Mechanisms of readthrough mitigation reveal principles of GCN1-mediated translational quality control.

Readthrough into the 3' untranslated region (3' UTR) of the mRNA results in the production of aberrant proteins. Metazoans efficiently clear readthrough proteins, but the underlying mechanisms remain unknown. Here, we show in Caenorhabditis elegans and mammalian cells that readthrough proteins are targeted by a coupled, two-level quality control pathway involving the BAG6 chaperone complex and the ribosome-collision-sensing protein GCN1. Readthrough proteins with hydrophobic C-terminal extensions (CTEs) are recognized by SGTA-BAG6 and ubiquitylated by RNF126 for proteasomal degradation. Additionally, cotranslational mRNA decay initiated by GCN1 and CCR4/NOT limits the accumulation of readthrough products. Unexpectedly, selective ribosome profiling uncovered a general role of GCN1 in regulating translation dynamics when ribosomes collide at nonoptimal codons, enriched in 3' UTRs, transmembrane proteins, and collagens. GCN1 dysfunction increasingly perturbs these protein classes during aging, resulting in mRNA and proteome imbalance. Our results define GCN1 as a key factor acting during translation in maintaining protein homeostasis.

Bacillus clausii UBBC-07 in the symptom management of upper respiratory tract infections in children: a double blind, placebo-controlled randomised study.

In children, upper respiratory tract infections (URTIs) are one of the most common causes of infections which often require outpatient consultations with the doctor. The purpose of this study was to evaluate the effect of probiotic Bacillus clausii UBBC-07 on symptom management of URTIs in children. In this double blind, randomised, placebo-controlled study, 90 children (age 4-7 years) with URTIs were equally divided into two groups, the probiotic and placebo. The children were instructed to take B. clausii UBBC-07 spores (2×109 per 5 ml vial) or placebo suspension daily twice for three months. The total duration of the study was 6 months, 3 months treatment and 3 months follow-up period. The parameters assessed were the mean number of URTIs, duration and severity of URTIs, absenteeism from school/childcare and immunity parameters, such as immunoglobulin (Ig)M, IgG, IgE and salivary IgA levels. At the end of treatment, there was a significant decrease in the number, duration and severity of URTIs in the probiotic treated group as compared to the baseline and placebo. IgE levels were significantly decreased and salivary IgA levels were significantly increased in the probiotic treated group suggesting probiotic mediated Th1/Th2 immune homeostasis to alleviate URTIs in children. In conclusion, B. clausii UBBC-07 may help in the reduction of symptoms of URTIs. The trial was registered prospectively with the Clinical Trial Registry of India (CTRI Reg. No: CTRI/2018/08/015282).

Glimepiride and Metformin Combinations in Diabetes Comorbidities and Complications: Real-World Evidence.

Objective To evaluate the usage of various strengths of glimepiride and metformin fixed-dose combinations in the management of type 2 diabetes mellitus (T2DM) patients with comorbidities and complications. Methods A retrospective, non-randomized, non-comparative, multi-centric real-world study included T2DM patients (age > 18 years) taking glimepiride and metformin fixed-dose combinations. Age, duration of diabetes, diabetes complications, comorbidities (hypertension and dyslipidemia), dosage frequency, and concomitant medications were analyzed from medical charts. Results A total of 4858 T2DM patients were included, with a mean age of 52.67 years and males being predominant in the study population (60.85%). The laboratory investigations showed a mean glycated hemoglobin (HbA1c) of 7.5, low-density lipoprotein (LDL) cholesterol of 104.81 ± 38.19 mg/dL, and serum creatinine of 0.88 ± 0.26 mg/dL. Around 2055 (42.30%) T2DM patients were hypertensive, and telmisartan alone and a telmisartan-based combination were the drugs of choice for hypertension management. Similarly, 1073 (22.08%) T2DM patients were having dyslipidemia and were primarily managed with rosuvastatin and its combination in 664 (62%) patients. Macrovascular complications were observed in 339 (6.97%) T2DM patients, among which coronary artery disease (CAD) had maximum prevalence, affecting 273 (5.61%) T2DM patients. Microvascular complications were 1010 (20.79%) T2DM patients, among which neuropathy had affected a maximum of 686 (14.12%) followed by retinopathy (2.34%) and nephropathy (1.81%). Among the available 11 strengths, the glimepiride 2 mg and metformin 500 mg combination were most widely prescribed in 1297 (26.69%), followed by glimepiride 1 mg and metformin 500 mg in 1193 (24.57%) patients, and the preferred dosage pattern was twice a daily in 2665 (54.85%) T2DM patients. An age-wise prescription analysis showed that glimepiride and metformin combinations were the preferred choice for the management of diabetes across all the age groups. Conclusion The real-world evidence in the Indian clinical setting indicates that glimepiride and metformin fixed-dose combinations are widely used in the management in T2DM patients with comorbidities like hypertension, dyslipidemia, and diabetes complications. Glimepiride and metformin fixed-dose combinations are suitable for early as well as long-standing diabetes.

