Conformational ensemble of yeast ATP synthase at low pH reveals unique intermediates and plasticity in F1-Fo coupling.

Mitochondrial adenosine triphosphate (ATP) synthase uses the proton gradient across the inner mitochondrial membrane to synthesize ATP. Structural and single molecule studies conducted mostly at neutral or basic pH have provided details of the reaction mechanism of ATP synthesis. However, pH of the mitochondrial matrix is slightly acidic during hypoxia and pH-dependent conformational changes in the ATP synthase have been reported. Here we use single-particle cryo-EM to analyze the conformational ensemble of the yeast (Saccharomyces cerevisiae) ATP synthase at pH 6. Of the four conformations resolved in this study, three are reaction intermediates. In addition to canonical catalytic dwell and binding dwell structures, we identify two unique conformations with nearly identical positions of the central rotor but different catalytic site conformations. These structures provide new insights into the catalytic mechanism of the ATP synthase and highlight elastic coupling between the catalytic and proton translocating domains.

Physio-biochemical and molecular stress regulators and their crosstalk for low-temperature stress responses in fruit crops: A review.

Low-temperature stress (LTS) drastically affects vegetative and reproductive growth in fruit crops leading to a gross reduction in the yield and loss in product quality. Among the fruit crops, temperate fruits, during the period of evolution, have developed the mechanism of tolerance, i.e., adaptive capability to chilling and freezing when exposed to LTS. However, tropical and sub-tropical fruit crops are most vulnerable to LTS. As a result, fruit crops respond to LTS by inducing the expression of LTS related genes, which is for climatic acclimatization. The activation of the stress-responsive gene leads to changes in physiological and biochemical mechanisms such as photosynthesis, chlorophyll biosynthesis, respiration, membrane composition changes, alteration in protein synthesis, increased antioxidant activity, altered levels of metabolites, and signaling pathways that enhance their tolerance/resistance and alleviate the damage caused due to LTS and chilling injury. The gene induction mechanism has been investigated extensively in the model crop Arabidopsis and several winter kinds of cereal. The ICE1 (inducer of C-repeat binding factor expression 1) and the CBF (C-repeat binding factor) transcriptional cascade are involved in transcriptional control. The functions of various CBFs and aquaporin genes were well studied in crop plants and their role in multiple stresses including cold stresses is deciphered. In addition, tissue nutrients and plant growth regulators like ABA, ethylene, jasmonic acid etc., also play a significant role in alleviating the LTS and chilling injury in fruit crops. However, these physiological, biochemical and molecular understanding of LTS tolerance/resistance are restricted to few of the temperate and tropical fruit crops. Therefore, a better understanding of cold tolerance's underlying physio-biochemical and molecular components in fruit crops is required under open and simulated LTS. The understanding of LTS tolerance/resistance mechanism will lay the foundation for tailoring the novel fruit genotypes for successful crop production under erratic weather conditions.

Biogenic Synthesis of Silver Nanoparticles (AgNPs) Using Aqueous Leaf Extract of Buchanania lanzan Spreng and Evaluation of Their Antifungal Activity against Phytopathogenic Fungi.

Nanoparticles show the multidisciplinary versatile utility and are gaining the prime place in various fields, such as medicine, electronics, pharmaceuticals, electrical designing, cosmetics, food industries, and agriculture, due to their small size and large surface to volume ratio. Biogenic or green synthesis methods are environmentally friendly, economically feasible, rapid, free of organic solvents, and reliable over conventional methods. Plant extracts are of incredible potential in the biosynthesis of metal nanoparticles owing to their bountiful availability, stabilizing, and reducing ability. In the present study, the aqueous leaf extract of Buchanania lanzan Spreng was mixed with 0.5 mM silver nitrate and incubated at 70°C for 1 h and synthesized a good quantity of AgNPs. The synthesized AgNPs were characterized using UV-visible spectroscopy, X-ray diffractometry (XRD), dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The maximum absorption of UV-visible spectra was obtained in the range of 420-430 nm. Furthermore, SEM and TEM results inferred that the size of the particles were 23-62 nm, spherical, crystalline, uniformly distributed, and negatively charged with the zeta potential of -27.6 mV. In addition, the antifungal activities of the AgNPs were evaluated against two phytopathogenic fungi Rhizoctonia solani and Fusarium oxysporum f. sp. lycopersici in vitro using poison food techniques on PDA media. The maximum rate of mycelia inhibition was found in 150 ppm concentration of AgNPs against both phytopathogenic fungi.

Mechanism of LolCDE as a molecular extruder of bacterial triacylated lipoproteins.

