Evaluation of millets for physio-chemical and root morphological traits suitable for resilient farming and nutritional security in Eastern Himalayas.

Introduction: Millets are nutritionally superior and climate-resilient short-duration crops and hold a prominent place in cropping sequences around the world. They have immense potential to grow in a marginal environment due to diverse adaptive mechanisms. Methods: An experiment was conducted in an organic production system in the North Eastern Himalayan foothills of India for 3 consecutive years by evaluating high-yielding varieties (HYVs) of different millets, viz., finger millet, foxtail millet, little millet, barnyard millet, proso millet, and browntop millet, along with local landraces of finger millets (Sikkim-1 and Sikkim-2; Nagaland-1 and Nagaland-2) to identify stable, high-yielding, and nutritionally superior genotypes suited for the region. Results: Among the various millets, finger millet, followed by little millet and foxtail millet, proved their superiority in terms of productivity (ranging between 1.16 and 1.43 Mg ha-1) compared to other millets. Among different varieties of finger millets, cv. VL Mandua 352 recorded the highest average grain yield (1.43 Mg ha-1) followed by local landraces, Nagaland-2 (1.31 Mg ha-1) and Sikkim-1 (1.25 Mg ha-1). Root traits such as total root length, root volume, average diameter of roots, and root surface area were significantly higher in finger millet landraces Nagaland-1, Nagaland-2, and Sikkim-1 compared to the rest of the millet genotypes. The different millets were found to be rich sources of protein as recorded in foxtail millet cv. SiA 3088 (12.3%), proso millet cv. TNAU 145 (11.5%), and finger millet landraces, Sikkim-1 and Nagaland-2 (8.7% each). Finger millet landrace Sikkim-2 recorded the highest omega-6 content (1.16%), followed by barnyard millet cv. VL 207 (1.09%). Barnyard millet cv. VL 207 recorded the highest polyunsaturated fatty acid (PUFA) content (1.23%), followed by foxtail millet cv. SiA 3088 (1.09%). The local finger millet landraces Sikkim-1 and Sikkim-2 recorded the highest levels of histidine (0.41%) and tryptophan (0.12%), respectively. Sikkim-1 and Nagaland-2 recorded the highest level of thiamine (0.32%) compared to the HYVs. Conclusion: These findings indicate that finger millet has great potential in the organic production system of the North Eastern Himalayan Region (NEHR) of India, and apart from HYVs like VL Mandua 352, local landraces, viz., Nagaland-2 and Sikkim-1, should also be promoted for ensuring food and nutritional security in this fragile ecosystem.

Converting primary forests to cultivated lands: Long-term effects on the vertical distribution of soil carbon and biological activity in the foothills of Eastern Himalaya.

The conversion of primary forests to cultivation brings a significant change in soil carbon (C) forms. In the foothills of the Eastern Himalayan Region of India (Manipur), such conversions are prevalent. However, little is known about the response of C forms, particularly in deep soil, to land use conversion in the region. We evaluated changes in soil C forms (total organic, inorganic, and pools) and microbiological properties (up to 1.0 m depth) mediated by C when the 45-year-old forest had been cultivated for 18-25 years. The cultivated land uses were tree-based agroforestry (LAF: legumes, NAF: non-legumes), horticultural fruits (WHF: woody, NHF: non-wood, mainly vegetables), and paddy agriculture system (AUS: upland, ALS: lowlands). Forest conversion significantly (p < 0.05) decreased the total carbon (TC) in the surface soil (0.0-0.15 m) from 4.88 % to 3.04-3.93 % in the tree-based land uses (LAF, NAF, and WHF). TC further declined to 2.05-2.81 % under seasonal crops (NHF, AUS, and ALS). Seasonal crop cultivation also caused a higher decline in microbial biomass carbon, soil enzymes, and carbon pools (active and passive) than the tree-based land use with the soil depth. The vertical distribution of C in the soil profile was inconsistent: organic C (including C pools) decreased, while inorganic C increased. The profile TC stock to a depth of 1.0 m in the forest was 358.8 Mg ha-1, of which 81 % were organic C, and 19 % were inorganic C. In comparison with forest soil, total soil C stocks (organic and inorganic) decreased more (-44.1 to -55.1 %) in seasonal crops than in tree-based (-15.4 to -36.3 %) land uses. The degradation index (DI) also confirmed that seasonal crop cultivation caused a larger decline in surface soil quality (DI: -423 % to -623 %) than tree-based land use (DI: -243 % to -317 %). The topsoil (up to 0.45 m) of seasonal crops was more degraded than that of the subsoil (>0.45 m-1.0 m). Forests converted to seasonal cultivation (upland rice and vegetables) caused higher degradation of soil C forms and overall soil health in the Himalayan foothills of northeastern India. We suggest the promotion of Agroforestry based on legumes (Parkia spp.) and woody fruits (mango/citrus/guava) in the uplands to minimize soil C degradation while ensuring nutritional security in the hill agro-ecosystems of the Indian Himalayas.

