Kinetics and Ligand-Binding Preferences of Mycobacterium tuberculosis Thymidylate Synthases, ThyA and ThyX

Mycobacterium tuberculosis kills approximately 2 million people each year and presents an urgent need to identify new targets and new antitubercular drugs. Thymidylate synthase (TS) enzymes from other species offer good targets for drug development and the M. tuberculosis genome contains two putative TS enzymes, a conventional ThyA and a flavin-based ThyX. In M. tuberculosis, both TS enzymes have been implicated as essential for growth, either based on drug-resistance studies or genome-wide mutagenesis screens. To facilitate future small molecule inhibitors against these proteins, a detailed enzymatic characterization was necessary.After cloning, overexpression, and purification, the thymidylate-synthesizing ability of ThyA and ThyX gene products were directly confirmed by HPLC analysis of reaction products and substrate saturation kinetics were established. 5-Fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) was a potent inhibitor of both ThyA and ThyX, offering important clues to double-targeting strategies. In contrast, the folate-based 1843U89 was a potent inhibitor of ThyA but not ThyX suggesting that it should be possible to find ThyX-specific antifolates. A turnover-dependent kinetic assay, combined with the active-site titration approach of Ackermann and Potter, revealed that both M. tuberculosis enzymes had very low k(cat) values. One possible explanation for the low catalytic activity of M. tuberculosis ThyX is that its true biological substrates remain to be identified. Alternatively, this slow-growing pathogen, with low demands for TMP, may have evolved to down-regulate TS activities by altering the turnover rate of individual enzyme molecules, perhaps to preserve total protein quantities for other purposes. In many organisms, TS is often used as a part of larger complexes of macromolecules that control replication and DNA repair.Thus, the present enzymatic characterization of ThyA and ThyX from M. tuberculosis provides a framework for future development of cell-active inhibitors and the biological roles of these TS enzymes in M. tuberculosis

Potential biological role of poly (ADP-ribose) polymerase (PARP) in male gametes

Maintaining the integrity of sperm DNA is vital to reproduction and male fertility. Sperm contain a number of molecules and pathways for the repair of base excision, base mismatches and DNA strand breaks. The presence of Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, and its homologues has recently been shown in male germ cells, specifically during stage VII of spermatogenesis. High PARP expression has been reported in mature spermatozoa and in proven fertile men. Whenever there are strand breaks in sperm DNA due to oxidative stress, chromatin remodeling or cell death, PARP is activated. However, the cleavage of PARP by caspase-3 inactivates it and inhibits PARP's DNA-repairing abilities. Therefore, cleaved PARP (cPARP) may be considered a marker of apoptosis. The presence of higher levels of cPARP in sperm of infertile men adds a new proof for the correlation between apoptosis and male infertility. This review describes the possible biological significance of PARP in mammalian cells with the focus on male reproduction. The review elaborates on the role played by PARP during spermatogenesis, sperm maturation in ejaculated spermatozoa and the potential role of PARP as new marker of sperm damage. PARP could provide new strategies to preserve fertility in cancer patients subjected to genotoxic stresses and may be a key to better male reproductive health

Genetic Studies of Leptin Concentrations Implicate Leptin in the Regulation of Early Adiposity.

Leptin influences food intake by informing the brain about the status of body fat stores. Rare LEP mutations associated with congenital leptin deficiency cause severe early-onset obesity that can be mitigated by administering leptin. However, the role of genetic regulation of leptin in polygenic obesity remains poorly understood. We performed an exome-based analysis in up to 57,232 individuals of diverse ancestries to identify genetic variants that influence adiposity-adjusted leptin concentrations. We identify five novel variants, including four missense variants, in LEP, ZNF800, KLHL31, and ACTL9, and one intergenic variant near KLF14. The missense variant Val94Met (rs17151919) in LEP was common in individuals of African ancestry only, and its association with lower leptin concentrations was specific to this ancestry (P = 2 × 10-16, n = 3,901). Using in vitro analyses, we show that the Met94 allele decreases leptin secretion. We also show that the Met94 allele is associated with higher BMI in young African-ancestry children but not in adults, suggesting that leptin regulates early adiposity

