Evolution of novel strains of Ensifer nodulating the invasive legume Leucaena leucocephala (Lam.) de Wit in different climatic regions of India through lateral gene transfer.

More than 200 root-nodule bacterial strains were isolated from Leucaena leucocephala growing at 42 sampling sites across 12 states and three union territories of India. Genetic diversity was observed among 114 strains from various climatic zones; based on recA, these were identified as strains of Ensifer, Mesorhizobium, Rhizobium, and Bradyrhizobium. In multilocus sequence analysis (MLSA) strains clustered into several novel clades and lineages. Ensifer were predominant nodulating genotype isolated from majority of alkaline soils, while Mesorhizobium and Rhizobium strains were isolated from a limited sampling in North-Eastern states with acidic soils. Positive nodulation assays of selected Ensifer representing different genetic combinations of housekeeping and sym genes suggested their broad host range within the closely related mimosoid genera Vachellia, Senegalia, Mimosa, and Prosopis. Leucaena selected diverse strains of Ensifer and Mesorhizobium as symbionts depending on available soil pH, climatic, and other edaphic conditions in India. Lateral gene transfer seems to play a major role in genetic diversification of Ensifer exhibited in terms of Old World vs. Neotropical genetic make-up and mixed populations at several sites. Although Neotropical Ensifer strains were most symbiotically effective on Leucaena, the native Ensifer are promiscuous and particularly well-adapted to a wide range of sampling sites with varied climates and edaphic factors.

Integrated physiological and comparative proteomics analysis of contrasting genotypes of pearl millet reveals underlying salt-responsive mechanisms.

Salinity stress poses a significant risk to plant development and agricultural yield. Therefore, elucidation of stress-response mechanisms has become essential to identify salt-tolerance genes in plants. In the present study, two genotypes of pearl millet (Pennisetum glaucum L.) with contrasting tolerance for salinity exhibited differential morpho-physiological and proteomic responses under 150 mM NaCl. The genotype IC 325825 was shown to withstand the stress better than IP 17224. The salt-tolerance potential of IC 325825 was associated with its ability to maintain intracellular osmotic, ionic, and redox homeostasis and membrane integrity under stress. The IC 325825 genotype exhibited a higher abundance of C4 photosynthesis enzymes, efficient enzymatic and non-enzymatic antioxidant system, and lower Na+ /K+ ratio compared with IP 17224. Comparative proteomics analysis revealed greater metabolic perturbation in IP 17224 under salinity, in contrast to IC 325825 that harbored pro-active stress-responsive machinery, allowing its survival and better adaptability under salt stress. The differentially abundant proteins were in silico characterized for their functions, subcellular-localization, associated pathways, and protein-protein interaction. These proteins were mainly involved in photosynthesis/response to light stimulus, carbohydrate and energy metabolism, and stress responses. Proteomics data were validated through expression profiling of the selected genes, revealing a poor correlation between protein abundance and their relative transcript levels. This study has provided novel insights into salt adaptive mechanisms in P. glaucum, demonstrating the power of proteomics-based approaches. The critical proteins identified in the present study could be further explored as potential objects for engineering stress tolerance in salt-sensitive major crops.

Psychological Morbidity among People in Quarantine.

Objective This study aimed to evaluate psychological distress of persons in quarantine and compare the same with a group of persons, who are currently in lockdown. Methodology Forty-four persons in quarantine and 45 subjects currently in lockdown were evaluated on Depression Anxiety Stress Scale II. Results About three-fourth (77.3%) of the participants in the quarantine group and one-third (37.8%) in the comparator group had depression. About one fourth (22.7%) in the quarantine group and one-third (35.6%) in the lockdown group had anxiety. Conclusion The present study suggests that lockdown and being in quarantine are associated with significantly higher psychiatric morbidity, especially anxiety.

Is age just a number: Exploring fear, anxiety, and coping in individuals during COVID-19.

