Deletion of murine astrocytic vesicular nucleotide transporter increases anxiety and depressive-like behavior and attenuates motivation for reward.

Astrocytes are multi-functional glial cells in the central nervous system that play critical roles in modulation of metabolism, extracellular ion and neurotransmitter levels, and synaptic plasticity. Astrocyte-derived signaling molecules mediate many of these modulatory functions of astrocytes, including vesicular release of ATP. In the present study, we used a unique genetic mouse model to investigate the functional significance of astrocytic exocytosis of ATP. Using primary cultured astrocytes, we show that loss of vesicular nucleotide transporter (Vnut), a primary transporter responsible for loading cytosolic ATP into the secretory vesicles, dramatically reduces ATP loading into secretory lysosomes and ATP release, without any change in the molecular machinery of exocytosis or total intracellular ATP content. Deletion of astrocytic Vnut in adult mice leads to increased anxiety, depressive-like behaviors, and decreased motivation for reward, especially in females, without significant impact on food intake, systemic glucose metabolism, cognition, or sociability. These behavioral alterations are associated with significant decreases in the basal extracellular dopamine levels in the nucleus accumbens. Likewise, ex vivo brain slices from these mice show a strong trend toward a reduction in evoked dopamine release in the nucleus accumbens. Mechanistically, the reduced dopamine signaling we observed is likely due to an increased expression of monoamine oxidases. Together, these data demonstrate a key modulatory role of astrocytic exocytosis of ATP in anxiety, depressive-like behavior, and motivation for reward, by regulating the mesolimbic dopamine circuitry.

Staphylococcus aureus ß-Toxin Exerts Anti-angiogenic Effects by Inhibiting Re-endothelialization and Neovessel Formation.

Staphylococcus aureus causes severe, life-threatening infections that often are complicated by severe local and systemic pathologies with non-healing lesions. A classic example is S. aureus infective endocarditis (IE), where the secreted hemolysin ß-toxin potentiates the disease via its sphingomyelinase and biofilm ligase activities. Although these activities dysregulate human aortic endothelial cell activation, ß-toxin effect on endothelial cell function in wound healing has not been addressed. With the use of the ex vivo rabbit aortic ring model, we provide evidence that ß-toxin prevents branching microvessel formation, highlighting its ability to interfere with tissue re-vascularization and vascular repair. We show that ß-toxin specifically targets both human aortic endothelial cell proliferation and cell migration and inhibits human umbilical vein endothelial cell rearrangement into capillary-like networks in vitro. Proteome arrays specific for angiogenesis-related molecules provided evidence that ß-toxin promotes an inhibitory profile in endothelial cell monolayers, specifically targeting production of TIMP-1, TIMP-4, and IGFBP-3 to counter the effect of a pro-angiogenic environment. Dysregulation in the production of these molecules is known to result in sprouting defects (including deficient cell proliferation, migration, and survival), vessel instability and/or vascular regression. When endothelial cells are grown under re-endothelialization/wound healing conditions, ß-toxin decreases the pro-angiogenic molecule MMP-8 and increases the anti-angiogenic molecule endostatin. Altogether, the data indicate that ß-toxin is an anti-angiogenic virulence factor and highlight a mechanism where ß-toxin exacerbates S. aureus invasive infections by interfering with tissue re-vascularization and vascular repair.

Insulin and IGF-1 elicit robust transcriptional regulation to modulate autophagy in astrocytes.

OBJECTIVE: Insulin is a principal metabolic hormone. It regulates a plethora of metabolic pathways in peripheral tissues. The highly homologous insulin-like growth factor 1 (IGF-1), on the other hand, is important for development and growth. Recent studies have shown that insulin and IGF-1 signaling plays fundamental roles in the brain. Loss of insulin or IGF-1 receptors in astrocytes leads to altered glucose handling, mitochondrial metabolism, neurovascular coupling, and behavioral abnormalities in mice. Here, we aim to investigate molecular mechanisms by which insulin and IGF-1 signaling regulates astrocyte functions. METHODS: IR-flox and IRKO primary astrocytes were treated with 100 nM insulin or IGF-1 for 6 h, and their transcriptomes were analyzed. Astrocytes with either IR deletion, IGF1R deletion or both were used to examine receptor-dependent transcriptional regulations using qPCR. Additional immunoblotting and confocal imaging studies were performed to functionally validate pathways involved in protein homeostasis. RESULTS: Using next-generation RNA sequencing, we show that insulin significantly regulates the expression of over 1,200 genes involved in multiple functional processes in primary astrocytes. Insulin-like growth factor 1 (IGF-1) triggers a similar robust transcriptional regulation in astrocytes. Thus, over 50% of the differentially expressed genes are regulated by both ligands. As expected, these commonly regulated genes are highly enriched in pathways involved in lipid and cholesterol biosynthesis. Additionally, insulin and IGF-1 induce the expression of genes involved in ribosomal biogenesis, while suppressing the expression of genes involved in autophagy, indicating a common role of insulin and IGF-1 on protein homeostasis in astrocytes. Insulin-dependent suppression of autophagy genes, including p62, Ulk1/2, and several Atg genes, is blunted only when both IR and IGF1R are deleted. CONCLUSIONS: In summary, insulin and IGF-1 potently suppress autophagy in astrocytes through transcriptional regulation. Both IR and IGF1R can elicit ligand-dependent transcriptional suppression of autophagy. These results demonstrate an important role of astrocytic insulin/IGF-1 signaling on proteostasis. Impairment of this regulation in insulin resistance and diabetes may contribute to neurological complications related to diabetes.

