Neuronal Zinc Transporter ZnT3 Modulates Cerebral Ischemia-Induced Blood-Brain Barrier Disruption.

Zinc plays important roles in both physiological and pathological processes in the brain. Accumulation of free zinc in ischemic tissue is recognized to contribute to blood-brain barrier (BBB) disruption following cerebral ischemia, but little is known either about the source of free zinc in microvessels or the mechanism by which free zinc mediates ischemia-induced BBB damage. We utilized cellular and animal models of ischemic stroke to determine the source of high levels of free zinc and the mechanism of free zinc-mediated BBB damage after ischemia. We report that cerebral ischemia elevated the level of extracellular fluid (ECF-Zn) of ischemic brain, leading to exacerbated BBB damage in a rat stroke model. Specifically suppressing zinc release from neurons, utilizing neuronal-specific zinc transporter 3 (ZnT3) knockout mice, markedly reduced ECF-Zn and BBB permeability after ischemia. Intriguingly, the activity of zinc-dependent metalloproteinase-2 (MMP-2) was modulated by ECF-Zn levels. Elevated ECF-Zn during ischemia directly bound to MMP-2 in extracellular fluid, increased its zinc content and augmented MMP-2 activity, leading to the degradation of tight junction protein in cerebral microvessels and BBB disruption. These findings suggest the role of neuronal ZnT3 in modulating ischemia-induced BBB disruption and reveal a novel mechanism of MMP-2 activation in BBB disruption after stroke, demonstrating ZnT3 as an effective target for stroke treatment.

Mammalian hybrid prophagophore is a precursor to autophagosomes.

The precursors to mammalian autophagosomes originate from preexisting membranes contributed by a number of sources, and subsequently enlarge through intermembrane lipid transfer, then close to sequester the cargo, and merge with lysosomes to degrade the cargo. Using cellular and in vitro membrane fusion analyses coupled with proteomic and biochemical studies we show that autophagosomes are formed from a hybrid membrane compartment referred to as a prophagophore or HyPAS (hybrid preautophagosomal structure). HyPAS is initially LC3-negative and subsequently becomes an LC3-positive phagophore. The prophagophore emerges through fusion of RB1CC1/FIP200-containing vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes. A specialized Ca2+-responsive apparatus controls prophagophore biogenesis and can be modulated by pharmacological agents such as SIGMAR1 agonists and antagonists including chloroquine. Autophagic prophagophore formation is inhibited during SARS-CoV-2 infection and is recapitulated by expression of SARS-CoV-2 nsp6. These findings show that mammalian autophagosomal prophagophores emerge via the convergence of secretory and endosomal pathways in a process that is targeted by microbial factors including coronaviral membrane proteins.Abbreviations: CLEM, correlative light and electron microscopy; CQ, chloroquine; HyPAS, hybrid preautophagosomal; strcuture/prophagophore; LC3, microtubule associated protein 1 light chain 3; RUPEX, a combination of RUSH and APEX2 systems; SARS-CoV-2, SARS-CoV-2 virus, causative agent of COVID19.

Mammalian hybrid pre-autophagosomal structure HyPAS generates autophagosomes.

The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.

Rapamycin modulates pulmonary pathology in a murine model of Mycobacterium tuberculosis infection.

Tuberculosis (TB) treatment regimens are lengthy, causing non-adherence to treatment. Inadequate treatment can lead to relapse and the development of drug resistance TB. Furthermore, patients often exhibit residual lung damage even after cure, increasing the risk for relapse and development of other chronic respiratory illnesses. Host-directed therapeutics are emerging as an attractive means to augment the success of TB treatment. In this study, we used C3HeB/FeJ mice as an experimental model to investigate the potential role of rapamycin, a mammalian target of rapamycin inhibitor, as an adjunctive therapy candidate during the treatment of Mycobacterium tuberculosis infection with moxifloxacin. We report that administration of rapamycin with or without moxifloxacin reduced infection-induced lung inflammation, and the number and size of caseating necrotic granulomas. Results from this study strengthen the potential use of rapamycin and its analogs as adjunct TB therapy, and importantly underscore the utility of the C3HeB/FeJ mouse model as a preclinical tool for evaluating host-directed therapy candidates for the treatment of TB.

