Monoallelic and Biallelic Variants in EMC1 Identified in Individuals with Global Developmental Delay,Hypotonia, Scoliosis,and Cerebellar Atrophy

The paradigm of a single gene associated with one specific phenotype and mode of inheritance has been repeatedly challenged. Genotype-phenotype correlations can often be traced to different mutation types, localization of the variants in distinct protein domains, or the trigger of or escape from nonsense-mediated decay. Using whole-exome sequencing, we identified homozygous variants in EMC1 that segregated with a phenotype of developmental delay, hypotonia, scoliosis, and cerebellar atrophy in three families. In addition, a de novo heterozygous EMC1 variant was seen in an individual with a similar clinical and MRI imaging phenotype. EMC1 encodes a member of the endoplasmic reticulum (ER)-membrane protein complex (EMC), an evolutionarily conserved complex that has been proposed to have multiple roles in ER-associated degradation, ER-mitochondria tethering, and proper assembly of multi-pass transmembrane proteins. Perturbations of protein folding and organelle crosstalk have been implicated in neurodegenerative processes including cerebellar atrophy. We propose EMC1 as a gene in which either biallelic or monoallelic variants might lead to a syndrome including intellectual disability and preferential degeneration of the cerebellum.     

Effect of chronic pesticide exposure on murine cornea: a histopathological,cytological and flow cytometric approach to study ocular damage by xenobiotics

Pesticide exposure can occur directly or indirectly in an occupational setting or otherwise. The health hazards of pesticides have long been studied; however, little is known about the ocular insult of these potent chemicals. In this study, we examined the consequences of long-term pesticide exposure on the ocular tissue in animal model with special focus on the cornea. Swiss Albino mice were sacrificed to obtain the eye globes and various cytological, cytotoxic and histological evaluations, in vitro growth kinetic studies and flow cytometric analyses of select cytokeratins were performed to determine the structural and functional damage due to pesticide exposure. Our study revealed the detrimental impact of this xenobiotic insult by cataloguing the damage to each layer of the cornea wherein it was discovered that all the functional layers as well as the membranes were compromised. We hope that our investigation will pave the way for future studies in this oft overlooked area of affront caused by pesticide exposure to the ocular surface.     

Cellular evidence of allelopathic interference of benzoic acid to mustard (Brassica juncea L.) seedling growth.

Cellular changes in the roots of mustard (Brassica juncea L.) grown in soil treated with 1.09, 1.46 and 1.83 mg benzoic acid per g soil, a known allelochemical, were analyzed after 7 days. The recoverable concentration of 1.09, 1.46 and 1.8 mg benzoic acid per g soil (measured by high performance liquid chromatography) was 68, 150 and 250 microg benzoic acid per g soil, respectively. The benzoic acid treatments suppressed root growth by 30.5%, 58.8% and 81.1% with increasing concentrations. Transmission electron microscopy studies of roots showed irregular shaped cells arranged in disorganized manner and disruption of cell organelles at cellular level. Root cells showed dissolution of middle lamella (at 68 and 150 microg benzoic acid per g soil) but intact middle lamella with increased wall deposits was observed with 250 microg benzoic acid per g soil. Damage to the mustard root at cellular level was evidenced by changes in cell morphology and internal organization.     

Some mechanistic aspects on chiral discrimination of organic acids by immobilized bovine serum albumin (BSA)

Although chiral anionic compounds, notably a large number of organic acids, have been found to be readily separated into enantiomers on BSA-based columns, the structural requirements for an efficient enantiomer discrimination by the protein is still not very well known. Since it is often observed that very hydrophobic acids, like many of the antiinflammatory “profens,” can be resolved with large separation factors for the enantiomers, a systematic study of a series of racemic α-substituted alkanoic acids was made. The series of analytes was prepared from α-amino acids, RCH(NH₂)CO₂H (where R = C₁-C₆), by reaction with N-(chloroformyl)-carbazole. A rapid increase in the capacity ratios of both enantiomers was found with increasing length of R. The effect, however, was larger for the last eluted enantiomer, leading to a substantial increase in the separation factor; this being 7.3 for R = C₆ in 20 mM phosphate buffer (pH 8.0) with 30% of acetonitrile. Further, the separation factor also increased with decreasing organic modifier content. Thus when the R = C₆-analyte was run at a mobile phase concentration of 20% acetonitrile and a flow rate of 1.5 ml/min, the time difference between the two eluted enantiomers exceeded 20 hr. A reasonable interpretation of our results seems to be that enantioselectivity is promoted by increased hydrophobic interaction. Since the anionic charge of the analyte is also taking part in the retention mechanism, a tight binding of the analyte will result from simultaneous electrostatic and hydrophobic interaction. When the latter is increased, less conformational freedom will be left for the analyte and the steric configuration at the α-carbon atom will become more and more important. Steric hindrance by the α-substituent in the first eluted enantiomer will counteract the tight binding caused by the combined binding interactions and lead to a smaller increase in the capacity ratio.     