Lipoproteins are important for bacterial growth and antibiotic resistance. These proteins use lipid acyl chains attached to the N-terminal cysteine residue to anchor on the outer surface of cytoplasmic membrane. In Gram-negative bacteria, many lipoproteins are transported to the outer membrane (OM), a process dependent on the ATP-binding cassette (ABC) transporter LolCDE which extracts the OM-targeted lipoproteins from the cytoplasmic membrane. Lipid-anchored proteins pose a unique challenge for transport machinery as they have both hydrophobic lipid moieties and soluble protein component, and the underlying mechanism is poorly understood. Here we determined the cryo-EM structures of nanodisc-embedded LolCDE in the nucleotide-free and nucleotide-bound states at 3.8-Å and 3.5-Å resolution, respectively. The structural analyses, together with biochemical and mutagenesis studies, uncover how LolCDE recognizes its substrate by interacting with the lipid and N-terminal peptide moieties of the lipoprotein, and identify the amide-linked acyl chain as the key element for LolCDE interaction. Upon nucleotide binding, the transmembrane helices and the periplasmic domains of LolCDE undergo large-scale, asymmetric movements, resulting in extrusion of the captured lipoprotein. Comparison of LolCDE and MacB reveals the conserved mechanism of type VII ABC transporters and emphasizes the unique properties of LolCDE as a molecule extruder of triacylated lipoproteins.

Efficacy of Autologous Serum in Fixing Conjunctival Autografts of Various Sizes in Different Types and Grades of Pterygium.

PURPOSE: To evaluate the efficacy of autologous serum in fixing conjunctival autografts of various sizes in different grades and types of pterygium and to determine the largest successfully secured graft size. METHODS: This prospective interventional study comprised 151 eyes of 151 patients belonging to age group of 21 to 64 years with different grades and types of primary and recurrent pterygium that underwent excision with inferior conjunctivo - limbal autograft secured with autologous serum. The autografts were measured with calipers and were grouped by size into three categories: Group A, small (5 × 5 mm); Group B, medium (5-7 × 5 mm); and Group C, large (>7 × 5 mm). The adhesive fixation power of autologous serum for the various conjunctival autograft sizes was determined for each group using the following criteria: graft stability, cosmetic appearance and complications in the immediate ( first week) and two-months postoperative follow-up visits. Descriptive statistical analysis was used to calculate the percentage frequency of the variables. RESULTS: The groups A, B, and C included 48%, 22%, and 30% of the autografts, respectively. Overall, 93.34% of the grafts were stable with good cosmetic appearance. However, subconjunctival hemorrhage (36%), graft edema (36%) and graft retraction (13.5%) were the most common complications. The largest successfully fixed graft was 14 × 5 mm in size. CONCLUSION: Autologous serum is efficient in securing conjunctival autografts of various sizes with minimal complications and satisfactory results, including good cosmesis.

Association of defecation disorders with suicidal ideation in young adult with chronic abdominal discomfort.

BACKGROUND: The role of physical symptoms on suicidal ideation in adolescents is incompletely understood. The purpose of this study was to discover the potential physical symptoms predictors for suicidal ideation in college freshmen. METHODS: This cross-sectional survey was conducted in a randomly selected freshmen population in Wuhan China (N = 2449). Questionnaire evaluated suicidal ideation as well as related psychosocial risks and gastrointestinal (GI) physical symptoms. The association between the suicidal ideation and potential predictors was analyzed by Mann-Whitney test and multiple logistic regression analysis. RESULTS: A total of 2053 (83.8%) completed this survey and 64 (3.1%) was reported to have suicidal ideation. Anxiety (OR 5.44(2.40-12.35)), depression (OR 8.36(3.90-17.94)) and chronic abdominal discomfort (OR 2.06(1.07-3.94)) were independent predictors for suicidal ideation. In freshmen with chronic abdominal discomfort, interestingly, defecation disorder(P = 0.01) and low satisfaction of defecation (P = 0.04) was associated with high risk for suicidal ideation. CONCLUSION: Defecation disorders were associated with high risk of suicidal intention in young adult with chronic abdominal discomfort. Our result implied that clinicians should be vigilant for suicidal ideation in chronic abdominal discomfort patients, especially those with defecation disorders.

Psychosocial Factors Associated With Irritable Bowel Syndrome Development in Chinese College Freshmen.

BACKGROUND/AIMS: The role of psychosocial factors on irritable bowel syndrome (IBS) in adolescents is incompletely understood. The aim of this study is to investigate the association between the psychosocial factors and the risk of developing IBS in college freshmen. METHODS: A cross-sectional survey was conducted in a randomly selected freshmen population in Wuhan China (n = 2449). Questionnaire evaluated demographics and psychosocial risks. The population was divided into 3 groups: non-discomfort, chronic abdominal discomfort and IBS. The association between the development of IBS and psychosocial factors was analyzed by ordinal and multiple logistic regression analysis. RESULTS: A total of 2053 (83.8%) completed this survey (mean age, 18.2 ± 0.9 years; female, 35.6%). Among them, 82 (4.0%) fulfilled the Rome III criteria for IBS. Female (odds ratio [OR], 3.31; 95% confidence interval [CI], 2.47-4.45), experience of abuse (OR, 2.44; 95% CI, 1.10-5.56), and suicidal intention (OR, 2.17; 95% CI, 1.15-4.17) were more likely to have IBS. Compared with chronic abdominal discomfort, however, depression (OR, 5.55; 95% CI, 1.36-22.71) was the only dependent risk factor for IBS. CONCLUSION: The prevalence of IBS in college freshmen is 4.0%, and to the freshmen, psychosocial factors such as experience of abuse, depression, and suicidal intention were associated with high risk of developing IBS.

