Phagocytosis and self-destruction break down dendrites of Drosophila sensory neurons at distinct steps of Wallerian degeneration.

After injury, severed dendrites and axons expose the "eat-me" signal phosphatidylserine (PS) on their surface while they break down. The degeneration of injured axons is controlled by a conserved Wallerian degeneration (WD) pathway, which is thought to activate neurite self-destruction through Sarm-mediated nicotinamide adenine dinucleotide (NAD+) depletion. While neurite PS exposure is known to be affected by genetic manipulations of NAD+, how the WD pathway coordinates both neurite PS exposure and self-destruction and whether PS-induced phagocytosis contributes to neurite breakdown in vivo remain unknown. Here, we show that in Drosophila sensory dendrites, PS exposure and self-destruction are two sequential steps of WD resulting from Sarm activation. Surprisingly, phagocytosis is the main driver of dendrite degeneration induced by both genetic NAD+ disruptions and injury. However, unlike neuronal Nmnat loss, which triggers PS exposure only and results in phagocytosis-dependent dendrite degeneration, injury activates both PS exposure and self-destruction as two redundant means of dendrite degeneration. Furthermore, the axon-death factor Axed is only partially required for self-destruction of injured dendrites, acting in parallel with PS-induced phagocytosis. Lastly, injured dendrites exhibit a unique rhythmic calcium-flashing that correlates with WD. Therefore, both NAD+-related general mechanisms and dendrite-specific programs govern PS exposure and self-destruction in injury-induced dendrite degeneration in vivo.

Versatile CRISPR/Cas9-mediated mosaic analysis by gRNA-induced crossing-over for unmodified genomes.

Mosaic animals have provided the platform for many fundamental discoveries in developmental biology, cell biology, and other fields. Techniques to produce mosaic animals by mitotic recombination have been extensively developed in Drosophila melanogaster but are less common for other laboratory organisms. Here, we report mosaic analysis by gRNA-induced crossing-over (MAGIC), a new technique for generating mosaic animals based on DNA double-strand breaks produced by CRISPR/Cas9. MAGIC efficiently produces mosaic clones in both somatic tissues and the germline of Drosophila. Further, by developing a MAGIC toolkit for 1 chromosome arm, we demonstrate the method's application in characterizing gene function in neural development and in generating fluorescently marked clones in wild-derived Drosophila strains. Eliminating the need to introduce recombinase-recognition sites into the genome, this simple and versatile system simplifies mosaic analysis in Drosophila and can in principle be applied in any organism that is compatible with CRISPR/Cas9.

Isolation and comparative analysis of culturable bacterial communities associated with life stages, breeding and rearing substrates of Culicoides oxystoma Kieffer (Diptera: Ceratopogonidae) vector of bluetongue virus.

Culicoides oxystoma Kieffer (Diptera: Ceratopogonidae) has been vectoring several arboviruses, protozoa and nematodes, leading to mortality and morbidity of livestock and wild ruminants in the tropics and subtropics. Insight into the bacterial communities associated with the vector species must be worked out. This work tries to inventorize the bacterial communities associated with this important vector species. Acquisition of gut microbiota may be the parental origin, while some are obtained through feeding during larval stages. Culicoides oxystoma possesses semi-aquatic life cycle strategies for egg-laying and larval survival. The bacteria associated with C. oxystoma were compared throughout (i) life stages: egg, larval instars, pupa, adult: male and female obtained from laboratory colony; (ii) field-collected adult: male and age-graded females; and (iii) natural breeding substrate and artificial rearing substrate. The culture-dependent bacteria were identified by Sanger sequencing of 16S rRNA, and haemolytic bacteria were screened on blood agar. Results show that Firmicutes and Proteobacteria are the predominant Phyla, of which Bacillus spp. was the most abundant across the life stages. Across the life history, Bacillus cereus, Bacillus pumilus, Bacillus tropicus, Lysinibacillus sp. and Paenibacillus sp. were retrieved routinely. Bacillus cereus and Alcaligenes faecalis were detected in the lab-reared specimens and shared between the natural breeding site and rearing medium. From the adults trapped across two locations, B. cereus, Bacillus flexus, A. faecalis, Enterococcus faecium and Pseudomonas sp. were isolated. The bacterial species associated with this vector may influence various physiological traits, such as vectorial capacity, digestion and larval development, which need further investigation.

