The proofreading exonuclease subunit ε of Escherichia coli DNA polymerase III is tethered to the polymerase subunit α via a flexible linker

Escherichia coli DNA polymerase III holoenzyme is composed of 10 different subunits linked by noncovalent interactions. The polymerase activity resides in the α-subunit. The ε-subunit, which contains the proofreading exonuclease site within its N-terminal 185 residues, binds to α via a segment of 57 additional C-terminal residues, and also to θ, whose function is less well defined. The present study shows that θ greatly enhances the solubility of ε during cell-free synthesis. In addition, synthesis of ε in the presence of θ and α resulted in a soluble ternary complex that could readily be purified and analyzed by NMR spectroscopy. Cell-free synthesis of ε from PCR-amplified DNA coupled with site-directed mutagenesis and selective 15N-labeling provided site-specific assignments of NMR resonances of ε that were confirmed by lanthanide-induced pseudocontact shifts. The data show that the proofreading domain of ε is connected to α via a flexible linker peptide comprising over 20 residues. This distinguishes the α : ε complex from other proofreading polymerases, which have a more rigid multidomain structure

Mamld1 Knockdown Reduces Testosterone Production and Cyp17a1 Expression in Mouse Leydig Tumor Cells

MAMLD1 is known to be a causative gene for hypospadias. Although previous studies have indicated that MAMLD1 mutations result in hypospadias primarily because of compromised testosterone production around the critical period for fetal sex development, the underlying mechanism(s) remains to be clarified. Furthermore, although functional studies have indicated a transactivation function of MAMLD1 for the non-canonical Notch target Hes3, its relevance to testosterone production remains unknown. To examine these matters, we performed Mamld1 knockdown experiments.Mamld1 knockdown was performed with two siRNAs, using mouse Leydig tumor cells (MLTCs). Mamld1 knockdown did not influence the concentrations of pregnenolone and progesterone but significantly reduced those of 17-OH pregnenolone, 17-OH progesterone, dehydroepiandrosterone, androstenedione, and testosterone in the culture media. Furthermore, Mamld1 knockdown significantly decreased Cyp17a1 expression, but did not affect expressions of other genes involved in testosterone biosynthesis as well as in insulin-like 3 production. Hes3 expression was not significantly altered. In addition, while 47 genes were significantly up-regulated (fold change >2.0×) and 38 genes were significantly down-regulated (fold change <0.5×), none of them was known to be involved in testosterone production. Cell proliferation analysis revealed no evidence for compromised proliferation of siRNA-transfected MLTCs.The results, in conjunction with the previous data, imply that Mamld1 enhances Cyp17a1 expression primarily in Leydig cells and permit to produce a sufficient amount of testosterone for male sex development, independently of the Hes3-related non-canonical Notch signaling

Recommendations for a core outcome set for measuring standing balance in adult populations: a consensus-based approach

Standing balance is imperative for mobility and avoiding falls. Use of an excessive number of standing balance measures has limited the synthesis of balance intervention data and hampered consistent clinical practice.To develop recommendations for a core outcome set (COS) of standing balance measures for research and practice among adults.A combination of scoping reviews, literature appraisal, anonymous voting and face-to-face meetings with fourteen invited experts from a range of disciplines with international recognition in balance measurement and falls prevention. Consensus was sought over three rounds using pre-established criteria.The scoping review identified 56 existing standing balance measures validated in adult populations with evidence of use in the past five years, and these were considered for inclusion in the COS.Fifteen measures were excluded after the first round of scoring and a further 36 after round two. Five measures were considered in round three. Two measures reached consensus for recommendation, and the expert panel recommended that at a minimum, either the Berg Balance Scale or Mini Balance Evaluation Systems Test be used when measuring standing balance in adult populations.Inclusion of two measures in the COS may increase the feasibility of potential uptake, but poses challenges for data synthesis. Adoption of the standing balance COS does not constitute a comprehensive balance assessment for any population, and users should include additional validated measures as appropriate.The absence of a gold standard for measuring standing balance has contributed to the proliferation of outcome measures. These recommendations represent an important first step towards greater standardization in the assessment and measurement of this critical skill and will inform clinical research and practice internationally

