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BackgroundInfectious disease-related stigma is a pervasive global issue that impedes disease control efforts by increasing reluctance to seek treatment or engagement in prevention behaviors for fear of ostracism. Despite this, there is limited research on COVID-19 stigma in Africa, specifically rural South Africa, which has faced infectious disease-related stigma throughout the HIV epidemic.MethodsPopulation-based surveys were conducted among 1,662 adults living in the Agincourt Health and Socio-Demographic Surveillance System (AHDSS) area in Mpumalanga, South Africa, in August-October 2020 and August-October 2021. Surveys measured anticipated COVID-19-related stigma from low to high levels. Changes in stigma between surveys were compared using Wilcoxon ranked sign tests, and log-binomial models estimated the association between socio-demographic factors and anticipated stigma at both intervals. Qualitative interviews were conducted in 2022 among 31 adults who completed the original surveys, and the data were analyzed thematically to describe anticipated, perceived, and enacted stigma.ResultsAnticipated stigma significantly decreased from the first to the second survey (p-value:ConclusionWhile COVID-19 stigma decreased over time in rural South Africa, different forms of stigma persisted and influenced participants' willingness to reveal their COVID-19 infection status. Given South Africa's history with infectious disease-related stigma hindering public health efforts, it is crucial that government bodies prioritize strategies to mitigate stigma in rural communities.

BackgroundHematopoietic malignancies are extremely common in pet dogs and represent nearly 30% of the malignancies diagnosed in this population each year. Clinicians commonly use existing tools such as physical exam findings, radiographs, ultrasound and baseline blood work to monitor these patients for treatment response and remission. Circulating biomarkers, such as prostate specific antigen or carcinoembryonic antigen, can be useful tools for monitoring treatment response and remission status in human cancer patients. To date, there has a been a lack of useful circulating biomarkers available to veterinary oncology patients.MethodsCirculating plasma nucleosome concentrations were evaluated at diagnosis, throughout treatment and during remission monitoring for 40 dogs with lymphoma, acute myelogenous leukemia and multiple myeloma. Additionally, C-reactive protein and thymidine kinase-1 levels were recorded.ResultsPlasma nucleosome concentrations were significantly higher at diagnosis and progressive disease than they were when dogs were in remission. All but two dogs had plasma nucleosome concentrations that returned to the low range during treatment. These two dogs had the shortest progression free and overall survival times. Dogs with the highest plasma nucleosome concentrations had a significantly shorter first progression free survival than dogs with lower plasma nucleosome concentrations at diagnosis. Plasma nucleosome concentrations correlated better with disease response and progression than either thymidine kinase or C reactive protein.ConclusionsPlasma nucleosome concentrations can be a useful tool for treatment monitoring and disease progression in dogs with hematopoietic malignancies.

Neuroendocrine prostate cancer (NEPrCa) arises de novo or after accumulation of genomic alterations in pre-existing adenocarcinoma tumors in response to androgen deprivation therapies. We have provided evidence that small extracellular vesicles released by PrCa cells and containing the αVβ3 integrin promote neuroendocrine differentiation of PrCa in vivo and in vitro. Here, we examined αVβ3 integrin expression in three murine models carrying a deletion of PTEN (SKO), PTEN and RB1 (DKO), or PTEN, RB1 and TRP53 (TKO) genes in the prostatic epithelium; of these three models, the DKO and TKO tumors develop NEPrCa with a gene signature comparable to those of human NEPrCa. Immunostaining analysis of SKO, DKO and TKO tumors shows that αVβ3 integrin expression is increased in DKO and TKO primary tumors and metastatic lesions, but absent in SKO primary tumors. On the other hand, SKO tumors show higher levels of a different αV integrin, αVβ6, as compared to DKO and TKO tumors. These results are confirmed by RNA-sequencing analysis. Moreover, TRAMP mice, which carry NEPrCa and adenocarcinoma of the prostate, also have increased levels of αVβ3 in their NEPrCa primary tumors. In contrast, the αVβ6 integrin is only detectable in the adenocarcinoma areas. Finally, analysis of 42 LuCaP patient-derived xenografts and primary adenocarcinoma samples shows a positive correlation between αVβ3, but not αVβ6, and the neuronal marker synaptophysin; it also demonstrates that αVβ3 is absent in prostatic adenocarcinomas. In summary, we demonstrate that αVβ3 integrin is upregulated in NEPrCa primary and metastatic lesions; in contrast, the αVβ6 integrin is confined to adenocarcinoma of the prostate. Our findings suggest that the αVβ3 integrin, but not αVβ6, may promote a shift in lineage plasticity towards a NE phenotype and might serve as an informative biomarker for the early detection of NE differentiation in prostate cancer.