INTRODUCTION: The current pandemic coronavirus (COVID-19) is a novel disease with no standardized course of treatment and cure. This novelty, origin, and dispersion of COVID-19 have been reported to cause chaos, anxiety, and fear among the general population. AIM: The present study aimed to explore the correlation between different age groups, in relation to their general coping strategies, fear, and anxiety due to COVID-19. METHODOLOGY: The research study employed a total sample of 135 participants. The sample was divided into four age groups: 12-18, 19-25, 26-55, and 56-85. Participants were selected through convenience sampling. General coping strategies employed by these participants were assessed using a brief COPE scale, the COVID-19 fear scale was used to assess fear, and coronavirus anxiety scale was used to screen anxiety. RESULTS: Our results indicated that age has a negative correlation with adaptive coping and positive correlation with problem-focused coping, emotion-focused coping, and fear of COVID-19. The most used coping strategy among the age groups 12-18 and 19-25 age group was adaptive coping; for the 26-55 age group, it was problem-focused coping; and for the age group 56-85, emotion-focused coping was used the most. CONCLUSION: The findings of the current research could help to make the intervention procedure tailor-made for different age groups, thereby ensuring adherence and relatability to the precautionary directions.

The experiential impact of isolation and quarantine on patients during the initial phase of the COVID-19 pandemic in India.

BACKGROUND: Most countries around the world have been affected by the COVID-19 pandemic. Although there are quantitative studies on the effects of the COVID-19 pandemic on health-care professionals and other population groups, there are few studies that have evaluated the experiences of patients in the initial phases of the pandemic. AIM: This study aims to conduct a qualitative study assessing the experiences of the patients in isolation and quarantine in the initial stage of the COVID-19 pandemic. METHODOLOGY: The present study was a qualitative study through telephonic interviews with patients in isolation and quarantine due to COVID-19 from April 4 to 12, 2020. Patients in isolation had confirmed COVID-19 and were mandatorily admitted in specially designated COVID hospitals. Patients in quarantine were suspected to have COVID-19 due to symptoms or contact with confirmed patients with COVID-19. RESULTS: The experiences could be classified as having psychological impact, interpersonal impact, social impact, behavioral changes and impact on occupational aspects. The experience was predominantly unpleasant and characterized by anxiety, stigma, ostracization, guilt, and worry about the future. CONCLUSION: The experiences of the individuals in both the groups emphasize the importance of addressing psychological stressors. It could be concluded that individuals and their families would accordingly benefit from effective interventions to deal with the negative experiences they have been through due to the present pandemic.

Single-Step Purification and Characterization of A Recombinant Serine Proteinase Inhibitor from Transgenic Plants.

Expression of recombinant therapeutic proteins in transgenic plants has a tremendous impact on safe and economical production of biomolecules for biopharmaceutical industry. The major limitation in their production is downstream processing of recombinant protein to obtain higher yield and purity of the final product. In this study, a simple and rapid process has been developed for purification of therapeutic recombinant α1-proteinase inhibitor (rα1-PI) from transgenic tomato plants, which is an abundant serine protease inhibitor in human serum and chiefly inhibits the activity of neutrophil elastase in lungs. We have expressed rα1-PI with modified synthetic gene in transgenic tomato plants at a very high level (≃3.2 % of total soluble protein). The heterologous protein was extracted with (NH4)2SO4 precipitation, followed by chromatographic separation on different matrices. However, only immunoaffinity chromatography resulted into homogenous preparation of rα1-PI with 54 % recovery. The plant-purified rα1-PI showed molecular mass and structural conformation comparable to native serum α1-PI, as shown by mass spectrometry and optical spectroscopy. The results of elastase inhibition assay revealed biological activity of the purified rα1-PI protein. This work demonstrates a simple and efficient one-step purification of rα1-PI from transgenic plants, which is an essential prerequisite for further therapeutic development.

Rice SAPs are responsive to multiple biotic stresses and overexpression of OsSAP1, an A20/AN1 zinc-finger protein, enhances the basal resistance against pathogen infection in tobacco.

Eukaryotic A20/AN1 zinc-finger proteins (ZFPs) play an important role in the regulation of immune and stress response. After elucidation of the role of first such protein, OsSAP1, in abiotic stress tolerance, 18 rice stress associated protein (SAP) genes have been shown to be regulated by multiple abiotic stresses. In the present study, expression pattern of all the 18 OsSAP genes have been analysed in response to different biotic stress simulators, in order to get insights into their possible involvement in biotic stress tolerance. Our results showed the upregulation of OsSAP1 and OsSAP11 by all biotic stress simulator treatments. Furthermore, the functional role of OsSAP1 in plant defence responses has been explored through overexpression in transgenic plants. Constitutive expression of OsSAP1 in transgenic tobacco resulted into enhanced disease resistance against virulent bacterial pathogen, together with the upregulation of known defence-related genes. Present investigation suggests that rice SAPs are responsive to multiple biotic stresses and OsSAP1 plays a key role in basal resistance against pathogen infection. This strongly supports the involvement of rice SAPs in cross-talk between biotic and abiotic stress signalling pathways, which makes them ideal candidate to design strategies for protecting crop plants against multiple stresses.

