Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer.

Single-cell RNA sequencing (scRNA-seq) is a powerful tool for defining cellular diversity in tumors, but its application toward dissecting mechanisms underlying immune-modulating therapies is scarce. We performed scRNA-seq analyses on immune and stromal populations from colorectal cancer patients, identifying specific macrophage and conventional dendritic cell (cDC) subsets as key mediators of cellular cross-talk in the tumor microenvironment. Defining comparable myeloid populations in mouse tumors enabled characterization of their response to myeloid-targeted immunotherapy. Treatment with anti-CSF1R preferentially depleted macrophages with an inflammatory signature but spared macrophage populations that in mouse and human expresses pro-angiogenic/tumorigenic genes. Treatment with a CD40 agonist antibody preferentially activated a cDC population and increased Bhlhe40+ Th1-like cells and CD8+ memory T cells. Our comprehensive analysis of key myeloid subsets in human and mouse identifies critical cellular interactions regulating tumor immunity and defines mechanisms underlying myeloid-targeted immunotherapies currently undergoing clinical testing.

Activation of orexin system facilitates anesthesia emergence and pain control.

Orexin (also known as hypocretin) neurons in the hypothalamus play an essential role in sleep-wake control, feeding, reward, and energy homeostasis. The likelihood of anesthesia and sleep sharing common pathways notwithstanding, it is important to understand the processes underlying emergence from anesthesia. In this study, we investigated the role of the orexin system in anesthesia emergence, by specifically activating orexin neurons utilizing the designer receptors exclusively activated by designer drugs (DREADD) chemogenetic approach. With injection of adeno-associated virus into the orexin-Cre transgenic mouse brain, we expressed the DREADD receptor hM3Dq specifically in orexin neurons and applied the hM3Dq ligand clozapine to activate orexin neurons. We monitored orexin neuronal activities by c-Fos staining and whole-cell patch-clamp recording and examined the consequence of orexin neuronal activation via EEG recording. Our results revealed that the orexin-DREADD mice with activated orexin neurons emerged from anesthesia with significantly shorter latency than the control mice. As an indication of reduced pain sensitivity, these orexin-DREADD mice took longer to respond to the 55 °C thermal stimuli in the hot plate test and exhibited significantly less frequent licking of the formalin-injected paw in the formalin test. Our study suggests that approaches to activate the orexin system can be beneficial in postoperative recovery.

TMEM16B Calcium-Activated Chloride Channels Regulate Action Potential Firing in Lateral Septum and Aggression in Male Mice.

The lateral septum (LS) plays an important role in regulating aggression. It is well recognized that LS lesions lead to a dramatic increase in aggressive behaviors. A better understanding of LS neurophysiology and its functional output is therefore important to assess LS involvement in regulating aggression. The LS is a heterogeneous structure that maintains inputs and outputs with multiple brain regions, and is also divided into subregions that innervate one another. Thus, it is challenging to identify the exact cell type and projections for characterization. In this study, we determined the expression pattern of the calcium-activated chloride channel, TMEM16B, in the LS of both male and female mice. We then investigated the physiological contribution of the calcium-activated chloride channel to LS neuronal signaling. By performing whole-cell patch-clamp recording, we showed that TMEM16B alters neurotransmitter release at the hippocampal-LS synapse, and regulates spike frequency and spike frequency adaptation in subpopulations of LS neurons. We further demonstrated that loss of TMEM16B function promotes lengthened displays of aggressive behaviors by male mice during the resident intruder paradigm. In conclusion, our findings suggest that TMEM16B function contributes to neuronal excitability in subpopulations of LS neurons and the regulation of aggression in male mice.SIGNIFICANCE STATEMENT Aggression is a behavior that arose evolutionarily from the necessity to compete for limited resources and survival. One particular brain region involved in aggression is the lateral septum (LS). In this study, we characterized the expression of the TMEM16B calcium-activated chloride channel in the LS and showed that TMEM16B regulates the action potential firing frequency of LS neurons. We discovered that loss of TMEM16B function lengthens the displays of aggressive behaviors in male mice. These findings suggest that TMEM16B plays an important role in regulating LS neuronal excitability and behaviors associated with LS function, thereby contributing to our understanding of how the LS may regulate aggression.

