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Background: Different pulp capping materials have different origins and compositions, require different preparations, and may vary in their bioactive properties., Aim: The purpose of this study was to evaluate the antibacterial activity, biocompatibility, and mineralization-inducing potential of calcium silicate-based pulp capping materials., Design: Six contemporary calcium silicate-based cements, ProRoot MTA, MTA Angelus, Biodentine, EndoSequence, NeoMTA 2, and NeoPutty, were evaluated. The antibacterial effects of these materials against Streptococcus mutans UA159 and Enterococcus faecalis ATCC 29212 were determined by the agar diffusion assay and the direct culture test. The biocompatibility and mineralization-inducing potential of these materials in preodontoblastic 17IIA11 cells were evaluated by the MTT assay and by Alizarin Red S staining, respectively., Results and Conclusion: In agar diffusion test, only Biodentine showed distinct antibacterial effects against S. mutans. All the tested materials, however, showed antibacterial effects against S. mutans and E. faecalis in the direct culture test, with Biodentine showing the strongest growth inhibition against both S. mutans and E. faecalis. All the tested materials showed acceptable biocompatibility and mineralization-supporting potential in our experimental conditions. In summary, ProRoot MTA, MTA Angelus, Biodentine, EndoSequence, NeoMTA 2, and NeoPutty demonstrated acceptable in vitro antimicrobial, biocompatible, and mineralization-supporting properties., (© 2024 The Authors. International Journal of Paediatric Dentistry published by BSPD, IAPD and John Wiley & Sons Ltd.)

Purpose: To evaluate the accuracy and consistency of chatbots in answering questions related to special needs dentistry. Methods: Nine publicly accessible chatbots, including Google Bard, ChatGPT 4, ChatGPT 3.5, Llama, Sage, Claude 2 100k, Claude-instant, Claude-instant-100k, and Google PaLM, were evaluated on their ability to answer a set of 25 true/false questions related to special needs dentistry and 15 questions for syndrome diagnosis based on their oral manifestations. Each chatbot was asked independently three times at a three-week interval from November to December 2023, and the responses were evaluated by dental professionals. The Wilcoxon exact test was used to compare accuracy rates among the chatbots while Cronbach's alpha was utilized to measure the consistency of the chatbots' responses. Results: Chatbots had an average accuracy of 55??4 percent in answering all questions, 37±6 percent in diagnosis, and 67±8 percent in answering true/false questions. No significant difference (P>0.05) in the accuracy proportion was detected between any pairwise chatbot comparison. All chatbots demonstrated acceptable reliability (Cronbach's alpha greater than 0.7), with Claude instant having the highest reliability of 0.93. Conclusion: Chatbots exhibit acceptable consistency in responding to questions related to special needs dentistry and better accuracy in responding to true/false questions than diagnostic questions. The clinical relevance is not fully established at this stage, but it may become a useful tool in the future.

Cocaine use disorder (CUD) is highly comorbid with HIV infection and worsens HIV outcomes. Preclinical research on the outcomes of HIV infection may yield crucial information on neurobehavioral changes resulting from chronic drug exposure in people living with HIV (PLWH). Repeated exposure to cocaine alters behavioral responses to cocaine. This includes development of cocaine locomotor sensitization - or increased locomotor responses to the same doses of cocaine - which depends on nucleus accumbens (NAc) neural plasticity. NAc astrocytes are key regulators of neural activity and plasticity, and their function can be impaired by cocaine exposure and HIV infection, thus implicating them as potential regulators of HIV-induced changes in behavioral response to cocaine. To characterize the effects of HIV infection on cocaine locomotor sensitization, we employed the EcoHIV mouse model to assess changes in locomotor responses after repeated cocaine (10mg/kg) exposure and challenge. EcoHIV infection potentiated expression of cocaine sensitization. We also identified EcoHIV-induced increases in expression of the astrocytic nuclear marker Sox9 selectively in the NAc core. To investigate whether modulation of NAc astrocytes could reverse EcoHIV-induced deficits, we employed a chemogenetic approach. We found that chemogenetic activation of NAc astrocyte Gq signaling attenuated EcoHIV-enhanced cocaine sensitization. We propose that HIV infection contributes to cocaine behavioral sensitization and induces adaptations in NAc astrocytes, while promoting NAc astrocytic Gq-signaling can recover EcoHIV-induced behavioral changes. These findings identify potential cellular substrates of disordered cocaine-driven behavior in the context of HIV infection and point toward strategies to reduce cocaine-related behavior in PLWH.

HIV-associated neurocognitive disorders (HAND) persist under antiretroviral therapy as a complex pathology that has been difficult to study in cellular and animal models. Therefore, we generated an ex vivo human brain slice model of HIV-1 infection from surgically resected adult brain tissue. Brain slice cultures processed for flow cytometry showed >90% viability of dissociated cells within the first three weeks in vitro, with parallel detection of astrocyte, myeloid, and neuronal populations. Neurons within brain slices showed stable dendritic spine density and mature spine morphologies in the first weeks in culture, and they generated detectable activity in multi-electrode arrays. We infected cultured brain slices using patient-matched CD4+ T-cells or monocyte-derived macrophages (MDMs) that were exposed to a GFP-expressing R5-tropic HIV-1 in vitro. Infected slice cultures expressed viral RNA and developed a spreading infection up to 9 days post-infection, which were significantly decreased by antiretrovirals. We also detected infected myeloid cells and astrocytes within slices and observed minimal effect on cellular viability over time. Overall, this human-centered model offers a promising resource to study the cellular mechanisms contributing to HAND (including antiretroviral toxicity, substance use, and aging), infection of resident brain cells, and new neuroprotective therapeutics.

HIV infection is an ongoing global health issue, despite increased access to antiretroviral therapy (ART). People living with HIV (PLWH) who are virally suppressed through ART still experience negative health outcomes, including neurocognitive impairment. It is increasingly evident that ART may act independently or in combination with HIV infection to alter the immune state, though this is difficult to disentangle in the clinical population. Thus, these experiments used multiplexed chemokine/cytokine arrays to assess peripheral (plasma) and brain (nucleus accumbens; NAc) expression of immune targets in the presence and absence of ART treatment in the EcoHIV mouse model. The findings identify the effects of EcoHIV infection and of treatment with bictegravir (B), emtricitabine (F), and tenofovir alafenamide (TAF) on the expression of numerous immune targets. In the NAc, this included EcoHIV-induced increases in IL-1α and IL-13 expression and B/F/TAF-induced reductions in KC/CXCL1. In the periphery, EcoHIV suppressed IL-6 and LIF expression, while B/F/TAF reduced IL-12p40 expression. In the absence of ART, IBA-1 expression was negatively correlated with CX3CL1 expression in the NAc of EcoHIV-infected mice. These findings identify distinct effects of ART and EcoHIV infection on peripheral and central immune factors and emphasize the need to consider ART effects on neural and immune outcomes.

