Insights into the differential proteome landscape of a newly isolated Paramecium multimicronucleatum in response to cadmium stress.

Employing microbial systems for the bioremediation of contaminated waters represent a potential option, however, limited understanding of the underlying mechanisms hampers the implication of microbial-mediated bioremediation. The omics tools offer a promising approach to explore the molecular basis of the bioremediation process. Here, a mass spectrometry-based quantitative proteome profiling approach was conducted to explore the differential protein levels in cadmium-treated Paramecium multimicronucleatum. The Proteome Discoverer software was used to identify and quantify differentially abundant proteins. The proteome profiling generated 7,416 peptide spectral matches, yielding 2824 total peptides, corresponding to 989 proteins. The analysis revealed that 29 proteins exhibited significant (p ≤ 0.05) differential levels, including a higher abundance of 6 proteins and reduced levels of 23 proteins in Cd2+ treated samples. These differentially abundant proteins were associated with stress response, energy metabolism, protein degradation, cell growth, and hormone processing. Briefly, a comprehensive proteome profile in response to cadmium stress of a newly isolated Paramecium has been established that will be useful in future studies identifying critical proteins involved in the bioremediation of metals in ciliates. SIGNIFICANCE: Ciliates are considered a good biological indicator of chemical pollution and relatively sensitive to heavy metal contamination. A prominent ciliate, Paramecium is a promising candidate for the bioremediation of polluted water. The proteins related to metal resistance in Paramecium species are still largely unknown and need further exploration. In order to identify and reveal the proteins related to metal resistance in Paramecia, we have reported differential protein abundance in Paramecium multimicronucleatum in response to cadmium stress. The proteins found in our study play essential roles during stress response, hormone processing, protein degradation, energy metabolism, and cell growth. It seems likely that Paramecia are not a simple sponge for metals but they could also transform them into less toxic derivatives or by detoxification by protein binding. This data will be helpful in future studies to identify critical proteins along with their detailed mechanisms involved in the bioremediation and detoxification of metal ions in Paramecium species.

Identification of Transcription Factors Differentially Expressed under Neuroinflammatory Conditions: Analysis of RNA-Seq Datasets Combined with an Unconventional Exploratory Approach.

Neuroinflammation, the inflammatory process of the brain or peripheral nervous system, is mediated by various chemokines, cytokines, and other secondary messengers. Just like any other physiological process, transcription factors (TFs) lie at the core of neuroinflammatory process too due to their direct effects on the control of gene expression. Although targeted studies are being done on some of the already known TFs involved in neuroinflammation, still the gap exists in profiling the whole repertoire from transcriptomics data which was the main aim of this study. Therefore, we retrieved RNA-sequencing (RNA-seq) datasets for lipopolysaccharide-treated mice brain tissues as well as three brain cell types - neurons, microglia, and astrocytes. The screening of differentially expressed genes resulted in identification of 15, 50, 98, and 29 TFs in brain, neurons, microglia, and astrocytes, respectively. Further exploration of the brain data with respect to the expression of identified TFs in normal tissues revealed interesting patterns of their expression along with the computational identification of the microRNAs (miRNAs) targeting the down-regulated TFs. Also, quite surprisingly, zf-C2H2 domain was found to be the most prevalent in all the TFs identified, i.e., brain tissue, neuronal, microglial, and astrocytic cells. Therefore, this study not only identified new TFs but also miRNA targets to explore in the process of neuroinflammation.

Glucose Deprivation Promotes Cancer Cell Invasion by Varied Expression of EMT Structural Proteins and Regulatory Molecules in MDA-MB-231 Triple-Negative Breast Cancer Cells.

