<Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> N-glycoproteome analysis: a small step towards sea buckthorn proteome mining

Hippophae rhamnoides is a hardy shrub capable of growing under extreme environmental conditions namely, high salt, drought and cold. Its ability to grow under extreme conditions and its wide application in pharmaceutical and nutraceutical industry calls for its in-depth analysis. N-glycoproteome mining by con A affinity chromatography from seedling was attempted. The glycoproteome was resolved on first and second dimension gel electrophoresis. A total of 48 spots were detected and 10 non-redundant proteins were identified by MALDI–TOF/TOF. Arabidopsis thaliana protein disulfide isomerase-like 1-4 (ATPDIL1-4) electron transporter, protein disulphide isomerase, calreticulin 1 (CRT1), glycosyl hydrolase family 38 (GH 38) protein, phantastica, maturase k, Arabidopsis trithorax related protein 6 (ATXR 6), cysteine protease inhibitor were identified out of which ATXR 6, phantastica and putative ATPDIL1-4 electron transporter are novel glycoproteins. Calcium binding protein CRT1 was validated for its calcium binding by stains all staining. GO analysis showed involvement of GH 38 and ATXR 6 in glycan and lysine degradation pathways. This is to our knowledge the first report of glycoproteome analysis for any Elaeagnaceae member.     

Mefloquine induces ROS mediated programmed cell death in malaria parasite: Plasmodium

Recent studies pioneer the existence of a novel programmed cell death pathway in malaria parasite plasmodium and suggest that it could be helpful in developing new targeted anti-malarial therapies. Considering this fact, we evaluated the underlying action mechanism of this pathway in mefloquine (MQ) treated parasite. Since cysteine proteases play a key role in apoptosis hence we performed preliminary computational simulations to determine binding affinity of MQ with metacaspase protein model. Binding pocket identified using computational studies, was docked with MQ to identify it’s potential to bind with the predicted protein model. We further determined apoptotic markers such as mitochondrial dysregulation, activation of cysteine proteases and in situ DNA fragmentation in MQ treated/untreated parasites by cell based assay. Our results showed low mitochondrial membrane potential, enhanced activity of cysteine protease and increased number of fragmented DNA in treated parasites compared to untreated ones. We next tested the involvement of oxidative stress in MQ mediated cell death and found significant increase in reactive oxygen species generation after 24 h of treatment. Therefore we conclude that apart from hemozoin inhibition, MQ is competent to induce apoptosis in plasmodium by activating metacaspase and ROS production.     

Endothelial cells are activated by angiopoeitin-1 gene transfer and produce coordinated sprouting in vitro and arteriogenesis in vivo

RATIONAL AND OBJECTIVES: Activation of fully differentiated vascular cells using angiogenic genes can lead to phenotypic changes resulting in formation of new blood vessels. We tested whether Ang-1 gene transfer to endothelial cells (EC) activates these cells. METHODS AND RESULTS: EC and SMC were transduced using retroviral or adenoviral vectors to produce Ang-1 or vascular endothelial growth factor (VEGF). EC Tie-2 receptor was phosphorilated by autologous secretion of Ang-1. Transduced EC and SMC sprouting capacity was tested using collagen embedded spheroids assay and capacity to produce arteriogenesis was tested in a hind limb model of ischemia. EC expressing Ang-1 in the presence of SMC expressing VEGF exhibited high levels of sprouting of the two cell types. Flow and numbers of arteries were increased after transduced cells implantation in vivo. CONCLUSIONS: Autologous secretion of Ang-1 by transduced EC resulted in Tie-2 activation and in the presence of SMC expressing VEGF resulted in coordinated sprouting in vitro and increase in flow and number of arteries in vivo.     

Identification of a 55-kDa ezrin-related protein that induces cytoskeletal changes and localizes to the nucleolus.

