Expression and Purification of Recombinant Human Mitochondrial RNA Polymerase (POLRMT) and the Initiation Factors TFAM and TFB2M.

Human mitochondrial DNA (mtDNA) encodes several components of oxidative phosphorylation responsible for the bulk of cellular energy production. The mtDNA is transcribed by a dedicated human mitochondrial RNA polymerase (POLRMT) that is structurally distinct from its nuclear counterparts, instead closely resembling the single-subunit viral RNA polymerases (e.g., T7 RNA polymerase). The initiation of transcription by POLRMT is aided by two initiation factors: transcription factor A, mitochondrial (TFAM), and transcription factor B2, mitochondrial (TFB2M). Although many details of human mitochondrial transcription initiation have been elucidated with in vitro biochemical and structural studies, much remains to be addressed relating to the mechanism and regulation of transcription. Studies of such mechanisms require reliable, high-yield, and high-purity methods for protein production, and this protocol provides the level of detail and troubleshooting tips that are necessary for a novice to generate meaningful amounts of proteins for experimental work. The current protocol describes how to purify recombinant POLRMT, TFAM, and TFB2M from Escherichia coli using techniques such as affinity column chromatography (Ni2+ and heparin), how to remove the solubility tags with TEV protease and recover untagged proteins of interest, and how to overcome commonly encountered challenges in obtaining high yield of each protein. Key features • This protocol builds upon purification methods developed by Patel lab (Ramachandran et al., 2017) and others with greater detail than previously published works. • The protocol requires several days to complete as various steps are designed to be performed overnight. • The recombinantly purified proteins have been successfully used for in vitro transcription experiments, allowing for finer control of experimental components in a minimalistic system.

Expression of advanced glycation end products and receptors in gingival tissues of patients with noninsulin-dependent diabetes mellitus-associated periodontitis.

Background/purpose: Advanced glycation end products (AGEs) are known to accumulate in the periodontal tissues of patients with diabetes mellitus (DM). Through this study, we aimed to investigate the distribution of AGEs and the receptor of AGEs (RAGE) in the gingival tissues of patients with chronic periodontitis with and without non-insulin-dependent diabetes mellitus (NIDDM). Materials and methods: Gingival biopsy samples from 13 patients with both NIDDM and periodontitis and 6 patients with both non-DM (NDM) and periodontitis were collected. The tissue sections were processed using immunohistochemical (IHC) staining to detect the distributions of AGEs and RAGE. Spearman correlation coefficients of all samples were calculated (P < 0.05) for the AGE and RAGE rankings of the following clinical parameters: plaque score (PI), probing depth (PD), bleeding on probing (BOP), and tooth loss (TL). Results: IHC analysis revealed that AGEs among patients with NIDDM had a significantly higher ranking than those of the NDM group (P < 0.05). Positive staining for RAGE was observed in both groups but was not significantly different (P > 0.05). A positive correlation between AGE ranking and TL was observed in the NIDDM group, but not between AGE ranking and PI, PD, or BOP. The distribution of RAGE was not correlated with PI, PD, BOP, or TL. Conclusion: AGEs were particularly distributed in the highly inflamed gingiva of patients with NIDDM-associated periodontitis and was statistically correlated with the long-term parameter TL.

Prevalence and risk factors for latent tuberculosis among diabetes patients in Taiwan: A cross-sectional study.

INTRODUCTION: Diabetes mellitus (DM) is a known risk factor for tuberculosis (TB), leading to an approximate three-fold higher risk of developing active TB. However, epidemiological studies on the prevalence of latent TB infection (LTBI) in DM patients are lacking. In this study, we investigated the presence of LTBI and determined risk factors for LTBI in DM patients. METHODOLOGY: We conducted a cross-sectional study at Taipei Medical University-Shuang Ho Hospital in northern Taiwan. The study population comprised DM patients (aged 20-70 years) attending a metabolism outpatient clinic between February 2011 and February 2013, excluding patients who were suspected or confirmed to have active TB. Venous blood samples were drawn from patients to detect LTBI using the QuantiFERON-TB Gold In-Tube (QFT-GIT) method. RESULTS: We enrolled 1120 patients with DM. The QFT-GIT showed positive results for 241 people (21.5%) and negative results for 879 people (78.5%). The mean age at QFT-GIT positivity was 58.2 years, which was significantly dissimilar to the mean age at QFT-GIT negativity, which was 55.0 years (p < 0.001). Multivariate logistic regression indicated that the trend of QFT-GIT positivity increased after the age of 50 years. Effective glycemic control did not differ significantly between QFT-GIT-positive and -negative patients. Moreover, men were predominant were predominant in both QFT-GIT-positive and -negative patients. CONCLUSIONS: More than one-fifth of DM patients have LTBI. Among the DM patients, those older than 50 years may have a higher risk of LTBI. Moreover, effective glycemic control did not differ significantly in patients with LTBI.