Neurotoxicity is caused by damage to the brain tissue by neurotoxic agents present in the environment and artificial substances produced by human beings. Acrylamide (ACR) is one such chemical substance that causes neurotoxicity, affecting the brain cells. This neurotoxicity causes damage to the sensory and metabolic functions. The current research investigates the favourable effect of probiotic EcN (Escherichia coli Nissle 1917) on ACR-induced neurotoxicity in zebrafish. The protective role of EcN against ACR induced toxicity was assessed based on behaviour, biochemical, and gene expression analysis. Initially, the colonisation period of EcN in the zebrafish gut was determined and EcN was given orally to the zebrafish only once prior to the ACR treatment. Very interestingly, this dosage was able to ameliorate the adverse effects of ACR significantly in the brain cells. Quantification of oxidative stress and neuronal cell death clearly vindicate the efficiency of probiotic EcN in reversing the damages caused by ACR. EcN is being explored largely in recent days for its therapeutic applications. This study strongly supports the view that EcN can be developed as a supplement to the patients diagnosed with neuronal cell toxicity.

RNA-binding motif protein 10 (RBM10) is a frequently mutated tumor suppressor in lung adenocarcinoma (LUAD). Yet, it remains unknown whether cancer-derived mutant RBM10 compromises its tumor suppression function and, if so, the molecular insight of the underlying mechanisms. Here, we show that wild-type RBM10 suppresses lung cancer cell growth and proliferation by inactivating c-Myc that is essential for cancer cell survival. RBM10 directly binds to c-Myc and promotes c-Myc's ubiquitin-dependent degradation, while RBM10 knockdown leads to the induction of c-Myc level and activity. This negative action on c-Myc is further boosted by ribosomal proteins (RPs) uL18 (RPL5) and uL5 (RPL11) via their direct binding to RBM10. Cancer-derived mutant RBM10-I316F fails to bind to uL18 and uL5 and to inactivate c-Myc, thus incapable of suppressing tumorigenesis. Our findings uncover RBM10 as a pivotal c-Myc repressor by cooperating with uL18 and uL5 in lung cancer cells, as its failure to do so upon mutation favors tumorigenesis., Competing Interests: Competing interests statement:The authors declare no competing interest.

After treatment and surgery, patient tumors can initially respond followed by a rapid relapse, or respond well and seemingly be cured, but then recur years or decades later. The state of surviving cancer cells during the long, undetected period is termed dormancy. By definition, the dormant tumor cells do not proliferate to create a mass that is detectable or symptomatic, but also never die. An intrinsic state and microenvironment that are inhospitable to the tumor would bias toward cell death and complete eradication, while conditions that favor the tumor would enable growth and relapse. In neither case would clinical dormancy be observed. Normal cells and tumor cells can enter a state of cellular senescence after stress such as that caused by cancer therapy. Senescence is characterized by a stable cell cycle arrest mediated by chromatin modifications that cause gene expression changes and a secretory phenotype involving many cytokines and chemokines. Senescent cell phenotypes have been shown to be both tumor promoting and tumor suppressive. The balance of these opposing forces presents an attractive model to explain tumor dormancy: phenotypes of stable arrest and immune suppression could promote survival, while reversible epigenetic programs combined with cytokines and growth factors that promote angiogenesis, survival, and proliferation could initiate the emergence from dormancy. In this review, we examine the phenotypes that have been characterized in different normal and cancer cells made senescent by various stresses and how these might explain the characteristics of tumor dormancy., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In the early avian embryo, the developing heart forms when bilateral fields of cardiac progenitor cells, which reside in the lateral plate mesoderm, move toward the embryonic midline, and fuse above the anterior intestinal portal (AIP) to form a straight, muscle-wrapped tube. During this process, the precardiac mesoderm remains in close contact with the underlying endoderm. Previous work has shown that the endoderm around the AIP actively contracts to pull the cardiac progenitors toward the midline. The morphogenetic deformations associated with this endodermal convergence, however, remain unclear, as do the signaling pathways that might regulate this process. Here, we fluorescently labeled populations of endodermal cells in early chicken embryos and tracked their motion during heart tube formation to compute time-varying strains along the anterior endoderm. We then determined how the computed endodermal strain distributions are affected by the pharmacological inhibition of either myosin II or fibroblast growth factor (FGF) signaling. Our data indicate that a mediolateral gradient in endodermal shortening is present around the AIP, as well as substantial convergence and extension movements both anterior and lateral to the AIP. These active endodermal deformations are disrupted if either actomyosin contractility or FGF signaling are inhibited pharmacologically. Taken together, these results demonstrate how active deformations along the anterior endoderm contribute to heart tube formation within the developing embryo., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Poor mental health of healthcare students is a cause for concern in many universities. Though previous research has identified mental health shame and self-compassion as critical in this student group, how these variables differ across different healthcare disciplines remains to be evaluated. Healthcare students ( n  = 344; counselling, occupational therapy, social work and nursing) completed measures regarding these variables. MANOVA and regression analyses were performed. (1) Counselling and nursing students were more depressed than occupational therapy students; (2) nursing students were more anxious than occupational therapy and social work students; (3) occupational therapy students had more positive attitudes towards mental health than the others; and (4) nursing students worried about their own reputation associated with their family more than counselling students. Self-compassion was the strongest predictor of mental health in all groups; however, the effect sizes varied: largest in nursing and smallest in social work students. Findings will help inform effective interventions for students in each healthcare discipline., Competing Interests: Conflict of InterestThe authors declare no competing interests., (© The Author(s) 2023.)

Breast cancer cells must avoid intrinsic and extrinsic cell death to relapse following chemotherapy. Entering senescence enables survival from mitotic catastrophe, apoptosis and nutrient deprivation, but mechanisms of immune evasion are poorly understood. Here we show that breast tumors surviving chemotherapy activate complex programs of immune modulation. Characterization of residual disease revealed distinct tumor cell populations. The first population was characterized by interferon response genes, typified by Cd274, whose expression required chemotherapy to enhance chromatin accessibility, enabling recruitment of IRF1 transcription factor. A second population was characterized by p53 signaling, typified by CD80 expression. Treating mammary tumors with chemotherapy followed by targeting the PD-L1 and/or CD80 axes resulted in marked accumulation of T cells and improved response; however, even combination strategies failed to fully eradicate tumors in the majority of cases. Our findings reveal the challenge of eliminating residual disease populated by senescent cells expressing redundant immune inhibitory pathways and highlight the need for rational immune targeting strategies., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Cancer cells survive chemotherapy and cause lethal relapse by entering a senescent state that facilitates expression of many phagocytosis/macrophage-related genes that engender a novel cannibalism phenotype. We used biosensors and live-cell imaging to reveal the basic steps and mechanisms of engulfment by senescent human and mouse tumor cells. We show filamentous actin in predator cells was localized to the prey cell throughout the process of engulfment. Biosensors to various phosphoinositide (PI) species revealed increased concentration and distinct localization of predator PI(4) P and PI(4,5)P2 at the prey cell during early stages of engulfment, followed by a transient burst of PI(3) P before and following internalization. PIK3C2B, the kinase responsible for generating PI(3)P, was required for complete engulfment. Inhibition or knockdown of Clathrin, known to associate with PIK3C2B and PI(4,5)P2, severely impaired engulfment. In sum, our data reveal the most fundamental cellular processes of senescent cell engulfment, including the precise localizations and dynamics of actin and PI species throughout the entire process., Competing Interests: The authors have declared that no competing interests exist.