Identification and differential expression of serotransferrin and apolipoprotein A-I in the plasma of HIV-1 patients treated with first-line antiretroviral therapy.

BACKGROUND: Plasma proteins are known to interfere the drug metabolism during therapy. As limited information is available regarding the role of plasma proteins in HIV drug resistance during ART in HIV/AIDS patients, the present study aimed to identify and characterize the differentially expressed plasma proteins in the drug resistant and drug respondent groups of HIV-1 infected patients with > 6 years of first line ART. METHODS: Four-drug resistant (treatment failure) and four-drug respondent (treatment responder) patients were selected for plasma proteomic analysis based on viral load and drug resistance associated mutations from a cohort study designed on the first line ART patients who were enrolled in the antiretroviral therapy center, Sarojini Naidu Medical College, Agra, India from December 2009 to November 2016. After depleting high abundant proteins, plasma proteins were resolved using two-dimensional gel electrophoresis on IPG strips, pH range of 3-10. Spots were selected in the gel based on the density of staining which was common in the drug resistant and drug respondent groups separately. The fold change of each spot was calculated using image-J. Each protein spot was identified using the matrix assisted laser desorption/ionization-time of flight/time of flight (MALDI-TOF/TOF) after tryptic digestion. Peptide peaks were identified through flex analysis version 3.3, and a search against a protein data base using the internal Mascot. Gene ontology study was completed through STRING v.11 and Panther15.0. RESULTS: Out of eight spots from 2D gel samples analyzed by MALDITOF/TOF, two proteins were found to have significant score (> 56) after Flex analysis. These two proteins were identified to be apolipoprotein A1 and serotransferrin. The fold change expression of these two proteins were analyzed in drug resistant and drug respondent group. Apolipoprotein-A1 and serotransferrin were observed to be expressed 1.76 and 1.13-fold more respectively in drug respondent group compared to drug resistant group. The gene ontology analysis revealed the involvement of these two proteins in various important physiological processes. CONCLUSION: Apolipoprotein A-I and serotransferrin were found to be expressed more in drug respondent group compared to drug resistant group.

Functional Modules of the Proteostasis Network.

Cells invest in an extensive network of factors to maintain protein homeostasis (proteostasis) and prevent the accumulation of potentially toxic protein aggregates. This proteostasis network (PN) comprises the machineries for the biogenesis, folding, conformational maintenance, and degradation of proteins with molecular chaperones as central coordinators. Here, we review recent progress in understanding the modular architecture of the PN in mammalian cells and how it is modified during cell differentiation. We discuss the capacity and limitations of the PN in maintaining proteome integrity in the face of proteotoxic stresses, such as aggregate formation in neurodegenerative diseases. Finally, we outline various pharmacological interventions to ameliorate proteostasis imbalance.

Antibiotics for tonsillitis: should the emergency department emulate general practice?