Endogenous zinc protoporphyrin formation critically contributes to hemorrhagic stroke-induced brain damage.

Hemorrhagic stroke is a leading cause of death. The causes of intracerebral hemorrhage (ICH)-induced brain damage are thought to include lysis of red blood cells, hemin release and iron overload. These mechanisms, however, have not proven very amenable to therapeutic intervention, and so other mechanistic targets are being sought. Here we report that accumulation of endogenously formed zinc protoporphyrin (ZnPP) also critically contributes to ICH-induced brain damage. ICH caused a significant accumulation of ZnPP in brain tissue surrounding hematoma, as evidenced by fluorescence microscopy of ZnPP, and further confirmed by fluorescence spectroscopy and supercritical fluid chromatography-mass spectrometry. ZnPP formation was dependent upon both ICH-induced hypoxia and an increase in free zinc accumulation. Notably, inhibiting ferrochelatase, which catalyzes insertion of zinc into protoporphyrin, greatly decreased ICH-induced endogenous ZnPP generation. Moreover, a significant decrease in brain damage was observed upon ferrochelatase inhibition, suggesting that endogenous ZnPP contributes to the damage in ICH. Our findings reveal a novel mechanism of ICH-induced brain damage through ferrochelatase-mediated formation of ZnPP in ICH tissue. Since ferrochelatase can be readily inhibited by small molecules, such as protein kinase inhibitors, this may provide a promising new and druggable target for ICH therapy.

Ambroxol and Ciprofloxacin Show Activity Against SARS-CoV2 in Vero E6 Cells at Clinically-Relevant Concentrations.

We studied the activity of a range of weakly basic and moderately lipophilic drugs against SARS CoV2 in Vero E6 cells, using Vero E6 survival, qPCR of viral genome and plaque forming assays. No clear relationship between their weakly basic and hydrophobic nature upon their activity was observed. However, the approved drugs ambroxol and ciprofloxacin showed potent activity at concentrations that are clinically relevant and within their known safety profiles, and so may provide potentially useful agents for preclinical and clinical studies in COVID-19.

Azithromycin and ciprofloxacin have a chloroquine-like effect on respiratory epithelial cells.

There is interest in the use of chloroquine/hydroxychloroquine (CQ/HCQ) and azithromycin (AZT) in COVID-19 therapy. Employing cystic fibrosis respiratory epithelial cells, here we show that drugs AZT and ciprofloxacin (CPX) act as acidotropic lipophilic weak bases and confer in vitro effects on intracellular organelles similar to the effects of CQ. These seemingly disparate FDA-approved antimicrobials display a common property of modulating pH of endosomes and trans-Golgi network. We believe this may in part help understand the potentially beneficial effects of CQ/HCQ and AZT in COVID-19, and that the present considerations of HCQ and AZT for clinical trials should be extended to CPX.

What are the challenges in commercial non-tuberculous mycobacteria (NTM) drug discovery and how should we move forward?

Introduction: Despite the great clinical need, the development of drugs for treating non-tuberculous mycobacteria lung disease (NTM-LD), partly from the scientific difficulty, but also because the eventual size and types of markets for commercially-developed drugs has been unclear.Areas Covered: Here, key questions regarding the markets for commercial NTM-LD drugs are discussed, together with potential solutions for resolving these questions.Expert Opinion: Many of these questions will become better resoled over time, but uncertainties will remain around likely competition and whether approval and reimbursement will be achieved for all NTM-LD or smaller subsections of the market. Additionally, both 'push' and 'pull' incentives should be considered by policymakers to ensure the NTM-LD market is successfully addressed.

Potential for breath test diagnosis of urease positive pathogens in lung infections.

Roles for urease in virulence are accepted for Helicobacter pylori and urinary tract pathogens. However, urease is widely expressed by bacterial and fungal lung pathogens causing emerging and opportunistic lung infections, organisms causing acute exacerbations of chronic bronchitis, mycobacterial lung diseases such as tuberculosis (TB), and ventilator associated pneumonia and health care associated pneumonia. Detection of urease provides a method for rapid in vivo detection of these lung pathogens by inhaled 13C- breath test, and this review discusses the range of lung pathogens that might be amenable to rapid diagnosis.