Defining the In Vivo Phenotype of Artemisinin-Resistant Falciparum Malaria: A Modelling Approach

BackgroundArtemisinin-resistant falciparum malaria has emerged in Southeast Asia, posing a major threat to malaria control. It is characterised by delayed asexual-stage parasite clearance, which is the reference comparator for the molecular marker ‘Kelch 13’ and in vitro sensitivity tests. However, current cut-off values denoting slow clearance based on the proportion of individuals remaining parasitaemic on the third day of treatment (''day-3''), or on peripheral blood parasite half-life, are not well supported. We here explore the parasite clearance distributions in an area of artemisinin resistance with the aim refining the in vivo phenotypic definitions.ConclusionsCharacterisation of overlapping distributions of parasite half-lives provides quantitative insight into the relationship between parasite clearance and artemisinin resistance, as well as the predictive value of the 10% cut-off in ''day-3'' parasitaemia. The findings are important for the interpretation of in vitro sensitivity tests and molecular markers for artemisinin resistance and for contextualising the ‘day 3’ threshold to account for initial parasitaemia and sample size.     

β-d-2'-α-F-2'-β-C-Methyl-6-O-substituted 3',5'-cyclic phosphate nucleotide prodrugs as inhibitors of hepatitis C virus replication: A structure-activity relationship study

The 3',5'-cyclic phosphate prodrug 9-[β-d-2'-deoxy-2'-α-fluoro-2'-β-C-methylribofuranosyl]-2-amino-6-ethoxypurine, PSI-352938 1, has demonstrated promising anti-HCV efficacy in vitro and in human clinical trials. A structure-activity relationship study of the nucleoside 3',5'-cyclic phosphate series of β-d-2'-deoxy-2'-α-fluoro-2'-β-C-methylribofuranosyl nucleoside prodrugs was undertaken and the anti-HCV activity and in vitro safety profile were assessed. Cycloalkyl 3',5'-cyclic phosphate prodrugs were shown to be significantly more potent as inhibitors of HCV replication than branched and straight chain alkyl 3',5'-cyclic phosphate prodrugs. No cytotoxicity and mitochondrial toxicity for prodrugs 12, 13 and 19 were observed at concentrations up to 100μm in vitro. Cycloalkyl esters of 3',5'-cyclic phosphate nucleotide prodrugs demonstrated the ability to produce high levels of active triphosphate in clone-A cells and primary human hepatocytes. Compounds 12, 13 and 19 also demonstrated the ability to effectively deliver in vivo high levels of active nucleoside phosphates to rat liver.     

Potential human cholesterol esterase inhibitor design: benefits from the molecular dynamics simulations and pharmacophore modeling studies

Human pancreatic cholesterol esterase (hCEase) is one of the lipases found to involve in the digestion of large and broad spectrum of substrates including triglycerides, phospholipids, cholesteryl esters, etc. The presence of bile salts is found to be very important for the activation of hCEase. Molecular dynamic simulations were performed for the apoform and bile salt complexed form of hCEase using the co-ordinates of two bile salts from bovine CEase. The stability of the systems throughout the simulation time was checked and two representative structures from the highly populated regions were selected using cluster analysis. These two representative structures were used in pharmacophore model generation. The generated pharmacophore models were validated and used in database screening. The screened hits were refined for their drug-like properties based on Lipinski's rule of five and ADMET properties. The drug-like compounds were further refined by molecular docking simulation using GOLD program based on the GOLD fitness score, mode of binding, and molecular interactions with the active site amino acids. Finally, three hits of novel scaffolds were selected as potential leads to be used in novel and potent hCEase inhibitor design. The stability of binding modes and molecular interactions of these final hits were re-assured by molecular dynamics simulations.     