OBJECTIVES: To determine whether antibiotics are prescribed appropriately for acute tonsillitis in an emergency department (ED). METHODS: Cross-sectional observational study in large district general hospital in London. Patients diagnosed and coded with 'acute tonsillitis' in the ED over a 3-month period in 2015. Medical records were reviewed for Centor criteria, which is a clinical scoring system to guide antibiotic prescribing in UK general practice. Drug charts were reviewed for the specific antibiotic(s) prescribed, and throat swab (TS) cultures were recorded. RESULTS: 273/389 patients with tonsillitis were analysed-186 children, 87 adults. Exclusions were missing patient records (86), patients had/awaiting tonsillectomy (22), receiving antibiotics (6) and immunocompromised (2). Centor score (CS) was not recorded for any patient. Based on derived CS from documented signs/symptoms, antibiotics were prescribed inappropriately to 196/273 patients (80%; 95% CI 74% to 85%) including broad-spectrum antibiotics to 25%. These included co-amoxiclav (18%), amoxicillin (6%), azithromycin (0.5%) and ceftriaxone (0.5%). TSs were taken in 66/273(24%) patients; 10/66 were positive for group A streptococcus (GAS). However, 48/56 GAS negative patients were prescribed antibiotics. CONCLUSIONS: CS was not being used in the ED to guide antibiotic prescribing for acute tonsillitis. Antibiotic prescribing was based on clinical judgement. Based on derived CS (<3), 80% of patients were inappropriately prescribed antibiotics, particularly broad-spectrum antibiotics. Further studies need to assess use of CS to guide antibiotic prescription in ED. TSs were commonly performed in the ED but did not either improve diagnosis or guide antibiotic prescription.

The RNA Stem-Loop to G-Quadruplex Equilibrium Controls Mature MicroRNA Production inside the Cell.

The biological role of the existence of overlapping structures in RNA is possible yet remains very unexplored. G-Rich tracts of RNA form G-quadruplexes, while GC-rich sequences prefer stem-loop structures. The equilibrium between alternate structures within RNA may occur and influence its functionality. We tested the equilibrium between G-quadruplex and stem-loop structure in RNA and its effect on biological processes using pre-miRNA as a model system. Dicer enzyme recognizes canonical stem-loop structures in pre-miRNA to produce mature miRNAs. Deviation from stem-loop leads to deregulated mature miRNA levels, providing readout of the existence of an alternate structure per se G-quadruplex-mediated structural interference in miRNA maturation. In vitro analysis using beacon and Dicer cleavage assays indicated that mature miRNA levels depend on relative amounts of K(+) and Mg(2+) ions, suggesting an ion-dependent structural shift. Further in cellulo studies with and without TmPyP4 (RNA G-quadruplex destabilizer) demonstrated that miRNA biogenesis is modulated by G-quadruplex to stem-loop equilibrium in a subset of pre-miRNAs. Our combined analysis thus provides evidence of the formation of noncanonical G-quadruplexes in competition with canonical stem-loop structure inside the cell and its effect on miRNA maturation in a comprehensive manner.

Sjögren Syndrome and Cryoglobulinemic Glomerulonephritis.

We report the case of a 53-year-old woman with Sjögren syndrome and cryoglobulinemia. The patient presented with nephrotic syndrome, hematuria, and reduced estimated glomerular filtration rate. The kidney biopsy revealed diffuse endocapillary proliferation and leukocyte exudation with focal intraluminal hyaline thrombi, prominent tubulointerstitial inflammation, and vasculitis. Diffuse granular mesangial and segmental to global capillary wall staining was observed on immunofluorescence with antisera to C3 and immunoglobulin M (IgM), with less intense staining indicative of IgG and κ and λ light chains. A biopsy diagnosis of Sjögren syndrome-related cryoglobulinemic membranoproliferative glomerulonephritis and vasculitis was rendered. Subsequent investigations revealed the presence of circulating type II cryoglobulins with cryocrit of 9%. Although rare, Sjögren syndrome is the most common cause of non-hepatitis C virus-related mixed cryoglobulinemia. We discuss the possible pathogenic mechanisms involved in the development of mixed cryoglobulinemia and its evolution to lymphoma, as best described in the setting of hepatitis C virus infection. Although the specific antigen involved is unknown, it is likely that the mixed cryoglobulinemia in Sjögren syndrome is triggered by the long-term B-cell stimulation, resulting in clonal proliferation of B cells. Additional chromosomal aberrations and cytokine milieu alterations, as seen in hepatitis C virus infection, may result in prolonged B-cell survival and progression to non-Hodgkin lymphoma.