Compensatory growth (CG), an accelerated growth phenomenon which occurs following a period of dietary restriction is utilised worldwide in animal production systems as a management practise to lower feed costs. The objective of this study was to evaluate the contribution of jejunal epithelial to CG in cattle through transcriptional profiling following a period of dietary restriction as well as subsequent re-alimentation induced CG. Sixty Holstein Friesian bulls were separated into two groups; RES and ADLIB, with 30 animals in each. RES animals were offered a restricted diet for 125 days (Period 1) followed by ad libitum feeding for 55 days (Period 2). ADLIB animals had ad libitum access to feed across both periods 1 and 2. At the end of each period, 15 animals from each treatment group were slaughtered, jejunal epithelium collected and RNAseq analysis performed. Animals that were previously diet restricted underwent CG, gaining 1.8 times the rate of their non-restricted counterparts. Twenty-four genes were differentially expressed in RES compared to ADLIB animals at the end of Period 1, with only one gene, GSTA1, differentially expressed between the two groups at the end of Period 2. When analysed within treatment (RES, Period 2 v Period 1), 31 genes were differentially expressed between diet restricted and animals undergoing CG. Dietary restriction and subsequent re-alimentation were associated with altered expression of genes involved in digestion and metabolism as well as those involved in cellular division and growth. Compensatory growth was also associated with greater expression of genes involved in cellular protection and detoxification in jejunal epithelium. This study highlights some of the molecular mechanisms regulating the response to dietary restriction and subsequent re-alimentation induced CG in cattle; however the gene expression results suggest that most of the CG in jejunal epithelium had occurred by day 55 of re-alimentation.

Compensatory growth (CG) is utilised worldwide in beef production systems as a management approach to reduce feed costs. However the underlying biology regulating the expression of CG remains to be fully elucidated. The objective of this study was to examine the effect of dietary restriction and subsequent re-alimentation induced CG on the global gene expression profile of ruminal epithelial papillae. Holstein Friesian bulls (n = 60) were assigned to one of two groups: restricted feed allowance (RES; n = 30) for 125 days (Period 1) followed by ad libitum access to feed for 55 days (Period 2) or (ii) ad libitum access to feed throughout (ADLIB; n = 30). At the end of each period, 15 animals from each treatment were slaughtered and rumen papillae harvested. mRNA was isolated from all papillae samples collected. cDNA libraries were then prepared and sequenced. Resultant reads were subsequently analysed bioinformatically and differentially expressed genes (DEGs) are defined as having a Benjamini-Hochberg P value of

Weaning of dairy calves is an early life husbandry management practice which involves the changeover from a liquid to a solid feed based diet. The objectives of the study were to use RNA-seq technology to examine the effect of (i) breed and (ii) gradual weaning, on the whole blood mRNA transcriptome of artificially reared Holstein-Friesian and Jersey calves. The calves were gradually weaned over 14 days (day (d) -13 to d 0) and mRNA transcription was examined one day before gradual weaning was initiated (d -14), one day after weaning (d 1), and 8 days after weaning (d 8). On d -14, 550 genes were differentially expressed between Holstein-Friesian and Jersey calves, while there were 490 differentially expressed genes (DEG) identified on d 1, and 411 DEG detected eight days after weaning (P < 0.05; FDR < 0.1). No genes were differentially expressed within breed, in response to gradual weaning (P > 0.05). The pathways, gene ontology terms, and biological functions consistently over-represented among the DEG between Holstein-Friesian and Jersey were associated with the immune response and immune cell signalling, specifically chemotaxis. Decreased transcription of several cytokines, chemokines, immunoglobulin-like genes, phagocytosis-promoting receptors and g-protein coupled receptors suggests decreased monocyte, natural killer cell, and T lymphocyte, chemotaxis and activation in Jersey compared to Holstein-Friesian calves. Knowledge of breed-specific immune responses could facilitate health management practices better tailored towards specific disease sensitivities of Holstein-Friesian and Jersey calves. Gradual weaning did not compromise the welfare of artificially-reared dairy calves, evidenced by the lack of alterations in the expression of any genes in response to gradual weaning.