Objectives: To improve clinical decision support (CDS) by allowing users to provide real-time feedback when they interact with CDS tools and by creating processes for responding to and acting on this feedback., Methods: Two organizations implemented similar real-time feedback tools and processes in their electronic health record and gathered data over a 30-month period. At both sites, users could provide feedback by using Likert feedback links embedded in all end-user facing alerts, with results stored outside the electronic health record, and provide feedback as a comment when they overrode an alert. Both systems are monitored daily by clinical informatics teams., Results: The two sites received 2,639 Likert feedback comments and 623,270 override comments over a 30-month period. Through four case studies, we describe our use of end-user feedback to rapidly respond to build errors, as well as identifying inaccurate knowledge management, user-interface issues, and unique workflows., Conclusion: Feedback on CDS tools can be solicited in multiple ways, and it contains valuable and actionable suggestions to improve CDS alerts. Additionally, end users appreciate knowing their feedback is being received and may also make other suggestions to improve the electronic health record. Incorporation of end-user feedback into CDS monitoring, evaluation, and remediation is a way to improve CDS., Competing Interests: None declared., (Thieme. All rights reserved.)

Previous studies have shown cognitive task performance to be affected by tinnitus severity, but also that the literature is conflicted. This study sought to identify neuroticism as a possible confound, since severe tinnitus distress is associated with higher levels of neuroticism. A total of 78 participants (39 with and 39 without tinnitus) undertook two cognitive tasks. It was found that when undertaking a Stroop paradigm, controlling for neuroticism rendered previously significant results not significant. It was also found that neuroticism was not a significant covariate for a change blindness task. Gender, age, anxiety, and depression were all controlled for, and future implications for the literature discussed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Edwards, Jackson and Evans.)

Purpose: The purpose of this study was to compare the chemical and physical properties of eight contemporary direct pulp capping materials. Methods: Materials included: calcium hydroxide-based (Dycal ® , Lime-Lite TM , and Ultra-Blend TM plus), bioceramic-based (MTA Angelus ® , Biodentine ® , TheraCal LC ® , Ceramir ® Protect LC), and resin ionomer-based materials (Bio-Cap ® ). Calcium release and pH changes were measured after one, seven, 28, and 90 days in 36.8 degrees Celsius of deionized water. Water sorption and solubility were calculated with weight loss after 90 days. Results: Biodentine ® (965.5 ppm) and MTA Angelus ® (921.2 ppm) released significantly more cumulative calcium ions (P<0.05), followed by Ceramir ® Protect LC (450.8 ppm) and Dycal ® (268.1 ppm); Lime-Lite TM had the least amount of calcium ions. After 90 days, Biodentine ® and MTA Angelus ® showed significantly higher pH (P<0.05), while Bio-Cap ® and Lime-Lite TM had limited effects in raising the pH to alkaline. Dycal ® presented the most weight loss (26 percent, P<0.05) after 90 days. Conclusions: Biodentine ® and MTA Angelus ® demonstrated more favorable in vitro characteristics for clinical pulp capping purposes, while Lime-Lite TM had no effect in releasing calcium ions and limited influence in raising pH value.

Objective: We describe the Clickbusters initiative implemented at Vanderbilt University Medical Center (VUMC), which was designed to improve safety and quality and reduce burnout through the optimization of clinical decision support (CDS) alerts., Materials and Methods: We developed a 10-step Clickbusting process and implemented a program that included a curriculum, CDS alert inventory, oversight process, and gamification. We carried out two 3-month rounds of the Clickbusters program at VUMC. We completed descriptive analyses of the changes made to alerts during the process, and of alert firing rates before and after the program., Results: Prior to Clickbusters, VUMC had 419 CDS alerts in production, with 488 425 firings (42 982 interruptive) each week. After 2 rounds, the Clickbusters program resulted in detailed, comprehensive reviews of 84 CDS alerts and reduced the number of weekly alert firings by more than 70 000 (15.43%). In addition to the direct improvements in CDS, the initiative also increased user engagement and involvement in CDS., Conclusions: At VUMC, the Clickbusters program was successful in optimizing CDS alerts by reducing alert firings and resulting clicks. The program also involved more users in the process of evaluating and improving CDS and helped build a culture of continuous evaluation and improvement of clinical content in the electronic health record., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Medical Informatics Association.)

Glioblastomas (GBMs) preferentially generate acetyl-CoA from acetate as a fuel source to promote tumor growth. O-GlcNAcylation has been shown to be elevated by increasing O-GlcNAc transferase (OGT) in many cancers and reduced O-GlcNAcylation can block cancer growth. Here, we identify a novel mechanism whereby OGT regulates acetate-dependent acetyl-CoA and lipid production by regulating phosphorylation of acetyl-CoA synthetase 2 (ACSS2) by cyclin-dependent kinase 5 (CDK5). OGT is required and sufficient for GBM cell growth and regulates acetate conversion to acetyl-CoA and lipids. Elevating O-GlcNAcylation in GBM cells increases phosphorylation of ACSS2 on Ser-267 in a CDK5-dependent manner. Importantly, we show that ACSS2 Ser-267 phosphorylation regulates its stability by reducing polyubiquitination and degradation. ACSS2 Ser-267 is critical for OGT-mediated GBM growth as overexpression of ACSS2 Ser-267 phospho-mimetic rescues growth in vitro and in vivo. Importantly, we show that pharmacologically targeting OGT and CDK5 reduces GBM growth ex vivo. Thus, the OGT/CDK5/ACSS2 pathway may be a way to target altered metabolic dependencies in brain tumors., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

In order to meet the energetic demands of cell-to-cell signaling, increases in local neuronal signaling are matched by a coordinated increase in local blood flow, termed neurovascular coupling. Multiple different signals from neurons, astrocytes, and pericytes contribute to this control of blood flow. Previously, several groups demonstrated that inhibition/ablation of glutamate transporters attenuates the neurovascular response. However, it was not determined if glutamate transporter activation was sufficient to increase blood flow. Here, we used multiphoton imaging to monitor the diameter of fluorescently labeled cortical arterioles in anesthetized C57/B6J mice. We delivered vehicle, glutamate transporter substrates, or a combination of a glutamate transporter substrate with various pharmacologic agents via a glass micropipette while simultaneously visualizing changes in arteriole diameter. We developed a novel image analysis method to automate the measurement of arteriole diameter in these time-lapse analyses. Using this workflow, we first conducted pilot experiments in which we focally applied L-glutamate, D-aspartate, or L- threo -hydroxyaspartate (L-THA) and measured arteriole responses as proof of concept. We subsequently applied the selective glutamate transport substrate L-THA (applied at concentrations that do not activate glutamate receptors). We found that L-THA evoked a significantly larger dilation than that observed with focal saline application. This response was blocked by co-application of the potent glutamate transport inhibitor, L-(2S,3S)-3-[3-[4-(trifluoromethyl)-benzoylamino]benzyloxy]-aspartate (TFB-TBOA). Conversely, we were unable to demonstrate a reduction of this effect through co-application of a cocktail of glutamate and GABA receptor antagonists. These studies provide the first direct evidence that activation of glutamate transport is sufficient to increase arteriole diameter. We explored potential downstream mechanisms mediating this transporter-mediated dilation by using a Ca 2+ chelator or inhibitors of reversed-mode Na + /Ca 2+ exchange, nitric oxide synthetase, or cyclo-oxygenase. The estimated effects and confidence intervals suggested some form of inhibition for a number of these inhibitors. Limitations to our study design prevented definitive conclusions with respect to these downstream inhibitors; these limitations are discussed along with possible next steps. Understanding the mechanisms that control blood flow are important because changes in blood flow/energy supply are implicated in several neurodegenerative disorders and are used as a surrogate measure of neuronal activity in widely used techniques such as functional magnetic resonance imaging (fMRI)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jackson, Krizman, Takano, Lee, Choi, Putt and Robinson.)

