Nitrogen Mustard-Induced Ex Vivo Human Cornea Injury Model and Therapeutic Intervention by Dexamethasone.

Sulfur mustard (SM), a vesicating agent first used during World War I, remains a potent threat as a chemical weapon to cause intentional/accidental chemical emergencies. Eyes are extremely susceptible to SM toxicity. Nitrogen mustard (NM), a bifunctional alkylating agent and potent analog of SM, is used in laboratories to study mustard vesicant-induced ocular toxicity. Previously, we showed that SM-/NM-induced injuries (in vivo and ex vivo rabbit corneas) are reversed upon treatment with dexamethasone (DEX), a US Food and Drug Administration-approved, steroidal anti-inflammatory drug. Here, we optimized NM injuries in ex vivo human corneas and assessed DEX efficacy. For injury optimization, one cornea (randomly selected from paired eyes) was exposed to NM: 100 nmoles for 2 hours or 4 hours, and 200 nmoles for 2 hours, and the other cornea served as a control. Injuries were assessed 24 hours post NM-exposure. NM 100 nmoles exposure for 2 hours was found to cause optimal corneal injury (epithelial thinning [∼69%]; epithelial-stromal separation [6-fold increase]). In protein arrays studies, 24 proteins displayed ≥40% change in their expression in NM exposed corneas compared with controls. DEX administration initiated 2 hours post NM exposure and every 8 hours thereafter until 24 hours post-exposure reversed NM-induced corneal epithelial-stromal separation [2-fold decrease]). Of the 24 proteins dysregulated upon NM exposure, six proteins (delta-like canonical Notch ligand 1, FGFbasic, CD54, CCL7, endostatin, receptor tyrosine-protein kinase erbB-4) associated with angiogenesis, immune/inflammatory responses, and cell differentiation/proliferation, showed significant reversal upon DEX treatment (Student's t test; P ≤ 0.05). Complementing our animal model studies, DEX was shown to mitigate vesicant-induced toxicities in ex vivo human corneas. SIGNIFICANCE STATEMENT: Nitrogen mustard (NM) exposure-induced injuries were optimized in an ex vivo human cornea culture model and studies were carried out at 24 h post 100 nmoles NM exposure. Dexamethasone (DEX) administration (started 2 h post NM exposure and every 8 h thereafter) reversed NM-induced corneal injuries. Molecular mediators of DEX action were associated with angiogenesis, immune/inflammatory responses, and cell differentiation/proliferation, indicating DEX aids wound healing via reversing vesicant-induced neovascularization (delta-like canonical Notch ligand 1 and FGF basic) and leukocyte infiltration (CD54 and CCL7).

Establishing a Dexamethasone Treatment Regimen To Alleviate Sulfur Mustard-Induced Corneal Injuries in a Rabbit Model.

Sulfur mustard (SM) is an ominous chemical warfare agent. Eyes are extremely susceptible to SM toxicity; injuries include inflammation, fibrosis, neovascularization (NV), and vision impairment/blindness, depending on the exposure dosage. Effective countermeasures against ocular SM toxicity remain elusive and are warranted during conflicts/terrorist activities and accidental exposures. We previously determined that dexamethasone (DEX) effectively counters corneal nitrogen mustard toxicity and that the 2-hour postexposure therapeutic window is most beneficial. Here, the efficacy of two DEX dosing frequencies [i.e., every 8 or 12 hours (initiated, as previously established, 2 hours after exposure)] until 28 days after SM exposure was assessed. Furthermore, sustained effects of DEX treatments were observed up to day 56 after SM exposure. Corneal clinical assessments (thickness, opacity, ulceration, and NV) were performed at the day 14, 28, 42, and 56 post-SM exposure time points. Histopathological assessments of corneal injuries (corneal thickness, epithelial degradation, epithelial-stromal separation, inflammatory cell, and blood vessel counts) using H&E staining and molecular assessments (COX-2, MMP-9, VEGF, and SPARC expressions) were performed at days 28, 42, and 56 after SM exposure. Statistical significance was assessed using two-way ANOVA, with Holm-Sidak post hoc pairwise multiple comparisons; significance was established if P < 0.05 (data represented as the mean ± S.E.M.). DEX administration every 8 hours was more potent than every 12 hours in reversing ocular SM injury, with the most pronounced effects observed at days 28 and 42 after SM exposure. These comprehensive results are novel and provide a comprehensive DEX treatment regimen (therapeutic-window and dosing-frequency) for counteracting SM-induced corneal injuries. SIGNIFICANCE STATEMENT: The study aims to establish a dexamethasone (DEX) treatment regimen by comparing the efficacy of DEX administration at 12 versus 8 hours initiated 2 hours after exposure. DEX administration every 8 hours was more effective in reversing sulfur mustard (SM)-induced corneal injuries. SM injury reversal during DEX administration (initial 28 days after exposure) and sustained [further 28 days after cessation of DEX administration (i.e., up to 56 days after exposure)] effects were assessed using clinical, pathophysiological, and molecular biomarkers.

