Staphylococcus aureus AbcA transporter enhances persister formation under ß-lactam exposure.

We evaluated the role of Staphylococcus aureus AbcA transporter in bacterial persistence and survival following exposure to the bactericidal agents nafcillin and oxacillin at both the population and single-cell levels. We show that AbcA overexpression resulted in resistance to nafcillin but not oxacillin. Using distinct fluorescent reporters of cell viability and AbcA expression, we found that over 6-14 hours of persistence formation, the proportion of AbcA reporter-expressing cells assessed by confocal microscopy increased sixfold as cell viability reporters decreased. Similarly, single-cell analysis in a high-throughput microfluidic system found a strong correspondence between antibiotic exposure and AbcA reporter expression. Persister cells grown in the absence of antibiotics showed neither an increase in nafcillin MIC nor in abcA transcript levels, indicating that survival was not associated with stable mutational resistance or abcA overexpression. Furthermore, persister cell levels on exposure to 1×MIC and 25×MIC of nafcillin decreased in an abcA knockout mutant. Survivors of nafcillin and oxacillin treatment overexpressed transporter AbcA, contributing to an enrichment of the number of persisters during treatment with pump-substrate nafcillin but not with pump-non-substrate oxacillin, indicating that efflux pump expression can contribute selectively to the survival of a persister population.

NorA efflux pump mediates Staphylococcus aureus response to Pseudomonas aeruginosa pyocyanin toxicity.

Endogenous transporters protect Staphylococcus aureus against antibiotics and also contribute to bacterial defense from environmental toxins. We evaluated the effect of overexpression of four efflux pumps, NorA, NorB, NorC, and Tet38, on S. aureus survival following exposure to pyocyanin (PYO) of Pseudomonas aeruginosa, using a well diffusion assay. We measured the PYO-created inhibition zone and found that only an overexpression of NorA reduced S. aureus susceptibility to pyocyanin killing. The MICPYO of the NorA overexpressor increased threefold compared to that of wild-type RN6390 and was reduced 2.5-fold with reserpine, suggesting that increased NorA efflux caused PYO resistance. The PYO-created inhibition zone of a ΔnorA mutant was consistently larger than that of a plasmid-borne NorA overexpressor. PYO also produced a modest increase in norA expression (1.8-fold at 0.25 µg/mL PYO) that gradually decreased with increasing PYO concentrations. Well diffusion assays carried out using P. aeruginosa showed that ΔnorA mutant was less susceptible to killing by PYO-deficient mutants PA14phzM and PA14phzS than to killing by PA14. NorA overexpression led to reduced killing by all tested P. aeruginosa. We evaluated the NorA-PYO interaction using a collection of 22 clinical isolates from adult and pediatric cystic fibrosis (CF) patients, which included both S. aureus (CF-SA) and P. aeruginosa (CF-PA). We found that when isolated alone, CF-PA and CF-SA expressed varying levels of PYO and norA transcripts, but all four CF-PA/CF-SA pairs isolated concurrently from CF patients produced a low level of PYO and low norA transcript levels, respectively, suggesting a partial adaptation of the two bacteria in circumstances of persistent co-colonization.

Targeting the αVß3/NgR2 pathway in neuroendocrine prostate cancer.

