International Classification of Disease (ICD) codes will be harmonised between ICD9-CM, ICD9, ICD10-CM and ICD10 for childhood adversity, mental health, and suicidal behaviour. RESULTS: ICD codes for childhood adversity from four key studies are reported in two categories, 1) Maltreatment or violence-related codes, and 2) Codes suggestive of maltreatment. 'Clinical Classifications Software' ICD codes to operationalise mental health codes are also reported. Harmonised lifespan ICD categories were achieved semi-automatically, but required labour-intensive supplementary manual coding. Cross-mapped codes are reported. CONCLUSION: There is a dearth of evidence about touchpoints prior to suicide. This study reports methods and harmonised ICD codes along the lifespan to understand hospital contact patterns for childhood adversity, which come to the attention of hospital practitioners. KEY WORDS: Childhood Adversity, Adverse Childhood Experiences, Mental Health, Self-harm, Suicide, Suicidality, Violence, Hospital episodes, Routine Data, Data Linkage, Study Protocol.

Familial hypercholesterolemia class II low-density lipoprotein receptor response to statin treatment.

Low-density lipoprotein (LDL) receptor (LDLR) mutations are the primary cause of familial hypercholesterolemia (FH). Class II LDLR mutations result in a misfolded LDLR retained in the endoplasmic reticulum (ER). We have developed a model of FH class II and CRISPR-corrected induced pluripotent stem cells (iPSC) capable of replicating mutant and repaired LDLR functions. We show here that iPSC and derived hepatocyte-like cells (HLC) replicate misfolded LDLR accumulation and restoration of LDLR function in CRISPR-corrected cells. It was reported that model cells overexpressing class II LDLR mutants result in endoplasmic reticulum (ER) accumulation of immature LDLR and activation of the unfolded protein response (UPR). We show here that statins induce a similar accumulation of immature LDLR that is resolved with class II correction. We also demonstrate that, although capable of UPR induction with tunicamycin treatment, unlike overexpression models, statin-treated class II iPSC and derived HLC do not induce the common UPR markers Grp78 (also known as HSPA5) or spliced XBP1 [XBP1 (S)]. Because statins are reported to inhibit UPR, we utilized lipoprotein-deficient serum (LPDS) medium, but still did not detect UPR induction at the Grp78 and XBP1 (S) levels. Our study demonstrates the recapitulation of mutant and corrected class II LDLR function and suggests that overexpression models may not accurately predict statin-mediated class II protein biology.

Enrichment and differentiation of human germ-like cells mediated by feeder cells and basic fibroblast growth factor signaling.

Human embryonic stem cells (hESCs) have recently demonstrated the potential for differentiation into germ-like cells in vitro. This provides a novel model for understanding human germ cell development and human infertility. Mouse embryonic fibroblast (MEF) feeders and basic fibroblast growth factor (bFGF) are two sources of signaling that are essential for primary culture of germ cells, yet their role has not been examined in the derivation of germ-like cells from hESCs. Here protein and gene expression demonstrated that both MEF feeders and bFGF can significantly enrich germ cell differentiation from hESCs. Under enriched differentiation conditions, flow cytometry analysis proved 69% of cells to be positive for DDX4 and POU5F1 protein expression, consistent with the germ cell lineage. Importantly, removal of bFGF from feeder-free cultures resulted in a 50% decrease in POU5F1- and DDX4-positive cells. Quantitative reverse transcription-polymerase chain reaction analysis established that bFGF signaling resulted in an upregulation of genes involved in germ cell differentiation with or without feeders; however, feeder conditions caused significant upregulation of premigratory/migratory (Ifitm3, DAZL, NANOG, and POU5F1) and postmigratory (PIWIL2, PUM2) genes, along with the meiotic markers SYCP3 and MLH1. After further differentiation, >90% of cells expressed the meiotic proteins SYCP3 and MLH1. This is the first demonstration that signaling from MEF feeders and bFGF can induce a highly enriched population of germ-like cells derived from hESCs, thus providing a critically needed model for further investigation of human germ cell development and signaling. Disclosure of potential conflicts of interest is found at the end of this article.

