Mamestra brassicae Multiple Nucleopolyhedroviruses Prevents Pupation of Helicoverpa armigera by Regulating Juvenile Hormone Titer.

Baculovirus infection can prevent the pupation of insects. Juvenile hormone (JH) plays a vital role in regulating insect molting and metamorphosis. However, the molecular mechanism of baculovirus preventing the pupation of larvae by regulating the Juvenile hormone (JH) pathway is still unclear. In this study, we found that the Mamestra brassicae multiple nucleopolyhedroviruses (MbMNPV) infection prolonged the larval stage of fourth instar Helicoverpa armigera (H. armigera) by 0.52 d and caused an increase in JH titer. To identify the genes that contribute to the JH increase in H. armigera-MbMNPV interaction, we analyzed mRNA expression profiles of the fat bodies of H. armigera infected by MbMNPV. A total of 3637 differentially expressed mRNAs (DE-mRNAs) were filtered out through RNA-seq analysis. These DE-mRNAs were mainly enriched in Spliceosome, Ribosome biogenesis in eukaryotes, Aminoacyl-tRNA biosynthesis, Mismatch repair, and RNA degradation signaling pathway, which are related to the virus infection. Real-time PCR was used to verify the RNA sequencing results. To find out which genes caused the increase in JH titer, we analyzed all the DE-mRNAs in the transcriptome and found that the JHE and JHEH genes, which were related to JH degradation pathway, were down-regulated. JHE and JHEH genes in the larvae of MbMNPV-infected group were significantly down-regulated compared with the control group by RT-qPCR. We further proved that the JH is degraded by JHE in H. armigera larvae by RNAi, ELISA, RT-qPCR and bioassay, while the hydrolysis of JH by JHEH in H. armigera larvae can almost be ignored. Knocking down of HaJHE promoted the expression of the JH receptor gene Met and the downstream gene Kr-h1, and the replication of MbMNPV. This study clarified that JH is mainly degraded by JHE in H. armigera larvae. The MbMNPV infection of H. armigera larvae leads to the increase of JH titer by inhibiting the expression of JHE. The increase in JH titer promotes the expression of the JH receptor gene Met and the downstream gene Kr-h1, which prevents the pupation of H. armigera, and promotes MbMNPV replication. This study provides new insights into H. armigera and MbMNPV interaction mechanisms.

Designed gRNAs for CRISPR-Cas9 based antifungal resistance in eggplant.

Eggplant is an important vegetable crop and is a good source of antioxidants, minerals, and vitamins. It has been used in ancient medicines for the treatment of multiple diseases. However, the cultivated varieties of eggplant are susceptible to numerous pathogens and pests that have a negative impact on vegetable crops. Increased resistance achieved through resistance genes (R genes) is limited in eggplant breeding due to the fact that R genes are typically specific to a pathogen race and can be quickly surpassed by pathogen evolution. The susceptibility genes (S genes) in plants facilitate pathogen entry and proliferation, thus disabling these genes might be beneficial for providing a broad range and durable resistance against pathogens. Reports on crops such as Arabidopsis, rice, wheat, citrus, and tomatoes have highlighted that the knockout mutants of the S genes are tolerant to multiple different pathogens. The CRISPR/Cas9 system facilitates plant genome editing that can be utilized efficiently for crop improvement. In the current work, we have identified the homologs of candidate S genes DMR1, DMR6, EDR1, and PMR4/5/6 in the eggplant genome and designed and screened putative gRNAs against the identified target loci. The gRNAs were screened and selected on the basis of recognition of the PAM sequence, the MIT score, their minimum free energy, and the secondary structure. Five gRNAs for each gene homolog were selected after an in-depth analysis of all the predicted gRNAs using the above-mentioned criterion.

A follow-up study of surgically managed benign vocal cord lesions using vocal handicap index - 10 score.