Compensatory growth (CG), an accelerated growth phenomenon which occurs following a period of dietary restriction is exploited worldwide in animal production systems as a method to lower feed costs. However the molecular mechanisms regulated CG expression remain to be elucidated fully. This study aimed to uncover the underlying biology regulating CG in cattle, through an examination of skeletal muscle transcriptional profiles utilising next generation mRNA sequencing technology. Twenty Holstein Friesian bulls were fed either a restricted diet for 125 days, with a target growth rate of 0.6 kg/day (Period 1), following which they were allowed feed ad libitum for a further 55 days (Period 2) or fed ad libitum for the entirety of the trial. M. longissimus dorsi biopsies were harvested from all bulls on days 120 and 15 of periods 1 and 2 respectively and RNAseq analysis was performed. During re-alimentation in Period 2, previously restricted animals displayed CG, growing at 1.8 times the rate of the ad libitum control animals. Compensating animals were also more feed efficient during re-alimentation and compensated for 48% of their previous dietary restriction. 1,430 and 940 genes were identified as significantly differentially expressed (Benjamini Hochberg adjusted P < 0.1) in periods 1 and 2 respectively. Additionally, 2,237 genes were differentially expressed in animals undergoing CG relative to dietary restriction. Dietary restriction in Period 1 was associated with altered expression of genes involved in lipid metabolism and energy production. CG expression in Period 2 occurred in association with greater expression of genes involved in cellular function and organisation. This study highlights some of the molecular mechanisms regulating CG in cattle. Differentially expressed genes identified are potential candidate genes for the identification of biomarkers for CG and feed efficiency, which may be incorporated into future breeding programmes.

Feed efficiency is an important trait in pig production, with evidence to suggest that the efficiencies of a variety of biological systems contribute to variation in this trait. Little work has been conducted on the contribution of the intestinal innate immune response to divergence in feed efficiency. Hence, the objective of this study was to examine select bacterial populations and gene expression profiles of a range of targets relating to gut health and immunity in the intestine of pigs phenotypically divergent in feed efficiency in: a) the basal state; and (b) following an ex-vivo lipopolysaccharide (LPS) challenge of ileal and colonic tissue. Male pigs (initial BW 22.4 kg (SD = 2.03)) were fed a standard finishing diet for the final 43 days prior to slaughter to evaluate feed intake and growth for the purpose of calculating residual feed intake (RFI). On day 115, 16 animals (average weight 85 kg, SEM 2.8 kg), designated high RFI (HRFI) and low RFI (LRFI) were slaughtered. The LRFI pigs had increased lactobacillus spp. in the caecum compared to HRFI pigs (P < 0.05). RFI groups did not differ in the expression of the measured genes involved in the innate immune system in the basal ileal or colonic tissues (P > 0.10). Interestingly, there was an interaction between RFI and LPS for the cytokines IL-8, IL-1, IL-6, TNF-α, Interferon-γ (IFN-γ) and SOCS3, with the LRFI group having consistently lower gene expression in the colon following the LPS challenge, compared to the HRFI group. The lower gene expression of SOCS and cytokines following an ex vivo LPS challenge supports the theory that a possible energy saving mechanism exists in the intestinal innate immune response to an immune challenge in more feed efficient pigs.

Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) has been instrumental in inferring the roles of histone post-translational modifications in the regulation of transcription, chromatin compaction and other cellular processes that require modulation of chromatin structure. However, analysis of ChIP-seq data is challenging when the manipulation of a chromatin-modifying enzyme significantly affects global levels of histone post-translational modifications. For example, small molecule inhibition of the methyltransferase EZH2 reduces global levels of histone H3 lysine 27 trimethylation (H3K27me3). However, standard ChIP-seq normalization and analysis methods fail to detect a decrease upon EZH2 inhibitor treatment. We overcome this challenge by employing an alternative normalization approach that is based on the addition of Drosophila melanogaster chromatin and a D. melanogaster-specific antibody into standard ChIP reactions. Specifically, the use of an antibody that exclusively recognizes the D. melanogaster histone variant H2Av enables precipitation of D. melanogaster chromatin as a minor fraction of the total ChIP DNA. The D. melanogaster ChIP-seq tags are used to normalize the human ChIP-seq data from DMSO and EZH2 inhibitor-treated samples. Employing this strategy, a substantial reduction in H3K27me3 signal is now observed in ChIP-seq data from EZH2 inhibitor treated samples.