Highly aggressive, metastatic, neuroendocrine prostate cancer, which typically develops from prostate cancer cells acquiring resistance to androgen deprivation therapy, is associated with limited treatment options and hence poor prognosis. We have previously demonstrated that the αVß3 integrin is over-expressed in neuroendocrine prostate cancer. We now show that LM609, a monoclonal antibody that specifically targets the human αVß3 integrin, hinders the growth of neuroendocrine prostate cancer patient-derived xenografts in vivo. Our group has recently identified a novel αVß3 integrin binding partner, NgR2, responsible for regulating the expression of neuroendocrine markers and for inducing neuroendocrine differentiation in prostate cancer cells. Through in vitro functional assays, we here demonstrate that NgR2 is crucial in promoting cell adhesion to αVß3 ligands. Moreover, we describe for the first time co-fractionation of αVß3 integrin and NgR2 in small extracellular vesicles derived from metastatic prostate cancer patients' plasma. These prostate cancer patient-derived small extracellular vesicles have a functional impact on human monocytes, increasing their adhesion to fibronectin. The monocytes incubated with small extracellular vesicles do not show an associated change in conventional polarization marker expression and appear to be in an early stage that may be defined as "adhesion competent". Overall, these findings allow us to better understand integrin-directed signaling and cell-cell communication during cancer progression. Furthermore, our results pave the way for new diagnostic and therapeutic perspectives for patients affected by neuroendocrine prostate cancer.

Delivery Models and Health Economics of Supportive Care Services in England: A Multicentre Analysis.

AIMS: Improvements in cancer treatment have led to more people living with and beyond cancer. These patients have symptom and support needs unmet by current services. The development of enhanced supportive care (ESC) services may meet the longitudinal care needs of these patients, including at the end of life. This study aimed to determine the impact and health economic benefits of ESC for patients living with treatable but not curable cancer. MATERIALS AND METHODS: A prospective observational evaluation was undertaken over 12 months across eight cancer centres in England. ESC service design and costs were recorded. Data relating to patients' symptom burden were collected using the Integrated Palliative Care Outcome Scale (IPOS). For patients in the last year of life, secondary care use was compared against an NHS England published benchmark. RESULTS: In total, 4594 patients were seen by ESC services, of whom 1061 died during follow-up. Mean IPOS scores improved across all tumour groups. In total, £1,676,044 was spent delivering ESC across the eight centres. Reductions in secondary care usage for the 1061 patients who died saved a total of £8,490,581. CONCLUSIONS: People living with cancer suffer with complex and unmet needs. ESC services appear to be effective at supporting these vulnerable people and significantly reduce the costs of their care.

Autoantibodies Drive Heart Damage Caused by Concomitant Radiation and PD-1 Blockade.

Concurrent PD-1 blockade and thoracic radiotherapy is being investigated in clinical trials for locally advanced, non-small cell lung cancer and small cell lung cancer, despite a potential overlapping risk of cardiotoxicity. Our prior studies demonstrate that cardiotoxicity from concurrent cardiac irradiation and anti-PD-1 administration in a mouse model is CD8+ T-cell dependent. The objective of this study was to determine whether humoral immunity contributed to the observed cardiac tissue damage, as measured by creatine kinase MB and cardiac troponin 1 release and decline in cardiac function. In the current study, we demonstrate the presence of cardiac autoantibodies, which were essential for the occurrence of cardiotoxicity from the combined therapy. Mice subjected to cardiac irradiation, while being treated with anti-PD-1, developed high levels of antibodies that reacted with cardiac tissues in vivo and cardiac antigens in vitro. Moreover, mice deficient in B cells were protected against cardiotoxicity, whereas the transfer of autoantibody-containing sera from mice that had received combined treatment reproduced the same pathologic phenotype in mice exposed to cardiac irradiation but was not observed in normal recipients. The cardiotoxic effect of the sera, which associated with CD8+ T-cell accumulation in cardiac tissue, was limited by IgG depletion. In conclusion, concurrent cardiac irradiation and PD-1 blockade leads to production of cardiac autoantibodies, likely due to antigen exposure within the irradiated cardiac tissues, which play a key role in the resulting cardiotoxicity.

Role of Staphylococcus aureus Tet38 in Transport of Tetracycline and Its Regulation in a Salt Stress Environment.

