Acute Ozone-Induced Transcriptional Changes in Markers of Oxidative Stress and Glucocorticoid Signaling in the Rat Hippocampus and Hypothalamus Are Sex-Specific.

Exposure to a prototypic air pollutant ozone (O3) has been associated with the activation of neuroendocrine stress response along with neural changes in oxidative stress (OS), inflammation, and Alzheimer's disease-like pathologies in susceptible animal models. We hypothesized that neural oxidative and transcriptional changes induced by O3 in stress responsive regions are sex-dependent. Male and female adult Long-Evans rats were exposed to filtered air or O3 for two consecutive days (0.8 ppm, 4 h/day) and brain regions were flash-frozen. Activities of cerebellar OS parameters and mitochondrial complex I, II, and IV enzymes were assessed to confirm prior findings. We assessed transcriptional changes in hypothalamus (HYP) and hippocampus (HIP) for markers of OS, microglial activity and glucocorticoid signaling using qPCR. Although there were no O3 or sex-related differences in the cerebellar activities of OS and mitochondrial enzymes, the levels of protein carbonyls and complex II activities were higher in females regardless of O3. There were no statistical differences in baseline expression of genes related to OS (Cat, Dhcr24, Foxm1, Gpx1, Gss, Nfe2l2, Sod1) except for lower HYP Sod1 expression in air-exposed females than males, and higher HIP Gss expression in O3-exposed females relative to matched males. Microglial marker Aif1 expression was higher in O3-exposed females relative to males; O3 inhibited Itgam only in males. The expression of Bdnf in HIP and HYP was inhibited by O3 in both sexes. Genes related to glucocorticoid signaling (Fkbp4, Fkbp5, Hsp90aa1, Hspa4, nr3c1, nr3c2) showed sex-specific effects due to O3 exposure. Baseline expression of HIP Fkbp4 was higher in females relative to males. O3 inhibited Nr3c1 in female HIP and male HYP, but Nr3c2 was inhibited in male HYP. Fkbp4 expression was higher in O3-exposed females when compared to matched males, whereas Fkbp5 was expressed at higher levels in both brain regions of males and females. These results indicate that sex-specific brain region responses to O3 might, in part, be caused by OS and regulation of glucocorticoid signaling.

Single bubble in-tube microextraction coupled with capillary electrophoresis.

Headspace (HS) extraction is a sample pretreatment technique for volatile and semivolatile organic compounds in a complex matrix. Recently, in-tube microextraction (ITME) coupled with CE using an acceptor plug placed in the capillary inlet was developed as a simple but powerful HS extraction method. Here, we present single bubble (SB) ITME using a bubble hanging to the capillary inlet immersed in a sample donor solution as a HS of submicroliter volume (∼200 nL). The analytes evaporated to the bubble were extracted into the acceptor phase through the capillary opening, then electrophoresis of the enriched extract was carried out. Since the bubble volume was much smaller than a conventional HS volume (∼1 mL), it was filled with the evaporated analytes rapidly and the analytes could be enriched much faster compared to conventional HS-ITME. Owing to the high surface-to-volume ratio of the SB, 5 min SB-ITME yielded the enrichment factor values similar to those of 10 min HS-ITME. When 5 min SB-ITME at room temperature was applied to a tap water sample, the enrichment factors of 2,4,6-trichlorophenol (TCP), 2,3,6-TCP, and 2,6-dichlorophenol were 53, 41, and 60, respectively, and the LOQs obtained by monitoring the absorbance at 214 nm were 5.6-8.3 ppb, much lower than 200 ppb, the World Health Organization guideline for the maximum permissible concentration of 2,4,6-TCP in drinking water.

The effects of ozone exposure and sedentary lifestyle on neuronal microglia and mitochondrial bioenergetics of female Long-Evans rats.