Impaired SNX9 Expression in Immune Cells during Chronic Inflammation: Prognostic and Diagnostic Implications.

Chronic inflammation is associated with immunosuppression and downregulated expression of the TCR CD247. In searching for new biomarkers that could validate the impaired host immune status under chronic inflammatory conditions, we discovered that sorting nexin 9 (SNX9), a protein that participates in early stages of clathrin-mediated endocytosis, is downregulated as well under such conditions. SNX9 expression was affected earlier than CD247 by the generated harmful environment, suggesting that it is a potential marker sensing the generated immunosuppressive condition. We found that myeloid-derived suppressor cells, which are elevated in the course of chronic inflammation, are responsible for the observed SNX9 reduced expression. Moreover, SNX9 downregulation is reversible, as its expression levels return to normal and immune functions are restored when the inflammatory response and/or myeloid-derived suppressor cells are neutralized. SNX9 downregulation was detected in numerous mouse models for pathologies characterized by chronic inflammation such as chronic infection (Leishmania donovani), cancer (melanoma and colorectal carcinoma), and an autoimmune disease (rheumatoid arthritis). Interestingly, reduced levels of SNX9 were also observed in blood samples from colorectal cancer patients, emphasizing the feasibility of its use as a diagnostic and prognostic biomarker sensing the host's immune status and inflammatory stage. Our new discovery of SNX9 as being regulated by chronic inflammation and its association with immunosuppression, in addition to the CD247 regulation under such conditions, show the global impact of chronic inflammation and the generated immune environment on different cellular pathways in a diverse spectrum of diseases.

Increased Expression of GM1 Detected by Electrospray Mass Spectrometry in Rat Primary Embryonic Cortical Neurons Exposed to Glutamate Toxicity.

Neurons within different brain regions have varying levels of vulnerability to external stress and respond differently to injury. A potential reason to explain this may lie within a key lipid class of the cell's plasma membrane called gangliosides. These glycosphingolipid species have been shown to play various roles in the maintenance of neuronal viability. The purpose of this study is to use electrospray ionization mass spectrometry (ESI-MS) and immunohistochemistry to evaluate the temporal expression profiles of gangliosides during the course of neurodegeneration in rat primary cortical neurons exposed to glutamate toxicity. Primary embryonic (E18) rat cortical neurons were cultured to DIV (days in vitro) 14. Glutamate toxicity was induced for 1, 3, 6, and 24 h to injure and kill neurons. Immunofluorescence was used to stain for GM1 and GM3 species, and ESI-MS was used to quantify the ganglioside species expressed within these injured neurons. ESI-MS data revealed that GM1, GM2, and GM3 were up-regulated in neurons exposed to glutamate. Interestingly, using immunofluorescence, we demonstrated that the GM1 increase following glutamate exposure occurred in viable neurons, possibly indicating a potential intrinsic neuroprotective response. To test this potential neuroprotective property, neurons were pretreated with GM1 for 24 h prior to glutamate exposure. Pretreatment with GM1 conferred significant neuroprotection against glutamate-induced cell death. Overall, work from this study validates the use of ESI-MS for cell-derived gangliosides and supports the further development of lipid based strategies to protect against neuron cell death.

Unique ζ-chain motifs mediate a direct TCR-actin linkage critical for immunological synapse formation and T-cell activation.

TCR-mediated activation induces receptor microclusters that evolve to a defined immune synapse (IS). Many studies showed that actin polymerization and remodeling, which create a scaffold critical to IS formation and stabilization, are TCR mediated. However, the mechanisms controlling simultaneous TCR and actin dynamic rearrangement in the IS are yet not fully understood. Herein, we identify two novel TCR ζ-chain motifs, mediating the TCR's direct interaction with actin and inducing actin bundling. While T cells expressing the ζ-chain mutated in these motifs lack cytoskeleton (actin) associated (cska)-TCRs, they express normal levels of non-cska and surface TCRs as cells expressing wild-type ζ-chain. However, such mutant cells are unable to display activation-dependent TCR clustering, IS formation, expression of CD25/CD69 activation markers, or produce/secrete cytokine, effects also seen in the corresponding APCs. We are the first to show a direct TCR-actin linkage, providing the missing gap linking between TCR-mediated Ag recognition, specific cytoskeleton orientation toward the T-cell-APC interacting pole and long-lived IS maintenance.

