Regulation of angiogenesis by endocytic trafficking mediated by cytoplasmic dynein 1 light intermediate chain 1.

Dynein cytoplasmic 1 light intermediate chain 1 (LIC1, DYNC1LI1) is a core subunit of the dynein motor complex. The LIC1 subunit also interacts with various cargo adaptors to regulate Rab-mediated endosomal recycling and lysosomal degradation. Defects in this gene are predicted to alter dynein motor function, Rab binding capabilities, and cytoplasmic cargo trafficking. Here, we have identified a dync1li1 zebrafish mutant, harboring a premature stop codon at the exon 12/13 splice acceptor site, that displays increased angiogenesis. In vitro, LIC1-deficient human endothelial cells display increases in cell surface levels of the pro-angiogenic receptor VEGFR2, SRC phosphorylation, and Rab11-mediated endosomal recycling. In vivo, endothelial-specific expression of constitutively active Rab11a leads to excessive angiogenesis, similar to the dync1li1 mutants. Increased angiogenesis is also evident in zebrafish harboring mutations in rilpl1/2, the adaptor proteins that promote Rab docking to Lic1 to mediate lysosomal targeting. These findings suggest that LIC1 and the Rab-adaptor proteins RILPL1 and 2 restrict angiogenesis by promoting degradation of VEGFR2-containing recycling endosomes. Disruption of LIC1- and RILPL1/2-mediated lysosomal targeting increases Rab11-mediated recycling endosome activity, promoting excessive SRC signaling and angiogenesis.

Mitochondrial uncoupling proteins protect human airway epithelial ciliated cells from oxidative damage.

Apical cilia on epithelial cells defend the lung by propelling pathogens and particulates out of the respiratory airways. Ciliated cells produce ATP that powers cilia beating by densely grouping mitochondria just beneath the apical membrane. However, this efficient localization comes at a cost because electrons leaked during oxidative phosphorylation react with molecular oxygen to form superoxide, and thus, the cluster of mitochondria creates a hotspot for oxidant production. The relatively high oxygen concentration overlying airway epithelia further intensifies the risk of generating superoxide. Thus, airway ciliated cells face a unique challenge of producing harmful levels of oxidants. However, surprisingly, highly ciliated epithelia produce less reactive oxygen species (ROS) than epithelia with few ciliated cells. Compared to other airway cell types, ciliated cells express high levels of mitochondrial uncoupling proteins, UCP2 and UCP5. These proteins decrease mitochondrial protonmotive force and thereby reduce production of ROS. As a result, lipid peroxidation, a marker of oxidant injury, decreases. However, mitochondrial uncoupling proteins exact a price for decreasing oxidant production; they decrease the fraction of mitochondrial respiration that generates ATP. These findings indicate that ciliated cells sacrifice mitochondrial efficiency in exchange for safety from damaging oxidation. Employing uncoupling proteins to prevent oxidant production, instead of relying solely on antioxidants to decrease postproduction oxidant levels, may offer an advantage for targeting a local area of intense ROS generation.

Bacillus spp. as Bio-factories for Antifungal Secondary Metabolites: Innovation Beyond Whole Organism Formulations.

Several fungi act as parasites for crops causing huge annual crop losses at both pre- and post-harvest stages. For years, chemical fungicides were the solution; however, their wide use has caused environmental contamination and human health problems. For this reason, the use of biofungicides has been in practice as a green solution against fungal phytopathogens. In the context of a more sustainable agriculture, microbial biofungicides have the largest share among the commercial biocontrol products that are available in the market. Precisely, the genus Bacillus has been largely studied for the management of plant pathogenic fungi because they offer a chemically diverse arsenal of antifungal secondary metabolites, which have spawned a heightened industrial engrossment of it as a biopesticide. In this sense, it is indispensable to know the wide arsenal that Bacillus genus has to apply these products for sustainable agriculture. Having this idea in our minds, in this review, secondary metabolites from Bacillus having antifungal activity are chemically and structurally described giving details of their action against several phytopathogens. Knowing the current status of Bacillus secreted antifungals is the base for the goal to apply these in agriculture and it is addressed in depth in the second part of this review.