Ozone (O3) is a widespread air pollutant that produces cardiovascular and pulmonary dysfunction possibly mediated by activation of central stress centers. Epidemiological data suggest that sedentary lifestyles may exacerbate responses to air pollutants such as O3. We sought to assess neurological changes in response to O3 exposure and an active lifestyle. We developed an animal model in which female Long-Evans rats were either sedentary or active with continuous access to running wheels starting at postnatal day (PND) 22 until the age of PND 100 and then exposed to O3 (0, 0.25, 0.5 or 1.0 ppm) 5 h/day for two consecutive days. We found significantly more reactive microglia within the hippocampus (HIP) in animals exposed to O3 in both sedentary and active rats. No changes were detected in astrocytic coverage. We next analyzed mitochondrial bioenergetic parameters (complex I, complex II and complex IV). Complex I activity was significantly affected by exercise in hypothalamus (HYP). Complex II activity was significantly affected by both exercise and O3 exposure in the HIP. Concomitant with the changes in enzymatic activity, there were also effects on expression of genes related to mitochondrial bioenergetics and antioxidant production. These results demonstrate that O3 induces microglia reactivity within stress centers of the brain and that mitochondrial bioenergetics are altered. Some of these effects may be augmented by exercise, suggesting a role for lifestyle in O3 effects on brain mitochondrial bioenergetics parameters in agreement with our previous reports on other endpoints.

Mitochondrial Bioenergetics in Brain Following Ozone Exposure in Rats Maintained on Coconut, Fish and Olive Oil-Rich Diets.

Dietary supplementation with omega-3 and omega-6 fatty acids offer cardioprotection against air pollution, but these protections have not been established in the brain. We tested whether diets rich in omega-3 or -6 fatty acids offered neuroprotective benefits, by measuring mitochondrial complex enzyme I, II and IV activities and oxidative stress measures in the frontal cortex, cerebellum, hypothalamus, and hippocampus of male rats that were fed either a normal diet, or a diet enriched with fish oil olive oil, or coconut oil followed by exposure to either filtered air or ozone (0.8 ppm) for 4 h/day for 2 days. Results show that mitochondrial complex I enzyme activity was significantly decreased in the cerebellum, hypothalamus and hippocampus by diets. Complex II enzyme activity was significantly lower in frontal cortex and cerebellum of rats maintained on all test diets. Complex IV enzyme activity was significantly lower in the frontal cortex, hypothalamus and hippocampus of animals maintained on fish oil. Ozone exposure decreased complex I and II activity in the cerebellum of rats maintained on the normal diet, an effect blocked by diet treatments. While diet and ozone have no apparent influence on endogenous reactive oxygen species production, they do affect antioxidant levels in the brain. Fish oil was the only diet that ozone exposure did not alter. Microglial morphology and GFAP immunoreactivity were assessed across diet groups; results indicated that fish oil consistently decreased reactive microglia in the hypothalamus and hippocampus. These results indicate that acute ozone exposure alters mitochondrial bioenergetics in brain and co-treatment with omega-6 and omega-3 fatty acids alleviate some adverse effects within the brain.

Interaction of Diet and Ozone Exposure on Oxidative Stress Parameters within Specific Brain Regions of Male Brown Norway Rats.

Oxidative stress (OS) contributes to the neurological and cardio/pulmonary effects caused by adverse metabolic states and air pollutants such as ozone (O3). This study explores the interactive effects of O3 and diet (high-fructose (FRUC) or high⁻fat (FAT)) on OS in different rat brain regions. In acute exposure, there was a decrease in markers of reactive oxygen species (ROS) production in some brain regions by diet and not by O3. Total antioxidant substances (TAS) were increased in the cerebellum (CER) and frontal cortex (FC) and decreased in the striatum (STR) by both diets irrespective of O3 exposure. Protein carbonyls (PC) and total aconitase decreased in some brain regions irrespective of exposure. Following subacute exposure, an increase in markers of ROS was observed in both diet groups. TAS was increased in the FC (FAT only) and there was a clear O3 effect where TAS was increased in the FC and STR. Diet increased PC formation within the CER in the FAT group, while the hippocampus showed a decrease in PC after O3 exposure in controls. In general, these results indicate that diet/O3 did not have a global effect on brain OS parameters, but showed some brain region- and OS parameter-specific effects by diets.

Lgr4 is a key regulator of prostate development and prostate stem cell differentiation.

