Chondrotinase ABC I thermal stability is enhanced by site-directed mutagenesis: a molecular dynamic simulations approach

Chondroitin sulfate proteoglycans (CSPGs) are potent inhibitors of growth in the adult central nervous system. Use of the enzyme chondroitinase ABC I (ChABC I) as a strategy to reduce CSPG inhibition in experimental models of spinal cord injury has led to observations of its remarkable capacity for repair. More importantly, ChABC therapy has been demonstrated to promote significant recovery of function to spinal injured animals. Despite this incomparable function of ChABC I, its clinical application has been limited because of its thermal instability as reported in the literature. In a recent study by Nazari-Robati et al., thermal stability of ChABC I was improved by protein engineering using site-directed mutagenesis method. Here, in this study, molecular dynamics simulations were used to take a closer look into the phenomenon leading to the experimentally observed thermal stability improvement followed by the corresponding site-directed mutagenesis. We concluded that the mutations induce local flexibility along with a re-conformation into the native structure which consequently increase the protein thermal stability.     

Modifying histones and initiating meiotic recombination; new answers to an old question

It is well documented that the formation of the DNA double-strand breaks (DSBs) that initiate meiotic recombination is influenced by chromatin and larger scale chromosome organization, but the molecular nature of this influence has remained elusive. Several recent studies, including (this issue of Cell), shed light on this issue by revealing roles for posttranslational histone modifications in promoting DSB formation.     

Immunological Characterization of cdc2 and wee1 Proteins during the Growth and Differentiation of Dictyostelium discoideum

Two different antibody preparations, raised independently against the conserved EGVPSTAIREISLLKE sequence of the protein kinases encoded by the Schizosaccharomyces pombe cdc2 gene and its species homologs, immunoblotted a Dictyostelium protein of 32 kDa (p32). This polypeptide bound to p13^suc1-agarose beads, suggesting that it represents the Dictyostelium cdc2 and / or cdk2 products. The amounts of p32 detectable in cell free extracts and bound to p13^suc1-agarose were unaltered during the growth of cells synchronized for division. Although there was also essentially no change in the level of p32 during differentiation, the protein from the pseudoplasmodial stage of development did not bind to p13^suc1-agarose, implicating a developmentally regulated modification of the kinase. One of the EGVPSTAIREISLLKE antibodies also recognized a protein of 49 kDa (p49) that increased in amount dramatically during aggregation and then remained at elevated levels throughout the remainder of the differentiation process. This protein was present in low amounts throughout the growth of cells synchronized for division and was not absorbed by p13^suc1-agarose.
A 103 kDa protein (p103) was detected by Western blot analysis using antibodies raised against two different peptides corresponding to sequences in the S. pombe protein kinase p105^wee1, which is a putative upstream negative regulator of p34^cdc2 in fission yeast. The levels of p103 were constant during differentiation and during the growth of cells synchronized for division.     

The role of electrostatic energy in prediction of obligate protein-protein interactions

Background

Prediction and analysis of protein-protein interactions (PPI) and specifically types of PPIs is an important problem in life science research because of the fundamental roles of PPIs in many biological processes in living cells. In addition, electrostatic interactions are important in understanding inter-molecular interactions, since they are long-range, and because of their influence in charged molecules. This is the main motivation for using electrostatic energy for prediction of PPI types.

Results

We propose a prediction model to analyze protein interaction types, namely obligate and non-obligate, using electrostatic energy values as properties. The prediction approach uses electrostatic energy values for pairs of atoms and amino acids present in interfaces where the interaction occurs. The main features of the complexes are found and then the prediction is performed via several state-of-the-art classification techniques, including linear dimensionality reduction (LDR), support vector machine (SVM), naive Bayes (NB) and k-nearest neighbor (k-NN). For an in-depth analysis of classification results, some other experiments were performed by varying the distance cutoffs between atom pairs of interacting chains, ranging from 5Å to 13Å. Moreover, several feature selection algorithms including gain ratio (GR), information gain (IG), chi-square (Chi2) and minimum redundancy maximum relevance (mRMR) are applied on the available datasets to obtain more discriminative pairs of atom types and amino acid types as features for prediction.