Natural therapeutics against SARS CoV2: the potentiality and challenges.

The incidence of the COVID-19 pandemic completely reoriented global socio-economic parameters and human civilization have experienced the worst situation in the recent past. The rapid mutation rates in viruses have continuously been creating emerging variants of concerns (VOCs) which devastated different parts of the world with subsequent waves of infection. Although, series of antiviral drugs and vaccines were formulated but cent percent effectiveness of these drugs is still awaited. Many of these drugs have different side effects which necessitate proper trial before release. Plants are the storehouse of antimicrobial metabolites which have also long been utilized as traditional medicines against different viral infections. Although, proper mechanism of action of these traditional medicines are unknown, they may be a potential source of effective anti-COVID drug for future implications. Advanced bioinformatic applications have opened up a new arena in predicting these repurposed drugs as a potential COVID mitigator. The present review summarizes brief accounts of the corona virus with their possible entry mechanism. This study also tries to classify different possible anti COVID-19 plant-derived metabolites based on their probable mode of action. This review will surely provide useful information on repurposed drugs to combat COVID-19 in this critical situation.

Microbe-fabricated nanoparticles as potent biomaterials for efficient food preservation.

In recent years, cutting-edge nanotechnology research has revolutionized several facets of the food business, including food processing, packaging, transportation, preservation, and functioning. Nanotechnology has beginning to loom large in the food business as the industry's demand for biogenic nanomaterial grows. The intracellular and extracellular synthesis of metal, metal oxide, and other essential NPs has recently been explored in a variety of microorganisms, including bacteria, actinomycetes, fungi, yeasts, microalgae, and viruses. These microbes produce a variety extracellular material, exopolysaccharides, enzymes, and secondary metabolites which play key roles in synthesizing as well as stabilizing the nanoparticle (NPs). Furthermore, genetic engineering techniques can help them to improve their capacity to generate NPs more efficiently. As a result, using microorganisms to manufacture NPs is unique and has a promising future. Microbial-mediated synthesis of NPs has lately been popular as a more environmentally friendly alternative to physical and chemical methods of nanomaterial synthesis, which require higher prices, more energy consumption, and more complex reaction conditions, as well as a potentially dangerous environmental impact. It is critical to consider regulatory measures implemented at all stages of the process, from production through refining, packaging, preservation, and storage, when producing bionanomaterials derived from culturable microbes for efficient food preservation. The current review discusses the synthesis, mechanism of action, and possible food preservation uses of microbial mediated NPs, which can assist to minimize food deterioration from the inside out while also ensuring that food is safe and free of contaminants. Despite the numerous benefits, there are looming debates concerning their usage in food items, particularly regarding its aggregation in human bodies and other risks to the environment. Other applications and impacts of these microbe-fabricated NPs in the context of future food preservation prospects connected with regulatory problems and potential hazards are highlighted.

WNK Inhibition Increases Surface Liquid pH and Host Defense in Cystic Fibrosis Airway Epithelia.

In cystic fibrosis (CF), reduced HCO3- secretion acidifies the airway surface liquid (ASL), and the acidic pH disrupts host defenses. Thus, understanding the control of ASL pH (pHASL) in CF may help identify novel targets and facilitate therapeutic development. In diverse epithelia, the WNK (with-no-lysine [K]) kinases coordinate HCO3- and Cl- transport, but their functions in airway epithelia are poorly understood. Here, we tested the hypothesis that WNK kinases regulate CF pHASL. In primary cultures of differentiated human airway epithelia, inhibiting WNK kinases acutely increased both CF and non-CF pHASL. This response was HCO3- dependent and involved downstream SPAK/OSR1 (Ste20/SPS1-related proline-alanine-rich protein kinase/oxidative stress responsive 1 kinase). Importantly, WNK inhibition enhanced key host defenses otherwise impaired in CF. Human airway epithelia expressed two WNK isoforms in secretory cells and ionocytes, and knockdown of either WNK1 or WNK2 increased CF pHASL. WNK inhibition decreased Cl- secretion and the response to bumetanide, an NKCC1 (sodium-potassium-chloride cotransporter 1) inhibitor. Surprisingly, bumetanide alone or basolateral Cl- substitution also alkalinized CF pHASL. These data suggest that WNK kinases influence the balance between transepithelial Cl- versus HCO3- secretion. Moreover, reducing basolateral Cl- entry may increase HCO3- secretion and raise pHASL, thereby improving CF host defenses.

