Cytotoxicity, Genotoxicity, and Phytotoxicity of Tannery Effluent Discharged into Palar River Basin, Tamil Nadu, India

Ambur, a town located on the banks of Palar River, is considered one of the most polluted areas in India and occupied by hundreds of tanneries and leather product units. The present study was designed to evaluate the toxic effect of discharged tannery effluent (TE) on model agricultural crops, ecofriendly microorganisms, and human blood cells. The phytotoxic effects of TE tested on Allium cepa and Lemna minor revealed inhibition of root growth and significant reduction in number of fronds, protein, and chlorophyll content. Moreover, TE induced chlorosis and tissue necrosis in Nostoc muscorum at low concentration (10%). TE has also negative impact on ecofriendly microorganisms, Bacillus thuringiensis, Rhizobium etli, and Aspergillus terreus which play an important role in the nutrition of plant growth. The genotoxicity of TE was investigated in human leukocytes which showed interference with normal mitotic division with subsequent cell lysis. It also intervened with the normal replication process and induced micronucleus formation in the healthy leukocyte. 5% concentration of TE has been revealed to be toxic to erythrocytes. From this study TE found in the Palar River of Ambur has adverse effects on all the three levels of organisms in ecosystem even at lower concentrations

Linking Ventilator Injury-Induced Leak across the Blood-Gas Barrier to Derangements in Murine Lung Function

Mechanical ventilation is vital to the management of acute respiratory distress syndrome, but it frequently leads to ventilator-induced lung injury (VILI). Understanding the pathophysiological processes involved in the development of VILI is an essential prerequisite for improving lung-protective ventilation strategies. The goal of this study was to relate the amount and nature of material accumulated in the airspaces to biomarkers of injury and the derecruitment behavior of the lung in VILI. Forty-nine BALB/c mice were mechanically ventilated with combinations of tidal volume and end-expiratory pressures to produce varying degrees of overdistension and atelectasis while lung function was periodically assessed. Total protein, serum protein, and E-Cadherin levels were measured in bronchoalveolar lavage fluid (BALF). Tissue injury was assessed by histological scoring. We found that both high tidal volume and zero positive end-expiratory pressure were necessary to produce significant VILI. Increased BALF protein content was correlated with increased lung derecruitability, elevated peak pressures, and histological evidence of tissue injury. Blood derived molecules were present in the BALF in proportion to histological injury scores and epithelial injury, reflected by E-Cadherin levels in BALF. We conclude that repetitive recruitment is an important factor in the pathogenesis of VILI that exacerbates injury associated with tidal overdistension. Furthermore, the dynamic mechanical behavior of the injured lung provides a means to assess both the degree of tissue injury and the nature and amount of blood-derived fluid and proteins that accumulate in the airspaces

Genome wide association study of uric acid in Indian population and interaction of identified variants with type 2 diabetes

Abnormal level of Serum Uric Acid (SUA) is an important marker and risk factor for complex diseases including Type 2 diabetes. Since genetic determinant of uric acid in Indians is totally unexplored, we tried to identify common variants associated with SUA in Indians using Genome Wide Association Study (GWAS). Association of five known variants in SLC2A9 and SLC22A11 genes with SUA level in 4,834 normoglycemics (1,109 in discovery and 3,725 in validation phase) was revealed with different effect size in Indians compared to other major ethnic population of the world. Combined analysis of 1,077 T2DM subjects (772 in discovery and 305 in validation phase) and normoglycemics revealed additional GWAS signal in ABCG2 gene. Differences in effect sizes of ABCG2 and SLC2A9 gene variants were observed between normoglycemics and T2DM patients. We identified two novel variants near long non-coding RNA genes AL356739.1 and AC064865.1 with nearly genome wide significance level. Meta-analysis and in silico replication in 11,745 individuals from AUSTWIN consortium improved association for rs12206002 in AL356739.1 gene to sub-genome wide association level. Our results extends association of SLC2A9, SLC22A11 and ABCG2 genes with SUA level in Indians and enrich the assemblages of evidence for SUA level and T2DM interrelationship