Effect of nonsteroidal anti-inflammatory drugs (aspirin and naproxen) on inflammation-associated proteomic profiles in mouse plasma and prostate during TMPRSS2-ERG (fusion)-driven prostate carcinogenesis.

Recent preclinical studies have shown that the intake of nonsteroidal anti-inflammatory drugs (NSAIDs) aspirin and naproxen could be an effective intervention strategy against TMPRSS2-ERG fusion-driven prostate tumorigenesis. Herein, as a follow-up mechanistic study, employing TMPRSS2-ERG (fusion) positive tumors and plasma from TMPRSS2-ERG. Ptenflox/flox mice, we profiled the stage specific proteomic changes (focused on inflammatory circulating and prostate tissue/tumor-specific cytokines, chemokines, and growth factors/growth signaling-associated molecules) that contribute to prostate cancer (PCa) growth and progression in the TMPRSS2-ERG fusion-driven mouse model of tumorigenesis. In addition, the association of the protective effects of NSAIDs (aspirin 1400 ppm and naproxen 400 ppm) with the modulation of these specific molecular pathways was determined. A sandwich Elisa based membrane array-proteome profiler identifying 111 distinct signaling molecules was employed. Overall, the plasma and prostate tissue sample analyses identified 54 significant and differentially expressed cytokines, chemokines, and growth factors/growth signaling-associated molecules between PCa afflicted mice (TMPRSS2-ERG. Ptenflox/flox, age-matched noncancerous controls, NSAIDs-supplemented and no-drug controls). Bioinformatic analysis of the array outcomes indicated that the protective effect of NSAIDs was associated with reduced expression of (a) tumor promoting inflammatory molecules (M-CSF, IL-33, CCL22, CCL12, CX3CL1, CHI3L1, and CD93), (b) growth factors- growth signaling-associated molecules (Chemerin, FGF acidic, Flt-3 ligand, IGFBP-5, and PEDF), and (c) tumor microenvironment/stromal remodeling proteins MMP2 and MMP9. Overall, our findings corroborate the pathological findings that protective effects of NSAIDs in TMPSS2-ERG fusion-driven prostate tumorigenesis are associated with antiproliferative and anti-inflammatory effects and possible modulation of the immune cell enriched microenvironment.

Dexamethasone targets actin cytoskeleton signaling and inflammatory mediators to reverse sulfur mustard-induced toxicity in rabbit corneas.

PURPOSE: Sulfur mustard (SM), a bi-functional alkylating agent, was used during World War I and the Iran-Iraq war. SM toxicity is ten times higher in eyes than in other tissues. Cornea is exceptionally susceptible to SM-injuries due to its anterior positioning and mucous-aqueous interphase. Ocular SM exposure induces blepharitis, photosensitivity, dry eye, epithelial defects, limbal ischemia and stem cell deficiency, and mustard gas keratopathy leading to temporary or permanent vision impairments. We demonstrated that dexamethasone (Dex) is a potent therapeutic intervention against SM-induced corneal injuries; however, its mechanism of action is not well known. Investigations employing proteomic profiling (LC-MS/MS) to understand molecular mechanisms behind SM-induced corneal injury and Dex efficacy were performed in the rabbit cornea exposed to SM and then received Dex treatment. PEAKS studio was used to extract, search, and summarize peptide identity. Ingenuity Pathway Analysis was used for pathway identification. Validation was performed using immunofluorescence. One-Way ANOVA (FDR < 0.05; p < 0.005) and Student's t-test (p < 0.05) were utilized for analyzing proteomics and IF data, respectively. Proteomic analysis revealed that SM-exposure upregulated tissue repair pathways, particularly actin cytoskeleton signaling and inflammation. Prominently dysregulated proteins included lipocalin2, coronin1A, actin-related protein2, actin-related protein2/3 complex subunit2, actin-related protein2/3 complex subunit4, cell division cycle42, ezrin, bradykinin/kininogen1, moesin, and profilin. Upregulated actin cytoskeleton signaling increases F-actin formation, dysregulating cell shape and motility. Dex reversed SM-induced increases in the aforementioned proteins levels to near control expression profiles. Dex aids corneal wound healing and improves corneal integrity via actin cytoskeletal signaling and anti-inflammatory effects following SM-induced injuries.