Silver nanostructure-modified graphite electrode for in-operando SERRS investigation of iron porphyrins during high-potential electrocatalysis.

In-operando spectroscopic observation of the intermediates formed during various electrocatalytic oxidation and reduction reactions is crucial to propose the mechanism of the corresponding reaction. Surface-enhanced resonance Raman spectroscopy coupled to rotating disk electrochemistry (SERRS-RDE), developed about a decade ago, proved to be an excellent spectroscopic tool to investigate the mechanism of heterogeneous oxygen reduction reaction (ORR) catalyzed by synthetic iron porphyrin complexes under steady-state conditions in water. The information about the formation of the intermediates accumulated during the course of the reaction at the electrode interface helped to develop better ORR catalysts with second sphere residues in the porphyrin rings. To date, the application of this SERRS-RDE setup is limited to ORR only because the thiol self-assembled monolayer (SAM)-modified Ag electrode, used as the working electrode in these experiments, suffers from stability issues at more cathodic and anodic potential, where H2O oxidation, CO2 reduction, and H+ reduction reactions occur. The current investigation shows the development of a second-generation SERRS-RDE setup consisting of an Ag nanostructure (AgNS)-modified graphite electrode as the working electrode. These electrodes show higher stability (compared to the conventional thiol SAM-modified Ag electrode) upon exposure to very high cathodic and anodic potential with a good signal-to-noise ratio in the Raman spectra. The behavior of this modified electrode toward ORR is found to be the same as the SAM-modified Ag electrode, and the same ORR intermediates are observed during electrochemical ORR. At higher cathodic potential, the signatures of Fe(0) porphyrin, an important intermediate in H+ and CO2 reduction reactions, was observed at the electrode-water interface.

Effect of substrate salinity and pH on life history traits of the bluetongue virus vector Culicoides peregrinus.

Habitat selection of Culicoides spp. (Diptera: Ceratopogonidae) is influenced by the physicochemical factors such as temperature, pH, salinity, moisture, conductivity, organic and inorganic compounds of substrates. These factors determine the life history traits of the vectors. We studied the influence of substrate salinity (0-40 parts per thousand, ppt) and pH (pH 1-13) on oviposition, egg hatching, larval survivability, and adult emergence of Culicoides peregrinus Kieffer under laboratory conditions. Most eggs (80.74%) were laid in 0 ppt and 95% in pH 7 but lowered with increased salinity and pH levels. It was observed that the females did not lay eggs in 30 ppt to 40 ppt salinity; pH 1 and pH 13 but interestingly up to 95% of the eggs were retained within the abdomen. Little effect of salinity and pH on egg hatching was observed up to 5 ppt and 10 ppt except at the extreme values of 40 ppt and pH 1, pH 13. Pupation did not occur in rearing plates with high salinities, 30 ppt and 40 ppt, although the few eggs hatched when exposed to such salinity. In low salinity (0 to 2 ppt), occurrence of adult emergence was more and then decreased with increasing salinity. Maximum emergence was seen when the rearing media was alkaline. This study deals with the suitability of breeding substrate of C. peregrinus when exposed to salinity and pH ranges. Our study suggests the ambient salinity and pH ranges to be maintained during laboratory rearing of this vector species.

Inactivation of Hippo and cJun-N-terminal Kinase (JNK) signaling mitigate FUS mediated neurodegeneration in vivo.