Utilization of agro-industrial waste for ß-galactosidase production under solid state fermentation using halotolerant Aspergillus tubingensis GR1 isolate.

A halotolerant fungal isolate Aspergillus tubingensis GR1 was isolated from the man-made solar saltern located at Khambhat, Gujarat, India, and identified using 28S rDNA partial genome sequencing. This isolate was studied for ß-galactosidase production under solid state fermentation using wheat bran and deproteinized acid cheese whey. The influence of various agro-industrial wastes, nitrogen source and other growth conditions on ß-galactosidase production was investigated using 'one-factor-at-a-time' approach. Among various variables screened along with wheat bran and deproteinized acid cheese whey as major growth substrate, corn steep liquor and MgSO4 were found to be most significant. The optimum concentrations of these significant parameters were determined employing the response surface central composite design, revealing corn steep liquor concentration (2 mL) and magnesium sulphate (50 mg) per 5 g of wheat bran and 20 mL of deproteinized acid cheese whey for highest enzyme production (15,936 U/gds). These results suggest the feasibility of industrial large-scale production of ß-galactosidase known to be valuable in whey hydrolysis and removal of galactosyl residue from polysaccharide.

Local properties of patterned vegetation: quantifying endogenous and exogenous effects.

Dryland ecosystems commonly exhibit periodic bands of vegetation, thought to form due to competition between individual plants for heterogeneously distributed water. In this paper, we develop a Fourier method for locally identifying the pattern wavenumber and orientation, and apply it to aerial images from a region of vegetation patterning near Fort Stockton, TX, USA. We find that the local pattern wavelength and orientation are typically coherent, but exhibit both rapid and gradual variation driven by changes in hillslope gradient and orientation, the potential for water accumulation, or soil type. Endogenous pattern dynamics, when simulated for spatially homogeneous topographic and vegetation conditions, predict pattern properties that are much less variable than the orientation and wavelength observed in natural systems. Our local pattern analysis, combined with ancillary datasets describing soil and topographic variation, highlights a largely unexplored correlation between soil depth, pattern coherence, vegetation cover and pattern wavelength. It also, surprisingly, suggests that downslope accumulation of water may play a role in changing vegetation pattern properties.

Local properties of patterned vegetation: quantifying endogenous and exogenous effects.

Dryland ecosystems commonly exhibit periodic bands of vegetation, thought to form due to competition between individual plants for heterogeneously distributed water. In this paper, we develop a Fourier method for locally identifying the pattern wavenumber and orientation, and apply it to aerial images from a region of vegetation patterning near Fort Stockton, TX, USA. We find that the local pattern wavelength and orientation are typically coherent, but exhibit both rapid and gradual variation driven by changes in hillslope gradient and orientation, the potential for water accumulation, or soil type. Endogenous pattern dynamics, when simulated for spatially homogeneous topographic and vegetation conditions, predict pattern properties that are much less variable than the orientation and wavelength observed in natural systems. Our local pattern analysis, combined with ancillary datasets describing soil and topographic variation, highlights a largely unexplored correlation between soil depth, pattern coherence, vegetation cover and pattern wavelength. It also, surprisingly, suggests that downslope accumulation of water may play a role in changing vegetation pattern properties.