Cancer is one of the biggest leading causes of death worldwide. With increased accumulation of environmental factors like radiation and mutagens, causative mutation in the gene pool is increasing day by day in the human population. Metastasis and its resistance to a wide variety of treatments are the reasons which make cancer difficult to treat. It has already been reported that cancer cells introduce smart modifications in their metabolism and signaling pathways which sense changes in cancer microenvironment and help them survive under harsh conditions. The pattern of expression of these signaling molecules and their structural counterparts involved in cancer metastasis need to be investigated. We used different approaches to study the effect of several levels of glucose deprivation on cancer metastasis in the highly resistant breast cancer model cell line MDA-MB-231 grown in high- (25-mM) and low- (5-mM) glucose medium. Microscopic observations have shown that these cancer cells attach to the surface faster in the presence of high-compared to low-glucose concentrations. These observations were supported by varied expression of genes involved in this morphological transition. Under low-glucose concentration, the expression of epithelial-to-mesenchymal transition (EMT) structural proteins and regulatory molecules such as SLUG, ZEB, HIFα1A, STAT3, and VIM was downregulated, whereas expression of G9a (a histone-modifying enzyme, histone methyl transferase inhibitor), Snail, FBPase, MMP13, and PKM2 was upregulated. This resulted in the turning on of resistance mechanisms in MDA-MB-231 cancer cells enabling them to cope with the stressed tumor microenvironments leading to increased cancer invasion and migration. The increased invasion as shown by trans-well study and loose spheroids in 3D spheroid study showed how metastasis is triggered in MDA-MB-231 under glucose starvation. Our data suggests the devastating outcome of nutrient deprivation on cancer progression, which was previously thought to be supportive in cancer control.

Association of IRGM promoter region polymorphisms and haplotype with pulmonary tuberculosis in Pakistani (Punjab) population.

Single nucleotide polymorphisms (SNPs) in IRGM are reported to affect Mycobacterium tuberculosis (M.tb) degradation pathway. Here, we aim to screen promoter-region regulatory SNPs of IRGM in Pakistani population. DNA extracted from blood of cohort containing 70 TB patients (TB) and 30 controls subjects (Ctrl), was amplified for IRGM promoter region, followed by DNA sequencing. Group-specific variations were found in allelic frequencies at four loci. Allele T (p-value = 0.03) at -1161T/C, allele G (p-value = 0.027) at -1133G/A; allele C (p-value = 0.029) at -1049C/T; and allele G (p-value = 0.02) at -708G/A, showed higher associations with TB in our cohort. These SNPs display strong linkage disequilibrium (LD) in Pakistani population. Haplotype analysis showed a significant association of haplotype -1161T/-1133G/-1049C/-708G (p-value = 0.007) to TB. This 4-SNP haplotype also represents an expression quantitative trait locus (eQTL), associated with Crohn's disease and chronic inflammatory diseases. Our findings show that variants -1161T/C, -1133G/A, -1049C/T, and -708G/A are associated with IRGM expression and susceptibility to TB in a Pakistani population.

Isolation and characterization of a highly effective bacterium Bacillus cereus b-525k for hexavalent chromium detoxification.

The chromate resistant Gram-positive Bacillus cereus strain b-525k was isolated from tannery effluents, demonstrating optimal propagation at 37 °C and pH 8. The minimum inhibitory concentration (MIC) test showed that B. cereus b-525k can tolerate up to 32 mM Cr6+, and also exhibit the ability to resist other toxic metal ions including Pb2+ (23 mM), As3+ (21 mM), Zn2+ (17 mM), Cd2+ (5 mM), Cu2+ (2 mM), and Ni2+ (3 mM) with the resistance order as Cr 6+ > Pb2+ > As3+ >Zn2+ >Cd2+ >Ni2+ >Cu2+. B. cereus b-525k showed maximum biosorption efficiency (q) of 51 mM Cr6+/g after 6 days. Chromate stress elicited pronounced production of antioxidant enzymes such as catalase (CAT) 191%, glutathione transferase (GST) 192%, superoxide dismutase (SOD) 161%, peroxidase (POX) 199%, and ascorbate peroxidase (APOX) (154%). Within B. cereus b-525k, the influence of Cr6+ stress (2 mM) did stimulate rise in levels of GSH (907%) and non-protein thiols (541%) was measured as compared to the control (without any Cr6+ stress) which markedly nullifies Cr6+ generated oxidative stress. The pilot scale experiments utilizing original tannery effluent showed that B. cereus b-525k could remove 99% Cr6+ in 6 days, thus, it could be a potential candidate to reclaim the chromate contaminated sites.