Genome-wide characterization leading to simple sequence repeat (SSR) markers development in Shorea robusta.

Tropical rainforests in Southeast Asia are enriched by multifarious biota dominated by Dipterocarpaceae. In this family, Shorea robusta is an ecologically sensitive and economically important timber species whose genomic diversity and phylogeny remain understudied due to lack of datasets on genetic resources. Smattering availability of molecular markers impedes population genetic studies indicating a necessity to develop genomic databases and species-specific markers in S. robusta. Accordingly, the present study focused on fostering de novo low-depth genome sequencing, identification of reliable microsatellites markers, and their validation in various populations of S. robusta in Uttarakhand Himalayas. With 69.88 million raw reads assembled into 1,97,489 contigs (read mapped to 93.2%) and a genome size of 357.11 Mb (29 × coverage), Illumina paired-end sequencing technology arranged a library of sequence data of ~ 10 gigabases (Gb). From 57,702 microsatellite repeats, a total of 35,049 simple sequence repeat (SSR) primer pairs were developed. Afterward, among randomly selected 60 primer pairs, 50 showed successful amplification and 24 were found as polymorphic. Out of which, nine polymorphic loci were further used for genetic analysis in 16 genotypes each from three different geographical locations of Uttarakhand (India). Prominently, the average number of alleles per locus (Na), observed heterozygosity (Ho), expected heterozygosity (He), and the polymorphism information content (PIC) were recorded as 2.44, 0.324, 0.277 and 0.252, respectively. The accessibility of sequence information and novel SSR markers potentially enriches the current knowledge of the genomic background for S. robusta and to be utilized in various genetic studies in species under tribe Shoreae.

Metabolomics for identifying pathways involved in vesicating agent lewisite-induced corneal injury.

Lewisite (LEW) is an arsenical vesicant that can be a potentially dangerous chemical warfare agent (CWA). Eyes are particularly susceptible to vesicant induced injuries and ocular LEW exposure can act swiftly, causing burning of eyes, edema, inflammation, cell death and even blindness. In our previous studies, we developed a LEW exposure-induced corneal injury model in rabbit and showed increased inflammation, neovascularization, cell death, and structural damage to rabbit corneas upon LEW exposure. In the present study, we further assessed the metabolomic changes to delineate the possible mechanisms underlying the LEW-induced corneal injuries. This information is vital and could help in the development of effective targeted therapies against ocular LEW injuries. Thus, the metabolomic changes associated with LEW exposures in rabbit corneas were assessed as a function of time, to delineate pathways from molecular perturbations at the genomic and proteomic levels. New Zealand white rabbit corneas (n = 3-6) were exposed to LEW vapor (0.2 mg/L; flow rate: 300 ml/min) for 2.5 min (short exposure; low dose) or 7.5 min (long-exposure; high dose) and then collected at 1, 3, 7, or 14 days post LEW exposure. Samples were prepared using the automated MicroLab STAR® system, and proteins precipitated to recover the chemically diverse metabolites. Metabolomic analysis was carried out by reverse phase UPLC-MS/MS and gas chromatography (GC)-MS. The data obtained were analyzed using Metabolon's software. The results showed that LEW exposures at high doses were more toxic, particularly at the day 7 post exposure time point. LEW exposure was shown to dysregulate metabolites associated with all the integral functions of the cornea and cause increased inflammation and immune response, as well as generate oxidative stress. Additionally, all important metabolic functions of the cells were also affected: lipid and nucleotide metabolism, and energetics. The high dose LEW exposures were more toxic, particularly at day 7 post LEW exposure (>10-fold increased levels of histamine, quinolinate, N-acetyl-ß-alanine, GMP, and UPM). LEW exposure dysregulated integral functions of the cornea, caused inflammation and heightened immune response, and generated oxidative stress. Lipid and nucleotide metabolism, and energetics were also affected. The novel information about altered metabolic profile of rabbit cornea following LEW exposure could assist in delineating complex molecular events; thus, aid in identifying therapeutic targets to effectively ameliorate ocular trauma.

Semi-microscopic Theory for the Current Rectification Phenomenon in Nanogap Molecular Devices.