Common variants in CLDN2 and MORC4 genes confer disease susceptibility in patients with chronic pancreatitis

A recent Genome-wide Association Study (GWAS) identified association with variants in X-linked CLDN2 and MORC4 and PRSS1-PRSS2 loci with Chronic Pancreatitis (CP) in North American patients of European ancestry. We selected 9 variants from the reported GWAS and replicated the association with CP in Indian patients by genotyping 1807 unrelated Indians of Indo-European ethnicity, including 519 patients with CP and 1288 controls. The etiology of CP was idiopathic in 83.62% and alcoholic in 16.38% of 519 patients. Our study confirmed a significant association of 2 variants in CLDN2 gene (rs4409525—OR 1.71, P = 1.38 x 10-09; rs12008279—OR 1.56, P = 1.53 x 10-04) and 2 variants in MORC4 gene (rs12688220—OR 1.72, P = 9.20 x 10-09; rs6622126—OR 1.75, P = 4.04x10-05) in Indian patients with CP. We also found significant association at PRSS1-PRSS2 locus (OR 0.60; P = 9.92 x 10-06) and SAMD12-TNFRSF11B (OR 0.49, 95% CI [0.31–0.78], P = 0.0027). A variant in the gene MORC4 (rs12688220) showed significant interaction with alcohol (OR for homozygous and heterozygous risk allele -14.62 and 1.51 respectively, P = 0.0068) suggesting gene-environment interaction. A combined analysis of the genes CLDN2 and MORC4 based on an effective risk allele score revealed a higher percentage of individuals homozygous for the risk allele in CP cases with 5.09 fold enhanced risk in individuals with 7 or more effective risk alleles compared with individuals with 3 or less risk alleles (P = 1.88 x 10-14). Genetic variants in CLDN2 and MORC4 genes were associated with CP in Indian patients

λ-integrase mediated seamless vector transgenesis platform

Genome engineering is an important component of gene/cell therapy and molecular medicine. In this respect, several DNA editing tools, such as ZFNs, TALEN and CRISPR-Cas system are being currently used for transgenesis. However, issues like insertional mutagenesis, off-target events, and small payload size have affected their usage as precise genome editing tools. A novel λ-Integrase based tool was recently developed in the laboratory of Professor Peter Dröge, where a λ-integrase variant could catalyze conservative site-specific integration of the large payload into a safe harbor site(s) of the human genome. The identified safe harbor site(s), termed attH4X, is present within human Long INterspersed Elements-1 (LINE-1) in about 1000 copies dispersed throughout the human genome (Chandra et al. 2016). One of the major requirements of this tool is a circular target vector for Int-mediated recombination into genomic attH4X for which the traditional Escherichia coli is used to produce a plasmid that carries attP4X. This requires the inclusion of bacterial backbone along with payload in the target vector construct that also get inserted into the human genome. However, the presence of bacterial sequences in the conventional plasmid reduces its overall efficiency by increasing its size, silencing transgene expression and activating immune responses in the host cell. Thus, to overcome these limitations and further to improve the safety and targeting efficiency of this tool, the λ-Integrase platform was utilized to eliminate such bacterial sequences by seamless vector generation via in vitro intramolecular recombination. For this, a λ-integrase variant protein was partially purified and a protocol for in vitro seamless vector generation was established. Additionally, to refine and scale up the production of seamless vector, an in vivo platform was developed using E. coli strain LZ41 that can produce a high amount of the seamless vector. λ-Int mediated seamless vector transgenesis into human LINE-1 element has been successfully validated in HT1080 cells and human embryonic stem cells. The switch from plasmid to seamless vector for λ-Int mediated site-specific genomic integration drastically increased the targeting efficiency and reduced off-target events. Furthermore, the seamless vector was utilized for successful transgenesis and expression of therapeutic protein like anti-CD19 chimeric antigen receptor (CAR) in human embryonic stem cells (hESCs). The targeted hES clone showed stable long-term expression of the therapeutic anti-CD19 CAR protein, demonstrating the potential utility of λ-integrase mediated seamless vector transgenesis platform in therapeutic application. Moreover, certain LINE-1 loci were identified as preferred insertion sites for Int-mediated seamless vector transgenesis. DNA sequence logo on these loci showed specificity in the target site sequence for Int-C3 mediated attL4X recombination. It can be further utilized to engineer Int variants for better recognition of this subset of attH4X sequence. To broaden the utility of the platform, the tool was further explored in the field of biologics testing and production. To achieve this, a master cell line of HEK293T was generated by random insertion of landing pads carrying 4X attH4X sequence with a reporter cassette. The landing pad of the master cell line was targeted with a seamless vector encoding a second reporter cassette. This resulted in the high reporter cassette expressing clones that harboured seamless vector in one of the four attH4X sequences of the landing pad. The master cell line was further utilized to integrate and express a large transgene of size ~14kb that encodes therapeutic factor VIII protein. The targeted bulk HEK293T showed a high expression of factor VIII protein with a yield of ~2.2µg/ml from 10000 cells indicating the potential application of this platform in the biopharmaceutical sector. Since this platform could allow integration of transgene(s) at a specific genomic locus, it can be further used to test different variants of a recombinant protein and select the best performing variant. In summary, a λ-integrase mediated seamless vector transgenesis platform was developed that manifests dual capability to generate seamless vectors as well as target the human genome for insertion and expression of large transgene cassette. This platform can be broadly utilized for various molecular medicine, gene therapy and synthetic biology applications.Doctor of Philosoph

