Flow Cytometric Assessment of CD26-Positive Leukemic Stem Cells: A Rapid and Valuable Tool in the Diagnosis and Follow-Up of Chronic Myeloid Leukemia.

Context Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm. Recent studies have suggested that CD26-positive leukemic stem cells (LSCs) circulating in peripheral blood are specific for CML. Objective This study was undertaken to determine the proportion of CD26-positive LSCs at diagnosis and its change during tyrosine kinase inhibitor therapy. Design This prospective study was conducted on 43 cases of CML at diagnosis. For flow cytometry, peripheral blood cells were stained with CD45, CD34, CD38, CD3, and CD26. A sequential gating strategy with CD45/SSC (side scatter), CD34/SSC, and CD34/CD38 was applied to identify CD45+/34+/38- populations, from which CD26-positive stem cells were identified and compared with controls. Data analysis was done with Kaluza software. Results All patients diagnosed with CML were detected with CD26-positive LSCs. The median percentage of CD26-positive CML LSCs was 0.02 with a range of 0.001 to 1.77. None of the control samples showed CD26 positivity. The percentage and absolute count of CD26-positive CML LSCs were reduced after six months of tyrosine kinase therapy in patients with complete hematological remission. Conclusion Flow cytometric analysis of circulating CD26-positive CML LSCs is a non-invasive, rapid, and useful tool in the diagnosis and follow-up of CML.

A multicenter prospective randomized controlled trial investigating the effects of combustion-free nicotine alternatives on cardiovascular risk factors and metabolic parameters in individuals with type 2 diabetes who smoke: the DiaSmokeFree study protocol.

Stopping smoking is crucial for public health and especially for individuals with diabetes. Combustion-free nicotine alternatives like e-cigarettes and heated tobacco products are increasingly being used as substitutes for conventional cigarettes, contributing to the decline in smoking prevalence. However, there is limited information about the long-term health impact of those products in patients with diabetes. This randomized controlled trial aims to investigate whether switching from conventional cigarettes to combustion-free nicotine alternatives will lead to a measurable improvement in cardiovascular risk factors and metabolic parameters over a period of 2 years in smokers with type 2 diabetes. The multicenter study will be conducted in seven sites across four countries. A total of 576 smokers with type 2 diabetes will be randomly assigned (1:2 ratio) to either standard of care with brief cessation advice (Control Arm) or combustion-free nicotine alternatives use (Intervention Arm). The primary end point is the change in the proportion of patients with metabolic syndrome between baseline and the 2-year follow-up. Additionally, the study will analyze the absolute change in the sum of the individual factors of metabolic syndrome at each study time point. Patient recruitment has started in September 2021 and enrollment is expected to be completed by December 2023. Results will be reported in 2026. This study may provide valuable insights into cardiovascular and metabolic health benefits or risks associated with using combustion-free nicotine alternatives for individuals with type 2 diabetes who are seeking alternatives to tobacco cigarette smoking. The study protocol, informed consent forms, and relevant documents were approved by seven ethical review boards. Study results will be disseminated through articles published in high-quality, peer-reviewed journals and presentations at conferences.

Improving the Health Literacy of Patients: A Qualitative Survey of Student Pharmacists.

