Multi-location evaluation of field pea in Indian climates: eco-phenological dynamics, crop-environment relationships, and identification of mega-environments.

Characterization of crop-growing environments in relation to crop's genotypic performance is crucial to harness positive genotype-by-environment interactions (GEI) in systematic breeding programs. Given that, the study aimed to delineate the impact of diverse environments on crop phenology and yield traits of dwarf-statured field pea, pinpointing location(s) favoring higher yield and distinctiveness within breeding lines. We tested twelve field pea breeding lines across twenty locations in India, covering Central Zone (CZ), North Western Plain Zone (NWPZ), North Eastern Plain Zone (NEPZ), and Northern Hill Zone (NHZ). Across these locations, maximum and minimum temperatures during flowering (TMAXF, TMINF) and reproductive period (TMAXRP, TMINRP) ranged 18.9-28.3, 3.3-18.0, 15.0-30.8, and 7.9-22.1oC, respectively. Meanwhile, notable variations in phenological and agronomic traits (coefficient of variation) were observed: flowering (31%), days to maturity (21%), reproductive period (18%), grain yield (48%), and 100-seed weight (18%). Combined ANOVA demonstrated an oversized impact of environment (81%) on yield, while genotype and GEI effects were 2% and 14%, respectively. The variables TMINF, TMINRP, and cumulative growing degree-day showed positive correlations with yield, while extended vegetative and maturity durations negatively influenced yield (p < 0.05). Additionally, linear mixed-models and PCA results explained that instability in crop phenology had significant influence on field pea yield. Seed weight was markedly varied within the locations (9.9-20.8 g) and both higher and lower seed weights were associated with lower yields (Optimal = 17.1 g). HA-GGE biplot-based on environment focus-scaling demonstrated three mega-environments and specific locations viz. Kota (CZ), SK Nagar (CZ), Raipur (CZ), Sehore (CZ), and Pantnagar (NWPZ) as the ideal testing-environments with high efficiency in selecting new genotypes with wider adaptability. The study findings highlight distinct impact of environments on crop phenology and agronomic traits of field pea (dwarf-type), hold substantial value in designing efficient field pea (dwarf-type) breeding program at mega-environment scale.

Seedling-stage salinity tolerance in rice: Decoding the role of transcription factors.

Rice is an important staple food crop that feeds over half of the human population, particularly in developing countries. Increasing salinity is a major challenge for continuing rice production. Though rice is affected by salinity at all the developmental stages, it is most sensitive at the early seedling stage. The yield thus depends on how many seedlings can withstand saline water at the stage of transplantation, especially in coastal farms. The rapid development of "omics" approaches has assisted researchers in identifying biological molecules that are responsive to salt stress. Several salinity-responsive quantitative trait loci (QTL) contributing to salinity tolerance have been identified and validated, making it essential to narrow down the search for the key genes within QTLs. Owing to the impressive progress of molecular tools, it is now clear that the response of plants toward salinity is highly complex, involving multiple genes, with a specific role assigned to the repertoire of transcription factors (TF). Targeting the TFs for improving salinity tolerance can have an inbuilt advantage of influencing multiple downstream genes, which in turn can contribute toward tolerance to multiple stresses. This is the first comparative study for TF-driven salinity tolerance in contrasting rice cultivars at the seedling stage that shows how tolerant genotypes behave differently than sensitive ones in terms of stress tolerance. Understanding the complexity of salt-responsive TF networks at the seedling stage will be helpful to alleviate crop resilience and prevent crop damage at an early growth stage in rice.

Characterizing plant trait(s) for improved heat tolerance in field pea (Pisum sativum L.) under subtropical climate.