Staphylococcus aureus Tet38 efflux pump has multiple functions, including conferring resistance to tetracycline and other compounds and enabling internalization and survival within epithelial cells. In this study, we evaluated the effects of sodium and potassium on tet38 expression. These monovalent cations are known to play a role in transport by the related S. aureus TetK and B. subtilis TetL transporters. tet38 transcription decreased with increasing sodium concentrations by means of direct repression by the salt stress-dependent KdpD/E regulator. tet38 transcription increased 20-fold and tetracycline minimum inhibitory concentration (MIC) increased 4-fold in a ΔkdpD mutant. KdpE bound specifically to the tet38 promoter. Under extreme salt stress, the survival of S. aureus with intact tet38 was reduced compared to that of a Δtet38 mutant. To study the effect of sodium on Tet38 function, we generated constructs overexpressing tet38 and tetK and introduced them into Escherichia coli TO114, which is deficient in major sodium transporters. Tet38 tetracycline efflux was directly demonstrated in a fluorescence assay, and tetracycline efflux of both Tet38 and TetK was abolished by the protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP). In contrast, NaCl inhibited efflux by Tet38 but not TetK, whereas KCl inhibited efflux by TetK but not Tet38. Cell-associated Na increased with heterologous overexpression of Tet38. These data indicate that S. aureus Tet38 is a tetracycline efflux pump regulated by the KdpD/E regulator. Under salt stress, S. aureus adjusted its survival in part by reducing the expression of tet38 through KdpD/E. The mechanisms by which Tet38 is detrimental to salt tolerance in S. aureus and inhibited by sodium remain to be determined. IMPORTANCE This study shows that S. aureus Tet38 is a tetracycline efflux pump regulated by KdpD/E regulator. These findings are the first direct demonstration of Tet38-mediated tetracycline efflux, which had previously been inferred from its ability to confer tetracycline resistance. Under salt stress, S. aureus adjusts its survival in part by reducing the expression of tet38 through KdpD/E. We demonstrated the differences in the respective functions of S. aureus Tet38 and other tetracycline efflux transporters (S. aureus TetK, B. subtilis TetL) regarding their transport of tetracycline and Na+/K+. Notably, sodium selectively reduced tetracycline efflux by Tet38, and potassium selectively reduced tetracycline efflux by TetK. The multiple functions of Tet38 emphasize its importance in bacterial adaptation to and survival in diverse environments.

Staphylococcus aureus Efflux Pumps and Tolerance to Ciprofloxacin and Chlorhexidine following Induction by Mupirocin.

Mupirocin induced expression of genes encoding efflux pumps NorA and MepA as well as a yellow fluorescent protein (YFP) fluorescence reporter of NorA. Mupirocin exposure also produced reduced susceptibility to pump substrates ciprofloxacin and chlorhexidine, a change that was dependent on intact norA and mepA, respectively.

Efficacy of Withania somnifera supplementation on adult's cognition and mood.

The present study examined the effects of a proprietary Ashwagandha (Withania somnifera) root and leaf extract (NooGandha® Specnova LLC, USA) supplement for improving cognitive abilities, cortisol levels, and self-reported mood, stress, food cravings, and anxiety with adults who have perceived stress. Healthy adults (n = 43 women and n = 17 men; mean age = 34.41 years) who reported experiencing perceived stress were randomized to the following groups: Ashwagandha (400 mg/d), Ashwagandha (225 mg/d), and placebo for 30 days. The following outcomes were assessed at Day 0, Day 15, and Day 30: saliva cortisol levels, cognitive performance (i.e., CNS vital signs), and the self-reported measures of Trait Anxiety Inventory, Depression Anxiety Stress Scale, Perceived Stress Scale, and Food Cravings Questionnaire-15. For the self-report assessments, significant main effects for time were evidenced for anxiety, depression, perceived stress, and food cravings, p's < 0.01. The main effect for group and the interactions were non-significant. For the CNS vital signs, significant differences were observed in cognitive flexibility, visual memory, reaction time, psychomotor speed, and executive functioning, p's < 0.05, with the Ashwagandha groups often out-performing the placebo group. Both Ashwagandha groups had reductions in cortisol levels over time, with significant reductions evidenced for the Ashwagandha 225 mg/d group from Day 0 to Day 15 to Day 30. The placebo group had a non-significant increase in cortisol levels from Day 0 to Day 15-30. No adverse events were reported. In conclusion, Ashwagandha supplementation may improve the physiological, cognitive, and psychological effects of stress.