Background Patients interacting with the multiple moving parts of the healthcare system may not fully understand all the information provided to them. They find themselves misinformed or unaware of certain facts pertaining to their health. Community pharmacists, who are readily accessible, are occupationally situated in such a way that makes them the most ideal candidates to impact and improve patients' health literacy. Aims and objectives The aim of this research is to identify and examine how pharmacists can aid their patients and help them toward adequate health literacy. Methods First-year pharmacy candidates enrolled in the Doctor of Pharmacy (PharmD) program at Temple University School of Pharmacy were given an optional, essay-based assignment titled, "How can pharmacists improve the health literacy of the patients they serve?" Students were given one week to respond to the prompt, and those who completed the assignment were awarded bonus points toward the final examination. Of the 145 students given the potential to complete the assignment, only 100 students participated in the assignment. These essays were subsequently read, categorized, and analyzed. Results The essays were categorized into six main perspectives: educating patients, using simple language, providing resources, creating a trusting patient-pharmacist relationship, sending medication reminders, and providing translation services. More than 30% of the student responses emphasized that educating patients about their medication side effects and disease states will help them better understand their medical needs. About another 30% of the students suggested that using simple, everyday language and providing translators, when necessary, will help patients deal with their health problems. The remaining suggested either creating a safe relationship with patients, providing reliable resources such as pamphlets and visual aids, or sending patient-specific reminders. Conclusion Although these suggestions are not new to the world of pharmacy and have been around before, the difficulty lies in practical application in a world that seems busier now than ever before. However, within the six student perspective response categories, most can be incorporated into one patient interaction. By creating a trusting relationship with the patient and counseling them while using simple language and translators, if necessary, teaching them about their medications, and providing the necessary outside resources, pharmacists can improve the health literacy of their patients.

Systemic prime exacerbates the ocular immune response to heat-killed Mycobacterium tuberculosis.

Post-infectious uveitis describes the condition of chronic immune mediated ocular inflammation associated with pathogens such as Mycobacterium tuberculosis (Mtb). Mtb associated post-infectious uveitis can be modeled in mice by intravitreal injection of heat-killed Mtb (HKMtb). To better understand how prior systemic exposure to the pathogen alters the local immune response to Mtb, we used flow cytometry and multiplex ELISAs to compare ocular responses to intravitreal HKMtb in the presence or absence of a systemic "prime" of HKMtb. Priming resulted in exacerbation of local inflammation with significantly increased clinical and histologic inflammation scores and increased vitreous cytokines concentrations one day after intravitreal injection of HKMtb. Seven days after injection, uveitis in unprimed animals had largely resolved. In contrast in primed animals, clinical signs of chronic inflammation were associated with a significant increase in the number of ocular T cells, NK cells, and Ly6Chi macrophages and increasing vitreous concentrations of IL-17, VEGF, MIG(CXCL9), IP-10(CXCL10), IL-12p40 and MIP-1α(CCL3). In mice lacking mature T and B cells (RAG2 deficient), the impact of priming on the ocular immune response was ameliorated with significantly lower vitreous cytokine concentrations and spontaneous resolution of uveitis. Altogether these results suggest that the ocular response to Mtb is exacerbated by prior systemic Mtb infection and chronic post-infectious uveitis is mediated by local production of cytokines and chemokines that amplify Th17 and Th1 responses. This mouse model of chronic Mtb associated uveitis will help elucidate mechanisms of disease in patients with post-infectious uveitis.

Data-driven readiness: A preliminary report on cataloging best practices in military civilian partnerships.

BACKGROUND: Between conflicts, many of the combat casualty care lessons learned are lost as the nation shifts priorities and providers leave the military. Solutions are needed to bridge the knowledge gap created by interwar periods. One of the foremost solutions is partnerships between civilian trauma centers and the military health system. Over the past two decades, a myriad of military-civilian partnerships (MCPs), which vary in their composition, duration, and focus, was created. The objective of this report is to describe the initial attempt of the Department of Defense to catalog existing MCPs to inform both civilian and military stakeholders. This initial catalog is intended as a reference to aid in future MCP development and facilitate the synchronization of efforts to improve trauma care delivery and readiness. METHODS: Using methodology from the Institute of Defense Analysis, the total number of eligible trauma centers in the United States was determined. The Institute of Defense Analysis determined eligibility-based American College of Surgeons Trauma Center verification or state trauma center designation. Each military service provided their list of MCPs, which were categorized. Military-civilian partnerships were cataloged by various characteristics and program components. Key variables include number and type of personnel trained, duration of training, and focus, for example, team versus individual focused and training versus maintaining proficiency focused. RESULTS: A total of 1,139 hospitals in the United States are potentially eligible for MCPs. There are at least 87 unique partnerships; the majority are part-time sustainment MCPs. The Air Force has the largest number of providers in MCPs. There are many challenges to maintain accurate and up to date data on MCPs. CONCLUSION: With the collated information, the Defense Health Agency, military services, special operations community, and civilian partners will be better empowered to optimize the readiness value of their programs and better prepare our military medical providers for the nation's and military's future needs.

