Temporal Variations in Plankton Communities and Environmental Factors in the Shipra, a Central Himalayan Tributary of the Kosi River in Uttarakhand, India.

The essential role of smaller streams and tributaries indigenous to the area is crucial in revitalizing and restoring the main river system. The present study unraveled the relationship between plankton communities and environmental variables in the Shipra River, a Central Himalayan tributary of the Kosi River in Uttarakhand, India. Plankton distribution, abundance, and presence were investigated at four locations: upstream (S1 and S2) and downstream (S3 and S4). Forty-eight genera of phytoplankton and zooplankton have been identified belonging to ten classes (Bacillariophyceae, Chlorophyceae, Zygnematophyceae, Cyanophyceae, Euglenopyceae, Protozoa, Rotifers, Copepoda, Cladocera, and Insecta) based on the findings. Winter was dominated by phytoplankton (Navicula spp., Nitzchia spp., Diatom spp.); summer and monsoon by zooplankton (Daphnia spp., Cyclops spp., Keratella spp., Brachionus spp.). Plankton communities of the tributary were assessed using diversity indices (Shannon-Weiner's index (H), Simpson's diversity index (1-D), and Evenness Index (E)). Seasonal variations in water temperature, specific conductivity, total dissolved solids, dissolved oxygen, nitrate, and ammonium ions were found to be statistically significant (p<0.05). Canonical Correspondence Analysis, K-dominance plots, cluster analysis, and NMDS analysis showed the dynamic nature of the plankton communities with seasonal physiochemical variations in the unexplored Himalayan tributary. The plankton communities exhibited significant temporal fluctuations with physicochemical factors.

Clinical signs, lethal dose and histopathological lesions in grass carp, Ctenopharyngodon idella experimentally infected with Edwardsiella tarda.

An experiment was conducted to study the lethal dose (LD50-96h) and histopathological changes occurring in several organs of grass carp challenged with different concentrations of Edwardsiella tarda. The healthy grass carps were challenged with the bacterial suspension of 106,107, 108, 109 and 1010 CFU ml-1. The study demonstrated that the lethal dose (LD50-96h) of E. tarda for grass carp is 1.3 × 109 CFU ml-1. The infected fish showed abnormal swimming behavior, slower movements, skin necrosis, hemorrhages, and open lesion on the fontanelle of the frontal bone of the skull during the initial phase of infection. About 60% of the fish which received the bacterial suspension of 1010 CFU ml-1 died within 24 h of infection. The histopathological examination of the infected tissue section demonstrated the severe damages in the internal organs. In gills, oedema, secondary lamellae fusion, and hyperplasia of basal epithelial lining between secondary lamellae were reported. The microscopic observation showed the disruption of submucosa to the mucosa, which finally led to degenerative changes in the intestine, necrosis of hepatocytes and infiltration of red blood cells in the liver. The tubular disintegration in kidney and loss of capsular boundary of red pulp in spleen were also reported. In conclusion, the result indicates that the infection caused by E. tarda can cause severe damages and alterations in grass carp tissues and potential mass mortality. Moreover, The bacteria isolated from the mobribund fish was characterized by biochemical tests and expression of five critical virulence genes like citC, fimA, gadB, mukF and gyrB were detected from the microorganism. The study aims to provide a research foundation for further studies on the susceptibility and pathological changes of grass carp induced by E. tarda infection.

Site-directed mutagenesis in the P-domain of calreticulin transacylase identifies Lys-207 as the active site residue.