Glioblastoma Contains Topologically Distinct Proliferative and Metabolically Defined Subpopulations of Nestin- and Glut1-Expressing Cells.

The difficulty in treatment of glioblastoma is a consequence of its natural infiltrative growth and the existence of a population of therapy-resistant glioma cells that contribute to growth and recurrence. To identify cells more likely to have these properties, we examined the expression in tumor specimens of several protein markers important for glioma progression including the intermediate filament protein, Nestin (NES), a glucose transporter (Glut1/SLC2A1), the glial lineage marker, glial fibrillary acidic protein, and the proliferative indicator, Ki-67. We also examined the expression of von Willebrand factor, a marker for endothelial cells as well as the macrophage/myeloid markers CD163 and CD15. Using a multicolor immunofluorescence and hematoxylin and eosin staining approach with archival formalin-fixed, paraffin embedded tissue from primary, recurrent, and autopsy IDH1 wildtype specimens combined with high-resolution tissue image analysis, we have identified highly proliferative NES(+)/Glut1(-) cells that are preferentially perivascular. In contrast, Glut1(+)/NES(-) cells are distant from blood vessels, show low proliferation, and are preferentially located at the borders of pseudopalisading necrosis. We hypothesize that Glut1(+)/NES(-) cells would be naturally resistant to conventional chemotherapy and radiation due to their low proliferative capacity and may act as a reservoir for tumor recurrence.

Broad Spectrum Polyphenol Supplementation from Tart Cherry Extract on Markers of Recovery from Intense Resistance Exercise.

BACKGROUND: Tart cherry supplementation has been shown to enhance recovery from strenuous exercise due to its antioxidant properties. The majority of these studies used tart cherry juice, with a significant calorie content. The primary purpose of this study was to assess whether powdered tart cherry extract with minimal calorie content reduces oxidative stress and enhances recovery following intense resistance exercise. METHODS: Thirteen men (mean age: 26.2 ± 5.3 years; height: 184.3 ± 8.2 cm; weight: 92.9 ± 15.6 kg) performed a demanding resistance exercise protocol consisting of 6 sets of 10 repetitions of barbell back squat with 80% 1RM. The protocol was performed once following 7 days of 500 mg of tart cherry extract and once following placebo. Serum protein carbonyl (PC) content, creatine kinase activity (CK) and creatine kinase myocardial band content (CK-MB) were used to assess oxidative stress, skeletal and cardiac muscle damage respectively. Muscle soreness was assessed by visual analog scale. Physical performance was measured by countermovement jump power and handgrip dynamometer strength. RESULTS: There was a significant increase in PC in the placebo (PL) condition when compared to the Tart Cherry (TC) condition at Immediate Post (IP) (PL: 0.4 ± 0.3 vs. TC: - 0.4 ± 0.2 nmol∙mg- 1; p < 0.001), 1 h (PL: 0.3 ± 0.3 vs. TC: - 0.7 ± 0.3 nmol∙mg- 1; p < 0.001) and 24 h (PL: 0.1 ± 0.4 vs. TC: - 0.3 ± 0.5 nmol∙mg- 1; p = 0.010). There was a significant increase in CK activity in PL when compared to the TC at IP (PL: 491.1 ± 280 vs. TC: 296.3 ± 178 U∙L- 1; p = 0.008) and 3 h (PL: - 87 ± 123 vs. TC: 43.1 ± 105.3 U∙L- 1; p = 0.006). There was a significant (p = 0.003) increase in CKMB concentration in PL when compared to the TC (PL: 21.6 ± 12.4 vs. TC: - 0.3 ± 11.8 ng∙ml- 1; p = 0.006) at 1 h post. There was a significant increase in handgrip strength in TC when compared to PL (PL: - 2 ± 5.1 vs. TC: 1.7 ± 3 kg; p = 0.017) at 24 h post. CONCLUSIONS: This study demonstrated that tart cherry extract reduced oxidative stress and markers of muscle and cardiac damage following intense resistance exercise. This occurred along with a prevention of the decrease in handgrip strength seen following the intense exercise protocol, indicating a potential reduction in central fatigue. These benefits were seen with minimal energy intake.