In silico mutational analysis of ACE2 to check the susceptibility of lung cancer patients towards COVID-19.

Being the second major cause of death worldwide, lung cancer poses a significant threat to the health of patients. This worsened during the era of pandemic since lung cancer is found to be more prone to SARS-CoV-2 infection. Many recent studies imply a high frequency of COVID-19 infection associated severe outcome. However, molecular studies are still lacking in this respect. Hence the current study is designed to investigate the binding affinities of ACE2 lung cancer mutants with the viral spike protein to find the susceptibility of respective mutants carrying patients in catching the virus. Quite interestingly, our study found lesser binding affinities of all the selected mutants thus implying that these cancer patients might be less affected by the virus than others. These results are opposed to the recent studies' propositions and open new avenues for more in-depth studies.

Repression of Cell-to-Matrix Adhesion by Metformin Chloride Supports Its Anti-Metastatic Potential in an In Vitro Study on Metastatic and Non-Metastatic Cancer Cells.

Glucose metabolism has significant impact on cancer cell survival and proliferation. Our previous studies have shown that level of glucose in the medium affects the cell attachment to and detachment from the substratum. Control of glucose metabolism in cancer cells has potential to serve as an anti-cancer therapy. Different anti-diabetic drugs have been reported to inhibit glucose uptake at cellular level by glucose transporters. Metformin chloride is commonly used as antidiabetic drug. It is known that use of metformin reduces chances of developing cancer in diabetic patients. Here we have investigated the effect of metformin on cell adhesion proteins and other related factors in different cancer cells, both metastatic and non-metastatic. The object was to evaluate the effect of different doses of metformin on the onset of metastasis after these are detached from their primary site of origin and re-attached at the secondary site. For this purpose, we grew different cancer cells (MDA-MB231, MCF7, HCT116, and SF767) in culturing media containing different concentrations of metformin chloride. Quantitative real-time PCR was used to evaluate the expression profile of the genes involved in cell adhesion. It was observed that metformin treatment increased the expression of epithelial isoforms of cell adhesion molecules along with integrins responsible for cell-to-matrix adhesion and inducing specific morphological changes such as development of cytoskeletal structures in different cancer cell lines which normally lead to attainment of mesenchymal phenotype. The effect of metformin appeared to be different in different doses. The glioblastoma cells (SF767) were observed to be the most sensitive cells among all cancer cells under study. Our data supports the idea that the metformin prevents the cancer cells from acquiring mesenchymal phenotype and hence prevents onset of metastasis, and if the process has already started then it has potential to prevent cancer cell attachment to the secondary site.

A computational and bioinformatic analysis of ACE2: an elucidation of its dual role in COVID-19 pathology and finding its associated partners as potential therapeutic targets.

Despite the continued global spread of the current COVID-19 pandemic, the nonavailability of a vaccine or targeted drug against this disease is still prevailing. The most established mechanism of viral entry into the body is considered to be via angiotensin-converting enzyme 2 (ACE2) acting as a receptor for viral spike protein thereby facilitating its entry in the cell. However, ACE2 is also involved in providing the protection from severe pathological changes. This article provides a computational and bioinformatics-based analysis of ACE2 with an objective of providing further insight into the earnest efforts to determine its true position in COVID-19 pathology. The results of this study show that ACE2 has strikingly low expression in healthy human lung tissue and was absent from the list of differentially expressed genes. However, when transcription factors were analyzed, we found a significant upregulation of FOS and downregulation of FOXO4 and FOXP2. Moreover, the miRNA prediction analysis revealed that miR-1246, whose upregulation has been experimentally established to be a cause of acute respiratory distress syndrome (ARDS), was found to be targeting the coding DNA sequence (CDS) of ACE2. This study presents a wide range of potentially important transcription factors as well as miRNA targets associated with ACE2 which can be potentially used for drug designing amid this challenging pandemic situation.Communicated by Ramaswamy H. Sarma.

Glucose Stress Induces Early Onset of Metastasis in Hormone-Sensitive Breast Cancer Cells.