Cross-Camera External Validation for Artificial Intelligence Software in Diagnosis of Diabetic Retinopathy.

Aims: To investigate the applicability of deep learning image assessment software VeriSee DR to different color fundus cameras for the screening of diabetic retinopathy (DR). Methods: Color fundus images of diabetes patients taken with three different nonmydriatic fundus cameras, including 477 Topcon TRC-NW400, 459 Topcon TRC-NW8 series, and 471 Kowa nonmyd 8 series that were judged as "gradable" by one ophthalmologist were enrolled for validation. VeriSee DR was then used for the diagnosis of referable DR according to the International Clinical Diabetic Retinopathy Disease Severity Scale. Gradability, sensitivity, and specificity were calculated for each camera model. Results: All images (100%) from the three camera models were gradable for VeriSee DR. The sensitivity for diagnosing referable DR in the TRC-NW400, TRC-NW8, and non-myd 8 series was 89.3%, 94.6%, and 95.7%, respectively, while the specificity was 94.2%, 90.4%, and 89.3%, respectively. Neither the sensitivity nor the specificity differed significantly between these camera models and the original camera model used for VeriSee DR development (p = 0.40, p = 0.065, respectively). Conclusions: VeriSee DR was applicable to a variety of color fundus cameras with 100% agreement with ophthalmologists in terms of gradability and good sensitivity and specificity for the diagnosis of referable DR.

Effects of the Method of Preparation and Dispersion Media on the Optical Properties and Particle Sizes of Aqueous Dispersions of a Double-Chain Cationic Surfactant.

As inferred from visual observations and turbidity measurements, the average radius of the unilamellar vesicles formed in water from the cationic double-chain surfactant didodecyldimethylammonium bromide (DDAB) varies with the method of preparation, being ∼24 nm after sonication (SS method) and ∼74 nm after extrusion/ultrafiltration (SE method). The radii were larger when the vesicles were produced in 10 mM NaBr, ∼65 nm for the SS method and ∼280 nm for the SE method. The specific turbidity, or turbidity per unit path length divided by the surfactant weight fraction, w, of these vesicular dispersions increased with decreasing w until a constant value was reached at w*, which depends on the preparation method and the dispersion medium. The constant specific turbidities are indicative of single and independent scattering and were used to estimate vesicle radii by solving the specific turbidity equations derived for the Rayleigh-Debye-Gans (RDG) regime. Two turbidity equations were used, one accounting for absorbance errors due to some scattered light reaching the detector and another with no correction. Estimates of the average distances between the vesicles and their corresponding Debye lengths were obtained for evaluating the importance of intervesicle electrostatic interactions, which could lead to dependent scattering at higher weight fractions.

Rayleigh and Rayleigh-Debye-Gans light scattering intensities and spetroturbidimetry of dispersions of unilamellar vesicles and multilamellar liposomes.

HYPOTHESIS: Since the volume fraction of the surfactant bilayer(s), of thickness db, in a vesicle and liposome is smaller than one, the dependences of the Rayleigh (R) scattering intensity and turbidity on the particle radius a are weaker than those for a homogeneous sphere, which are proportional to a3. The dependences of the Rayleigh-Debye-Gans (RDG) scattering intensity and turbidity on a are also weaker. Work done: The dependences of the effective relative refractive index on a, db, and dw (water layer thickness) were derived. The specific Rayleigh ratio [Formula: see text] and the specific turbidity τ** for single and independent scattering were derived analytically for R and RDG scattering. Spectroturbidimetry data at 25°C for a cationic double-chain surfactant, didodecyldimethylammonium bromide (DDAB) were compared to the turbidity predictions. FINDINGS: For R scattering, [Formula: see text] and τ** are proportional to a2db for vesicles, and to a3dbdw+db for liposomes. For RDG and particle radii 20-1000 nm, τ** is proportional to an, where n is 2 to 0.4 for vesicles and 2 to 1.1 for liposomes. Turbidity data for DDAB vesicles are consistent with the RDG predictions, which are also used to estimate the vesicles' sizes. RDG applies to liposomes < 800 nm and to much larger sizes for vesicles.

A Metastable Contact and Structural Disorder in the Estrogen Receptor Transactivation Domain.