Thalamic control of neocortical area formation in mice.

The mammalian neocortex undergoes dramatic transformation during development, from a seemingly homogenous sheet of neuroepithelial cells into a complex structure that is tangentially divided into discrete areas. This process is thought to be controlled by a combination of intrinsic patterning mechanisms within the cortex and afferent axonal projections from the thalamus. However, roles of thalamic afferents in the formation of areas are still poorly understood. In this study, we show that genetically increasing or decreasing the size of the lateral geniculate nucleus of the mouse thalamus resulted in a corresponding change in the size of the primary visual area. Furthermore, elimination of most thalamocortical projections from the outset of their development resulted in altered areal gene expression patterns, particularly in the primary visual and somatosensory areas, where they lost sharp boundaries with adjacent areas. Together, these results demonstrate the critical roles of thalamic afferents in the establishment of neocortical areas.

What is the work environment of orthopaedic surgeons in China?

BACKGROUND: Physicians in China face heavy demands from patients and the government for services but deal with the threat of unpredictable legal and physical conflicts with patients, some ending with the death of doctors. More than 40 doctors and nurses have been killed by patients since 2001. QUESTIONS/PURPOSES: We sought to evaluate (1) the demographics of orthopaedic practice, (2) duty periods, (3) practice support, and (4) job satisfaction among orthopaedic surgeons in China. METHODS: Questionnaires were posted online at www.OrthoChina.org for download by orthopaedic surgeons in 2006 to 2007, and sent to those attending meetings in 2013. In 2013, a total of 1350 surgeons were invited and 456 participated in the survey at meetings. In 2007, during the period of the survey, 9759 individuals were qualified orthopaedic surgeons, and 334 participated in the survey at www.OrthoChina.org . RESULTS: Ninety-one percent of orthopaedic surgeons work in public and 9% in private hospitals. Ninety-four percent work more than 8 hours per day 6 to 7 days a week. Twenty-five percent work more than 12 hours per day 6 to 7 days a week without extra compensation. The majority of orthopaedic surgeons must work on national statutory holidays. Almost none received contractually mandated income for weekends and national holidays. Approximately 80% of participants reported an attack of some kind, including physical or psychologic harm. With respect to job satisfaction, 73% stated they would not choose to be a physician again and 86% reported that they do not want their children to become a physician. CONCLUSIONS: China's rapid economic growth and resulting demands for modern health care have resulted in heavy pressure on orthopaedic surgeons, financially and personally. Chinese orthopaedic surgeons are overworked, suffer lack of respect, and face the possibility of serious personal harm. As a consequence, they are demoralized and unsatisfied. Significant reforms are needed.

Efficacy of meropenem extended infusion vs intermittent bolus monotherapy and in combination with colistin against Pseudomonas aeruginosa biofilm.