Mutations in the exonuclease domain of POLE are associated with tumors harboring very high mutation burdens. The mechanisms linking this significant mutation accumulation and tumor development remain poorly understood. Pole +/P286R ; Trp53 +/- mice showed accelerated cancer mortality compared to Pole +/P286R ; Trp53 +/+ mice. Cells from Pole +/P286R mice showed increased p53 activation, and subsequent loss of p53 permitted rapid growth, implicating canonical p53 loss of heterozygosity in POLE mutant tumor growth. However, p53 status had no effect on tumor mutation burden or single base substitution signatures in POLE mutant tumors from mice or humans. Pten has important roles in maintaining genome stability. We find that PTEN mutations are highly enriched in human POLE mutant tumors, including many in POLE signature contexts. One such signature mutation, PTEN-F341V , was previously shown in a mouse model to specifically decrease nuclear Pten and lead to increased DNA damage. We found tumors in Pole +/P286R mice that spontaneously acquired Pten F341V mutations and were associated with significantly reduced nuclear Pten and elevated DNA damage. Re-analysis of human TCGA (The Cancer Genome Atlas) data showed that all PTEN-F341V mutations occurred in tumors with mutations in POLE . Taken together with recent published work, our results support the idea that development of POLE mutant tumors may involve disabling surveillance of nuclear DNA damage in addition to POLE-mediated hypermutagenesis., (© The Author(s) 2022. Published by Oxford University Press on behalf of NAR Cancer.)

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient's breast cancer brain metastatic growth within the brain microenvironment.

Background: The relationship between tuberculosis (TB), one of the leading infectious causes of death worldwide, and the microbiome, which is critical for health, is poorly understood., Methods: To identify potential microbiome-host interactions, profiling of the oral, sputum and stool microbiota [n = 58 cases, n = 47 culture-negative symptomatic controls (SCs)] and whole blood transcriptome were done in pre-treatment presumptive pulmonary TB patients. This was a cross-sectional study. Microbiota were also characterised in close contacts of cases (CCCs, n = 73) and close contacts of SCs (CCSCs, n = 82) without active TB., Findings: Cases and SCs each had similar α- and β-diversities in oral washes and sputum, however, β-diversity differed in stool (PERMANOVA p = 0•035). Cases were enriched with anaerobes in oral washes, sputum (Paludibacter, Lautropia in both) and stool (Erysipelotrichaceae, Blautia, Anaerostipes) and their stools enriched in microbial genes annotated as amino acid and carbohydrate metabolic pathways. In pairwise comparisons with their CCCs, cases had Megasphaera-enriched oral and sputum microbiota and Bifidobacterium-, Roseburia-, and Dorea-depleted stools. Compared to their CCSCs, SCs had reduced α-diversities and many differential taxa per specimen type. Cases differed transcriptionally from SCs in peripheral blood (PERMANOVA p = 0•001). A co-occurrence network analysis showed stool taxa, Erysipelotrichaceae and Blautia, to negatively co-correlate with enriched "death receptor" and "EIF2 signalling" pathways whereas Anaerostipes positively correlated with enriched "interferon signalling", "Nur77 signalling" and "inflammasome" pathways; all of which are host pathways associated with disease severity. In contrast, none of the taxa enriched in SCs correlated with host pathways., Interpretation: TB-specific microbial relationships were identified in oral washes, induced sputum, and stool from cases before the confounding effects of antibiotics. Specific anaerobes in cases' stool predict upregulation of pro-inflammatory immunological pathways, supporting the gut microbiota's role in TB., Funding: European & Developing Countries Clinical Trials Partnership, South African-Medical Research Council, National Institute of Allergy and Infectious Diseases., Competing Interests: Declaration of Competing Interest GT received in-kind donations (GeneXpert cartridges and machines) from Cepheid and FIND. Cepheid had no role in study design or interpretation of results. BWPR received travel support from Cepheid to attend a conference and present unrelated data. All other authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

POLE mutations are a major cause of hypermutant cancers, yet questions remain regarding mechanisms of tumorigenesis, genotype-phenotype correlation, and therapeutic considerations. In this study, we establish mouse models harboring cancer-associated POLE mutations P286R and S459F, which cause rapid albeit distinct time to cancer initiation in vivo , independent of their exonuclease activity. Mouse and human correlates enabled novel stratification of POLE mutations into three groups based on clinical phenotype and mutagenicity. Cancers driven by these mutations displayed striking resemblance to the human ultrahypermutation and specific signatures. Furthermore, Pole -driven cancers exhibited a continuous and stochastic mutagenesis mechanism, resulting in intertumoral and intratumoral heterogeneity. Checkpoint blockade did not prevent Pole lymphomas, but rather likely promoted lymphomagenesis as observed in humans. These observations provide insights into the carcinogenesis of POLE -driven tumors and valuable information for genetic counseling, surveillance, and immunotherapy for patients. SIGNIFICANCE: Two mouse models of polymerase exonuclease deficiency shed light on mechanisms of mutation accumulation and considerations for immunotherapy. See related commentary by Wisdom and Kirsch p. 5459 ., (©2020 American Association for Cancer Research.)

TP53 wild-type breast tumors rarely undergo a complete pathological response after chemotherapy treatment. These patients have an extremely poor survival rate and studies show these tumors preferentially undergo senescence instead of apoptosis. These senescent cells persist after chemotherapy and secrete cytokines and chemokines comprising the senescence associated secretory phenotype, which promotes survival, proliferation, and metastasis. We hypothesized that eliminating senescent tumor cells would improve chemotherapy response and extend survival. Previous studies have shown "senolytic" agents selectively kill senescent normal cells, but their efficacy in killing chemotherapy-induced senescent cancer cells is unknown. We show that ABT-263, a BH3 mimetic that targets antiapoptotic proteins BCL2/BCL-XL/BCL-W, had no effect on proliferating cells, but rapidly and selectively induced apoptosis in a subset of chemotherapy-treated cancer cells, though sensitivity required days to develop. Low NOXA expression conferred resistance to ABT-263 in some cells, necessitating additional MCL1 inhibition. Gene editing confirmed breast cancer cells relied on BCL-XL or BCL-XL/MCL1 for survival in senescence. In a mouse model of breast cancer, ABT-263 treatment following chemotherapy led to apoptosis, greater tumor regression, and longer survival. Our results reveal cancer cells that have survived chemotherapy by entering senescence can be eliminated using BH3 mimetic drugs that target BCL-XL or BCL-XL/MCL1. These drugs could help minimize residual disease and extend survival in breast cancer patients that otherwise have a poor prognosis and are most in need of improved therapies.