In silico-docking studies from previous work have suggested that Lys-206 and lys-207 of calreticulin (CR) play a pivotal key role in its well-established transacetylation activity. To experimentally validate this prediction, we introduced three mutations at lysine residues of P-domain of CR: K → A, P mut-1 (K -206, -209), P mut-2 (K -206, -207) and P mut-3 (K -207, -209) and analyzed their immunoreactivity and acetylation potential. The clones of wild-type P-domain (P wt ) and three mutated P-domain (P mut-1, P mut-2 and P mut-3) were expressed in pTrcHis C vector and the recombinant P wt , P mut-1 , P mut-2 and P mut-3 proteins were purified by Ni-NTA affinity chromatography. Screening of the transacylase activity (TAase) by the Glutathione S Transferase (GST) assay revealed that the TAase activity was associated with the P wt and P mut-1 while P mut-2 and P mut-3 did not show any activity. The immune-reactivity to anti-lysine antibody was also retained only by the P mut-1 in which the Lys-207 was intact. Retention of the TAase activity and immunoreactivity of P mut-1 with mutations introduced at Lys-206, Lys-209, while its loss with a mutation at Lys-207 residue indicated that lysine-207 of P-domain constitutes the active site residue controlling TAase activity. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02659-1.

Microbial-assisted and genomic-assisted breeding: a two way approach for the improvement of nutritional quality traits in agricultural crops.

Both human and animals, for their nutritional requirements, mainly rely on the plant-based foods, which provide a wide range of nutrients. Minerals, proteins, vitamins are among the nutrients which are essential and need to be available in adequate amount in edible portion of the staple crops. Increasing nutritional content in staple crops either through agronomic biofortification or through conventional plant-breeding strategies continue to be a huge task for scientists around the globe. Although some success has been achieved in recent past, in most cases, we have fallen short of expected targets. To maximize the nutrient uptake and partitioning to different economic part of plants, scientists have employed and tailored several biofortification strategies. But in present agricultural and environmental concerns, these approaches are not much effective. Henceforth, we are highlighting the recent developments and promising aspects of microbial-assisted and genomic-assisted breeding as candidate biofortification approach, that have contributed significantly in increasing nutritional content in grains of different crops. The methods used to date to accomplish nutrient enrichment with recently emerging strategies that we believe could be the most promising and holistic approach for future biofortification program. Results are encouraging, but for future perspective, the existing knowledge about the strategies needs to be confined. Concerted scientific investment are required to widen up these biofortification strategies, so that it could play an important role in ensuring nutritional security of ever-growing population in growing agricultural and environmental constraints.

Role of calreticulin in biotic and abiotic stress signalling and tolerance mechanisms in plants.

Calreticulin (CRT) is calcium binding protein of endoplasmic reticulum (ER) which performs plethora of functions besides it's role as molecular chaperone. Among the three different isoforms of this protein, CRT3 is most closely related to primitive CRT gene of higher plants. Based on their distinct structural and functional organisation, the plant CRTs have been known to contain three different domains: N, P and the C domain. The domain organisation and various biochemical characterstics of plant and animal CRTs are common with the exception of some differences. In plant calreticulin, the important N-glycosylation site(s) are replaced by the glycan chain(s) and several consensus sequences for in vitro phosphorylation by protein kinase CK2 (casein kinase-2), are also present unlike the animal calreticulin. Biotic and abiotic stresses play a significant role in bringing down the crop production. The role of various phytohormones in defense against fungal pathogens is well documented. CRT3 has been reported to play important role in protecting the plants against fungal and bacterial pathogens and in maintaining plant innate immunity. There is remarkable crosstalk between CRT mediated signalling and biotic, abiotic stress, and phytohormone mediated signalling pathways The role of CRT mediated pathway in mitigating biotic and abiotic stress can be further explored in plants so as to strategically modify it for development of stress tolerant plants.

Corrigendum to The Competence of 7,8-Diacetoxy-4-Methylcoumarin and Other Polyphenolic Acetates in Mitigating the Oxidative Stress and their Role in Angiogenesis.

