Prevalence and Intensity of Cornudiscoides Agarwali (Monogenoidea) on the Gills of Day's Mystus (Mystus Bleekeri) in Relation to Some Ecological and Biological Factors from Arunachal Pradesh, India.

This work investigated the relationship of host size, seasons, and water quality parameters with the prevalence and intensity of Cornudiscoides agarwali on Mystus bleekeri collected from the Dikrong River in Arunachal Pradesh, India from February 2016 to January 2017. A total of 2760 specimens of C. agarwali were recovered from 114 individuals of M. bleekeri. The levels of mean intensity, but not the prevalence, of infection of C. agarwali were positively correlated with fish host size, peaking in the largest size class (45.20 ± 5.69 parasites/fi sh). The prevalence values had a statistically significant seasonal trend, reaching highest (100 %) during the pre-monsoon season, followed by 91.8% during the post-monsoon period and 87.5 % during the monsoon season. The levels of mean intensity of infection were also dependent on the seasons, reaching significantly higher levels during the pre-monsoon season (42.75 ± 4.18 parasites/fi sh). All water quality parameters measured were within the safety value recommended for freshwater aquaculture. Cornudiscoides agarwali maintained its prevalence above 87.5 % throughout the annual cycle, which means it was able to reproduce year-round in a non-polluted river. This could be an indication of monogenoidean community and population dynamics thriving best under optimum water quality parameters. Also, this article draws the attention of parasitologists and ichthyologists to a taxonomic problem of the misidentification of Mystus spp., and therefore, possibly of their parasitic monogenoids.

Preparation, biological evaluation and molecular docking study of imidazolyl dihydropyrimidines as potential Mycobacterium tuberculosis dihydrofolate reductase inhibitors.

A series of novel dihydropyrimidine derivatives bearing an imidazole nucleus at C-4 position were synthesized in excellent yields via Biginelli multi-component reaction. The newly synthesized compounds were characterized by IR, (1)H NMR, (13)C NMR and Mass spectroscopy. In vitro antitubercular evaluation of all the newly synthesized compounds 4a-p against Mycobacterium tuberculosis (Mtb) H37Rv showed, 4j (MIC: 0.39µg/mL; SI: >25.64), 4m (MIC: 0.78µg/mL; SI: >12.82) and 4p (MIC: 0.39µg/mL; SI: 24.10) as the most promising lead analogues. Compounds 4j, 4m and 4p displayed effective reduction in residual Mtb growth within the tuberculosis-infected macrophage model. Further, molecular docking study of active molecules 4j, 4m and 4p against Mycobacterium tuberculosis dihydrofolate reductase (Mtb DHFR) proved their potency as Mtb DHFR inhibitors acting as potential leads for further development. Pharmacokinetic properties leading to drug-likeness were also predicted for most active molecules 4j, 4m and 4p.

SWI/SNF chromatin remodeling enzymes in melanocyte differentiation and melanoma.

Epidermal melanocytes are pigment-producing cells derived from the neural crest that protects skin from the damaging effects of solar radiation. Malignant melanoma, a highly aggressive cancer, arises from melanocytes. SWI/SNF enzymes are multiprotein complexes that remodel chromatin structure and have extensive roles in cellular differentiation. Components of the complex have been found to be mutated or lost in several human cancers. This review focuses on studies that implicate SWI/SNF enzymes in melanocyte differentiation and in melanoma.

Studies on molecular properties prediction, antitubercular and antimicrobial activities of novel quinoline based pyrimidine motifs.

In the present study, a series of 3-((6-(2,6-dichloroquinolin-3-yl)-4-aryl-1,6-dihydro-pyrimidin-2-yl)thio)propanenitriles 5a-o were synthesized and subjected to molecular properties prediction and drug-likeness model score by Molinspiration property calculation toolkit and MolSoft software, respectively. Compound 5m (4-OCH3) was found to be maximum drug-likeness model score (0.42). Among the screened compounds, 5m showed the most promising antitubercular activity with MIC of 0.20 µg/mL, while compounds 5g, 5k and 5m displayed broad spectrum antibacterial activity against all the bacterial strains. Moreover, compound 5k was found to be the most potent antifungal agent. Further, the results of preliminary MTT cytotoxicity studies on HeLa cells suggested that potent antimicrobial activity of 5g, 5k and 5m was escorted by low cytotoxicity.

Optimising dynamic mechanical analysis experiments on soft rubbers for use in time temperature superposition.