Treatment of hypertension: postmarketing surveillance study results of telmisartan monotherapy, fixed dose combination of telmisartan + hydrochlorothiazide/amlodipine.

Elevated blood pressure is a staggering health problem as it is very common and it remains asymptomatic. Hypertension is most important risk factor in coronary heart disease and cerebrovascular accidents. The present study was undertaken to evaluate the efficacy and safety of telmisartan monotherapy (TELMED 20/40) and fixed dose combination of telmisartan + hydrochlorothiazide/amlodipine (TELMED-H/AM) in adult, Indian hypertensive patients. A total 350 patients were enrolled in this open, multicentric, prospective, non-comparative, phase IV, postmarketing surveillance study by 28 physicians in India. Patients were treated with telmisartan monotherapy or fixed dose combination of telmisartan + hydrochlorothiazide/amlodipine once daily for 12 weeks. Total 318 patients completed the study and 32 patients failed to follow-up and were considered as drop outs. At the end of 12 weeks therapy, 106 patients (33.33%) remained on telmisartan monotherapy while 212 patients (66.67%) received of telmisartan + hydrochlorothiazidelamlodipine. On telmisartan monotherapy a significant (p < 0.001) reduction of systolic and diastolic blood pressure was noted ie, 27.75 and 13.83 mm Hg respectively. In patients on telmisartan + hydrochlorothiazide/amlodipine reduction in systolic and diastolic blood pressure was even greater than telmisartan monotherapy ie, 38.5 and 16.06 mm Hg respectively (p < 0.001). Joint National Committee VII goal for hypertension was achieved in 82.08% patients. 'Good to excellent' efficacy and safety were reported in 97.1% patients. The present study demonstrates that telmisartan with or without combination therapy significantly reduces systolic and diastolic blood pressure and is very well tolerated and accepted by all hypertensive patients with or without diabetes, ischaemic heart diseases.

CDC27 protein is involved in radiation response in squamous cell cervix carcinoma.

In the present study, an attempt was made to identify genes involved in radiation response in cervix carcinoma. Differential display technique was used to study the expression profiles of tumour biopsy samples obtained from patients, responding and not responding to treatment. The samples were obtained prior to radiotherapy and subsequent to treatment with Tele-radiation at 10 Gray (Gy). One of the differentially expressed cDNAs, when sequenced was identified to be CDC27. Immuno-histochemical analysis of pre- and post-treated tumour samples from fifteen patients showed the downregulation of expression of CDC27 protein in seven patients. Down-regulation was associated with poorer response to radiotherapy. Cervical cancer cell lines SiHa and C33A were irradiated and their nuclei were stained for expression of CDC27 and analyzed using fluorescent-activated cell sorting (FACS). Down-regulation of CDC27 protein in the irradiated SiHa cell line was associated with greater survival fraction, compared to the irradiated C33A cell line, which had only slight fall in the level of CDC27 protein. This is the first study to suggest a role for CDC27 in radiation response. However, a larger cohort is needed to further confirm the value of CDC27 protein as a predictive marker, for radiation response in cervix cancer.

Variant of mitochondrial ribosomal protein s28 (mrps28) gene is differentially expressed in response to radiation in a cervical carcinoma derived cell line.

Gene expression before and after radiation treatment (10 Gy) in a cervical carcinoma derived cell line SiHa was studied. Differential display performed on non-irradiated and irradiated cells (10 Gy) using a combination of 3 anchor primers and 16 arbitrary primers yielded 20 differentially expressed fragments. Differential expression of one of the fragments differentially expressed in the irradiated sample was also confirmed in Northern blots. Cloning and sequencing of the fragment revealed it to be similar to a splice variant of the MRPS28 gene. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed using primers designed to amplify the splice variant and confirmed the expression. In conclusion, the study shows that the splice variant kDec03 of the MRPS28 gene is differentially expressed in response to radiation in SiHa cells.