The N-terminal transactivation domain (NTD) of estrogen receptor alpha, a well-known member of the family of intrinsically disordered proteins, mediates the receptor's transactivation function. However, an accurate molecular dissection of NTD's structure-function relationships remains elusive. Here, we show that the NTD adopts a mostly disordered, unexpectedly compact conformation that undergoes structural expansion on chemical denaturation. By combining small-angle X-ray scattering, hydroxyl radical protein footprinting, and computational modeling, we derive the ensemble-structures of the NTD and determine its ensemble-contact map revealing metastable long-range contacts, e.g., between residues I33 and S118. We show that mutation at S118, a known phosphorylation site, promotes conformational changes and increases coactivator binding. We further demonstrate via fluorine-19 (19F) nuclear magnetic resonance that mutations near I33 alter 19F chemical shifts at S118, confirming the proposed I33-S118 contact in the ensemble of structural disorder. These findings extend our understanding of how specific contact metastability mediates critical functions of disordered proteins.

Correction: Effect of body mass index on diabetogenesis factors at a fixed fasting plasma glucose level.

[This corrects the article DOI: 10.1371/journal.pone.0189115.].

Efficacy and Safety of Rituximab for the Treatment of Graves' Orbitopathy: A Meta-analysis of Randomized Controlled Trials.

STUDY OBJECTIVE: To investigate the efficacy and safety of rituximab in patients with Graves' orbitopathy (GO). DESIGN: Systematic review and meta-analysis of four randomized controlled trials. PATIENTS: A total of 293 patients with GO who received rituximab or control (either glucocorticoids, the established first-line therapy [three trials], or saline [one trial]). MEASUREMENTS AND RESULTS: Relevant studies published before February 2018 were identified from the PubMed, EMBASE, Cochrane Library, and Scopus databases and the ClinicalTrials.gov registry. Individual effect sizes were standardized, and a meta-analysis was conducted to calculate the pooled effect size by using a random-effects model. Treatment efficacy was assessed by measuring the following outcomes: clinical activity score (CAS), sight visual acuity reduction (NOSPECS) score, proptosis, diplopia, changes in eye volume, quality of life, and adverse events. In the four included trials, 113 patients in the rituximab group and 108 patients in the control group were evaluated. Compared with the control group, CAS (weighted mean difference 0.57, 95% confidence interval 0.25-0.89) was significantly reduced at 24 weeks in the rituximab group. Compared with the control group, considerable proptosis reduction was also observed in the rituximab group; however, the difference was not significant. The proportion of adverse events in the rituximab group was not significantly higher than that in the glucocorticoid control group, but one of the included trials indicated that the rituximab group had more serious adverse events than the saline control group. CONCLUSION: Rituximab is a relatively safe and viable treatment that is superior to glucocorticoids or saline for patients with moderate to severe GO. However, the incidence of serious adverse events was disparate among the included trials. Additional studies involving a larger sample size and investigating the optimal rituximab dosage, frequency, and method of administration are warranted.

Effect of body mass index on diabetogenesis factors at a fixed fasting plasma glucose level.

AIM: The present study evaluated the relative influence of body mass index (BMI) on insulin resistance (IR), first-phase insulin secretion (FPIS), second-phase insulin secretion (SPIS), and glucose effectiveness (GE) at a fixed fasting plasma glucose level in an older ethnic Chinese population. METHODS: In total, 265 individuals aged 60 years with a fasting plasma glucose level of 5.56 mmol/L were enrolled. Participants had BMIs of 20.0-34.2 kg/m2. IR, FPIS, SPIS, and GE were estimated using our previously developed equations. Pearson correlation analysis was conducted to assess the correlations between the four diabetogenesis factors and BMI. A general linear model was used to determine the differences in the percentage of change among the four factor slopes against BMI. RESULTS: Significant correlations were observed between BMI and FPIS, SPIS, IR, and GE in both women and men, which were higher than those reported previously. In men, BMI had the most profound effect on SPIS, followed by IR, FPIS, and GE, whereas in women, the order was slightly different: IR, followed by FPIS, SPIS, and GE. Significant differences were observed among all these slopes, except for the slopes between FPIS and SPIS in women (p = 0.856) and IR and FPIS in men (p = 0.258). CONCLUSIONS: The contribution of obesity to all diabetes factors, except GE, was higher than that reported previously. BMI had the most profound effect on insulin secretion in men and on IR in women in this 60-year-old cohort, suggesting that lifestyle modifications for obesity reduction in women remain the most important method for improving glucose metabolism and preventing future type 2 diabetes mellitus.

A Practical Guide to iSPOT Modeling: An Integrative Structural Biology Platform.

