Fusogenic potential of sperm membrane lipids: nature's wisdom to accomplish targeted gene delivery

The membrane-membrane fusion during fertilization of oocyte by spermatozoa is believed to be mainly mediated by so called "fusion proteins". In the present study we have tried to demonstrate that beside the proteins, lipid components of membrane may play an important role in fusion of oocyte with spermatozoa. Conventional membrane-membrane fusion assays were used as means to demonstrate fusogenic potential of human sperm membrane lipids. The liposomes (spermatosomes) made of the lipids isolated from sperm membrane were found to undergo strong membrane-membrane fusion as evident from fluorescence dequenching and resonance energy transfer assays. Furthermore, the fusion of these liposomes with living cells (J774 A.1 macrophage cell line) was demonstrated to result in an effective transfer of a water-soluble fluorescent probe (calcein) to cytosol of the target cell. Lastly, the liposomes were demonstrated to behave like efficient vehicles for the in vivo cytosolic delivery of the antigens to target cells resulting in elicitation of antigen specific CD8(+) T cell responses.     

Bioactivity studies of Huh-7 cells derived human epidermal growth factor expressed in Pichia pastoris

Previously, we have reported cloning of human epidermal growth factor gene from Huh-7 cells and its extracellular expression in Pichia pastoris. The presented work is a detailed report regarding molecular characterization of Huh-7 cells-derived hEGF expressed in Pichia pastoris with special reference to its glycosylation profiling and bioactivity studies. Densitometric scanning of SDS-PAGE separated extracellular proteins from hEGF recombinant Pichia pastoris strain indicated that about 84% of the extracellular proteins were glycosylated. Size exclusion chromatography using Superdex 75 prep grade column was successfully utilized to separate fractions containing glycosylated and non-glycosylated extracellular proteins. In dot blot assay, hEGF was detected in both glycosylated and non-glycosylated fractions. Bioactivity assays revealed that both glycosylated and non-glycosylated fractions were bioactive as determined by cell viability assay. It was also observed that hEGF present in non-glycosylated fraction was relatively more bioactive than hEGF present in glycosylated fraction.     

Atomic Insight into the Altered O6-Methylguanine-DNA Methyltransferase Protein Architecture in Gastric Cancer

O⁶-methylguanine-DNA methyltransferase (MGMT) is one of the major DNA repair protein that counteracts the alkalyting agent-induced DNA damage by replacing O⁶-methylguanine (mutagenic lesion) back to guanine, eventually suppressing the mismatch errors and double strand crosslinks. Exonic alterations in the form of nucleotide polymorphism may result in altered protein structure that in turn can lead to the loss of function. In the present study, we focused on the population feared for high exposure to alkylating agents owing to their typical and specialized dietary habits. To this end, gastric cancer patients pooled out from the population were selected for the mutational screening of a specific error prone region of MGMT gene. We found that nearly 40% of the studied neoplastic samples harbored missense mutation at codon¹⁵¹ resulting into Serine to Isoleucine variation. This variation resulted in bringing about the structural disorder, subsequently ensuing into a major stoichiometric variance in recognition domain, substrate binding and selectivity loop of the active site of the MGMT protein, as observed under virtual microscope of molecular dynamics simulation (MDS). The atomic insight into MGMT protein by computational approach showed a significant change in the intra molecular hydrogen bond pattern, thus leading to the observed structural anomalies. To further examine the mutational implications on regulatory plugs of MGMT that holds the protein in a DNA-Binding position, a MDS based analysis was carried out on, all known physically interacting amino acids essentially clustered into groups based on their position and function. The results generated by physical-functional clustering of protein indicated that the identified mutation in the vicinity of the active site of MGMT protein causes the local and global destabilization of a protein by either eliminating the stabilizing salt bridges in cluster C3, C4, and C5 or by locally destabilizing the “protein stabilizing hing” mapped on C3-C4 cluster, preceding the active site.     

Fumonisin B1-Induced Oxidative Burst Perturbed Photosynthetic Activity and Affected Antioxidant Enzymatic Response in Tomato Plants in Ethylene-Dependent Manner

Fumonisin B1 (FB1) is a harmful mycotoxin produced by Fusarium species, which results in oxidative stress leading to cell death in plants. FB1 perturbs the metabolism of sphingolipids and causes growth and yield reduction. This study was conducted to assess the role of ethylene in the production and metabolism of reactive oxygen species in the leaves of wild type (WT) and ethylene receptor mutant Never ripe (Nr) tomato and to elucidate the FB1-induced phytotoxic effects on the photosynthetic activity and antioxidant mechanisms triggered by FB1 stress. FB1 exposure resulted in significant ethylene emission in a concentration-dependent manner in both genotypes. Moreover, FB1 significantly affected the photosynthetic parameters of PSII and PSI and activated photoprotective mechanisms, such as non-photochemical quenching in both genotypes, especially under 10 µM FB1 concentration. Further, the net photosynthetic rate and stomatal conductance were significantly reduced in both genotypes in a FB1 dose-dependent manner. Interestingly, lipid peroxidation and loss of cell viability were also more pronounced in WT as compared to Nr leaves indicating the role of ethylene in cell death induction in the leaves. Thus, FB1-induced oxidative stress affected the working efficiency of PSI and PSII in both tomato genotypes. However, ethylene-dependent antioxidant enzymatic defense mechanisms were activated by FB1 and showed significantly elevated levels of superoxide dismutase (18.6%), ascorbate peroxidase (129.1%), and glutathione S-transferase activities (66.62%) in Nr mutants as compared to WT tomato plants confirming the role of ethylene in the regulation of cell death and defense mechanisms under the mycotoxin exposure.