INTRODUCTION: Device-related infections are difficult to treat due to biofilms. In this setting, optimizing antibiotic efficacy is difficult as most pharmacokinetic/pharmacdynamic (PK/PD) studies have been performed on planktonic cells, and therapies are limited when multi-drug-resistant bacteria are involved. This study aimed to analyse the PK/PD indices of meropenem predicting anti-biofilm efficacy against meropenem-susceptible and meropenem-resistant strains of Pseudomonas aeruginosa. MATERIALS AND METHODS: Pharmacodynamics of meropenem dosages mimicking those of clinical practice (intermittent bolus of 2 g every 8 h; extended infusion of 2 g over 4 h every 8 h), with and without colistin, were evaluated with the CDC Biofilm Reactor in-vitro model for susceptible (PAO1) and extensively-drug-resistant (XDR-HUB3) P. aeruginosa. Efficacy was correlated with the PK/PD indices for meropenem. RESULTS: For PAO1, both meropenem regimens were bactericidal, with higher killing for extended infusion [∆log10 colony-forming units (CFU)/mL 54-0h=-4.66±0.93 for extended infusion vs ∆log10 CFU/mL 54-0h=-3.4±0.41 for intermittent bolus; P<0.001]. For XDR-HUB3, the intermittent bolus regimen was non-active, but extended infusion showed bactericidal effect (∆log10 CFU/mL 54-0h=-3.65±0.29; P<0.001). Time above minimum inhibitory concentration (f%T>MIC) had the best correlation with efficacy for both strains. The addition of colistin always improved meropenem activity, and resistant strains did not emerge. CONCLUSION: f%T>MIC was the PK/PD index that best correlated with the anti-biofilm efficacy of meropenem; it was better optimized when using the extended infusion regimen, allowing recovery of bactericidal activity in monotherapy, including activity against meropenem-resistant P. aeruginosa. Combining meropenem by extended infusion with colistin offered the most effective therapy for both strains. Optimizing meropenem dosing by extended infusion should be encouraged when treating biofilm-related infections.

Transcriptomics of angiotensin II-induced long noncoding and coding RNAs in endothelial cells.

OBJECTIVE: Angiotensin II (Ang II)-induced endothelial dysfunction plays an important role in the pathogenesis of cardiovascular diseases such as systemic hypertension, cardiac hypertrophy and atherosclerosis. Recently, long noncoding RNAs (lncRNAs) have been shown to play an essential role in the pathobiology of cardiovascular diseases; however, the effect of Ang II on lncRNAs and coding RNAs expression in endothelial cells has not been evaluated. Accordingly, we sought to evaluate the expression profiles of lncRNAs and coding RNAs in endothelial cells following treatment with Ang II. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured and treated with Ang II (10-6 mol/l) for 24 h. The cells were then profiled for the expression of lncRNAs and mRNAs using the Arraystar Human lncRNA Expression Microarray V3.0. RESULTS: In HUVECs following Ang II treatment, from a total of 30 584 lncRNA targets screened, 25 targets were significantly upregulated, while 69 were downregulated. In the same HUVECs samples, from 26 106 mRNA targets screened, 28 targets were significantly upregulated and 67 were downregulated. Of the differentially expressed lncRNAs, RP11-354P11.2 and RP11-360F5.1 were the most upregulated (11-fold) and downregulated (three-fold) lncRNAs, respectively. Assigning the differentially regulated genes into functional groups using bioinformatics reveals numerous genes involved in the nucleotide excision repair and ECM-receptor interaction. CONCLUSION: This is the first study to profile the Ang II-induced differentially expressed lncRNAs and mRNAs in human endothelial cells. Our results reveal novel targets and substantially extend the list of potential candidate genes involved in Ang II-induced endothelial dysfunction and cardiovascular diseases.

Oral antibiotics perturbation on gut microbiota after prostate biopsy.

Introduction: The use of antibiotics may induce the changes in gut microbiota. Previous studies have shown conflicting results on whether the changed gut microbiota by antibiotics can be recovered. Our study aims to investigate whether the gut microbiota could be recovered after a single dose of oral co-amoxiclav before transrectal ultrasound-guided transperineal prostate biopsy (TPPBx) in 5 weeks' time. Methods: Fifteen patients with elevated serum prostate-specific antigen (PSA) were recruited to provide pre-antibiotic and post-antibiotic fecal samples. The V4 region of 16S rRNA was sequenced. Analysis was performed by QIIME2. Alpha- and beta-diversities were analyzed, as well as the differential enrichment by Linear discriminant analysis Effect Size (LEfSe) analysis. Results: Both the alpha- and beta-diversities of the pre- and post-antibiotic fecal samples were significantly different. Genera that are associated with alleviation of inflammation were enriched in the pre-antibiotic fecal samples, while the inflammation-associated genera were more enriched in the post-antibiotic fecal samples. Conclusion: A single dose of oral co-amoxiclav before TPPBx could have led to a change of gut microbiota that cannot be recovered in 5 weeks' time. Microbiome studies on prostate cancer patients should be cautioned on the use of post-prostate biopsy fecal sampling. Further studies should be conducted for the impact on gut microbiome for TPPBx alone.