Mechanisms modulating prostate cell fate determination remain unexplored. The leucine-rich repeat containing G-protein-coupled receptors (Lgr) have been identified as important stem cell markers in various tissues. Here, we investigated the roles of Lgr4/Gpr48 in prostate stem cells (PSCs) and development. Lgr4 was ubiquitously expressed during early prostate development prior to lineage specification, with adult expression restricted to a few basal cells (principally Lin(-)Sca1(+)CD49f(+)). Lgr4(-/-) mice had compromised branching morphogenesis and delayed epithelial differentiation, leading to decreased prostate size and impaired luminal cell function. In vitro prostate sphere culture revealed that Lgr4(-/-) Lin(-)/Sca1(+)/CD49f(+) cells failed to generate p63(low) cells, indicating a differentiation deficiency. Furthermore, Lgr4 ablation arrested PSC differentiation of in vivo kidney capsule prostate grafts, suggesting that Lgr4 modulates PSC properties independent of hormonal and mesenchymal effects. Analysis of neonatal prostates and prostate spheres revealed a decrease in Wnt, Sonic Hedgehog, and Notch1 expression in Lgr4(-/-) cells. Lgr4 loss blocked differentiation of prostate sphere p63(hi) cells to p63(low). Treatment with exogenous Sonic Hedgehog partially restored the differentiation of p63(hi) cells in Lgr4(-/-) spheres. Taken together, our data revealed the roles of Lgr4 in early prostate development and in stem cell differentiation through regulation of the Wnt, Notch, and Sonic Hedgehog signaling pathways.

Influence of Mild Chronic Stress and Social Isolation on Acute Ozone-Induced Alterations in Stress Biomarkers and Brain-Region-Specific Gene Expression in Male Wistar-Kyoto Rats.

Individuals with psychosocial stress often experience an exaggerated response to air pollutants. Ozone (O3) exposure has been associated with the activation of the neuroendocrine stress-response system. We hypothesized that preexistent mild chronic stress plus social isolation (CS), or social isolation (SI) alone, would exacerbate the acute effects of O3 exposure on the circulating adrenal-derived stress hormones, and the expression of the genes regulating glucocorticoid stress signaling via an altered stress adaptation in a brain-region-specific manner. Male Wistar-Kyoto rats (5 weeks old) were socially isolated, plus were subjected to either CS (noise, confinement, fear, uncomfortable living, hectic activity, and single housing), SI (single housing only, restricted handling and no enrichment) or no stress (NS; double housing, frequent handling and enrichment provided) for 8 weeks. The rats were then exposed to either air or O3 (0.8 ppm for 4 h), and the samples were collected immediately after. The indicators of sympathetic and hypothalamic-pituitary axis (HPA) activation (i.e., epinephrine, corticosterone, and lymphopenia) increased with O3 exposure, but there were no effects from CS or SI, except for the depletion of serum BDNF. CS and SI revealed small changes in brain-region-specific glucocorticoid-signaling-associated markers of gene expression in the air-exposed rats (hypothalamic Nr3c1, Nr3c2 Hsp90aa1, Hspa4 and Cnr1 inhibition in SI; hippocampal HSP90aa1 increase in SI; and inhibition of the bed nucleus of the stria terminalis (BNST) Cnr1 in CS). Gene expression across all brain regions was altered by O3, reflective of glucocorticoid signaling effects, such as Fkbp5 in NS, CS and SI. The SI effects on Fkbp5 were greatest for SI in BNST. O3 increased Cnr2 expression in the hypothalamus and olfactory bulbs of the NS and SI groups. O3, in all stress conditions, generally inhibited the expression of Nr3c1 in all brain regions, Nr3c2 in the hippocampus and hypothalamus and Bdnf in the hippocampus. SI, in general, showed slightly greater O3-induced changes when compared to NS and CS. Serum metabolomics revealed increased sphingomyelins in the air-exposed SI and O3-exposed NS, with underlying SI dampening some of the O3-induced changes. These results suggest a potential link between preexistent SI and acute O3-induced increases in the circulating adrenal-derived stress hormones and brain-region-specific gene expression changes in glucocorticoid signaling, which may partly underlie the stress dynamic in those with long-term SI.

Wnt and Notch pathways have interrelated opposing roles on prostate progenitor cell proliferation and differentiation.