Effect of land-use change along altitudinal gradients on soil micronutrients in the mountain ecosystem of Indian (Eastern) Himalaya.

Management of soil micronutrients for better crop production needs a sound understanding of their status and causes of variability. This is more relevant for acid soils of the mountain ecosystem of Eastern Himalaya (Northeast India). We assessed the status, and the effect of land uses along altitudinal gradients (14 to 4090 masl) on soil properties and micronutrient concentrations (DTPA extractable Fe, Mn, Cu, and Zn) across the region. Soils varied widely in micronutrient concentrations: Fe from 0.665 to 257.1 mg kg-1 while Mn, Cu, and Zn from traces to 93.4, 17.1, and 34.2 mg kg-1, respectively. On conversion of evergreen forests (EF) to upland agriculture (Shifting-SC and Settled-SA) and plantation (PH), Mn, Cu, and Zn concentrations decreased significantly from 30.5, 1.74, and 2.13 mg kg-1 to 6.44-17.8, 0.68-0.81, and 1.06-1.42 mg kg-1, respectively. Grassland (GL) and lowland paddy (LP) had comparable Fe, Mn, and Cu concentrations (except Zn). Degradation of EF to scrubland (SL) recorded the lowest Mn (5.91 mg kg-1), Cu (0.59 mg kg-1), and Zn (0.68 mg kg-1) concentrations. Fe concentration was however increased in degraded SL (+ 73%) over EF (48.7 mg kg-1). The distribution of micronutrients among the land uses was inconsistent and followed the order: (i) Fe: SL > PH > LP > EF > GL > SC > SA, (ii) Mn: EF > GL > LP > PH > SC > SA > SL; (iii) Cu: EF > GL > LP > SC > SA = PH > SL; and (iv) Zn: GL > EF > LP > SC > SA > PH > SL. Four micronutrients responded differently and followed a non-linear, 6th-order polynomial trend along the altitudinal gradients (< 500 to 4100 masl). Peak concentrations of Fe, Mn, and Cu were recorded at 1001-2000 m while Zn was recorded at > 4000 masl. The variability (54-64%) in soil micronutrients was mainly controlled by three key soil properties: acidity, clay, and organic carbon contents. Thus, altitude-specific land-use management holds significance in the distribution of available soil micronutrients in hilly ecosystems.

ROS induces NETosis by oxidizing DNA and initiating DNA repair.

Reactive oxygen species (ROS) are essential for neutrophil extracellular trap (NET) formation or NETosis. Nevertheless, how ROS induces NETosis is unknown. Neutrophil activation induces excess ROS production and a meaningless genome-wide transcription to facilitate chromatin decondensation. Here we show that the induction of NADPH oxidase-dependent NETosis leads to extensive DNA damage, and the subsequent translocation of proliferating cell nuclear antigen (PCNA), a key DNA repair protein, stored in the cytoplasm into the nucleus. During the activation of NETosis (e.g., by phorbol myristate acetate, Escherichia coli LPS, Staphylococcus aureus (RN4220), or Pseudomonas aeruginosa), preventing the DNA-repair-complex assembly leading to nick formation that decondenses chromatin causes the suppression of NETosis (e.g., by inhibitors to, or knockdown of, Apurinic endonuclease APE1, poly ADP ribose polymerase PARP, and DNA ligase). The remaining repair steps involving polymerase activity and PCNA interactions with DNA polymerases ß/δ do not suppress agonist-induced NETosis. Therefore, excess ROS produced during neutrophil activation induces NETosis by inducing extensive DNA damage (e.g., oxidising guanine to 8-oxoguanine), and the subsequent DNA repair pathway, leading to chromatin decondensation.

Orchestration of Neutrophil Extracellular Traps (Nets), a Unique Innate Immune Function during Chronic Obstructive Pulmonary Disease (COPD) Development.