Cellular and molecular architecture of submucosal glands in wild-type and cystic fibrosis pigs.

Submucosal glands (SMGs) protect lungs but can also contribute to disease. For example, in cystic fibrosis (CF), SMGs produce abnormal mucus that disrupts mucociliary transport. CF is an ion transport disease, yet knowledge of the ion transporters expressed by SMG acini, which produce mucus, and SMG ducts that carry it to the airway lumen is limited. Therefore, we isolated SMGs from newborn pigs and used single-cell messenger RNA sequencing, immunohistochemistry, and in situ hybridization to identify cell types, gene expression, and spatial distribution. Cell types and transcript levels were the same in non-CF and CF SMGs, suggesting that loss of epithelial anion secretion rather than an intrinsic cell defect causes CF mucus abnormalities. Gene signatures of acinar mucous and acinar serous cells revealed specialized functions in producing mucins and antimicrobials, respectively. However, surprisingly, these two cell types expressed the same ion transporters and neurohumoral receptors, suggesting the importance of balancing mucin and liquid secretion to produce optimal mucus properties. SMG duct cell transcripts suggest that they secrete HCO3- and Cl-, and thus have some similarity to pancreatic ducts that are also defective in CF. These and additional findings suggest the functions of the SMG acinus and duct and provide a baseline for understanding how environmental and genetic challenges impact their contribution to lung disease.

RUSC2 and WDR47 oppositely regulate kinesin-1-dependent distribution of ATG9A to the cell periphery.

Autophagy-related protein 9 (ATG9) is a transmembrane protein component of the autophagy machinery that cycles between the trans-Golgi network (TGN) in the perinuclear area and other compartments in the peripheral area of the cell. In mammalian cells, export of the ATG9A isoform from the TGN into ATG9A-containing vesicles is mediated by the adaptor protein 4 (AP-4) complex. However, the mechanisms responsible for the subsequent distribution of these vesicles to the cell periphery are unclear. Herein we show that the AP-4-accessory protein RUSC2 couples ATG9A-containing vesicles to the plus-end-directed microtubule motor kinesin-1 via an interaction between a disordered region of RUSC2 and the kinesin-1 light chain. This interaction is counteracted by the microtubule-associated protein WDR47. These findings uncover a mechanism for the peripheral distribution of ATG9A-containing vesicles involving the function of RUSC2 as a kinesin-1 adaptor and WDR47 as a negative regulator of this function.

Deciphering plant-microbe crosstalk through proteomics studies.

Proteomic approaches are being used to elucidate a better discretion of interactions occurring between host, pathogen, and/or beneficial microorganisms at the molecular level. Application of proteomic techniques, unravel pathogenicity, stress-related, and antioxidant proteins expressed amid plant-microbe interactions and good information have been generated. It is being perceived that a fine regulation of protein expression takes place for effective pathogen recognition, induction of resistance, and maintenance of host integrity. However, our knowledge of molecular plant-microbe interactions is still incomplete and inconsequential. This review aims to provide insight into numerous ways used for proteomic investigation including peptide/protein identification, separation, and quantification during host defense response. Here, we highlight the current progress in proteomics of defense responses elicited by bacterial, fungal, and viral pathogens in plants along with which the proteome level changes induced by beneficial microorganisms are also discussed.

Procedures in COVID-19 Patients: Part-I.

