Identification, characterization of Apyrase (APY) gene family in rice (Oryza sativa) and analysis of the expression pattern under various stress conditions.

Apyrase (APY) is a nucleoside triphosphate (NTP) diphosphohydrolase (NTPDase) which is a member of the superfamily of guanosine diphosphatase 1 (GDA1)-cluster of differentiation 39 (CD39) nucleoside phosphatase. Under various circumstances like stress, cell growth, the extracellular adenosine triphosphate (eATP) level increases, causing a detrimental influence on cells such as cell growth retardation, ROS production, NO burst, and apoptosis. Apyrase hydrolyses eATP accumulated in the extracellular membrane during stress, wounds, into adenosine diphosphate (ADP) and adenosine monophosphate (AMP) and regulates the stress-responsive pathway in plants. This study was designed for the identification, characterization, and for analysis of APY gene expression in Oryza sativa. This investigation discovered nine APYs in rice, including both endo- and ecto-apyrase. According to duplication event analysis, in the evolution of OsAPYs, a significant role is performed by segmental duplication. Their role in stress control, hormonal responsiveness, and the development of cells is supported by the corresponding cis-elements present in their promoter regions. According to expression profiling by RNA-seq data, the genes were expressed in various tissues. Upon exposure to a variety of biotic as well as abiotic stimuli, including anoxia, drought, submergence, alkali, heat, dehydration, salt, and cold, they showed a differential expression pattern. The expression analysis from the RT-qPCR data also showed expression under various abiotic stress conditions, comprising cold, salinity, cadmium, drought, submergence, and especially heat stress. This finding will pave the way for future in-vivo analysis, unveil the molecular mechanisms of APY genes in stress response, and contribute to the development of stress-tolerant rice varieties.

Identification and characterization of aldehyde dehydrogenase (ALDH) gene superfamily in garlic and expression profiling in response to drought, salinity, and ABA.

In response to biotic and abiotic stressors, aldehydes are detoxified and converted to carboxylic acids by aldehyde dehydrogenases (ALDHs), which are enzymes that use NAD+/NADP+ as cofactors. Garlic (Allium sativum L.) has not yet undergone a systematic examination of the ALDH superfamily, despite the genome sequence having been made public. In this investigation, we identified, characterized, and profiled the expression of the garlic ALDH gene family over the entire genome. The ALDH Gene Nomenclature Committee (AGNC) classification was used to classify and name the 34 ALDH genes that were discovered. Except for chromosome 8, all AsALDH genes were dispersed across the chromosomes. AsALDH genes have various localizations, according to predictions about subcellular localization. The AsALDH proteins are more varied and closely related to rice than to Arabidopsis, according to a study of conserved motifs and phylogenetic relationships. The presence of stress modulation pathways is indicated by the abundance of stress-related cis-elements in the AsALDH genes' promoter regions. Analysis of the RNA-seq data showed that AsALDHs expressed differently in various tissues and at various developmental stages. Nine AsALDHs were chosen for study using RT-qPCR, and the results revealed that the majority of the genes were upregulated in response to ABA and downregulated in response to salinity and drought. The results of this study improved our knowledge of the traits, evolutionary background, and biological functions of AsALDHs genes in growth and development.

Genome wide analysis of the heavy-metal-associated (HMA) gene family in tomato and expression profiles under different stresses.

The heavy-metal-associated (HMA) family plays a major role in the transportation of metals. Despite having the genome sequence of the tomato (Solanum lycopersicum), the HMA gene family has not been studied yet. In this study, we identified 48 HMA genes and categorized them into Cu/Ag P1B-ATPase and Zn/Co/Cd/Pb P1BATPase sub-families according to their phylogenic relationship with Arabidopsis and rice. The SlHMA genes were distributed throughout the 12 chromosomes. Analysis of gene structure, chromosomal position, and synteny, revealed that segmental duplications bestowed their evolution. The high numbers of stress-related cis-elements were found to be present in the putative promoter regions indicate the involvement of SlHMAs in stress modulation pathways. RNA-seq data revealed that SlHMAs had divergent expression in different tissues and developmental stages, where members of Cu/Ag P1B-ATPase subfamily were strongly expressed in the roots. RT-qPCR analysis of nine selected SlHMAs showed that most of the genes were up-regulated in response to heavy metals and moderately regulated in response to different abiotic stresses such as salt, drought, and cold.