Morbidity, mortality and economic burden caused by chronic obstructive pulmonary disease (COPD) is a significant global concern. Surprisingly, COPD is already the third leading cause of death worldwide, something that WHO had not predicted to occur until 2030. It is characterized by persistent respiratory symptoms and airway limitation due to airway and/or alveolar abnormalities usually caused by significant exposure to noxious particles of gases. Neutrophil is one of the key infiltrated innate immune cells in the lung during the pathogenesis of COPD. Neutrophils during pathogenic attack or injury decide to undergo for a suicidal death by releasing decondensed chromatin entangled with antimicrobial peptides to trap and ensnare pathogens. Casting neutrophil extracellular traps (NETs) has been widely demonstrated to be an effective mechanism against invading microorganisms thus controlling overwhelming infections. However, aberrant and massive NETs formation has been reported in several pulmonary diseases, including chronic obstructive pulmonary disease. Moreover, NETs can directly induce epithelial and endothelial cell death resulting in impairing pulmonary function and accelerating the progression of the disease. Therefore, understanding the regulatory mechanism of NET formation is the need of the hour in order to use NETs for beneficial purpose and controlling their involvement in disease exacerbation. For example, DNA neutralization of NET proteins using protease inhibitors and disintegration with recombinant human DNase would be helpful in controlling excess NETs. Targeting CXC chemokine receptor 2 (CXCR2) would also reduce neutrophilic inflammation, mucus production and neutrophil-proteinase mediated tissue destruction in lung. In this review, we discuss the interplay of NETs in the development and pathophysiology of COPD and how these NETs associated therapies could be leveraged to disrupt NETopathic inflammation as observed in COPD, for better management of the disease.

Comparing and Contrasting MERS, SARS-CoV, and SARS-CoV-2: Prevention, Transmission, Management, and Vaccine Development.

The COVID-19 pandemic is responsible for an unprecedented disruption to the healthcare systems and economies of countries around the world. Developing novel therapeutics and a vaccine against SARS-CoV-2 requires an understanding of the similarities and differences between the various human coronaviruses with regards to their phylogenic relationships, transmission, and management. Phylogenetic analysis indicates that humans were first infected with SARS-CoV-2 in late 2019 and the virus rapidly spread from the outbreak epicenter in Wuhan, China to various parts of the world. Multiple variants of SARS-CoV-2 have now been identified in particular regions. It is apparent that MERS, SARS-CoV, and SARS-CoV-2 present with several common symptoms including fever, cough, and dyspnea in mild cases, but can also progress to pneumonia and acute respiratory distress syndrome. Understanding the molecular steps leading to SARS-CoV-2 entry into cells and the viral replication cycle can illuminate crucial targets for testing several potential therapeutics. Genomic and structural details of SARS-CoV-2 and previous attempts to generate vaccines against SARS-CoV and MERS have provided vaccine targets to manage future outbreaks more effectively. The coordinated global response against this emerging infectious disease is unique and has helped address the need for urgent therapeutics and vaccines in a remarkably short time.

Potential Mechanism of Dermal Wound Treatment With Preparations From the Skin Gel of Arabian Gulf Catfish: A Unique Furan Fatty Acid (F6) and Cholesta-3,5-Diene (S5) Recruit Neutrophils and Fibroblasts to Promote Wound Healing.

Preparations from Arabian Gulf catfish (Arius bilineatus, Val) epidermal gel secretion (PCEGS) effectively heal chronic wounds in diabetic patients. However, specific lipid components of PCEGS that are responsible for various aspects of wound healing are unknown. Here, we report for the first time that, i) a unique preparation containing only proteins and lipids (Fraction B, FB), derived from the PCEGS accelerated the healing of experimental dermal wounds in female rats (transdermal punch biopsy) in vivo. Histological analyses showed that topical treatment of these wounds with FB promoted the migration of fibroblasts, facilitated the production of extracellular matrix (collagen, fibronectin), induced capillary formation and recruitment of immune cells, and accelerated overall wound healing by day 4 (tested at 1, 2, 3, 4, and 10 days; n=15 for vehicle; n=15 for FB treatment), ii) the lipids responsible for different stages of wound healing were separated into a protein-free bioactive lipid fraction, Ft, which contained a few common long-chain fatty acids, a unique furan fatty acid (F6) and a cholesterol metabolite, cholesta-3,5-diene (S5). Ft (the partially purified lipid fraction of PCEGS), and F6 and S5 present in Ft, proved to be bioactive for wound healing in human dermal fibroblasts. Ft increased the production and extracellular deposition of collagen and fibronectin, ex vivo, iii) Ft and its subcomponents, pure F6 and S5, also promoted human dermal fibroblast migration into the scratch wound gaps, ex vivo, iv) Ft, F6, and S5 promoted the recruitment of neutrophils (Green fluorescence protein labeled) to the site of injury in the transected tailfins of transgenic zebrafish, in vivo, v) Ft, but not F6 or S5, promoted the regeneration of tissues at the wound site in the transgenic zebrafish tailfin, in vivo. Therefore, we conclude that lipid fraction Ft from PCEGS contains the components necessary to promote complete wound healing, and F6 and S5 are responsible for promoting fibroblast and neutrophil recruitment to the site of wounds.