Background and Objectives: Benign lesions of the vocal cords not only impair the patient's ability to communicate due to poor pronunciation and voice capabilities, but they also cause a variety of psychological and social problems, worsening their quality of life. To assess voice handicap, Voice Handicap Index (VHI)-10 is an easy-to-administer, valid and reliable tool. The present study was conducted to compare the pre-operative and post-operative well-being of patients with benign vocal cord lesions using VHI-10 among patients attending our Outpatient department. Materials and Methods: The study was a hospital based observational study with prospective study design, conducted over a period of 17 months (August 2019 to December 2020) on 53 patients who were clinically diagnosed to have benign vocal cord lesion and underwent micro laryngeal surgery followed by histopathological confirmation. Follow-up of the participants was done on 4th and 8th week post-operatively. Visualization of the condition of the laryngeal structures using laryngoscope assessment of VHI-10 score was done at each follow-up visit. Results: In our study, out of 53 study participants, approximately one-third of the cases were seen in the 5th decade, that is, 40-49 years. The mean (±SD) age of the study participants was 43.2 (±12.1) years. There was male predominance (73.6%) with male to female ratio of 2.78:1. Majority of the patients had voice-demanding profession with history of vocal abuse. In cases of non-professional voice users, the highest incidence was noted in homemakers (11.4%). Smoking was noted in nine male patients while all the female patients were non-smokers. Habit of alcohol consumption was seen in 15.1% patients and 7.5% patients had habit of other substance abuse such as tobacco chewing, betel nut chewing, etc. Vocal polyps were the most common benign lesions found in 43.3% of the patients. Preoperatively mean (± SD) VHI-10 score was 20.7 (±3.5) which significantly decreased to 12.6 (±2.5) at 4th week post-operatively and 8.5 (±2.3) at 8th week post-operatively. Mean differences between pre-operative and post-operative scores were statistically significant (P < 0.01), depicting that there was improvement in the voice of the patients post-operatively. Conclusion: The VHI-10 scale is shown to be a good and convenient tool for assessing patient voice handicap and determining improvement post-operatively. Wider studies with larger sample size in different parts of the country may be recommended to validate the findings of the study. In addition to surgical excision of lesion, speech therapy, and patient counselling are indubitably essential measures that should be undertaken for each patient so as to alleviate the handicapping effect of voice disorder and improve the overall quality of life of the individuals.

Astrocytic and microglial cells as the modulators of neuroinflammation in Alzheimer's disease.

Neuroinflammation is instigated by the misfiring of immune cells in the central nervous system (CNS) involving microglia and astrocytes as key cell-types. Neuroinflammation is a consequence of CNS injury, infection, toxicity, or autoimmunity. It is favorable as well as a detrimental process for neurodevelopment and associated processes. Transient activation of inflammatory response involving release of cytokines and growth factors positively affects the development and post-injury tissue. However, chronic or uncontrolled inflammatory responses may lead to various neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis, and multiple sclerosis. These diseases have variable clinical and pathological features, but are underlaid by the aggregation of misfolded proteins with a cytotoxic effect. Notably, abnormal activation of glial cells could mediate neuroinflammation, leading to the neurodegenerative condition. Microglia, a type of glial cell, a resident immune cell, form the forefront defense of the CNS immune system. Dysfunctional microglia and astrocyte, a different kind of glial cell with homeostatic function, impairs the protein aggregate (amyloid-beta plaque) clearance in AD. Studies have shown that microglia and astrocytes undergo alterations in their genetic profile, cellular and molecular responses, and thus promote dysfunctional immune cross-talk in AD. Hence, targeting microglia and astrocytes-driven molecular pathways could resolve the particular layers of neuroinflammation and set a reliable therapeutic intervention in AD progression.

CRISPR/Cas9 System: A Potential Tool for Genetic Improvement in Floricultural Crops.

Demand of flowers is increasing with time worldwide. Floriculture has become one of the most important commercial trades in agriculture. Although traditional breeding methods like hybridization and mutation breeding have contributed significantly to the development of important flower varieties, flower production and quality of flowers can be significantly improved by employing modern breeding approaches. Novel traits of significance have interest to consumers and producers, such as fragrance, new floral color, change in floral architecture and morphology, vase life, aroma, and resistance to biotic and abiotic stresses, have been introduced by genetic manipulation. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has recently emerged as a powerful genome-editing tool for accurately changing DNA sequences at specific locations. It provides excellent means of genetically improving floricultural crops. CRISPR/Cas system has been utilized in gene editing in horticultural cops. There are few reports on the utilization of the CRISPR/Cas9 system in flowers. The current review summarizes the research work done by employing the CRISPR/Cas9 system in floricultural crops including improvement in flowering traits such as color modification, prolonging the shelf life of flowers, flower initiation, and development, changes in color of ornamental foliage by genome editing. CRISPR/Cas9 gene editing could be useful in developing novel cultivars with higher fragrance and enhanced essential oil and many other useful traits. The present review also highlights the basic mechanism and key components involved in the CRISPR/Cas9 system.