Versatile seamless DNA vector production in E. coli using enhanced phage lambda integrase

Seamless DNA vectors derived from bacterial plasmids are devoid of bacterial genetic elements and represent attractive alternatives for biomedical applications including DNA vaccines. Larger scale production of seamless vectors employs engineered Escherichia coli strains in order to enable tightly regulated expression of site-specific DNA recombinases which precisely delete unwanted sequences from bacterial plasmids. As a novel component of a developing lambda integrase genome editing platform, we describe here strain MG1655-ISC as a means to easily produce different scales of seamless vectors, ranging in size from a few hundred base pairs to more than ten kilo base pairs. Since we employed an engineered lambda integrase that is able to efficiently recombine pairs of DNA crossover sites that differ in sequence, the resulting seamless vectors will be useful for subsequent genome editing in higher eukaryotes to accommodate variations in target site sequences. Future inclusion of single cognate sites for other genome targeting systems could enable modularity. These features, together with the demonstrated simplicity of in vivo seamless vector production, add to their utility in the biomedical space.National Research Foundation (NRF)Published versionThis work was supported through a grant from the National Research Foundation-Competitive Research Programme, Singapore to P.D. (NRF-CRP21-2018-0002)

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Production of mini-seamless vectors in <i>MG1655-ISC</i>.

(A) Map of recombination substrate pattPhae2 (attL). The attB and attL (PT2) sequences are derivatives from the corresponding wild-type attB and attL and were placed about 500 bp apart as direct repeats. Recombination deletes the DNA segment flanked by the two att sites. (B) A suspension culture (10ml) was induced for integrase expression by arabinose and further incubation for 70 minutes. Plasmid DNA was purified and analysed by agarose gel electrophoresis. Lane L: marker ladder; Lane 1: substrate vector attPhae2 (attL) undigested; Lane 2: substrate vector attPhae2(attL) after induction, undigested; Lane 3: substrate vector NdeI digested; Lane 4: substrate vector ScaI digested; Lane 5: purified plasmid DNA after arabinose induction was digested with NdeI and T5 exonuclease; 3μl of 100μl total sample loaded; Lane 6: same as lane 5, 6 μl loaded; Lane 7: Purified plasmid DNA after arabinose induction digested with ScaI. Note that the supercoiled and linearized seamless vectors run at about the same position.</p