A semi-microscopic theory is developed for heterogeneous electron transfer (HET) kinetics based on the energy level alignment approach at self-assembled monolayer (SAM) covered metal electrodes. Theory provides the electronic and molecular property-dependent equations for the HET rate constant (k0) and the transfer coefficient (α) for potential. k0 is formulated using the activation free energy as a product of the SAM covered metal work function (WF) and fractional electronic charge exchanged at the transition state (attained through the alignment of the frontier molecular orbital (FMO) energy level of the electroactive group with the WF of metal). k0 is a function of the metal jellium electronic screening length and dielectric and of the molecular self-assembly (through its dipole moment, size, and packing density) and the FMO energies of electroactive groups. The operative potential at the transition state is governed by α, which is a function of molecular spacer length and characteristic electronic-dipolar coupling length. The current rectification phenomenon in nanogap molecular devices is theoretically analyzed using equations for k0 and α for SAM covered source and drain electrodes. Theory unravels the LUMO or HOMO dichotomy for a given metal: (i) for the HOMO assisted ET, the metal with a high WF has a high current rectification ratio (RR), while (ii) for the LUMO assisted ET, the metal with a low WF has a high current RR in asymmetrical devices. Theory predicts the reversal in current rectification by altering the dipole moment of the anchoring molecule, the HOMO/LUMO energy of the electroactive groups, and the nature of the metal. Finally, theory shows qualitative and quantitative coherence with the reported experimental current-potential response of molecular device.

Ecological niche modelling and population genetic analysis of Indian temperate bamboo Drepanostachyum falcatum in the western Himalayas.

The present study was conducted to understand the key ecological and biological questions of conservation importance in Drepanostachyum falcatum which aimed to map potential distribution in the western Himalayas and decipher spatial genetic structure. Eco-distribution maps were generated through ecological niche modelling using the Maximum Entropy (MaxEnt) algorithm implemented with 228 geocoordinates of species presence and 12 bioclimatic variables. Concomitantly, 26 natural populations in the western Himalayas were genetically analysed using ten genomic sequence-tagged microsatellite (STMS) markers. Model-derived distribution was adequately supported with appropriate statistical measures, such as area under the 'receiver operating characteristics (ROC)' curve (AUC; 0.917 ± 0.034)", Kappa (K; 0.418), normalized mutual information (NMI; 0.673) and true skill statistic (TSS; 0.715). Further, Jackknife test and response curves showed that the precipitation (pre- and post-monsoon) and temperature (average throughout the year and pre-monsoon) maximize the probabilistic distribution of D. falcatum. We recorded a wide and abundant (4096.86 km2) distribution of D. falcatum in the western Himalayas with maximum occurrence at 1500 to 2500 m asl. Furthermore, marker analysis exemplified high gene diversity with low genetic differentiation in D. falcatum. Relatively, the populations of Uttarakhand are more genetically diverse than Himachal Pradesh, whereas within the Uttarakhand, the Garhwal region captured a higher allelic diversity than Kumaon. Clustering and structure analysis indicated two major gene pools, where genetic admixing appeared to be controlled by long-distance gene flow, horizontal geographical distance, aspect, and precipitation. Both the species distribution map and population genetic structure derived herein may serve as valuable resources for conservation and management of Himalayan hill bamboos.

Chemopreventive efficacy of silibinin against basal cell carcinoma growth and progression in UVB-irradiated Ptch+/- mice.

The factors (environmental and genetic) contributing to basal cell carcinoma (BCC) pathogenesis are well-established; however, effective agents for BCC prevention are marred by toxic side-effects. Herein, we assessed the efficacy of flavonolignan silibinin against ultraviolet B (UVB)-induced BCC in Ptch+/- (heterozygous patched homolog 1 gene) mouse model. Both male and female Ptch+/- mice were irradiated with a 240 mJ/cm2 UVB dose 3 times/week for 26 or 46 weeks, with or without topical application of silibinin (9 mg/200 µl in acetone, applied 30 min before or after UVB exposure). Results indicated that silibinin application either pre- or post-UVB exposure for 26 weeks significantly decreased the number of BCC lesions by 65% and 39% (P < 0.001 for both) and the area covered by BCCs (72% and 45%, P < 0.001 for both), respectively, compared to UVB alone. Furthermore, continuous UVB exposure for 46 weeks increased the BCC lesion number and the BCC area covered by ~6 and ~3.4 folds (P < 0.001), respectively. Notably, even in this 46 week prolonged UVB exposure, silibinin (irrespective of pre- or post-UVB treatment) significantly halted the growth of BCCs by 81-94% (P < 0.001) as well as other epidermal lesions; specifically, silibinin treated tissues had less epidermal dysplasia, fibrosarcoma, and squamous cell carcinoma. Immunohistochemistry and immunofluorescence studies revealed that silibinin significantly decreased basal cell proliferation (Ki-67) and the expression of cytokeratins (14 and 15), and Hedgehog signaling mediators Smo and Gli1 in the BCC lesions. Together, our findings demonstrate strong potential of silibinin to be efficacious in preventing the growth and progression of UVB-induced BCC.