Mitochondria are essential organelles involved in energy supply and calcium homeostasis. The regulated distribution of mitochondria in polarized cells, particularly neurons, is thought to be essential to these roles. Altered mitochondrial function and impairment of mitochondrial distribution and dynamics is implicated in a number of neurologic disorders. Several recent reports have described mechanisms regulating the activity-dependent distribution of mitochondria within astrocyte processes and the functional consequences of altered mitochondrial transport. Here we provide an ex vivo method for monitoring the transport of mitochondria within the processes of astrocytes using organotypic "slice" cultures. These methods can be easily adapted to investigate a wide range of mitochondrial behaviors, including fission and fusion dynamics, mitophagy, and calcium signaling in astrocytes and other cell types of the central nervous system. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of brain slices Basic Protocol 2: Preparation of gene gun bullets Basic Protocol 3: Gene gun transfection of slices Basic Protocol 4: Visualization and tracking of mitochondrial movement Alternate Protocol: Transduction of EGFP-mito via viral injection of the neonatal mouse brain., (© 2020 John Wiley & Sons, Inc.)

Background: Xpert MTB/RIF Ultra (Ultra) is a new test for tuberculosis undergoing global roll-out. We assessed the performance of Ultra compared with Xpert MTB/RIF (Xpert) in an HIV-endemic setting where previous tuberculosis is frequent and current test performance is suboptimal., Methods: In this two-cohort diagnostic accuracy study, we used sputum samples from patients in South Africa to evaluate the accuracy of Ultra and Xpert against a single culture reference standard. For the first cohort (cohort A), we recruited adults (aged ≥18 years) with symptoms of presumptive tuberculosis at Scottsdene clinic in Cape Town, South Africa. We collected three sputum samples from each patient in cohort A, two at the first visit of which one was tested using Xpert and the other was tested using culture, and one sample the next morning which was tested using Ultra. In a separate cohort of patients with presumptive tuberculosis and recent previous tuberculosis (≤2 years) who had submitted sputum samples to the National Health Laboratory Services (cohort B), decontaminated sediments were, after processing, randomly allocated (1:1) for testing with Ultra or Xpert. For both cohorts we calculated the sensitivity and specificity of Ultra and Xpert and evaluated the effects of different methods of interpreting Ultra trace results., Findings: Between Feb 6, 2016, and Feb 2, 2018, we recruited 302 people into cohort A, all of whom provided sputum samples and 239 were included in the head-to-head analyses of Ultra and Xpert. For cohort B, we collected sputum samples from eligible patients who had submitted samples between Dec 6, 2016, and Dec 21, 2017, to give a cohort of 831 samples, of which 352 were eligible for inclusion in analyses and randomly assigned to Ultra (n=173) or Xpert (n=179). In cohort A, Ultra gave more non-actionable results (not positive or negative) than did Xpert (28 [10%] 275 vs 14 [5%] 301; p=0·011). In the head-to-head analysis, in smear-negative patients, sensitivity of Ultra was 80% (95% CI 64-90) and of Xpert was 73% (57-85; p=0·45). Overall, specificity of Ultra was lower than that of Xpert (90% [84-94] vs 99% [95-100]; p=0·001). In cohort B, overall sensitivity was 92% (81-98) for Xpert versus 86% (73-95; p=0·36) for Ultra and overall specificity was 69% (60-77) for Ultra versus 84% (78-91; p=0·005) for Xpert. Ultra specificity estimates improved after reclassification of results with the lowest Ultra-positive semiquantitation category (trace) to negative (15% [8-22]). In cohort A, the positive predictive value (PPV) for Ultra was 78% (67-87) and for Xpert was 96% (87-99; p=0·004); in cohort B, the PPV for Ultra was 50% (43-57) and for Xpert was 70% (61-78; p=0·014). Ultra PPV estimates in previously treated patients were low: at 15% tuberculosis prevalence, half of Ultra-positive patients with presumptive tuberculosis would be culture negative, increasing to approximately 70% in patients with recent previous tuberculosis. In cohort B, 21 (28%) of 76 samples that were Ultra positive were rifampicin indeterminate (all trace) and, like cohort A, most were culture negative (19 [90%] of 21)., Interpretation: In a setting with a high burden of previous tuberculosis, Ultra generated more non-actionable results and had diminished specificity compared with Xpert. In patients with recent previous tuberculosis, a quarter of Ultra-positive samples were indeterminate for rifampicin resistance and culture negative, suggesting that additional drug-resistance testing will probably be unsuccessful. Our data have implications for the handling of Ultra-positive results in patients with previous tuberculosis in high burden settings., Funding: South African Medical Research Council, the EDCTP2 program, and the Faculty of Medicine and Health Sciences, Stellenbosch University., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

TP53 is the most frequently mutated gene in breast cancer, but its role in survival is confounded by different studies concluding that TP53 mutations are associated with negative, neutral, or positive outcomes. Closer examination showed that many studies were limited by factors such as imprecise methods to detect TP53 mutations and small cohorts that combined patients treated with drugs having very different mechanisms of action. When only studies of patients receiving the same treatment(s) were compared, they tended to agree. These analyses reveal a role for TP53 in response to different treatments as complex as its different biological activities. We discuss studies that have assessed the role of TP53 mutations in breast cancer treatment and limitations in interpreting reported results., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The breast tumors that are most difficult to eradicate with chemotherapy have wild-type TP53 and preferentially enter senescence after treatment. One factor contributing to the persistence of senescent cells in residual disease: acquisition of a novel phenotype that allows cannibalism of entire cells and engulfment of other substrates., (© 2019 Taylor & Francis Group, LLC.)

In chemotherapy-treated breast cancer, wild-type p53 preferentially induces senescence over apoptosis, resulting in a persisting cell population constituting residual disease that drives relapse and poor patient survival via the senescence-associated secretory phenotype. Understanding the properties of tumor cells that allow survival after chemotherapy treatment is paramount. Using time-lapse and confocal microscopy to observe interactions of cells in treated tumors, we show here that chemotherapy-induced senescent cells frequently engulf both neighboring senescent or nonsenescent tumor cells at a remarkable frequency. Engulfed cells are processed through the lysosome and broken down, and cells that have engulfed others obtain a survival advantage. Gene expression analysis showed a marked up-regulation of conserved macrophage-like program of engulfment in chemotherapy-induced senescent cell lines and tumors. Our data suggest compelling explanations for how senescent cells persist in dormancy, how they manage the metabolically expensive process of cytokine production that drives relapse in those tumors that respond the worst, and a function for their expanded lysosomal compartment., (© 2019 Tonnessen-Murray et al.)