Normal and transformed human cells when stained for ezrin, an F-actin-binding ERM (ezrin/radixin/moesin) family protein, revealed a faint and intense immunofluorescence, respectively. Surprisingly, nuclear staining that was assigned to the nucleolus by confocal laser and immunoelectron microscopy was detected in both cell types and was more prominent in normal cells due to the absence of glistering cytoplasmic fluorescence. By Western analysis the nuclear fraction was seen to have a 55-kDa ezrin-reactive protein that did not react to the antibodies raised against the C-terminus of the protein, suggesting that it may correspond to an endogenously cleaved N-terminus of the protein. Transfections of cells with a cDNA encoding full-length ezrin tagged with green fluorescent protein (GFP) at its N-terminus indeed resulted in two GFP-tagged products corresponding to full-length and 55-kDa endogenously cleaved forms. Transfection with a cDNA encoding approximately 55 kDa of the ezrin N-terminus (N-ezrin) showed that it can translocate to the nucleus. N-ezrin transfected cells exhibited irregular cell edges and collapse of actin fibers. Similar changes were seen following microinjection of anti-p81/ezrin antibody, suggesting that N-ezrin may function as a dominant negative competitor of ezrin. These data demonstrate the existence of an N-terminal cleavage form of ezrin that localizes to the nucleolus and that its overexpression induces cytoskeletal changes.     

Nitric oxide synthase immunoreactivity in the developing and adult human retina

Nitric oxide synthase (NOS) catalyzes the formation of nitric oxide (NO) from L-arginine. In this study, the cellular localization of neuronal NOS (nNOS) activity in the human retina since fetal development was examined by immunohistochemistry. No detectable staining in the fetal retina was present at 14 weeks of gestation (wg), the earliest age group examined. A centro-peripheral gradient of development of nNOS immunoreactivity was evident at 16–17 wg, with the midperipheral retina showing nNOS immunoreactivity in most of the cell types and the inner plexiform layer while the peripheral part demonstrated moderate immunoreactivity only in the ganglion cell layer and photoreceptor precursors. A transient increase in nNOS immunoreactivity in the ganglion cells and Müller cell endfeet between 18–19 and 24–25 wg was observed at the time when programmed cell death in the ganglion cell layer, loss of optic nerve fibres as well as increase in glutamate immunoreactivity and parvalbumin (a calcium binding protein) immunoreactivity in the ganglion cells was reported. These observations indicate that programmed cell death of ganglion cells in the retina may be linked to glutamate toxicity and NO activity, as also suggested by others in the retina and cerebral cortex. The presence of nNOS immunoreactivity in the photoreceptors from 16–17 weeks of fetal life to adulthood indicates other functions, besides their involvement in photoreceptor function of transduction and information processing.     

The futuristic applications of transition metal dichalcogenides for cancer therapy

The second-most common cause of death resulting from genetic mutations in DNA sequences is cancer. The difficulty in the field of anticancer research is the application of the traditional methods, which also affects normal cells. Mutations, genetic replication alterations, and chromosomal abnormalities have a direct impact on the effectiveness of anticancer drugs at different stages. Presently, therapeutic techniques utilize nanotechnology, transition metal dichalcogenides (TMDCs), and robotics. TMDCs are being increasingly employed in tumor therapy and biosensing applications due to their biocompatibility, adjustable bandgap, versatile functionality, exceptional photoelectric properties, and wide range of applications. This study reports the advancement of nanoplatforms based on TMDCs that are specifically engineered for responsive and intelligent cancer therapy. This article offers a thorough examination of the current challenges, future possibilities for theranostic applications using TMDCs, and recent progress in employing TMDCs for cancer therapy. Currently, there is significant interest in two-dimensional (2D) TMDCs nanomaterials as ultrathin unique physicochemical properties. These materials have attracted attention in various fields, including biomedicine. Due to their inherent ability to absorb near-infrared light and their exceptionally large surface area, significant efforts are being made to prepare multifunctional nanoplatforms based on 2D TMDCs.     

Role of Se(VI) in counteracting oxidative damage in <Emphasis Type="Italic">Brassica juncea</Emphasis> L. under Cr(VI) stress

Chromium (Cr) is considered to be one of the major environmental hazards and poses a threat to both plant and animal health. Selenium (Se), however, has been recognized as an essential micronutrient in plants. To understand the role of Se(VI) in oxidative stress management and regulation of antioxidative defence mechanism against heavy metal stress, the seedlings of Brassica juncea L. were raised in Petri plates containing nutrient media supplemented with only with Se(VI) and Cr(VI), or their combination. It was observed that of Cr(VI) causes an increase in reactive oxygen species (ROS) in the seedlings leading to oxidative stress. Histological studies using confocal and visible microscopy confirmed the biochemical results. Supplementation of up to 4 µM of Se(VI) to media containing 300 µM of Cr(VI) reduced the contents of ROS and increased enzymatic and non-enzymatic antioxidants in the seedlings. At a concentration of 6 µM, however, Se(VI) was toxic. The results suggested that at appropriate concentrations, the exogenous application of Se(VI) enabled the B. juncea seedlings to counteract the effects of Cr(VI), thereby increasing the resistance of plants.     