The number of cases with novel coronavirus disease-2019 (COVID-19) infection is increasing every day in the world, and India contributes a substantial proportion of this burden. Critical care specialists have accepted the challenges associated with the COVID-19 pandemic and are frontline warriors in this war. They have worked hard in streamlining workflow isolation of positive patients, clinical management of critically ill patients, and infection prevention practices. With no end in sight for this pandemic, intensive care unit (ICU) practitioners, hospital administrators, and policy makers have to join hands to prepare for the surge in critical care bed capacity. In this position article, we offer several suggestions on important interventions to the ICU practitioners for better management of critically ill patients. This position article highlights key interventions for COVID-19 treatment and covers several important issues such as endotracheal intubation and tracheostomy (surgical vs PCT), nebulization, bronchoscopy, and invasive procedures such as central venous catheters, arterial lines, and HD catheters. How to cite this article: Pande RK, Bhalla A, SN Myatra, Yaddanpuddi LN, Gupta S, Sahoo TK, et al. Procedures in COVID-19 Patients: Part-I. Indian J Crit Care Med 2020;24(Suppl 5):S263-S271.

Procedures in COVID-19 Patients: Part-II.

Critical care in the era of novel coronavirus disease-2019 (COVID-19) infection has multiple challenges including management of the patient, underlying comorbidities, and the complications. With no end in sight to the pandemic, intensive care unit (ICU) practitioners and hospital administrators have to join hands to prepare for the long battle ahead. Critically ill COVID-19 patients need imaging or image-guided interventions in one form or the other including X-rays, ultrasonography (USG), echocardiography (ECHO), and CT scan. These patients often require renal replacement therapy (RRT) for either the preexisting chronic renal insufficiency or acutely developing kidney injury. Another important component of care is transfer of the patient to and fro from the ICU or to higher care centers. Most of the ICUs are equipped with modern facilities but with increasing number of patients a large number of makeshift arrangements are being made for managing these patients. This position paper outlines important tips to formulate protocols and procedures for critically ill patients, who are managed in the ICU. How to cite this article: Pande RK, Bhalla A, Myatra SN, Yaddanpuddi LN, Gupta S, Sahoo TK, et al. Procedures in COVID-19 Patients: Part-II. Indian J Crit Care Med 2020;24(Suppl 5):S272-S279.

Synergistic consortium of beneficial microorganisms in rice rhizosphere promotes host defense to blight-causing Xanthomonas oryzae pv. oryzae.

MAIN CONCLUSION: Rice plants primed with beneficial microbes Bacillus amyloliquefaciens and Aspergillus spinulosporus with biocontrol potential against Xanthomonas oryzae pv. oryzae, provided protection from disease by reprogramming host defence response under pathogen challenge. Plant-beneficial microbe interactions taking place in the rhizosphere are widely used for growth promotion and mitigation of biotic stresses in plants. The present study aims to evaluate the defense network induced by beneficial microorganisms in the rice rhizosphere, and the three-way interaction involved upon inoculation with dreadful bacteria Xanthomonas oryzae pv. oryzae (Xoo). Differential expression of defense-related enzymes, proteins, and genes in rice variety Swarna primed with a microbial consortium of Bacillus amyloliquefaciens and Aspergillus spinulosporus were quantified in the presence and absence of Xoo. The time-based expression profile alterations in leaves under the five distinct treatments "(unprimed unchallenged, unprimed Xoo challenged, B. amyloliquefaciens primed and challenged, A. spinulosporus primed and challenged, B. amyloliquefaciens and A. spinulosporus consortium primed and challenged)" revealed differential early upregulation of SOD, PAL, PO, PPO activities and TPC content in beneficial microbes primed plants in comparison to unprimed challenged plants. The enhanced defense response in all the rice plants recruited with beneficial microbe was also reflected by reduced plant mortality and an increased plant dry biomass and chlorophyll content. Also, more than 550 protein spots were observed per gel by PD Quest software, a total of 55 differentially expressed protein spots were analysed used MALDI-TOF MS, out of which 48 spots were recognized with a significant score with direct or supporting roles in stress alleviation and disease resistance. qRT-PCR was carried out to compare the biochemical and proteomic data to mRNA levels. We conclude that protein biogenesis and alleviated resistance response may contribute to improved biotic stress adaptation. These results might accelerate the functional regulation of the Xoo-receptive proteins in the presence of beneficial rhizospheric microbes and their computation as promising molecular markers for superior disease management.