Hypoxia regulates cross-talk between Syk and Lck leading to breast cancer progression and angiogenesis

Hypoxia is a key parameter that controls tumor angiogenesis and malignant progression by regulating the expression of several oncogenic molecules. The nonreceptor protein-tyrosine kinases Syk and Lck play crucial roles in the signaling mechanism of various cellular processes. The enhanced expression of Syk in normal breast tissue but not in malignant breast carcinoma has prompted us to investigate its potential role in mammary carcinogenesis. Accordingly, we hypothesized that hypoxia/reoxygenation (H/R) may play an important role in regulating Syk activation, and Lck may be involved in this process. In this study, we have demonstrated that H/R differentially regulates Syk phosphorylation and its subsequent interaction and cross-talk with Lck in MCF-7 cells. Moreover, Syk and Lck play differential roles in regulating Sp1 activation and expressions of melanoma cell adhesion molecule (MelCAM), urokinase-type plasminogen activator (uPA), matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor (VEGF) in response to H/R. Overexpression of wild type Syk inhibited the H/R-induced uPA, MMP-9, and VEGF expression but up-regulated MelCAM expression. Our data also indicated that MelCAM acts as a tumor suppressor by negatively regulating H/R-induced uPA secretion and MMP-9 activation. The mice xenograft study showed the cross-talk between Syk and Lck regulated H/R-induced breast tumor progression and further correlated with the expressions of MelCAM, uPA, MMP-9, and VEGF. Human clinical specimen analysis supported the in vitro and in vivo findings. To our knowledge, this is first report that the cross-talk between Syk and Lck regulates H/R-induced breast cancer progression and further suggests that Syk may act as potential therapeutic target for the treatment of breast cancer.     

Binding of myotrophin/V-1 to actin-capping protein: implications for how capping protein binds to the filament barbed end

The heterodimeric actin-capping protein (CP) regulates actin assembly and cell motility by binding tightly to the barbed end of the actin filament. Here we demonstrate that myotrophin/V-1 binds directly to CP in a 1:1 molar ratio with a Kd of 10-50 nm. V-1 binding inhibited the ability of CP to cap the barbed ends of actin filaments. The actin-binding COOH-terminal region, the "tentacle," of the CP beta subunit was important for binding V-1, with lesser contributions from the alpha subunit COOH-terminal region and the body of the protein. V-1 appears to be unable to bind to CP that is on the barbed end, based on the observations that V-1 had no activity in an uncapping assay and that the V-1.CP complex had no capping activity. Two loops of V-1, which extend out from the alpha-helical backbone of this ankyrin repeat protein, were necessary for V-1 to bind CP. Parallel computational studies determined a bound conformation of the beta tentacle with V-1 that is consistent with these findings, and they offered insight into experimentally observed differences between the alpha1 and alpha2 isoforms as well as the mutant lacking the alpha tentacle. These results support and extend our "wobble" model for CP binding to the actin filament, in which the two COOH-terminal regions of CP bind independently to the actin filament, and bound CP is able to wobble when attached only via its mobile beta-subunit tentacle. This model is also supported by molecular dynamics simulations of CP reported here. The existence of the wobble state may be important for actin dynamics in cells.     

Biology of vascular endothelial growth factors

Angiogenesis is the process by which new blood vessels are formed from existing vessels. The vascular endothelial growth factors (VEGFs) are considered as key molecules in the process of angiogenesis. The VEGF family currently includes VEGF-A, -B, -C, -D, -E, -F and placenta growth factor (PlGF), that bind in a distinct pattern to three structurally related receptor tyrosine kinases, denoted VEGF receptor-1, -2, and -3. VEGF-C and VEGF-D also play a crucial role in the process of lymphangiogenesis. Here, we review the biology of VEGFs and evaluate their role in pathological angiogenesis and lymphangiogenesis.