Identification and <Emphasis Type="Italic">In Silico</Emphasis> Analysis of Major Redox Modulated Proteins from <Emphasis Type="Italic">Brassica juncea</Emphasis> Seedlings Using 2D Redox SDS PAGE (2-Dimensional Diagonal Redox Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)

The thiol–disulphide exchange regulates the activity of proteins by redox modulation. Many studies to analyze reactive oxygen species (ROS), particularly, hydrogen peroxide (H₂O₂) induced changes in the gene expression have been reported, but efforts to detect H₂O₂ modified proteins are comparatively few. Two-dimensional diagonal redox sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was used to detect polypeptides which undergo thiol–disulphide exchange in Brassica juncea seedlings following H₂O₂ (10 mM) treatment for 30 min. Eleven redox responsive polypeptides were identified which included cruciferin, NLI [Nuclear LIM (Lin11, Isl-1 & Mec-3 domains)] interacting protein phosphatase, RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) large subunit, and myrosinase. Redox modulation of RuBisCO large subunit was further confirmed by western blotting. However, the small subunit of RuBisCO was not affected by these redox changes. All redox modulated targets except NLI interacting protein (although it contains two cysteines) showed oxidation sensitive cysteines by in silico analysis. Interestingly, interactome of cruciferin and myrosinase indicated that they may have additional function(s) beside their well-known roles in the seedling development and abiotic stress respectively. Cruciferin showed interactions with stress associated proteins like defensing-like protein 192 and 2-cys peroxiredoxin. Similarly, myrosinase showed interactions with nitrilase and cytochrome p450 which are involved in nitrogen metabolism and/or hormone biosynthesis. This simple procedure can be used to detect major stress mediated redox changes in other plants.     

Morphological,phytochemical and genetic diversity of threatened Polygonatum verticillatum (L.) All. populations of different altitudes and habitat types in Himalayan region

Polygonatum verticillatum (L.) All. is an important medicinal herb that belongs to the family Asparagaceae. The rhizome of the species is used in Chyavanprash preparation and several other ayurvedic formulations. Numerous active constituents like saponins, alkaloids, phytohormones, flavonoids, antioxidants, lysine, serine, aspartic acid, diosgenin, β-sitosterol, etc. have been reported from this species. In this study, morphological, phytochemical, antioxidant and genetic variations of 11 distant populations of P. verticillatum were measured. Considerably (P < 0.05) higher variations were recorded among different populations of P. verticillatum using morphological, phytochemical and genetic diversity parameters. AGFW (above ground fresh weights); flavonols, FRAP (Ferric ion reducing antioxidant power) and NO (Nitric Oxide scavenging activity) were recorded maximum in Kafni population. Similarly, a significantly higher above and below ground dry weight was recorded in Mayawati and Surmoli populations respectively. Maximum phenolic content, tannins, and DPPH (2,2-diphenyl-1-picrylhydrazyl) activity were recorded in Milam population. A total of 165 individuals from 11 populations were assessed for genetic diversity using inter-simple sequence repeats (ISSR) marker. High genetic diversity (He = 0.35) was recorded in Himkhola and Surmoli populations while it was observed minimum (0.28) in the Mayawati population. Altitude showed a significant positive correlation with tannins (r = 0.674; P < 005) and DPPH (r = 0.820; P < 0.01). Phenol content exhibited a considerably positive relationship with He (r = 0.606; P < 0.05) and BGFW (r = 0.620; P < 0.05), flavonol displayed a positive correlation with Pp% (r = 0.606; P < 0.05). The population structure of P. verticillatum, exhibited that the optimal value of the K was 3 for its populations as determined by the ΔK statistic structure. Among populations, the amount of gene flow is higher (Nm = 1.717) among all sites. Hence, it can be concluded that P. verticillatum populations possess considerable variability in the collected populations. Likewise, the populations from Kafni, Satbunga and Himkhola with higher morphological, phytochemicals and genetic variability were prioritized and therefore recommended for cultivation and mass multiplication to meet the industrial demand for target species.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01044-9.