Tissue stem cells are capable of both self-renewal and differentiation to maintain a constant stem cell population and give rise to the plurality of cells within a tissue. Wnt signaling has been previously identified as a key mediator for the maintenance of tissue stem cells; however, possible cross-regulation with other developmentally critical signaling pathways involved in adult tissue homeostasis, such as Notch, is not well understood. By using an in vitro prostate stem cell colony ("prostasphere") formation assay and in vivo prostate reconstitution experiments, we demonstrate that Wnt pathway induction on Sca-1(+) CD49f(+) basal/stem cells (B/SCs) promotes expansion of the basal epithelial compartment with noticeable increases in "triple positive" (cytokeratin [CK] 5(+), CK8(+), p63(+)) prostate progenitor cells, concomitant with upregulation of known Wnt target genes involved in cell-cycle induction. Moreover, Wnt induction affects expression of epithelial-to-mesenchymal transition signature genes, suggesting a possible mechanism for priming B/SC to act as potential tumor-initiating cells. Interestingly, induction of Wnt signaling in B/SCs results in downregulation of Notch1 transcripts, consistent with its postulated antiproliferative role in prostate cells. In contrast, induction of Notch signaling in prostate progenitors inhibits their proliferation and disrupts prostasphere formation. In vivo prostate reconstitution assays further demonstrate that induction of Notch in B/SCs disrupts proper acini formation in cells expressing the activated Notch1 allele, Notch-1 intracellular domain. These data emphasize the importance of Wnt/Notch cross-regulation in adult stem cell biology and suggest that Wnt signaling controls the proliferation and/or maintenance of epithelial progenitors via modulation of Notch signaling.

Dicer ablation impairs prostate stem cell activity and causes prostate atrophy.

Dicer is an RNase III enzyme essential for microRNA maturation. Dicer ablation in diverse tissues has been shown to block tissue differentiation, induce cell apoptosis, impair specialized cellular function, and perturb organ structures. To gain insight into the role of microRNAs in prostate tissue function and homeostasis, we conditionally disrupted Dicer activity in the mouse prostate using an ARR2PB-Cre. We demonstrated that Dicer activity is disrupted in both prostatic basal/stem cells and differentiated luminal cells. Dicer knockout murine prostates are smaller in size and mass and develop epithelial hypotrophy in ventral prostates by 4 months. Dicer ablation induces increased apoptosis in the prostate, predominantly in the differentiated luminal cells. Paradoxically, a concurrent increase in proliferation is observed in both basal/stem cells and luminal cells, presumably due to compensatory growth of the cells devoid of homologous recombination in response to the elevated cellular apoptosis. We have previously shown that Lin(CD31CD45Ter119)(-)Sca-1(+)CD49f(high) (LSC) cells enrich for prostate stem cell activity. Through proliferation and differentiation, some LSC cells are capable of forming prostate spheres composed of cells at various stages of differentiation. Although LSC cells were expanded by threefold in Dicer knockout mice, the sphere-forming units of Dicer knockout prostate cells decreased by more than half compared with wild-type cells. In addition, most prostate spheres in the Dicer knockout culture were derived from cells that did not undergo homologous recombination. Our results demonstrate a critical role of microRNAs for the proliferative capacity of prostate stem cells and the maintenance of prostate homeostasis.

Facile and highly efficient three-phase single drop microextraction in-line coupled with capillary electrophoresis.

A high-performance version of in-line, three-phase direct immersion-single drop microextraction (DI-SDME) coupled with capillary electrophoresis (CE) was demonstrated using a commercial CE instrument, and all the major and minor details were described to provide an easy-to-follow and user-friendly protocol. The excellent sample cleanup and enrichment power of this method was demonstrated with nonsteroidal anti-inflammatory drugs (NSAIDs) in human urine. The only preparation of urine samples was the addition of HCl to acidify the urine sample to pH 2. The acidic NSAIDs in the acidified urine sample were extracted into a basic acceptor drop covered with a thin organic layer attached to the inlet tip of a capillary immersed in the sample. A simple but powerful DI-SDME-CE method could be carried out automatically without any modification of the existing CE instrument. For improved performance, sample agitation and heating were employed by installing a microstirrer and a thermostating jacket in the sample tray. With 10 min of DI-SDME at 35°C with stirring, NSAIDs such as ketoprofen, ibuprofen, and naproxen in urine were enriched 340-970-fold with intraday and interday RSDs of 0.8-2.4% and 1.1-3.6%, respectively. The LODs obtained with in-line coupled CE/UV were 10-50 nM (2-10 µg/L). The performance of DI-SDME-CE/UV was also demonstrated by determining the naproxen level in human urine collected 24 h after taking a single oral dose of the drug. The spike recovery of naproxen from a single-point standard addition to the urine sample was 80%. Our high-performance three-phase DI-SDME-CE method is quite promising for the analysis of ionizable trace analytes in a complex sample matrix.