In the Original Research Article entitled "The Competence of 7, 8-Diacetoxy-4-methylcoumarin and other Polyphenolic Acetates in Mitigating the Oxidative Stress and their Role in Angiogenesis" Published in Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 2, on page no. 179, the order of author names was rearranged because second authorship is acceptable as they only acknowledge the first and the second authorship as per the new policies of Medical Council of India. The order of authors should be read as follows: Rini Joshi, Vishwajeet Rohil, Shvetambri Arora, Sushma Manral, Ajit Kumar, Sanjay Goel, Nivedita Priya, Prabhjoth Singh, Prija Ponnan, Suvro Chatterji, Bilikere S. Dwarakanath, Daman Saluja, Diwan S. Rawat, Ashok K. Prasad, Luciano Saso, Ekta Kohli, Anthony L. DePass, Marc E. Bracke, Virinder S. Parmar and Hanumantharao G. Raj.

The Competence of 7,8-Diacetoxy-4-Methylcoumarin and Other Polyphenolic Acetates in Mitigating the Oxidative Stress and their Role in Angiogenesis.

The potential role of polyphenolic acetate (PA) in causing diverse biological and pharmacological actions has been well studied in our laboratory. Our investigations, for the first time, established the role of calreticulin transacetylase (CRTAase) in catalyzing the acetylation of nitric oxide synthase (NOS) by Pas leading to robust activation of NOS. 7, 8- Diacetoxy-4-methylcoumarin (DAMC) and other acetoxycoumarins augmented the expression of thioredoxin (TRX) and vascular endothelial growth factor (VEGF) in human peripheral blood mononuclear cells (PBMCs). These findings substantiated our earlier observations that DAMC was a superb inducer of angiogenesis. The enhanced expression of thioredoxin reductase (TRXR) and diminished expression of thioredoxin interacting protein (TRXIP) leading to increased expression and activity of TRX in PBMCs due to the action of DAMC was revealed by real time RT-PCR analysis. The possible activation of TRX due to acetylation was confirmed by the fact that TRX activity of PBMCs was enhanced by various acetoxycoumarins in tune with their affinities to CRTAase as substrates. DAMC caused enhanced production of NO by way of acetylation of NOS as mentioned above and thereby acted as an inducer of VEGF. Real time RT-PCR and VEGF ELISA results also revealed the overexpression of TRX. DAMC and other PAs were found to reduce the oxidative stress in cells as proved by significant reduction of intracellular ROS levels. Thus, the crucial role of TRX in DAMC-induced angiogenesis with the involvement of VEGF was established.

The Competence Of 7,8-Diacetoxy-4-Methylcoumarinand Other Polyphenolic Acetates In Mitigating The Oxidative Stress And Their Role In Angiogenesis.

The potential role of polyphenolic acetate (PA) in causing diverse biological and pharmacological actions has been well studied in our laboratory. Our investigations, for the first time, established the role of calreticulin transacetylase (CRTAase) in catalyzing the acetylation of nitric oxide synthase (NOS) by Pas leading to robust activation of NOS. 7, 8-Diacetoxy-4-methylcoumarin (DAMC) and other acetoxycoumarins augmented the expression of thioredoxin (TRX) and vascular endothelial growth factor (VEGF) in human peripheral blood mononuclear cells (PBMCs). These findings substantiated our earlier observations that DAMC was a superb inducer of angiogenesis. The enhanced expression of thioredoxin reductase (TRXR) and diminished expression of thioredoxin interacting protein (TRXIP) leading to increased expression and activity of TRX in PBMCs due to the action of DAMC was revealed by real time RT-PCR analysis. The possible activation of TRX due to acetylation was confirmed by the fact that TRX activity of PBMCs was enhanced by variousacetoxycoumarins in tune with their affinities to CRTAase as substrates. DAMC caused enhanced production of NO by way of acetylation of NOS as mentioned above and thereby acted as an inducer of VEGF. Real time RT-PCR and VEGF ELISA results also revealed the overexpression of TRX. DAMC and other PAs were found to reduce the oxidative stress in cells as proved by significant reduction of intracellular ROS levels. Thus, the crucial role of TRX in DAMC-induced angiogenesis with the involvement of VEGF was established.

Comparison of protein acetyltransferase action of CRTAase with the prototypes of HAT.