Enhancement of lung levels of antibiotics by ambroxol and bromhexine.

INTRODUCTION: Major unmet needs remain for improved antibiotic treatment in lung infections. While development of new antibiotics is needed to overcome resistance, other approaches to optimize therapy using existing agents are also attractive. Ambroxol induces lung autophagy at human-relevant doses and improves lung levels of several approved antibiotics. Areas covered: This review discusses preclinical and clinical studies of the effects of ambroxol (and its prodrug precursor bromhexine) co-treatment upon levels of antibiotics in lung tissue, sputum, and bronchoalveolar lavage fluid. Expert opinion: Ambroxol co-treatment is associated with significant increases in lung tissue and airway surface fluid levels of a range of antibiotics including beta lactams, glycopeptides, macrolides, nitrofurans, and rifamycins. In most cases, the increased levels are only modest and are insufficient to overcome high-level resistance against that same antibiotic class, and so co-treatment with ambroxol is unlikely to alter clinical outcomes. Additionally, for most antibiotics there is no evidence that outcomes in non-resistant disease are improved by higher drug levels, and there is limited efficacy of co-treatment of antibiotics with ambroxol for most pathogens. The two cases where ambroxol may improve therapy are rifampin-sensitive tuberculosis and non-tuberculous mycobacterial infection, and vancomycin sensitive methicillin resistant Staphylococcus aureus pneumonia.

Pharmacist perceptions of the New Mexico pharmacist-performed tuberculosis testing program.

OBJECTIVE: This study evaluated pharmacists' perceptions of the New Mexico pharmacist-performed tuberculosis skin testing (PPTST) program. METHODS: This cross-sectional study was conducted using a telephone survey. New Mexico pharmacists who completed the tuberculin skin test (TST) training from March 2011 to June 2016 were eligible for inclusion. Data collected included demographics, years since licensure, pharmacy setting and location, reasons for obtaining certification, training time, training quality, self-perceived competency after training, whether the participant was performing TSTs, number of tests performed, time required to administer or interpret the test, and reasons for not testing. RESULTS: We attempted to contact all 209 pharmacists who completed the TST training during the evaluation period. Ninety-four of the 99 pharmacists contacted consented to participate (overall study response rate of 45%). The chain community pharmacy was the most common practice setting of respondents. After training completion, greater than 95% agreed or strongly agreed they felt confident in administering the TST. The percent of respondents working in New Mexico who were actively testing was 50.6%, with 42% of those pharmacists providing TSTs in small cities. Eleven pharmacists reported that they were performing TSTs in locations where testing would not otherwise have been available. An initial TST visit was approximately 6-15 minutes, and follow-up visits were typically 5 minutes or less. The most common reason reported for not testing was lack of employer support (61%). The strongest association with testing was training requirement by employer (odds ratio [OR], 20.4; 95% CI 4.2-99.2), followed by strong confidence in their ability to perform the TST (OR, 14.2; 95% CI 2.8-71.2). CONCLUSION: PPTST is positively perceived by New Mexico pharmacists and provides testing in non-urban areas where access may be low. Survey respondents were confident in their ability to perform the TST and report that testing typically takes less than 15 minutes. The main hindrance to implementing PPTST was lack of employer support.

Ambroxol Induces Autophagy and Potentiates Rifampin Antimycobacterial Activity.

Host-directed therapy in tuberculosis is a potential adjunct to antibiotic chemotherapy directed at Mycobacterium tuberculosis Ambroxol, a lead compound, emerged from a screen for autophagy-inducing drugs. At clinically relevant doses, ambroxol induced autophagy in vitro and in vivo and promoted mycobacterial killing in macrophages. Ambroxol also potentiated rifampin activity in a murine tuberculosis model.

Normobaric Hyperoxia Reduces Blood Occludin Fragments in Rats and Patients With Acute Ischemic Stroke.