Rapid detection of SARS-CoV-2 antibodies using electrochemical impedance-based detector.

Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was classified as a pandemic by the World Health Organization and has caused over 550,000 deaths worldwide as of July 2020. Accurate and scalable point-of-care devices would increase screening, diagnosis, and monitoring of COVID-19 patients. Here, we demonstrate rapid label-free electrochemical detection of SARS-CoV-2 antibodies using a commercially available impedance sensing platform. A 16-well plate containing sensing electrodes was pre-coated with receptor binding domain (RBD) of SARS-CoV-2 spike protein, and subsequently tested with samples of anti-SARS-CoV-2 monoclonal antibody CR3022 (0.1 µg/ml, 1.0 µg/ml, 10 µg/ml). Subsequent blinded testing was performed on six serum specimens taken from COVID-19 and non-COVID-19 patients (1:100 dilution factor). The platform was able to differentiate spikes in impedance measurements from a negative control (1% milk solution) for all CR3022 samples. Further, successful differentiation and detection of all positive clinical samples from negative control was achieved. Measured impedance values were consistent when compared to standard ELISA test results showing a strong correlation between them (R2=0.9). Detection occurs in less than five minutes and the well-based platform provides a simplified and familiar testing interface that can be readily adaptable for use in clinical settings.

Low-density Taqman miRNA array reveals miRNAs differentially expressed in prostatic stem cells and luminal cells.

BACKGROUND: miRNAs are a class of naturally occurring small RNAs that generally repress gene expression. They have been shown to actively control diverse biological processes including stem cell differentiation and lineage commitment. METHODS: Fluorescence-activated cell sorting was utilized to isolate murine prostate stem cells and differentiated luminal cells. The expression levels of Drosha and Dicer1, the two key RNAseIII enzymes for miRNA maturation, were evaluated by quantitative RT-PCR. Low-density Taqman miRNA array analyses were also performed to identify miRNAs that are differentially expressed in individual lineages. RESULTS: Drosha and Dicer1 are expressed at comparable transcriptional levels in murine prostate stem cells and differentiated luminal cells. Twenty-nine miRNAs were discovered to be differentially expressed in prostate stem cells and luminal cells. Many of these miRNAs are coded in clusters, suggesting a cell-specific transcriptional regulation. Some of these differentially expressed miRNAs have been reported to regulate genes relevant to the molecular and phenotypic features of each lineage. CONCLUSIONS: miRNAs may play a potentially critical role in fine regulation of prostatic lineage identity.

Dysregulation of Hypothalamic Gene Expression and the Oxytocinergic System by Soybean Oil Diets in Male Mice.

Soybean oil consumption has increased greatly in the past half-century and is linked to obesity and diabetes. To test the hypothesis that soybean oil diet alters hypothalamic gene expression in conjunction with metabolic phenotype, we performed RNA sequencing analysis using male mice fed isocaloric, high-fat diets based on conventional soybean oil (high in linoleic acid, LA), a genetically modified, low-LA soybean oil (Plenish), and coconut oil (high in saturated fat, containing no LA). The 2 soybean oil diets had similar but nonidentical effects on the hypothalamic transcriptome, whereas the coconut oil diet had a negligible effect compared to a low-fat control diet. Dysregulated genes were associated with inflammation, neuroendocrine, neurochemical, and insulin signaling. Oxt was the only gene with metabolic, inflammation, and neurological relevance upregulated by both soybean oil diets compared to both control diets. Oxytocin immunoreactivity in the supraoptic and paraventricular nuclei of the hypothalamus was reduced, whereas plasma oxytocin and hypothalamic Oxt were increased. These central and peripheral effects of soybean oil diets were correlated with glucose intolerance but not body weight. Alterations in hypothalamic Oxt and plasma oxytocin were not observed in the coconut oil diet enriched in stigmasterol, a phytosterol found in soybean oil. We postulate that neither stigmasterol nor LA is responsible for effects of soybean oil diets on oxytocin and that Oxt messenger RNA levels could be associated with the diabetic state. Given the ubiquitous presence of soybean oil in the American diet, its observed effects on hypothalamic gene expression could have important public health ramifications.