In cattle, conceptus-maternal interactions are critical for the establishment and maintenance of pregnancy. A major component of this early interaction involves the transport of nutrients and secretion of key molecules by uterine epithelial cells to help support conceptus development during the peri-implantation period of pregnancy. Objectives were to: 1) analyze temporal changes in the amino acid (AA) content of uterine luminal fluid (ULF) during the bovine estrous cycle; 2) understand conceptus-induced alterations in AA content; 3) determine expression of AA transporters in the endometrium and conceptus; and 4) determine how these transporters are modulated by (Progesterone) P4. Concentrations of aspartic acid, arginine, glutamine, histidine, lysine, isoleucine, leucine, phenylalanine and tyrosine decreased on Day 16 of the estrous cycle but increased on Day 19 in pregnant heifers (P

The transition from vegetative to reproductive stages marks a major milestone in plant development. It is clear that global change factors (e.g., increasing [CO(2)] and temperature) have already had and will continue to have a large impact on plant flowering times in the future. Increasing atmospheric [CO(2)] has recently been shown to affect flowering time, and may produce even greater responses than increasing temperature. Much is known about the genes influencing flowering time, although their relevance to changing [CO(2)] is not well understood. Thus, we present the first study to identify QTL (Quantitative Trait Loci) that affect flowering time at elevated [CO(2)] in Arabidopsis thaliana.We developed our mapping population by crossing a genotype previously selected for high fitness at elevated [CO(2)] (SG, Selection Genotype) to a Cape Verde genotype (Cvi-0). SG exhibits delayed flowering at elevated [CO(2)], whereas Cvi-0 is non-responsive to elevated [CO(2)] for flowering time. We mapped one major QTL to the upper portion of chromosome 1 that explains 1/3 of the difference in flowering time between current and elevated [CO(2)] between the SG and Cvi-0 parents. This QTL also alters the stage at which flowering occurs, as determined from higher rosette leaf number at flowering in RILs (Recombinant Inbred Lines) harboring the SG allele. A follow-up study using Arabidopsis mutants for flowering time genes within the significant QTL suggests MOTHER OF FT AND TFL1 (MFT) as a potential candidate gene for altered flowering time at elevated [CO(2)].This work sheds light on the underlying genetic architecture that controls flowering time at elevated [CO(2)]. Prior to this work, very little to nothing was known about these mechanisms at the genomic level. Such a broader understanding will be key for better predicting shifts in plant phenology and for developing successful crops for future environments.

Neural stem cells (NSCs) can be isolated from different regions of the central nervous system. There has been controversy whether regional differences amongst stem and progenitor cells are cell intrinsic and whether these differences are maintained during expansion in culture. The identification of inherent regional differences has important implications for the use of these cells in neural repair. Here, we compared NSCs derived from the spinal cord and embryonic cortex. We found that while cultured cortical and spinal cord derived NSCs respond similarly to mitogens and are equally neuronogenic, they retain and maintain through multiple passages gene expression patterns indicative of the region from which they were isolated (e.g Emx2 and HoxD10). Further microarray analysis identified 229 genes that were differentially expressed between cortical and spinal cord derived neurospheres, including many Hox genes, Nuclear receptors, Irx3, Pace4, Lhx2, Emx2 and Ntrk2. NSCs in the cortex express LeX. However, in the embryonic spinal cord there are two lineally related populations of NSCs: one that expresses LeX and one that does not. The LeX negative population contains few markers of regional identity but is able to generate LeX expressing NSCs that express markers of regional identity. LeX positive cells do not give rise to LeX-negative NSCs. These results demonstrate that while both embryonic cortical and spinal cord NSCs have similar self-renewal properties and multipotency, they retain aspects of regional identity, even when passaged long-term in vitro. Furthermore, there is a population of a LeX negative NSC that is present in neurospheres derived from the embryonic spinal cord but not the cortex.