Field pea is highly sensitive to climatic vagaries, particularly high-temperature stress. The crop often experiences terminal heat stress in tropical climates indicating the need for the development of heat-tolerant cultivars. Characterization and identification of stress-adaptive plant traits are pre-requisites for breeding stress-tolerant/adaptive cultivar(s). In the study, a panel of 150 diverse field pea genotypes was tested under three different temperature environments (i.e., normal sowing time or non-heat stress environment (NSTE), 15 days after normal sowing time or heat stress environment-I (LSHTE-I), and 30 days after normal sowing time or heat stress environment-II (LSHTE-II)) to verify the effect of high-temperature environment, genotype, and genotype × environment interaction on different plant traits and to elucidate their significance in heat stress adaptation/tolerance. The delayed sowing had exposed field pea crops to high temperatures during flowering stage by + 3.5 °C and + 8.1 °C in the LSHTE-I and LSHTE-II, respectively. Likewise, the maximum ambient temperature during the grain-filling period was + 3.3 °C and + 6.1 °C higher in the LSHTE-I and LSHTE-II over the NSTE. The grain yield loss with heat stress was 25.8 ± 2.2% in LSHTE-I, and 59.3 ± 1.5% in LSHTE-II compared to the NSTE. Exposure of crops to a high-temperature environment during the flowering stage had a higher impact on grain yield than the heat stress at the grain filling period. Results suggested that the reduced sink capacity (pod set (pod plant-1), seed set (seed pod-1)) was the primary cause of yield loss under the heat stress environments, while, under the NSTE, yield potential was mostly attributed to the source capacity (plant biomass). The high-temperature stress resulted in forced maturity as revealed by shrinkage in crop period (5-11%) and reproductive period (15-36%), prominently in long-duration genotypes. The failure of pod set in the upper nodes and higher ovule abortion (7-16%) was noticed under the high-temperature environments, particularly in the LSHTE-II. Multivariate analysis results revealed seed set, pods plant-1, last pod bearing node, and plant biomass as a critical yield determinant under the heat stress. The GGE biplot suggested that the genotypes G-112, G-114, and G-33 had higher potential to sustain yield coupled with higher stability across the environments and, thus, could serve as a source for breeding heat-tolerant high yielding cultivars.

Takinib Inhibits Inflammation in Human Rheumatoid Arthritis Synovial Fibroblasts by Targeting the Janus Kinase-Signal Transducer and Activator of Transcription 3 (JAK/STAT3) Pathway.

TGF ß-activated kinase 1 (TAK1) is an important participant in inflammatory pathogenesis for diseases such as rheumatoid arthritis (RA) and gouty arthritis. The central position it occupies between the mitogen activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways makes it an attractive therapeutic target. As this field has developed in recent years, several novel inhibitors have been presented as having specific activity that reduces the TAK1 function either covalently as in the case of 5Z-7-oxozeanol (5Z7O) or reversibly (NG-25). However, the mechanism through which takinib elicits its anti-inflammatory activity remains elusive. While this inhibitor shows great promise, a thorough analysis of its inhibitor function and its potential off-target effects is necessary before addressing its clinical potential or its use in inflammatory conditions. An analysis through Western blot showed an unexpected increase in IL-1ß-induced TAK1 phosphorylation-a prerequisite for and indicator of its functional potential-by takinib while simultaneously demonstrating the inhibition of the JAK/STAT pathway in human rheumatoid arthritis synovial fibroblasts (RASFs) in vitro. In THP-1 monocyte-derived macrophages, takinib again led to the lipopolysaccharide-induced phosphorylation of TAK1 without a marked inhibition of the TAK1 downstream effectors, namely, of c-Jun N-terminal kinase (JNK), phospho-c-Jun, NF-κB phospho-p65 or phospho-IκBα. Taken together, these findings indicate that takinib inhibits inflammation in these cells by targeting multiple signaling pathways, most notably the JAK/STAT pathway in human RASFs.