SEC is an antiangiogenic virulence factor that promotes Staphylococcus aureus endocarditis independent of superantigen activity.

The superantigen staphylococcal enterotoxin C (SEC) is critical for Staphylococcus aureus infective endocarditis (SAIE) in rabbits. Superantigenicity, its hallmark function, was proposed to be a major underlying mechanism driving SAIE but was not directly tested. With the use of S. aureus MW2 expressing SEC toxoids, we show that superantigenicity does not sufficiently account for vegetation growth, myocardial inflammation, and acute kidney injury in the rabbit model of native valve SAIE. These results highlight the critical contribution of an alternative function of superantigens to SAIE. In support of this, we provide evidence that SEC exerts antiangiogenic effects by inhibiting branching microvessel formation in an ex vivo rabbit aortic ring model and by inhibiting endothelial cell expression of one of the most potent mediators of angiogenesis, VEGF-A. SEC's ability to interfere with tissue revascularization and remodeling after injury serves as a mechanism to promote SAIE and its life-threatening systemic pathologies.

Evolution of antibiotic cross-resistance and collateral sensitivity in Staphylococcus epidermidis using the mutant prevention concentration and the mutant selection window.

In bacteria, evolution of resistance to one antibiotic is frequently associated with increased resistance (cross-resistance) or increased susceptibility (collateral sensitivity) to other antibiotics. Cross-resistance and collateral sensitivity are typically evaluated at the minimum inhibitory concentration (MIC). However, these susceptibility changes are not well characterized with respect to the mutant prevention concentration (MPC), the antibiotic concentration that prevents a single-step mutation from occurring. We measured the MIC and the MPC for Staphylococcus epidermidis and 14 single-drug resistant strains against seven antibiotics. We found that the MIC and the MPC were positively correlated but that this correlation weakened if cross-resistance did not evolve. If any type of resistance did evolve, the range of concentrations between the MIC and the MPC tended to shift right and widen. Similar patterns of cross-resistance and collateral sensitivity were observed at the MIC and MPC levels, though more symmetry was observed at the MIC level. Whole-genome sequencing revealed mutations in both known-target and nontarget genes. Moving forward, examining both the MIC and the MPC may lead to better predictions of evolutionary trajectories in antibiotic-resistant bacteria.

Functional equivalence of stem cell and stem cell-derived extracellular vesicle transplantation to repair the irradiated brain.

Cranial radiotherapy, although beneficial for the treatment of brain tumors, inevitably leads to normal tissue damage that can induce unintended neurocognitive complications that are progressive and debilitating. Ionizing radiation exposure has also been shown to compromise the structural integrity of mature neurons throughout the brain, an effect believed to be at least in part responsible for the deterioration of cognitive health. Past work has shown that cranially transplanted human neural stem cells (hNSCs) or their extracellular vesicles (EVs) afforded long-term beneficial effects on many of these cognitive decrements. To provide additional insight into the potential neuroprotective mechanisms of cell-based regenerative strategies, we have analyzed hippocampal neurons for changes in structural integrity and synaptic remodeling after unilateral and bilateral transplantation of hNSCs or EVs derived from those same cells. Interestingly, hNSCs and EVs similarly afforded protection to host neurons, ameliorating the impact of irradiation on dendritic complexity and spine density for neurons present in both the ipsilateral and contralateral hippocampi 1 month following irradiation and transplantation. These morphometric improvements were accompanied by increased levels of glial cell-derived growth factor and significant attenuation of radiation-induced increases in postsynaptic density protein 95 and activated microglia were found ipsi- and contra-lateral to the transplantation sites of the irradiated hippocampus treated with hNSCs or hNSC-derived EVs. These findings document potent far-reaching neuroprotective effects mediated by grafted stem cells or EVs adjacent and distal to the site of transplantation and support their potential as therapeutic agents to counteract the adverse effects of cranial irradiation.