Amyotrophic Lateral Sclerosis (ALS), a late-onset neurodegenerative disorder characterized by the loss of motor neurons in the central nervous system, has no known cure to-date. Disease causing mutations in human Fused in Sarcoma (FUS) leads to aggressive and juvenile onset of ALS. FUS is a well-conserved protein across different species, which plays a crucial role in regulating different aspects of RNA metabolism. Targeted misexpression of FUS in Drosophila model recapitulates several interesting phenotypes relevant to ALS including cytoplasmic mislocalization, defects at the neuromuscular junction and motor dysfunction. We screened for the genetic modifiers of human FUS-mediated neurodegenerative phenotype using molecularly defined deficiencies. We identified hippo (hpo), a component of the evolutionarily conserved Hippo growth regulatory pathway, as a genetic modifier of FUS mediated neurodegeneration. Gain-of-function of hpo triggers cell death whereas its loss-of-function promotes cell proliferation. Downregulation of the Hippo signaling pathway, using mutants of Hippo signaling, exhibit rescue of FUS-mediated neurodegeneration in the Drosophila eye, as evident from reduction in the number of TUNEL positive nuclei as well as rescue of axonal targeting from the retina to the brain. The Hippo pathway activates c-Jun amino-terminal (NH2) Kinase (JNK) mediated cell death. We found that downregulation of JNK signaling is sufficient to rescue FUS-mediated neurodegeneration in the Drosophila eye. Our study elucidates that Hippo signaling and JNK signaling are activated in response to FUS accumulation to induce neurodegeneration. These studies will shed light on the genetic mechanism involved in neurodegeneration observed in ALS and other associated disorders.

Spectroscopic and magnetic investigations of a spin-frustrated Mn-doped CoAl2O4 spinel.

Single-phase polycrystalline spin-frustrated spinel oxides Co1-xMnxAl2O4 (0 ≤ x ≤ 0.3) have been prepared to investigate the optical and magnetic properties. Linear variation of the lattice parameter along with the characteristic hyperfine electron paramagnetic resonance (EPR) signal establish the fact that the Mn2+ ions are incorporated at Co2+ sites of the CoAl2O4 lattice. Optical absorption spectra reveal three absorption features in wavelength regions: 250-400 nm, 500-700 nm and 1000-1700 nm. The optical band gap associated with the d-d transition increases from 1.84 eV to 1.88 eV with 30% Mn substitution. Temperature dependent magnetization measurements indicate a clear transformation of the magnetic ground state from the collinear antiferromagnetic state (for x = 0) to the spin-glass-like state (for x = 0.1) to the cluster-glass-like state (for x = 0.2 and 0.3) with the increase of Mn concentration. In addition, our time dependent isothermal remanent magnetization (IRM) study further fortifies the above transformation of the magnetic ground state. The value of the magnetic frustration parameter moderately decreases with Mn substitution, but the compositional variation is not monotonous.

The Hippo pathway effector Yki downregulates Wg signaling to promote retinal differentiation in the Drosophila eye.

The evolutionarily conserved Hippo signaling pathway is known to regulate cell proliferation and maintain tissue homeostasis during development. We found that activation of Yorkie (Yki), the effector of the Hippo signaling pathway, causes separable effects on growth and differentiation of the Drosophila eye. We present evidence supporting a role for Yki in suppressing eye fate by downregulation of the core retinal determination genes. Other upstream regulators of the Hippo pathway mediate this effect of Yki on retinal differentiation. Here, we show that, in the developing eye, Yki can prevent retinal differentiation by blocking morphogenetic furrow (MF) progression and R8 specification. The inhibition of MF progression is due to ectopic induction of Wingless (Wg) signaling and Homothorax (Hth), the negative regulators of eye development. Modulating Wg signaling can modify Yki-mediated suppression of eye fate. Furthermore, ectopic Hth induction due to Yki activation in the eye is dependent on Wg. Last, using Cut (Ct), a marker for the antennal fate, we show that suppression of eye fate by hyperactivation of yki does not change the cell fate (from eye to antenna-specific fate). In summary, we provide the genetic mechanism by which yki plays a role in cell fate specification and differentiation - a novel aspect of Yki function that is emerging from multiple model organisms.