EGCG, a Green Tea Catechin, as a Potential Therapeutic Agent for Symptomatic and Asymptomatic SARS-CoV-2 Infection.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged to be the greatest threat to humanity in the modern world and has claimed nearly 2.2 million lives worldwide. The United States alone accounts for more than one fourth of 100 million COVID-19 cases across the globe. Although vaccination against SARS-CoV-2 has begun, its efficacy in preventing a new or repeat COVID-19 infection in immunized individuals is yet to be determined. Calls for repurposing of existing, approved, drugs that target the inflammatory condition in COVID-19 are growing. Our initial gene ontology analysis predicts a similarity between SARS-CoV-2 induced inflammatory and immune dysregulation and the pathophysiology of rheumatoid arthritis. Interestingly, many of the drugs related to rheumatoid arthritis have been found to be lifesaving and contribute to lower COVID-19 morbidity. We also performed in silico investigation of binding of epigallocatechin gallate (EGCG), a well-known catechin, and other catechins on viral proteins and identified papain-like protease protein (PLPro) as a binding partner. Catechins bind to the S1 ubiquitin-binding site of PLPro, which might inhibit its protease function and abrogate SARS-CoV-2 inhibitory function on ubiquitin proteasome system and interferon stimulated gene system. In the realms of addressing inflammation and how to effectively target SARS-CoV-2 mediated respiratory distress syndrome, we review in this article the available knowledge on the strategic placement of EGCG in curbing inflammatory signals and how it may serve as a broad spectrum therapeutic in asymptomatic and symptomatic COVID-19 patients.

Critical role of IL-1α in IL-1ß-induced inflammatory responses: cooperation with NF-κBp65 in transcriptional regulation.

The IL-1 cytokines are considered among the first family of cytokines that orchestrate acute and chronic inflammatory diseases. Both IL-1ß and IL-1α are members of the IL-1 family; however, their distinct roles in the inflammatory processes remain poorly understood. We explored the role of IL-1α in IL-1ß-activated signaling pathways causing synovial inflammation in rheumatoid arthritis (RA). Using synovial fibroblasts isolated from RA joints, we found that IL-1ß significantly stimulated IL-1α expression, which was selectively inhibited by blocking the NF-κB pathway. Knockdown of IL-1α using small interfering RNA abolished IL-1ß-induced pro-IL-1α and pro-IL-1ß expression and suppressed inflammation. Native and chromatin immunoprecipitation studies showed that IL-1α cooperates in NF-κBp65 binding to the distal region of IL-1α promoter and to the proximal region of IL-1ß promoter upstream of the transcription start site to stabilize their gene transcription. Molecular dynamics simulation of IL-1α or IL-1ß binding to IL-1 receptor showed distinct interaction sites that corroborate with the ability of IL-1α to differentially activate phosphorylation of signaling proteins compared with IL-1ß. Our study highlights the importance of IL-1α in mediating IL-1ß-induced inflammation in addition to maintaining its expression and providing a rationale for targeting IL-1α to minimize the role of IL-1ß in inflammatory diseases like RA.-Singh, A. K., Fechtner, S., Chourasia, M., Sicalo, J., Ahmed, S. Critical role of IL-1α in IL-1ß-induced inflammatory responses: cooperation with NF-κBp65 in transcriptional regulation.

Bacteria associated with marine macroorganisms as potential source of quorum-sensing antagonists.

Samples were collected from different undisturbed areas along the coast of Gujarat like Okha, Diu, Veraval, and Somnath. A total of 68 marine isolates were obtained out of which 53 were associated with various marine macroorganisms like sponges, gastropods, and algae, whereas 15 were free living. Quorum-quenching ability of all the isolates was tested against Chromobacterium violaceum MK by co-culture technique as a way to simultaneously detect signal-degrading as well as nondegrading quorum-sensing inhibitors. Nineteen macroorganism-associated bacteria and eight free-living bacteria were found to possess quorum-sensing inhibitory activity against C. violaceum MK without affecting its growth. Isolate OA22 from grape alga and OA10 from purple sponge (Haliclona sp.) were found to possess the highest C6-HSL degradation activity and extracellular non-N-acyl-homoserine lactone degrading QSI activity, respectively. OA22 was also found to degrade 3-oxo-C12 homoserine lactone. Acid recovery of both the C6- and C12-HSL after degradation by OA22 indicated the presence of lactonase enzyme in the isolate. Cell-free supernatant of OA10 was extracted with ethyl acetate to obtain the quorum-quenching compound. Pigment inhibition in C. violaceum MK treated with OA10 extract was demonstrated in various ways and was indicative of QSI activity of the extract without degradation of the quorum-sensing signaling molecule. The isolates OA22 and OA10 were identified as Desemzia incerta and Bacillus sp., respectively, by 16S ribosomal DNA sequence analysis.

