Efficient formation of single-copy human artificial chromosomes.

Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through ~125-base pair DNA sequence-defined centromeres, mammals and many other eukaryotes use large, epigenetic centromeres. Harnessing centromere epigenetics permits human artificial chromosome (HAC) formation but is not sufficient to avoid rampant multimerization of the initial DNA molecule upon introduction to cells. We describe an approach that efficiently forms single-copy HACs. It employs a ~750-kilobase construct that is sufficiently large to house the distinct chromatin types present at the inner and outer centromere, obviating the need to multimerize. Delivery to mammalian cells is streamlined by employing yeast spheroplast fusion. These developments permit faithful chromosome engineering in the context of metazoan cells.

Multiple Adaptive Strategies of Himalayan Iodobacter sp. PCH194 to High-Altitude Stresses.

Bacterial adaption to the multiple stressed environments of high-altitude niches in the Himalayas is intriguing and is of considerable interest to biotechnologists. Previously, we studied the culturable and unculturable metagenome microbial diversity from glacial and kettle lakes in the Western Himalayas. In this study, we explored the adaptive strategies of a unique Himalayan eurypsychrophile Iodobacter sp. PCH194, which can synthesize polyhydroxybutyrate (PHB) and violacein pigment. Whole-genome sequencing and analysis of Iodobacter sp. PCH194 (4.58 Mb chromosome and three plasmids) revealed genetic traits associated with adaptive strategies for cold/freeze, nutritional fluctuation, defense against UV, acidic pH, and the kettle lake's competitive environment. Differential proteome analysis suggested the adaptive role of chaperones, ribonucleases, secretion systems, and antifreeze proteins under cold stress. Antifreeze activity inhibiting the ice recrystallization at -9°C demonstrated the bacterium's survival at subzero temperature. The bacterium stores carbon in the form of PHB under stress conditions responding to nutritional fluctuations. However, violacein pigment protects the cells from UV radiation. Concisely, genomic, proteomic, and physiological studies revealed the multiple adaptive strategies of Himalayan Iodobacter to survive the high-altitude stresses.

A ricin-based peptide BRIP from Hordeum vulgare inhibits Mpro of SARS-CoV-2.

COVID-19 pandemic caused by SARS-CoV-2 led to the research aiming to find the inhibitors of this virus. Towards this world problem, an attempt was made to identify SARS-CoV-2 main protease (Mpro) inhibitory peptides from ricin domains. The ricin-based peptide from barley (BRIP) was able to inhibit Mpro in vitro with an IC50 of 0.52 nM. Its low and no cytotoxicity upto 50 µM suggested its therapeutic potential against SARS-CoV-2. The most favorable binding site on Mpro was identified by molecular docking and steered molecular dynamics (MD) simulations. The Mpro-BRIP interactions were further investigated by evaluating the trajectories for microsecond timescale MD simulations. The structural parameters of Mpro-BRIP complex were stable, and the presence of oppositely charged surfaces on the binding interface of BRIP and Mpro complex further contributed to the overall stability of the protein-peptide complex. Among the components of thermodynamic binding free energy, Van der Waals and electrostatic contributions were most favorable for complex formation. Our findings provide novel insight into the area of inhibitor development against COVID-19.

Ice structuring protein extract of Hordeum vulgare var. dolma grain reduces drip loss and loss of soluble vitamin content in peas during frozen storage.

The Himalayan plants face low temperature during their life cycle and are expected to possess antifreeze proteins. The present work describes screening of six plant species that grow in Himalayas, for their ice structuring properties with an aim to provide a vegetarian source of ice structuring proteins. The ice-structuring protein extract of barley [Hordeum vulgare; variety Dolma (HvISE)] and wheat [Triticum aestivum; variety Him Pratham DH 114 (TaISE) ]showed inhibition of growth of ice crystals during the process of recrystallization. This property was analyzed for its application in frozen peas by pretreatment with HvISE before freezing. The drip loss was measured in frozen peas after thawing. Further, the water-soluble vitamins; thiamine, pyridoxine, riboflavin, and ascorbic acid were quantified before and after freezing of green peas by UHPLC-PDA. The pretreatment with HvISE reduced the loss of ascorbic acid, riboflavin, and pyridoxine during their frozen storage. The present study identified barley as a remarkable source of ice structuring proteins and validated its potential for frozen peas. The work has enormous implications in frozen food industry, in general.

