Nicotinamide restores tissue NAD+ and improves survival in rodent models of cardiac arrest.

Metabolic suppression in the ischemic heart is characterized by reduced levels of NAD+ and ATP. Since NAD+ is required for most metabolic processes that generate ATP, we hypothesized that nicotinamide restores ischemic tissue NAD+ and improves cardiac function in cardiomyocytes and isolated hearts, and enhances survival in a mouse model of cardiac arrest. Mouse cardiomyocytes were exposed to 30 min simulated ischemia and 90 min reperfusion. NAD+ content dropped 40% by the end of ischemia compared to pre-ischemia. Treatment with 100 µM nicotinamide (NAM) at the start of reperfusion completely restored the cellular level of NAD+ at 15 min of reperfusion. This rescue of NAD+ depletion was associated with improved contractile recovery as early as 10 min post-reperfusion. In a mouse model of cardiac arrest, 100 mg/kg NAM administered IV immediately after cardiopulmonary resuscitation resulted in 100% survival at 4 h as compared to 50% in the saline group. In an isolated rat heart model, the effect of NAM on cardiac function was measured for 20 min following 18 min global ischemia. Rate pressure product was reduced by 26% in the control group following arrest. Cardiac contractile function was completely recovered with NAM treatment given at the start of reperfusion. NAM restored tissue NAD+ and enhanced production of lactate and ATP, while reducing glucose diversion to sorbitol in the heart. We conclude that NAM can rapidly restore cardiac NAD+ following ischemia and enhance glycolysis and contractile recovery, with improved survival in a mouse model of cardiac arrest.

Dissecting the RecA-(In)dependent Response to Mitomycin C in Mycobacterium tuberculosis Using Transcriptional Profiling and Proteomics Analyses.

Mycobacteria exploit at least two independent global systems in response to DNA damage: the LexA/RecA-dependent SOS response and the PafBC-regulated pathway. Intracellular pathogens, such as Mycobacterium tuberculosis, are exposed to oxidative and nitrosative stress during the course of infection while residing inside host macrophages. The current understanding of RecA-independent responses to DNA damage is based on the saprophytic model of Mycobacterium smegmatis, a free-living and nonpathogenic mycobacterium. The aim of the present study was to identify elements of RecA-independent responses to DNA damage in pathogenic intracellular mycobacteria. With the help of global transcriptional profiling, we were able to dissect RecA-dependent and RecA-independent pathways. We profiled the DNA damage responses of an M. tuberculosis strain lacking the recA gene, a strain with an undetectable level of the PafBC regulatory system, and a strain with both systems tuned down simultaneously. RNA-Seq profiling was correlated with the evaluation of cell survival in response to DNA damage to estimate the relevance of each system to the overall sensitivity to genotoxic agents. We also carried out whole-cell proteomics analysis of the M. tuberculosis strains in response to mitomycin C. This approach highlighted that LexA, a well-defined key element of the SOS system, is proteolytically inactivated during RecA-dependent DNA repair, which we found to be transcriptionally repressed in response to DNA-damaging agents in the absence of RecA. Proteomics profiling revealed that AlkB was significantly overproduced in the ΔrecA pafBCCRISPRi/dCas9 strain and that Holliday junction resolvase RuvX was a DNA damage response factor that was significantly upregulated regardless of the presence of functional RecA and PafBC systems, thus falling into a third category of DNA damage factors: RecA- and PafBC-independent. While invisible to the mass spectrometer, the genes encoding alkA, dnaB, and dnaE2 were significantly overexpressed in the ΔrecA pafBCCRISPRi/dCas9 strain at the transcript level.

Cobalamin is present in cells of non-tuberculous mycobacteria, but not in Mycobacterium tuberculosis.

Cobalamin (vitamin B12) is a structurally complex molecule that acts as a cofactor for enzymes and regulates gene expression through so-called riboswitches. The existing literature on the vitamin B12 synthesis capacity in Mycobacterium tuberculosis is ambiguous, while in non-tuberculous mycobacteria (NTM) is rather marginal. Here we present the results of our investigation into the occurrence of vitamin B12 in mycobacteria. For detection purposes, immunoassay methods were applied to cell lysates of NTM and M. tuberculosis clinical and laboratory strains grown under different conditions. We show that whereas vitamin B12 is present in cells of various NTM species, it cannot be evidenced in strains of differently cultured M. tuberculosis, even though the genes responsible for vitamin B12 synthesis are actively expressed based on RNA-Seq data. In summary, we conclude that the production of vitamin B12 does occur in mycobacteria, with the likely exception of M. tuberculosis. Our results provide direct evidence of vitamin B12 synthesis in a clinically important group of bacteria.

TAT delivery of a PTEN peptide inhibitor has direct cardioprotective effects and improves outcomes in rodent models of cardiac arrest.