Ursolic acid facilitates apoptosis in rheumatoid arthritis synovial fibroblasts by inducing SP1-mediated Noxa expression and proteasomal degradation of Mcl-1.

Rheumatoid arthritis (RA) is characterized by hyperplastic pannus formation mediated by activated synovial fibroblasts (RASFs) that cause joint destruction. We have shown earlier that RASFs exhibit resistance to apoptosis, primarily as a result of enhanced expression of myeloid cell leukemia-1 (Mcl-1). In this study, we discovered that ursolic acid (UA), a plant-derived pentacyclic triterpenoid, selectively induces B-cell lymphoma 2 homology 3-only protein Noxa in human RASFs. We observed that UA-induced Noxa expression was followed by a consequent decrease in Mcl-1 expression in a dose-dependent manner. Subsequent evaluation of the signaling pathways showed that UA-induced Noxa is primarily mediated by the JNK pathway in human RASFs. Chromatin immunoprecipitation (IP) studies into the promoter region of Noxa indicated the role of transcription factor specificity protein 1 in JNK-mediated Noxa expression. Furthermore, the results from IP studies and proximity ligation assays indicated that UA-induced Noxa colocalizes and associates with Mcl-1 to prime it for proteasomal degradation through K48-linked ubiquitination by the selective recruitment of Mcl-1 ubiquitin ligase E3, a homologous to E6-associated protein C terminus domain-containing E3 ubiquitin ligase. These findings unveil a novel mechanism of inducing apoptosis in RASFs and a potential adjunct therapeutic strategy of regulating synovial hyperplasia in RA.-Kim, E. Y., Sudini, K., Singh, A. K., Haque, M., Leaman, D., Khuder, S., Ahmed, S. Ursolic acid facilitates apoptosis in rheumatoid arthritis synovial fibroblasts by inducing SP1-mediated Noxa expression and proteasomal degradation of Mcl-1.

Role of cannabinoid receptor 2 in mediating interleukin-1ß-induced inflammation in rheumatoid arthritis synovial fibroblasts.

OBJECTIVES: Recent studies showed that the expression of cannabinoid receptor 2 (CB2), not CB1, is upregulated at both the mRNA and protein levels in rheumatoid arthritis synovial fibroblasts (RASFs), however, little is known about its endogenous role in pro-inflammatory cytokine signalling in RASFs. Our aim was to investigate the role of CB2 receptor in mediating IL-1ß-induced inflammation in human RASFs. METHODS: Human RASFs were pretreated with CB2 selective agonist (JWH-133), followed by stimulation with interleukin-1ß (IL-1ß, 10 ng/mL). The role of CB2 in IL-1ß signalling was examined using small interfering RNA (siRNA) or an overexpression plasmid specific for CB2. RESULTS: Pretreatment with JWH-133 did not reduce IL-1ß-induced IL-6 and IL-8 production and amplified the cellular expression of cyclooxygenase-2 (COX-2) by >2-fold in human RASFs. Furthermore, the knockdown of CB2 using siRNA markedly inhibited IL-1ß-induced IL-6, IL-8, ENA-78, and RANTES production by more than 50% and completely abrogated COX-2 expression in human RASFs. MMP-2 and MMP-9 activity was also reduced by 50% with CB2 knockdown. On the contrary, overexpression of CB2 in human RASFs further increased IL-1ß-induced IL-6, IL-8, and RANTES by approximately 3-fold whereas ENA-78 expression increased by 1.5-fold. Immunoprecipitation analysis to study the protein-protein interactions revealed that JWH-133 coordinates CB2 association with TGFß-activated kinase 1 (TAK1), a key signalling molecule, to increase IL-1ß-induced nuclear translocation of transcription factors nuclear factor-κBp65 (NF-κBp65) and activation protein-1 (AP-1). CONCLUSIONS: Overall, our results indicate for the first time that CB2 mediates IL-1ß-induced signalling pathways in RASFs and may serve as a potential target to manage pain and inflammation in RA.

Manipulation of glyoxalase pathway confers tolerance to multiple stresses in rice.