Integrative structure modeling is an emerging method for structural determination of protein-protein complexes that are challenging for conventional structural techniques. Here, we provide a practical protocol for implementing our integrated iSPOT platform by integrating three different biophysical techniques: small-angle X-ray scattering (SAXS), hydroxyl radical footprinting, and computational docking simulations. Specifically, individual techniques are described from experimental and/or computational perspectives, and complementary structural information from these different techniques are integrated for accurate characterization of the structures of large protein-protein complexes.

The Association of Thyrotropin and Autoimmune Thyroid Disease in Developing Papillary Thyroid Cancer.

BACKGROUND: Papillary thyroid carcinoma (PTC) is the most common type of malignant thyroid neoplasm. However, the incidence of PTC with autoimmune thyroid disease (AITD) varies between studies. This study aims to investigate whether patients with AITD have increased incidence of PTC. We also analyzed the relationship of serum thyroid-stimulating hormone (TSH) levels and PTC in relation to AITD based on histopathological data. METHODS: A total of 533 participants who underwent thyroidectomy were enrolled in this retrospective study based on clinicohistopathological data and known thyroid autoantibodies. Patients were divided into PTC and benign groups according to histopathologic diagnosis. Age, gender, body mass index, and serum TSH level before thyroidectomy were recorded. RESULTS: Of the 533 enrolled patients, 159 (29.8%) were diagnosed with PTC, of which 38 (35.5%) had Hashimoto's thyroiditis (HT). More patients with HT were female, and patients with HT, Graves' disease, and thyroid nodules with higher TSH level had a higher incidence of PTC. CONCLUSIONS: A high proportion of the patients with PTC had HT. There was a trend that a higher serum TSH level was associated with a greater risk of thyroid cancer.

Efficacy and safety of alogliptin in patients with type 2 diabetes mellitus: A multicentre randomized double-blind placebo-controlled Phase 3 study in mainland China, Taiwan, and Hong Kong.

BACKGROUND: This study determined the efficacy and safety of once-daily oral alogliptin in patients from mainland China, Taiwan, and Hong Kong with type 2 diabetes mellitus. METHODS: In this Phase 3 multicenter double-blind placebo-controlled 16-week trial, 506 patients were randomized to receive once-daily alogliptin 25 mg or placebo: 185 in the monotherapy group, 197 in the add-on to metformin group, and 124 in the add-on to pioglitazone group. The primary efficacy variable was the change from baseline (CFB) in HbA1c at Week 16; other efficacy measures included CFB to Week 16 in fasting plasma glucose (FPG), incidence of marked hyperglycemia (FPG ≥11.1 mmol/L), and the incidence of clinical HbA1c ≤6.5 % (48 mmol/mol) and ≤7.0 % (53 mmol/mol) at Week 16. Safety was assessed throughout the trial. RESULTS: Alogliptin monotherapy provided a significantly greater decrease in HbA1c from baseline to Week 16 compared with placebo (-0.58 %; 95 % confidence interval [CI] -0.78 %, -0.37 %; P < 0.001). As an add-on to metformin or pioglitazone, alogliptin also significantly decreased HbA1c compared with placebo (-0.69 % [95 % CI -0.87 %, -0.51 %; P < 0.001] and -0.52 % [95 % CI -0.75 %, -0.28 %; P < 0.001], respectively). In any treatment group versus placebo, alogliptin led to greater decreases in FPG (P ≤ 0.004) and a higher percentage of patients who achieved an HbA1c target of ≤6.5 % and ≤7.0 % (P ≤ 0.003). No weight gain was observed in any treatment group. A similar percentage of patients experienced drug-related, treatment-emergent adverse events in the alogliptin and placebo arms. Four and two patients in the alogliptin and placebo arms, respectively, experienced mild or moderate hypoglycemia. CONCLUSIONS: Alogliptin 25 mg once daily reduced HbA1c and FPG and enhanced clinical response compared with placebo when used as monotherapy or as an add-on to metformin or pioglitazone. Therapy with alogliptin was well tolerated.

A diagnostic dilemma: acute abdomen presenting as segmental arterial mediolysis masked by a ruptured hepatocellular carcinoma.

A 65-year-old male was brought to our hospital with right upper abdominal fullness sensation and recent body weight loss of about 3 kg. The patient had developed episodes of melena following progressive abdominal muscular guarding and drop of haemoglobin level to 6.3 g/dL. An abdominal computed tomography scan disclosed a ruptured hepatocellular cell carcinoma. A segmental arterial mediolysis was found on the superior mesenteric artery in the process of repairing the ruptured right hepatic artery with the assistance of angiography. Transarterial embolization was carried out and permanent haemostasis was achieved.