Astrobiological Potential of Fe/Mg Smectites with Special Emphasis on Jezero Crater, Mars 2020 Landing Site.

Life is known to adapt in accordance with its surrounding environment and sustainable resources available to it. Since harsh conditions would have precluded any possible aerobic evolution of life at the martian surface, it is plausible that martian life, should it exist, would have evolved in such a way as to derive energy from more optimum resources. Iron is one of the most abundant elements present in the martian crust and occurs at about twice the amount present on Earth. Clay minerals contribute to about half the iron found in soils and sediments. On Earth, clay acts as an electron donor as well as an acceptor in the carbon cycles and thereby supports a wide variety of metabolic reactions. In this context, we consider the potential of Fe/Mg smectites, one of the most widely reported hydrated minerals on Mars, for preservation of macro- and microscopic biosignatures. We proceed by understanding the environmental conditions during the formation of smectites and various microbes and metabolic processes associated with them as indicated in Earth-based studies. We also explore the possibility of biosignatures and their identification within the Mars 2020 landing site (Jezero Crater) by using the astrobiological payloads on board the Perseverance rover.

Integrated Analysis of MicroRNA and mRNA Expression Profiles in the Fat Bodies of MbMNPV-Infected Helicoverpa armigera.

MicroRNAs (miRNAs), are a novel class of gene expression regulators, that have been found to participate in regulating host-virus interactions. However, the function of insect-derived miRNAs in response to virus infection is poorly understood. We analyzed miRNA expression profiles in the fat bodies of Helicoverpa armigera (H. armigera) infected with Mamestra brassicae multiple nucleopolyhedroviruses (MbMNPV). A total of 52 differentially expressed miRNAs (DEmiRNAs) were filtered out through RNA-seq analysis. The targets of 52 DEmiRNAs were predicted and 100 miRNA-mRNA interaction pairs were obtained. The predicted targets of DEmiRNAs were mainly enriched in the Wnt signaling pathway, phagosome, and mTOR signaling pathway, which are related to the virus infection. Real-time PCR was used to verify the RNA sequencing results. ame-miR-317-3p, mse-miR-34, novel1-star, and sfr-miR-6094-5p were shown to be involved in the host response to MbMNPV infection. Results suggest that sfr-miR-6094-5p can negatively regulate the expression of four host genes eIF3-S7, CG7583, CG16901, and btf314, and inhibited MbMNPV infection significantly. Further studies showed that RNAi-mediated knockdown of eIF3-S7 inhibited the MbMNPV infection. These findings suggest that sfr-miR-6094-5p inhibits MbMNPV infection by negatively regulating the expression of eIF3-S7. This study provides new insights into MbMNPV and H. armigera interaction mechanisms.

Autonomous regulation of inducible nitric oxide synthase and cytochrome P450 2E1-mediated oxidative stress in maneb- and paraquat-treated rat polymorphs.