Performance of antifreeze protein HrCHI4 from Hippophae rhamnoides in improving the structure and freshness of green beans upon cryopreservation.

Antifreeze proteins restrict the growth of ice crystals during recrystallization and therefore find application in the protection of food products from damage upon freezing. Hippophae rhamnoides (seabuckthorn) is a freeze tolerant Himalayan shrub exhibiting antifreeze properties. Here, ~39 kDa class IV chitinase (HrCHI4) was purified from seabuckthorn seeds using chitin-affinity chromatography that showed antifreeze property by ice recrystallization inhibition. The application of HrCHI4 in cryopreservation of green beans was analyzed to verify its antifreeze potential. HrCHI4 pretreatment reduced the drip loss and electrolytic leakage in frozen beans, revealing that it preserved the membrane integrity upon cryopreservation. The texture analysis and SEM further validated structural maintenance. The volatile component analysis using GC-MS was performed to evaluate the quality of frozen beans. HrCHI4 contributed positively towards the retention of volatile components after freeze-thaw. In conclusion, a class IV chitinase HrCHI4 was purified from seabuckthorn seeds and its cryoprotective function was reported.

Two ICE isoforms showing differential transcriptional regulation by cold and hormones participate in Brassica juncea cold stress signaling.

C-repeat binding factor (CBF) dependent cold stress signaling cascade is well studied in the model plant arabidopsis but is relatively lesser studied in the crop plants. In the present study, two novel isoforms of an upstream regulator of CBF, Inducer of CBF expression (ICE), BjICE46 (1314 bp, accession number HQ446510) and BjICE53 (1494 bp, accession number HQ857208) were isolated from Brassica juncea seedlings. Genomic clones of both the isoforms (accession numbers HQ433510 and JX571043) showed three introns, out of which one intron was spanning the bHLH (basic helix-loop-helix) domain. Interestingly, the constitutive expression of BjICE53 was 21 fold higher than BjICE46. Real time quantitative expression (RT-qPCR) showed BjICE53 to be cold induced but non-responsive to phytohormones. Interestingly, BjICE46 was salinity stress induced and showed upregulation with methyl jasmonate (MeJa) and abscisic acid (ABA). This was supported by the presence of ABA, MeJa and defense related cis- acting regulatory elements in the promoter region of BjICE46. The downstream transcription factor BjCBF (645 bp) was also isolated. The promoter region of BjCBF showed three E-boxes, the binding site for ICE. BjCBF was expressed and purified from E. coli and binding of purified BjCBF with the DRE/CRT elements (present in the promoter of cold responsive genes) was EMSA confirmed. Overall, this study shows that ICE-CBF pathway is conserved in Brassica juncea along with the differential regulation of the ICE isoforms indicating cross-talk between cold and defense signaling.

A novel class I Chitinase from Hippophae rhamnoides: Indications for participating in ICE-CBF cold stress signaling pathway.

Plant chitinases are the members of PR (Pathogenesis related) proteins family and protect plants from biotic and abiotic stress. A novel chitinase HrCHI1 (Accession number JQ289153) of 954bp ORF encoding 317 amino acids protein was cloned, expressed and characterized from seabuckthorn, a cold/freeze tolerant shrub. The 3D structure (predicted with I-TASSER server) showed highest homology with Oryza sativa class I chitinase (PDB 2dkvA). Putative promoter region (obtained by genome walking) showed GCC box, E-boxes, the binding site for bHLH proteins and DRE elements, the CBF (C-repeat binding factor) binding site besides TATA and CAAT boxes. The gel shift assay with the nuclear extract indicated that the HrCHI1 might be participating in CBF/ERF dependent cold stress signaling pathway. The quantitative transcript profiling supported this observation as cold induced expression of HrCBF peaked earlier (at 1h) while HrCHI1 peaked latter (after 3h) indicating HrCHI1 expression might be induced by HrCBF. Further, HrCHI1 expression was methyl jasmonate (MeJa) dependent and salicylic acid (SA) independent. HrCHI1 was expressed in E. coli and purified using chitin affinity chromatography. It showed 512U/mg chitinase hydrolytic activity and resolved as a 34kDa spot with a slightly basic pI (8.5) on a 2-D gel. The E. coli cells containing recombinant chitinase showed higher rate of growth in cold in comparison with the cells containing the empty vector. In conclusion, we have isolated and characterized a cold responsive basic class I chitinase which is regulated by MeJa and seems to be functioning via CBF/ERF dependent cold stress signaling pathway.