A potent betulinic acid analogue ascertains an antagonistic mechanism between autophagy and proteasomal degradation pathway in HT-29 cells.

BACKGROUND: Betulinic acid (BA), a member of pentacyclic triterpenes has shown important biological activities like anti-bacterial, anti-malarial, anti-inflammatory and most interestingly anticancer property. To overcome its poor aqueous solubility and low bioavailability, structural modifications of its functional groups are made to generate novel lead(s) having better efficacy and less toxicity than the parent compound. BA analogue, 2c was found most potent inhibitor of colon cancer cell line, HT-29 cells with IC50 value 14.9 µM which is significantly lower than standard drug 5-fluorouracil as well as parent compound, Betulinic acid. We have studied another mode of PCD, autophagy which is one of the important constituent of cellular catabolic system as well as we also studied proteasomal degradation pathway to investigate whole catabolic pathway after exploration of 2c on HT-29 cells. METHODS: Mechanism of autophagic cell death was studied using fluorescent dye like acridine orange (AO) and monodansylcadaverin (MDC) staining by using fluorescence microscopy. Various autophagic protein expression levels were determined by Western Blotting, qRT-PCR and Immunostaining. Confocal Laser Scanning Microscopy (CLSM) was used to study the colocalization of various autophagic proteins. These were accompanied by formation of autophagic vacuoles as revealed by FACS and transmission electron microscopy (TEM). Proteasomal degradation pathway was studied by proteasome-Glo™ assay systems using luminometer. RESULTS: The formation of autophagic vacuoles in HT-29 cells after 2c treatment was determined by fluorescence staining--confirming the occurrence of autophagy. In addition, 2c was found to alter expression levels of different autophagic proteins like Beclin-1, Atg 5, Atg 7, Atg 5-Atg 12, LC3B and autophagic adapter protein, p62. Furthermore we found the formation of autophagolysosome by colocalization of LAMP-1 with LC3B, LC3B with Lysosome, p62 with lysosome. Finally, as proteasomal degradation pathway downregulated after 2c treatment colocalization of ubiquitin with lysosome and LC3B with p62 was studied to confirm that protein degradation in autophagy induced HT-29 cells follows autolysosomal pathway. CONCLUSIONS: In summary, betulinic acid analogue, 2c was able to induce autophagy in HT-29 cells and as proteasomal degradation pathway downregulated after 2c treatment so protein degradation in autophagy induced HT-29 cells follows autolysosomal pathway.

Cationic dextrin nanoparticles for effective intracellular delivery of cytochrome C in cancer therapy.

Intracellular protein delivery shows promise as a selective and specific approach to cancer therapy. However, a major challenge is posed by delivering proteins into the target cells. Despite the development of nanoparticle (NP)-based approaches, a versatile and biocompatible delivery system that can deliver active therapeutic cargo into the cytosol while escaping endosome degradation remains elusive. In order to overcome these challenges, a polymeric nanocarrier was prepared using cationic dextrin (CD), a biocompatible and biodegradable polymer, to encapsulate and deliver cytochrome C (Cyt C), a therapeutic protein. The challenge of endosomal escape of the nanoparticles was addressed by co-delivering the synthesized NP construct with chloroquine, which enhances the endosomal escape of the therapeutic protein. No toxicity was observed for both CD NPs and chloroquine at the concentration tested in this study. Spectroscopic investigations confirmed that the delivered protein, Cyt C, was structurally and functionally active. Additionally, the delivered Cyt C was able to induce apoptosis by causing depolarization of the mitochondrial membrane in HeLa cells, as evidenced by flow cytometry and microscopic observations. Our findings demonstrate that an engineered delivery system using CD NPs is a promising platform in nanomedicine for protein delivery applications.

NIR-Active Gold Dogbone Nanorattles Impregnated in Cationic Dextrin Nanoparticles for Cancer Nanotheranostics.