Understanding the interaction between α-1-acid glycoprotein (AGP) and potential Cu/Zn metallo-drugs of benzimidazole derived organic motifs: A multi-spectroscopic and molecular docking study.

Drug-binding and interactions with plasma proteins strongly affect their efficiency of delivery, hence considered as a key factor in determining the overall pharmacological action. Alpha-1-acid glycoprotein (AGP), a second most abundant plasma protein in blood circulation, has unique drug binding ability and involved in the transportation of various compounds. Here, we have investigated the mechanism of interaction between AGP and potential Cu/Zn metallo-drugs of benzimidazole derived organic motifs (CuL2 and ZnL2, where L is Schiff base ligand) by applying integrated spectroscopic, biophysical techniques and computational molecular docking analyses. We found that both the metallo-drugs (CuL2 and ZnL2) were bound at the central cavity of AGP interacting with the residues of lobe I, lobe II as well as lobe III. The binding of metallo-drugs to AGP occurs in 1:1 M ratios. Hydrogen bonding, electrostatic and hydrophobic interactions played a significant role in stabilizing the AGP-metallo-drug complexes. Binding affinities of both the metallo-drugs towards AGP at 298 K were of the order of 104-105 M-1, corresponding to Gibbs free energy of stabilization of approximately -5.50 to -6.62 kcal mol-1. Furthermore, the spectroscopic investigation by circular dichroism and synchronous fluorescence analyses suggest conformational changes in AGP upon the binding of metallic compounds.

Genetic Variability Based on Tandem Repeat Numbers in a Genomic Locus of 'Candidatus Liberibacter asiaticus' Prevalent in North East India.

The genetic variability of 'Candidatus Liberibacter asiaticus' (CLas) population associated with huanglongbing (HLB) disease of citrus in North Eastern (NE) region of India, a geographically locked region, and home for the diversity of many citrus species was analyzed on the basis of tandem repeat numbers (TRN) in variable CLIBASIA_01645 genomic loci. Fifty-five CLas strains sampled from different groves of NE Hill (NEH) region of India were in single amplicon group, but there was remarkable genetic variability in TRNs. The TRN in HLB-associated CLas strains varied from 0-21 and two novel repeat motifs were also identified. Among the NE population of CLas, TRN5 and TRN9 were most frequent (total frequency of 36.36%) followed by TRN4 (14.55%) and TRN6, TNR7 with a frequency of 12.73% each. Class II type CLas genotypes (5 < TRN ≤ 10) had highest prevalence (frequency of 60.00%) in the samples characterized in present study. Class I (TRN ≤ 5) genotypes were second highest prevalent (29.09%) in the NEH region. Further analysis of genetic diversity parameters using Nei's measure (H value) indicated wide genetic diversity in the CLas strains of NE India (H value of 0.58-0.86). Manipur CLas strains had highest genetic variability (0.86) as compared to Eastern, Southern and Central India. The R10 values (TRN ≤ 10/TRN > 10) of NE CLas population was 10.43 (73/7), higher from other regions of India. Present study conclusively reported the occurrence of high genetic variability in TRN of CLas population in North East Indian citrus groves which have evolved to adapt to the specific ecological niche.

Anthracyclines Suppress Both NADPH Oxidase- Dependent and -Independent NETosis in Human Neutrophils.

Neutrophil extracellular traps (NETs) are cytotoxic DNA-protein complexes that play positive and negative roles in combating infection, inflammation, organ damage, autoimmunity, sepsis and cancer. However, NETosis regulatory effects of most of the clinically used drugs are not clearly established. Several recent studies highlight the relevance of NETs in promoting both cancer cell death and metastasis. Here, we screened the NETosis regulatory ability of 126 compounds belonging to 39 classes of drugs commonly used for treating cancer, blood cell disorders and other diseases. Our studies show that anthracyclines (e.g., epirubicin, daunorubicin, doxorubicin, and idarubicin) consistently suppress both NADPH oxidase-dependent and -independent types of NETosis in human neutrophils, ex vivo. The intercalating property of anthracycline may be enough to alter the transcription initiation and lead NETosis inhibition. Notably, the inhibitory doses of anthracyclines neither suppress the production of reactive oxygen species that are necessary for antimicrobial functions nor induce apoptotic cell death in neutrophils. Therefore, anthracyclines are a major class of drug that suppresses NETosis. The dexrazoxane, a cardioprotective agent, used for limiting the side effects of anthracyclines, neither affect NETosis nor alter the ability of anthracyclines to suppress NETosis. Hence, at correct doses, anthracyclines together with dexrazoxane could be considered as a therapeutic candidate drug for suppressing unwanted NETosis in NET-related diseases.

