Novel components of the Toxoplasma inner membrane complex revealed by BioID.

UNLABELLED:The inner membrane complex (IMC) of Toxoplasma gondii is a peripheral membrane system that is composed of flattened alveolar sacs that underlie the plasma membrane, coupled to a supporting cytoskeletal network. The IMC plays important roles in parasite replication, motility, and host cell invasion. Despite these central roles in the biology of the parasite, the proteins that constitute the IMC are largely unknown. In this study, we have adapted a technique named proximity-dependent biotin identification (BioID) for use in T. gondii to identify novel components of the IMC. Using IMC proteins in both the alveoli and the cytoskeletal network as bait, we have uncovered a total of 19 new IMC proteins in both of these suborganellar compartments, two of which we functionally evaluate by gene knockout. Importantly, labeling of IMC proteins using this approach has revealed a group of proteins that localize to the sutures of the alveolar sacs that have been seen in their entirety in Toxoplasma species only by freeze fracture electron microscopy. Collectively, our study greatly expands the repertoire of known proteins in the IMC and experimentally validates BioID as a strategy for discovering novel constituents of specific cellular compartments of T. gondii. IMPORTANCE:The identification of binding partners is critical for determining protein function within cellular compartments. However, discovery of protein-protein interactions within membrane or cytoskeletal compartments is challenging, particularly for transient or unstable interactions that are often disrupted by experimental manipulation of these compartments. To circumvent these problems, we adapted an in vivo biotinylation technique called BioID for Toxoplasma species to identify binding partners and proximal proteins within native cellular environments. We used BioID to identify 19 novel proteins in the parasite IMC, an organelle consisting of fused membrane sacs and an underlying cytoskeleton, whose protein composition is largely unknown. We also demonstrate the power of BioID for targeted discovery of proteins within specific compartments, such as the IMC cytoskeleton. In addition, we uncovered a new group of proteins localizing to the alveolar sutures of the IMC. BioID promises to reveal new insights on protein constituents and interactions within cellular compartments of Toxoplasma

Engineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues.

PublishedJournal ArticleThis is the final version of the article. Available from American Chemical Society via the DOI in this record.Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.Work is supported by the National Institute of Malaria Research, Indian Council of Medical Research, New Delhi and Dept. of Biotechnology, New Delhi. K.C.P. is a recipient of the Prof. Ramalingaswami Fellowship (Department of Biotechnology, Government of India (BT/HRD/35/02/2006), K.V. is a recipient of UGC Senior Research Fellowship, M.v.d.G. is grateful for support from the University of Exeter and the Wellcome Trust (078566/A/05/Z)

The MBD7 complex promotes expression of methylated transgenes without significantly altering their methylation status.

DNA methylation is associated with gene silencing in eukaryotic organisms. Although pathways controlling the establishment, maintenance and removal of DNA methylation are known, relatively little is understood about how DNA methylation influences gene expression. Here we identified a METHYL-CpG-BINDING DOMAIN 7 (MBD7) complex in Arabidopsis thaliana that suppresses the transcriptional silencing of two LUCIFERASE (LUC) reporters via a mechanism that is largely downstream of DNA methylation. Although mutations in components of the MBD7 complex resulted in modest increases in DNA methylation concomitant with decreased LUC expression, we found that these hyper-methylation and gene expression phenotypes can be genetically uncoupled. This finding, along with genome-wide profiling experiments showing minimal changes in DNA methylation upon disruption of the MBD7 complex, places the MBD7 complex amongst a small number of factors acting downstream of DNA methylation. This complex, however, is unique as it functions to suppress, rather than enforce, DNA methylation-mediated gene silencing

PatientExploreR: an extensible application for dynamic visualization of patient clinical history from electronic health records in the OMOP common data model.

MotivationElectronic health records (EHRs) are quickly becoming omnipresent in healthcare, but interoperability issues and technical demands limit their use for biomedical and clinical research. Interactive and flexible software that interfaces directly with EHR data structured around a common data model (CDM) could accelerate more EHR-based research by making the data more accessible to researchers who lack computational expertise and/or domain knowledge.ResultsWe present PatientExploreR, an extensible application built on the R/Shiny framework that interfaces with a relational database of EHR data in the Observational Medical Outcomes Partnership CDM format. PatientExploreR produces patient-level interactive and dynamic reports and facilitates visualization of clinical data without any programming required. It allows researchers to easily construct and export patient cohorts from the EHR for analysis with other software. This application could enable easier exploration of patient-level data for physicians and researchers. PatientExploreR can incorporate EHR data from any institution that employs the CDM for users with approved access. The software code is free and open source under the MIT license, enabling institutions to install and users to expand and modify the application for their own purposes.Availability and implementationPatientExploreR can be freely obtained from GitHub: https://github.com/BenGlicksberg/PatientExploreR. We provide instructions for how researchers with approved access to their institutional EHR can use this package. We also release an open sandbox server of synthesized patient data for users without EHR access to explore: http://patientexplorer.ucsf.edu.Supplementary informationSupplementary data are available at Bioinformatics online