Theranostic systems, which integrate therapy and diagnosis into a single platform, have gained significant attention as a promising approach for noninvasive cancer treatment. The field of image-guided therapy has revolutionized real-time tumor detection, and within this domain, plasmonic nanostructures have garnered significant attention. These structures possess unique localized surface plasmon resonance (LSPR), allowing for enhanced absorption in the near-infrared (NIR) range. By leveraging the heat generated from plasmonic nanoparticles upon NIR irradiation, target cancer cells can be effectively eradicated. This study introduces a plasmonic gold dogbone-nanorattle (AuDB NRT) structure that exhibits broad absorption in the NIR region and demonstrates a photothermal conversion efficiency of 35.29%. When exposed to an NIR laser, the AuDB NRTs generate heat, achieving a maximum temperature rise of 38 °C at a concentration of 200 µg/mL and a laser power density of 3 W/cm2. Additionally, the AuDB NRTs possess intrinsic electromagnetic hotspots that amplify the signal of a Raman reporter molecule, making them an excellent probe for surface-enhanced Raman scattering-based bioimaging of cancer cells. To improve the biocompatibility of the nanorattles, the AuDB NRTs were conjugated with mPEG-thiol and successfully encapsulated into cationic dextrin nanoparticles (CD NPs). Biocompatibility tests were performed on HEK 293 A and MCF-7 cell lines, revealing high cell viability when exposed to AuDB NRT-CD NPs. Remarkably, even at a low laser power density of 1 W/cm2, the application of the NIR laser resulted in a remarkable 80% cell death in cells treated with a nanocomposite concentration of 100 µg/mL. Further investigation elucidated that the cell death induced by photothermal heat followed an apoptotic mechanism. Overall, our findings highlight the significant potential of the prepared nanocomposite for cancer theranostics, combining effective photothermal therapy along with the ability to image cancer cells.

Tumor antigen presentation and the associated signal transduction during carcinogenesis.

Numerous developments have been achieved in the study and treatment of cancer throughout the decades that it has been common. After decades of research, about 100 different kinds of cancer have been found, each with unique subgroups within certain organs. This has significantly expanded our understanding of the illness. A mix of genetic, environmental, and behavioral variables contribute to the complicated and diverse process of cancer formation. Mutations, or changes in the DNA sequence, are crucial to the development of cancer. These mutations have the ability to downregulate the expression and function of Major Histocompatibility Complex class I (MHC I) and MHCII receptors, as well as activate oncogenes and inactivate tumor suppressor genes. Cancer cells use this tactic to avoid being recognized by cytotoxic CD8+T lymphocytes, which causes issues with antigen presentation and processing. This review goes into great length into the PI3K pathway, changes to MHC I, and positive impacts of tsMHC-II on disease-free survival and overall survival and the involvement of dendritic cells (DCs) in different tumor microenvironments. The vital functions that the PI3K pathway and its link to the mTOR pathway are highlighted and difficulties in developing effective cancer targeted therapies and feedback systems has also been mentioned, where resistance mechanisms include RAS-mediated oncogenic changes and active PI3K signalling.

Immunomodulatory effects of Diospyros peregrina fruit preparation (DFP) in non-small cell lung cancer (NSCLC) by utilizing dendritic cell-mediated antigen presentation and T helper (TH) cell differentiation.

Diospyros peregrina is a dioecious plant which is native to India. It belongs to the family of Ebenaceae and is extensively used to treat various ailments, such as leucorrhoea and other uterine-related problems. Though few studies have been on D. peregrina for their anti-tumour response, little is known. Therefore, this intrigued us to understand its immunomodulator capabilities on various types of cancer extensively. Our primary focus is on NSCLC (Non-Small Cell Lung Cancer), which is ranked as the second largest form of cancer in the world, and the treatments demand non-invasive agents to target NSCLC effectively. In an objective to generate an efficient Lung Cancer Associated Antigen (LCA) specific anti-tumour immune response, LCA was presented using dendritic cells (DCs) in the presence of D. peregrina fruit preparation (DFP). Moreover, we also investigated DFP's role in the differentiation of T-helper (TH) cells. Therefore, this study aimed at better LCA presentation mediated by DFP by activating the LCA pulsed DCs and T helper cell differentiation for better immune response. DCs were pulsed with LCA for tumour antigen presentation in vitro, with and without DFP. Differentially pulsed DCs were irradiated to co-culture with autologous and allogeneic lymphocytes. Extracellular supernatants were collected for the estimation of cytokine levels by ELISA. LDH release assay was performed to test Cytotoxic T lymphocytes (CTLs) mediated lung tumour cell cytotoxicity. Thus, DFP may be a potential vaccine to generate anti-LCA immune responses to restrict NSCLC.