Systemic Immune Bias Delineates Malignant Astrocytoma Survival Cohorts.

Patients with grade III anaplastic astrocytomas (AA) separate into survival cohorts based on the presence or absence of mutations in isocitrate dehydrogenase (IDH). Progression to glioblastoma (GBM), morphologically distinguishable by elevated microvascular proliferation, necrosis, and cell division in tumor tissues, is considerably more rapid in IDH wild-type tumors such that their diagnosis as AA is relatively rare. More often initially presenting as GBM, these contain higher numbers of tumor-associated macrophages (TAMs) than most AA, and GBM patients also have higher levels of circulating M2 monocytes. TAM and M2 monocytes share functional properties inhibitory for antitumor immunity. Yet, although there is a wealth of data implicating TAM in tumor-immune evasion, there has been limited analysis of the impact of the circulating M2 monocytes. In the current study, immune parameters in sera, circulating cells, and tumor tissues from patients with primary gliomas morphologically diagnosed as AA were assessed. Profound differences in serum cytokines, glioma extracellular vesicle cross-reactive Abs, and gene expression by circulating cells identified two distinct patient cohorts. Evidence of type 2-immune bias was most often seen in patients with IDH wild-type AA, whereas a type 1 bias was common in patients with tumors expressing the IDH1R132H mutation. Nevertheless, a patient's immune profile was better correlated with the extent of tumor vascular enhancement on magnetic resonance imaging than IDH mutational status. Regardless of IDH genotype, AA progression appears to be associated with a switch in systemic immune bias from type 1 to type 2 and the loss of tumor vasculature integrity.

Staphylococcus aureus Tet38 Efflux Pump Structural Modeling and Roles of Essential Residues in Drug Efflux and Host Cell Internalization.

The Staphylococcus aureus Tet38 membrane protein has distinct functions, including drug efflux and host cell attachment and internalization mediated by interaction with host cell CD36. Using structural modeling and site-directed mutagenesis, we identified key amino acids involved in different functions. Tet38, a member of the major facilitator superfamily, is predicted to have 14 transmembrane segments (TMS), 6 cytoplasmic loops, and 7 external loops. Cysteine substitutions of arginine 106 situated at the junction of TMS 4 and external loop L2, and glycine 151 of motif C on TMS 5, resulted in complete or near-complete (8- to 16-fold) reductions in Tet38-mediated resistance to tetracycline, with minimal to no effect on A549 host cell internalization. In contrast, a three-amino-acid deletion, F411P412G413, in external loop L7 situated between TMS 13 and 14 led to a decrease of 4-fold in S. aureus internalization by A549 cells and a partial effect on tetracycline resistance (4-fold reduction). A three-amino-acid deletion, D38D39L40, in external loop L1 situated between TMS-1 and TMS-2, had a similar partial effect on tetracycline resistance but did not affect cell internalization. Using an Ni column retention assay, we showed further that the L7, but not the L1, deletion impaired binding to CD36. Thus, the L7 domain of Tet38 is key for interaction with CD36 and host cell internalization, and amino acids R106 and G151 (TMSs 4 and 5) are particularly important for tetracycline resistance without affecting internalization.

Phase Ib Clinical Trial of IGV-001 for Patients with Newly Diagnosed Glioblastoma.