BACKGROUND AND PURPOSE: Damage of the blood-brain barrier (BBB) increases the incidence of neurovascular complications, especially for cerebral hemorrhage after tPA (tissue-type plasminogen activator) therapy. Currently, there is no effective method to evaluate the extent of BBB damage to guide tPA use. Herein, we investigated whether blood levels of tight junction proteins could serve as biomarker of BBB damages in acute ischemic stroke (AIS) in both rats and patients. We examined whether this biomarker could reflect the extent of BBB permeability during cerebral ischemia/reperfusion and the effects of normobaric hyperoxia (NBO) on BBB damage. METHODS: Rats were exposed to NBO (100% O2) or normoxia (21% O2) during middle cerebral artery occlusion. BBB permeability was determined. Occludin and claudin-5 in blood and cerebromicrovessels were measured. Patients with AIS were assigned to oxygen therapy or room air for 4 hours, and blood occludin and claudin-5 were measured at different time points after stroke. RESULTS: Cerebral ischemia/reperfusion resulted in the degradation of occludin and claudin-5 in microvessels, leading to increased BBB permeability in rats. In blood samples, occludin increased with 4-hour ischemia and remained elevated during reperfusion, correlating well with its loss from ischemic cerebral microvessels. NBO treatment both prevented occludin degradation in microvessels and reduced occludin levels in blood, leading to improved neurological functions in rats. In patients with AIS receiving intravenous tPA thrombolysis, the blood occludin was already elevated when patients arrived at hospital (within 4.5 hours since symptoms appeared) and remained at a high level for 72 hours. NBO significantly lowered the level of blood occludin and improved neurological functions in patients with AIS. CONCLUSIONS: Blood occludin may be a clinically viable biomarker for evaluating BBB damage during ischemia/reperfusion. NBO therapy has the potential to reduce blood occludin, protect BBB, and improve outcome in AIS patients with intravenous tPA thrombolysis. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02974283.

Deuterated drugs; updates and obviousness analysis.

INTRODUCTION: The pharmacokinetics and/or toxicity of many known drugs can be modified by selective deuteration, an area of significant commercial interest and scientific and regulatory progress. Areas covered: This review firstly discusses recent developments in deuterated drugs including the FDA approval of deutetrabenazine. Secondly, it discusses 35 U.S.C. §103 'obviousness' as it relates to recent patent prosecution, and also to Inter Partes Review (IPR). IPR is a new post-award review of patentability under §102 or §103, two IPR petitions upon deuterated drugs have been instituted and included §103 arguments. Finally, an extended analysis of §103 obviousness based upon the practices of major pharmaceutical companies is provided, that supports rather late priority dates, while §102 is also discussed. Expert opinion: The total value of transactions involving deuterated drugs is close to $5 billion. While the importance of §103 'obviousness' rejections remains in patent applications under current prosecution, IPR of issued patents is developing and will affect likely affect §103 interpretations in this area. However, patents are still issuing with later priority dates, and further litigation will likely occur.

Stool microbiome reveals diverse bacterial ureases as confounders of oral urea breath testing for Helicobacter pylori and Mycobacterium tuberculosis in Bamako, Mali.

Detection of bacterial urease activity has been utilized successfully to diagnose Helicobacter pylori (H. pylori). While Mycobacterium tuberculosis (M. tuberculosis) also possesses an active urease, it is unknown whether detection of mycobacterial urease activity by oral urease breath test (UBT) can be exploited as a rapid point of care biomarker for tuberculosis (TB) in humans. We enrolled 34 individuals newly diagnosed with pulmonary TB and 46 healthy subjects in Bamako, Mali and performed oral UBT, mycobacterial sputum culture and H. pylori testing. Oral UBT had a sensitivity and specificity (95% CI) of 70% (46-88%) and 11% (3-26%), respectively, to diagnose culture-confirmed M. tuberculosis disease among patients without H. pylori, and 100% sensitivity (69-100%) and 11% specificity (3-26%) to diagnose H. pylori among patients without pulmonary TB. Stool microbiome analysis of controls without TB or H. pylori but with positive oral UBT detected high levels of non-H. pylori urease producing organisms, which likely explains the low specificity of oral UBT in this setting and in other reports of oral UBT studies in Africa.

Stable isotope biomarker breath tests for human metabolic and infectious diseases: a review of recent patent literature.