Our laboratory is credited for the discovery of enzymatic acetylation of protein, a phenomenon unknown till we identified an enzyme termed acetoxy drug: protein transacetylase (TAase), catalyzing the transfer of acetyl group from polyphenolic acetates to receptor proteins (RP). Later, TAase was identified as calreticulin (CR), an endoplasmic reticulum luminal protein. CR was termed calreticulin transacetylase (CRTAase). Our persistent study revealed that CR like other families of histone acetyltransferases (HATs) such as p300, Rtt109, PCAF, and ESA1, undergoes autoacetylation. The autoacetylated CR was characterized as a stable intermediate in CRTAase catalyzed protein acetylation, and similar was the case with ESA1. The autoacetylation of CR like that of HATs was found to enhance protein-protein interaction. CR like HAT-1, CBP, and p300 mediated the acylation of RP utilizing acetyl CoA and propionyl CoA as the substrates. The similarities between CRTAase and HATs in mediating protein acylation are highlighted in this review.

Calreticulin transacetylase mediated upregulation of thioredoxin by 7,8-diacetoxy-4-methylcoumarin enhances the antioxidant potential and the expression of vascular endothelial growth factor in peripheral blood mononuclear cells.

Extensive research carried out in our group on polyphenolic acetates (PAs) substantiated the potential role of PAs in causing diverse biological and pharmacological actions. Our earlier investigations firmly established the calreticulin transacetylase (CRTAase) catalyzed activation of nitric oxide synthase (NOS) by PAs. In this report, we have studied the effect of 7,8-diacetoxy-4-methylcoumarin (DAMC, a model PA) and other acetoxy coumarins on the thioredoxin and VEGF expression in human peripheral blood mononuclear cells (PBMCs), with a view to substantiate our earlier observation that DAMC was a superb inducer of angiogenesis. Real time RT-PCR analysis revealed the enhanced expression of thioredoxin reductase (TRXR) and diminished expression of thioredoxin interacting protein (TRXIP) leading to the increased expression and activity of thioredoxin (TRX) in PBMCs due to the the action of DAMC. The fact that TRX activity of PBMCs was enhanced by various acetoxy coumarins in tune with their affinity to CRTAase as substrate, suggested the possible activation of TRX due to acetylation. The overexpression of thioredoxin was found to correlate with that of VEGF as proved by real time RT-PCR and VEGF -ELISA results, apart from the DAMC-caused enhanced production of NO acting as an inducer of VEGF. Moreover, the intracellular ROS levels were also found to be reduced drastically, by DAMC thus reducing the oxidative stress in cells. These observations strongly evidenced the crucial role of TRX in DAMC-induced tissue angiogenesis with the involvement of VEGF.

Calreticulin Transacetylase mediated activation of human platelet nitric oxide synthase by acetyl group donor compounds.

Polyphenols have attracted immense interest because of their diverse biological and pharmacological activities. Surprisingly, not much is documented about the biological activities of acetoxy derivatives of polyphenol called polyphenolic acetates (PA). In our previous reports, we have conclusively established the Calreticulin Transacetylase (CRTAase) catalyzed activation of neuronal nitric oxide synthase (nNOS) and tumor necrosis factor-α (TNF-α) induced nitric oxide synthase (iNOS) by PA. In the present work, specificity of CRTAase to various classes of PA was characterized in human platelet. The effect of PA, on platelet NOS and intracellular cyclic guanosine monophosphate (cGMP), and adenosine diphosphate (ADP)-induced platelet aggregation were studied in an elaborated manner. Platelet CRTAase exhibited differential specificities to polyphenolic acetates upon incubation with l-arginine leading to activation of NOS. The intraplatelet generation of NO was studied by flowcytometry using DCFH-DA. The differential specificities of CRTAase to PA were found to positively correlate with increased production of NO upon incubation of PRP with PA and l-arginine. Further, the inhibitory effect of l-NAME on PA induced NO formation in platelets substantiated the CRTAase catalyzed activation of NOS. The real-time RT-PCR profile of NOS isoforms confirmed the preponderance of eNOS over iNOS in human platelets on treatment with PA. Western blot analysis also reiterated the differential pattern of acetylation of eNOS by PA. PA were also found effective in increasing the intraplatelet cGMP levels and inhibiting ADP-induced platelet aggregation. It is worth mentioning that the effects of PA were found to be in tune with the specificities of platelet CRTAase to PA as the substrates.