Conclusions

Our results on two well-known datasets of obligate and non-obligate complexes confirm that electrostatic energy is an important property to predict obligate and non-obligate protein interaction types on the basis of all the experimental results, achieving accuracies of over 98%. Furthermore, a comparison performed by changing the distance cutoff demonstrates that the best values for prediction of PPI types using electrostatic energy range from 9Å to 12Å, which show that electrostatic interactions are long-range and cover a broader area in the interface. In addition, the results on using feature selection before prediction confirm that (a) a few pairs of atoms and amino acids are appropriate for prediction, and (b) prediction performance can be improved by eliminating irrelevant and noisy features and selecting the most discriminative ones.

     

Efficiency of Plasmocin™ on various mammalian cell lines infected by mollicutes in comparison with commonly used antibiotics in cell culture: a local experience

Mycoplasma contamination is a deleterious event for cell culture laboratories. Plasmocin™ is used to prevent and eradicate mycoplasma infections from cell. In this study, 80 different mammalian cell lines from various sources; human, monkey, mice, hamster and rat were used to study and evaluate plasmocin™ efficiency and compare it to commonly used antibiotics such as BM-cyclin, ciprofloxacin and mycoplasma removal agent (MRA). It was shown that mycoplasma infections were eradicated by plasmocin™, BM-cyclin, ciprofloxacin and MRA in 65%, 66.25%, 20%, and 31.25%, respectively, of infected cell cultures. However, re-infection with mycoplasmas after the period of 4 months occurred in 10–80% of the studied cell lines. Cell cytotoxicity and culture death was observed in 25, 17.5 and 10% of the treated cells, for plasmocin™, BM-cyclin and MRA, respectively. In this study, Plasmocin™ showed strong ability to eradicate mollicutes from our cell lines with minimal percentage of regrowth. However, due to its high cell cytotoxicity it should be used with caution especially when dealing with expensive or hard-to-obtain cell lines. Amongst the antibiotics tested, BM-cyclin was shown to remove mycoplasma with the highest efficiency.     

Extracellular vesicles as novel approaches for the treatment of osteoarthritis: a narrative review on potential mechanisms

Journal of Molecular Histology - Osteoarthritis (OA) is a progressive degeneration of articular cartilage with involvement of synovial membrane, and subchondral bone. Current treatment approaches...     

The role of bla(OXA-like carbapenemase) and their insertion sequences (ISS) in the induction of resistance against carbapenem antibiotics among Acinetobacter baumannii isolates in Tehran hospitals

This study aimed to evaluate the occurrence and dissemination of bla(OXA-like) carbapenemase genes and their insertion sequences among Acinetobacter baumannii isolates, taken from different hospitals in Tehran city and also their roles in the induction of resistance to carbapenem drugs. A total number of 100 non duplicate Acinetobacter baumannii with different origins, were isolated from patients with proved nosocomial infections at eight university hospital in Tehran city. Antimicrobial susceptibility of these strains was done by E-test against 7 antimicrobial agents according to CLSI guideline. PCR of bla(OXA-51-like), bla(OXA-23-like), bla(OXA-24-like), bla(OXA-58-like), IS(ABA-1), IS(1133) was carried out by specialized primers and then these strains were typed by REP-fingerprinting. Colistin, imipenem and meropenem were the most sensitive antibiotics against Acinetobacter baumannii isolates with 96%, 51% and 51% sensitivity respectively. All the isolates had a bla(OXA-51-like) intrinsic to these species. The rates of bla(OXA-23), 23 and 58-like were 38%, 32% and 1% respectively. Coexistence of bla(OXA-51/23/24-like) was observed among 16% of these isolates. All bla(OXA-23-like) carbapenemase genes had only one IS(ABA1). REP fingerprinting showed 5 genotypes among carbapenem resistant isolates, 16 of them being genotype A. This study emphasized on the major role of bla(OXA-like) carbapenemase, particularly bla(OXA-23-like) carbapenemase and their IS(ABA1), in the dissemination of carbapenem resistant Acinetobacter baumannii. This study confirmed a presumptive role of IS element neighboring the carbapenemase gene in the elevation of resistance to carbapenem drug among Acinetobacter baumannii isolates for the first time in Iran.     