     

Corrugation Enabled Asymmetrically Ultrastretchable (95%) Monocrystalline Silicon Solar Cells with High Efficiency (19%)

Stretchable solar cells are of growing interest due their key role in realizing many applications such as wearables and biomedical devices. Ultrastretchability, high energy‐efficiency, biocompatibility, and mechanical resilience are essential characteristics of such energy harvesting devices. Here, the development of wafer‐scale monocrystalline silicon solar cells with world‐record ultrastretchability (95%) and efficiency (19%) is demonstrated using a laser‐patterning based corrugation technique. The demonstrated approach transforms interdigitated back contacts (IBC) based rigid solar cells into mechanically reliable but ultrastretchable cells with negligible degradation in the electric performance in terms of current density, open‐circuit voltage, and fill factor. The corrugation method is based on the creation of alternating grooves resulting in silicon islands with different shapes. The stretchability is achieved by orthogonally aligning the active silicon islands to the applied tensile stress and using a biocompatible elastomer (Ecoflex) as a stretchable substrate. The resulting mechanics ensure that the brittle silicon areas do not experience significant mechanical stresses upon asymmetrical stretching. Different patterns are studied including linear, diamond, and triangular patterns, each of which results in a different stretchability and loss of active silicon area. Finally, finite element method based simulation is conducted to study the generated deformation in the different patterned solar cells.     

Recent advances in engineering of microbial cell factories for intelligent pH regulation and tolerance

pH regulation is a serious concern in the industrial fermentation process as pH adjustment heavily utilizes acid/base and pollutes the environment. Under pH-stress conditions, microbial growth and production of valuable target products may be severely affected. Furthermore, some strains generating acidic or alkaline products require self pH regulation and increased tolerance against pH-stress. For pH control, synthetic biology has provided advanced engineering approaches to construct robust and more intelligent microbial strains, exhibiting tolerance to pH-stress to cope with limitations of pH regulation. This study reviewed the current progress of advanced strain evolution strategies to engineer pH-stress tolerant strains via synthetic biology. In addition, a large number of pH-responsive elements, including promoters, riboswitches, and some proteins have been investigated and applied for construction of pH-responsive genetic circuits and intelligent pH-responsive microbial strains.     

Neurons Refine the Caenorhabditis elegans Body Plan by Directing Axial Patterning by Wnts

Metazoans display remarkable conservation of gene families, including growth factors, yet somehow these genes are used in different ways to generate tremendous morphological diversity. While variations in the magnitude and spatio-temporal aspects of signaling by a growth factor can generate different body patterns, how these signaling variations are organized and coordinated during development is unclear. Basic body plans are organized by the end of gastrulation and are refined as limbs, organs, and nervous systems co-develop. Despite their proximity to developing tissues, neurons are primarily thought to act after development, on behavior. Here, we show that in Caenorhabditis elegans, the axonal projections of neurons regulate tissue progenitor responses to Wnts so that certain organs develop with the correct morphology at the right axial positions. We find that foreshortening of the posteriorly directed axons of the two canal-associated neurons (CANs) disrupts mid-body vulval morphology, and produces ectopic vulval tissue in the posterior epidermis, in a Wnt-dependent manner. We also provide evidence that suggests that the posterior CAN axons modulate the location and strength of Wnt signaling along the anterior–posterior axis by employing a Ror family Wnt receptor to bind posteriorly derived Wnts, and hence, refine their distributions. Surprisingly, despite high levels of Ror expression in many other cells, these cells cannot substitute for the CAN axons in patterning the epidermis, nor can cells expressing a secreted Wnt inhibitor, SFRP-1. Thus, unmyelinated axon tracts are critical for patterning the C. elegans body. Our findings suggest that the evolution of neurons not only improved metazoans by increasing behavioral complexity, but also by expanding the diversity of developmental patterns generated by growth factors such as Wnts.     

Root membrane lipids as potential biomarkers to discriminate silage-corn genotypes cultivated on podzolic soils in boreal climate

Root membrane lipids are important biomolecules determining plant's ability to adapt to different growing environmental or climatic conditions. Herein, we demonstrate the potential use of root membrane lipids as biomarkers to discriminate silage-corn genotypes based on herbicide and insect/pest resistance genetic traits when cultivated on podzolic soils under short growing and moderately warm summer season in boreal climate. Lipids in root membranes of field grown silage-corn genotypes were previously quantified at crop maturity by ultra-high-performance liquid chromatography-hydrophilic interaction chromatography-heated electrospray ionization mass spectrometry. The lipid identified and quantified in silage-corn roots were phospholipids, glycolipids and sphingolipids. Following hierarchical cluster analysis, three groups of membrane lipids were observed to be very effective in segregating the five silage-corn genotypes. The first group consisted of hexosylceramide (HexCer), phosphatidylcholine (PC) and phosphatidylinositol (PI). The second group consisted of lysophosphatidic acid (LPA16:0) and lysophosphatidylcholine (LPC16:0), while the third group consisted of 37 molecular species from observed lipids (phospholipids, glycolipids, sphingolipids). Partial least squares-discriminant analysis (PLS-DA) based on 37 membrane lipid species, as well as principal component analysis using the variables important in projection derived from the PLS-DA segregated the five silage-corn genotypes into three groups according to their pesticide/herbicide resistant traits. This study is second to none using root lipidomics in discriminating different silage-corn genotypes based on their herbicide and insect/pest resistance genetic traits for cultivation in boreal climates. The segregated genotypes possess three different genetic traits for herbicide and insect/pest resistance including VT Double Pro (VT2P), VT Triple Pro Roundup Ready (VT3P/RR) and Roundup Ready-2 corn (RR2). These findings demonstrate that root membrane lipids could serve as appropriate chemical biosignatures to identify silage-corn genotypes based on herbicide and insect/pest resistance genetic traits suitable for cultivation in boreal climates.