Antidepressants during and after Menopausal Transition: A Systematic Review and Meta-Analysis

To assess the therapeutic benefits of antidepressants in depressive women during and after menopausal transition, PubMed, Cochrane Library, EMBASE and Science Direct were systematically searched from inception to February 1, 2020 for randomized controlled trials examining antidepressants compared to placebo. Primary outcome was change in depressive symptom severity, while secondary outcomes were rates of response/remission rates and dropout/discontinuation due to adverse events. Seven trials involving 1,676 participants (mean age = 52.6 years) showed significant improvement in depressive symptoms (k = 7, Hedges’ g = 0.44, 95% confidence interval (CI) = 0.32 to 0.57, p < 0.001) relative to that in controls. Furthermore, response (k = 3, odds ratio (OR) = 2.53, 95% CI = 1.24 to 5.15, p = 0.01) and remission (k = 3, OR = 1.84, 95% CI = 1.32 to 2.57, p < 0.001) rates were significantly higher in antidepressant-treated groups compared to those with controls. Although dropout rates did not differ between antidepressant and control groups (k = 6, OR = 0.93, 95% CI = 0.70 to 1.26, p = 0.68), the rate of discontinuation due to adverse events was significantly higher in antidepressant-treated groups (k = 6, OR = 0.55, 95% CI = 0.35 to 0.86, p = 0.01). Subgroup analysis indicated that antidepressants were also efficacious for depressive symptoms in those without diagnosis of MDD. The results demonstrated that antidepressants were efficacious for women with depressive syndromes during and after menopausal transition but associated with a higher risk of discontinuation due to adverse events

Indices of insulin sensitivity and secretion from a standard liquid meal test in subjects with type 2 diabetes, impaired or normal fasting glucose

<p>Abstract</p> <p>Background</p> <p>To provide an initial evaluation of insulin sensitivity and secretion indices derived from a standard liquid meal tolerance test protocol in subjects with normal (NFG), impaired fasting glucose (IFG) or type 2 diabetes mellitus.</p> <p>Methods</p> <p>Areas under the curve (AUC) for glucose, insulin and C-peptide from pre-meal to 120 min after consumption of a liquid meal were calculated, as were homeostasis model assessments of insulin resistance (HOMA2-IR) and the Matsuda index of insulin sensitivity.</p> <p>Results</p> <p>Subjects with NFG (n = 19), IFG (n = 19), and diabetes (n = 35) had mean ± SEM HOMA2-IR values of 1.0 ± 0.1, 1.6 ± 0.2 and 2.5 ± 0.3 and Matsuda insulin sensitivity index values of 15.6 ± 2.0, 8.8 ± 1.2 and 6.0 ± 0.6, respectively. The log-transformed values for these variables were highly correlated overall and within each fasting glucose category (r = -0.91 to -0.94, all p < 0.001). Values for the product of the insulin/glucose AUC ratio and the Matsuda index, an indicator of the ability of the pancreas to match insulin secretion to the degree of insulin resistance, were 995.6 ± 80.7 (NFG), 684.0 ± 57.3 (IFG) and 188.3 ± 16.1 (diabetes) and discriminated significantly between fasting glucose categories (p < 0.001 for each comparison).</p> <p>Conclusion</p> <p>These results provide initial evidence to support the usefulness of a standard liquid meal tolerance test for evaluation of insulin secretion and sensitivity in clinical and population studies.</p

Incomplete gastric metaplasia in children with insulin-dependent diabetes mellitus and celiac disease. An ultrastructural study