Purpose Tinnitus is a chronic medical condition that can result in distress, concentration difficulties, and clinical depression. An effective, objective measure of tinnitus distress does not currently exist. Endocrinal studies into the condition have been few, with those investigating the cortisol awakening response limited in scope. It was hypothesized that distressed individuals with tinnitus would awaken and be unable to effectively prepare for the day ahead due to a blunted cortisol response. Method Twenty individuals with varying tinnitus distress were compared with a control group ( n = 10) in a pilot study, which measured salivary cortisol concentrations on awakening. Multiple exclusion variables were applied. Results In line with previous studies, total cortisol volume (as measured by area under the curve) was not found to be significantly different in the most distressed individuals with tinnitus, F (2, 26) = 0.254, p = .777ns. However, a separate measure of changing cortisol levels-the area under the curve with respect to increase (or AUC i )-was found to be significantly less robust in those individuals reporting the most severe tinnitus distress, F (2, 26) = 7.671, p = .002. This indicates that fewer resources would be available to cope with the demands of the day ahead. Additionally, the AUC i correlated negatively with tinnitus distress later the same day. Conclusions Relationships between proposed objective and self-reported components of self-reported tinnitus distress are considered, with some aspects of tinnitus distress more closely related to physiological mechanisms than others. It is suggested that, with further research, the cortisol awakening response (AUC i ) may be put forward as a credible objective biomarker of tinnitus distress.

Missense mutations in the TP53 -binding domain predominate, and >30% of these occur in just eight codons. Dominant negative properties of mutant p53, taken together with the mutation susceptibility of the nucleotides in the codon, are believed to explain the prevalence of specific mutations, including hot spots. We analyzed multiple tumor types and found no difference in clinical characteristics or survival between patients with dominant negative p53 mutant tumors and those with TP53 mutations that are predicted to be non-dominant negative. The rate tumors underwent loss of heterozygosity in these respective mutation classes was nearly identical, suggesting that presence of stable, mutant protein with predicted dominant negative activity does not reduce selective pressure to inactivate the wild-type allele. Our data suggest all inactivating mutations of TP53 are equal, and the frequency of dominant negative, hot spot mutations is likely driven more by the relative mutability of the DNA at specific codons., Competing Interests: The authors declare no competing interests.

Background: Previous studies on the role of TP53 mutation in breast cancer treatment response and survival are contradictory and inconclusive, limited by the use of different endpoints to determine clinical significance and by small sample sizes that prohibit stratification by treatment., Methods: We utilized large datasets to examine overall survival according to TP53 mutation status in patients across multiple clinical features and treatments., Results: Confirming other studies, we found that in all patients and in hormone therapy-treated patients, TP53 wild-type status conferred superior 5-year overall survival, but survival curves crossed at 10 or more years. In contrast, further stratification within the large dataset revealed that in patients receiving chemotherapy and no hormone therapy, wild-type TP53 status conferred remarkably poor overall survival. This previously unrecognized inferior survival is consistent with p53 inducing arrest/senescence instead of apoptosis. Addition of hormone therapy to chemotherapy improved survival notably in patients with TP53 wild-type tumors, but not mutant, suggesting hormone therapy could eradicate arrested/senescent cells. Testing this, we found that estrogen receptor-positive, TP53 wild-type breast cancer cells that were made senescent by doxorubicin treatment were sensitive to tamoxifen., Conclusions: The poor survival of chemotherapy-treated patients with TP53 wild-type tumors may be improved by strategies to eliminate senescent cells, including the addition of hormone therapy when appropriate.

p53 is a transcription factor that regulates expression of genes involved in cell cycle arrest, senescence, and apoptosis. TP53 harbors mutations that inactivate its transcriptional activity in roughly 30% of breast cancers, and these tumors are much more likely to undergo a pathological complete response to chemotherapy. Thus, the gene expression program activated by wild-type p53 contributes to a poor response. We used an in vivo genetic model system to comprehensively define the p53- and p21-dependent genes and pathways modulated in tumors following doxorubicin treatment. We identified genes differentially expressed in spontaneous mammary tumors harvested from treated MMTV-Wnt1 mice that respond poorly (Trp53+/+) or favorably (Trp53-null) and those that lack the critical senescence/arrest p53 target gene Cdkn1a. Trp53 wild-type tumors differentially expressed nearly 10-fold more genes than Trp53-null tumors after treatment. Pathway analyses showed that genes involved in cell cycle, senescence, and inflammation were enriched in treated Trp53 wild-type tumors; however, no genes/pathways were identified that adequately explain the superior cell death/tumor regression observed in Trp53-null tumors. Cdkn1a-null tumors that retained arrest capacity (responded poorly) and those that proliferated (responded well) after treatment had remarkably different gene regulation. For instance, Cdkn1a-null tumors that arrested upregulated Cdkn2a (p16), suggesting an alternative, p21-independent route to arrest. Live animal imaging of longitudinal gene expression of a senescence/inflammation gene reporter in Trp53+/+ tumors showed induction during and after chemotherapy treatment, while tumors were arrested, but expression rapidly diminished immediately upon relapse., (Copyright © 2018 BAYLOR COLLEGE OF MEDICINE. Published by Elsevier Inc. All rights reserved.)

Astrocytes are the major glial cell in the central nervous system. These polarized cells possess numerous processes that ensheath the vasculature and contact synapses. Astrocytes play important roles in synaptic signaling, neurotransmitter synthesis and recycling, control of nutrient uptake, and control of local blood flow. Many of these processes depend on local metabolism and/or energy utilization. While astrocytes respond to increases in neuronal activity and metabolic demand by upregulating glycolysis and glycogenolysis, astrocytes also possess significant capacity for oxidative (mitochondrial) metabolism. Mitochondria mediate energy supply and metabolism, cellular survival, ionic homeostasis, and proliferation. These organelles are dynamic structures undergoing extensive fission and fusion, directed movement along cytoskeletal tracts, and degradation. While many of the mechanisms underlying the dynamics of these organelles and their physiologic roles have been characterized in neurons and other cells, the roles that mitochondrial dynamics play in glial physiology is less well understood. Recent work from several laboratories has demonstrated that mitochondria are present within the fine processes of astrocytes, that their movement is regulated, and that they contribute to local Ca 2+ signaling within the astrocyte. They likely play a role in local ATP production and metabolism, particularly that of glutamate. Here we will review these and other findings describing the mechanism by which mitochondrial dynamics are regulated in astrocytes, how mitochondrial dynamics might influence astrocyte and brain metabolism, and draw parallels to mitochondrial dynamics in neurons. Additionally, we present new analyses of the size, distribution, and dynamics of mitochondria in astrocytes performed using in vivo using 2-photon microscopy., (© 2017 Wiley Periodicals, Inc.)

Transformed cells have properties that allow them to survive and proliferate inappropriately. These characteristics often arise as a result of mutations caused by DNA damage. p53 suppresses transformation by removing the proliferative or survival capacity of cells with DNA damage or inappropriate cell-cycle progression. Cellular senescence, marked by morphological and gene expression changes, is a critical component of p53-mediated tumor suppression. In response to stress, p53 can facilitate an arrest and senescence program in cells exposed to stresses such as DNA damage and oncogene activation, preventing transformation. Senescent cells are evident in precancerous adenoma-type lesions, whereas proliferating, malignant tumors have bypassed senescence, either by p53 mutation or inactivation of the p53 pathway by other means. Tumors that have retained wild-type p53 often show a p53-mediated senescence response to chemotherapy. This response is actually detrimental in some tumor types, as senescent cells can drive relapse by persisting and producing cytokines and chemokines through an acquired secretory phenotype., (Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.)

