Membrane topology of yeast alkaline ceramidase YPC1

Ypc1p and Ydc1p are alkaline ceramide hydrolases, which reside in the ER. Ypc1p can catalyze the reverse reaction, i.e. the condensation of free fatty acids with phytosphingosine or dihydrosphingosine and overexpression of YPC1 or YDC1 can provide enough ceramide synthesis as to rescue the viability of cells lacking the normal acyl-CoA-dependent ceramide synthases. To better understand the coexistence of acyl-CoA dependent ceramide synthases and ceramidases in the ER we investigated the membrane topology of Ypc1p by probing cysteine accessibility of natural and substituted cysteines with membrane non-permeating mass-tagged probes. The N- and C-terminal ends of Ypc1p are oriented towards the lumen and cytosol, respectively. Two of the 5 natural cysteines, Cys27 and Cys219, are essential for enzymatic activity and form a disulfide bridge. The data allow inferring that all amino acids of Ypc1p that are conserved in the pfam PF05875 ceramidase motif and the CREST superfamily are located in or near the ER lumen. Microsomal assays using a lysine-specific reagent show that the reverse ceramidase activity can only be blocked when the reagent has access to Ypc1p from the lumenal side. Overall the data suggest that the active site of Ypc1p resides at the lumenal side of the ER membrane

Adaptation of low-resolution methods for the study of yeast microsomal polytopic membrane proteins: a methodological review

Most integral membrane proteins of yeast with two or more membrane-spanning sequences have not yet been crystallized and for many of them the side on which the active sites or ligand-binding domains reside is unknown. Also, bioinformatic topology predictions are not yet fully reliable. However, so-called low-resolution biochemical methods can be used to locate hydrophilic loops or individual residues of polytopic membrane proteins at one or the other side of the membrane. The advantages and limitations of several such methods for topological studies with yeast ER integral membrane proteins are discussed. We also describe new tools that allow us to better control and validate results obtained with SCAM (substituted cysteine accessibility method), an approach that determines the position of individual residues with respect to the membrane plane, whereby only minimal changes in the primary sequence have to be introduced into the protein of interest

GREEN ALLOY OF SILVER NANOPARTICLES FROM ENDOPHYTIC EXTRACTS OF WITHANIA SOMNIFERA AND STUDIES OF ANTIBACTERIAL AND ANTIMITOTIC ACTIVITY

  Objectives: The main aim is to elaborate a cost-effective and environmentally friendly synthesis of silver nanoparticles (AgNPs) by endophytic extracts isolated from Withania somnifera as a reducing and capping agent, which has proven antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Klebsiella sp.Methods: Characterization of AgNPs was carried out employing ultraviolet-visible spectrophotometry, scanning electron microscopy (SEM), and X-ray diffraction studies (XRD). Antibacterial activity of AgNPs was conducted by disc diffusion method antimitotic activity was also evaluated by determining mitotic index in Allium cepa root tips.Results: Ultraviolet-visible spectroscopy was given a peak at 400 nm confirmed the AgNPs. The images of the SEM have confirmed the formation of AgNPs with an average size of 40 nm. XRD results were remarkable in confirmation of synthesized AgNPs with distinct XRD peaks at 2θ values of 38, 44, 64, and 77 lattice planes were observed which indexed the facts of silver (111), (200), (220), and (311), respectively. The AgNPs showed effective antibacterial activity against tested microorganisms at 100 μg/discs concentrations. A significant mitotic index (22.8±1.4b and 26.9±0.9b) was observed in A. cepa root tips at 10 mg/ml, 5 mg/ml concentration, respectively.Conclusion: It can be concluded that the endophytes of W. somnifera can be a good source for AgNP synthesis and showed a significant antimicrobial activity against tested microorganisms, especially E. coli followed by S. aureus and P. aeruginosa. Suggestive results were found in antimitotic activity which one of screening methods for development of anticancer drugs. An important outcome of our study will be the extension of value-added products for the industries of biomedical and nanotechnology based