Laryngotracheal Microbiota in Adult Laryngotracheal Stenosis.

Laryngotracheal stenosis is an obstructive respiratory disease that leads to voicing difficulties and dyspnea with potential life-threatening consequences. The majority of incidences are due to iatrogenic etiology from endotracheal tube intubation; however, airway scarring also has idiopathic causes. While recent evidence suggests a microbial contribution to mucosal inflammation, the microbiota associated with different types of stenosis has not been characterized. High-throughput sequencing of the V4 region of the16S rRNA gene was performed to characterize the microbial communities of 61 swab samples from 17 iatrogenic and 10 adult idiopathic stenosis patients. Nonscar swabs from stenosis patients were internal controls, and eight swabs from four patients without stenosis represented external controls. Significant differences in diversity were observed between scar and nonscar samples and among sample sites, with decreased diversity detected in scar samples and the glottis region. Permutational analysis of variance (PERMANOVA) results revealed significant differences in community composition for scar versus nonscar samples, etiology type, sample site, groups (iatrogenic, idiopathic, and internal and external controls), and individual patients. Pairwise Spearman's correlation revealed a strong inverse correlation between Prevotella and Streptococcus among all samples. Finally, bacteria in the family Moraxellaceae were found to be distinctly associated with idiopathic stenosis samples in comparison with external controls. Our findings suggest that specific microbiota and community shifts are present with laryngotracheal stenosis in adults, with members of the family Moraxellaceae, including the known pathogens Moraxella and Acinetobacter, identified in idiopathic scar. Further work is warranted to elucidate the contributing role of bacteria on the pathogenesis of laryngotracheal stenosis.IMPORTANCE The laryngotracheal region resides at the intersection between the heavily studied nasal cavity and lungs; however, examination of the microbiome in chronic inflammatory conditions of the subglottis and trachea remains scarce. To date, studies have focused on the microbiota of the vocal folds, or the glottis, for laryngeal carcinoma, as well as healthy larynges, benign vocal fold lesions, and larynges exposed to smoking and refluxate. In this study, we seek to examine the structure and composition of the microbial community in adult laryngotracheal stenosis of various etiologies. Due to the heterogeneity among the underlying pathogenesis mechanisms and clinical outcomes seen in laryngotracheal stenosis disease, we hypothesized that different microbial profiles will be detected among various stenosis etiology types. Understanding differences in the microbiota for subglottic stenosis subtypes may shed light upon etiology-specific biomarker identification and offer novel insights into management approaches for this debilitating disease.

Timing of Voice Therapy: A Primary Investigation of Voice Outcomes for Surgical Benign Vocal Fold Lesion Patients.

OBJECTIVE: This study aims to examine the role of timing of speech-language pathology intervention on outcomes for surgical patients with benign vocal fold lesions as measured by a patient self-reported scale (Voice Handicap Index [VHI]) and objective acoustic measures (jitter %, Dysphonia Severity Index, noise-to-harmonics ratio). For the purpose of this study, interventions were categorized into three groups: preoperative (preoperative counseling session followed by postoperative therapy), pre- and postoperative therapy (multiple therapy sessions preoperatively and postoperatively), and postoperative therapy alone. STUDY DESIGN: Retrospective review of a prospective disease-specific outcomes database. METHODS: Subjects identified in the database consisted of 12 preoperative counseling participants (six male, six female), 11 pre- and postoperative therapy participants (three male, eight female), and eight postoperative therapy only participants (three male, five female). Preoperative and postoperative VHI, Dysphonia Severity Index, jitter, and noise-to-harmonics ratio scores were compared between groups using paired t tests with a multiple comparison Bonferroni correction (α = 0.016). RESULTS: Despite lack of statistically significant changes in acoustic measures, groups receiving preoperative intervention whether with postoperative therapy or not, had statistically significant improvements in their VHI total scores (P = 0.001 for preoperative only and P = 0.0002 for pre- and postoperative therapy). CONCLUSIONS: Patients receiving speech pathology services before surgery for benign vocal fold lesions with or without postoperative therapy demonstrated greater gains in their subjective view of vocal function and quality as measured by the VHI.

Insights Into the Role of Collagen in Vocal Fold Health and Disease.

As one of the key fibrous proteins in the extracellular matrix, collagen plays a significant role in the structural and biomechanical characteristics of the vocal fold. Anchored fibrils of collagen create secure structural regions within the vocal folds and are strong enough to sustain vibratory impact and stretch during phonation. This contributes tensile strength, density, and organization to the vocal folds and influences health and pathogenesis. This review offers a comprehensive summary for a current understanding of collagen within normal vocal fold tissues throughout the life span as well as vocal pathology and wound repair. Further, collagen's molecular structure and biosynthesis are discussed. Finally, collagen alterations in tissue injury and repair and the incorporation of collagen-based biomaterials as a method of treating voice disorders are reviewed.