Cell adhesion is the backbone of many events in the cancer cell life cycle, including proliferation, metastasis, migration, invasion and even cell survival. In a tumor, usually the cells in the core have high migratory potential though they constantly suffer from glucose starvation. Our study was aimed at understanding events such as attachment to the surfaces at one site and then mobility to the secondary sites during progression of cancer in the hormone sensitive breast cancer cells MCF7, following their exposure to different concentrations of glucose in the environment. We have shown that low glucose availability is detected within 3 h of shortage which is then translated into variable expression of genes for cell-to-cell adhesion such as cadherins and Ig-like cell adhesion molecules, and matrix-associated genes such as integrins and metalloproteases. We also found that low glucose concentrations induced cell adhesion, whereas higher concentrations stimulated cell migration. In addition, several regulatory molecules involved in mitochondrial metabolism, proliferation, and glucose uptake as demonstrated respectively by MTT assay, BrdU uptake, glucose uptake and pyruvate kinase activity showed varied expression during epithelial to mesenchymal transition. Cytoskeleton staining demonstrated development of lamellipodia in glucose starved medium indicating cascade of physiological and molecular events in the cells to find a more nutrient-rich environment for the development of secondary tumor. Further studies on protein markers with a 3D spheroid culturing approach are likely to expand our understanding of onset of metastasis in tumor tissues.

Induction of Apoptosis in Human Lung Epithelial Cell by Sphingomonas sp. Shah, a Recently Identified Cell Culture Contaminant.

Sphingomonas sp. Shah is a bacterium that was first isolated from mammalian cell cultures. According to ribotyping data it is very much homologous to the clinically important pathogen Sphingomonas paucimobilis, which has generated pseudo-outbreaks. Using a tissue culture system, Sphingomonas sp. Shah was discovered to induce apoptosis in human lung epithelial carcinoma. Apoptosis of infected cells was determined by numerous criteria including (1) visual alterations in cellular morphology, (2) initiation of nuclear marginalization and chromatin compaction condensation, (3) the attendance of a high percentage of cells with subG1 DNA content, and (4) caspase-3 activation. In the current study we demonstrate the induction of apoptosis in mammalian lung epithelial cells upon infection with Sphingomonas sp. Shah and provide insight into the molecular processes triggering apoptosis.

Interplay of immune components and their association with recurrent pregnancy loss.

Maintenance and progression of pregnancy is an intricate process governed by a variety of developmental cues. Recurrent pregnancy loss (RPL) is a complication experienced by expecting mothers that is defined as three or more consecutive pregnancy losses. This review focuses on the dysfunctions of the immune system as one of the key contributors towards RPL. The current data suggests that the alloimmune and autoimmune factors contribute to the loss of fetus. Such causes despite being recognized as a definitive reason for recurrent pregnancy loss, are still under extensive investigation with new parameters being discovered and scrutinized for their association with RPLs. More in-depth and high throughput studies are required for devising better diagnostic tools and management strategies for the affected female so that they can carry their pregnancy to term.

SHISA3, an antagonist of the Wnt/ß-catenin signaling, is epigenetically silenced and its ectopic expression suppresses growth in breast cancer.

Breast cancer (BC) is the foremost cause of cancer related deaths in women globally. Currently there is a scarcity of reliable biomarkers for its early stage diagnosis and theranostics monitoring. Altered DNA methylation patterns leading to the silencing of tumor suppressor genes are considered as an important mechanism underlying tumor development and progression in various cancer types, including BC. Very recently, epigenetic silencing of SHISA3, an antagonist of ß-catenin, has been reported in various types of tumor. However, the role of SHISA3 in BC has not been investigated yet. Therefore, we aimed at evaluating the contribution of SHISA3 in BC causation by analyzing its expression and methylation levels in BC cell lines (MDA-MB231, MCF-7 and BT-474) and in 103 paired BC tissue samples. The SHISA3 expression and methylation status was determined by qPCR and methylation specific PCR (MSP) respectively. The role of SHISA3 in BC tumorigenesis was evaluated by proliferation and migration assays after ectopic expression of SHISA3. The association between SHISA3 hypermethylation and clinicopathological parameters of BC patients was also studied. The downregulation of SHISA3 expression was found in three BC cell lines used and in all BC tissue samples. However, SHISA3 promoter region was hypermethylated in 61% (63/103) tumorous tissues in comparison to the 18% of their matched normal tissues. The 5-aza-2'-deoxycytidine treatment restored SHISA3 expression by reversing promoter hypermethylation in both MDA-MB231 and MCF-7 cells. Furthermore, ectopic expression of SHISA3 significantly reduced the proliferation and migration ability of these cells. Taken together, our findings for the first time reveal epigenetic silencing and tumor suppressing role of SHISA3 in BC. Henceforth, this study has identified SHISA3 as potentially powerful target for the development of new therapies against BC, as well as novel diagnostic and therapy response monitoring approaches.