Ectopic expression of Vigna radiata's vacuolar Na+/H+ antiporter gene (VrNHX1) in indica rice (Oryza sativa L.).

It is essential to develop high salt-tolerant rice varieties in order to cultivate the salt-affected lands. In this study, Na+/H+ exchanger 1 (NHX1) gene isolated from Vigna radiata L. Wilczek was transferred in Bangladesh Rice Research Institute (BRRI) developed two indica rice genotypes BRRI Dhan28 and BRRI Dhan29 using in-planta approach for improvement of salinity tolerance. Embryonic axes of matured dehusked rice seeds were injured and co-cultivated with Agrobacterium strain harboring VrNHX1 gene and finally regenerated. GUS histochemical assay and PCR amplification of GUS-a and VrNHX1 were performed to confirm the transformation. Expression confirmation was done by semi-quantitative RT-PCR. Under salinity stress, transgenic lines showed higher chlorophyll, relative water content and decreased electrolyte leakage, proline content, lipid peroxidation level, and catalase enzyme activity which represent the better physiology than control plants. Moreover, under salinity stress (150 mM), transgenic lines exhibited superior growth and salt tolerant than non-transgenic plants.

Computational and structural based approach to identify malignant nonsynonymous single nucleotide polymorphisms associated with CDK4 gene.

Cycline-dependent kinase 4 (CDK4), an enzyme of the cycline dependent or Ser/Thr protein kinase family, plays a role in cell cycle progression (G1 phase) by phosphorylating a tumor suppressor protein called pRB. Alteration of this enzyme due to missense mutation/ nonsynonymous single nucleotide polymorphisms (nsSNPs) are responsible for various types of cancer progression, e.g. melanoma, lung cancer, and breast cancer. Hence, this study is designed to identify the malignant missense mutation of CDK4 from the single nucleotide polymorphism database (dbSNP) by incorporating computational algorithms. Out of 239 nsSNPs; G15S, D140Y and D140H were predicted to be highly malignant variants which may have a devastating impact on protein structure or function. We also found defective binding motif of these three mutants with the CDK4 inhibitor ribociclib and ATP. However, by incorporating molecular dynamic simulation, our study concludes that the superiority of G15S than the other two mutants (D140Y and D140H) in destabilizing proteins nature.

Characterization of transcription factor MYB59 and expression profiling in response to low K+ and NO3- in indica rice (Oryza sativa L.).

BACKGROUND: Nitrogen and potassium are crucial supplements for plant development and growth. Plants can detect potassium and nitrate ions in soils and in like way, they modify root-to-shoot transport of these ions to adjust the conveyance among roots and shoots. Transcription factor MYB59 plays essential roles in numerous physiological processes inclusive of hormone response, abiotic stress tolerance, plant development, and metabolic regulation. In this study, we retrieved 56 MYB59 proteins from different plant species. Multiple sequence alignment, phylogenetic tree, conserved motif, chromosomal localization, and cis-regulatory elements of the retrieved sequences were analyzed. Gene structure, protein 3D structure, and DNA binding of OsMYB59 indica were also predicted. Finally, we characterized OsMYB59 and its function under low K+/NO3- conditions in Oryza sativa subsp. indica. RESULTS: Data analysis showed that MYB59s from various groups separated in terms of conserved functional domains and gene structure, where members of genus Oryza clustered together. Plants showed reduced height and yellowish appearance when grown on K+ and NO3- deficient medium. Quantitative real-time PCR uncovered that the OsMYB59 reacted to abiotic stresses where its expression was increased in BRRI dhan56 but decreased in other varieties on K+ deficient medium. In addition, OsMYB59 transcript level increased on NO3- deficient medium. CONCLUSIONS: Our results can help to explain the biological functions of indica rice MYB59 protein and gave a theoretical premise to additionally describe its biological roles in response to abiotic stresses particularly drought.

Genome-wide identification, evolution, and transcript profiling of Aldehyde dehydrogenase superfamily in potato during development stages and stress conditions.

The Aldehyde dehydrogenase (ALDH) superfamily comprises a group of enzymes involved in the scavenging of toxic aldehyde molecules by converting them into their corresponding non-toxic carboxylic acids. A genome-wide study in potato identified a total of 22 ALDH genes grouped into ten families that are presented unevenly throughout all the 12 chromosomes. Based on the evolutionary analysis of ALDH proteins from different plant species, ALDH2 and ALDH3 were found to be the most abundant families in the plant, while ALDH18 was found to be the most distantly related one. Gene expression analysis revealed that the expression of StALDH genes is highly tissue-specific and divergent in various abiotic, biotic, and hormonal treatments. Structural modelling and functional analysis of selected StALDH members revealed conservancy in their secondary structures and cofactor binding sites. Taken together, our findings provide comprehensive information on the ALDH gene family in potato that will help in developing a framework for further functional studies.