Functional Analysis of the Genes Encoding Diaminopropionate Ammonia Lyase in Escherichia coli and Salmonella enterica Serovar Typhimurium

Diaminopropionate ammonia lyase (DAPAL) is a pyridoxal-5'phosphate (PLP)-dependent enzyme that catalyzes the conversion of diaminopropionate (DAP) to pyruvate and ammonia and plays an important role in cell metabolism. We have investigated the role of the ygeX gene of Escherichia coli K-12 and its ortholog, STM1002, in Salmonella enterica serovar Typhimurium LT2, presumed to encode DAPAL, in the growth kinetics of the bacteria. While Salmonella Typhimurium LT2 could grow on DL-DAP as a sole carbon source, the wild-type E. coli K-12 strain exhibited only marginal growth on DL-DAP, suggesting that DAPAL is functional in S. Typhimurium. The expression of ygeX in E. coli was low as detected by reverse transcriptase PCR (RT-PCR), consistent with the poor growth of E. coli on DL-DAP. Strains of S. Typhimurium and E. coli with STM1002 and ygeX, respectively, deleted showed loss of growth on DL-DAP, confirming that STM1002 (ygeX) is the locus encoding DAPAL. Interestingly, the presence of DL-DAP caused a growth inhibition of the wild-type E. coli strain as well as the knockout strains of S. Typhimurium and E. coli in minimal glucose/glycerol medium. Inhibition by DL-DAP was rescued by transforming the strains with plasmids containing the STM1002 (ygeX) gene encoding DAPAL or supplementing the medium with Casamino Acids. Growth restoration studies using media lacking specific amino acid supplements suggested that growth inhibition by DL-DAP in the absence of DAPAL is associated with auxotrophy related to the inhibition of the enzymes involved in the biosynthetic pathways of pyruvate and aspartate and the amino acids derived from them

Efficacy and safety of a modified- ‘modified Ponticelli’ regimen for treatment of primary membranous nephropathy

Background: Modified Ponticelli regimen (mPR), consisting of cyclical steroids and cyclophosphamide, is the most established therapy for primary membranous nephropathy (MN). Yet, the potential toxicity of this treatment regimen poses a significant concern. Objectives: The aim of this study was to assess the efficacy and safety of a modified version of the conventional mPR for primary MN using lower-than-standard dose pulse steroids. Patients and Methods: This was a retrospective single-center analysis of patients admitted between January 2008 to December 2017. All treatment-naive patients with biopsy-proven primary MN treated with a lower-than-standard dose pulse steroid-based modification of the conventional mPR (intravenous pulse of 500 mg methyl-prednisolone, instead of 1000 mg) were included. We report the remission rates at the end of 6 months (both complete and partial), relapses and adverse effects of treatment at the end of follow-up. Results: A total of 41 individuals were included. Of 31 individuals who completed six months of treatment (six were lost to follow-up, while four discontinued immunosuppression due to infections), 71% (n=22) responded to treatment [complete remission in 25.8% (n=8), partial remission in 45.2% (n=14)]. Most common complications detected throughout the treatment were steroid induced diabetes mellitus in 40% (n=14/35), infections in 25.7% (of which immunosuppression was discontinued for four participants), and leucopenia in 8.5% (n=3/35). Relapses were seen in 29% (n=9) during follow-up (mean follow-up period: 36 months). Conclusions: The modified- ‘modified Ponticelli’ regimen with lower-than-standard dose intravenous steroids and cyclophosphamide was efficient in attaining remission in primary MN

Intradialytic hypertension prevalence and predictive factors: A single centre study