Origin, Potential Therapeutic Targets and Treatment for Coronavirus Disease (COVID-19).

The ongoing episode of coronavirus disease 19 (COVID-19) has imposed a serious threat to global health and the world economy. The disease has rapidly acquired a pandemic status affecting almost all populated areas of the planet. The causative agent of COVID-19 is a novel coronavirus known as SARS-CoV-2. The virus has an approximate 30 kb single-stranded positive-sense RNA genome, which is 74.5% to 99% identical to that of SARS-CoV, CoV-pangolin, and the coronavirus the from horseshoe bat. According to available information, SARS-CoV-2 is inferred to be a recombinant virus that originated from bats and was transmitted to humans, possibly using the pangolin as the intermediate host. The interaction of the SARS-CoV-2 spike protein with the human ACE2 (angiotensin-converting enzyme 2) receptor, and its subsequent cleavage by serine protease and fusion, are the main events in the pathophysiology. The serine protease inhibitors, spike protein-based vaccines, or ACE2 blockers may have therapeutic potential in the near future. At present, no vaccine is available against COVID-19. The disease is being treated with antiviral, antimalarial, anti-inflammatory, herbal medicines, and active plasma antibodies. In this context, the present review article provides a cumulative account of the recent information regarding the viral characteristics, potential therapeutic targets, treatment options, and prospective research questions.

Bioactivity-Guided Isolation and Identification of Anti-adipogenic Constituents from the n-Butanol Fraction of Cissus quadrangularis.

Obesity is marked by the buildup of fat in adipose tissue that increases body weight and the risk of many associated health problems, including diabetes and cardiovascular disease. Treatment options for obesity are limited, and available medications have many side effects. Thus there is a great need to find alternative medicines for treating obesity. This study explores the anti-adipogenic potential of the n-butanol fraction of Cissus quadrangularis (CQ-B) on 3T3-L1 mouse preadipocyte cell line. The expression of various lipogenic marker genes such as adiponectin, peroxisome proliferator-activated receptor gamma, leptin, fatty acid-binding proteins, sterol regulatory element-binding proteins, fetal alcohol syndrome, steroyl-CoA desaturase-1, lipoproteins, acetyl-CoA carboxylase alpha, and acetyl-CoA carboxylase beta were variously significantly downregulated. After establishing the anti-adipogenic potential of CQ-B, it was fractionated to isolate anti-adipogenic compounds. We observed significant reduction in neutral lipid content of differentiated cells treated with various fractions of CQ-B. Gas chromatography-mass spectrometry analysis revealed the presence of thirteen compounds with reported anti-adipogenic activities. Further studies to purify these compounds can offer efficacious and viable treatment options for obesity and related complications.

Low glucose availability alters the expression of genes involved in initial adhesion of human glioblastoma cancer cell line SF767.