A synthetic lipopeptide targeting top-priority multidrug-resistant Gram-negative pathogens.

The emergence of multidrug-resistant (MDR) Gram-negative pathogens is an urgent global medical challenge. The old polymyxin lipopeptide antibiotics (polymyxin B and colistin) are often the only therapeutic option due to resistance to all other classes of antibiotics and the lean antibiotic drug development pipeline. However, polymyxin B and colistin suffer from major issues in safety (dose-limiting nephrotoxicity, acute toxicity), pharmacokinetics (poor exposure in the lungs) and efficacy (negligible activity against pulmonary infections) that have severely limited their clinical utility. Here we employ chemical biology to systematically optimize multiple non-conserved positions in the polymyxin scaffold, and successfully disconnect the therapeutic efficacy from the toxicity to develop a new synthetic lipopeptide, structurally and pharmacologically distinct from polymyxin B and colistin. This resulted in the clinical candidate F365 (QPX9003) with superior safety and efficacy against lung infections caused by top-priority MDR pathogens Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae.

Development and characterization of a mouse floxed Bmp2 osteoblast cell line that retains osteoblast genotype and phenotype.

Bone morphogenetic protein 2 (Bmp2) is essential for osteoblast differentiation and osteogenesis. Generation of floxed Bmp2 osteoblast cell lines is a valuable tool for studying the effects of Bmp2 on osteoblast differentiation and its signaling pathways during skeletal metabolism. Due to relatively limited sources of primary osteoblasts, we have developed cell lines that serve as good surrogate models for the study of osteoblast cell differentiation and bone mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 osteoblast cell lines. Primary mouse floxed Bmp2 osteoblasts were transfected with pSV3-neo and clonally selected. These transfected cells were verified by PCR and immunohistochemistry. To determine the genotype and phenotype of the immortalized cells, cell morphology, proliferation, differentiation and mineralization were analyzed. Also, expression of osteoblast-related gene markers including Runx2, Osx, ATF4, Dlx3, bone sialoprotein, dentin matrix protein 1, osteonectin, osteocalcin and osteopontin were examined by quantitative RT-PCR and immunohistochemistry. These results showed that immortalized floxed Bmp2 osteoblasts had a higher proliferation rate but preserved their genotypic and phenotypic characteristics similar to the primary cells. Thus, we, for the first time, describe the development of immortalized mouse floxed Bmp2 osteoblast cell lines and present a useful model to study osteoblast biology mediated by BMP2 and its downstream signaling transduction pathways.

Abnormalities in the enamel in bmp2-deficient mice.

Tooth development is regulated by epithelial-mesenchymal interactions and their reciprocal molecular signaling. Bone morphogenetic protein 2 (Bmp2) is essential for tooth formation. However, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in the regulation of postnatal enamel formation was investigated via the conditional ablation of Bmp2 in enamel using the (Osx-Cre) mouse. Bmp2 gene ablation was confirmed by PCR analysis in Osx-Cre, Bmp2(flox/flox) mice. Bmp2-null mice displayed a severe and profound tooth phenotype with asymmetric and open forked incisors. Microradiographs revealed broken incisor tips and dental pulp chamber exposure. The enamel layer of incisors and molars was thin with hypomineralization. Scanning electron microscopy analysis showed that the enamel surface was rough with chipping and the enamel lacked a typical prismatic architecture. These results demonstrate that Bmp2 is essential for enamel formation.

A cross-sectional study on gut microbiota in prostate cancer patients with prostatectomy or androgen deprivation therapy.