Characterization of stage-specific tumor progression in TMPRSS2-ERG (fusion)-driven and non-fusion-driven prostate cancer in GEM models.

In the present study, we performed a comparative stage-specific pathological and molecular marker evaluation of TMPRSS2-ERG fusion and PTEN loss-driven (TMPRSS2-ERG. Ptenflox/flox ) versus non-fusion-driven prostate tumorigenesis (Hi-Myc) in mice. Anterior, ventral, and dorsolateral prostates were collected from mice at different ages (or time points post-Cre induction). Results indicated that growth and progression of prostatic intraepithelial lesions to adenocarcinoma stages occurred in both mice models albeit at different rates. In the TMPRSS2-ERG. Ptenflox/flox mice, the initiation of tumorigenesis was slow, but subsequent progression through different stages became increasingly faster. Adenocarcinoma stage was reached early on; however, no high-grade undifferentiated tumors were observed. Conversely, in the Hi-Myc+/- mice, tumorigenesis initiation was rapid; however, progression through different stages was relatively slower and it took a while to reach the more aggressive phenotype stage. Nevertheless, at the advanced stages in the Hi-Myc+/- mice, high-grade undifferentiated tumors were observed compared to the later stage tumors observed in the fusion-driven TMPRSS2-ERG. Ptenflox/flox mice. These results were corroborated by the stage specific-pattern in the molecular expression of proliferation markers (PCNA and c-Myc); androgen receptor (AR); fusion-resultant overexpression of ERG; Prostein (SLC45-A3); and angiogenesis marker (CD-31). Importantly, there was a significant increase in immune cell infiltrations, which increased with the stage of tumorigenesis, in the TMPRSS2-ERG fusion-positive tumors relative to fusion negative tumors. Together, these findings are both novel and highly significant in establishing a working preclinical model for evaluating the efficacy of interventions during different stages of tumorigenesis in TMPRSS2-ERG fusion-driven PCa.

Gender-based effect of absence of gut microbiota on the protective efficacy of Bifidobacterium longum-fermented rice bran diet against inflammation-associated colon tumorigenesis.

Dietary rice bran (RB) has shown capacity to influence metabolism by modulation of gut microbiota in individuals at risk for colorectal cancer (CRC), which warranted attention for delineating mechanisms for bidirectional influences and cross-feeding between the host and RB-modified gut microbiota to reduce CRC. Accordingly, in the present study, fermented rice bran (FRB, fermented with a RB responsive microbe Bifidobacterium longum), and non-fermented RB were fed as 10% w/w (diet) to gut microbiota-intactspf or germ-free micegf to investigate comparative efficacy against inflammation-associated azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC. Results indicated both microbiota-dependent and independent mechanisms for RB meditated protective efficacy against CRC that was associated with reduced neoplastic lesion size and local-mucosal/systemic inflammation, and restoration of colonic epithelial integrity. Enrichment of beneficial commensals (such as, Clostridiales, Blautia, Roseburia), phenolic metabolites (benzoate and catechol metabolism), and dietary components (ferulic acid-4 sulfate, trigonelline, and salicylate) were correlated with anti-CRC efficacy. Germ-free studies revealed gender-specific physiological variables could differentially impact CRC growth and progression. In the germ-free females, the RB dietary treatment showed a ∼72% reduction in the incidence of colonic epithelial erosion when compared to the ∼40% reduction in FRB-fed micegf . Ex vivo fermentation of RB did not parallel the localized-protective benefits of gut microbial metabolism by RB in damaged colonic tissues. Findings from this study suggest potential needs for safety considerations of fermented fiber rich foods as dietary strategies against severe inflammation-associated colon tumorigenesis (particularly with severe damage to the colonic epithelium).

Effect of dexamethasone treatment at variable therapeutic windows in reversing nitrogen mustard-induced corneal injuries in rabbit ocular in vivo model.