Maneb (MB)- and paraquat (PQ)-induced oxidative stress in rat polymorphonuclear leukocytes (PMNs) is regulated in parallel by cytochrome P450 2E1 (CYP2E1) and inducible nitric oxide synthase (iNOS). However, mechanism underlying their regulation is not yet understood. The study investigated the role of nuclear factor- kappa B (NF-κB) and mitogen-activated protein kinase/extracellular signal regulated kinase/protein kinase C (MEK/ERK/PKC) pathway in the regulation of iNOS- and CYP2E1-induced oxidative stress in PMNs. MB + PQ-induced changes in nitrite content, lipid peroxidation (LPO), iNOS expression/activity and inflammatory mediators were alleviated by aminoguanidine (AG), an iNOS inhibitor, without any change in CYP2E1. Alternatively, diallyl sulphide (DAS), a CYP2E1 inhibitor, rescued from MB + PQ-induced changes in CYP2E1 activity/expression, free radical generation, superoxide dismutase (SOD) activity, LPO and pro-inflammatory cytokines without any alterations in nitrite content and iNOS activity/expression. Pyrrolidine dithiocarbamate (PDTC), NF-κB inhibitor, did not alter CYP2E1 but mitigated free radical generation, SOD activity, LPO, nitrite content, iNOS activity/expression and levels of pro-inflammatory cytokines (tumor necrosis factor-α, interleukine-1ß and interleukine-4). Ex-vivo treatment with MEK inhibitor (PD98059), ERK1/2 inhibitor (AG126) or PKC inhibitor (rottlerin) ameliorated MB + PQ-induced increase in free radical generation and CYP2E1 activity/expression in PMNs. While PD98059 and AG126 abated MB + PQ-induced increase in ERK1/2, PKC-α/δ and CYP2E1 levels, rottlerin restored PKC-α/δ and CYP2E1 towards normalcy without affecting ERK1/2 level in MB + PQ-treated group. The results suggest that iNOS and CYP2E1 contributing to MB + PQ-induced oxidative stress in rat PMNs exhibit differential regulatory mechanisms. The inflammatory mediators regulate iNOS expression while CYP2E1 expression is triggered via MEK-ERK1/2-PKC pathway.

Integrative analysis of microRNA and mRNA expression profiles in MARC-145 cells infected with PRRSV.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes tremendous economic losses to the swine industry worldwide. miRNAs are crucial regulators of gene expression and a wide range of complex interactions of miRNAs-mRNAs is possible during virus infection. However, there is no comprehensive integrated study of miRNA and mRNA networks in MARC-145 cells after infection with PRRSV. We analyzed the differential expressions, co-relations, annotations, and putative functions of miRNA and mRNA networks in PRRSV-infected MARC-145 cells. Based on the filtering criterion, 22 differentially expressed miRNAs (DEmiRs) (15 up- and 7 downregulated) were filtered out. miRNA-mRNA interaction networks were constructed. For the 18 selected miRNAs, 390 potential target genes were predicted from the differentially expressed mRNAs (DEmRs). GO and KEGG pathway annotations predicted 34 KEGG pathways, 12 of which are known to be involved in virus infection. Real-time PCR validated the RNA-seq results. Our analysis showed that miR-27a-5p and miR-21-3p downregulate the expression of two of their potential target genes-SPARC, CLIC1, and cofilin-1, COX7A2, respectively. Further experiments proved that miR-21-3p and miR-27a-5p can promote PRRSV replication significantly. It is the first report that these two miRNAs participate in the interaction of host cells with PRRSV. Our results provide insights into the role of miRNAs in response to PRRSV infection, which will aid the research for developing novel therapies against PRRSV.

Sinomenine inhibits amyloid beta-induced astrocyte activation and protects neurons against indirect toxicity.

Amyloid beta is a major constituent of the plaques found in the brains of patients suffering from Alzheimer's disease (AD). A growing body of research work suggests that neuroinflammation plays important roles in the development of AD. Thus, considerable efforts are directed towards identification of compounds that can reduce or inhibit neuroinflammation. Here, we show that sinomenine, a compound present in a Chinese medicinal plant, Sinomenium acutum, inhibits oligomeric amyloid beta-induced production of reactive oxygen species (ROS), nitric oxide (NO) and inflammation-related molecules from astrocytic cells. The conditioned medium from oligomeric amyloid beta-treated astrocytic cells induces cell death in the hippocampal neuronal cells. Importantly, sinomenine inhibits this cell death. In addition, this compound has inhibitory effects on the production of ROS, NO and inflammation-related factors from oligomeric amyloid-beta treated human astrocytes. Finally, the conditioned medium from oligomeric amyloid beta-treated human astrocytes induces cell death in the primary culture of human neurons, which is inhibited by sinomenine. Thus, sinomenine inhibits amyloid beta-induced production of toxic factors from astrocytes, and confers protection to hippocampal neuronal cells as well as human neurons against indirect toxicity. The results suggest that this compound could provide beneficial effects in AD and other neurodegenerative conditions by reducing inflammation and neuronal cell death.