PURPOSE: Despite standard of care (SOC) established by Stupp, glioblastoma remains a uniformly poor prognosis. We evaluated IGV-001, which combines autologous glioblastoma tumor cells and an antisense oligonucleotide against IGF type 1 receptor (IMV-001), in newly diagnosed glioblastoma. PATIENTS AND METHODS: This open-label protocol was approved by the Institutional Review Board at Thomas Jefferson University. Tumor cells collected during resection were treated ex vivo with IMV-001, encapsulated in biodiffusion chambers with additional IMV-001, irradiated, then implanted in abdominal acceptor sites. Patients were randomized to four exposure levels, and SOC was initiated 4-6 weeks later. On the basis of clinical improvements, randomization was halted after patient 23, and subsequent patients received only the highest exposure. Safety and tumor progression were primary and secondary objectives, respectively. Time-to-event outcomes were compared with the SOC arms of published studies. RESULTS: Thirty-three patients were enrolled, and median follow-up was 3.1 years. Six patients had adverse events (grade ≤3) possibly related to IGV-001. Median progression-free survival (PFS) was 9.8 months in the intent-to-treat population (vs. SOC, 6.5 months; P = 0.0003). In IGV-001-treated patients who met Stupp-eligible criteria, PFS was 11.6 months overall (n = 22; P = 0.001) and 17.1 months at the highest exposure (n = 10; P = 0.0025). The greatest overall survival was observed in Stupp-eligible patients receiving the highest exposure (median, 38.2 months; P = 0.044). Stupp-eligible patients with methylated O6-methylguanine-DNA methyltransferase promoter (n = 10) demonstrated median PFS of 38.4 months (P = 0.0008). Evidence of immune activation was noted. CONCLUSIONS: IGV-001 was well tolerated, PFS compared favorably with SOC, and evidence suggested an immune-mediated mechanism (ClinicalTrials.gov: NCT02507583).

Brain tissue-resident immune memory cells are required for long-term protection against CNS infection with rabies virus.

Immune memory cells residing in previously infected, nonlymphoid tissues play a role in immune surveillance. In the event that circulating antibodies fail to prevent virus spread to the tissues in a secondary infection, these memory cells provide an essential defense against tissue reinfection. CNS tissues are isolated from circulating immune cells and antibodies by the blood-brain barrier, making the presence of tissue-resident immune memory cells particularly needed to combat recurrent infection by neurotropic viruses. Wild-type and laboratory-engineered rabies viruses are neurotropic, differ in pathogenicity, and have varying effects on BBB functions. These viruses have proven invaluable tools in demonstrating the importance of tissue-resident immune memory cells in the reinfection of CNS tissues. Only Type 1 immune memory is effective at therapeutically clearing a secondary infection with wild-type rabies viruses from the CNS and does so despite the maintenance of blood-brain barrier integrity.

Dual Role of Toll-Like Receptor 7 in the Pathogenesis of Rabies Virus in a Mouse Model.

Rabies, caused by rabies virus (RABV), is a fatal encephalitis in humans and other mammals, which continues to present a public health threat in most parts of the world. Our previous study demonstrated that Toll-like receptor 7 (TLR7) is essential in the induction of anti-RABV antibodies via the facilitation of germinal center formation. In the present study, we investigated the role of TLR7 in the pathogenicity of RABV in a mouse model. Using isolated plasmacytoid dendritic cells (pDCs), we demonstrated that TLR7 is an innate recognition receptor for RABV. When RABV invaded from the periphery, TLR7 detected viral single-stranded RNA and triggered immune responses that limited the virus's entry into the central nervous system (CNS). When RABV had invaded the CNS, its detection by TLR7 led to the production of cytokines and chemokines and an increase the permeability of the blood-brain barrier. Consequently, peripheral immune cells, including pDCs, macrophages, neutrophils, and B cells infiltrated the CNS. While this immune response, triggered by TLR7, helped to clear viruses, it also increased neuroinflammation and caused immunopathology in the mouse brain. Our results demonstrate that TLR7 is an innate recognition receptor for RABV, which restricts RABV invasion into the CNS in the early stage of viral infection but also contributes to immunopathology by inducing neuroinflammation.IMPORTANCE Developing targeted treatment for RABV requires understanding the innate immune response to the virus because early virus clearance is essential for preventing the fatality when the infection has progressed to the CNS. Previous studies have revealed that TLR7 is involved in the immune response to RABV. Here, we establish that TLR7 recognizes RABV and facilitates the production of some interferon-stimulated genes. We also demonstrated that when RABV invades into the CNS, TLR7 enhances the production of inflammatory cytokines which contribute to immunopathology in the mouse brain. Taken together, our findings suggest that treatments for RABV must consider the balance between the beneficial and harmful effects of TLR7-triggered immune responses.