Continuous Inhalation Exposure to Fungal Allergen Particulates Induces Lung Inflammation While Reducing Innate Immune Molecule Expression in the Brainstem.

Continuous exposure to aerosolized fine (particle size ≤2.5 µm) and ultrafine (particle size ≤0.1 µm) particulates can trigger innate inflammatory responses in the lung and brain depending on particle composition. Most studies of manmade toxicants use inhalation exposure routes, whereas most studies of allergens use soluble solutions administered via intranasal or injection routes. Here, we tested whether continuous inhalation exposure to aerosolized Alternaria alternata particulates (a common fungal allergen associated with asthma) would induce innate inflammatory responses in the lung and brain. By designing a new environmental chamber able to control particle size distribution and mass concentration, we continuously exposed adult mice to aerosolized ultrafine Alternaria particulates for 96 hr. Despite induction of innate immune responses in the lung, induction of innate immune responses in whole brain samples was not detected by quantitative polymerase chain reaction or flow cytometry. However, exposure did trigger decreases in Arginase 1, inducible nitric oxide synthase, and tumor necrosis factor alpha mRNA in the brainstem samples containing the central nervous system respiratory circuit (the dorsal respiratory group, ventral respiratory group, and the pre-Bötzinger and Bötzinger complexes). In addition, a significant decrease in the percentage of Toll-like receptor 2-expressing brainstem microglia was detected by flow cytometry. Histologic analysis revealed a significant decrease in Iba1 but not glial fibrillary acidic protein immunoreactivity in both the brainstem and the hippocampus. Together these data indicate that inhalation exposure to a natural fungal allergen under conditions sufficient to induce lung inflammation surprisingly causes reductions in baseline expression of select innate immune molecules (similar to that observed during endotoxin tolerance) in the region of the central nervous system controlling respiration.

Social defeat stress-induced behavioral responses are mediated by the endogenous kappa opioid system.

Previous studies have demonstrated that repeated forced-swim stress-induced behaviors (including analgesia, immobility, and increased drug reward) were mediated by the release of endogenous prodynorphin-derived opioid peptides and subsequent activation of the kappa opioid receptor (KOR). We tested the generality of these effects using a different type of stressful situation: repeated social defeat. C57Bl/6 mice subjected to social defeat stress (SDS) over 3 days showed a characteristic stress-induced immobility and defeated-postural response, as well as stress-induced analgesia (SIA). Daily pretreatment with the KOR antagonist nor-binaltorphimine (nor-BNI, 10 mg/kg, i.p.) blocked the SIA and significantly reduced the stress-induced immobility on the second and third days of SDS exposure. In contrast, prodynorphin gene-disrupted mice showed no significant increase in immobility, socially defeated postures, or SIA following repeated exposure to SDS. Since both stress and the kappa opioid system can modulate the response to drugs of abuse, we tested the effects of SDS on cocaine-conditioned place preference (CPP). SDS-exposed mice conditioned with cocaine (15 mg/kg, s.c.) showed significant potentiation of place-preference for the drug-paired chamber over the responses of unstressed mice. Nor-BNI pretreatment blocked stress-induced potentiation of cocaine-CPP. Consistent with this result, mice lacking the prodynorphin gene did not show stress-induced potentiation of cocaine-CPP, whereas wild-type littermates did. The findings suggest that chronic SDS may activate the kappa opioid system to produce analgesia, immobility, social defeat postures, and resulting in a potentiation of the acute rewarding properties of cocaine.

Increased Notch signalling inhibits anoikis and stimulates proliferation of prostate luminal epithelial cells.

The prostate epithelial lineage hierarchy remains inadequately defined. Recent lineage-tracing studies have implied the existence of prostate luminal epithelial progenitors with extensive regenerative capacity. However, this capacity has not been demonstrated in prostate stem cell activity assays, probably owing to the strong susceptibility of luminal progenitors to anoikis. Here we show that constitutive expression of Notch1 intracellular domain impairs secretory function of mouse prostate luminal cells, suppresses anoikis of luminal epithelial cells by augmenting NF-κB activity independent of Hes1, stimulates luminal cell proliferation by potentiating PI3K-AKT signalling, and rescues the capacities of the putative prostate luminal progenitors for unipotent differentiation in vivo and short-term self-renewal in vitro. Epithelial cell autonomous AR signalling is dispensable for the Notch-mediated effects. As Notch activity is increased in prostate cancers, and anoikis resistance is a hallmark for metastatic cancer cells, this study suggests a pro-metastatic function of Notch signalling during prostate cancer progression.