Rubbers are ubiquitous in engineering applications where they are often subjected to loading leading to high strain rate deformation. The strong rate and temperature dependence of rubbers and their composites motivates research into understanding their mechanical response under a wide range of conditions. However, experimental characterisation of the rate-temperature dependence of soft rubbers is challenging. In this methods paper, an improved methodology is proposed for conducting Dynamic Mechanical Analysis (DMA) experiments on rubbers. The higher quality data produced can be used in time-temperature superposition (TTS) applications to derive a more accurate definition of the rubber's rate-temperature dependence. Overall, the improvements obtained can be summarised as follows:•Overall, the proposed methodology can be summarised with the following improvements:•Reducing clamping artefacts due to volume expansion•Ensuring high quality temperature stability•Improving the contact area between the specimen and the clamps.

Laparoscopic kidney transplantation: an initial experience.

Laparoscopic donor nephrectomy has the advantages of less pain, early ambulation and shorter hospitalization compared to open donor nephrectomy. Kidney recipient surgery is, however, traditionally performed by open surgery. Our aim was to study feasibility and safety of laparoscopic kidney transplantation (LKT). After permission from Internal Review Board, LKT was performed in four patients. All kidneys were procured from deceased donors. Left kidney was used for LKT and transplanted in left iliac fossa while right kidney was used for standard open kidney transplantation (OKT). All transplantation procedures were performed successfully. Cold ischemia time varied between 4 h and 14 h. For LKT, mean time for anastomosis was 65 (range 62-72) min, mean operative time was 3.97 (range 3.5-5) h, mean blood loss was 131.25 mL (range 45-350) mL. Mean wound length was 7 cm in LKT group and 18.4 cm in OKT group. Delayed graft function was observed in one patient in each group. One patient was lost in OKT group due to posttransplant bacterial meningitis. At 6 months, both groups have comparable value of serum creatinine. In conclusion, LKT is technically feasible and safe. Long term outcome needs to be evaluated in a larger study.

Mutagenic, cytotoxic, and genotoxic properties of tobacco smoke produced by cigarillos available on the Canadian market.

Cigarillos (aka little cigars) have been increasing in popularity unlike cigarettes; but relatively little is known about the toxicology of the mainstream smoke (MSS) from such products. Therefore, the objective of this work was to compare the toxicological properties of the MSS (Health Canada Intensive smoking conditions) from a range of cigarillo products with the toxicological properties of MSS of cigarettes. Three in vitro assays were used to evaluate the toxicities of the MSS total particulate matter (TPM): (1) mutagenicity using Ames assay with Salmonella strains TA98 and TA100 with S9 metabolic activation (+S9); (2) cytotoxicity using the Neutral Red Uptake (NRU) assay with CHO (Chinese Hamster Ovary) cells; and (3) genotoxicity using the micronucleus assay with CHO cells and short-term exposures (3-h ± S9). The Ames assay (TA100+S9) and the NRU assay were also applied to the gas/vapour phase of the MSS that passed through the Cambridge pad. On a per-milligram-nicotine basis, the preferred way of comparing toxicities of different types of tobacco products, the MSS from cigarillos was not less toxic, and in some cases more toxic (TPM fraction TA98+S9, NRU), than the MSS from cigarettes. Thus, our findings support our prior work on smoke mutagenicity that showed MSS from cigarillos was not less toxic than MSS from cigarettes.

Identification and metabolite profiling of Sitophilus oryzae L. by 1D and 2D NMR spectroscopy.

The polyphagous insect Sitophilus oryzae L. (Coleoptera:Curculionidae) has a tremendous adaptability in feeding behaviour, making it a serious invasive pest of stored cereals. The present study identifies the metabolite composition of Sitophilus oryzae (S. oryzae) using Nuclear Magnetic Resonance (NMR) spectroscopy. Assignment of 1D-proton by NMR, 1H-1H COSY, 2D-TOCSY 1H-1H, had been done. Amongst the various biochemically important metabolites isoleucine, valine, leucine, beta-hydroxybutyrate, lysine, glutamate, glutamine, proline, lactate, alanine, di-methylamine, alpha-glucose, beta-glucose, choline, glycerophosphorylcholine and tyrosine are present in S. oryzae. In wheat-fed S. oryzae, the presence of threonine and the absence of lactate is observed. In rice-fed S. oryzae, however, the presence of lactate and the absence of threonine were observed. Barley-fed S. oryzae shows presence of both tyrosine and lactate. It is concluded that the pest S. oryzae has adaptability on different stored cereals and grains, depicting the presence of earlier reported metabolites. The present study aims to identify the key metabolic components and associated enzymes in Sitophilus oryzae fed on different cereals.