Lysosome trafficking plays a significant role in tumor invasion, a key event for the development of metastasis. Previous studies from our laboratory have demonstrated that the anterograde (outward) movement of lysosomes to the cell surface in response to certain tumor microenvironment stimulus, such as hepatocyte growth factor (HGF) or acidic extracellular pH (pHe), increases cathepsin B secretion and tumor cell invasion. Anterograde lysosome trafficking depends on sodium-proton exchanger activity and can be reversed by blocking these ion pumps with Troglitazone or EIPA. Since these drugs cannot be advanced into the clinic due to toxicity, we have designed a high-content assay to discover drugs that block peripheral lysosome trafficking with the goal of identifying novel drugs that inhibit tumor cell invasion. An automated high-content imaging system (Cellomics) was used to measure the position of lysosomes relative to the nucleus. Among a total of 2210 repurposed and natural product drugs screened, 18 "hits" were identified. One of the compounds identified as an anterograde lysosome trafficking inhibitor was niclosamide, a marketed human anti-helminthic drug. Further studies revealed that niclosamide blocked acidic pHe, HGF, and epidermal growth factor (EGF)-induced anterograde lysosome redistribution, protease secretion, motility, and invasion of DU145 castrate resistant prostate cancer cells at clinically relevant concentrations. In an effort to identify the mechanism by which niclosamide prevented anterograde lysosome movement, we found that this drug exhibited no significant effect on the level of ATP, microtubules or actin filaments, and had minimal effect on the PI3K and MAPK pathways. Niclosamide collapsed intralysosomal pH without disruption of the lysosome membrane, while bafilomycin, an agent that impairs lysosome acidification, was also found to induce JLA in our model. Taken together, these data suggest that niclosamide promotes juxtanuclear lysosome aggregation (JLA) via modulation of pathways involved in lysosome acidification. In conclusion, we have designed a validated reproducible high-content assay to screen for drugs that inhibit lysosome trafficking and reduce tumor invasion and we summarize the action of one of these drugs.

Type II diabetes is an established cause of vascular impairment. Particulate air pollution is known to exacerbate cardiovascular and respiratory conditions, particularly in susceptible populations. This study set out to determine the impact of exposure to traffic pollution, with and without particle filtration, on vascular endothelial function in Type II diabetes. Endothelial production of nitric oxide (NO) has previously been linked to vascular health. Reactive hyperemia induces a significant increase in plasma nitrite, the proximal metabolite of NO, in healthy subjects, while diabetics have a lower and more variable level of response. Twenty type II diabetics and 20 controls (ages 46-70 years) were taken on a 1.5 hr roadway traffic air pollution exposure as passengers. We analyzed plasma nitrite, as a measure of vascular function, using forearm ischemia to elicit a reactive hyperemic response before and after exposure to one ride with and one without filtration of the particle components of pollution. Control subjects displayed a significant increase in plasma nitrite levels during reactive hyperemia. This response was no longer present following exposure to traffic air pollution, but did not vary with whether or not the particle phase was filtered out. Diabetics did not display an increase in nitrite levels following reactive hyperemia. This response was not altered following pollution exposure. These data suggest that components of acute traffic pollution exposure diminish vascular reactivity in non-diabetic individuals. It also confirms that type II diabetics have a preexisting diminished ability to appropriately respond to a vascular challenge, and that traffic pollution exposure does not cause a further measureable acute change in plasma nitrite levels in Type II diabetics.

Background: Many prevention of mother-to-child HIV transmission (PMTCT) programs currently prioritize antiretroviral therapy (ART) for women with advanced HIV. Point-of-care (POC) CD4 assays may expedite the selection of three-drug ART instead of zidovudine, but are costlier than traditional laboratory assays., Methods: We used validated models of HIV infection to simulate pregnant, HIV-infected women (mean age 26 years, gestational age 26 weeks) in a general antenatal clinic in South Africa, and their infants. We examined two strategies for CD4 testing after HIV diagnosis: laboratory (test rate: 96%, result-return rate: 87%, cost: $14) and POC (test rate: 99%, result-return rate: 95%, cost: $26). We modeled South African PMTCT guidelines during the study period (WHO "Option A"): antenatal zidovudine (CD4 ≤350/μL) or ART (CD4>350/μL). Outcomes included MTCT risk at weaning (age 6 months), maternal and pediatric life expectancy (LE), maternal and pediatric lifetime healthcare costs (2013 USD), and cost-effectiveness ($/life-year saved)., Results: In the base case, laboratory led to projected MTCT risks of 5.7%, undiscounted pediatric LE of 53.2 years, and undiscounted PMTCT plus pediatric lifetime costs of $1,070/infant. POC led to lower modeled MTCT risk (5.3%), greater pediatric LE (53.4 years) and lower PMTCT plus pediatric lifetime costs ($1,040/infant). Maternal outcomes following laboratory were similar to POC (LE: 21.2 years; lifetime costs: $23,860/person). Compared to laboratory, POC improved clinical outcomes and reduced healthcare costs., Conclusions: In antenatal clinics implementing Option A, the higher initial cost of a one-time POC CD4 assay will be offset by cost-savings from prevention of pediatric HIV infection.