Studying the metabolic pathways of cancer cells is considered as a key to control cancer malignancies and open windows for effective drug discovery against cancer. Of all the properties of a tumor, metastasis potential is a defining characteristic. Metastasis is controlled by a variety of factors that directly control the expression of cell adhesion proteins. In this study we have investigated the expression of cell to cell and cell to matrix adhesion protein genes during the initial phases of attachment of human glioblastoma cancer cell line SF767 (66Y old human female: UCSF Neurosurgery Tissue Bank) to the attachment surface under (Cell culture treated polystyrene plate bottom) glucose-rich and glucose-starved conditions. The aim was to imitate the natural microenvironment of glucose availability to cancer cells inside a tumor that triggers epithelial to mesenchymal transition (EMT). In this study, we have observed the gene expression of epithelial and mesenchymal isoforms of cadherin (E-CAD and N-CAD) and Ig like cell adhesion molecules (E-CAM and N-CAM) along with Integrin family subunits for the initial attachment of cancer cells. We observed that high glucose environments promoted cell survival and cell adhesion, whereas low glucose accelerated EMT by downregulating the expression level of integrin, E-CAD, and N-CAD, and upregulation of N-CAM during early period of cell adhesion. Low glucose availability also downregulated variety of structural and regulatory genes, such as zinc finger E-box binding home box 1A), cytokeratin, Snail, and ß catenin, and upregulation of hypoxia-inducible factor 1, matrix metalloprotease 13/Collagenase 3, vimentim, p120, and fructose 1,6 bisphosphatase. Glucose conditions are more efficient for cancer studies in this case glioblastoma cells.

Synergistic effect of piperonyl butoxide and emamectin benzoate on enzymatic activities in resistant populations of red flour beetle, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae).

Resistance to grain protectants in Tribolium castaneum (Herbst) is a serious threat to international grain trade. Frequent and overdose application of chemical insecticides is becoming a serious health hazard and cause environmental pollution. Resistance management approaches by using various synergists along with novel compounds has become more imperative to increase efficacy of environmentally safe insecticides. We have evaluated piperonyl butoxide (PBO) and emamectin benzoate mixtures for management of resistant field populations of T. castaneum. The collected strains had demonstrated 50 to 200% resistance already developed against emamectin benzoate as compared with deltamethrin susceptible reference strain. The inclusion of PBO along with emamectin significantly reduced this resistance by at least 28% and the LC50 were lowered from 5.12 to 1.9 µg/ml with the highest synergism ration of 2.7 in resistant strain. Enzymatic assays clearly demonstrated that the specific activities of catalase and acetylcholinesterases were significantly decreased at an average of 80% and 60%, respectively, when PBO was included as a synergist at 1:2 ratio with emamectin benzoate. The results highlight the mechanism that renders the field population resistant to emamectin benzoate and suggests the synergistic role of piperonyl butoxide as a potent additive in grain protectants for resistance management.

Ethyl acetate and n-butanol fraction of Cissus quadrangularis promotes the mineralization potential of murine pre-osteoblast cell line MC3T3-E1 (sub-clone 4).

In a sequel to investigate osteogenic potential of ethanolic extract of Cissus quadrangularis (CQ), the present study reports the osteoblast differentiation and mineralization potential of ethyl acetate (CQ-EA) and butanol (CQ-B) extracts of CQ on mouse pre-osteoblast cell line MC3T3-E1 (sub-clone 4) with an objective to isolate an antiosteoporotic compound. Growth curve, proliferation, and viability assays showed that both the extracts were nontoxic to the cells even at high concentration (100 µg/ml). The cell proliferation was enhanced at low concentrations (0.1 µg/ml and 1 µg/ml) of both the extracts. They also upregulated the osteoblast differentiation and mineralization processes in MC3T3-E1 cells as reflected by expression profile of osteoblast marker genes such as RUNX2, Osterix, Collagen (COL1A1), Alkaline Phosphatase (ALP), Integrin-related Bone Sialoprotein (IBSP), Osteopontin (OPN), and Osteocalcin (OCN). CQ-EA treatment resulted in early differentiation and mineralization as compared with the CQ-B treatment. These findings suggest that low concentrations of CQ-EA and CQ-B have proliferative and osteogenic properties. CQ-EA, however, is more potent osteogenic than CQ-B.

Multiple upstream start codons (AUG) in 5' untranslated region enhance translation efficiency of cry2Ac11 without helper protein.