Single amino acid substitutions in recombinant plant-derived human α1-proteinase inhibitor confer enhanced stability and functional efficacy.

BACKGROUND: Human α1-proteinase inhibitor (α1-PI) is the most abundant serine protease inhibitor in the blood and the heterologous expression of recombinant α1-PI has great potential for possible therapeutic applications. However, stability and functional efficacy of the recombinant protein expressed in alternate hosts are of major concern. METHODS: Five variants of plant-expressed recombinant α1-PI protein were developed by incorporating single amino acid substitutions at specific sites, namely F51C, F51L, A70G, M358V and M374I. Purified recombinant α1-PI variants were analyzed for their expression, biological activity, oxidation-resistance, conformational and thermal stability by DAC-ELISA, porcine pancreatic elastase (PPE) inhibition assays, transverse urea gradient (TUG) gel electrophoresis, fluorescence spectroscopy and far-UV CD spectroscopy. RESULTS: Urea-induced unfolding of recombinant α1-PI variants revealed that the F51C mutation shifted the mid-point of transition from 1.4M to 4.3M, thus increasing the conformational stability close to the human plasma form, followed by F51L, A70G and M374I variants. The variants also exhibited enhanced stability for heat denaturation, and the size-reducing substitution at Phe51 slowed down the deactivation rate ~5-fold at 54°C. The M358V mutation at the active site of the protein did not significantly affect the conformational or thermal stability of the recombinant α1-PI but provided enhanced resistance to oxidative inactivation. CONCLUSIONS: Our results suggest that single amino acid substitutions resulted in improved stability and oxidation-resistance of the plant-derived recombinant α1-PI protein, without inflicting the inhibitory activity of the protein. GENERAL SIGNIFICANCE: Our results demonstrate the significance of engineered modifications in plant-derived recombinant α1-PI protein molecule for further therapeutic development.

Differential subcellular targeting of recombinant human α1-proteinase inhibitor influences yield, biological activity and in planta stability of the protein in transgenic tomato plants.

The response of protein accumulation site on yield, biological activity and in planta stability of therapeutic recombinant human proteinase inhibitor (α1-PI) was analyzed via targeting to different subcellular locations, like endoplasmic reticulum (ER), apoplast, vacuole and cytosol in leaves of transgenic tomato plants. In situ localization of the recombinant α1-PI protein in transgenic plant cells was monitored by immunohistochemical staining. Maximum accumulation of recombinant α1-PI in T0 and T1 transgenic tomato plants was achieved from 1.5 to 3.2% of total soluble protein (TSP) by retention in ER lumen, followed by vacuole and apoplast, whereas cytosolic targeting resulted into degradation of the protein. The plant-derived recombinant α1-PI showed biological activity for elastase inhibition, as monitored by residual porcine pancreatic elastase (PPE) activity assay and band-shift assay. Recombinant α1-PI was purified from transgenic tomato plants with high yield, homogeneity and biological activity. Purified protein appeared as a single band of ∼48-50 kDa on SDS-PAGE with pI value ranging between 5.1 and 5.3. Results of mass spectrometry and optical spectroscopy of purified recombinant α1-PI revealed the structural integrity of the recombinant protein comparable to native serum α1-PI. Enzymatic deglycosylation and lectin-binding assays with the purified recombinant α1-PI showed compartment-specific N-glycosylation of the protein targeted to ER, apoplast and vacuole. Conformational studies based on urea-induced denaturation and circular dichroism (CD) spectroscopy revealed relatively lower stability of the recombinant α1-PI protein, compared to its serum counterpart. Pharmacokinetic evaluation of plant derived recombinant and human plasma-purified α1-PI in rat, by intravenous route, revealed significantly faster plasma clearance and lower area under curve (AUC) of recombinant protein. Our data suggested significance of protein sorting sequences and feasibility to use transgenic plants for the production of stable, glycosylated and biologically active recombinant α1-PI for further therapeutic applications.

Expression and purification of recombinant human alpha1-proteinase inhibitor and its single amino acid substituted variants in Escherichia coli for enhanced stability and biological activity.