Functional reconstitution of vacuolar H+-ATPase from Vo proton channel and mutant V1-ATPase provides insight into the mechanism of reversible disassembly.

The vacuolar H+-ATPase (V-ATPase; V1Vo-ATPase) is an ATP-dependent proton pump that acidifies subcellular compartments in all eukaryotic organisms. V-ATPase activity is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel subcomplexes, a process that is poorly understood on the molecular level. V-ATPase is a rotary motor, and recent structural analyses have revealed different rotary states for disassembled V1 and Vo, a mismatch that is likely responsible for their inability to reconstitute into holo V-ATPase in vitro Here, using the model organism Saccharomyces cerevisiae, we show that a key impediment for binding of V1 to Vo is the conformation of the inhibitory C-terminal domain of subunit H (HCT). Using biolayer interferometry and biochemical analyses of purified mutant V1-ATPase and Vo proton channel reconstituted into vacuolar lipid-containing nanodiscs, we further demonstrate that disruption of HCT's V1-binding site facilitates assembly of a functionally coupled and stable V1Vo-ATPase. Unlike WT, this mutant enzyme was resistant to MgATP hydrolysis-induced dissociation, further highlighting HCT's role in the mechanism of V-ATPase regulation. Our findings provide key insight into the molecular events underlying regulation of V-ATPase activity by reversible disassembly.

MgATP hydrolysis destabilizes the interaction between subunit H and yeast V1-ATPase, highlighting H's role in V-ATPase regulation by reversible disassembly.

Vacuolar H+-ATPases (V-ATPases; V1Vo-ATPases) are rotary-motor proton pumps that acidify intracellular compartments and, in some tissues, the extracellular space. V-ATPase is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel sectors. An important player in V-ATPase regulation is subunit H, which binds at the interface of V1 and Vo H is required for MgATPase activity in holo-V-ATPase but also for stabilizing the MgADP-inhibited state in membrane-detached V1 However, how H fulfills these two functions is poorly understood. To characterize the H-V1 interaction and its role in reversible disassembly, we determined binding affinities of full-length H and its N-terminal domain (HNT) for an isolated heterodimer of subunits E and G (EG), the N-terminal domain of subunit a (aNT), and V1 lacking subunit H (V1ΔH). Using isothermal titration calorimetry (ITC) and biolayer interferometry (BLI), we show that HNT binds EG with moderate affinity, that full-length H binds aNT weakly, and that both H and HNT bind V1ΔH with high affinity. We also found that only one molecule of HNT binds V1ΔH with high affinity, suggesting conformational asymmetry of the three EG heterodimers in V1ΔH. Moreover, MgATP hydrolysis-driven conformational changes in V1 destabilized the interaction of H or HNT with V1ΔH, suggesting an interplay between MgADP inhibition and subunit H. Our observation that H binding is affected by MgATP hydrolysis in V1 points to H's role in the mechanism of reversible disassembly.

Breaking up and making up: The secret life of the vacuolar H+ -ATPase.

The vacuolar ATPase (V-ATPase; V1 Vo -ATPase) is a large multisubunit proton pump found in the endomembrane system of all eukaryotic cells where it acidifies the lumen of subcellular organelles including lysosomes, endosomes, the Golgi apparatus, and clathrin-coated vesicles. V-ATPase function is essential for pH and ion homeostasis, protein trafficking, endocytosis, mechanistic target of rapamycin (mTOR), and Notch signaling, as well as hormone secretion and neurotransmitter release. V-ATPase can also be found in the plasma membrane of polarized animal cells where its proton pumping function is involved in bone remodeling, urine acidification, and sperm maturation. Aberrant (hypo or hyper) activity has been associated with numerous human diseases and the V-ATPase has therefore been recognized as a potential drug target. Recent progress with moderate to high-resolution structure determination by cryo electron microscopy and X-ray crystallography together with sophisticated single-molecule and biochemical experiments have provided a detailed picture of the structure and unique mode of regulation of the V-ATPase. This review summarizes the recent advances, focusing on the structural and biophysical aspects of the field.

Diagnostic performance of confocal laser endomicroscopy for atrophy and gastric intestinal metaplasia: A meta-analysis.