Crop plants face a multitude of diverse abiotic and biotic stresses in the farmers' fields. Although there now exists a considerable knowledge of the underlying mechanisms of response to individual stresses, the crosstalk between response pathways to various abiotic and biotic stresses remains enigmatic. Here, we investigated if the cytotoxic metabolite methylglyoxal (MG), excess of which is generated as a common consequence of many abiotic and biotic stresses, may serve as a key molecule linking responses to diverse stresses. For this, we generated transgenic rice plants overexpressing the entire two-step glyoxalase pathway for MG detoxification. Through assessment of various morphological, physiological and agronomic parameters, we found that glyoxalase-overexpression imparts tolerance towards abiotic stresses like salinity, drought and heat and also provides resistance towards damage caused by the sheath blight fungus (Rhizoctonia solani) toxin phenylacetic acid. We show that the mechanism of observed tolerance of the glyoxalase-overexpressing plants towards these diverse abiotic and biotic stresses involves improved MG detoxification and reduced oxidative damage leading to better protection of chloroplast and mitochondrial ultrastructure and maintained photosynthetic efficiency under stress conditions. Together, our findings indicate that MG may serve as a key link between abiotic and biotic stress response in plants.

Fluorescent Organic Nanoparticles of 3-Styrylindoles: Synthesis and Characterization.

Nanoparticles of 3-styrylindoles viz. 3-styryl-1H-indole (1), 3-(4-nitrostyryl)-1H-indole (2), 3-(4-nitrostyryl)-5-methoxy-1H-indole (3) and 3-(4-nitrostyryl)-5-bromo-1H-indole (4) have been synthesized by re-precipitation method. The nanoparticles are formed in the shape of a sphere with mean diameter of about 20­30 nm. The size of nanoparticles is controlled mainly by the fraction of added water and final concentration of the nanoparticles dispersion. The nanoparticles of 1, 2 and 4 showed little change in their fluorescence spectra as compared to the fluorescence spectra of the corresponding isolated molecules in THF. However, nanoparticles of 3 show enhanced fluorescence emission. This is attributed to the presence of methoxy and nitro groups in 3, which may be effective in reducing face-to-face intermolecular interaction in the nanoparticles of 3, inducing formation of J-type morphology responsible for enhanced fluorescence emission. Over a period of time, the nanoparticles progressively tend to clump in to bigger sizes, and become less fluorescent.

Haloarchaea: worth exploring for their biotechnological potential.

Halophilic archaea are unique microorganisms adapted to survive under high salt conditions and biomolecules produced by them may possess unusual properties. Haloarchaeal metabolites are stable at high salt and temperature conditions that are useful for industrial applications. Proteins and enzymes of this group of archaea are functional under salt concentrations at which bacterial counterparts fail to be active. Such properties makes haloarchaeal enzymes suitable for salt-based applications and their use under dehydrating conditions. For example, bacteriorhodopsin or the purple membrane protein present in halophilic archaea has the most recognizable applications in photoelectric devices, artificial retinas, holograms etc. Haloarchaea are also useful for bioremediation of polluted hypersaline areas. Polyhydroxyalkanoates and exopolysccharides produced by these microorganisms are biodegradable and have the potential to replace commercial non-degradable plastics and polymers. Moreover, halophilic archaea have excellent potential to be used as drug delivery systems and for nanobiotechnology by virtue of their gas vesicles and S-layer glycoproteins. Despite of possible applications of halophilic archaea, laboratory-to-industrial transition of these potential candidates is yet to be established.

Unique miRNome during anthesis in drought-tolerant indica rice var. Nagina 22.