Genomic rearrangements commonly occur in many types of cancers and often initiate or alter the progression of disease. Here we describe an in vivo mouse model that recapitulates the most frequent rearrangement in prostate cancer, the fusion of the promoter region of TMPRSS2 with the coding region of the transcription factor, ERG. A recombinant bacterial artificial chromosome including an extended TMPRSS2 promoter driving genomic ERG was constructed and used for transgenesis in mice. TMPRSS2-ERG expression was evaluated in tissue sections and FACS-fractionated prostate cell populations. In addition to the anticipated expression in luminal cells, TMPRSS2-ERG was similarly expressed in the Sca-1(hi)/EpCAM(+) basal/progenitor fraction, where expanded numbers of clonogenic self-renewing progenitors were found, as assayed by in vitro sphere formation. These clonogenic cells increased intrinsic self renewal in subsequent generations. In addition, ERG dependent self-renewal and invasion in vitro was demonstrated in prostate cell lines derived from the model. Clinical studies have suggested that the TMPRSS2-ERG translocation occurs early in prostate cancer development. In the model described here, the presence of the TMPRSS2-ERG fusion alone was not transforming but synergized with heterozygous Pten deletion to promote PIN. Taken together, these data suggest that one function of TMPRSS2-ERG is the expansion of self-renewing cells, which may serve as targets for subsequent mutations. Primary prostate epithelial cells demonstrated increased post transcriptional turnover of ERG compared to the TMPRSS2-ERG positive VCaP cell line, originally isolated from a prostate cancer metastasis. Finally, we determined that TMPRSS2-ERG expression occurred in both castration-sensitive and resistant prostate epithelial subpopulations, suggesting the existence of androgen-independent mechanisms of TMPRSS2 expression in prostate epithelium.

Background: Breast cancer (BrCa) is a complex disease driven by aberrant gene alterations and environmental factors. Recent studies reveal that abnormal epigenetic gene regulation also plays an important role in its pathogenesis. Ubiquitin carboxyl- terminal esterase L1 (UCHL1) is a tumor suppressor silenced by promoter methylation in multiple cancers, but its role and alterations in breast tumorigenesis remain unclear., Methodology/principal Findings: We found that UCHL1 was frequently downregulated or silenced in breast cancer cell lines and tumor tissues, but readily expressed in normal breast tissues and mammary epithelial cells. Promoter methylation of UCHL1 was detected in 9 of 10 breast cancer cell lines (90%) and 53 of 66 (80%) primary tumors, but rarely in normal breast tissues, which was statistically correlated with advanced clinical stage and progesterone receptor status. Pharmacologic demethylation reactivated UCHL1 expression along with concomitant promoter demethylation. Ectopic expression of UCHL1 significantly suppressed the colony formation and proliferation of breast tumor cells, through inducing G0/G1 cell cycle arrest and apoptosis. Subcellular localization study showed that UCHL1 increased cytoplasmic abundance of p53. We further found that UCHL1 induced p53 accumulation and reduced MDM2 protein level, and subsequently upregulated the expression of p21, as well as cleavage of caspase3 and PARP, but not in catalytic mutant UCHL1 C90S-expressed cells., Conclusions/significance: UCHL1 exerts its tumor suppressive functions by inducing G0/G1cell cycle arrest and apoptosis in breast tumorigenesis, requiring its deubiquitinase activity. Its frequent silencing by promoter CpG methylation may serve as a potential tumor marker for breast cancer.