Summarizing data through a piecewise linear growth curve model.

Most of the research in clinical trials is based on longitudinal designs, which involve repeated measurements of a variable of interest. Such designs are very powerful, both statistically and scientifically. Recent advances in statistical theory and software development incorporate the covariance structures such as unstructured, compound symmetry, auto-regressive and random effects, etc., for analysing longitudinal data. Hathaway et al. propose a technique for summarizing longitudinal data using linear growth curve model and establish that the number of summary statistics is fixed as four irrespective of the length of study. In this paper, we develop a procedure for analysing the longitudinal data through a piecewise linear growth curve model on the lines of Hathaway et al. Under different covariance structures, the linear model is fitted for Leprosy data and the residual sum of squares computed. Goodness of fit has also been considered for various models. In order to prove that the proposed method is robust and better than the others in terms of goodness of fit, simulation studies are carried out and the results presented.

Live three-dimensional transthoracic echocardiographic identification of discrete subaortic membranous stenosis.

We describe an adult patient in whom live three-dimensional transthoracic echocardiography illustrated the exact site and full extent of the subaortic membrane as well as the narrow opening within the membrane, indicative of severe stenosis. To our knowledge this has not been reported previously.

Pattern recognition technique in immunological antigenic tests to identify Mycobacterium tuberculosis infection.

UNLABELLED: The importance of a diagnostic test that is simple and quick to identify Mycobacterium tuberculosis infection needs no emphasis. The tuberculin skin test (TST - 1 TU RT23) is the diagnostic tool for identifying M. tuberculosis infection at present. The test reaction on the skin is measured after 48-72 h. It is observed that often multi-modes are seen, when the reactions are drawn as a graph and the bimodality is seen very feebly. Because of the difficulties in the administration of TST, several serological tests were developed over three decades, but none of the studies showed the desired results. One study to evaluate purified protein derivative (PPD) antigen resulted in a claim of 80% sensitivity and 4% false-positivity rate (14), while other researchers were not able to obtain similar results. In addition, several problems were encountered due to the non-availability of antigens, and data analyses from an ELISA-based diagnostic test showed considerable overlap of distributions of optical density (OD) values among patients and healthy individuals (10). Classical statistical techniques cannot explain the cause of these overlaps. Hence, an attempt is made in this article to resolve these difficulties by the pattern recognition technique (PRT). The technique lies in splitting the data into clusters using a supervised algorithm. The data set is normally split into a training set, a test set and a validation set. The PRT gets "trained" through the training data set until the infected and uninfected groups of individuals are correctly classified. The training occurs based on an algorithm on the training set. On successful completion of the training, this technique is further tested and validated in the respective data sets. SETTING: A total of 273 finger-prick specimens were collected from five categories (Al, A2, B, C, D, E) of subjects not vaccinated with (bacille Calmette Guerin) (BCG) from Trivellore BCG Trial area adopted by the Tuberculosis Research Center, Chennai, India. OBJECTIVE: The study was conducted with the primary aim of evaluating purified antigens--r38 kDa, PPD and 30 kDa--for their usefulness as diagnostic tools and to test the applicability of the PRT in the evaluation of diagnostic tests. Individuals in two main categories (definitely not infected categories Al, A2 and D, and definitely infected categories B, E and C based on reaction to TST) were assembled for the purpose. RESULTS: The overall PRT performance of 30 kDa was 72.3% sensitivity and 90.9% specificity for identifying M. tuberculosis infection, while the r38 kDa antigen recorded a sensitivity of 73.8% and a specificity of 84.6%. In the case of PPD, the results were not promising. CONCLUSION: This paper on ELISA-based diagnostic tests attempts to implement an optimal decision support system through PRT that would identify the outcome (as infected or non-infected) based on the OD values. The PRT was able to predict the outcome for individual suspects. Further, Kullback-Leibler distance measurement has validated the PRT in distinguishing infected individuals from healthy subjects (based on the OD values).