Macrophage Response to Allogeneic Adipose Tissue-Derived Stromal Cells in Hyaluronan-Based Hydrogel in a Porcine Vocal Fold Injury Model.

OBJECTIVE: Adipose tissue-derived stromal cells (ASC) embedded in hyaluronan scaffold is a beneficial prophylactic treatment for vocal fold (VF) surgical scar. Here, we investigated the macrophage inflammatory response to allogeneic ASC-constructs and identified changes in lamina propria extracellular matrix. METHOD: Pig ASC were characterized and transfected with GFP+ lentivirus. Thirty-three pigs underwent VF biopsies, and after 3 days, gel alone, gel+pASC, placebo, or pASC alone was injected into wound bed. Animals were sacrificed 3, 7, or 26 days post-injection. Flow cytometry; qPCR for NF-α, TGFß, IL-10, IL-4, IFNγ, IL-12, FGF2, Col1A1, and HGF; and immunohistochemistry for collagen, elastin, HA, and fibronectin were performed to characterize macrophage phenotype, quantify cytokine transcription, analyze extracellular matrix remodeling, and track GFP+ cells. RESULTS: No significant differences were found in SWC3+/SWC9+ phenotype or mRNA expression between cells+gel, gel, or placebo. The ASC alone exhibited significantly greater collagen, gel alone resulted in significantly less hyaluronan, and gel+pASC significantly more fibronectin (all P < .05). The pASC-GFP+ were detected 26 days post-injection. CONCLUSIONS: The ASC-constructs were biocompatible; they did not influence the macrophage inflammatory response or provoke increases in collagen expression. Long-term engraftment was confirmed.

Characterization of sporulation histidine kinases of Bacillus anthracis.

The initiation of sporulation in Bacillus species is regulated by the phosphorelay signal transduction pathway, which is activated by several histidine sensor kinases in response to cellular and metabolic signals. Comparison of the protein components of the phosphorelay between Bacillus subtilis and Bacillus anthracis revealed high homology in the phosphorelay orthologs of Spo0F, Spo0B, and Spo0A. The sensor domains of sensor histidine kinases are poorly conserved between species, making ortholog recognition tenuous. Putative sporulation sensor histidine kinases of B. anthracis were identified by homology to the HisKA domain of B. subtilis sporulation sensor histidine kinases, which interacts with Spo0F. Nine possible kinases were uncovered, and their genes were assayed for complementation of kinase mutants of B. subtilis, for ability to drive lacZ expression in B. subtilis and B. anthracis, and for the effect of deletion of each on the sporulation of B. anthracis. Five of the nine sensor histidine kinases were inferred to be capable of inducing sporulation in B. anthracis. Four of the sensor kinases could not be shown to induce sporulation; however, the genes for two of these were frameshifted in all B. anthracis strains and one of these was also frameshifted in the pathogenic pXO1-bearing Bacillus cereus strain G9241. It is proposed that acquisition of plasmid pXO1 and pathogenicity may require a dampening of sporulation regulation by mutational selection of sporulation sensor histidine kinase defects. The sporulation of B. anthracis ex vivo appears to result from any one or a combination of the sporulation sensor histidine kinases remaining.

Biflavonoids with cytotoxic and antibacterial activity from Ochna macrocalyx.

Six biflavonoid and related compounds were isolated from the ethanolic extract of Ochna macrocalyx bark. One is a new compound, the isoflavanone dimer dehydroxyhexaspermone C ( 1). Previously isolated compounds obtained from the bark are biisoflavonoid hexaspermone C ( 2). tetrahydrofuran derivative ochnone ( 3). furobenzopyran derivative cordigol ( 4). and biflavonoids calodenin B ( 5). and dihydrocalodenin B ( 6). Although 3 has already been isolated, its spectral data are presented here for the first time. Isolated compounds were tested for cytotoxic activity on MCF-7 breast cancer cells using the MTT reduction assay method. Compound 5 showed cytotoxic activity (7 +/- 0.5 microM) and 6 showed moderate cytotoxicity (35 +/- 7 microM). In antibacterial assays performed using three strains of multi-drug resistant (mdr) Staphylococcus aureus (RN4220, XU212 and SA-1199-B) compounds 5 and in particular 6 showed strong antibacterial activity (MICs 5 : 64, 8, 16 microg/mL 6 : 8, 8, 8 microg/mL, respectively). The ethanolic extract of the bark also showed NF-kappaB inhibitory activity.