MAIN CONCLUSION: Drought-tolerant rice variety, Nagina 22 (N22), has a unique spikelet miRNome during anthesis stage drought as well as transition from heading to anthesis. Molecular characterization of genetic diversity of rice is essential to understand the evolution and molecular basis of various agronomically important traits such as drought tolerance. miRNAs play an important role in regulating plant development as well as stress response such as drought. In this study, we characterized the yet unexplored dynamics of the spikelet miRNA population during developmental transition from 'heading' to 'anthesis' as well as anthesis stage drought stress in a drought-tolerant indica rice variety, N22. A significant proportion of miRNA population (~20 %) in N22 spikelets is modulated during transition from heading to anthesis indicating a unique miRNome at anthesis, a developmental stage highly sensitive to stress (drought/heat). Based on the analysis of degradome data, majority of differentially regulated miRNAs appear to regulate transcription factors, some of which are implicated in regulation of development and fertilization. Similarly, drought during anthesis leads to a global change in miRNA expression pattern including those which regulate ROS homeostasis. It was possible to identify several miRNAs that were not reported to be drought responsive in earlier studies. Interestingly, a significant proportion of the drought-regulated miRNAs co-localize within QTLs related to drought tolerance and associated traits. Comparison of the expression profiles between N22 and Pusa Basmati 1 (drought sensitive) identified miRNAs with variety-specific expression patterns during phase transition (miR164, miR396, miR812, and miR1881) as well as drought stress (miR1881) indicating an evolution of a distinct and variety-specific regulatory mechanism. The promoters of these miRNAs contain LREs (light-responsive elements) and are induced by dark treatment. It was also possible to identify 4 novel miRNAs including an intronic miRNA that was conserved in both rice varieties.

Thymoquinone inhibits TNF-α-induced inflammation and cell adhesion in rheumatoid arthritis synovial fibroblasts by ASK1 regulation.

Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine produced by monocytes/macrophage that plays a pathological role in rheumatoid arthritis (RA). In this study, we investigate the effect of thymoquinone (TQ), a phytochemical found in Nigella sativa, in regulating TNF-α-induced RA synovial fibroblast (RA-FLS) activation. Treatment with TQ (1-5µM) had no marked effect on the viability of human RA-FLS. Pre-treatment of TQ inhibited TNF-α-induced interleukin-6 (IL-6) and IL-8 production and ICAM-1, VCAM-1, and cadherin-11 (Cad-11) expression in RA-FLS (p<0.01). Evaluation of the signaling events showed that TQ inhibited TNF-α-induced phospho-p38 and phospho-JNK expression, but had no inhibitory effect on NF-κB pathway, in RA-FLS (p<0.05; n=4). Interestingly, we observed that selective down-regulation of TNF-α-induced phospho-p38 and phospho-JNK activation by TQ is elicited through inhibition of apoptosis-regulated signaling kinase 1 (ASK1). Furthermore, TNF-α selectively induced phosphorylation of ASK1 at Thr845 residue in RA-FLS, which was inhibited by TQ pretreatment in a dose dependent manner (p<0.01). Pre-treatment of RA-FLS with ASK1 inhibitor (TC ASK10), blocked TNF-α induced expression of ICAM-1, VCAM-1, and Cad-11. Our results suggest that TNF-α-induced ASK1-p38/JNK pathway is an important mediator of cytokine synthesis and enhanced expression of adhesion molecule in RA-FLS and TQ, by selectively inhibiting this pathway, may have a potential therapeutic value in regulating tissue destruction observed in RA.

Design, synthesis and biological evaluation of functionalized phthalimides: a new class of antimalarials and inhibitors of falcipain-2, a major hemoglobinase of malaria parasite.

Phthalimides functionalized with cyclic amines were synthesized, characterized and screened for their in vitro antimalarial efficacy against Plasmodium falciparum (Pf3D7). Of all the listed phthalimides evaluated, 14 and 24 were identified as potent antimalarial agents as advocated by assessment of their ability to inhibit [(3)H] hypoxanthine incorporation in the nucleic acid of parasites. In addition, phthalimides 14 and 24 were incubated for 60 and 90h and an enhanced antimalarial effect was noticed with increase in time to great extent. A reduction in IC50 values was observed with increase in exposure time of the parasite to the compounds. A symmetric phthalimide, 24 possessing piperazine as linker unit was identified as the most potent antimalarial agent with IC50 values of 5.97±0.78, 2.0±1.09 and 1.1±0.75µM on incubation period of 42, 60 and 90h, respectively. The abnormal morphologies such as delay in developmental stages, growth arrest and condensed nuclei of parasite were observed with the aid of microscopic studies upon exposure with 14 and 24. The evaluation of 14 and 24 against chloroquine resistant strain, (Pf7GB) of P. falciparum afforded IC50 values, 13.29±1.20 and 7.21±0.98µM, respectively. The combination of 24 with artemisinin (ART) showed enhanced killing of parasite against Pf3D7. Further, all phthalimides were evaluated for their activity against falcipain-2 (FP2), a major hemoglobinase of malarial parasite. The enzymatic assay afforded 6 as most active member against FP2. To the best of our knowledge this is the initial study represents phthalimide protected amino acids functionalized with cyclic amines as potent antimalarial agents.