Notch and TGFß form a reciprocal positive regulatory loop that suppresses murine prostate basal stem/progenitor cell activity.

The role of Notch signaling in the maintenance of adult murine prostate epithelial homeostasis remains unclear. We found that Notch ligands are mainly expressed within the basal cell lineage, while active Notch signaling is detected in both the prostate basal and luminal cell lineages. Disrupting the canonical Notch effector Rbp-j impairs the differentiation of prostate basal stem cells and increases their proliferation in vitro and in vivo, but does not affect luminal cell biology. Conversely, ectopic Notch activation in adult prostates results in a decrease in basal cell number and luminal cell hyperproliferation. TGFß dominates over Notch signaling and overrides Notch ablation-induced proliferation of prostate basal cells. However, Notch confers sensitivity and positive feedback by upregulating a plethora of TGFß signaling components including TgfßR1. These findings reveal crucial roles of the self-enforced positive reciprocal regulatory loop between TGFß and Notch in maintaining prostate basal stem cell dormancy.

ROCK inhibitor Y-27632 suppresses dissociation-induced apoptosis of murine prostate stem/progenitor cells and increases their cloning efficiency.

Activation of the RhoA/ROCK signaling pathway has been shown to contribute to dissociation-induced apoptosis of embryonic and neural stem cells. We previously demonstrated that approximately 1 out of 40 Lin(-)Sca-1(+)CD49f(high) (LSC) prostate basal epithelial cells possess the capacities of stem cells for self-renewal and multi-lineage differentiation. We show here that treating LSC cells with the ROCK kinase inhibitor Y-27632 increases their cloning efficiency by 8 fold in an in vitro prostate colony assay. Y-27632 treatment allows prostate colony cells to replate efficiently, which does not occur otherwise. Y-27632 also increases the cloning efficiency of prostate stem cells in a prostate sphere assay and a dissociated prostate cell regeneration assay. The increased cloning efficiency is due to the suppression of the dissociation-induced, RhoA/ROCK activation-mediated apoptosis of prostate stem cells. Dissociation of prostate epithelial cells from extracellular matrix increases PTEN activity and attenuates AKT activity. Y-27632 treatment alone is sufficient to suppress cell dissociation-induced activation of PTEN activity. However, this does not contribute to the increased cloning efficiency, because Y-27632 treatment increases the sphere-forming unit of wild type and Pten null prostate cells to a similar extent. Finally, knocking down expression of both ROCK kinases slightly increases the replating efficiency of prostate colony cells, corroborating that they play a major role in the Y-27632 mediated increase in cloning efficiency. Our study implies that the numbers of prostate cells with stem/progenitor activity may be underestimated based on currently employed assays, supports that dissociation-induced apoptosis is a common feature of embryonic and somatic stem cells with an epithelial phenotype, and highlights the significance of environmental cues for the maintenance of stem cells.

Mutagenesis and repair by low doses of alpha radiation in mammalian cells.

Low doses of alpha radiation in basements have been causally implicated in lung cancer. Previous studies have concentrated on high dose effects, for which no significant repair was found. In the present study, the methodology for measuring mutation by quantitating mitotic breaks and gaps was found to be applicable to G2-phase Chinese hamster ovary cells irradiated with 10-50 cGy of alpha radiation. The mutation yield in such cells closely resembles that of gamma irradiation. Caffeine, which inhibits repair, produces the same straight line increase of alpha and gamma mutation yields plotted against the dose. In the absence of caffeine, the repair of alpha radiation lesions is almost twice as great as for gamma radiation. Mitotic index changes substantiate these interpretations. It is proposed that the higher ion density associated with alpha radiation may result in fewer lesions being missed by the repair processes. The quantitation of chromosomal lesions for G2 cells exposed to low doses of alpha radiation, gamma radiation, or chemical mutagens in the presence and absence of caffeine is a rapid and reproducible methodology. Protection from mutational disease in a fashion similar to the use of sanitation for infectious disease appears practical.