Chemical and toxicological characterization of commercial smokeless tobacco products available on the Canadian market.

Some health experts are recommending that smokers who refuse to quit or refuse to use nicotine replacement therapy (NRT) such as nicotine-containing chewing gum switch to certain types of smokeless tobacco products (STP) such as Swedish snus. Other health experts disagree citing the uncertainty in the composition of commercially available STP, the lack of governmental regulations to ensure that STP advertised to meet certain standards (i.e., GothiaTek) do actually meet such standards, and the uncertainty that any STP can provide as safe as alternative to smoking as NRT. One reason for uncertainty is the dearth of detailed chemical and toxicological information on contemporary STP. Unlike the situation with cigarettes, there are few standardized methods for analytical and toxicological studies of STP. Consequently, the objective for this work was to characterize several types of STP available on the Canadian market using the modifications of the Official Health Canada chemical and toxicological methods developed for cigarettes. Moist snuff samples tested had TSNA and B[a]P levels somewhat above the GothiaTek standard while samples of Swedish snus, low-moisture snuff, and US-style chewing tobacco did not. Use of in vitro assays to assess STP toxicity was of limited utility in distinguishing product types.

Overexpression of Human Mutant PANK2 Proteins Affects Development and Motor Behavior of Zebrafish Embryos.

Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a genetic and early-onset neurodegenerative disorder characterized by iron accumulation in the basal ganglia. It is due to mutations in Pantothenate Kinase 2 (PANK2), an enzyme that catalyzes the phosphorylation of vitamin B5, first and essential step in coenzyme A (CoA) biosynthesis. Most likely, an unbalance of the neuronal levels of this important cofactor represents the initial trigger of the neurodegenerative process, yet a complete understanding of the connection between PANK2 malfunctioning and neuronal death is lacking. Most PKAN patients carry mutations in both alleles and a loss of function mechanism is proposed to explain the pathology. When PANK2 mutants were analyzed for stability, dimerization capacity, and enzymatic activity in vitro, many of them showed properties like the wild-type form. To further explore this aspect, we overexpressed the wild-type protein, two mutant forms with reduced kinase activity and two retaining the catalytic activity in zebrafish embryos and analyzed the morpho-functional consequences. While the wild-type protein had no effects, all mutant proteins generated phenotypes that partially resembled those observed in pank2 and coasy morphants and were rescued by CoA and vitamin B5 supplementation. The overexpression of PANK2 mutant forms appears to be associated with perturbation in CoA availability, irrespective of their catalytic activity.

Proteomic identification of C/EBP-DBD multiprotein complex: JNK1 activates stem cell regulator C/EBPalpha by inhibiting its ubiquitination.

Functional inactivation of transcription factors in hematopoietic stem cell development is involved in the pathogenesis of acute myeloid leukemia (AML). Stem cell regulator C/enhancer binding protein (EBP)alpha is among such transcription factors known to be inactive in AML. This is either due to mutations or inhibition by protein-protein interactions. Here, we applied a mass spectrometry-based proteomic approach to systematically identify putative co-activator proteins interacting with the DNA-binding domain (DBD) of C/EBP transcription factors. In our proteomic screen, we identified c-Jun N-terminal kinase (JNK) 1 among others such as PAK6, MADP-1, calmodulin-like skin proteins and ZNF45 as proteins interacting with DBD of C/EBPs from nuclear extract of myelomonocytic U937 cells. We show that kinase JNK1 physically interacts with DBD of C/EBPalpha in vitro and in vivo. Furthermore, we show that active JNK1 inhibits ubiquitination of C/EBPalpha possibly by phosphorylating in its DBD. Consequently, JNK1 prolongs C/EBPalpha protein half-life leading to its enhanced transactivation and DNA-binding capacity. In certain AML patients, however, the JNK1 mRNA expression and its kinase activity is decreased which suggests a possible reason for C/EBPalpha inactivation in AML. Thus, we report the first proteomic screen of C/EBP-interacting proteins, which identifies JNK1 as positive regulator of C/EBPalpha.

C/EBPalpha S248A mutation reduces granulocytic differentiation in human leukemic K562 cells.

Phosphorylation of C/EBPalpha can either lead to granulocytic differentiation or a block in granulopoiesis. This dichotomy in effect is dependent on the upstream signaling pathway and the phosphorylation site in C/EBPalpha. Ras signaling induced phosphorylation of S248 residue of C/EBPalpha is known to enhance granulocytic differentiation. In this study, using beta-estradiol inducible stable cell lines we show that the point mutation of phosphorylation site S248 in C/EBP disrupts the CD11b and GCSFr expression and subsequently reduces the differentiation of leukemic K562 cells. Based on our observations in the present study, we conclude that S248A mutation of C/EBPalpha leads to a reduction of granulocytic differentiation markers and a block in differentiation at the morphological level.