Case 301: Traumatic Subarachnoid-Pleural Fistula.

HISTORY: A 38-year-old man who had been in a motor vehicle collision was referred to our institute. He was suspected of having left-sided pneumothorax. This necessitated intercostal drainage with a chest tube, which had been placed elsewhere prior to his arrival. Paraparesis was noted at the initial clinical examination, with adequately maintained vital signs, while the blood work-up revealed a mildly reduced hemoglobin level of 10.1 mg/dL (normal range, 13.8-17.2 mg/dL); however, the rest of the laboratory values were within normal limits. The patient was then immediately referred for further evaluation with CT of the brain, cervical spine, and thorax. Thereafter, serial chest radiography was performed for follow-up.

Case 301.

History A 38-year-old man who had been in a motor vehicle collision was referred to our institute. He was suspected of having left-sided pneumothorax. This necessitated intercostal drainage with a chest tube, which had been placed elsewhere prior to his arrival. Paraparesis was noted on the initial clinical examination, with adequately maintained vital signs, while the blood work-up revealed a mildly reduced hemoglobin level of 10.1 mg/dL (normal range, 13.8-17.2 mg/dL); however, the rest of the laboratory values were within normal limits (Figs 1-5). The patient was then immediately referred for further evaluation with CT of the brain, cervical spine, and thorax. Thereafter, serial chest radiography was performed for follow-up.

Clinical efficacy of nitric oxide nasal spray (NONS) for the treatment of mild COVID-19 infection.

Baek et al.1 investigated the duration of COVID-19 virus shedding in infected patients and demonstrated that even in patients demonstrating prolonged viral clearance, the virus was no longer viable after 15 days post onset of symptoms. Our study aimed to measure whether nitric oxide nasal spray (NONS) further accelerates this reduction in SARS-CoV-2 RNA load versus a control arm with saline spray. Our study recruited 80 participants who were divided into a NONS treatment arm or a placebo arm to test the efficacy of NONS as a treatment for mild COVID-19 infection.

International randomised controlled trial evaluating metabolic syndrome in type 2 diabetic cigarette smokers following switching to combustion-free nicotine delivery systems: the DIASMOKE protocol.

INTRODUCTION: Reducing exposure to cigarette smoke is an imperative for public health and for patients with diabetes. Increasingly, combustion-free nicotine delivery systems (C-F NDS) such as e-cigarettes and heated tobacco products are substituting conventional cigarettes and accelerating the downward trends in smoking prevalence. However, there is limited information about the long-term health impact in patients with diabetes who use C-F NDS. This randomised trial of type 2 diabetic cigarette smokers will test the hypothesis that following a switch from conventional cigarettes to C-F NDS a measurable improvement in metabolic syndrome (MetS) factors will be shown over the course of 2 years. METHODS AND ANALYSIS: The study is multicentre and thus will take place in five locations in four countries in an ambulatory setting. A total of 576 patients with diabetes will be randomised (1:2 ratio) to either a control arm (Study Arm A), in which they will be offered referral to smoking cessation programmes or to an intervention arm (Study Arm B) assigned to C-F NDS use. Participants will be at least 23 years old and of any gender. Patient recruitment will start in February 2021 and is expected to be completed by December 2021. Primary outcome measures include fasting plasma glucose, blood pressure, triglycerides, high-density lipoprotein and waist circumference, while secondary feature absolute change in the sum of the individual factors of MetS and change in each individual factor of MetS measured at each study time point. ETHICS AND DISSEMINATION: The approval of research ethics committee (REC) regarding the trial protocol, informed consent forms and other relevant documents is required to commence the study. Substantial amendments to the study protocol cannot be implemented until the REC grants a favourable opinion. The results of the study are intended to be published as articles in high quality peer-reviewed journals and disseminated through conference papers. TRIAL REGISTRATION NUMBER: NCT04231838. Pre-results stage.

Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis.

The term 'uveitis' describes a heterogeneous set of conditions that all feature intraocular inflammation. Broadly, uveitis is defined by etiology: infection or autoimmunity. Infectious uveitis requires treatment with the appropriate antimicrobial agents, while autoimmune uveitis requires treatment with corticosteroids or other immunosuppressive agents. Post-infectious uveitis is a form of chronic uveitis that requires corticosteroids to control immune sequela following the initial infection. Uveitis associated with Mycobacterium tuberculosis (Mtb) infection is a well-recognized form of post-infectious uveitis, but the mechanisms of disease are not fully understood. To understand the role mycobacterial antigens and innate ligands play in stimulating chronic ocular inflammation following mTB infection, the model Primed Mycobacterial Uveitis (PMU) was developed for use in mice. This manuscript outlines the methods for generating PMU and monitoring the clinical course of inflammation using color fundus and optical coherence tomography (OCT) imaging. PMU is induced by immunization with heat-killed mycobacterial extract followed by intravitreal injection of the same extract into one eye seven days later. Ocular inflammation is monitored longitudinally using in vivo imaging and followed by sample collection for a wide range of assays, including histology, flow cytometry, cytokine analysis, qPCR, or mRNA sequencing. The mouse model of PMU is a useful new tool for studying the ocular responses to mTB, the mechanism of chronic uveitis, and for preclinical effectiveness tests of new anti-inflammatory therapies.

A Methodology for Estimating the Cost of Educating Military Physicians at the Uniformed Services University.

INTRODUCTION: The Department of Defense (DoD) operates a large, multi-channeled physician accession pipeline to maintain a professional workforce of over 10,000 active duty physicians. The Uniformed Services University (USU) operates the nation's only federal medical school providing trained doctors to the Army, Navy, Air Force, and Public Health Service. Although the school serves an essential purpose, policymakers question the cost of operating the University's medical school. One challenge is to develop reproducible and transparent costing methods that can be used to evaluate the University's value and efficiency. METHODS: This work proposes a replicable methodology for estimating the cost per student-year at USU. Using detailed data from USU encompassing facility use, budgeting and expenditures, and faculty and student rosters, we break out and attribute costs to the University's component schools. Using faculty and staff time-use surveys, we further break out education-related personnel costs from other University activities such as research and service. We can then calculate the School of Medicine's annual cost to educate a uniformed physician. RESULTS: In Fiscal Year 2017, it cost the DoD approximately $253,000 per year (more than $1 million dollars total over a 4-year curriculum) to directly educate a physician though the USU School of Medicine. Data from the following Fiscal Year show that education costs grew a modest 2.1% per student-year. CONCLUSIONS: This work provides a foundational framework and approach to estimate the costs of accessioning a physician at USU. This methodology can be replicated for subsequent value analyses of physician accession and retention as budgetary pressures change to match the DoD operating environment. Uniformed Services University's costs should be periodically reassessed against those of alternative accession sources.

COVID-19 outcomes in UK centre within highest health and wealth band: a prospective cohort study.

OBJECTIVES: To describe the characteristics and outcomes of hospitalised patients with COVID-19 from UK in the highest decile of health and gross regional products per capita. DESIGN: Prospective cohort study. SETTING: Recruited all adult inpatients with laboratory-confirmed COVID-19 symptoms admitted to a single Surrey centre between March and April 2020. Extensive demographic details were documented. OUTCOME MEASURE: COVID-19 status of alive/dead and intensive care unit (ICU) status of yes/no. PARTICIPANTS: Patients with COVID-19 from Surrey centre UK (n=429). RESULTS: 429 adult inpatients (mean age 70±18 years; men 56.4%) were included in this study, of whom, 19.1% required admission to ICU and 31.9% died. Adverse outcomes were associated with age (OR with each decade of years: 1.78, 95% CI 1.53 to 2.11, p<0.001 for mortality); male gender (OR=1.08, 95% CI 0.72 to 1.63, p=0.72, present in 70.7%, of admissions to ICU versus 53% of other cases, p=0.004); cardiac disease (OR=3.43, 95% CI 2.10 to 5.63, p<0.001), diabetes mellitus (OR=2.37, 95% CI 1.09 to 5.17, p=0.028) and dementia (OR=5.06, 95% CI 2.79 to 9.44, p<0.001). There was no significant impact of ethnicity or body mass index on disease outcome. CONCLUSIONS: Despite reports of worse outcomes in deprived regions, we show similar complication and mortality rates due to COVID-19 in an affluent and high life expectancy region.