Nitrogen mustard (NM) is an analogue of the potent vesicating agent sulfur mustard, with well-established ocular injury models in rabbit eyes to study vesicant-induced ocular toxicity. The effects of NM-exposure to eyes may include irritation, redness, inflammation, fibrosis, epithelial degradation, blurred vision, partial/complete blindness, which may be temporary or permanent, depending on the route, duration, and dosage of exposure. Effective countermeasures against vesicant exposure are presently not available and are warranted in case of any terrorist activity or accidental leakage from stockpiles. Herein, our focus was to evaluate whether dexamethasone (DEX), an FDA approved potent corticosteroid with documented anti-inflammatory activities, could be an effective treatment modality. Accordingly, utilizing NM-induced corneal injuries in rabbit ocular in vivo model, we examined and compared the efficacy of DEX treatments when administration was started at early (2 h), intermediate (4 h), and late (6 h) therapeutic windows of intervention after NM-exposure and administered every 8 h thereafter. The effects of NM-exposure and DEX treatments were evaluated on clinical (corneal opacity, ulceration, and neovascularization), biological (epithelial thickness, epithelial-stromal separation, blood vessels density, and inflammatory cell and keratocyte counts) and molecular (COX-2 and VEGF expression) parameters, at day 1, 3, 7 and 14. Results indicated that DEX treatment markedly and effectively reversed the NM-induced injury markers in rabbit corneas. Early administration of DEX at 2 h was found to be most effective in reversing NM-induced corneal injuries, followed by DEX 4 h and DEX 6 h administration initiation, indicating that DEX has best efficacy at the early therapeutic window in our study model.

Global Forestry Perspective: COVID-19 Impact and Assessment.

Corona virus disease 2019 (COVID-19) caused by corona virus SARS-CoV-2 is believed to be originated in Wuhan province of China, which has spread all over the world. It has severely impacted the social, cultural, educational, research and development programmes throughout the globe. Since the inception of various lifeforms on planet earth, forests' plays an important role in the evolutionary history of organisms and fitness persuades the existence of modern-day species in different ecosystems. The visible effect of pandemic leads to the lockdown with positive implications on the earth biosphere as a whole; while on other hand, lockdown seized and reduced the urbanization and developmental projects, especially the research and development progress in the field of forestry and allied sciences. This viewpoint addresses key questions and point-out with detailed exploration of the problem - as how COVID-19 pandemic impacts forestry research all over the world? We also suggest possible solutions, and define the role and association of disciplines, such as bioinformatics, remote sensing and statistical modules along with previously explored and collected field data as a sustainable measure to resolve the problems of forestry sector of this mighty challenge.

Genome skimming-based simple sequence repeat (SSR) marker discovery and characterization in Grevillea robusta.

Proteaceae, a largely southern hemisphere family consisting of 80 genera distributed in Australia and southern Africa as its centres of greatest diversity, also extends well in northern and southern America. Under this family, Grevillea robusta is a fast-growing species got popularity in farm and avenue plantations. Despite the ecological and economic importance, the species has not yet been investigated for its genetic improvement and genome-based studies. Only a few molecular markers are available for the species or its close relatives, which hinders  genomic and population genetics studies. Genetic markers have been intensively applied for the main strategies in breeding programs, especially for the economically important traits. Hence, it is of utmost priority to develop genomic database resources and species-specific markers for studying quantitative genetics in G. robusta. Given this, the present study aimed to develop de novo genome sequencing, robust microsatellites markers, sequence annotation and their validation in different stands of G. robusta in northern India. Library preparation and sequencing were carried out using Illumina paired-end sequencing technology. Approximately, ten gigabases (Gb) sequence data with 70.87 million raw reads assembled into 425,923 contigs (read mapped to 76.48%) comprising 455 Mb genome size (23 × coverage) generated through genome skimming approach. In total, 9421 simple sequence repeat (SSR) primer pairs were successfully designed from 13,335 microsatellite repeats. Afterward, a subset of 161 primer pairs was randomly selected, synthesized and validated. All the tested primers showed successful amplification but only 13 showed polymorphisms. The polymorphic SSRs were further used to estimate the measures of genetic diversity in 12 genotypes each from the states of Punjab, Haryana, Himachal Pradesh and Uttarakhand. Importantly, the average number of alleles (Na), observed heterozygosity (Ho), expected heterozygosity (He), and the polymorphism information content (PIC) were recorded as 2.69, 0.356, 0.557 and 0.388, respectively. The availability of sequence information and newly developed SSR markers could potentially be used in various genetic analyses and improvements through molecular breeding strategies for G. robusta. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01035-w.

First de novo genome specific development, characterization and validation of simple sequence repeat (SSR) markers in Genus Salvadora.