Introduction: Intradialytic hypertension (IDH) is associated with significant vascular and cardiac adverse outcomes. Objectives: This study was performed to know the prevalence and factors predicting IDH. Patients and Methods: A single-center cross-sectional observational study at a tertiary care hospital. After ethics committee approval and informed consent, all patients over 18 years on twice weekly hemodialysis were included, those on peritoneal dialysis and acute kidney injury excluded. Primary outcome was prevalence of IDH based on three definitions and secondary outcome was predictive factors. IDH was defined as ≥10 mm Hg surge in systolic blood pressure (SBP) between pre-and postdialysis in 4 of 6 successive sessions or >15 mm Hg rise in mean arterial pressure (MAP) between start and end of dialysis or symptomatic rise in blood pressure requiring intervention. SBP and MAP were measured on standardized monitors before, hourly and 30 minutes post dialysis. Results: Of 136 patients, prevalence of intra-dialytic hypertension was 78/136 (57%), 33/136 (24%), 15/136 (11%) based on systolic rise, rise in MAP and symptomatic rise in BP respectively. Among those with systolic rise, diabetes mellitus (P= 0.03), undernourishment (P=0.03), inter-dialytic weight gain >3 kg (P 3 years (P3 kg and dialysis vintage >3 years predicted IDH

COVID-19 in kidney transplant recipients; an Indian experience

Introduction: Kidney transplant recipients appear to be at high risk for severe COVID-19 illness due to chronic immunosuppression and coexisting conditions. Objectives: We aimed to study the clinical characteristics, laboratory and radiological results, treatment aspects and clinical outcomes of kidney transplant patients with COVID-19. Patient and Methods: Twenty consecutive kidney transplant patients with COVID-19 pneumonia from two tertiary care centers from India were retrospectively studied from July 1 to Oct 31, 2020. Results: Of 20 patients, 18 required admission; mean age was 42.8±9.39 years and 18 out of 20 (90%) were male. Symptom onset to testing time was a mean of 3.05±1.47 days. All patients were on triple immunosuppression. The median time since transplantation to COVID-19 was 3.75 years (IQR 2.37-5.41). Fever, cough and breathlessness were the most common presenting symptoms. Nine out of twenty (45%) had severe COVID-19 while six out of 20 (30%) required intensive care. Twelve (60%) patients had lymphopenia. Additionally mycophenolate was withheld in seventeen out of twenty (85 %) and enoxaparin and intravenous methylprednisolone were administered in all hospitalized patients while remdesivir was prescribed in 16 out of 20 (80%). Moreover, acute kidney injury (AKI) was seen in five out of 20 (25%) since one of died (5%). After a median hospital stay of 8.5 days (IQR 6.75-15.5), seventeen patients were discharged from the hospital. Conclusion: COVID-19 infection in kidney transplant recipients is usually a moderate-severe form. COVID-19 should be a differential diagnosis for fever in this high-risk population however lymphopenia may not be seen in all. Antimetabolite withdrawal, intravenous steroid, anticoagulation and early remdesivir were all found to be safe and effective strategies for improving outcomes. Early diagnosis and timely treatment may decrease mortality in this high-risk population

Genomic introgression in laboratory evolved hybrid races, Cytorace 1 and Fissioncytorace-1 of Nasuta-albomicans

Nasuta-albomicans complex (NAC) of Drosophila is an artificial hybrid zone comprising of Drosophila nasuta nasuta, Drosophila nasuta albomicans and 16 Cytoraces, which are the evolutionary products of a long range hybridization experiment conducted in the laboratory environment. Occurrence of centric fission in the X3 chromosome of Cytorace 1 led to the derivation of Fissioncytorace-1. Molecular techniques have emerged as powerful and valuable tools for detection and exploitation of genetic polymorphism. In the present study, Cytorace 1 and Fissioncytorace-1 were subjected to Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) analyses to determine the introgression of D. n. nasuta and D. n. albomicans genomes. It was found that Cytorace 1 and Fissioncytorace-1 exhibit similarities in RAPD and ISSR profiles although different combinations of genomic regions could have favoured Fissioncytorace-1, for better morphophenotypes and fitness, when compared to Cytorace 1, which has existed for over 15 years from the time of its evolution in the laboratory environment