Chickpea seed endophyte Enterobacter sp. mediated yield and nutritional enrichment of chickpea for improving human and livestock health.

Chickpeas (Cicer arietinum L.) are used as a good source of proteins and energy in the diets of various organisms including humans and animals. Chickpea straws can serve as an alternative option for forage for different ruminants. This research mainly focussed on screening the effects of adding beneficial chickpea seed endophytes on increasing the nutritional properties of the different edible parts of chickpea plants. Two efficient chickpea seed endophytes (Enterobacter sp. strain BHUJPCS-2 and BHUJPCS-8) were selected and applied to the chickpea seeds before sowing in the experiment conducted on clay pots. Chickpea seeds treated with both endophytes showed improved plant growth and biomass accumulation. Notably, improvements in the uptake of mineral nutrients were found in the foliage, pericarp, and seed of the chickpea plants. Additionally, nutritional properties such as total phenolics (0.47, 0.25, and 0.55 folds), total protein (0.04, 0.21, and 0.18 folds), carbohydrate content (0.31, 0.32, and 0.31 folds), and total flavonoid content (0.45, 027, and 0.8 folds) were increased in different parts (foliage, pericarp, and seed) of the chickpea plants compared to the control plants. The seed endophyte-treated plants showed a significant increase in mineral accumulation and improvement in nutrition in the different edible parts of chickpea plants. The results showed that the seed endophyte-mediated increase in dietary and nutrient value of the different parts (pericarp, foliage, and seeds) of chickpea are consumed by humans, whereas the other parts (pericarp and foliage) are used as alternative options for forage and chaff in livestock diets and may have direct effects on their nutritional conditions.

miR-277 targets the proapoptotic gene-hid to ameliorate Aß42-mediated neurodegeneration in Alzheimer's model.

Alzheimer's disease (AD), an age-related progressive neurodegenerative disorder, exhibits reduced cognitive function with no cure to date. One of the reasons for AD is the accumulation of Amyloid-beta 42 (Aß42) plaque(s) that trigger aberrant gene expression and signaling, which results in neuronal cell death by an unknown mechanism(s). Misexpression of human Aß42 in the developing retina of Drosophila exhibits AD-like neuropathology. Small non-coding RNAs, microRNAs (miRNAs), post-transcriptionally regulate the expression of their target genes and thereby regulate different signaling pathways. In a forward genetic screen, we identified miR-277 (human ortholog is hsa-miR-3660) as a genetic modifier of Aß42-mediated neurodegeneration. Loss-of-function of miR-277 enhances the Aß42-mediated neurodegeneration. Whereas gain-of-function of miR-277 in the GMR > Aß42 background downregulates cell death to maintain the number of neurons and thereby restores the retinal axonal targeting defects indicating the functional rescue. In addition, gain-of-function of miR-277 rescues the eclosion- and climbing assays defects observed in GMR > Aß42 background. Thus, gain-of-function of miR-277 rescues both structurally as well as functionally the Aß42-mediated neurodegeneration. Furthermore, we identified head involution defective (hid), an evolutionarily conserved proapoptotic gene, as one of the targets of miR-277 and validated these results using luciferase- and qPCR -assays. In the GMR > Aß42 background, the gain-of-function of miR-277 results in the reduction of hid transcript levels to one-third of its levels as compared to GMR > Aß42 background alone. Here, we provide a novel molecular mechanism where miR-277 targets and downregulates proapoptotic gene, hid, transcript levels to rescue Aß42-mediated neurodegeneration by blocking cell death. These studies shed light on molecular mechanism(s) that mediate cell death response following Aß42 accumulation seen in neurodegenerative disorders in humans and provide new therapeutic targets for neurodegeneration.