INTRODUCTION: Stable isotope breath tests can rapidly and quantitatively report metabolic phenotypes and disease in both humans and microbes in situ. The labelled compound is administered and acted upon by human or microbial metabolism, producing a labelled gas that is detected in exhaled breath. Areas covered: This review details the unique advantages (and disadvantages) of phenotypic stable isotope based breath tests. A review of recent US patent applications and prosecutions since 2010 is conducted. Finally, current clinical trials, product pipelines and approved products are discussed. Expert opinion: Stable isotope breath tests offer new approaches for rapid and minimally invasive detection and study of metabolic phenotypes, both human and microbial. The patent literature has developed considerably in the last 6 years, with over 30 patent applications made. Rates of issuance remain high, although rejections citing 35 U.S.C. §101(subject matter eligibility), §102 (novelty), §103 (obviousness) and §112 (description, enablement and best mode) have occurred. The prior art is significantly greater for human metabolism than microbial, and may drive differing rates of future issuance. These biomarker and diagnostic tools can enable optimization of drug doses, diagnosis of metabolic disease and its progression, and detection of infectious disease and optimize its treatment.

Pharmaceutical screen identifies novel target processes for activation of autophagy with a broad translational potential.

Autophagy is a conserved homeostatic process active in all human cells and affecting a spectrum of diseases. Here we use a pharmaceutical screen to discover new mechanisms for activation of autophagy. We identify a subset of pharmaceuticals inducing autophagic flux with effects in diverse cellular systems modelling specific stages of several human diseases such as HIV transmission and hyperphosphorylated tau accumulation in Alzheimer's disease. One drug, flubendazole, is a potent inducer of autophagy initiation and flux by affecting acetylated and dynamic microtubules in a reciprocal way. Disruption of dynamic microtubules by flubendazole results in mTOR deactivation and dissociation from lysosomes leading to TFEB (transcription factor EB) nuclear translocation and activation of autophagy. By inducing microtubule acetylation, flubendazole activates JNK1 leading to Bcl-2 phosphorylation, causing release of Beclin1 from Bcl-2-Beclin1 complexes for autophagy induction, thus uncovering a new approach to inducing autophagic flux that may be applicable in disease treatment.

Spray-Dried Multiscale Nano-biocomposites Containing Living Cells.

Three-dimensional encapsulation of cells within nanostructured silica gels or matrices enables applications as diverse as biosensors, microbial fuel cells, artificial organs, and vaccines; it also allows the study of individual cell behaviors. Recent progress has improved the performance and flexibility of cellular encapsulation, yet there remains a need for robust scalable processes. Here, we report a spray-drying process enabling the large-scale production of functional nano-biocomposites (NBCs) containing living cells within ordered 3D lipid-silica nanostructures. The spray-drying process is demonstrated to work with multiple cell types and results in dry powders exhibiting a unique combination of properties including highly ordered 3D nanostructure, extended lipid fluidity, tunable macromorphologies and aerodynamic diameters, and unexpectedly high physical strength. Nanoindentation of the encasing nanostructure revealed a Young's modulus and hardness of 13 and 1.4 GPa, respectively. We hypothesized this high strength would prevent cell growth and force bacteria into viable but not culturable (VBNC) states. In concordance with the VBNC state, cellular ATP levels remained elevated even over eight months. However, their ability to undergo resuscitation and enter growth phase greatly decreased with time in the VBNC state. A quantitative method of determining resuscitation frequencies was developed and showed that, after 36 weeks in a NBC-induced VBNC, less than 1 in 10,000 cells underwent resuscitation. The NBC platform production of large quantities of VBNC cells is of interest for research in bacterial persistence and screening of drugs targeting such cells. NBCs may also enable long-term preservation of living cells for applications in cell-based sensing and the packaging and delivery of live-cell vaccines.

Detecting virulence and drug-resistance mycobacterial phenotypes in vivo.

Bacterial phenotypes are predominantly studied in culture because detection of their specific metabolic pathways in the host is challenging. Development of stable-isotope breath tests, allowing in situ phenotype analyses, may endow diagnostics with new modalities based upon direct monitoring of in vivo microbial metabolism and host-pathogen phenotypic interactions.