Salvadoraceae constitutes ecologically imperative desert families of 3 genera-Azima, Dobera and Salvadora. Under genus Salvadora of this family, S. oleoides is a keystone species of socio-economic and medicinal value. This species naturally grows in the arid zones but currently experiencing severe fragmentation due to land use change and reduced regeneration, which may have resulted in the depletion of genetic diversity. Hence, it is up-most important to develop genomic resources for studying the population genetics in S. oleoides. This study aims to develop robust microsatellites markers, which were not yet reported in genus Salvodora due to lack of genome sequence information. We developed novel microsatellites markers in S. oleoides using Illumina paired-end sequencing technology. In total, 14,552 simple sequence repeat (SSR) markers were successfully designed from 21,055 microsatellite repeats detected in the 13 Gb raw sequence data. Afterwards, a subset of 101 SSRs were randomly selected and validated, 94 primers were successfully amplified and 34 showed polymorphisms. These SSRs were used to estimate the measures of genetic diversity in three natural populations of state Rajasthan and Gujarat. Importantly, average number of alleles (Na), observed heterozygosity (Ho), expected heterozygosity (He), and polymorphism information content (PIC) were recorded as 2.4, 0.529, 0.357, and 0.326, respectively. Furthermore, 15 primers were evaluated in S. persica for cross-transferability, and all were successfully amplified but only eight showed polymorphisms. This study has been conducted first time for S. oleoides and pioneer among the native species of arid-zone in India.

Bitter melon juice-intake modulates glucose metabolism and lactate efflux in tumors in its efficacy against pancreatic cancer.

The established role of bitter melon juice (BMJ), a natural product, in activating master metabolic regulator adenosine monophosphate-activated protein kinase in pancreatic cancer (PanC) cells served as a basis for pursuing deeper investigation into the underlying metabolic alterations leading to BMJ efficacy in PanC. We investigated the comparative metabolic profiles of PanC cells with differential KRAS mutational status on BMJ exposure. Specifically, we employed nuclear magnetic resonance (NMR) metabolomics and in vivo imaging platforms to understand the relevance of altered metabolism in PanC management by BMJ. Multinuclear NMR metabolomics was performed, as a function of time, post-BMJ treatment followed by partial least square discriminant analysis assessments on the quantitative metabolic data sets to visualize the treatment group clustering; altered glucose uptake, lactate export and energy state were identified as the key components responsible for cell death induction. We next employed PANC1 xenograft model for assessing in vivo BMJ efficacy against PanC. Positron emission tomography ([18FDG]-PET) and magnetic resonance imaging on PANC1 tumor-bearing animals reiterated the in vitro results, with BMJ-associated significant changes in tumor volumes, tumor cellularity and glucose uptake. Additional studies in BMJ-treated PanC cells and xenografts displayed a strong decrease in the expression of glucose and lactate transporters GLUT1 and MCT4, respectively, supporting their role in metabolic changes by BMJ. Collectively, these results highlight BMJ-induced modification in PanC metabolomics phenotype and establish primarily lactate efflux and glucose metabolism, specifically GLUT1 and MCT4 transporters, as the potential metabolic targets underlying BMJ efficacy in PanC.

Acute corneal injury in rabbits following nitrogen mustard ocular exposure.

Sulfur mustard (SM), a potent vesicating chemical warfare agent, and its analog nitrogen mustard (NM), are both strong bi-functional alkylating agents. Eyes, skin, and the respiratory system are the main targets of SM and NM exposure; however, ocular tissue is most sensitive, resulting in severe ocular injury. The mechanism of ocular injury from vesicating agents' exposure is not completely understood. To understand the injury mechanism from exposure to vesicating agents, NM has been previously employed in our toxicity studies on primary human corneal epithelial cells and ex vivo rabbit cornea organ culture model. In the current study, corneal toxicity from NM ocular exposure (1%) was analyzed for up to 28 days post-exposure in New Zealand White male rabbits to develop an acute corneal injury model. NM exposure led to conjunctival and eyelid swelling within a few hours after exposure, in addition to significant corneal opacity and ulceration. An increase in total corneal thickness and epithelial degradation was observed starting at day 3 post-NM exposure, which was maximal at day 14 post-exposure and did not resolve until 28 days post-exposure. There was an NM-induced increase in the number of blood vessels and inflammatory cells, and a decrease in keratocytes in the corneal stroma. NM exposure resulted in increased expression levels of cyclooxygenase-2, Interleukin-8, vascular endothelial growth factor and Matrix Metalloproteinase 9 indicating their involvement in NM-induced corneal injury. These clinical, biological, and molecular markers could be useful for the evaluation of acute corneal injury and to screen for therapies against NM- and SM-induced ocular injury.

Cutaneous exposure to vesicant phosgene oxime: Acute effects on the skin and systemic toxicity.