Cosmic-ray antinuclei as messengers of new physics: status and outlook for the new decade.

The precise measurement of cosmic-ray antinuclei serves as an important means for identifying the nature of dark matter and other new astrophysical phenomena, and could be used with other cosmic-ray species to understand cosmic-ray production and propagation in the Galaxy. For instance, low-energy antideuterons would provide a "smoking gun" signature of dark matter annihilation or decay, essentially free of astrophysical background. Studies in recent years have emphasized that models for cosmic-ray antideuterons must be considered together with the abundant cosmic antiprotons and any potential observation of antihelium. Therefore, a second dedicated Antideuteron Workshop was organized at UCLA in March 2019, bringing together a community of theorists and experimentalists to review the status of current observations of cosmic-ray antinuclei, the theoretical work towards understanding these signatures, and the potential of upcoming measurements to illuminate ongoing controversies. This review aims to synthesize this recent work and present implications for the upcoming decade of antinuclei observations and searches. This includes discussion of a possible dark matter signature in the AMS-02 antiproton spectrum, the most recent limits from BESS Polar-II on the cosmic antideuteron flux, and reports of candidate antihelium events by AMS-02; recent collider and cosmic-ray measurements relevant for antinuclei production models; the state of cosmic-ray transport models in light of AMS-02 and Voyager data; and the prospects for upcoming experiments, such as GAPS. This provides a roadmap for progress on cosmic antinuclei signatures of dark matter in the coming years.

Low testosterone: Androgen deficiency, endurance exercise training, and competitive performance.

PURPOSE: Our intent was to (a) characterize weekly changes in resting testosterone (T), cortisol (C), and the T:C ratio in males during an intensive endurance training program, and (b) determine if clinical androgen deficiency (AD) based upon T-level criteria developed. METHODS: An 18-week training program in which individual training volume (km/week) increased at 25% increments over baseline (BL) levels observed prior to the study beginning at 4-week intervals throughout the first 12 weeks. After 12 weeks, the volume was reduced to that of the first 4 weeks until the study end (week 18). Competitive performance running tests were assessed at BL and every 4 weeks, while blood T and C were assessed weekly. RESULTS: Performance improved from BL at weeks 4-16 (p < 0.01). T was reduced (p < 0.01) from BL at weeks 3, and 5-18. The greatest reduction from BL was at week 13, subsequently T returned toward BL at week 18. C was highly variable, and no significant changes from BL were noted. The T:C ratio at weeks 5, 6, and 8-16 was significantly less than at BL (p < 0.01), the greatest reduction at week 13. The T:C ratio values also returned toward BL by week 18. Finally, ∼50% of the subjects reached T levels to be classified as AD. CONCLUSIONS: Sports scientists should recognize decreases in T or T:C ratio is not always indicative of compromised competitive performance potential. Clinicians should be aware increased training loads can lead to AD in men.

Tet38 of Staphylococcus aureus Binds to Host Cell Receptor Complex CD36-Toll-Like Receptor 2 and Protects from Teichoic Acid Synthesis Inhibitors Tunicamycin and Congo Red.