Bioluminescence for in vivo detection of cell-type-specific inflammation in a mouse model of uveitis.

This study reports the use of cell-type-specific in vivo bioluminescence to measure intraocular immune cell population dynamics during the course of inflammation in a mouse model of uveitis. Transgenic lines expressing luciferase in inflammatory cell subsets (myeloid cells, T cells, and B cells) were generated and ocular bioluminescence was measured serially for 35 days following uveitis induction. Ocular leukocyte populations were identified using flow cytometry and compared to the ocular bioluminescence profile. Acute inflammation is neutrophilic (75% of ocular CD45 + cells) which is reflected by a significant increase in ocular bioluminescence in one myeloid reporter line on day 2. By day 7, the ocular T cell population increases to 50% of CD45 + cells, leading to a significant increase in ocular bioluminescence in the T cell reporter line. While initially negligible (< 1% of CD45 + cells), the ocular B cell population increases to > 4% by day 35. This change is reflected by a significant increase in the ocular bioluminescence of the B cell reporter line starting on day 28. Our data demonstrates that cell-type-specific in vivo bioluminescence accurately detects changes in multiple intraocular immune cell populations over time in experimental uveitis. This assay could also be useful in other inflammatory disease models.

In Vitro and In Vivo Characterization of Potent Antileishmanial Methionine Aminopeptidase 1 Inhibitors.

Leishmania major is the causative agent of cutaneous leishmaniasis (CL). No human vaccine is available for CL, and current drug regimens present several drawbacks, such as emerging resistance, severe toxicity, medium effectiveness, and/or high cost. Thus, the need for better treatment options against CL is a priority. In the present study, we validate the enzyme methionine aminopeptidase 1 of L. major (MetAP1Lm), a metalloprotease that catalyzes the removal of N-terminal methionine from peptides and proteins, as a chemotherapeutic target against CL infection. The in vitro antileishmanial activities of eight novel MetAP1 inhibitors (OJT001 to OJT008) were investigated. Three compounds, OJT006, OJT007, and OJT008, demonstrated potent antiproliferative effects in macrophages infected with L. major amastigotes and promastigotes at submicromolar concentrations, with no cytotoxicity against host cells. Importantly, the leishmanicidal effect in transgenic L. major promastigotes overexpressing MetAP1Lm was diminished by almost 10-fold in comparison to the effect in wild-type promastigotes. Furthermore, the in vivo activities of OJT006, OJT007, and OJT008 were investigated in L. major-infected BALB/c mice. In comparison to the footpad parasite load in the control group, OJT008 decreased the footpad parasite load significantly, by 86%, and exhibited no toxicity in treated mice. We propose MetAP1 inhibitor OJT008 as a potential chemotherapeutic candidate against CL infection caused by L. major infection.

Galanin suppresses visceral afferent responses to noxious mechanical and inflammatory stimuli.