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Preoperative cerebrospinal fluid cytokine levels and the risk of postoperative delirium in elderly hip fracture patients

Aging and neurodegenerative disease predispose to delirium and are both associated with increased activity of the innate immune system resulting in an imbalance between pro- and anti-inflammatory mediators in the brain. We examined whether hip fracture patients who develop postoperative delirium have altered levels of inflammatory mediators in cerebrospinal fluid (CSF) prior to surgery. Patients were 75 years and older and admitted for surgical repair of an acute hip fracture. CSF samples were collected preoperatively. In an exploratory study, we measured 42 cytokines and chemokines by multiplex analysis. We compared CSF levels between patients with and without postoperative delirium and examined the association between CSF cytokine levels and delirium severity. Delirium was diagnosed with the Confusion Assessment Method; severity of delirium was measured with the Delirium Rating Scale Revised-98. Mann-Whitney U tests or Student t-tests were used for between-group comparisons and the Spearman correlation coefficient was used for correlation analyses. Sixty-one patients were included, of whom 23 patients (37.7%) developed postsurgical delirium. Concentrations of Fms-like tyrosine kinase-3 (P=0.021), Interleukin-1 receptor antagonist (P=0.032) and Interleukin-6 (P=0.005) were significantly lower in patients who developed delirium postoperatively. Our findings fit the hypothesis that delirium after surgery results from a dysfunctional neuroinflammatory response: stressing the role of reduced levels of anti-inflammatory mediators in this process. The Effect of Taurine on Morbidity and Mortality in the Elderly Hip Fracture Patient.Registration number: NCT00497978. Local ethical protocol number: NL16222.094.0

Differentiation of human adipose-derived stem cells into neuron/motoneuron-like cells for cell replacement therapy of spinal cord injury

Human adipose-derived stem cells (hADSCs) are increasingly presumed to be a prospective stem cell source for cell replacement therapy in various degenerative and/or traumatic diseases. The potential of trans-differentiating hADSCs into motor neuron cells indisputably provides an alternative way for spinal cord injury (SCI) treatment. In the present study, a stepwise and efficient hADSC trans-differentiation protocol with retinoic acid (RA), sonic hedgehog (SHH), and neurotrophic factors were developed. With this protocol hADSCs could be converted into electrophysiologically active motoneuron-like cells (hADSC-MNs), which expressed both a cohort of pan neuronal markers and motor neuron specific markers. Moreover, after being primed for neuronal differentiation with RA/SHH, hADSCs were transplanted into SCI mouse model and they survived, migrated, and integrated into injured site and led to partial functional recovery of SCI mice. When ablating the transplanted hADSC-MNs harboring HSV-TK-mCherry overexpression system with antivirial Ganciclovir (GCV), functional relapse was detected by motor-evoked potential (MEP) and BMS assays, implying that transplanted hADSC-MNs participated in rebuilding the neural circuits, which was further confirmed by retrograde neuronal tracing system (WGA). GFP-labeled hADSC-MNs were subjected to whole-cell patch-clamp recording in acute spinal cord slice preparation and both action potentials and synaptic activities were recorded, which further confirmed that those pre-conditioned hADSCs indeed became functionally active neurons in vivo. As well, transplanted hADSC-MNs largely prevented the formation of injury-induced cavities and exerted obvious immune-suppression effect as revealed by preventing astrocyte reactivation and favoring the secretion of a spectrum of anti-inflammatory cytokines and chemokines. Our work suggests that hADSCs can be readily transformed into MNs in vitro, and stay viable in spinal cord of the SCI mouse and exert multi-therapeutic effects by rebuilding the broken circuitry and optimizing the microenvironment through immunosuppression