OBJECTIVE: To systematically evaluate the diagnostic efficacy of confocal laser endomicroscopy (CLE) for gastric atrophy (GA) and gastric intestinal metaplasia (GIM). METHODS: Literature search was performed in PubMed and the Cochrane Library for CLE, GA and GIM. The sensitivity, specificity and diagnostic odds ratio (DOR) in diagnosing GA and GIM were pooled for analysis. A summary receiver operating curve (SROC) was documented and the area under the curve was calculated. RESULTS: Of the 10 studies included in this current analysis, the pooled sensitivity, specificity and DOR of CLE to diagnose GA and GIM were found to be 88%, 98% and 330.85, and 93%, 98% and 439.97, respectively. The area under the SROC were 0.9491 and 0.9812 for the diagnosis of GA and GIM, respectively. Higher sensitivity and specificity of this technique in diagnosing GA and GIM were found in patients without representative disease spectrum and those received pCLE by subgroup analysis. CONCLUSION: CLE is of great value and may be considered an alternative modality for the early diagnosis of GA and GIM.

Biolayer interferometry of lipid nanodisc-reconstituted yeast vacuolar H+ -ATPase.

Vacuolar H+ -ATPase (V-ATPase) is a large, multisubunit membrane protein complex responsible for the acidification of subcellular compartments and the extracellular space. V-ATPase activity is regulated by reversible disassembly, resulting in cytosolic V1 -ATPase and membrane-integral V0 proton channel sectors. Reversible disassembly is accompanied by transient interaction with cellular factors and assembly chaperones. Quantifying protein-protein interactions involving membrane proteins, however, is challenging. Here we present a novel method to determine kinetic constants of membrane protein-protein interactions using biolayer interferometry (BLI). Yeast vacuoles are solubilized, vacuolar proteins are reconstituted into lipid nanodiscs with native vacuolar lipids and biotinylated membrane scaffold protein (MSP) followed by affinity purification of nanodisc-reconstituted V-ATPase (V1 V0 ND). We show that V1 V0 ND can be immobilized on streptavidin-coated BLI sensors to quantitate binding of a pathogen derived inhibitor and to measure the kinetics of nucleotide dependent enzyme dissociation.

Antiovulatory and abortifacient effects of Areca catechu (betel nut) in female rats.

OBJECTIVES: To study the antiovulatory and abortifacient effects of ethanolic extract of Areca catechu in female rats. MATERIALS AND METHODS: For antiovulatory effect, ethanolic extract of A. catechu at 100 and 300 mg/kg doses was administered orally for 15 days. Vaginal smears were examined daily microscopically for estrus cycle. Rats were sacrificed on 16(th) day. Ovarian weight, cholesterol estimation, and histopathological studies were done. Abortifacient activity was studied in rats at 100 and 300 mg/kg doses administered orally from 6(th) to 15(th) day of pregnancy. Rats were laparotomised on 19(th) day. The number of implantation sites and live fetuses were observed in both horns of the uterus. RESULTS: The extract of A. catechu showed a significant decrease in the duration of estrus at 100 mg/kg (P = 0.015) and 300 mg/kg doses (P = 0.002) as compared with control. Metestrus phase was also significantly reduced at 100 mg/kg (P = 0.024) and 300 mg/kg doses (P = 0.002). There was a significant increase in proestrus (P < 0.001) phase. However, diestrus phase was unchanged. Histopathological study of the ovaries showed mainly primordial, primary, and secondary follicles in the test groups as compared to control. There was also a significant (P = 0.002) decrease in ovarian weight and a significant (P = 0.021) increase in ovarian cholesterol level at 100 mg/kg dose. In the study to evaluate abortifacient effect, the mean percentage of abortion with 100 and 300 mg/kg doses were 75.5% and 72.22%, respectively, which was significantly (P = 0.008 and P = 0.006, respectively) increased when compared with control. CONCLUSION: The ethanolic extract of A. catechu at doses of 100 and 300 mg/kg has antiovulatory and abortifacient effects.

Sporadic Creutzfeldt-Jakob disease--a review.

The objective is to study a patient with sporadic Creutzfeldt-Jakob disease (CJD). The patient, a 70-year-old woman with a history spanning over 1 month, with acute onset, progressive abnormal behavior, and cognitive decline with generalized asymmetrical myoclonic jerking, startle phenomenon, and cortical blindness, was referred to the hospital. On observation of clinical symptoms, metabolic and hematological investigations, MRI (magnetic resonance imaging), and EEG (electroencephalogram) were done. The clinical symptoms, MRI, and diagnostic EEG were suggestive of sporadic CJD. Other metabolic encephalopathies were ruled out. With sodium valproate and clonezepam, her myoclonic jerks improved slightly. As CJD is an incurable disease, no definitive treatment could be given.