Neutrophil Extracellular Trap Formation: Physiology, Pathology, and Pharmacology.

Neutrophil extracellular traps (NETs), a unique DNA framework decorated with antimicrobial peptides, have been in the scientific limelight for their role in a variety of pathologies ranging from cystic fibrosis to cancer. The formation of NETs, as well as relevant regulatory mechanisms, physiological factors, and pharmacological agents have not been systematically discussed in the context of their beneficial and pathological aspects. Novel forms of NET formation including vital NET formation continue to be uncovered, however, there remain fundamental questions around established mechanisms such as NADPH-oxidase (Nox)-dependent and Nox-independent NET formation. Whether NET formation takes place in the tissue versus the bloodstream, internal factors (e.g. reactive oxygen species (ROS) production and transcription factor activation), and external factors (e.g. alkaline pH and hypertonic conditions), have all been demonstrated to influence specific NET pathways. Elements of neutrophil biology such as transcription and mitochondria, which were previously of unknown significance, have been identified as critical mediators of NET formation through facilitating chromatin decondensation and generating ROS, respectively. While promising therapeutics inhibiting ROS, transcription, and gasdermin D are being investigated, neutrophil phagocytosis plays a critical role in host defense and any therapies targeting NET formation must avoid impairing the physiological functions of these cells. This review summarizes what is known in the many domains of NET research, highlights the most relevant challenges in the field, and inspires new questions that can bring us closer to a unified model of NET formation.

Furanoic Lipid F-6, A Novel Anti-Cancer Compound that Kills Cancer Cells by Suppressing Proliferation and Inducing Apoptosis.

Identifying novel anti-cancer drugs is important for devising better cancer treatment options. In a series of studies designed to identify novel therapeutic compounds, we recently showed that a C-20 fatty acid (12,15-epoxy-13,14-dimethyleicosa-12,14-dienoic acid, a furanoic acid or F-6) present in the lipid fraction of the secretions of the Arabian Gulf catfish skin (Arius bilineatus Val.; AGCS) robustly induces neutrophil extracellular trap formation. Here, we demonstrate that a lipid mix (Ft-3) extracted from AGCS and F-6, a component of Ft-3, dose dependently kill two cancer cell lines (leukemic K-562 and breast MDA MB-231). Pure F-6 is approximately 3.5 to 16 times more effective than Ft-3 in killing these cancer cells, respectively. Multiplex assays and network analyses show that F-6 promotes the activation of MAPKs such as Erk, JNK, and p38, and specifically suppresses JNK-mediated c-Jun activation necessary for AP-1-mediated cell survival pathways. In both cell lines, F-6 suppresses PI3K-Akt-mTOR pathway specific proteins, indicating that cell proliferation and Akt-mediated protection of mitochondrial stability are compromised by this treatment. Western blot analyses of cleaved caspase 3 (cCasp3) and poly ADP ribose polymerase (PARP) confirmed that F-6 dose-dependently induced apoptosis in both of these cell lines. In 14-day cell recovery experiments, cells treated with increasing doses of F-6 and Ft-3 fail to recover after subsequent drug washout. In summary, this study demonstrates that C-20 furanoic acid F-6, suppresses cancer cell proliferation and promotes apoptotic cell death in leukemic and breast cancer cells, and prevents cell recovery. Therefore, F-6 is a potential anti-cancer drug candidate.

Progression of Cystic Fibrosis Lung Disease from Childhood to Adulthood: Neutrophils, Neutrophil Extracellular Trap (NET) Formation, and NET Degradation.