Cry2Ac11, a 65 kDa insecticidal protein produced by Bacillus thuringiensis, shows toxicity against dipteran and lepidopteran larvae. It is encoded by cry2Ac11 gene ( orf3), which is part of an operon comprising orf1, orf2, and orf3. Orf2, a helper protein, helps in proper folding and prevents aberrant aggregation of newly produced molecules. In this study, we have elucidated the effect of different mutations in translation initiation region (TIR), particularly the ribosomal binding site and the start codon (RBS-ATG) on cry2Ac11 gene expression without helper protein. All recombinant constructs were expressed in acrystalliferous B. thuringiensis subsp israelensis 4Q7 under the control of strong chimeric promoter cyt1AP/STAB. Of all the mutants, mut/RBS2, with two consecutive AUGs after the spacer region in TIR, exhibited 89- and 2246-fold higher transcript levels compared with 4Q7-operSalI/RBS ( cry2Ac11 operon) and 4Q7-w-RBS ( cry2Ac11 gene), respectively. The analysis of mut/RBS2 messenger RNA (mRNA) structure in the RBS-AUG region showed the presence of RBS in the single-stranded part of the moderately stable hairpin loop. The high expression efficiency of Cry2Ac11 mutant without helper protein is a cumulative and cooperative result of chimeric promoter cyt1AP/STAB-SD with the optimal context of RBS-AUG region provided by multiple AUGs and stabilizer sequence at 3' ends.

Identification of variants in the mitochondrial lysine-tRNA (MT-TK) gene in myoclonic epilepsy-pathogenicity evaluation and structural characterization by in silico approach.

Variations in mitochondrial genes have an established link with myoclonic epilepsy. In the present study we evaluated the nucleotide sequence of MT-TK gene of 52 individuals from 12 unrelated families and reported three variations in 2 of the 13 epileptic patients. The DNA sequences coding for MT-TK gene were sequenced and mutations were detected in all participants. The mutations were further analyzed by the in silico analysis and their structural and pathogenic effects were determined. All the investigated patients had symptoms of myoclonus, 61.5% were positive for ataxia, 23.07% were suffering from hearing loss, 15.38% were having mild to severe dementia, 69.23% were males, and 61.53% had cousin marriage in their family history. DNA extracted from saliva was used for the PCR amplification of a 440 bp DNA fragment encompassing complete MT-TK gene. The nucleotide sequence analysis revealed three mutations, m.8306T>C, m.8313G>C, and m.8362T>G that are divergent from available reports. The identified mutations designate the heteroplasmic condition. Furthermore, pathogenicity of the identified variants was predicted by in silico tools viz., PON-mt-tRNA and MitoTIP. Secondary structure of altered MT-TK was predicted by RNAStructure web server. Studies by MitoTIP and PON-mt-tRNA tools have provided strong evidences of pathogenic effects of these mutations. Single nucleotide variations resulted in disruptive secondary structure of mutant MT-TK models, as predicted by RNAStructure. In vivo confirmation of structural and pathogenic effects of identified mutations in the animal models can be prolonged on the basis of these findings.

Domain III of Cry1Ac Is Critical to Binding and Toxicity against Soybean Looper (Chrysodeixis includens) but Not to Velvetbean Caterpillar (Anticarsia gemmatalis).

Insecticidal proteins Cry1Ac and Cry2Ac7 from the bacterium Bacillus thuringiensis (Bt) belong to the three-domain family of Bt toxins. Commercial transgenic soybean hybrids produce Cry1Ac to control the larvae of the soybean looper (Chrysodeixis includens) and the velvet bean caterpillar (Anticarsia gemmatalis). The specificity of Cry1Ac is determined by loops extending from domain II and regions of domain III in the three-dimensional structure of the toxin. In this study, we constructed a hybrid toxin (H1.2Ac) containing domains I and II of Cry1Ac and domain III of Cry2Ac7, in an attempt to obtain a protein with enhanced toxicity compared to parental toxins. Bioassays with H1.2Ac revealed toxicity against the larvae of A. gemmatalis but not against C. includens. Saturation binding assays with radiolabeled toxins and midgut brush border membrane vesicles demonstrated no specific H1.2Ac binding to C. includens, while binding in A. gemmatalis was specific and saturable. Results from competition binding assays supported the finding that Cry1Ac specificity against A. gemmatalis is mainly dictated by domain II. Taken together, these distinct interactions with binding sites may help explain the differential susceptibility to Cry1Ac in C. includens and A. gemmatalis, and guide the design of improved toxins against soybean pests.