Galanin is a neuropeptide expressed by sensory neurones innervating the gastrointestinal (GI) tract. Galanin displays inhibitory effects on vagal afferent signaling within the upper GI tract, and the goal of this study was to determine the actions of galanin on colonic spinal afferent function. Specifically, we sought to evaluate the effect of galanin on lumbar splanchnic nerve (LSN) mechanosensitivity to noxious distending pressures and the development of hypersensitivity in the presence of inflammatory stimuli and colitis. Using ex vivo electrophysiological recordings we show that galanin produces a dose-dependent suppression of colonic LSN responses to mechanical stimuli and prevents the development of hypersensitivity to acutely administered inflammatory mediators. Using galanin receptor (GalR) agonists, we show that GalR1 activation, but not GalR2/3 activation, suppresses mechanosensitivity. The effect of galanin on colonic afferent activity was not observed in tissue from mice with dextran sodium sulfate-induced colitis. We conclude that galanin has a marked suppressive effect on colonic mechanosensitivity at noxious distending pressures and prevents the acute development of mechanical hypersensitivity to inflammatory mediators, an effect not seen in the inflamed colon. These actions highlight a potential role for galanin in the regulation of mechanical nociception in the bowel and the therapeutic potential of targeting galaninergic signaling to treat visceral hypersensitivity.

Pharmacokinetics of isoniazid: The good, the bad, and the alternatives.

Although isoniazid (INH) has been successful in treating Tuberculosis (TB) since its introduction in 1952, there has been continual reports of drug-associated hepatotoxicity in TB patients. These toxic side effects may reveal more about the recipient of the drug, than the drug itself. A combination of pharmacogenetic and pharmacokinetic studies have identified polymorphisms within enzymes involved in INH metabolism and detoxification. These essential metabolic enzymes include N-acetyltransferase 2, Cytochrome P450 2E1, and glutathione S transferases. Different phenotypes of these enzymes can affect the rate of INH metabolism, resulting in production of hepatotoxic metabolites. This review is intended to elucidate the pharmacokinetics of INH by examining its Administration, Distribution, Metabolism, and Elimination, while suggesting potential alternatives within INH personalized treatment to help reduce hepatotoxicity.

Targeted disruption of PI3K/Akt/mTOR signaling pathway, via PI3K inhibitors, promotes growth inhibitory effects in oral cancer cells.

PURPOSE: The phosphoinositide-3-kinase (PI3K) pathway is the frequently altered in human cancer. This has led to the development and study of novel PI3K inhibitors for targeted therapy and also to overcome resistance to radiotherapy. METHOD: The anti-tumour effects of PI3K inhibitors (PI-828, PI-103 and PX-866) in terms of cell proliferation, colony formation, induction of apoptosis, cell cycle arrest, invasion, autophagy, and pNF-κB/p65 translocation in SCC-4, SCC-9 and SCC-25 cells were studied by performing MTT, clonogenic, DAPI staining, propidium iodide staining, annexin-V binding, matrigel invasion, acridine orange staining and immuno-fluorescence assay. Western blot assay was performed to assess the alteration in the expression of various proteins. RESULT: PI-828 and PI-103 treatment exhibited dose-dependent inhibition of growth and proliferation of OSCC cells with a concomitant induction of apoptosis, altered cell cycle regulation and decreased invasiveness (p < 0.01). PX-866 induced apoptosis, cell cycle arrest, autophagy and a significant decrease in the invasiveness of oral cancer cells as compared to untreated cells (p < 0.01). These compounds significantly reduced expression of COX-2, cyclin-D1 and VEGF in the treated cells besides cytoplasmic accumulation of pNF-κB/p65 protein. In addition to PI3Kα, inactivation of downstream components, i.e. Akt and mTOR was seen. CONCLUSION: PI3K inhibitors such as PI-103, PI-828 and PX-866 may be developed as potential therapeutic agents for effective treatment of oral squamous cell carcinoma (OSCC) patients, associated with activated PI3K/Akt pathway.

Socialização de idosos institucionalizados: oficina de pintura em uma ILPI de Rio Grande, RS / Socialization of institutionalized elderly: painting workshop in a ILPI of Rio Grande, RS / Socialización de ancianos institucionalizados: taller de pintura en una ILPI de Rio Grande, RS

A Oficina de Pintura é um projeto de extensão do Programa Núcleo Universitário da Terceira Idade da Universidade Federal do Rio Grande (NUTI/FURG), realizado em uma Instituição de Longa Permanência para Idosos. O objetivo do presente trabalho é relatar a experiência de acadêmicas do Curso de Psicologia na implementação desse projeto com idosos institucionalizados. Em encontros semanais foram disponibilizados desenhos e materiais de pintura. A intervenção mostrou-se um espaço de possibilidades para o desenvolvimento de interações sociais entre os idosos participantes.