The curious case of SARM1: Dr. Jekyll and Mr. Hyde in cell death and immunity?

Sterile alpha and toll/interleukin-1 receptor motif-containing protein 1 (SARM1) was first identified as a novel ortholog of Drosophila protein CG7915 and was subsequently placed as the fifth member of the human TIR-containing adaptor protein. SARM1 holds a unique position in this family where, unlike other members, it downregulates NFκB activity in response to immunogenic stimulation, interacts with another member of the family, TRIF, to negatively regulate its function, and it also mediates cell death responses. Over the past decade, SARM1 has emerged as one of the primary mediators of programmed axonal degeneration and this robust regulation of axonal degeneration-especially in models of peripheral neuropathy and traumatic injury-makes it an attractive target for therapeutic intervention. The TIR domain of SARM1 possesses an intrinsic NADase activity resulting in cellular energy deficits within the axons, a striking deviation from its other family members of human TLR adaptors. Interestingly, the TIR NADase activity, as seen in SARM1, is also observed in several prokaryotic TIR-containing proteins where they are involved in immune evasion once within the host. Although the immune function of SARM1 is yet to be conclusively discerned, this closeness in function with the prokaryotic TIR-domain containing proteins, places it at an interesting juncture of evolution raising questions about its origin and function in cell death and immunity. In this review, we discuss how a conserved immune adaptor protein like SARM1 switches to a pro-neurodegenerative function and the evolutionarily significance of the process.

Binding of stress-responsive OsWRKY proteins through WRKYGQK heptapeptide residue with the promoter region of two rice blast disease resistance genes Pi2 and Pi54 is important for development of blast resistance.

Molecular docking was done to investigate the interactions between five differentially expressed rice WRKY proteins when challenged with the rice blast disease caused by Magnaporthe oryzae and drought stresses applied either individually or overlapped, with the promoter region of two blast resistance genes (Pi2 and Pi54). Molecular docking was performed using the HDOCK server. Initially, the homology models for each of the five rice WRKY proteins were prepared using I-TASSER server, and then the secondary structure as well as the DNA-binding pockets were predicted using PSIPRED and BindUP servers, respectively. The molecular docking study revealed a differential binding pattern of the rice WRKYs with the two blast resistance genes. The WRKY proteins (OsWRKY88 and OsWRKY102), whose transcript levels decrease when drought and blast stresses are overlapped, interact with the two resistance genes mostly involving the residues of the zinc finger structure. On the other hand, the WRKY proteins (OsWRKY53-1 and OsWRKY113), whose transcript levels did not reduce significantly when challenged by drought and blast overlapped condition compared to individual treatment of blast, interact mostly involving the residues of the conserved WRKYGQK heptapeptide sequence. Interestingly, the protein OsWRKY74 whose transcript levels are unaffected in both individual and overlapped stresses, interacts with both the blast resistance genes involving few residues of both WRKYGQK heptapeptide and the zinc finger structure. The findings thus indicate that the interaction of OsWRKY proteins involving the conserved WRKYGQK heptapeptide sequence with the blast resistance genes Pi2 and Pi54 is important to mitigate the blast challenge in rice even during overlapping challenges of drought. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03711-y.

A taxonomic revision of the Indian species of the 'Aterinervis' group of Culicoides Latreille Subgenus Hoffmania Fox (Diptera: Ceratopogonidae).