Cellular senescence is a permanent growth arrest in cells with damage or stress that could lead to transformation. Some tumor cells also undergo senescence in response to chemotherapy. Senescent cells secrete cytokines and other factors of the senescence-associated secretory phenotype (SASP) that contribute to tumor suppression by enforcing arrest and recruiting immune cells that remove these damaged or oncogene-expressing cells from organisms. However, some cells can develop a SASP comprising factors that are immunosuppressive and protumorigenic by paracrine mechanisms. Likewise, the SASP in treated cancers can either contribute to durable responses or drive relapse. Here, we discuss the studies that have demonstrated a complex and often conflicting role for the SASP in tumorigenesis and treatment response., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In the mammalian brain, a family of sodium-dependent transporters maintains low extracellular glutamate and shapes excitatory signaling. The bulk of this activity is mediated by the astroglial glutamate transporters GLT-1 and GLAST (also called EAAT2 and EAAT1). In this review, we will discuss evidence that these transporters co-localize with, form physical (co-immunoprecipitable) interactions with, and functionally couple to various 'energy-generating' systems, including the Na(+)/K(+)-ATPase, the Na(+)/Ca(2+) exchanger, glycogen metabolizing enzymes, glycolytic enzymes, and mitochondria/mitochondrial proteins. This functional coupling is bi-directional with many of these systems both being regulated by glutamate transport and providing the 'fuel' to support glutamate uptake. Given the importance of glutamate uptake to maintaining synaptic signaling and preventing excitotoxicity, it should not be surprising that some of these systems appear to 'redundantly' support the energetic costs of glutamate uptake. Although the glutamate-glutamine cycle contributes to recycling of neurotransmitter pools of glutamate, this is an over-simplification. The ramifications of co-compartmentalization of glutamate transporters with mitochondria for glutamate metabolism are discussed. Energy consumption in the brain accounts for ∼20% of the basal metabolic rate and relies almost exclusively on glucose for the production of ATP. However, the brain does not possess substantial reserves of glucose or other fuels. To ensure adequate energetic supply, increases in neuronal activity are matched by increases in cerebral blood flow via a process known as 'neurovascular coupling'. While the mechanisms for this coupling are not completely resolved, it is generally agreed that astrocytes, with processes that extend to synapses and endfeet that surround blood vessels, mediate at least some of the signal that causes vasodilation. Several studies have shown that either genetic deletion or pharmacologic inhibition of glutamate transport impairs neurovascular coupling. Together these studies strongly suggest that glutamate transport not only coordinates excitatory signaling, but also plays a pivotal role in regulating brain energetics., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Unlabelled: Recently, mitochondria have been localized to astrocytic processes where they shape Ca(2+) signaling; this relationship has not been examined in models of ischemia/reperfusion. We biolistically transfected astrocytes in rat hippocampal slice cultures to facilitate fluorescent confocal microscopy, and subjected these slices to transient oxygen/glucose deprivation (OGD) that causes delayed excitotoxic death of CA1 pyramidal neurons. This insult caused a delayed loss of mitochondria from astrocytic processes and increased colocalization of mitochondria with the autophagosome marker LC3B. The losses of neurons in area CA1 and mitochondria in astrocytic processes were blocked by ionotropic glutamate receptor (iGluR) antagonists, tetrodotoxin, ziconotide (Ca(2+) channel blocker), two inhibitors of reversed Na(+)/Ca(2+) exchange (KB-R7943, YM-244769), or two inhibitors of calcineurin (cyclosporin-A, FK506). The effects of OGD were mimicked by NMDA. The glutamate uptake inhibitor (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartate increased neuronal loss after OGD or NMDA, and blocked the loss of astrocytic mitochondria. Exogenous glutamate in the presence of iGluR antagonists caused a loss of mitochondria without a decrease in neurons in area CA1. Using the genetic Ca(2+) indicator Lck-GCaMP-6S, we observed two types of Ca(2+) signals: (1) in the cytoplasm surrounding mitochondria (mitochondrially centered) and (2) traversing the space between mitochondria (extramitochondrial). The spatial spread, kinetics, and frequency of these events were different. The amplitude of both types was doubled and the spread of both types changed by ∼2-fold 24 h after OGD. Together, these data suggest that pathologic activation of glutamate transport and increased astrocytic Ca(2+) through reversed Na(+)/Ca(2+) exchange triggers mitochondrial loss and dramatic increases in Ca(2+) signaling in astrocytic processes., Significance Statement: Astrocytes, the most abundant cell type in the brain, are vital integrators of signaling and metabolism. Each astrocyte consists of many long, thin branches, called processes, which ensheathe vasculature and thousands of synapses. Mitochondria occupy the majority of each process. This occupancy is decreased by ∼50% 24 h after an in vitro model of ischemia/reperfusion injury, due to delayed fragmentation and mitophagy. The mechanism appears to be independent of neuropathology, instead involving an extended period of high glutamate uptake into astrocytes. Our data suggest that mitochondria serve as spatial buffers, and possibly even as a source of calcium signals in astrocytic processes. Loss of mitochondria resulted in drastically altered calcium signaling that could disrupt neurovascular coupling and gliotransmission., (Copyright © 2016 the authors 0270-6474/16/367110-19$15.00/0.)

We recently showed that inhibition of neuronal activity, glutamate uptake, or reversed-Na(+)/Ca(2+)-exchange with TTX, TFB-TBOA, or YM-244769, respectively, increases mitochondrial mobility in astrocytic processes. In the present study, we examined the interrelationships between mitochondrial mobility and Ca(2+) signaling in astrocyte processes in organotypic cultures of rat hippocampus. All of the treatments that increase mitochondrial mobility decreased basal Ca(2+). As recently reported, we observed spontaneous Ca(2+) spikes with half-lives of ∼1 s that spread ∼6 μm and are almost abolished by a TRPA1 channel antagonist. Virtually all of these Ca(2+) spikes overlap mitochondria (98%), and 62% of mitochondria are overlapped by these spikes. Although tetrodotoxin, TFB-TBOA, or YM-244769 increased Ca(2+) signaling, the specific effects on peak, decay time, and/or frequency were different. To more specifically manipulate mitochondrial mobility, we explored the effects of Miro motor adaptor proteins. We show that Miro1 and Miro2 are both expressed in astrocytes and that exogenous expression of Ca(2+)-insensitive Miro mutants (KK) nearly doubles the percentage of mobile mitochondria. Expression of Miro1(KK) had a modest effect on the frequency of these Ca(2+) spikes but nearly doubled the decay half-life. The mitochondrial proton ionophore, FCCP, caused a large, prolonged increase in cytosolic Ca(2+) followed by an increase in the decay time and the spread of the spontaneous Ca(2+) spikes. Photo-ablation of mitochondria in individual astrocyte processes has similar effects on Ca(2+). Together, these studies show that Ca(2+) regulates mitochondrial mobility, and mitochondria in turn regulate Ca(2+) signals in astrocyte processes., Significance Statement: In neurons, the movement and positioning of mitochondria at sites of elevated activity are important for matching local energy and Ca(2+) buffering capacity. Previously, we demonstrated that mitochondria are immobilized in astrocytes in response to neuronal activity and glutamate uptake. Here, we demonstrate a mechanism by which mitochondria are immobilized in astrocytes subsequent to increases in intracellular [Ca(2+)] and provide evidence that mitochondria contribute to the compartmentalization of spontaneous Ca(2+) signals in astrocyte processes. Immobilization of mitochondria at sites of glutamate uptake in astrocyte processes provides a mechanism to coordinate increases in activity with increases in mitochondrial metabolism., (Copyright © 2015 the authors 0270-6474/15/3515199-15$15.00/0.)