BACKGROUND: Androgen deprivation therapy (ADT), either by medical or surgical castration, is the backbone for standard treatment of locally advanced or metastatic prostate cancer, yet it is also associated with various metabolic and cardiovascular complications. Recent evidence have shown that obesity, insulin resistance, or metabolic disturbances can be associated with changes in the gut microbiome, while animal studies also show that castration is associated with changes in the gut microbiome. This study aims to investigate whether the fecal microbiota in prostate cancer patients who had undergone prostatectomy or ADT are different, and explore changes in phylogeny and pathways that may lead to side effects from ADT. METHODS: A total of 86 prostate cancer patients (56 patients on ADT and 30 patients with prostatectomy) were recruited. The fecal microbiota was analyzed by the 16S rRNA gene for alpha- and beta-diversities by QIIME2, as well as the predicted metabolic pathways by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2. RESULTS: The alpha-diversity was significantly lower in the ADT group. The beta-diversity was significantly different between the groups, in which Ruminococcus gnavus and Bacteroides spp were having higher relative abundance in the ADT group, whereas Lachnospira and Roseburia were reduced. The Firmicutes-to-Bacteroidetes ratio is noted to be lower in the ADT group as well. The functional pathway prediction showed that the biosynthesis of lipopolysaccharide (endotoxin) and propanoate was enriched in the ADT as well as the energy cycle pathways. This study is limited by the cross-sectional design and the clinical heterogeneity. CONCLUSIONS: There is a significant difference in gut microbiome between prostate cancer patients on ADT and prostatectomy. We theorize that this difference may contribute to the development of metabolic complications from ADT. Further longitudinal studies are awaited.

TCF-1 regulates HIV-specific CD8+ T cell expansion capacity.

Although many HIV cure strategies seek to expand HIV-specific CD8+ T cells to control the virus, all are likely to fail if cellular exhaustion is not prevented. A loss in stem-like memory properties (i.e., the ability to proliferate and generate secondary effector cells) is a key feature of exhaustion; little is known, however, about how these properties are regulated in human virus-specific CD8+ T cells. We found that virus-specific CD8+ T cells from humans and nonhuman primates naturally controlling HIV/SIV infection express more of the transcription factor TCF-1 than noncontrollers. HIV-specific CD8+ T cell TCF-1 expression correlated with memory marker expression and expansion capacity and declined with antigenic stimulation. CRISPR-Cas9 editing of TCF-1 in human primary T cells demonstrated a direct role in regulating expansion capacity. Collectively, these data suggest that TCF-1 contributes to the regulation of the stem-like memory property of secondary expansion capacity of HIV-specific CD8+ T cells, and they provide a rationale for exploring the enhancement of this pathway in T cell-based therapeutic strategies for HIV.

Immortalized mouse floxed Bmp2 dental papilla mesenchymal cell lines preserve odontoblastic phenotype and respond to BMP2.

Bone morphogenetic protein 2 (Bmp2) is essential for odontogensis and dentin mineralization. Generation of floxed Bmp2 dental mesenchymal cell lines is a valuable application for studying the effects of Bmp2 on dental mesenchymal cell differentiation and its signaling pathways during dentinogenesis. Limitation of the primary culture of dental mesenchymal cells has led to the development of cell lines that serve as good surrogate models for the study of dental mesenchymal cell differentiation into odontoblasts and mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 dental papilla mesenchymal cell lines, which were isolated from 1st mouse mandibular molars at postnatal day 1 and immortalized with pSV40 and clonally selected. These transfected cell lines were characterized by RT-PCR, immunohistochemistry, and analyzed for alkaline phosphatase activity and mineralization nodule formation. One of these immortalized cell lines, iBmp2-dp, displayed a higher proliferation rate, but retained the genotypic and phenotypic characteristics similar to primary cells as determined by expression of tooth-specific markers as well as demonstrated the ability to differentiate and form mineralized nodules. In addition, iBmp2-dp cells were inducible and responded to BMP2 stimulation. Thus, we for the first time described the establishment of an immortalized mouse floxed Bmp2 dental papilla mesenchyma cell line that might be used for studying the mechanisms of dental cell differentiation and dentin mineralization mediated by Bmp2 and other growth factor signaling pathways.