Primary sclerosing cholangitis in India

Six patients with PSC have been diagnosed and followed up at a centre in Northern India for periods upto 4 years. They all presented with cholestatic jaundice and cholangitis, but one of them subsequently presented with variceal haemorrhage. Cholangiographic features were the most characteristic and included irregular narrowing and segmental dilatation of the biliary radicles giving them a beaded appearance. Treating them was most frustrating. Two of them died of hepatic encephalopathy, others have also continued to be sick during the follow-up

COMPUTATIONAL APPROACHES IN THE ESTIMATION AND ANALYSIS OF TRANSCRIPTS DIFFERENTIAL EXPRESSION AND SPLICING: APPLICATION TO SPINAL MUSCULAR ATROPHY

Spinal Muscular Atrophy (SMA) is among the most common genetic neurological diseases that cause infant mortality. SMA is caused by deletion or mutations in the survival motor neuron 1 gene (SMN1), which are expected to generate alterations in RNA transcription, or splicing and most importantly reductions in mRNA transport within the axons of motor neurons (MNs). SMA ultimately results in the selective degeneration of MNs in spinal cord, but the underlying reason is still not clear entirely. The aim of this study is to investigate splicing abnormalities in SMA, and to identify genes presenting differential splicing possibly involved in the pathogenesis of SMA at genome-wide level. We performed RNA-Sequencing data analysis on 2 SMA patients and 2 controls, with 2 biological replicates each sample, derived from their induced Pluripotent Stem Cells-differentiated-MNs. Three types of analyses were executed. Firstly, differential expression analysis was performed to identify possibly mis-regulated genes using Cufflinks. Secondly, alternative splicing analysis was conducted to find differentially-used exons (DUEs; using DEXSeq) as splicing patterns are known to be altered in MNs by the suboptimal levels of SMN protein. Thirdly, we did RNA-binding protein (RBP) - motif discovery for the set of identified alternative cassette-DUEs, to pinpoint possible mechanisms of such alterations, specific to MNs. The gene ontology enrichment analysis of significant DEGs and alternative cassette-DUEs revealed various interesting terms including axon-guidance, muscle-contraction, microtubule-based transport, axon-cargo transport, synapse etc. which suggests their involvement in SMA. Further, promising results were obtained from motif analysis which has identified 22 RBPs out of which 7 RBPs namely, PABPC1, PABPC3, PABPC4, PABPC5, PABPN1, SART3 and KHDRBS1 are known for mRNAs stabilization and mRNA transport across MN-axon. Five RBPs from PABP family are known to interact directly with SMN protein that enhance mRNA transport in MNs. To validate our results specific wet-lab experiments are required, involving precise recognition of RNA-binding sites correspondent with our findings. Our work has provided a promising set of putative targets which might offer potential therapeutic role towards treating SMA. During the course of our study, we have observed that current methods for an effective understanding of differential splicing events within the transcriptomic landscape at high resolution are insufficient. To address this problem, we developed a computational model which has a potential to precisely estimate the \u201ctranscript expression levels\u201d within a given gene locus by disentangling mature and nascent transcription contributions for each transcript at per base resolution. We modeled exonic and intronic read coverages by applying a non-linear computational model and estimated expression for each transcript, which best approximated the observed expression in total RNA-Seq data. The performance of our model was good in terms of computational processing time and memory usage. The application of our model is in the detection of differential splicing events. At exon level, differences in the ratio of the sum of mature and the sum of nascent transcripts over all the transcripts in a gene locus gives an indication of differential splicing. We have implemented our model in R-statistical language

Management of fracture upper end humerus with proximal humerus internal locking system

Background: Fracture of proximal humerus is the second most common fracture of the upper extremity, following distal forearm fractures. Aim of the present study was to evaluate the efficacy of proximal humerus internal locking system (PHILOS) plate fixation for proximal humerus fractures.Methods: Functional outcomes of 12 men and 18 women aged 22 to 78 (mean, 58) years who underwent PHILOS plate fixation for proximal humeral fractures were reviewed. Indications for surgery were 2 part (n=9), 3 part (n=14) or 4-part (n=7) closed proximal humeral fractures with angulation of more than 45 degrees or displacement of more than 1 cm. Functional outcomes and shoulder range of movement were assessed based on the Constant scoring system.Results: All patients will be followed up at monthly intervals for 6 months. During this period patient, will be motivated for physiotherapy and gradual normal use of the affected limb. Fracture union will be assessed clinically and radiologically. In our study of fracture proximal humerus union in most of the cases (24) occurring between 10-14 weeks. Range of union time was 8 to 18 weeks. one case of avascular necrosis of head occur in our case. In out of 30 cases excellent result in 7 cases, Good in16 cases Satisfactory in 5 cases and Poor in 2 cases were obtained.Conclusions: The PHILOS plate fixation is effective treatment for proximal humeral fractures particularly in osteoporotic bones

