Biology subjects

Taneja, P.

Publications and source records attributed to Taneja, P..

5 recordsLinked to original sources

bioRxiv2020

Cloning of Human ABCB11 Gene in E. coli required the removal of an Intragenic Pribnow-Schaller Box before its Insertion into Genomic Safe Harbor AAVS1 Site using CRISPR Cas9

BackgroundGenomic safe harbors are sites in the genome which are safe for gene insertion such that the inserted gene will function properly, and the disruption of the genomic location doesnt cause any foreseeable risk to the host. The AAVS1 site is the site which is disrupted upon integration of Adeno Associated Virus (AAV) and is considered a safe-harbor in human genome because about one third of humans are infected with AAV and so far there is no apodictic evidence that AAV is pathogenic or disruption of AAVS1 causes any disease in man. Therefore, we chose to target AAVS1 site for the insertion of ABCB11, a bile acid transporter which is defective in Progressive Familial Intra Hepatic Cholestasis Type-2 (PFIC-2), a lethal disease of children where cytotoxic bile salts accumulate inside hepatocytes killing them and eventually the patient. MethodsWe used CRISPR Cas9 a genome editing tool to insert ABCB11 gene at AAVS1 site in human cell-lines. ResultsWe found that human ABCB11 sequence has a "Pribnow- Schaller Box" which allows its expression in bacteria and expression of ABCB11 protein which is toxic to E. coli and the removal of the same was required for successful cloning. We inserted ABCB11 at AAVS1 site in HEK 293T using CRISPR-Cas9 tool. We also found that ABCB11 protein has similarity with E. coli Endotoxin (Lipid A) Transporter MsbA. ConclusionWe inserted ABCB11 at AAVS1 site using CRISPR-Cas9, however, the frequency of homologous recombination was very low for this approach to be successful in-vivo (Figure: pictorial abstract). Pictorial Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/284125v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1ae72ddorg.highwire.dtl.DTLVardef@1f72ee8org.highwire.dtl.DTLVardef@128a226org.highwire.dtl.DTLVardef@adacfb_HPS_FORMAT_FIGEXP M_FIG ABCB11 gene (which codes the transporter of human bile salts) is targeted to AAVS1 site using a construct which has 5 and 3 overhangs which are homologous to the AAVS1 site. A Pribnow box was detected inside ABCB11 gene which allowed the gene to transcribe in E. Coli causing bacterial lysis probably through competitive replacement of a homologous transporter protein in E. Coli (E. coli Endotoxin (Lipid A) Transporter) MsbA, resulting in Lipid A (L) accumulation inside the bacteria. C_FIG

cell biology↗

bioRxiv2020

Glibenclamide and Metformin Increases the Expression of Human Bile Salt Export Pump ABCB11

BackgroundBile Salt Export Pump (BSEP/ABCB11) is important in the maintenance of the enterohepatic circulation of bile acids and drugs. Drugs such as rifampicin, glibenclamide inhibit BSEP. Progressive Familial Intrahepatic Cholestasis Type-2, a lethal pediatric disease, some forms of intrahepatic cholestasis of pregnancy, and drug-induced cholestasis are associated with BSEP dysfunction. MethodsWe started with a bioinformatic approach to identify the relationship between ABCB11 and other proteins, microRNAs, and drugs. Microarray data set of the liver samples from ABCB11 knockout mice was analyzed by GEO2R tool. Differentially expressed gene pathway enrichment analysis was done by ClueGo v2.5.5 app from Cytoscape. Protein-protein interaction network was constructed by STRING application in Cytoscape. Networks were analyzed using the Cytoscape software v3.7.1. CyTargetLinker v4.1.0 was used to screen the transcription factors, microRNAs and drugs. Predicted drugs were validated on human liver cell line, HepG2. BSEP expression was quantified by Real Time PCR and Western Blot. ResultsABCB11 knockout in mice was associated with a predominant upregulation and downregulation of genes associated with cellular component movement and sterol metabolism respectively. We further identified the hub genes in the network. Genes related to immune activity, cell signaling and fatty acid metabolism were dysregulated. We further identified drugs (glibenclamide and ATP) and a total of 14 microRNAs targeting the gene. Western Blot and Real Time PCR analysis confirmed the upregulation of BSEP on the treatment of HepG2 cells with glibenclamide, ATP, and metformin. ConclusionThe differential expression of cell signaling genes and those related to immune activity in ABCB11 KO animals may be secondary to cell injury. We have found glibenclamide, ATP, and metformin upregulates BSEP. The mechanisms involved and the clinical relevance of these findings need to be investigated.

molecular biology↗

bioRxiv2020

Anti-microbial activity of potential probiotic lactic acid bacteria against Methicillin-Resistant Staphylococcus Aureus (MRSA)