Antibacterial activity of graphene oxide nanosheet against multidrug resistant superbugs isolated from infected patients.

Graphene oxide (GO) is a derivative of graphene nanosheet which is the most promising material of the decade in biomedical research. In particular, it has been known as an antimicrobial nanomaterial with good biocompatibility. In this study, we have synthesized and characterize GO and checked its antimicrobial property against different Gram-negative and Gram-positive multidrug drug resistant (MDR) hospital superbugs grown in solid agar-based nutrient plates with and without human serum through the utilization of agar well diffusion method, live/dead fluorescent staining and genotoxicity analysis. No significant changes in antibacterial activity were found in these two different conditions. We also compare the bactericidal capability of GO with some commonly administered antibiotics and in all cases the degree of inhibition is found to be higher. The data presented here are novel and show that GO is an effective bactericidal agent against different superbugs and can be used as a future antibacterial agent.

Genetic variability analysis of partially salt tolerant local and inbred rice (Oryza sativa L.) through molecular markers.

Random Amplified Polymorphic DNA (RAPD) analysis was performed to assess the genetic variability in sixteen selected germplasms of rice, Oryza sativa L. using eight decamer RAPD primers. The data obtained from this investigation reveals a high level of polymorphism between cultivars. The primers produced a total of 255 bands of which all 255 bands were polymorphic indicating 100% polymorphism. The size of the amplified bands ranged from 220 bp to 2290 bp. The number of polymorphic fragments ranged from 24 to 49 with an average of 32 polymorphic fragments for each primer. The primer OPX04 produced the maximum number (49) of polymorphic bands while the OPB04 and OPB17 produced the minimum number (24) of polymorphic bands. The polymorphic information content (PIC) values ranged from 0.6616 to 0.8845 with an average of 0.832. The highest PIC value (0.8845) was obtained for primer OPL03. The RAPD data was analyzed to determine the pair-wise genetic similarity coefficients which ranged from 0.00 to 0.83. The BRRIdhan 23 and the BRRIdhan 41 varieties were the closest genotypes with the highest similarity index of 83%. This was followed by 77% similarity between a pair of cultivars Kalamona and Horkuch. On the other hand, 100% dissimilarity was seen between BRRIdhan 53, BRRIdhan 50, BRRIdhan 10, BRRIdhan 70, BRRIdhan 54, BRRIdhan 40, BRRIdhan 23, BRRIdhan 47, BRRIdhan 41 and Dadsail respectively and between BRRIdhan 53 and Horkuch; indicating a high level of variability between paired genotypes. Cluster analysis was performed using Unweighted Paired Group of Arithmetic Means (UPGMA). The UPGMA dendrogram resolved the selected rice cultivars into four clusters.

Development of partial abiotic stress tolerant Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck through Agrobacterium-mediated transformation method.

BACKGROUND: Recent studies indicate that farmers are facing several challenges due to biotic and abiotic stresses like diseases, drought, cold, and soil salinity which are causing declined Citrus production. Thus, it is essential to improve these varieties which would be resistant against biotic and abiotic stresses as well as high yielding. The transformation of abiotic stress tolerant genes in Citrus species is essential for using areas affected by abiotic stresses. This study was aimed to improve resistance of Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck to abiotic stresses by transferring PsCBL and PsCIPK genes through Agrobacterium-mediated transformation. RESULTS: Abiotic stress tolerant PsCBL and PsCIPK genes isolated from Pisum sativum were transformed into two Citrus species, Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck, through Agrobacterium-mediated transformation method. Mature seed-derived calli of two Species were infected with Agrobacterium tumefaciens LBA4404 harboring PsCBL and PsCIPK genes. The infected calli were co-cultured in dark condition and later on washed with antibiotic solution and transferred to selection medium. Preliminary resistant calli were recovered and regenerated to plantlets. Maximum regeneration rate was 61.11 ± 1.35% and 55.55 ± 1.03%, respectively. The genetic transformation was confirmed by performing ß glucuronidase (GUS) assays and subsequent PCR amplification of the GUS gene. The transformation rates of the two cultivated species were higher than previous reports. Maximum transformation frequencies were found when bacterial OD600 was 0.5 and concentration of acetosyringone was 150 µM. In-vitro evaluation of drought and salt tolerance of transgenic plantlets were done, and transgenic plantlets showed better performance than the control plants. CONCLUSIONS: The present study demonstrates that transformation of Citrus plants with PsCBL and PsCIPK genes result in improved abiotic stress tolerance.

