Mycobacterium Survival Strategy Translated to Develop a Lipo-Peptide Based Fusion Inhibitor

The entry of enveloped viruses requires fusion of viral and host cell membranes. An effective fusion inhibitor aiming at impeding such virus-host cell membrane fusion may emerge as a broad-spectrum antiviral agent to neutralize the infection from an increasing diversity of harmful new viruses. Mycobacterium survives inside the phagosome of the host cells by inhibiting phagosome-lysosome fusion with the help of a coat protein coronin 1. Structural analysis of coronin 1 and other WD40-repeat containing protein suggest that the tryptophan-aspartic acid (WD) sequence is placed at distorted β-meander motif (more exposed) whereas the WD resides in regular β-meander motif in other WD40 proteins. The unique structural feature of coronin 1 was explored to identify a simple lipo-peptide sequence (lipid-WD), which effectively inhibit the membrane fusion by increasing interfacial order and decreasing water penetration, surface potential. The effective fusion inhibitory role of mycobacterium inspired lipo-dipeptide was applied to combat type 1 influenza virus (H1N1) infection as a ‘broad spectrum’ antiviral agent.<br /

Heterogeneity and Compositional Diversities of <i>Campylobacter jejuni</i> Outer Membrane Vesicles (OMVs) Drive Multiple Cellular Uptake Processes

Naturally secreted outer membrane vesicles (OMVs) from gut microbes carry diverse cargo, including proteins, nucleic acids, toxins, and many unidentified secretory factors. Bacterial OMVs can shuttle molecules across different cell types as a generalized secretion system, facilitating bacterial pathogenicity and self-survival. Numerous mucosal pathogens, including Campylobacter jejuni (C. jejuni), share a mechanism of harmonized secretion of major virulence factors. Intriguingly, as a common gut pathogen, C. jejuni lacks some classical virulence-associated secretion systems; alternatively, it often employs nanosized lipid-bound OMVs as an intensive strategy to deliver toxins, including secretory proteins, into the target cells. To better understand how the biophysical and compositional attributes of natural OMVs of C. jejuni regulate their cellular interactions to induce a biologically relevant host response, we conducted an in-depth morphological and compositional analysis of naturally secreted OMVs of C. jejuni. Next, we focused on understanding the mechanism of host cell-specific OMVs uptake from the extracellular milieu. We showed that intracellular perfusion of OMVs is mediated by cytosolic as well as multiple endocytic uptake processes due to the heterogenic nature, abundance of surface proteins, and membrane phospholipids acquired from the source bacteria. Furthermore, we used human and avian cells as two different host targets to provide evidence of target cell-specific preferential uptake of OMVs. Together, the present study provides insight into the unique functionality of natural OMVs of C. jejuni at the cellular interface, upholding their potential for multimodal use as prophylactic and therapeutic carriers

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Not AvailableTo develop a successful cultivar-independent in vitro plant regeneration protocol in wheat, mature embryos of nine Indian elite wheat (Triticum aestivum) cultivars were taken to establish a reliable effective, reproducible callus culture and plant regeneration procedure. Two different auxins, naphtalenacetic acid (NAA) and dichlorophenoxyacetic acid (2,4-D) and wo different cytokinins, 6-Benzylaminopurine (BAP) and kinetin (Kn) were assessed in several combinations for their effect on callus induction and plant regeneration from mature embryos in nine elite Indian wheat cultivars. The cultivar-dependent substantial variances were noted in per cent of regeneration response. The callus induction was assessed in terms of size of callus induced in seven days of initiation. Induction of callus did happen in all cultivars on all media combinations tried, though there was difference in size of callus induced in different cultivars cultured with different combination of growth regulators. However, the induced callus, irrespective of cultivar when nurtured on MS media supplemented with 2,4-D (4.0 mg/L) and NAA (1.0 mg/L or 2.0 mg/L) showed prominent callus growth. There were significant differences among the nine cultivars in regeneration of shoots from embryonal callus on the various shooting media used. In spite of cultivar dependent response, the MS media supplemented with kn (2.0 mg/L), NAA (0.5mg/L) and BAP (0.5 mg/L) was identified as the best media for shoot regeneration for some of the elite Indian cultivars.Not Availabl

<span style="font-size:11.0pt;mso-bidi-font-size: 12.0pt;font-family:"Times New Roman";mso-fareast-font-family:"Times New Roman"; mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-GB;mso-fareast-language: EN-US;mso-bidi-language:AR-SA" lang="EN-GB">Development and characterization of a high temperature stress responsive subtractive cDNA library in Pearl Millet <i style="mso-bidi-font-style:normal">Pennisetum glaucum </i><span style="mso-bidi-font-style:italic">(L.) R.Br.</span></span>