Management of intra-articular fracture of distal femur with LCP and Lag screws in adults

Background: Accurate reconstruction of articular surface of distal femur by closed manipulation is not possible. The recent trend for displaced intra-articular fractures of the distal femur is open reduction and stable osteosynthesis with early rehabilitation. The best exposure is achieved through Swashbuckler approach with good results. Purpose of the study was to review the surgical management with LCP and lag screws of complex and most challenging intra-articular fracture of distal femur.Methods: 30 cases of intra-articular fractures of distal femur were treated with LCP and Lag screws in adults. Regular follow-up with all records was carried out for a period of 36 months. Most of patients were treated with Swashbuckler approach. Final outcome was carried based on Neer’s criteria.Results: Union was achieved in all the cases and mean time was 15 weeks. Patients have more range of motion in C1 and C2 types of fracture as compare to C3 Types. Mean range of motion was 1140. Early physiotherapy has big role to achieve good range of motion.Conclusions: LCP is better implant in comminuted I/A fractures of distal end of femur and in elderly patients with osteoporotic bone. In spite of the worst fracture anatomy of the comminuted fracture of distal femur and the poor quality of bone in elderly patients, this can provide better post-operative range of knee motion with overall better Knee score, achieving bony union in all the cases with early physiotherapy

Negative Feedback Regulation of the Yeast Cth1 and Cth2 mRNA Binding Proteins Is Required for Adaptation to Iron Deficiency and Iron Supplementation

Iron (Fe) is an essential element for all eukaryotic organisms because it functions as a cofactor in a wide range of biochemical processes. Cells have developed sophisticated mechanisms to tightly control Fe utilization in response to alterations in cellular demands and bioavailability. In response to Fe deficiency, the yeast Saccharomyces cerevisiae activates transcription of the CTH1 and CTH2 genes, which encode proteins that bind to AU-rich elements (AREs) within the 3′ untranslated regions (3′UTRs) of many mRNAs, leading to metabolic reprogramming of Fe-dependent pathways and decreased Fe storage. The precise mechanisms underlying Cth1 and Cth2 function and regulation are incompletely understood. We report here that the Cth1 and Cth2 proteins specifically bind in vivo to AREs located at the 3′UTRs of their own transcripts in an auto- and cross-regulated mechanism that limits their expression. By mutagenesis of the AREs within the CTH2 transcript, we demonstrate that a Cth2 negative-feedback loop is required for the efficient decline in Cth2 protein levels observed upon a rapid rise in Fe availability. Importantly, Cth2 autoregulation is critical for the appropriate recovery of Fe-dependent processes and resumption of growth in response to a change from Fe deficiency to Fe supplementation

Management of Arbeitsgemeinschaft fur osteosynthesefragen type C fractures of distal humerus in adults with two column fixation

Background: Accurate reconstruction of articular surface of distal humerus by closed manipulation is not possible. The recent trend for displaced intra-articular fractures of the distal humerus is open reduction and stable osteosynthesis with early rehabilitation. The best exposure of both columns of the distal part of the humerus and articular surface is achieved through trans-olecranon approach. Objective of the study was to evaluate the management and outcome of two column fixation of intra-articular fracture of distal humerus in adults by posterior approach via olecanon osteotomy and to restore early elbow joint function.Methods: Twenty-five cases of intra-articular fractures of distal humerus were treated by open reduction and internal fixation. All the patients were of adult age group. Chevron type olecranon osteotomy was performed and fixed with tension band wiring in all cases and follow-up period was upto 36 months. Regular clinical examination and periodical radiological evaluation were done.Results: All fractures united within average duration of 3 months. Results were evaluated as per Mayo Elbow Performance Score. According to this criteria, excellent result was achieved in 8 patients (32%), good in 13 (52%), fair in 3 (12%) and poor in 1 (4%) patients. Thus satisfactory result was obtained in 96% cases.Conclusions: The critical factors for a successful outcome of intra-articular fractures of the distal humerus depend upon severity of fracture, meticulous surgical technique, stable internal fixation, surgical experimentation and early controlled postoperative mobilization