Renal cortical necrosis is a disappearing entity in obstetric acute kidney injury in developing countries: our three decade of experience from India.

RATIONAL: Obstetrical complications are the commonest causes of Renal Cortical Necrosis (RCN). However, the overall incidence of RCN in obstetric acute kidney injury in developing countries has been decreasing in recent years. OBJECTIVE: The aim of this study was to evaluate the changing profile of RCN in obstetric AKI over the last three decades. METHODS: This single center study included patients with biopsy proven renal cortical necrosis over a period of 32 years from 1982 to 2014. The diagnosis of RCN was suspected in patients with prolonged AKI (>4 weeks) with absolute anuria in the setting of hemorrhage, hypotension and sepsis; and was confirmed by renal biopsy. The changing pattern in the incidence, etiology and outcome of RCN in patients with obstetric AKI was compared in the three study periods, namely 1982-1991, 1992-2002 and 2003-2014. RESULTS: Over a period of 32 years, RCN was diagnosed in 15/259(5.8%) cases of obstetric AKI. Diffuse and patchy cortical necrosis were noted in 8(53.3%) and 7(46.7%) patients, respectively. RCN occurred in 17%(11/65), 2.4%(3/125) and 1.44%(1/69) patients in 1982-1991, 1992-2002 and 2003-2014, respectively. Septic abortion was commonest cause of RCN in the first two study periods but no case was observed in last decade. The decrease in incidence of RCN over the three decades was statistically significant (p-value < 0.001). Maternal mortality decreased to zero in 2003-2014 from 72.7% in 1982-1991. CONCLUSIONS: The incidence of RCN in obstetric AKI in developing countries has declined low enough to label it as a disappearing entity.

Characterization of pandemic influenza A (H1N1) virus hemagglutinin specific polyclonal antibodies for biosensor applications.

In this study, recombinant hemagglutinin protein (rH1N1HA) of Pandemic influenza virus and polyclonal antibodies against it for biosensor applications have been characterized. For rapid and high sensitive detection of H1N1 virus or its antibodies, PCR-free and label free detection method based on a surface plasmon resonance technique has been proposed. The glycosylated H1N1HA protein was expressed in yeast and the authenticity of the expressed protein was confirmed by Western blotting. Rabbit polyclonal antibodies developed against rH1N1HA protein were evaluated for their ability to neutralize H1N1 virus through plaque reduction neutralization test and indirect ELISA. Affinity purified anti-H1N1HA IgG were characterized further for their specificity, affinity of interaction, the association and dissociation rates at which they interact through surface plasmon resonance technique. The equilibrium constant and maximum binding capacity of analyte was found to be 49.7 nM and 47.28m°, respectively. The assay could detect a lowest IgG of 0.5 ng on a rH1N1HA coated chip. Combined with the high sensitivity of surface plasmon resonance technique and specificity of the reagents, it is possible to develop a rapid detection assay for monitoring influenza infections.

Labda-8(17),12,14-trien-19-oic acid contained in fruits of Cupressus sempervirens suppresses benign prostatic hyperplasia in rat and in vitro human models through inhibition of androgen and STAT-3 signaling.