Clinical governance implementation in a selected teaching emergency department: a systems approach

ABSTRACT: BACKGROUND: Clinical governance (CG) is among the different frameworks proposed to improve the quality of healthcare. Iran, like many other countries, has put healthcare quality improvement in its top health policy priorities. In November 2009, implementation of CG became a task for all hospitals across the country. However, it has been a challenge to clarify the notion of CG and the way to implement it in Iran. The purpose of this action research study is to understand how CG can be defined and implemented in a selected teaching emergency department (ED).Methods/designWe will use Soft Systems Methodology for both designing the study and inquiring into its content. As we considered a complex problem situation regarding the quality of care in the selected ED, we initially conceptualized CG as a cyclic set of purposeful activities designed to explore the situation and find relevant changes to improve the quality of care. Then, implementation of CG will conceptually be to carry out that set of purposeful activities. The activities will be about: understanding the situation and finding out relevant issues concerning the quality of care; exploring different stakeholders' views and ideas about the situation and how it can be improved; and defining actions to improve the quality of care through structured debates and development of accommodations among stakeholders. We will flexibly use qualitative methods of data collection and analysis in the course of the study. To ensure the study rigor, we will use different strategies. DISCUSSION: Successful implementation of CG, like other quality improvement frameworks, requires special consideration of underlying complexities. We believe that addressing the complex situation and reflections on involvement in this action research will make it possible to understand the concept of CG and its implementation in the selected setting. By describing the context and executed flexible methods of implementation, the results of this study would contribute to the development of implementation science and be employed by boards and executives governing other clinical settings to facilitate CG implementation.     

CD1d-independent activation of mouse and human iNKT cells by bacterial superantigens

Invariant NKT (iNKT) cells are infrequent but important immunomodulatory lymphocytes that exhibit CD1d-restricted reactivity with glycolipid Ags. iNKT cells express a unique T-cell receptor (TCR) composed of an invariant α-chain, paired with a limited range of β-chains. Superantigens (SAgs) are microbial toxins defined by their ability to activate conventional T cells in a TCR β-chain variable domain (Vβ)-specific manner. However, whether iNKT cells are directly activated by bacterial SAgs remains an open question. Herein, we explored the responsiveness of mouse and human iNKT cells to a panel of staphylococcal and streptococcal SAgs and examined the contribution of major histocompatibility complex (MHC) class II and CD1d to these responses. Bacterial SAgs that target mouse Vβ8, such as staphylococcal enterotoxin B (SEB), were able to activate mouse hybridoma and primary hepatic iNKT cells in the presence of mouse APCs expressing human leukocyte antigen (HLA)-DR4. iNKT cell-mediated cytokine secretion in SEB-challenged HLA-DR4-transgenic mice was CD1d-independent and accompanied by a high interferon-γ:interleukin-4 ratio consistent with an in vivo Th1 bias. Furthermore, iNKT cells from SEB-injected HLA-DR4-transgenic mice, and iNKT cells from SEB-treated human PBMCs, showed early activation by intracellular cytokine staining and CD69 expression. Unlike iNKT cell stimulation by α-galactosylceramide, stimulation by SEB did not induce TCR downregulation of either mouse or human iNKT cells. We conclude that Vβ8-targeting bacterial SAgs can activate iNKT cells by utilizing a novel pathway that requires MHC class II interactions, but not CD1d. Therefore, iNKT cells fulfill important effector functions in response to bacterial SAgs and may provide attractive targets in the management of SAg-induced illnesses.