The seven species of Culicoides spp. belonging to the Aterinervis Group of subgenus Hoffmania Fox reported from India are revised. The study is based on type material and fresh specimens trapped during the Annual Biodiversity Assessment (2nd & 4th) of Neora Valley National Park (NVNP) in the Darjeeling-Sikkim Himalaya of India. Comparative redescriptions of adult male and female of Culicoides isoregalis, C. neoregalis, C. pararegalis, C. pseudoregalis, C. quasiregalis, C. regalis and C. subregalis are provided along with the formal transfer of the nominate species, Culicoides aterinervis from subgenus Culicoides Latreille to Hoffmania. A key to the Indian species belonging to the Aterinervis group is provided along with a list of the Culicoides species present in the Darjeeling-Sikkim Himalayas.

Role of distal arginine residue in the mechanism of heme nitrite reductases.

Heme nitrite reductases reduce NO2- by 1e-/2H+ to NO or by 6e-/8H+ to NH4+ which are key steps in the global nitrogen cycle. Second-sphere residues, such as arginine (with a guanidine head group), are proposed to play a key role in the reaction by assisting substrate binding and hydrogen bonding and by providing protons to the active site for the reaction. The reactivity of an iron porphyrin with a NO2- covalently attached to a guanidinium arm in its 2nd sphere was investigated to understand the role of arginine residues in the 2nd sphere of heme nitrite reductases. The presence of the guanidinium residue allows the synthetic ferrous porphyrin to reduce NO2- and produce a ferrous nitrosyl species ({FeNO}7), where the required protons are provided by the guanidinium group in the 2nd sphere. However, in the presence of additional proton sources in solution, the reaction of ferrous porphyrin with NO2- results in the formation of ferric porphyrin and the release of NO. Spectroscopic and kinetic data indicated that re-protonation of the guanidine group in the 2nd sphere by an external proton source causes NO to dissociate from a ferric nitrosyl species ({FeNO}6) at rates similar to those observed for enzymatic sites. This re-protonation of the guanidine group mimics the proton recharge mechanism in the active site of NiR. DFT calculations indicated that the lability of the Fe-NO bond in the {FeNO}6 species is derived from the greater binding affinity of anions (e.g. NO2-) to the ferric center relative to neutral NO due to hydrogen bonding and electrostatic interaction of these bound anions with the protonated guanidium group in the 2nd sphere. The reduced {FeNO}7 species, once formed, is not affected significantly by the re-protonation of the guanidine residue. These results provide direct insight into the role of the 2nd sphere arginine residue present in the active sites of heme-based NiRs in determining the fate of NO2- reduction. Specifically, the findings using the synthetic model suggest that rapid re-protonation of these arginine residues may trigger the dissociation of NO from the {FeNO}6, which may also be the case in the protein active site.

A Metagenomic Based Approach on Abundance and Diversity of Bacterial Communities Across the Life Stages of Culicoides peregrinus (Diptera: Ceratopogonidae) a Vector of Bluetongue Virus.

During larval rearing of Culicoides peregrinus Kieffer (Diptera: Ceratopogonidae) it was obligatory to add a small quantity of mud from larval habitat to nutrient broth in culture plates. This initiated microbial growth in rearing plates which facilitated growth and development of immature. The primary aim was to enumerate gut microbial communities across the different life stages of C. peregrinus. Amplicon sequencing of the V3-V4 hypervariable region (16S rDNA) was done on Illumina Miseq platform to detect gut bacterial communities at different life stages, while ITS regions (18S rRNA) were targeted for fungal communities of the 4th instar larvae. The major findings were: 1) Phylum Proteobacteria and Firmicutes were the most abundant throughout the life stages, along with the highest bacterial alpha diversity in the egg, 2) bacterial compositions were similar to laboratory reared and field collected adults, and 3) abundant fungal phyla associated with the larval gut were Ascomycota and Basidiomycota. Furthermore, analyses of the gut microbiome with METAGENassist might be indicative of their likely function in the natural habitat. Abundant gut-associated bacteria and/or fungal genera detected in the present study could be used as dietary supplements to establish laboratory colonies for further vectorial research. While, individual roles of the bacteria or fungi in paratransgenesis are warned for their possible utilization to frame the management strategy in upcoming works.