Inhibition of multiple defense responsive pathways by CaWRKY70 transcription factor promotes susceptibility in chickpea under Fusarium oxysporum stress condition.

BACKGROUND: Suppression and activation of plant defense genes is comprehensively regulated by WRKY family transcription factors. Chickpea, the non-model crop legume suffers from wilt caused by Fusarium oxysporum f. sp. ciceri Race1 (Foc1), defense response mechanisms of which are poorly understood. Here, we attempted to show interaction between WRKY70 and several downstream signaling components involved in susceptibility/resistance response in chickpea upon challenge with Foc1. RESULTS: In the present study, we found Cicer arietinum L. WRKY70 (CaWRKY70) negatively governs multiple defense responsive pathways, including Systemic Acquired Resistance (SAR) activation in chickpea upon Foc1 infection. CaWRKY70 is found to be significantly accumulated at shoot tissues of susceptible (JG62) chickpea under Foc1 stress and salicylic acid (SA) application. CaWRKY70 overexpression promotes susceptibility in resistant chickpea (WR315) plants to Foc1 infection. Transgenic plants upon Foc1 inoculation demonstrated suppression of not only endogenous SA concentrations but expression of genes involved in SA signaling. CaWRKY70 overexpressing chickpea roots exhibited higher ion-leakage and Foc1 biomass accumulation compared to control transgenic (VC) plants. CaWRKY70 overexpression suppresses H2O2 production and resultant reactive oxygen species (ROS) induced cell death in Foc1 infected chickpea roots, stem and leaves. Being the nuclear targeted protein, CaWRKY70 suppresses CaMPK9-CaWRKY40 signaling in chickpea through its direct and indirect negative regulatory activities. Protein-protein interaction study revealed CaWRKY70 and CaRPP2-like CC-NB-ARC-LRR protein suppresses hyper-immune signaling in chickpea. Together, our study provides novel insights into mechanisms of suppression of the multiple defense signaling components in chickpea by CaWRKY70 under Foc1 stress. CONCLUSION: CaWRKY70 mediated defense suppression unveils networking between several immune signaling events negatively affecting downstream resistance mechanisms in chickpea under Foc1 stress.

Initial report of the genitourinary and gastrointestinal toxicity of post-prostatectomy proton therapy for prostate cancer patients undergoing adjuvant or salvage radiotherapy.

PURPOSE: To report acute and late genitourinary (GU) and gastrointestinal (GI) toxicities associated with post-prostatectomy proton therapy (PT). METHODS: The first 100 consecutive patients from 2010 to 2016 were retrospectively assessed. Baseline characteristics, prospectively graded CTCAE v4.0 toxicities, and patient-reported outcomes were reported. Late outcomes were reported for 79 patients with 3 months minimum follow up. Toxicity-free survival Kaplan-Meier curves were estimated. Logistic regression assessed associations between toxicities and clinical and treatment characteristics (p < .05 significance). RESULTS: Median age, months after surgery, and months of follow-up were respectively 64 years (range 42-77), 25 (5-216), and 25 (0-47). PT received was 70.2 Gy (RBE) (89%), salvage (93%), prostate bed only (80%), pencil beam scanning (86%), with IMRT (31%), and with androgen deprivation (34%). Acute and late maximum toxicities, respectively were: GU grade 0 (14%; 18%), 1 (71%; 62%), 2 (15%; 20%), ≥3 (0), and GI: grade 0 (66%; 73%), 1 (34%; 27%), ≥2 (0). Toxicity-free survival at 24 months was GU grade 2 (83%) and GI grade 1 (74%). Mean (±std dev) baseline International Prostate Symptom Score (IPSS), International Index of Erectile Function, and Expanded Prostate Cancer Index Composite bowel function and bother were 6.6 ± 6.1, 10.5 ± 7.3, 90.9 ± 10.8, 93.3 ± 11.2, respectively, and largely unchanged at 2 years: 6.3 ± 3.6, 11.1 ± 6.3, 92.8 ± 5.8, and 90.9 ± 10.3. On multivariate analysis, baseline IPSS (p = .009) associated with GU grade 2 acute toxicity. Bladderless-CTV median dose, V30, and V40 associated with GU grade 2 acute toxicity and maximum dose with late (Ps <0.05). For GI, on multivariate analysis, baseline bowel function (p = .033) associated with acute grade 1 toxicity. Rectal minimum and median dose, V10, and V20, and anterior rectal wall median dose and V10 through V65 associated with acute grade 1 GI toxicity (Ps < .05). CONCLUSIONS: Post-prostatectomy PT for prostate cancer is feasible with a favorable GU and GI toxicity profile acutely and through early follow up.