Fruit extract of Cupressus sempervirens (CS), which is used traditionally to treat Benign Prostatic Hyperplasia (BPH)-like urinary symptoms in patients, was scientifically validated for anti-BPH activity. The ethanolic fruit extract of CS inhibited proliferation of human BPH-stromal cells and the activity was localized to its chloroform-soluble, diterpene-rich fraction. Eight major diterpenes isolated from this fraction exhibited moderate to potent activity and the most active diterpene (labda-8(17),12,14-trien-19-oic acid) exhibited an IC50 of 37.5 µM (antiproliferative activity against human BPH-stromal cells). It significantly inhibited activation (phosphorylation) of Stat-3 in BPH-stromal cells and prevented transactivation of androgen sensitive KLK3/PSA and TMPRSS2 genes in LNCaP cells. Labda-8(17),12,14-trien-19-oic acid-rich CS fraction prevented prostatic hyperplasia in rat model and caused TUNEL labeling of stromal cells with lower expressions of IGF-I, TGF-ß and PCNA, and bcl-2/bax ratio. Human BPH tissues exhibited precise lowering of stromal component after incubation in labda-8(17),12,14-trien-19-oic acid, ex vivo. We conclude that labda-8(17),12,14-trien-19-oic acid contained in CS exhibits anti-BPH activity through inhibition of stromal proliferation and suppression of androgen action in the prostate, presenting a unique lead structure for further optimization of anti-BPH activity.

Dual role of Response gene to complement-32 in multiple sclerosis.

Response gene to complement (RGC)-32 is a novel molecule that plays an important role in cell proliferation. We investigated the expression of RGC-32 in multiple sclerosis (MS) brain and in peripheral blood mononuclear cells (PBMCs) obtained from patients with relapsing-remitting multiple sclerosis. We found that CD3(+), CD68(+), and glial fibrillar acidic protein (GFAP)(+) cells in MS plaques co-localized with RGC-32. Our results show a statistically significant decrease in RGC-32 mRNA expression in PBMCs during relapses when compared to the levels in stable MS patients. This decrease might be useful in predicting disease activity in patients with relapsing-remitting MS. RGC-32 expression was also correlated with that of FasL mRNA during relapses. FasL mRNA expression was significantly reduced after RGC-32 silencing, indicating a role for RGC-32 in the regulation of FasL expression. In addition, the expression of Akt1, cyclin D1, and IL-21 mRNA was significantly increased during MS relapses when compared to levels in healthy controls. Furthermore, we investigated the role of RGC-32 in TGF-ß-induced extracellular matrix expression in astrocytes. Blockage of RGC-32 using small interfering RNA significantly inhibits TGF-ß induction of procollagen I, fibronectin and of the reactive astrocyte marker α-smooth muscle actin (α-SMA). Our data suggest that RGC-32 plays a dual role in MS, both as a regulator of T-cells mediated apoptosis and as a promoter of TGF-ß-mediated profibrotic effects in astrocytes.

Optimization of Dengue-3 recombinant NS1 protein expression in E. coli and in vitro refolding for diagnostic applications.

Dengue non-structural protein (NS1) is known to be protective antigen and also has immense application for serodiagnosis. Several serodiagnostic assays available for dengue viral infection are dependent on tissue culture-grown viral proteins. This task is unsafe, laborious, more expensive that makes it unsuitable for routine large-scale production. Although bacterial expression is relatively simple and easy for recombinant protein expression, it is more challenging to make NS1 protein with native structural and immunological features using bacterial expression system. We have successfully developed a method leading to the purification and refolding of recombinant dengue virus type 3 (DENV3) NS1. The gene encoding NS1 was amplified and cloned in pET28a (+) vector. In order to increase the purity of the recombinant NS1, the transgene was engineered to carry 6× Histidine tags at both N and C-terminal ends. The recombinant construct (pETNS1) was transformed into E. coli Rosetta-gami cells and the expression conditions viz IPTG concentration, media type, temperature, and harvest time were optimized. The size of the expressed protein was found to be ~45 kDa and the authenticity of the expressed protein was confirmed using anti-His and anti-NS1 monoclonal antibodies. The NS1 protein was purified under denaturing conditions, to attain the native conformation, NS1 protein was in vitro refolded and dialyzed. The refolded NS1 protein was detected by commercial Immuno chromatographic strip and NS1 specific monoclonal antibodies. IgM antibody capture ELISA was performed using refolded recombinant NS1 protein which recognized the IgM antibodies in dengue-positive samples of acute phase of infection. Our result suggests that rNS1 protein has immense diagnostic potential and can be used in developing point of care diagnostic assays.