Structural analysis and molecular docking of potential ligands with chorismate synthase of Listeria monocytogenes: a novel antibacterial drug target.

Listeriosis, in particular that caused by Listeria monocytogenes, is a major foodborne pathogen, and its control is becoming difficult because of widespread emergence of drug resistance strains. Chorismate synthase (CS), an essential enzyme of shikimate pathway present only in bacteria, fungi, plant and some apicomplexan parasites, is a validated potential antimicrobial drug target. Antimicrobial development through the elucidation of essential structural features of the CS of L. monocytogenes (LmCS), identification and prioritization of potential lead compounds targeted against LmCS were done. Structure-based virtual screening and docking studies were performed using Autodock tools to retrieve potential candidates with high affinity binding against LmCS model from several ligand repositories. The potency of binding was also checked with other structurally similar CS from Streptococcus pneumoniae (SpCS) and Mycobacterium tuberculosis (MtCS). The sequence and structural studies revealed LmCS was similar to be other CS structures (1Q1L, 1QXO, 1R52, 1R53, 1SQ1, 1UMO, 1UMF, 1ZTB, 2011, 2012, 4ECD and 2G85) with each monomer presenting ß-α-ß sandwich topology with a central helical core. Molecular docking studies and ADME/Tox results revealed that ZINC03803450 and ZINC20149031 were most potent molecules binding into the active site of LmCS. Other two ligands ZINC13387711-and ZINC16052528 showed a strong binding affinity score against all three structures (LmCS, SpCS and MtCS) and bind to LmCS with the predicted inhibition constant (K(i)) values of 22.94 nM and 35.84 nM, respectively. A reported benzofuran-3[2H]-one analog CHEMBL135212 with good ADME/Tox properties and experimental IC50 (nM) value of 7000 nM with SpCS could also be considered as a potential inhibitor of LmCS, as compared to previously reported 41 benzofuran-3[2H]-one analogs against SpCS. This information will assist in discovering those compounds that may act as potent CS inhibitors. Further experimental studies and evaluation of structure-activity relationship could help in the development of potential inhibitors against listeriosis, as well as antibacterial chemotherapy.

Antagonism of endothelin action normalizes altered levels of VEGF and its signaling in the brain of stroke-prone spontaneously hypertensive rat.

Stroke-prone spontaneously hypertensive rats (SHRSP) often suffer from spontaneous stroke, in part, due to abnormalities in the cerebrovasculature. Here, we investigate the profile of key angiogenic factors and their basic signaling molecules in the brain of SHRSP during the age-dependent stages of hypertension. The profile of VEGF and its receptor, Flk-1, was dependent on age and stage of hypertension (i.e., down regulated at pre-hypertensive and malignant hypertensive stages, but up regulated at typical hypertensive stage), while that of its downstream components, pAkt and eNOS, were down regulated in a time-dependent manner in the frontal cortex of SHRSP compared to age-matched genetic control, normotensive WKY rats. On the other hand, the expression of endothelin-1 and its type A receptor (endothelin ETA receptor) were up regulated, depending on age and stage of hypertension. In contrast, levels of endothelin type B receptor were down regulated. The regional cerebral blood flow decreased during the development of malignant hypertension. Thus, subsequent experiments were designed to investigate whether endothelin-1 receptor antagonism, using endothelin-A/-B dual receptor antagonist SB209670, could normalize the molecular profile of these factors in SHRSP brain. Interestingly, blockage of endothelin-1 receptor restored to normal, levels of cerebral endothelin-1, endothelin ETA receptor and endothelin ETB receptor; VEGF and Flk-1; endothelial nitric oxide synthase (eNOS) and pAkt, in SHRSP, compared to age-matched WKY. Endothelin receptor blocker might be important to prevent the progression in the defect in VEGF and its angiogenic signaling cascade in the pathogenesis of hypertension-induced vascular remodeling in frontal cortex of SHRSP rats.