Objectives: Worldwide studies were combined to examine two hypotheses: (i) bipolar disorder is associated with smoking behaviors, compared with the general population; and (ii) smoking behavior prevalences in bipolar disorder are intermediate between those in major depressive disorder and those in schizophrenia., Methods: Combined analyses used 56 articles on adults obtained from a PubMed search or the senior author's article collection. Odds ratios (ORs) and their 95% confidence intervals (CIs) compared current smoking, heavy smoking among current smokers, smoking cessation in ever smokers, and ever smoking in bipolar disorder versus control groups., Results: The combined OR was 3.5 (CI: 3.39-3.54) in 51 current smoking studies of bipolar disorder versus the general population from 16 countries. More limited data provided an OR = 0.34 (CI: 0.31-0.37) for smoking cessation and an OR = 3.6 (CI: 3.30-3.80) for ever smoking. The combined OR was 0.76 (CI: 0.74-0.79) for current smoking in bipolar disorder versus schizophrenia in 20 studies from ten countries. Ever smoking may be lower in bipolar disorder than in schizophrenia (OR = 0.83, CI: 0.75-0.91). The OR was 2.05 (CI: 2.00-2.10) for current smoking in bipolar disorder versus major depression in 18 studies from seven countries. Ever smoking may be higher (OR = 1.5, CI: 1.40-1.70) and smoking cessation lower (OR = 0.51, CI: 0.45-0.59) in bipolar disorder than in major depression., Conclusions: Increased current smoking in bipolar disorder versus the general population reflected increased ever smoking (initiation) and decreased smoking cessation. Smoking behavior frequencies in bipolar disorder may be between those in depressive disorder and schizophrenia, with schizophrenia showing the highest severity level., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Nitric oxide (NO) is a signaling intermediate during glutamatergic neurotransmission in the central nervous system (CNS). NO signaling is in part accomplished through cysteine S-nitrosylation, a posttranslational modification by which NO regulates protein function and signaling. In our investigation of the protein targets and functional impact of S-nitrosylation in the CNS under physiological conditions, we identified 269 S-nitrosocysteine residues in 136 proteins in the wild-type mouse brain. The number of sites was significantly reduced in the brains of mice lacking endothelial nitric oxide synthase (eNOS(-/-)) or neuronal nitric oxide synthase (nNOS(-/-)). In particular, nNOS(-/-) animals showed decreased S-nitrosylation of proteins that participate in the glutamate/glutamine cycle, a metabolic process by which synaptic glutamate is recycled or oxidized to provide energy. (15)N-glutamine-based metabolomic profiling and enzymatic activity assays indicated that brain extracts from nNOS(-/-) mice converted less glutamate to glutamine and oxidized more glutamate than those from mice of the other genotypes. GLT1 [also known as EAAT2 (excitatory amino acid transporter 2)], a glutamate transporter in astrocytes, was S-nitrosylated at Cys(373) and Cys(561) in wild-type and eNOS(-/-) mice, but not in nNOS(-/-) mice. A form of rat GLT1 that could not be S-nitrosylated at the equivalent sites had increased glutamate uptake compared to wild-type GLT1 in cells exposed to an S-nitrosylating agent. Thus, NO modulates glutamatergic neurotransmission through the selective, nNOS-dependent S-nitrosylation of proteins that govern glutamate transport and metabolism., (Copyright © 2015, American Association for the Advancement of Science.)

The glutamate transporter GLT-1 is the major route for the clearance of extracellular glutamate in the forebrain, and most GLT-1 protein is found in astrocytes. This protein is coupled to the Na(+) electrochemical gradient, supporting the active intracellular accumulation of glutamate. We recently used a proteomic approach to identify proteins that may interact with GLT-1 in rat cortex, including the Na(+)/K(+) -ATPase, most glycolytic enzymes, and several mitochondrial proteins. We also showed that most GLT-1 puncta (∼ 70%) are overlapped by mitochondria in astroglial processes in organotypic slices. From this analysis, we proposed that the glycolytic enzyme hexokinase (HK)-1 might physically form a scaffold to link GLT-1 and mitochondria because HK1 is known to interact with the outer mitochondrial membrane protein voltage-dependent anion channel (VDAC). The current study validates the interactions among HK-1, VDAC, and GLT-1 by using forward and reverse immunoprecipitations and provides evidence that a subfraction of HK1 colocalizes with GLT-1 in vivo. A peptide known to disrupt the interaction between HK and VDAC did not disrupt interactions between GLT-1 and several mitochondrial proteins. In parallel experiments, displacement of HK from VDAC reduced GLT-1-mediated glutamate uptake. These results suggest that, although HK1 forms coimmunoprecipitatable complexes with both VDAC and GLT-1, it does not physically link GLT-1 to mitochondrial proteins. However, the interaction of HK1 with VDAC supports GLT-1-mediated transport activity., (© 2014 Wiley Periodicals, Inc.)

p53(R172H/+) mice inherit a p53 mutation found in Li-Fraumeni syndrome and develop metastatic tumors at much higher frequency than p53(+/-) mice. To explore the mutant p53 metastatic phenotype, we used expression arrays to compare primary osteosarcomas from p53(R172H/+) mice with metastasis to osteosarcomas from p53(+/-) mice lacking metastasis. For this study, 213 genes were differentially expressed with a P value <0.05. Of particular interest, Pla2g16, which encodes a phospholipase that catalyzes phosphatidic acid into lysophosphatidic acid and free fatty acid (both implicated in metastasis), was increased in p53(R172H/+) osteosarcomas. Functional analyses showed that Pla2g16 knockdown decreased migration and invasion in mutant p53-expressing cells, and vice versa: overexpression of Pla2g16 increased the invasion of p53-null cells. Furthermore, Pla2g16 levels were increased upon expression of mutant p53 in both mouse and human osteosarcoma cell lines, indicating that Pla2g16 is a downstream target of the mutant p53 protein. ChIP analysis revealed that several mutant p53 proteins bind the Pla2g16 promoter at E26 transformation-specific (ETS) binding motifs and knockdown of ETS2 suppressed mutant p53 induction of Pla2g16. Thus, our study identifies a phospholipase as a transcriptional target of mutant p53 that is required for metastasis.