A Cross-Sectional Study to Assess the Quality of Life of Perimenopausal and Post menopausal Women in Rural Etawah, Uttar Pradesh, India.

BACKGROUND: Perimenopause is the period in a woman's life during which she passes from the reproductive to the nonreproductive stage. According to the 2008 estimates, the number of menopausal women in India was 43 million. Projected values in 2026 depict the menopausal population at 103 million. Due to the increasing life expectancy, improved quality of life is imperative to decrease the disability and frailty of a society. OBJECTIVES: To study the quality of life of perimenopausal women in rural areas of Etawah district, Uttar Pradesh, and the various factors associated with it. MATERIALS AND METHODS: This is a community-based cross-sectional study conducted in 4 villages in the Saifai block of Etawah district, Uttar Pradesh, India. One hundred and ninety-nine healthy, perimenopausal women of the age group 45-55 years were included in the study. Data were collected on sociodemographic variables, and Menopause Specific Quality of Life Questionnaire - Intervention version questionnaire was used to assess the quality of life. RESULTS: The mean age of attainment of menopause was 45.38 ± 3.58 years. Majority of the women experienced physical (100%) and psychosocial (94.5%) symptoms; the most common symptom being "decrease in physical strength" (86.4%) and being able to "accomplish less than previously" (80.4%). Women who handled stress poorly showed severe vasomotor (P = 0.047) and psychosocial (P = 0.014) symptoms. Postmenopausal women who regularly exercised were 52.6% less likely to have vasomotor symptoms (odd's ratio OR 0.474 (0.235-0.960), P = 0.038). CONCLUSION: The quality of life among the study population was affected by the physical and psychosocial problems they experienced. By taking appropriate preventive measures, these can be ameliorated, and further deterioration can be checked.

Visualizing the Transport of Porcine Reproductive and Respiratory Syndrome Virus in Live Cells by Quantum Dots-Based Single Virus Tracking.

Quantum dots (QDs)-based single particle analysis technique enables real-time tracking of the viral infection in live cells with great sensitivity over a long period of time. The porcine reproductive and respiratory syndrome virus (PRRSV) is a small virus with the virion size of 40-60 nm which causes great economic losses to the swine industry worldwide. A clear understanding of the viral infection mechanism is essential for the development of effective antiviral strategies. In this study, we labeled the PRRSV with QDs using the streptavidin-biotin labeling system and monitored the viral infection process in live cells. Our results indicated that the labeling method had negligible effect on viral infectivity. We also observed that prior to the entry, PRRSV vibrated on the plasma membrane, and entered the cells via endosome mediated cell entry pathway. Viruses moved in a slow-fast-slow oscillatory movement pattern and finally accumulated in a perinuclear region of the cell. Our results also showed that once inside the cell, PRRSV moved along the microtubule, microfilament and vimentin cytoskeletal elements. During the transport process, virus particles also made contacts with non-muscle myosin heavy chain II-A (NMHC II-A), visualized as small spheres in cytoplasm. This study can facilitate the application of QDs in virus infection imaging, especially the smaller-sized viruses and provide some novel and important insights into PRRSV infection mechanism.

Correction to: CYP2E1-mediated oxidative stress regulates HO-1 and GST expression in maneb- and paraquat-treated rat polymorphonuclear leukocytes.

In the original publication of the article, the wrong ß-actin blots were pasted in Figs. 1b and 2c. The correct versions of Figs. 1b and 2c are given in this correction.

Genome-Wide Identification of the Alba Gene Family in Plants and Stress-Responsive Expression of the Rice Alba Genes.