Genetic defects in cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene cause CF. Infants with CFTR mutations show a peribronchial neutrophil infiltration prior to the establishment of infection in their lung. The inflammatory response progressively increases in children that include both upper and lower airways. Infectious and inflammatory response leads to an increase in mucus viscosity and mucus plugging of small and medium-size bronchioles. Eventually, neutrophils chronically infiltrate the airways with biofilm or chronic bacterial infection. Perpetual infection and airway inflammation destroy the lungs, which leads to increased morbidity and eventual mortality in most of the patients with CF. Studies have now established that neutrophil cytotoxins, extracellular DNA, and neutrophil extracellular traps (NETs) are associated with increased mucus clogging and lung injury in CF. In addition to opportunistic pathogens, various aspects of the CF airway milieux (e.g., airway pH, salt concentration, and neutrophil phenotypes) influence the NETotic capacity of neutrophils. CF airway milieu may promote the survival of neutrophils and eventual pro-inflammatory aberrant NETosis, rather than the anti-inflammatory apoptotic death in these cells. Degrading NETs helps to manage CF airway disease; since DNAse treatment release cytotoxins from the NETs, further improvements are needed to degrade NETs with maximal positive effects. Neutrophil-T cell interactions may be important in regulating viral infection-mediated pulmonary exacerbations in patients with bacterial infections. Therefore, clarifying the role of neutrophils and NETs in CF lung disease and identifying therapies that preserve the positive effects of neutrophils, while reducing the detrimental effects of NETs and cytotoxic components, are essential in achieving innovative therapeutic advances.

Biosynthesized Silver Nanoparticle (AgNP) From Pandanus odorifer Leaf Extract Exhibits Anti-metastasis and Anti-biofilm Potentials.

Cancer and the associated secondary bacterial infections are leading cause of mortality, due to the paucity of effective drugs. Here, we have synthesized silver nanoparticles (AgNPs) from organic resource and confirmed their anti-cancer and anti-microbial potentials. Microwave irradiation method was employed to synthesize AgNPs using Pandanus odorifer leaf extract. Anti-cancer potential of AgNPs was evaluated by scratch assay on the monolayer of rat basophilic leukemia (RBL) cells, indicating that the synthesized AgNPs inhibit the migration of RBL cells. The synthesized AgNPs showed MIC value of 4-16 µg/mL against both Gram +ve and Gram -ve bacterial strains, exhibiting the anti-microbial potential. Biofilm inhibition was recorded at sub-MIC values against Gram +ve and Gram -ve bacterial strains. Violacein and alginate productions were reduced by 89.6 and 75.6%, respectively at 4 and 8 µg/mL of AgNPs, suggesting anti-quorum sensing activity. Exopolysaccharide production was decreased by 61-79 and 84% for Gram -ve and Gram +ve pathogens respectively. Flagellar driven swarming mobility was also reduced significantly. Furthermore, In vivo study confirmed their tolerability in mice, indicating their clinical perspective. Collective, we claim that the synthesized AgNPs have anti-metastasis as well as anti-microbial activities. Hence, this can be further tested for therapeutic options to treat cancer and secondary bacterial infections.

High-Throughput Virtual Screening, Molecular Dynamics Simulation, and Enzyme Kinetics Identified ZINC84525623 as a Potential Inhibitor of NDM-1.

The bacteria expressing New Delhi Metallo-ß-lactamase-1 (NDM-1) can hydrolyze all ß-lactam antibiotics including carbapenems, causing multi-drug resistance. The worldwide emergence and dissemination of gene blaNDM-1 (produces NDM-1) in hospital and community settings, rising problems for public health. Indeed, there is an urgent need for NDM-1 inhibitors to manage antibiotic resistance. Here, we have identified novel non-ß-lactam ring-containing inhibitors of NDM-1 by applying a high-throughput virtual screening of lead-like subset of ZINC database. The screened compounds were followed for the molecular docking, the molecular dynamics simulation, and then enzyme kinetics assessment. The adopted screening procedure funnels out five novel inhibitors of NDM-1 including ZINC10936382, ZINC30479078, ZINC41493045, ZINC7424911, and ZINC84525623. The molecular mechanics-generalized born surface area and molecular dynamics (MD) simulation showed that ZINC84525623 formed the most stable complex with NDM-1. Furthermore, analyses of the binding pose after MD simulation revealed that ZINC84525623 formed two hydrogen bonds (electrostatic and hydrophobic interaction) with key amino acid residues of the NDM-1 active site. The docking binding free energy and docking binding constant for the ZINC84525623 and NDM-1 interaction were estimated to be -11.234 kcal/mol, and 1.74 × 108 M-1 respectively. Steady-state enzyme kinetics in the presence of ZINC84525623 show the decreased catalytic efficiency (i.e., kcat/Km) of NDM-1 on various antibiotics. The findings of this study would be helpful in identifying novel inhibitors against other ß-lactamases from a pool of large databases. Furthermore, the identified inhibitor (ZINC84525623) could be developed as efficient drug candidates.