Functional diversification of structurally alike NLR proteins in plants.

In due course of evolution many pathogens alter their effector molecules to modulate the host plants' metabolism and immune responses triggered upon proper recognition by the intracellular nucleotide-binding oligomerization domain containing leucine-rich repeat (NLR) proteins. Likewise, host plants have also evolved with diversified NLR proteins as a survival strategy to win the battle against pathogen invasion. NLR protein indeed detects pathogen derived effector proteins leading to the activation of defense responses associated with programmed cell death (PCD). In this interactive process, genome structure and plasticity play pivotal role in the development of innate immunity. Despite being quite conserved with similar biological functions in all eukaryotes, the intracellular NLR immune receptor proteins happen to be structurally distinct. Recent studies have made progress in identifying transcriptional regulatory complexes activated by NLR proteins. In this review, we attempt to decipher the intracellular NLR proteins mediated surveillance across the evolutionarily diverse taxa, highlighting some of the recent updates on NLR protein compartmentalization, molecular interactions before and after activation along with insights into the finer role of these receptor proteins to combat invading pathogens upon their recognition. Latest information on NLR sensors, helpers and NLR proteins with integrated domains in the context of plant pathogen interactions are also discussed.

Sociodemographic disparities in the utilization of proton therapy for prostate cancer at an urban academic center.

PURPOSE: Despite increasing use, proton therapy (PT) remains a relatively limited resource. The purpose of this study was to assess clinical and demographic differences in PT use for prostate cancer compared to intensity modulated radiation therapy (IMRT) at a single institution. METHODS AND MATERIALS: All patients with low- and intermediate-risk prostate cancer (N = 633) who underwent definitive radiation therapy between 2010 and 2015 were divided into PT (n = 508) and IMRT (n = 125) comparison groups and compared using χ2 and independent sample t tests. Univariable and multivariable logistic regression analyses were conducted to assess the associations between PT use and demographic, clinical, and treatment characteristics. RESULTS: The PT and IMRT cohorts varied by age, race, poverty, distance, treatment year, and treating physician. Patients who underwent IMRT were more likely to be older (mean age, 66 vs. 68 years), black (51% vs. 75%), and living in poverty or close to the facility (mean distance between residence and facility, 90 vs. 21 miles; P < .05). Prostate-specific antigen, prostate volume, and International Index of Erectile Function were significantly higher in the IMRT cohort (P < .05), but insurance type, risk group, tumor stage, Gleason score, and patient-reported urinary and bowel scores did not differ significantly (P > .05). Patients who underwent PT were more likely to receive hypofractionated therapy and less likely to receive androgen deprivation therapy (P < .01). On multivariable analysis, black (odds ratio [OR], 0.29; 95% confidence interval [CI], 0.15-0.57) and other race (OR, 0.42; 95% CI, 0.20-0.90); distance (OR, 1.14; 95% CI, 1.06-1.24); treatment years 2011 (OR, 4.87; 95% CI, 2.23-10.6), 2012 (OR, 8.27; 95% CI, 3.43-19.9), and 2014 (OR, 4.44; 95% CI, 1.94-10.2) relative to 2010; and a single treating physician (OR, 0.38; 95% CI, 0.18-0.81) relative to the reference physician with the highest rate of use were associated with PT use, whereas clinical factors such as prostate-specific antigen, prostate volume, International Index of Erectile Function, and androgen deprivation therapy were not. CONCLUSION: Sociodemographic disparities exist in PT use for prostate cancer at an urban academic institution. Further investigation of potential barriers to access is warranted to ensure equitable distribution across all demographic groups.