Chloroquine Restores Ganglioside Homeostasis and Improves Pathological and Behavioral Outcomes Post-stroke in the Rat.

Perturbations of ganglioside homeostasis have been observed following stroke whereby toxic simple gangliosides GM2 and GM3 accumulate, while protective complex species GM1 and GD1 are reduced. Thus, there is a need for therapeutic interventions which can prevent ganglioside dysregulation after stroke. A pharmacological intervention using chloroquine was selected for its transient lysosomotropic properties which disrupt the activity of catabolic ganglioside enzymes. Chloroquine was administered both in vitro (0.1 µM), to primary cortical neurons exposed to GM3 toxicity, and in vivo (45 mg/kg i.p.), to 3-month-old male Wistar rats that underwent a severe stroke injury. Chloroquine was administered for seven consecutive days beginning 3 days prior to the stroke injury. Gangliosides were examined using MALDI imaging mass spectrometry at 3 and 21 days after the injury, and motor deficits were examined using the ladder task. Chloroquine treatment prevented ganglioside dysregulation 3 days post-stroke and partially prevented complex ganglioside depletion 21 days post-stroke. Exogenous GM3 was found to be toxic to primary cortical neurons which was protected by chloroquine treatment. Motor deficits were prevented in the forelimbs of stroke-injured rats with chloroquine treatment and was associated with decreased inflammation, neurodegeneration, and an increase in cell survival at the site of injury. Chloroquine administration prevents ganglioside dysregulation acutely, protects against GM3 toxicity in neurons, and is associated with long-term functional and pathological improvements after stroke in the rat. Therefore, targeting lipid dysregulation using lysosomotropic agents such as chloroquine may represent a novel therapeutic avenue for stroke injuries.

The Sixth Transmembrane Segment Is a Major Gating Component of the TMEM16A Calcium-Activated Chloride Channel.

Calcium-activated chloride channels (CaCCs) formed by TMEM16A or TMEM16B are broadly expressed in the nervous system, smooth muscles, exocrine glands, and other tissues. With two calcium-binding sites and a pore within each monomer, the dimeric CaCC exhibits voltage-dependent calcium sensitivity. Channel activity also depends on the identity of permeant anions. To understand how CaCC regulates neuronal signaling and how CaCC is, in turn, modulated by neuronal activity, we examined the molecular basis of CaCC gating. Here, we report that voltage modulation of TMEM16A-CaCC involves voltage-dependent occupancy of calcium- and anion-binding site(s) within the membrane electric field as well as a voltage-dependent conformational change intrinsic to the channel protein. These gating modalities all critically depend on the sixth transmembrane segment.

Ultraviolet Radiation Inhibits Mammary Carcinogenesis in an ER-Negative Murine Model by a Mechanism Independent of Vitamin D3.

Three decades ago, the Garlands postulated that vitamin D3 produced in the skin by ultraviolet radiation (UVR)-induced conversion of 7-dehydrocholesterol to pre-D3 has anticancer effects, thus triggering more than 9,500 publications on D3 and cancer. Here, we report that UVR treatment of transgenic mice of the well-established C3(1)/SV40 Tag mammary cancer model significantly inhibits both autochthonous carcinogenesis and allograft tumor growth, but in contrast neither dietary nor topical D3 influences mammary carcinogenesis in this specific mouse model. Furthermore, UVR's inhibitory effects occur irrespective of whether or not the treatment increases circulating D3 in the mice. The inhibitory effect of UVR on autochthonous tumors occurs at or before the stage of ductal carcinoma in situ. Our studies indicate clearly that UVR can exert D3-independent anticancer effects in C3(1)/SV40 Tag mice. Therefore, supplemental D3 may not mimic all possible beneficial effects of UVR, and uncovering non-D3-mediated mechanisms of UVR tumor inhibition may lead to novel strategies for cancer prevention. Cancer Prev Res; 11(7); 383-92. ©2018 AACR.