Architectural proteins play key roles in genome construction and regulate the expression of many genes, albeit the modulation of genome plasticity by these proteins is largely unknown. A critical screening of the architectural proteins in five crop species, viz., Oryza sativa, Zea mays, Sorghum bicolor, Cicer arietinum, and Vitis vinifera, and in the model plant Arabidopsis thaliana along with evolutionary relevant species such as Chlamydomonas reinhardtii, Physcomitrella patens, and Amborella trichopoda, revealed 9, 20, 10, 7, 7, 6, 1, 4, and 4 Alba (acetylation lowers binding affinity) genes, respectively. A phylogenetic analysis of the genes and of their counterparts in other plant species indicated evolutionary conservation and diversification. In each group, the structural components of the genes and motifs showed significant conservation. The chromosomal location of the Alba genes of rice (OsAlba), showed an unequal distribution on 8 of its 12 chromosomes. The expression profiles of the OsAlba genes indicated a distinct tissue-specific expression in the seedling, vegetative, and reproductive stages. The quantitative real-time PCR (qRT-PCR) analysis of the OsAlba genes confirmed their stress-inducible expression under multivariate environmental conditions and phytohormone treatments. The evaluation of the regulatory elements in 68 Alba genes from the 9 species studied led to the identification of conserved motifs and overlapping microRNA (miRNA) target sites, suggesting the conservation of their function in related proteins and a divergence in their biological roles across species. The 3D structure and the prediction of putative ligands and their binding sites for OsAlba proteins offered a key insight into the structure-function relationship. These results provide a comprehensive overview of the subtle genetic diversification of the OsAlba genes, which will help in elucidating their functional role in plants.

Alpha-synuclein aggregation, Ubiquitin proteasome system impairment, and L-Dopa response in zinc-induced Parkinsonism: resemblance to sporadic Parkinson's disease.

Alpha-synuclein (α-synuclein) aggregation and impairment of the Ubiquitin proteasome system (UPS) are implicated in Parkinson's disease (PD) pathogenesis. While zinc (Zn) induces dopaminergic neurodegeneration resulting in PD phenotype, its effect on protein aggregation and UPS has not yet been deciphered. The current study investigated the role of α-synuclein aggregation and UPS in Zn-induced Parkinsonism. Additionally, levodopa (L-Dopa) response was assessed in Zn-induced Parkinsonian model to establish its closeness with idiopathic PD. Male Wistar rats were treated with zinc sulfate (Zn; 20 mg/kg; i.p.) twice weekly for 12 weeks along with respective controls. In few subsets, animals were subsequently treated with L-Dopa for 21 consecutive days following Zn exposure. A significant increase in total and free Zn content was observed in the substantia nigra of the brain of exposed groups. Zn treatment caused neurobehavioral anomalies, striatal dopamine decline, and dopaminergic neuronal cell loss accompanied with a marked increase in α-synuclein expression/aggregation and Ubiquitin-conjugated protein levels in the exposed groups. Zn exposure substantially reduced UPS-associated trypsin-like, chymotrypsin-like, and caspase-like activities along with the expression of SUG1 and ß-5 subunits of UPS in the nigrostriatal tissues of exposed groups. L-Dopa treatment rescued from Zn-induced neurobehavioral deficits and restored dopamine levels towards normalcy; however, Zn-induced dopaminergic neuronal loss, reduction in tyrosine hydroxylase expression, and increase in oxidative stress were unaffected. The results suggest that Zn caused UPS impairment, resulting in α-synuclein aggregation subsequently leading to dopaminergic neurodegeneration, and that Zn-induced Parkinsonism exhibited positive L-Dopa response similar to sporadic PD.

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution.

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes. The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

Neuronal Nitric Oxide Synthase Negatively Regulates Zinc-Induced Nigrostriatal Dopaminergic Neurodegeneration.

The study aimed to investigate the role of NO and neuronal NO synthase (nNOS) in Zn-induced neurodegeneration. Animals were treated with zinc sulfate (20 mg/kg), twice a week, for 2-12 weeks along with control. In a few sets, animals were also treated with/without a NO donor, sodium nitroprusside (SNP), or S-nitroso-N-acetyl penicillamine (SNAP) for 12 weeks. Moreover, human neuroblastoma (SH-SY-5Y) cells were also employed to investigate the role of nNOS in Zn-induced toxicity in in vitro in the presence/absence of nNOS inhibitor, 7-nitroindazole (7-NI). Zn caused time-dependent reduction in nitrite content and total/nNOS activity/expression. SNP/SNAP discernibly alleviated Zn-induced neurobehavioral impairments, dopaminergic neurodegeneration, tyrosine hydroxylase (TH) expression, and striatal dopamine depletion. NO donors also salvage from Zn-induced increase in lipid peroxidation (LPO), mitochondrial cytochrome c release, and caspase-3 activation. While Zn elevated LPO content, it attenuated nitrite content, nNOS activity, and glutathione level along with the expression of TH and nNOS in SH-SY-5Y cells. 7-NI further augmented Zn-induced changes in the cell viability, oxidative stress, and expression of TH and nNOS. The results obtained thus demonstrate that Zn inhibits nNOS that partially contributes to an increase in oxidative stress, which subsequently leads to the nigrostriatal dopaminergic neurodegeneration.