Lactic acid bacteria (LAB)are the essential ingredients in probiotic foods, intestinal microflora, and dairy products able to cope up and exist in diverse environmental ranges. Samples were collected using sterile test tubes and transported to a laboratory in the icebox for further biochemical characterization. Gram test and catalase activity were examined after microscopically distinct colonies were sub-cultured to pure colonies based on standard gram and catalase test protocols. Subsequently, these bacteria were characterized for their ability to grow at various salt concentrations (5%,10%, and15%) and temperature gradients (15{degrees}C, 30{degrees}C, 45{degrees}C). Acid-tolerance was analyzed by growing the colonies in MRS broth adjusted to acidic pH (pH 3) and pH 7.2 (control). Bile tolerance of LAB isolates was assayed by growing in 0.3% bile-supplemented MRS agar. Bile salt hydrolase (BSH) activity was studied by growing 10 l of the prepared overnight culture on BSH screening media containing MRS agar plate supplemented with bile salts. The LAB isolates were checked for antimicrobial activity by agar well diffusion assay. All isolates found gram-positive, catalase-negative and non-motile, convex elevation and entire margin. All LAB isolates were able to grow at 5-10% Nacl concentration, whereas moderately grow at 10% concentration but rarely grow at 15% Nacl concentration. BCM2, BBM3 and BGM1 record the highest acidic resistance viability percentage 94.9%, 92.7%, and 91.8% respectively. BCM3 has the lowest acidic resistance with a viable percentage of 87.4%. BBM1 records the highest bile tolerance activity whereas BCM2 has the lowest bile tolerance. All isolates were found BSH positive. The study reveals LAB isolates showed a putative probiotic potential. Article highlightsLAB are the main ingredients of probiotic products commercially available in the market nowadays. To effectively functioning the host gastrointestinal tract probiotics, need to have certain criteria like acid and bile tolerance this study reveals O_LIAcid tolerance activity of LAB isolated from bovine milk C_LIO_LIBile tolerance activity of LAB isolated from bovine milk C_LIO_LIBile salt hydrolase (BSH) activity of LAB isolates C_LIO_LIAntimicrobial activity of LAB against MRSA C_LI

microbiology↗

bioRxiv2020

Effects of substituents on anticancer activity of thiosemicarbazone against MCF-7 human breast cancer cell line

Background/AimBreast cancer is one of the worlds leading cause of deaths in women. This study evaluated the in-vitro anticancer activity of different thiosemicarbazones (HacTSc, HSTsc, 3-MBTSc, 4-NBTSc and 4-HBTSc) against MCF-7 human breast cancer cell line and MCF-10 normal cell. Materials and MethodsThe ligands were prepared and characterized by UV vis, IR, 1H NMR. MTT assay was used to determine cell viability. Then data were analyzed using two-way ANOVA with Tukey post-hoc test. Result: The ligands have IC50 value ranging from 2.271{micro}g/ml to 7.081{micro}g/ml. Acetone thiosemicarbazone and 3-Methoxybenzaldehyde thiosemicarbazone, were identified as the most potent against MCF-7 breast cancer cells with IC50 value of 2.271{micro}g/ml and 2.743{micro}g/ml respectively. Whereas 4-Nitrobenzaldehyde thiosemicarbazone was the least potent. Also, the IC50 of the normal MCF-10 cell indicated their activity were selective. Conclusion: The activity of the ligands were dose, position and substituents dependent. Acetone thiosemicarbazone and 3-Methoxybenzaldehyde thiosemicarbazone are promising anticancer agents for further study.

cancer biology↗

bioRxiv2019

Cortical RORβ is required for layer 4 transcriptional identity and barrel integrity.

Retinoic Acid-Related Orphan Receptor Beta (ROR{beta}) is a transcription factor (TF) and marker of layer 4 (L4) neurons, which are distinctive both in transcriptional identity and the ability to form aggregates such as barrels in rodent somatosensory cortex. However, the relationship between transcriptional identity and L4 cytoarchitecture is largely unknown. We find ROR{beta} is required in the cortex for L4 aggregation into barrels and thalamocortical afferent (TCA) segregation. Interestingly, barrel organization also degrades with age. Loss of ROR{beta} delays excitatory input and disrupts gene expression and chromatin accessibility, with downregulation of L4 and upregulation of L5 genes, suggesting a shift in cellular identity. Expression and binding site accessibility change for many other TFs, including closure of neurodevelopmental TF binding sites and increased expression and binding capacity of activity-regulated TFs. Lastly, a putative target of ROR{beta}, Thsd7a, is downregulated without ROR{beta}, and Thsd7a knockout alone disrupts TCA organization in adult barrels.

neuroscience↗