Multiple ways of C/EBPalpha inhibition in myeloid leukaemia.

Transcription factors play a crucial role in myeloid differentiation and lineage determination. Tumour suppressor protein C/EBPalpha is a key regulator of granulocytic differentiation whose functional inactivation has become a pathophysiological signature of myeloid leukaemia. In this review we describe various mechanisms such as antagonistic protein-protein interaction, mutation and posttranslational modifications of C/EBPalpha which lead to its transcriptional inhibition and render C/EBPalpha inactive in its functions.

Early Targeting of L-Selectin on Leukocytes Promotes Recovery after Spinal Cord Injury, Implicating Novel Mechanisms of Pathogenesis.

L-selectin, a lectin-like receptor on all leukocyte classes, functions in adhesive and signaling roles in the recruitment of myeloid cells from the blood to sites of inflammation. Here, we consider L-selectin as a determinant of neurological recovery in a murine model of spinal cord injury (SCI). Spinal cord-injured, L-selectin knock-out (KO) mice (male) showed improved long-term recovery with greater white matter sparing relative to wild-type (WT) mice and reduced oxidative stress in the injured cord at 72 h post-SCI. There was a partial and transient reduction in accumulation of neutrophils in the injured spinal cords of KOs at 24 h post-injury. To complement these findings with KO mice, we sought a pharmacologic means for lowering L-selectin levels. We found that diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), induced the shedding of L-selectin from the cell surface of myeloid subsets, specifically neutrophils and non-classical monocytes, in the blood and the injured spinal cord. Diclofenac administration to injured WT mice enhanced neurological recovery to a level comparable to that of KOs but did not improve recovery in KOs. While diclofenac treatment had no effect on myeloid cell accumulation, there was a reduction in oxidative stress at 72 h post-SCI. These findings implicate L-selectin in secondary pathogenesis beyond a role in leukocyte recruitment and raise the possibility of repurposing diclofenac for the treatment of SCI.

Differential Proteomics for Distinguishing Ischemic Stroke from Controls: a Pilot Study of the SpecTRA Project.

A diagnostic blood test for stroke is desirable but will likely require multiple proteins rather than a single "troponin." Validating large protein panels requires large patient numbers. Mass spectrometry (MS) is a cost-effective tool for this task. We compared differences in the abundance of 147 protein markers to distinguish 20 acute cerebrovascular syndrome (ACVS) patients who presented to the Emergency Department of one urban hospital within < 24 h from onset) and from 20 control patients who were enrolled via an outpatient neurology clinic. We targeted proteins from the stroke literature plus cardiovascular markers previously studied in our lab. One hundred forty-one proteins were quantified using MS, 8 were quantified using antibody protein enrichment with MS, and 32 were measured using ELISA, with some proteins measured by multiple techniques. Thirty proteins (4 by ELISA and 26 by the MS techniques) were differentially abundant between mimic and stroke after adjusting for age in robust regression analyses (FDR < 0.20). A logistic regression model using the first two principal components of the proteins significantly improved discrimination between strokes and controls compared to a model based on age alone (p < 0.001, cross-validated AUC 0.93 vs. 0.78). Significant proteins included markers of inflammation (47%), coagulation (40%), atrial fibrillation (7%), neurovascular unit injury (3%), and other (3%). These results suggest the potential value of plasma proteins as biomarkers for ACVS diagnosis and the role of plasma-based MS in this area.

Canadian Stroke Best Practice Recommendations for Acute Stroke Management: Prehospital, Emergency Department, and Acute Inpatient Stroke Care, 6th Edition, Update 2018.