Histone Acetylation Promotes Neutrophil Extracellular Trap Formation.

Neutrophils undergo a unique form of cell death to generate neutrophil extracellular traps (NETs). It is well established that citrullination of histones (e.g., CitH3) facilitates chromatin decondensation during NET formation (NETosis), particularly during calcium-induced NETosis that is independent of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activation. However, the importance of other forms of histone modifications in NETosis has not been established. We considered that acetylation of histones would also facilitate NETosis. To test this hypothesis, we induced NOX-dependent NETosis in human neutrophils with phorbol myristate acetate or lipopolysaccharide (from Escherichia coli 0128), and NOX-independent NETosis with calcium ionophores A23187 or ionomycin (from Streptomycesconglobatus) in the presence or absence of two pan histone deacetylase inhibitors (HDACis), belinostat and panobinostat (within their half maximal inhibitory concentration (IC50) range). The presence of these inhibitors increased histone acetylation (e.g., AcH4) in neutrophils. Histone acetylation was sufficient to cause a significant increase (~20%) in NETosis in resting neutrophils above baseline values. When acetylation was promoted during NOX-dependent or -independent NETosis, the degree of NETosis additively increased (~15⁻30%). Reactive oxygen species (ROS) production is essential for baseline NETosis (mediated either by NOX or mitochondria); however, HDACis did not promote ROS production. The chromatin decondensation step requires promoter melting and transcriptional firing in both types of NETosis; consistent with this point, suppression of transcription prevented the NETosis induced by the acetylation of histones. Collectively, this study establishes that histone acetylation (e.g., AcH4) promotes NETosis at baseline, and when induced by both NOX-dependent or -independent pathway agonists, in human neutrophils. Therefore, we propose that acetylation of histone is a key component of NETosis.

Furanoid F-Acid F6 Uniquely Induces NETosis Compared to C16 and C18 Fatty Acids in Human Neutrophils.

Various biomolecules induce neutrophil extracellular trap (NET) formation or NETosis. However, the effect of fatty acids on NETosis has not been clearly established. In this study, we focused on the NETosis-inducing ability of several lipid molecules. We extracted the lipid molecules present in Arabian Gulf catfish (Arius bilineatus, Val) skin gel, which has multiple therapeutic activities. Gas chromatography⁻mass spectrometry (GC-MS) analysis of the lipid fraction-3 from the gel with NETosis-inducing activity contained fatty acids including a furanoid F-acid (F6; 12,15-epoxy-13,14-dimethyleicosa-12,14-dienoic acid) and common long-chain fatty acids such as palmitic acid (PA; C16:0), palmitoleic acid (PO; C16:1), stearic acid (SA; C18:0), and oleic acid (OA; C18:1). Using pure molecules, we show that all of these fatty acids induce NETosis to different degrees in a dose-dependent fashion. Notably, F6 induces a unique form of NETosis that is rapid and induces reactive oxygen species (ROS) production by both NADPH oxidase (NOX) and mitochondria. F6 also induces citrullination of histone. By contrast, the common fatty acids (PA, PO, SA, and OA) only induce NOX-dependent NETosis. The activation of the kinases such as ERK (extracellular signal-regulated kinase) and JNK (c-Jun N-terminal kinase) is important for long-chain fatty acid-induced NETosis, whereas, in F-acid-induced NETosis, Akt is additionally needed. Nevertheless, NETosis induced by all of these compounds requires the final chromatin decondensation step of transcriptional firing. These findings are useful for understanding F-acid- and other fatty acid-induced NETosis and to establish the active ingredients with therapeutic potential for regulating diseases involving NET formation.

Feather degrading, phytostimulating, and biocontrol potential of native actinobacteria from North Eastern Indian Himalayan Region.