543-550Pearl millet (<i style="mso-bidi-font-style: normal">Pennisetum glaucum L. R. Br.) is an important cereal crop grown mainly in the arid and semi-arid regions of India known to possess the natural ability to withstand thermal stress. To elucidate the molecular basis of high temperature response in pearl millet, 12 days old seedlings of P. glaucum cv. 841A were subjected to heat stress at 46°C for different time durations (30 min, 2, 4, 8, 12 and 24 h) and a forward subtractive cDNA library was constructed from pooled RNA of heat stressed seedlings. A total of 331 high quality Expressed Sequence Tags (ESTs) were obtained from randomly selected 1050 clones. Sequences were assembled into 103 unique sequences consisting of 37 contigs and 66 singletons. Of these, 92 unique sequences were submitted to NCBI dbEST database. Gene Ontology through RGAP data base and BLASTx analysis revealed that about 18% of the ESTs showed homology to genes for “response to abiotic and biotic stimulus”. About 2% of the ESTs showed no homology with genes in dbEST, indicating the presence of uncharacterized candidate genes involved in heat stress response in P. glaucum. Differential expression of selected genes (<i style="mso-bidi-font-style: normal">hsp101 and CRT) from the SSH library were validated by qRT-PCR analysis. The ESTs thus generated are a rich source of heat stress responsive genes, which can be utilized in improving thermotolerance of other food crops. </span

De novo design of lipopeptide-based fusion inhibitor as potential broad-spectrum antiviral agent

The recent surge in emerging viral infections warrants the need to design broad-spectrum antivirals. We aimed to develop a lead molecule that targets the membrane to block fusion, an obligate step of enveloped virus infection. The approach is based on the Coronin-1 protein of Mycobacterium, which presumably inhibits the phagosome-lysosome fusion, and a unique Trp-Asp (WD) sequence is placed at the distorted -meander motif. We have designed a WD-based branched lipopeptide that supports C=OHN hydrogen-bonding, the tryptophan-tryptophan - stacking, and the intermolecular H-bonding between COO and CO2H groups. These cooperative interactions are expected to create a -sheet-like supramolecular assembly at the membrane surface, which increases the interfacial order, and decreases the water penetration. Myr-D(WD)2 was shown to block artificial membrane fusion completely. We demonstrated that the Myr-D(WD)2 supramolecular organization can restrict the infection from H1N1, H9N2, murine coronavirus, and human coronavirus (HCoV-OC43). Together, the present study provided an evidence-based broad-spectrum antiviral potential of a designed small lipopeptide

Lipidated Lysine and Fatty Acids Assemble into Protocellular Membranes to Assist Regioselective Peptide Formation: Correlation to the Natural Selection of Lysine over Nonproteinogenic Lower Analogues

The self-assembly of prebiotically plausible amphiphiles (fatty acids) to form a bilayer membrane for compartmentalization is an important factor during protocellular evolution. Such fatty acid-based membranes assemble at relatively high concentrations, and they lack robust stability. We have demonstrated that a mixture of lipidated lysine (cationic) and prebiotic fatty acids (decanoic acid, anionic) can form protocellular membranes (amino acid-based membranes) at low concentrations via electrostatic, hydrogen bonding, and hydrophobic interactions. The formation of vesicular membranes was characterized by dynamic light scattering (DLS), pyrene and Nile Red partitioning, cryo-transmission electron microscopy (TEM) images, and glucose encapsulation studies. The lipidated nonproteinogenic analogues of lysine (Lys), such as ornithine (Orn) and 2,4-diaminobutyric acid (Dab), also form membranes with decanoate (DA). Time-dependent turbidimetric and 1H NMR studies suggested that the Lys-based membrane is more stable than the membranes prepared from nonproteinogenic lower analogues. The Lys-based membrane embeds a model acylating agent (aminoacyl-tRNA mimic) and facilitates the colocalization of substrates to support regioselective peptide formation via the α-amine of Lys. These membranes thereby assist peptide formation and control the positioning of the reactants (model acylating agent and −NH2 of amino acids) to initiate biologically relevant reactions during early evolution

A quick, easy and cost-effective in planta method to develop direct transformants in wheat

Not AvailableAgrobacterium mediated in planta method was used to transform Indian elite wheat genotype HD2894 with herbicide-tolerant CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene. The apical meristems of germinated seeds were targeted for introgression of transgene. The obtained T1 plants were screened by spraying 1% glyphosate and only positive transformants survived. The presence of transgene was also confirmed by PCR and Southern hybridization. Using this method, 3.07% transformation rate was observed. To identify transgenic lines carrying stably integrated CP4-EPSPS gene, the transgenic populations were screened in T3 generation using 1% glyphosate and lines with 100% survival were considered as homozygous. No significant morpho-physiological variations were observed within the transgenic lines as compared to non-transgenic plants. The present study resulted in herbicide-tolerant transgenic wheat and provides a valuable tool for development of wheat genetic transformation.Not Availabl