Using an affinity column retention assay, we showed that the purified Tet38 membrane transporter of Staphylococcus aureus bound specifically to host cell CD36 and to the complex CD36-Toll-like receptor 2 (TLR-2), but not to TLR-2 alone or TLR-2 and S. aureus lipoteichoic acid (LTA). We tested the effect of LTA on the internalization of S. aureustet38 mutant QT7 versus RN6390 by A549 epithelial cells. Addition of anti-LTA antibody to the bacteria prior to adding to A549 cells reduced internalization of QT7 2-fold compared to that with nonspecific antibody treatment. QT7 internalized 4- to 6-fold less than RN6390 with or without anti-LTA antibody. These data suggested that Tet38 and LTA were independently involved in the invasion process. The wall teichoic acid (WTA) inhibitor tunicamycin had an 8-fold decrease in activity with overexpression of tet38 and a 2-fold increase in activity in QT7 (tet38). Reserpine (an inhibitor of efflux pumps) reduced the effect of tet38 overexpression on tunicamycin resistance 4-fold. In addition, tet38 affected growth in the presence of LTA inhibitor Congo red, with overexpression increasing growth and deletion of tet38 reducing growth. In conclusion, Tet38 contributes to S. aureus invasion of A549 via direct binding to CD36 of the complex CD36-TLR-2, and LTA independently bound to TLR-2. The reduction of tunicamycin resistance in the presence of reserpine and the survival ability of the tet38 overexpressor in the presence of Congo red suggest that Tet38 can also protect the synthesis of LTA and WTA in S. aureus against their inhibitors, possibly functioning as an efflux pump.

Sphenopalatine ganglion stimulation is a reversible and frequency-dependent modulator of the blood-brain barrier.

BACKGROUND: The sphenopalatine ganglion (SPG) is a vasoactive mediator of the anterior intracranial circulation in mammals. SPG stimulation has been demonstrated to alter blood-brain barrier (BBB) permeability, although this phenomenon is not well characterized. OBJECTIVE: To determine the effect of SPG stimulation on the BBB using rat models. METHODS: Extravasation of fluorescent tracer 70 kDa FITC-dextran into rat brain specimens was measured across a range of stimulation parameters to assess BBB permeability. Tight junction (TJ) morphology was compared by assessing differences in the staining of proteins occludin and ZO-1 and analyzing ultrastructural changes on transmission electron microscopy (TEM) between stimulated and unstimulated specimens. RESULTS: SPG stimulation at 10 Hz maximally increased BBB permeability, exhibiting a 6-fold increase in fluorescent traceruptake (1.66% vs 0.28%, p < 0.0001). This effect was reversed 4-hours after stimulation (0.36% uptake, p = 0.99). High-frequency stimulation at 20 Hz and 200 Hz did not increase tracer extravasation, (0.26% and 0.28% uptake, p = >0.999 and p = 0.998, respectively). Stimulation was associated a significant decrease in the colocalization of occludin and ZO-1 with endothelial markers in stimulated brains compared to control (74.6% vs. 39.7% and 67.2% vs. 60.4% colocalization, respectively, p < 0.0001), and ultrastructural changes in TJ morphology associated with increased BBB permeability were observed on TEM. CONCLUSION: This study is the first to show a reversible, frequency-dependent increase in BBB permeability with SPG stimulation and introduces a putative mechanism of action through TJ disruption. Bypassing the BBB with SPG stimulation could enable new paradigms in delivering therapeutics to the CNS. Further study of this technology is needed.

Activated Extracellular Vesicles as New Therapeutic Targets?

A recent study by Genschmer et al. (Cell 2019;176:113-126) presents provocative new evidence that extracellular vesicles/exosomes released by activated neutrophils can degrade extracellular proteins. Elastase that is produced by neutrophils both coats the exosomes and is protected by the exosomes from inactivation by α1 antitrypsin, ultimately causing the pathological degradation of extracellular protein fibrils in lung alveoli.