Human neural progenitor cells derived from embryonic stem cells in feeder-free cultures.

Derivation of human neural progenitors (hNP) from human embryonic stem (hES) cells in culture has been reported with the use of feeder cells or conditioned media. This introduces undefined components into the system, limiting the ability to precisely investigate the requirement for factors that control the process. Also, the use of feeder cells of non-human origin introduces the potential for zoonotic transmission, limiting its clinical usefulness. Here we report a feeder-free system to produce hNP from hES cells and test the effects of various media components involved in the process. Five protocols using defined media components were compared for efficiency of hNP generation. Based on this analysis, we discuss the role of basic fibroblast growth factor (FGF2), N2 supplement, non-essential amino acids (NEAA), and knock-out serum replacement (KSR) on the process of hNP generation. All protocols led to down-regulation of Oct4/POU5F1 expression (from 90.5% to <3%), and up-regulation of neural progenitor markers to varying degrees. Media with N2 but not KSR and NEAA produced cultures with significantly higher (p<0.05) expression of the neural progenitor marker Musashi 1 (MSI1). Approximately 89% of these cells were Nestin (NES)+ after 3 weeks, but they did not proliferate. In contrast, differentiation media supplemented with KSR and NEAA produced fewer NES+ (75%) cells, but these cells were proliferative, and by five passages the culture consisted of >97% NES+ cells. This suggests that KSR and NEAA supplements did not enhance early differentiation but did promote proliferating of hNP cell cultures. This resulted in an efficient, robust, repeatable differentiation system suitable for generating large populations of hNP cells. This will facilitate further study of molecular and biochemical mechanisms in early human neural differentiation and potentially produce uniform neuronal cells for therapeutic uses without concern of zoonotic transmission from feeder layers.

WormBot, an open-source robotics platform for survival and behavior analysis in C. elegans.

Caenorhabditis elegans is a popular organism for aging research owing to its highly conserved molecular pathways, short lifespan, small size, and extensive genetic and reverse genetic resources. Here we describe the WormBot, an open-source robotic image capture platform capable of conducting 144 parallel C. elegans survival and behavioral phenotyping experiments. The WormBot uses standard 12-well tissue culture plates suitable for solid agar media and is built from commercially available robotics hardware. The WormBot is controlled by a web-based interface allowing control and monitoring of experiments from any internet connected device. The standard WormBot hardware features the ability to take both time-lapse bright field images and real-time video micrographs, allowing investigators to measure lifespan, as well as heathspan metrics as worms age. The open-source nature of the hardware and software will allow for users to extend the platform and implement new software and hardware features. This extensibility, coupled with the low cost and simplicity of the system, allows the automation of C. elegans survival analysis even in small laboratory settings with modest budgets.

Will histone deacetylase inhibitors require combination with other agents to fulfil their therapeutic potential?

Histone deacetylase inhibitors have progressed rapidly from the laboratory to clinical testing. This review highlights the promising data for their combination with a wide range of established and novel anticancer agents and discusses the mechanisms that underpin these interactions.

The Adipose Stromal Vascular Fraction as a Complex Cellular Source for Tissue Engineering Applications.

A major challenge in tissue engineering is the generation of sufficient volumes of viable tissue for organ transplant. The development of a stable, mature vasculature is required to sustain the metabolic and functional activities of engineered tissues. Adipose stromal vascular fraction (SVF) cells are an easily accessible, heterogeneous cell system comprised of endothelial cells, macrophages, pericytes, and various stem cell populations. Collectively, SVF has been shown to spontaneously form vessel-like networks in vitro and robust, patent, and functional vasculatures in vivo. Capitalizing on this ability, we and others have demonstrated adipose SVF's utility in generating and augmenting engineered liver, cardiac, and vascular tissues, to name a few. This review highlights the scientific origins of SVF, the use of SVF as a clinically relevant vascular source, various SVF constituents and their roles, and practical considerations associated with isolating SVF for various tissue engineering applications.