Twelve actinobacterial strains were isolated from tomato rhizospheric soil from Manipur, a state in North East Indian Himalayan Region and screened for keratinolytic and plant growth promoting traits. Nine promising isolates were identified as Streptomyces species using partial 16S rRNA gene sequencing. Among the seven isolates showing chicken feather degradation activity, three keratinolytic strains RCM-SSR-2, -6, and -12 were found to be the most efficient feather degrading strains achieving 90% feather weight loss within 48 h of incubation. They also showed maximum keratinase and soluble peptide production. Strain RCM-SSR-2, -5, -6, -8, and -11 showed positive results for all plant growth promoting traits tested. Maximum indole-3-acetic acid production was exhibited by RCM-SSR-6. Strain RCM-SSR-1, -2, -5, -6, -9, and -11 showed antagonistic activity against three important plant pathogens. Feather hydrolysate of RCM-SSR-6 was also evaluated for in vitro seed germination test using garden pea seeds. Higher concentration of feather protein hydrolysate (3 mg ml-1 ) inhibited shoot and root length of the germinating embryo. However, lower concentration (0.01 mg ml-1 ) of feather protein hydrolysate promoted seed germination. Among the 12 strains, four isolates namely RCM-SSR-1, -2, -5, and -6 were found to be promising as multi-traits plant growth promoting rhizobacteria for development of organic fertilizer, phytostimulator, and biocontrol agents.

Two-in-one: UV radiation simultaneously induces apoptosis and NETosis.

NETosis is a unique form of neutrophil death that differs from apoptosis and necrosis. However, whether NETosis and apoptosis can occur simultaneously in the same neutrophil is unknown. In this paper, we show that increasing doses of ultraviolet (UV) irradiation increases NETosis, which is confirmed by myeloperoxidase colocalisation to neutrophil extracellular DNA. Increasing UV irradiation increases caspase 3 activation, mitochondrial reactive oxygen species (ROS) generation and p38, but not ERK, phosphorylation. Inhibition of mitochondrial ROS production and p38 activation, but not NADPH oxidase (NOX) activity, suppresses UV-induced NETosis, indicating that UV induces NOX-independent NETosis. Like classical NOX-dependent and -independent NETosis, UV-induced NETosis requires transcriptional firing for chromatin decondensation. Cell death-specific inhibitor studies indicate that UV-mediated NETosis is not apoptosis, necrosis or necroptosis. Collectively, these studies indicate that increasing doses of UV irradiation induce both apoptosis and NETosis simultaneously, but the ultimate outcome is the induction of a novel form of NOX-independent NETosis, or "ApoNETosis".

Hypertonic Saline Suppresses NADPH Oxidase-Dependent Neutrophil Extracellular Trap Formation and Promotes Apoptosis.

Tonicity of saline (NaCl) is important in regulating cellular functions and homeostasis. Hypertonic saline is administered to treat many inflammatory diseases, including cystic fibrosis. Excess neutrophil extracellular trap (NET) formation, or NETosis, is associated with many pathological conditions including chronic inflammation. Despite the known therapeutic benefits of hypertonic saline, its underlying mechanisms are not clearly understood. Therefore, we aimed to elucidate the effects of hypertonic saline in modulating NETosis. For this purpose, we purified human neutrophils and induced NETosis using agonists such as diacylglycerol mimetic phorbol myristate acetate (PMA), Gram-negative bacterial cell wall component lipopolysaccharide (LPS), calcium ionophores (A23187 and ionomycin from Streptomyces conglobatus), and bacteria (Pseudomonas aeruginosa and Staphylococcus aureus). We then analyzed neutrophils and NETs using Sytox green assay, immunostaining of NET components and apoptosis markers, confocal microscopy, and pH sensing reagents. This study found that hypertonic NaCl suppresses nicotinamide adenine dinucleotide phosphate oxidase (NADPH2 or NOX2)-dependent NETosis induced by agonists PMA, Escherichia coli LPS (0111:B4 and O128:B12), and P. aeruginosa. Hypertonic saline also suppresses LPS- and PMA- induced reactive oxygen species production. It was determined that supplementing H2O2 reverses the suppressive effect of hypertonic saline on NOX2-dependent NETosis. Many of the aforementioned suppressive effects were observed in the presence of equimolar concentrations of choline chloride and osmolytes (d-mannitol and d-sorbitol). This suggests that the mechanism by which hypertonic saline suppresses NOX2-dependent NETosis is via neutrophil dehydration. Hypertonic NaCl does not significantly alter the intracellular pH of neutrophils. We found that hypertonic NaCl induces apoptosis while suppressing NOX2-dependent NETosis. In contrast, hypertonic solutions do not suppress NOX2-independent NETosis. Although hypertonic saline partially suppresses ionomycin-induced NETosis, it enhances A23187-induced NETosis, and it does not alter S. aureus-induced NETosis. Overall, this study determined that hypertonic saline suppresses NOX2-dependent NETosis induced by several agonists; in contrast, it has variable effects on neutrophil death induced by NOX2-independent NETosis agonists. These findings are important in understanding the regulation of NETosis and apoptosis in neutrophils.