Effects of total fat intake on body fatness in adults

Background: The ideal proportion of energy from fat in our food and its relation to body weight is not clear. In order to prevent overweight and obesity in the general population, we need to understand the relationship between the proportion of energy from fat and resulting weight and body fatness in the general population. Objectives: To assess the effects of proportion of energy intake from fat on measures of body fatness (including body weight, waist circumference, percentage body fat and body mass index) in people not aiming to lose weight, using all appropriate randomised controlled trials (RCTs) of at least six months duration. Search methods: We searched CENTRAL, MEDLINE, Embase, Clinicaltrials.gov and the WHO International Clinical Trials Registry Platform (ICTRP) to October 2019. We did not limit the search by language. Selection criteria: Trials fulfilled the following criteria: 1) randomised intervention trial, 2) included adults aged at least 18 years, 3) randomised to a lower fat versus higher fat diet, without the intention to reduce weight in any participants, 4) not multifactorial and 5) assessed a measure of weight or body fatness after at least six months. We duplicated inclusion decisions and resolved disagreement by discussion or referral to a third party. Data collection and analysis: We extracted data on the population, intervention, control and outcome measures in duplicate. We extracted measures of body fatness (body weight, BMI, percentage body fat and waist circumference) independently in duplicate at all available time points. We performed random-effects meta-analyses, meta-regression, subgrouping, sensitivity, funnel plot analyses and GRADE assessment. Main results: We included 37 RCTs (57,079 participants). There is consistent high-quality evidence from RCTs that reducing total fat intake results in small reductions in body fatness; this was seen in almost all included studies and was highly resistant to sensitivity analyses (GRADE high-consistency evidence, not downgraded). The effect of eating less fat (compared with higher fat intake) is a mean body weight reduction of 1.4 kg (95% confidence interval (CI) -1.7 to -1.1 kg, in 53,875 participants from 26 RCTs, I2 = 75%). The heterogeneity was explained in subgrouping and meta-regression. These suggested that greater weight loss results from greater fat reductions in people with lower fat intake at baseline, and people with higher body mass index (BMI) at baseline. The size of the effect on weight does not alter over time and is mirrored by reductions in BMI (MD -0.5 kg/m2, 95% CI -0.6 to -0.3, 46,539 participants in 14 trials, I2 = 21%), waist circumference (MD -0.5 cm, 95% CI -0.7 to -0.2, 16,620 participants in 3 trials; I2 = 21%), and percentage body fat (MD -0.3% body fat, 95% CI -0.6 to 0.00, P = 0.05, in 2350 participants in 2 trials; I2 = 0%). There was no suggestion of harms associated with low fat diets that might mitigate any benefits on body fatness. The reduction in body weight was reflected in small reductions in LDL (-0.13 mmol/L, 95% CI -0.21 to -0.05), and total cholesterol (-0.23 mmol/L, 95% CI -0.32 to -0.14), with little or no effect on HDL cholesterol (-0.02 mmol/L, 95% CI -0.03 to 0.00), triglycerides (0.01 mmol/L, 95% CI -0.05 to 0.07), systolic (-0.75 mmHg, 95% CI -1.42 to -0.07) or diastolic blood pressure(-0.52 mmHg, 95% CI -0.95 to -0.09), all GRADE high-consistency evidence or quality of life (0.04, 95% CI 0.01 to 0.07, on a scale of 0 to 10, GRADE low-consistency evidence). Authors' conclusions: Trials where participants were randomised to a lower fat intake versus a higher fat intake, but with no intention to reduce weight, showed a consistent, stable but small effect of low fat intake on body fatness: slightly lower weight, BMI, waist circumference and percentage body fat compared with higher fat arms. Greater fat reduction, lower baseline fat intake and higher baseline BMI were all associated with greater reductions in weight. There was no evidence of harm to serum lipids, blood pressure or quality of life, but rather of small benefits or no effect