The Painting Workshop is an extension program of the Third Age University Center of the Federal University of Rio Grande (NUTI/FURG) which takes place at a Long Term Care Institution for the Aged. The objective of this paper is to report the experience of academics of the Psychology Course in the implementation of this project with elder persons that live in these institutions. Drawings and painting supplies were provided in weekly meetings. The experience created manifold possibilities for the development of social interactions among the elderly participants.

El Taller de Pintura es un proyecto de extensión del Programa Núcleo Universitario de la Tercera Edad de la Universidad Federal de Rio Grande (NUTI / FURG), realizado en una Institución de Larga Permanencia para ancianos. El objetivo del presente trabajo es relatar la experiencia de académicas del Curso de Psicología en la implementación de ese proyecto con ancianos institucionalizados. En encuentros semanales se pusieron a disposición dibujos y materiales de pintura. La intervención se mostró un espacio de posibilidades para el desarrollo de interacciones sociales entre los ancianos participantes.

Characterization of 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone as a novel inhibitor of methionine aminopeptidases from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) and the Human Immunodeficiency Virus (HIV) pose a major public health threat. The 2015 World Health Organization (WHO) report estimates that one in three HIV deaths is due to Mtb, the causative agent of Tuberculosis (TB). The lethal synergy between these two pathogens leads to a decline in the immune function of infected individuals as well as a rise in morbidity and mortality rates. The deadly interaction between TB and HIV, along with the heightened emergence of drug resistance, drug-drug interactions, reduced drug efficacy and increased drug toxicity, has made the therapeutic management of co-infected individuals a major challenge. Hence, the development of new drug targets and/or new drug leads are imperative for the effective therapeutic management of co-infected patients. Here, we report the characterization of 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone (311), a known inhibitor of HIV-1 replication and transcription as a new inhibitor of methionine aminopeptidases (MetAPs) from Mycobacterium tuberculosis: MtMetAP1a and MtMetAP1c. MetAP is a metalloprotease that removes the N-terminal methionine during protein synthesis. The essential role of MetAP in microbes makes it a promising chemotherapeutic target. We demonstrated that 311 is a potent and selective inhibitor of MtMetAP1a and MtMetAP1c. Furthermore, we found that 311 is active against replicating and aged non-growing Mtb at low micromolar concentrations. These results suggest that 311 is a promising lead for the development of novel class of therapeutic agents with dual inhibition of TB and HIV for the treatment of TB-HIV co-infection.

X-Ray Structure and Inhibition of 3C-like Protease from Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that infects pigs and can have mortality rates approaching 100% in piglets, causing serious economic impact. The 3C-like protease (3CL(pro)) is essential for the coronaviral life cycle and is an appealing target for the development of therapeutics. We report the expression, purification, crystallization and 2.10 Å X-ray structure of 3CL(pro) from PEDV. Analysis of the PEDV 3CL(pro) structure and comparison to other coronaviral 3CL(pro)'s from the same alpha-coronavirus phylogeny shows that the overall structures and active site architectures across 3CL(pro)'s are conserved, with the exception of a loop that comprises the protease S2 pocket. We found a known inhibitor of severe acute respiratory syndrome coronavirus (SARS-CoV) 3CL(pro), (R)-16, to have inhibitor activity against PEDV 3CL(pro), despite that SARS-3CL(pro) and PEDV 3CL(pro) share only 45.4% sequence identity. Structural comparison reveals that the majority of residues involved in (R)-16 binding to SARS-3CL(pro) are conserved in PEDV-3CL(pro); however, the sequence variation and positional difference in the loop forming the S2 pocket may account for large observed difference in IC50 values. This work advances our understanding of the subtle, but important, differences in coronaviral 3CL(pro) architecture and contributes to the broader structural knowledge of coronaviral 3CL(pro)'s.