Mycobacterium tuberculosis Rv3802c Encodes a Phospholipase/Thioesterase and Is Inhibited by the Antimycobacterial Agent Tetrahydrolipstatin

The cell wall of M. tuberculosis is central to its success as a pathogen. Mycolic acids are key components of this cell wall. The genes involved in joining the α and mero mycolates are located in a cluster, beginning with Rv3799c and extending at least until Rv3804c. The role of each enzyme encoded by these five genes is fairly well understood, except for Rv3802c. Rv3802 is one of seven putative cutinases encoded by the genome of M. tuberculosis. In phytopathogens, cutinases hydrolyze the waxy layer of plants, cutin. In a strictly mammalian pathogen, such as M. tuberculosis, it is likely that these proteins perform a different function. Of the seven, we chose to focus on Rv3802c because of its location in a mycolic acid synthesis gene cluster, its putative essentiality, its ubiquitous presence in actinomycetes, and its conservation in the minimal genome of Mycobacterium leprae. We expressed Rv3802 in Escherichia coli and purified the enzymatically active form. We probed its activities and inhibitors characterizing those relevant to its possible role in mycolic acid biosynthesis. In addition to its reported phospholipase A activity, Rv3802 has significant thioesterase activity, and it is inhibited by tetrahydrolipstatin (THL). THL is a described anti-tuberculous compound with an unknown mechanism, but it reportedly targets cell wall synthesis. Taken together, these data circumstantially support a role for Rv3802 in mycolic acid synthesis and, as the cell wall is integral to M. tuberculosis pathogenesis, identification of a novel cell wall enzyme and its inhibition has therapeutic and diagnostic implications

Meta-analysis: Neither quick nor easy

BACKGROUND: Meta-analysis is often considered to be a simple way to summarize the existing literature. In this paper we describe how a meta-analysis resembles a conventional study, requiring a written protocol with design elements that parallel those of a record review. METHODS: The paper provides a structure for creating a meta-analysis protocol. Some guidelines for measurement of the quality of papers are given. A brief overview of statistical considerations is included. Four papers are reviewed as examples. The examples generally followed the guidelines we specify in reporting the studies and results, but in some of the papers there was insufficient information on the meta-analysis process. CONCLUSIONS: Meta-analysis can be a very useful method to summarize data across many studies, but it requires careful thought, planning and implementation

Total and corrected antioxidant capacity in hemodialyzed patients

BACKGROUND: Oxidative stress may play a critical role in the vascular disease of end stage renal failure and hemodialysis patients. Studies, analyzing either discrete analytes and antioxidant substances, or the integrated total antioxidant activity of human plasma during hemodialysis, give contradictory results. METHODS: Recently, we have introduced a new automated method for the determination of Total Antioxidant Capacity (TAC) of human plasma. We have serially measured TAC and corrected TAC (cTAC: after subtraction of the interactions due to endogenous uric acid, bilirubin and albumin) in 10 patients before the onset of the dialysis session, 10 min, 30 min, 1 h, 2 h and 3 h into the procedure and after completion of the session. RESULTS: Our results indicate that TAC decreases, reaching minimum levels at 2 h. However, corrected TAC increases with t(1/2 )of about 30 min. We then repeated the measurements in 65 patients undergoing dialysis with different filters (36 patients with ethylene vinyl alcohol copolymer resin filter -Eval-, 23 patients with two polysulfone filters -10 with F6 and 13 with PSN140-, and 6 patients with hemophan filters). Three specimens were collected (0, 30, 240 min). The results of this second group confirm our initial results, while no significant difference was observed using either filter. CONCLUSIONS: Our results are discussed under the point of view of possible mechanisms of modification of endogenous antioxidants, and the interaction of lipid- and water-soluble antioxidants

Canagliflozin and Renal Outcomes in Type 2 Diabetes and Nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to 300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m 2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Causes of molecular convergence and parallelism in protein evolution

To what extent is the convergent evolution of protein function attributable to convergent or parallel changes at the amino acid level? The mutations that contribute to adaptive protein evolution may represent a biased subset of all possible beneficial mutations owing to mutation bias and/or variation in the magnitude of deleterious pleiotropy. A key finding is that the fitness effects of amino acid mutations are often conditional on genetic background. This context dependence (epistasis) can reduce the probability of convergence and parallelism because it reduces the number of possible mutations that are unconditionally acceptable in divergent genetic backgrounds. Here, I review factors that influence the probability of replicated evolution at the molecular level