The 2018 update of the Canadian Stroke Best Practice Recommendations for Acute Stroke Management, 6th edition, is a comprehensive summary of current evidence-based recommendations, appropriate for use by healthcare providers and system planners caring for persons with very recent symptoms of acute stroke or transient ischemic attack. The recommendations are intended for use by a interdisciplinary team of clinicians across a wide range of settings and highlight key elements involved in prehospital and Emergency Department care, acute treatments for ischemic stroke, and acute inpatient care. The most notable changes included in this 6th edition are the renaming of the module and its integration of the formerly separate modules on prehospital and emergency care and acute inpatient stroke care. The new module, Acute Stroke Management: Prehospital, Emergency Department, and Acute Inpatient Stroke Care is now a single, comprehensive module addressing the most important aspects of acute stroke care delivery. Other notable changes include the removal of two sections related to the emergency management of intracerebral hemorrhage and subarachnoid hemorrhage. These topics are covered in a new, dedicated module, to be released later this year. The most significant recommendation updates are for neuroimaging; the extension of the time window for endovascular thrombectomy treatment out to 24 h; considerations for treating a highly selected group of people with stroke of unknown time of onset; and recommendations for dual antiplatelet therapy for a limited duration after acute minor ischemic stroke and transient ischemic attack. This module also emphasizes the need for increased public and healthcare provider's recognition of the signs of stroke and immediate actions to take; the important expanding role of paramedics and all emergency medical services personnel; arriving at a stroke-enabled Emergency Department without delay; and launching local healthcare institution code stroke protocols. Revisions have also been made to the recommendations for the triage and assessment of risk of recurrent stroke after transient ischemic attack/minor stroke and suggested urgency levels for investigations and initiation of management strategies. The goal of this updated guideline is to optimize stroke care across Canada, by reducing practice variations and reducing the gap between current knowledge and clinical practice.

Proteomics of acute myeloid leukaemia: Cytogenetic risk groups differ specifically in their proteome, interactome and post-translational protein modifications.

Acute myeloid leukaemia (AML) is characterized by specific cytogenetic aberrations that are strong determinants of prognostic outcome and therapeutic response. Because the pathological outcome of AML patients with cytogenetic abnormalities differs considerably, we hypothesized that their proteome may also differ specifically in their expression pattern, protein interaction pathways and post-translational modifications (PTM). We performed this study using 42 AML patients diagnosed for various cytogenetic abnormalities based on two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MS) and MSMS tandem MS. We could identify significant differences in the proteome and PTM of peptides, later confirmed by other methods, between cytogenetic groups. The interactome analysis based on computational bioinformatics reveals major regulating networks: MAPK8 and MYC for complex aberrant karyotype, TP53 for t(8;21), TP53-MYC-PRKAC for 11q23 and JUN and MYC for Inv(16). Further, we analysed 42 MS spectra representative of hnRNPH1, calreticulin and hnRNPA2/B1 in a peak explorer, which reveals a cytogenetic-specific PTM of beta-O-linked N-acetyl glucosamine (O-GlcNAc) of hnRNPH1 in AML patients with 11q23 translocation, an acetylation of calreticulin in t(8;21) translocation and methylation of hnRNPA2/B1 in patients with translocations of t(8;21) and inv(16). This report may lead to a new thinking about AML pathogenesis, as differences at PTM level could be used to distinguish different subtypes of AML.

Hepatitis B virus X protein induces RNA polymerase III-dependent gene transcription and increases cellular TATA-binding protein by activating the Ras signaling pathway.

Our previous studies have shown that the hepatitis B virus protein, X, activates all three classes of RNA polymerase III (pol III)-dependent promoters by increasing the cellular level of TATA-binding protein (TBP) (H.-D. Wang et al., Mol. Cell. Biol. 15:6720-6728, 1995), a limiting transcription component (A. Trivedi et al., Mol. Cell. Biol. 16:6909-6916, 1996). We have investigated whether these X-mediated events are dependent on the activation of the Ras/Raf-1 signaling pathway. Transient expression of a dominant-negative mutant Ras gene (Ras-ala15) in a Drosophila S-2 stable cell line expressing X (X-S2), or incubation of the cells with a Ras farnesylation inhibitor, specifically blocked both the X-dependent activation of a cotransfected tRNA gene and the increase in cellular TBP levels. Transient expression of a constitutively activated form of Ras (Ras-val12) in control S2 cells produced both an increase in tRNA gene transcription and an increase in cellular TBP levels. These events are not cell type specific since X-mediated gene induction was also shown to be dependent on Ras activation in a stable rat 1A cell line expressing X. Furthermore, increases in RNA pol III-dependent gene activity and TBP levels could be restored in X-S2 cells expressing Ras-ala15 by coexpressing a constitutively activated form of Raf-1. These events are serum dependent, and when the cells are serum deprived, the X-mediated effects are augmented. Together, these results demonstrate that the X-mediated induction of RNA pol III-dependent genes and increase in TBP are both dependent on the activation of the Ras/Raf-1 signaling cascade. In addition, these studies define two new and important consequences mediated by the activation of the Ras signal transduction pathway: an increase in the central transcription factor, TBP, and the induction of RNA pol III-dependent gene activity.