IFN-γ regulates xanthine oxidase-mediated iNOS-independent oxidative stress in maneb- and paraquat-treated rat polymorphonuclear leukocytes.

Maneb (MB) and paraquat (PQ) provoke oxidative stress-mediated cell damage. Role of xanthine oxidase (XO) in oxidative stress and its association with nitric oxide (NO)/NO synthase (NOS) have been widely reported. While inducible NOS (iNOS) is implicated in MB+PQ-induced toxicity in rat polymorphonuclear leukocytes (PMNs), role of XO and its alliance with iNOS have not yet been established. The study investigated the role of XO in MB+PQ-induced oxidative stress in rat PMNs and its regulation by iNOS and inflammatory cytokines. MB+PQ-augmented reactive oxygen species (ROS), superoxide, nitro-tyrosine, lipid peroxidation (LPO), and nitrite levels along with the catalytic activity of iNOS, superoxide dismutase (SOD), and XO. XO inhibitor, allopurinol (AP), alleviated MB+PQ-induced changes except nitrite content and iNOS activity. Conversely, an iNOS inhibitor, aminoguanidine, mitigated MB+PQ-induced LPO, nitrite, iNOS, and nitro-tyrosine levels; however, no change was observed in ROS, SOD, and XO. Nuclear factor-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), tumor necrosis factor-alpha (TNF-α) inhibitor, pentoxyfylline, and an anti-inflammatory agent, dexamethasone, attenuated MB+PQ-induced increase in XO, superoxide, and ROS with parallel reduction in the expression of interferon-gamma (IFN-γ), TNF-α, and interleukin-1ß (IL-1ß) in rat PMNs. Exogenous IFN-γ, TNF-α, and IL-1ß enhanced superoxide, ROS, and XO in the PMNs of control and MB+PQ-treated rats; however, IFN- γ was found to be the most potent inducer. Moreover, AP ameliorated cytokine-induced free radical generation and restored XO activity towards normalcy. The results thus demonstrate that XO mediates oxidative stress in MB+PQ-treated rat PMNs via iNOS-independent but cytokine (predominantly IFN-γ)-dependent mechanism.

Essential RNA-Based Technologies and Their Applications in Plant Functional Genomics.

Genome sequencing has not only extended our understanding of the blueprints of many plant species but has also revealed the secrets of coding and non-coding genes. We present here a brief introduction to and personal account of key RNA-based technologies, as well as their development and applications for functional genomics of plant coding and non-coding genes, with a focus on short tandem target mimics (STTMs), artificial microRNAs (amiRNAs), and CRISPR/Cas9. In addition, their use in multiplex technologies for the functional dissection of gene networks is discussed.

Isolation of Cognate Cellular and Viral Ribonucleoprotein Complexes of HIV-1 RNA Applicable to Proteomic Discovery and Molecular Investigations.

All decisions affecting the life cycle of human immunodeficiency virus (HIV-1) RNA are executed by ribonucleoprotein complexes (RNPs). HIV-1 RNA cycles through a progression of host RNPs composed of RNA-binding proteins regulating all stages of synthesis, processing, nuclear transport, translation, decay, and co-localization with assembling virions. RNA affinity chromatography is a versatile method to identify RNA-binding proteins to investigate the molecular basis of viral and cellular posttranscriptional control of gene expression. The bait is a HIV-1 RNA motif immobilized on a solid support, typically magnetic or Sepharose beads. The prey is pre-formed RNPs admixed in lysate from cells or concentrated virus particles. The methodology distinguishes high-affinity RNA-protein interactions from low-affinity complexes by increases in ionic strength during progressive elution cycles. Here, we describe RNA affinity chromatography of the 5' untranslated region of HIV-1, obtaining mixtures of high-affinity RNA binding proteins suitable for mass spectrometry and proteome identification.