BACKGROUND: The association of insulin-dependent diabetes mellitus (IDDM) and celiac disease (CD) has been widely reported in children but the relationship between the two conditions is incompletely understood. Moreover, specific studies on intestinal biopsies of patients with the association of the two diseases are still lacking. METHODS: We studied the ultrastructure of the duodenal mucosa in 12 patients with both IDDM and CD. RESULTS: All patients had either total or partial atrophy of duodenal mucosa. In seven subjects, an accumulation of electrondense granules in the apical cytoplasm of groups of enterocytes was found. In four of them, a double population of granules existed (mean diameter: 400-800 nm and 100-200 nm respectively) showing a biphasic pattern. In the other three patients, only smaller granules (100- 200 nm) were found in the enterocytes. CONCLUSIONS: The present work suggests that patients with IDDM/CD may represent a subgroup in the context of the CD population. Intestinal biopsies of such individuals often show accumulation of electrondense granules in the apical cytoplasm of enterocytes that can be interpreted as incomplete gastric metaplasia

Metabolic Effects of Krill Oil are Essentially Similar to Those of Fish Oil but at Lower Dose of EPA and DHA, in Healthy Volunteers

The purpose of the present study is to investigate the effects of krill oil and fish oil on serum lipids and markers of oxidative stress and inflammation and to evaluate if different molecular forms, triacylglycerol and phospholipids, of omega-3 polyunsaturated fatty acids (PUFAs) influence the plasma level of EPA and DHA differently. One hundred thirteen subjects with normal or slightly elevated total blood cholesterol and/or triglyceride levels were randomized into three groups and given either six capsules of krill oil (N = 36; 3.0 g/day, EPA + DHA = 543 mg) or three capsules of fish oil (N = 40; 1.8 g/day, EPA + DHA = 864 mg) daily for 7 weeks. A third group did not receive any supplementation and served as controls (N = 37). A significant increase in plasma EPA, DHA, and DPA was observed in the subjects supplemented with n-3 PUFAs as compared with the controls, but there were no significant differences in the changes in any of the n-3 PUFAs between the fish oil and the krill oil groups. No statistically significant differences in changes in any of the serum lipids or the markers of oxidative stress and inflammation between the study groups were observed. Krill oil and fish oil thus represent comparable dietary sources of n-3 PUFAs, even if the EPA + DHA dose in the krill oil was 62.8% of that in the fish oil

Discovery of the inhibitory effect of a phosphatidylinositol derivative on P-glycoprotein by virtual screening followed by <i>in vitro</i> cellular studies

P-glycoprotein is capable of effluxing a broad range of cytosolic and membrane penetrating xenobiotic substrates, thus leading to multi-drug resistance and posing a threat for the therapeutic treatment of several diseases, including cancer and central nervous disorders. Herein, a virtual screening campaign followed by experimental validation in Caco-2, MDKCII, and MDKCII mdr1 transfected cell lines has been conducted for the identification of novel phospholipids with P-gp transportation inhibitory activity. Phosphatidylinositol-(1,2-dioctanoyl)-sodium salt (8∶0 PI) was found to significantly inhibit transmembrane P-gp transportation in vitro in a reproducible-, cell line-, and substrate-independent manner. Further tests are needed to determine whether this and other phosphatidylinositols could be co-administered with oral drugs to successfully increase their bioavailability. Moreover, as phosphatidylinositols and phosphoinositides are present in the human diet and are known to play an important role in signal transduction and cell motility, our finding could be of substantial interest for nutrition science as well

Precise measurement of the W-boson mass with the CDF II detector

We have measured the W-boson mass MW using data corresponding to 2.2/fb of integrated luminosity collected in proton-antiproton collisions at 1.96 TeV with the CDF II detector at the Fermilab Tevatron collider. Samples consisting of 470126 W->enu candidates and 624708 W->munu candidates yield the measurement MW = 80387 +- 12 (stat) +- 15 (syst) = 80387 +- 19 MeV. This is the most precise measurement of the W-boson mass to date and significantly exceeds the precision of all previous measurements combined