Human alpha(1)-proteinase inhibitor (alpha(1)-PI) is the most abundant protease inhibitor found in the blood and expression of biologically active recombinant alpha(1)-PI has great potential in therapeutic applications. We report here the expression of a synthetic alpha(1)-PI gene and its variants in Escherichia coli. Modified alpha(1)-PI gene and its single amino acid variants were cloned in pMAL-c2X vector, which allowed expression of recombinant protein(s) as a fusion of maltose-binding protein (MBP) with factor Xa protease recognition site between the fusion partners. The synthetic gene(s) were expressed in different E. coli strains and maximum expression of recombinant alpha(1)-PI and variants up to 24% of total soluble protein (TSP) was achieved with engineered strain carrying extra copies of tRNAs for rare codons. Recombinant alpha(1)-PI protein(s) were purified by amylose affinity chromatography with high homogeneity and overall yield of about 7-9 mg l(-1) of bacterial culture (approximately 5.2 g wet cell mass). E. coli expressed recombinant alpha(1)-PI showed specific anti-elastase activity and appeared as a single band of approximately 45.0 kDa on SDS-PAGE. Primary structure of purified protein and integrity of N-terminus has been verified by mass spectrometric analysis. Recombinant alpha(1)-PI expressed in E. coli was fully intact having molecular mass similar to native unglycosylated protein purified from human plasma. Increased thermostability and specific activities of purified alpha(1)-PI variant proteins confirmed the stabilizing effect of incorporated mutations. Our results demonstrate efficient expression and purification of stable and biologically active alpha(1)-PI and its variants in E. coli for further therapeutic applications.

Effect of point mutations in translation initiation context on the expression of recombinant human alpha(1)-proteinase inhibitor in transgenic tomato plants.

The functional and biological significance of translation initiation context sequence in determining high-level expression of modified synthetic human alpha(1)-proteinase inhibitor (alpha(1)-PI) gene was documented in stable transgenic tomato plants. Context sequence of initiator ATG codon derived from statistical analysis of databases was identified as taaA(A/C)aATGGCt in highly expressed dicot plant genes. Removal of initiator ATG context sequence reduced the expression of recombinant alpha(1)-PI protein to fourfolds. The mutation of consensus base at +4 position to a pyrimidine either alone or with substitution at -3 position eliminated most of the alpha(1)-PI expression, while mutation at -3 alone resulted in about sevenfold reduction. The presence of steady-state levels of alpha(1)-PI transcript in transgenic plants indicated that the variation in expression is entirely due to the point mutations incorporated in translation initiation context. These results indicated the significance of conserved nucleotide sequence around initiator ATG codon in augmenting post-transcriptional events and high-level expression of heterologous genes in transgenic plants.

Study of matrix metalloproteinase-2 in inguinal hernia.

BACKGROUND: Abnormal collagen metabolism is thought to play an important role in the development of primary inguinal and ventral hernia. The detection of an impaired collagen balance both in the tissue as well as in cultured fibroblasts underlines the suspicion that the development of hernia is likely to be implemented primarily by a disturbance of the fibroblast function and their collagen genes. Based on these results we assume that the altered collagen synthesis in hernia patients can be regarded as a genetically linked deregulation serving as a basic initiating or promoting factor for the development of primary inguinal hernias. With the hypothesis that hernia is a local manifestation of a systemic disease manifested by increased expression of matrix metallo-proteinase-2 (MMP-2), a study was planned with following aims: 1) to establish a causal association between inguinal hernia and MMP-2; 2) to test the hypothesis that hernia is a local manifestation of a systemic disorder rather than a mere local mechanical defect. METHODS: A case control study was conducted on 30 subjects of each direct and indirect inguinal hernia and 30 controls. DAC-ELISA test was used for analysis of serum (preoperative) and tissue samples (fascia transversalis) in patients as well as controls. RESULTS: Statistically, serum levels of MMP-2 were significantly increased in all the hernia patients as compared to controls. This increment was maximum in patients of direct hernia. MMP-2 was not detectable in tissue samples. CONCLUSIONS: Hernia is a local manifestation of a systemic disease rather than a mere local mechanical defect. KEYWORDS: MMP-2; Matrix Metalloproteinase-2; Inguinal hernia; DAC-ELISA; Collagen metabolism; PBST-Phosphate Buffer Saline Tween-20.