Phosgene Oxime (CX), an urticant or nettle agent categorized as a vesicant, is a potential chemical warfare and terrorist weapon. Its exposure can result in widespread and devastating effects including high mortality due to its fast penetration and ability to cause immediate severe cutaneous injury. It is one of the least studied chemical warfare agents with no effective therapy available. Thus, our goal was to examine the acute effects of CX following its cutaneous exposure in SKH-1 hairless mice to help establish a relevant injury model. Results from our study show that topical cutaneous exposure to CX vapor causes blanching of exposed skin with an erythematous ring, necrosis, edema, mild urticaria and erythema within minutes after exposure out to 8h post-exposure. These clinical skin manifestations were accompanied with increases in skin thickness, apoptotic cell death, mast cell degranulation, myeloperoxidase activity indicating neutrophil infiltration, p53 phosphorylation and accumulation, and an increase in COX-2 and TNFα levels. Topical CX-exposure also resulted in the dilatation of the peripheral vessels with a robust increase in RBCs in vessels of the liver, spleen, kidney, lungs and heart tissues. These events could cause a drop in blood pressure leading to shock, hypoxia and death. Together, this is the first report on effects of CX cutaneous exposure, which could help design further comprehensive studies evaluating the acute and chronic skin injuries from CX topical exposure and elucidate the related mechanism of action to aid in the identification of therapeutic targets and mitigation of injury.

Clinical progression of ocular injury following arsenical vesicant lewisite exposure.

Ocular injury by lewisite (LEW), a potential chemical warfare and terrorist agent, results in edema of eyelids, inflammation, massive corneal necrosis and blindness. To enable screening of effective therapeutics to treat ocular injury from LEW, useful clinically-relevant endpoints are essential. Hence, we designed an efficient exposure system capable of exposing up to six New-Zealand white rabbits at one time, and assessed LEW vapor-induced progression of clinical ocular lesions mainly in the cornea. The right eye of each rabbit was exposed to LEW (0.2 mg/L) vapor for 2.5, 5.0, 7.5 and 10.0 min and clinical progression of injury was observed for 28 days post-exposure (dose-response study), or exposed to same LEW dose for 2.5 and 7.5 min and clinical progression of injury was observed for up to 56 days post-exposure (time-response study); left eye served as an unexposed control. Increasing LEW exposure caused corneal opacity within 6 h post-exposure, which increased up to 3 days, slightly reduced thereafter till 3 weeks, and again increased thereafter. LEW-induced corneal ulceration peaked at 1 day post-exposure and its increase thereafter was observed in phases. LEW exposure induced neovascularization starting at 7 days which peaked at 22-35 days post-exposure, and remained persistent thereafter. In addition, LEW exposure caused corneal thickness, iris redness, and redness and swelling of the conjunctiva. Together, these findings provide clinical sequelae of ocular injury following LEW exposure and for the first time establish clinically-relevant quantitative endpoints, to enable the further identification of histopathological and molecular events involved in LEW-induced ocular injury.

The rigidity and chelation effect of ligands on the hydrogen evolution reaction catalyzed by Ni(II) complexes.

With increasing interest in nickel-based electrocatalysts, three heteroleptic Ni(II) dithiolate complexes with the general formula [Ni(II)L(L')2] (1-3), L = 2-(methylene-1,1'-dithiolato)-5,5'-dimethylcyclohexane-1,3-dione and L' = triphenylphosphine (1), 1,1'-bis(diphenylphosphino)ferrocene (DPPF) (2), and 1,2-bis(diphenylphosphino)ethane (DPPE) (3), have been synthesized and characterized by various spectroscopic techniques (UV-vis, IR, 1H, and 31P{1H} NMR) as well as the electrochemical method. The molecular structure of complex 2 has also been determined by single-crystal X-ray crystallography. The crystal structure of complex 2 reveals a distorted square planar geometry around the nickel metal ion with a NiP2S2 core. The cyclic voltammograms reveal a small difference in the redox properties of complexes (ΔE° = 130 mV) while the difference in the catalytic half-wave potential becomes substantial (ΔEcat/2 = 670 mV) in the presence of 15 mM CF3COOH. The common S^S-dithiolate ligand provides stability, while the rigidity effect of other ligands (DPPE (3) > DPPF (2) > PPh3 (1)) regulates the formation of the transition state, resulting in the NiIII-H intermediate in the order of 1 > 2 > 3. The foot-of-the-wave analysis supports the widely accepted ECEC mechanism for Ni-based complexes with the first protonation step as a rate-determining step. The electrocatalytic proton reduction activity follows in the order of complex 1 > 2 > 3. The comparatively lower overpotential and higher turnover frequency of complex 1 are attributed to the flexibility of the PPh3 ligand, which favours the easy formation of a transition state.