CD19-negative B-lineage acute lymphoblastic leukemia: A diagnostic and therapeutic challenge.

B-lineage acute lymphoblastic leukemia (B-ALL) is an aggressive neoplasm of B-lymphocyte precursors that express the pan B-cell marker CD19 in all the cases. Rarely, a case may be assigned as B-lineage even if CD19 is negative. Here, a 16-year-old male presented with complaints of pain abdomen, on and off fever, joint pain, and hepatosplenomegaly for 2 months. Bone marrow examination was suggestive of acute leukemia with numerous leukoblasts on aspiration. On flow cytometry, gated blast population was negative for CD19, cytoCD3, and myeloperoxidase MPO and positive for CD34, TdT, HLA-DR, CD22, CD79a, and CD10. Immunohistochemistry study showed positivity for TdT, CD34, CD10 (focal), and PAX 5 and negativity for CD20, CD3, MPO, CD117, and CD68. Lack of awareness of negative CD19 expression in B-ALL can lead to incorrect immunophenotypic diagnosis, treatment, and monitoring of B-ALL. Proper diagnosis should be based on clinical features, immunophenotypic profiles, immunohistochemistry findings, and molecular analysis.

Unorthodox Acetylcholine Binding Sites Formed by α5 and ß3 Accessory Subunits in α4ß2* Nicotinic Acetylcholine Receptors.

All nicotinic acetylcholine receptors (nAChRs) evolved from homomeric nAChRs in which all five subunits are involved in forming acetylcholine (ACh) binding sites at their interfaces. Heteromeric α4ß2* nAChRs typically have two ACh binding sites at α4/ß2 interfaces and a fifth accessory subunit surrounding the central cation channel. ß2 accessory subunits do not form ACh binding sites, but α4 accessory subunits do at the α4/α4 interface in (α4ß2)2α4 nAChRs. α5 and ß3 are closely related subunits that had been thought to act only as accessory subunits and not take part in forming ACh binding sites. The effect of agonists at various subunit interfaces was determined by blocking homologous sites at these interfaces using the thioreactive agent 2-((trimethylammonium)ethyl) methanethiosulfonate (MTSET). We found that α5/α4 and ß3/α4 interfaces formed ACh binding sites in (α4ß2)2α5 and (α4ß2)2ß3 nAChRs. The α4/α5 interface in (ß2α4)2α5 nAChRs also formed an ACh binding site. Blocking of these sites with MTSET reduced the maximal ACh evoked responses of these nAChRs by 30-50%. However, site-selective agonists NS9283 (for the α4/α4 site) and sazetidine-A (for the α4/ß2 site) did not act on the ACh sites formed by the α5/α4 or ß3/α4 interfaces. This suggests that unorthodox sites formed by α5 and ß3 subunits have unique ligand selectivity. Agonists or antagonists for these unorthodox sites might be selective and effective drugs for modulating nAChR function to treat nicotine addiction and other disorders.

Comparative proteomic analysis in pea treated with microbial consortia of beneficial microbes reveals changes in the protein network to enhance resistance against Sclerotinia sclerotiorum.

Microbial consortia may provide protection against pathogenic ingress via enhancing plant defense responses. Pseudomonas aeruginosa PJHU15, Trichoderma harzianum TNHU27 and Bacillus subtilis BHHU100 were used either singly or in consortia in the pea rhizosphere to observe proteome level changes upon Sclerotinia sclerotiorum challenge. Thirty proteins were found to increase or decrease differentially in 2-DE gels of pea leaves, out of which 25 were identified by MALDI-TOF MS or MS/MS. These proteins were classified into several functional categories including photosynthesis, respiration, phenylpropanoid metabolism, protein synthesis, stress regulation, carbohydrate and nitrogen metabolism and disease/defense-related processes. The respective homologue of each protein identified was trapped in Pisum sativum and a phylogenetic tree was constructed to check the ancestry. The proteomic view of the defense response to S. sclerotiorum in pea, in the presence of beneficial microbes, highlights the enhanced protection that can be provided by these microbes in challenged plants.