We have recently shown that pharmacologic inhibition of PTEN significantly increases cardiac arrest survival in a mouse model, however, this protection required pretreatment 30 min before the arrest. To improve the onset of PTEN inhibition during cardiac arrest treatment, we have designed a TAT fused cell-permeable peptide (TAT-PTEN9c) based on the C-terminal PDZ binding motif of PTEN for rapid tissue delivery and protection. Western blot analysis demonstrated that TAT-PTEN9c peptide significantly enhanced Akt activation in mouse cardiomyocytes in a concentration- and time-dependent manner. Mice were subjected to 8 min asystolic arrest followed by CPR, and 30 mice with successful CPR were then randomly assigned to receive either saline or TAT-PTEN9c treatment. Survival was significantly increased in TAT-PTEN9c-treated mice compared with that of saline control at 4 h after CPR. The treated mice had increased Akt phosphorylation at 30 min resuscitation with significantly decreased sorbitol content in heart or brain tissues and reduced release of taurine and glutamate in blood, suggesting improved glucose metabolism. In an isolated rat heart Langendorff model, direct effects of TAT-PTEN9c on cardiac function were measured for 20 min following 20 min global ischemia. Rate pressure product was reduced by >20% for both TAT vehicle and nontreatment groups following arrest. Cardiac contractile function was completely recovered with TAT-PTEN9c treatment given at the start of reperfusion. We conclude that TAT-PTEN9c enhances Akt activation and decreases glucose shunting to the polyol pathway in critical organs, thereby preventing osmotic injury and early cardiovascular collapse and death.NEW & NOTEWORTHY We have designed a cell-permeable peptide, TAT-PTEN9c, to improve cardiac arrest survival. It blocked endogenous PTEN binding to its adaptor and enhanced Akt signaling in mouse cardiomyocytes. It improved mouse survival after cardiac arrest, which is related to improved glucose metabolism and reduced glucose shunting to sorbitol in critical organs.

ATP-Dependent Ligases and AEP Primases Affect the Profile and Frequency of Mutations in Mycobacteria under Oxidative Stress.

The mycobacterial nonhomologous end-joining pathway (NHEJ) involved in double-strand break (DSB) repair consists of the multifunctional ATP-dependent ligase LigD and the DNA bridging protein Ku. The other ATP-dependent ligases LigC and AEP-primase PrimC are considered as backup in this process. The engagement of LigD, LigC, and PrimC in the base excision repair (BER) process in mycobacteria has also been postulated. Here, we evaluated the sensitivity of Mycolicibacterium smegmatis mutants defective in the synthesis of Ku, Ku-LigD, and LigC1-LigC2-PrimC, as well as mutants deprived of all these proteins to oxidative and nitrosative stresses, with the most prominent effect observed in mutants defective in the synthesis of Ku protein. Mutants defective in the synthesis of LigD or PrimC/LigC presented a lower frequency of spontaneous mutations than the wild-type strain or the strain defective in the synthesis of Ku protein. As identified by whole-genome sequencing, the most frequent substitutions in all investigated strains were T→G and A→C. Double substitutions, as well as insertions of T or CG, were exclusively identified in the strains carrying functional Ku and LigD proteins. On the other hand, the inactivation of Ku/LigD increased the efficiency of the deletion of G in the mutant strain.

A Cross-Sectional Survey on Telemedicine Use for Doctor-Patient Communication.

Introduction Use of computers for doctor-patient communication is increasing. Considering effective doctor-patient communication is important for good health outcomes. This study helps to determine the level of acceptance of telemedicine in general public and factors associated with it. Methods: This survey with cross-sectional analysis comprised a brief survey with 15 questions. The survey was distributed in public places to determine the opinions of the general public. Results Randomly selected 125 participants completed the questionnaire. Synchronous telemedicine was favored by young people (82% in the 18-34 age group vs 37.5% of participants aged >55 years; p<0.01), those with a higher education level (46.7% of non-college-educated persons vs 80.6% of college-educated persons; p<0.01), and frequent computer users (67% who used a computer for less than two hours a month vs 86.5% of those who used a computer more than hours a month; p=0.03). Asynchronous communication, like sending health information to doctors via a safe portal was acknowledged mostly by people who had used patient portals in the past (84.1% vs 65.4%; p=0.02). Use of patient portals was less among older users and senior citizens (20.8% use in the age group >55 vs. 51.3% in the age group 35-53 years vs. 71% in age group 18-34 years). Receiving video education for specific health concerns was favored by those who used a computer frequently (94.6% who used a computer more than two hours a month vs 77% who used a computer less than two hours a month; p =0.02). Conclusion Telemedicine is generally favored, but physicians should be mindful about older people as they may not feel comfortable. Step by step guidance should be provided especially to senior citizens for telemedicine and portal use.

Early Drug Development and Evaluation of Putative Antitubercular Compounds in the -Omics Era.

Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis. According to the WHO, the disease is one of the top 10 causes of death of people worldwide. Mycobacterium tuberculosis is an intracellular pathogen with an unusually thick, waxy cell wall and a complex life cycle. These factors, combined with M. tuberculosis ability to enter prolonged periods of latency, make the bacterium very difficult to eradicate. The standard treatment of TB requires 6-20months, depending on the drug susceptibility of the infecting strain. The need to take cocktails of antibiotics to treat tuberculosis effectively and the emergence of drug-resistant strains prompts the need to search for new antitubercular compounds. This review provides a perspective on how modern -omic technologies facilitate the drug discovery process for tuberculosis treatment. We discuss how methods of DNA and RNA sequencing, proteomics, and genetic manipulation of organisms increase our understanding of mechanisms of action of antibiotics and allow the evaluation of drugs. We explore the utility of mathematical modeling and modern computational analysis for the drug discovery process. Finally, we summarize how -omic technologies contribute to our understanding of the emergence of drug resistance.