Prostate cancers of luminal adenocarcinoma histology display a range of clinical behaviors. Although most prostate cancers are slow-growing and indolent, a proportion is aggressive, developing metastasis and resistance to androgen deprivation treatment. One hypothesis is that a portion of aggressive cancers initiate from stem-like, androgen-independent tumor-propagating cells. Here we demonstrate the in vitro creation of a mouse cell line, selected for growth as self-renewing stem/progenitor cells, which manifests many in vivo properties of aggressive prostate cancer. Normal mouse prostate epithelium containing floxed Pten and TP53 alleles was subjected to CRE-mediated deletion in vitro followed by serial propagation as protospheres. A polyclonal cell line was established from dissociated protospheres and subsequently a clonal daughter line was derived. Both lines demonstrate a mature luminal phenotype in vitro. The established lines contain a stable minor population of progenitor cells with protosphere-forming ability and multi-lineage differentiation capacity. Both lines formed orthotopic adenocarcinoma tumors with metastatic potential to lung. Intracardiac inoculation resulted in brain and lung metastasis, while intra-tibial injection induced osteoblastic bone formation, recapitulating the bone metastatic phenotype of human prostate cancer. The cells showed androgen receptor dependent growth in vitro. Importantly, in vivo, the deprivation of androgens from established orthotopic tumors resulted in tumor regression and eventually castration-resistant growth. These data suggest that transformed prostate progenitor cells preferentially differentiate toward luminal cells and recapitulate many characteristics of the human disease.

Quantification of microvascular network structure is important in a myriad of emerging research fields including microvessel remodeling in response to ischemia and drug therapy, tumor angiogenesis, and retinopathy. To mitigate analyst-specific variation in measurements and to ensure that measurements represent actual changes in vessel network structure and morphology, a reliable and automatic tool for quantifying microvascular network architecture is needed. Moreover, an analysis tool capable of acquiring and processing large data sets will facilitate advanced computational analysis and simulation of microvascular growth and remodeling processes and enable more high throughput discovery. To this end, we have produced an automatic and rapid vessel detection and quantification system using a MATLAB graphical user interface (GUI) that vastly reduces time spent on analysis and greatly increases repeatability. Analysis yields numerical measures of vessel volume fraction, vessel length density, fractal dimension (a measure of tortuosity), and radii of murine vascular networks. Because our GUI is open sourced to all, it can be easily modified to measure parameters such as percent coverage of non-endothelial cells, number of loops in a vascular bed, amount of perfusion and two-dimensional branch angle. Importantly, the GUI is compatible with standard fluorescent staining and imaging protocols, but also has utility analyzing brightfield vascular images, obtained, for example, in dorsal skinfold chambers. A manually measured image can be typically completed in 20 minutes to 1 hour. In stark comparison, using our GUI, image analysis time is reduced to around 1 minute. This drastic reduction in analysis time coupled with increased repeatability makes this tool valuable for all vessel research especially those requiring rapid and reproducible results, such as anti-angiogenic drug screening.

Background: In patients with melanoma, knowledge of regional lymph node status provides important information on outlook. Since lymph node status can influence treatment, surgery for sentinel lymph node (SLN) biopsy became a standard staging procedure for these patients. Current imaging modalities have a limited sensitivity for detection of micrometastases in lymph nodes and, therefore, there is a need for a better technique that can accurately identify occult SLN metastases., Methodology/principal Findings: B16-F10 murine melanoma cells were infected with replication-competent herpes simplex virus (HSV) NV1023. The presence of tumor-targeting and reporter-expressing virus was assessed by [(18)F]-2'-fluoro-2'-deoxy-1-beta-D-beta-arabinofuranosyl-5-ethyluracil ([(18)F]FEAU) positron emission tomography (PET) and confirmed by histochemical assays. An animal foot pad model of melanoma lymph node metastasis was established. Mice received intratumoral injections of NV1023, and 48 hours later were imaged after i.v. injection of [(18)F]FEAU. NV1023 successfully infected and provided high levels of lacZ transgene expression in melanoma cells. Intratumoral injection of NV1023 resulted in viral trafficking to melanoma cells that had metastasized to popliteal and inguinal lymph nodes. Presence of virus-infected tumor cells was successfully imaged with [(18)F]FEAU-PET, that identified 8 out of 8 tumor-positive nodes. There was no overlap between radioactivity levels (lymph node to surrounding tissue ratio) of tumor-positive and tumor-negative lymph nodes., Conclusion/significance: A new approach for imaging SLN metastases using NV1023 and [(18)F]FEAU-PET was successful in a murine model. Similar studies could be translated to the clinic and improve the staging and management of patients with melanoma.