Nitric oxide modulates Lycopersicon esculentum C-repeat binding factor 1 (LeCBF1) transcriptionally as well as post-translationally by nitrosylation.

Nitric oxide (NO) production increases in the cold stress. This cold enhanced NO manifests its effect either by regulating the gene expression or by modulating proteins by NO based post-translational modifications (PTMs) including S-nitrosylation. CBF (C-repeat binding factor) dependent cold stress signaling is most studied cold stress-signaling pathway in plants. SNP (sodium nitroprusside, a NO donor) treatment to tomato seedlings showed four fold induction of LeCBF1 (a cold inducible CBF) transcript in cold stress. S-nitrosylation as PTM of CBF has not been analyzed till date. In silico analysis using GPS-SNO 1.0 software predicted Cys 68 as the probable site for nitrosylation in LeCBF1. The 3D structure and motif prediction showed it to be present in the beta hairpin loop and hence available for S-nitrosylation. LeCBF1 was cloned and expressed in Escherichia coli. LeCBF1 accumulated in the inclusion bodies, which were solubilized under denaturing conditions and purified after on column refolding by Ni-NTA His tag affinity chromatography. Purified LeCBF1 resolved as a 34 kDa spot with a slightly basic pI (8.3) on a 2-D gel. MALDI-TOF mass spectrometry identified it as LeCBF1 and western blotting using anti-LeCBF1 antibodies confirmed its purification. Biotin switch assay and neutravidin affinity chromatography showed LeCBF1 to be S-nitrosylated in presence of GSNO (NO donor) as well as endogenously (without donor) in cold stress treated tomato seedlings. Dual regulation of LeCBF1 by NO at both transcriptional as well as post-translational level (by S-nitrosylation) is shown for the first time.

Dealing with emerging waste streams: used tyre assessment in Thailand using material flow analysis.

Increasing urbanisation and automobile use have given rise to an increase in global tyre waste generation. A tyre becomes waste once it wears out and is no longer fit for its original purpose, and is thus in its end-of-life state. Unlike in developed countries, where waste tyre management has already become a significant issue, it is rarely a priority waste stream in developing countries. Hence, a large quantity of waste tyres ends up either in the open environment or in landfill. In Thailand, waste tyre management is in its infancy, with increased tyre production and wider use of vehicles, but low levels of recycling, leaving scope for more appropriate policies, plans and strategies to increase waste tyre recycling. This article describes the journey of waste tyres in Thailand in terms of recycling and recovery, and disposal. Material flow analysis was used as a tool to quantify the flows and accumulation of waste tyres in Thailand in 2012. The study revealed that, in Thailand in 2012, waste tyre management was still biased towards destructive technologies (48.9%), rather than material recovery involving rubber reclamation, retreading tyres and whole and shredded tyre applications (6.7%). Despite having both economic and environmental benefits, 44.4% of used tyres in 2012 were dumped in the open environment, and the remaining 0.05% in landfills.

First systematic plant proteomics workshop in Botany Department, University of Delhi: transferring proteomics knowledge to next-generation researchers and students.

International Plant Proteomics Organization (INPPO) outlined ten initiatives to promote plant proteomics in each and every country. With greater emphasis in developing countries, one of those was to "organize workshops at national and international levels to train manpower and exchange information". This third INPPO highlights covers the workshop organized for the very first time in a developing country, India, at the Department of Botany in University of Delhi on December 26-30, 2013 titled - "1(st) Plant Proteomics Workshop / Training Program" under the umbrella of INPPO India-Nepal chapter. Selected 20 participants received on-hand training mainly on gel-based proteomics approach along with manual booklet and parallel lectures on this and associated topics. In house, as well as invited experts drawn from other Universities and Institutes (national and international), delivered talks on different aspects of gel-based and gel-free proteomics. Importance of gel-free proteomics approach, translational proteomics, and INPPO roles were presented and interactively discussed by a group of three invited speakers Drs. Ganesh Kumar Agrawal (Nepal), Randeep Rakwal (Japan), and Antonio Masi (Italy). Given the output of this systematic workshop, it was proposed and thereafter decided to be organized every alternate year; the next workshop will be held in 2015. Furthermore, possibilities on providing advanced training to those students / researchers / teachers with basic knowledge in proteomics theory and experiments at national and international levels were discussed. INPPO is committed to generating next-generation trained manpower in proteomics, and it would only happen by the firm determination of scientists to come forward and do it.