Normalization of deranged signal transduction in lymphocytes of COPD patients by the novel calcium channel blocker H-DHPM.

Investigations on the role of intracellular Ca(2+) ion concentration in the mechanism of development of COPD in smokers and non-smokers were carried out. The intracellular Ca(2+) levels were found to be increased in human lymphocytes in patients with COPD as compared to non-smokers and smokers without COPD. The investigations reveal an association in altered intracellular Ca(2+) regulation in lymphocytes and severity of COPD, by means of significant activation of Protein kinase C and inducible nitric oxide synthase (iNOS). The effect of a novel calcium channel blocker ethyl 4-(4'-heptanoyloxyphenyl)-6-methyl-3,4-dihydropyrimidin-2-one-5-carboxylate (H-DHPM) as a potential candidate for the treatment of COPD was also investigated. H-DHPM treated cells showed a decrease in intracellular Ca(2+) level as compared to the control cells. Molecular studies were carried out to evaluate the expression profile of NOS isoforms in human lymphocytes and it was shown that H-DHPM decreases the increased iNOS in COPD along with reestablishing the normal levels of endothelial nitric oxide synthase (eNOS). The results of H-DHPM were comparable with those of Amlodipine, a known calcium channel blocker. Calcium channel blocker H-DHPM proves to be a potential candidate for the treatment of COPD and further clinical studies are required to prove its role in the treatment of pulmonary hypertension (PH).

Calreticulin transacylase: genesis, mechanism of action and biological applications.

Our earlier investigations have identified a unique enzyme in the endoplasmic reticulum (ER) termed Acetoxy Drug: Protein Transacetylase (TAase) catalyzing the transfer of acetyl group from polyphenolic acetates (PA) to certain receptor proteins (RP). An elegant assay procedure for TAase was developed based on the inhibition of glutathione S-transferase (GST) due to acetylation by a model acetoxycoumarin, 7, 8-Diacetoxy-4-methylcoumarin (DAMC). TAase purified from various mammalian tissue microsomes to homogeneity exhibited a molecular weight (M.wt) of 55kDa. Further, by N-terminal sequencing TAase was identified as Calreticulin (CR), a multifunctional Ca2+-binding protein in ER lumen. The identity of TAase with CR was evidenced by proteomics studies such as immunoreactivity with anti-CR antibody and mass spectrometry. This function of CR was termed Calreticulin transacetylase (CRTAase). CRTAase was also found to mediate the transfer of acetyl group from DAMC to RP such as NADPH Cytochrome c Reductase (CYPR) and Nitric Oxide Synthase (NOS). The autoacetylation of purified human placental CRTAase concomitant with the acetylation of RP by DAMC was observed. CRTAase activity was found to be inhibited by Ca2+. Our investigations on the individual domains (N, P and C) of CR from a nematode Haemonchus contortus revealed that the P-domain alone was found to possess CRTAase activity. Based on the observation that the autoacetylated CR was a stable intermediate in the CRTAase catalyzed protein acetylation by PA, a putative mechanism was proposed. Further, CRTAase was also found capable of transferring propionyl group from a propoxy derivative of polyphenol, 7,8-Dipropoxy-4-methylcoumarin (DPMC) to RP and concomitant autopropionylation of CR was encountered. Hence, CRTAase was assigned the general term Calreticulin Transacylase. Also, CRTAase was found to act upon the biological acyl group donors, acetyl CoA and propionyl CoA. CRTAase mediated modulation of specific functional proteins by way of acylation was exploited to elicit the biological applications of PA.