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Introduction: Erectile dysfunction (ED) is a well-studied entity in diabetes mellitus (DM). As with DM, prediabetes also appears to be associated with changes in microvascular-related comorbidities. These include retinopathy, neuropathy, nephropathy, and ED. ED is a condition characterized by an inability to maintain penile erection for adequate sexual activity, either consistently or recurrently. Endothelial damage due to any cause corresponding to hyperglycemia signifies the relationship between prediabetes and ED. While the evaluation of ED in diabetics has been extensively studied worldwide, there is a paucity of knowledge about ED among prediabetics., Materials and Methods: The study type was observational and was conducted at a tertiary center in India. Adult males aged 18-50 years with prediabetes were included in the study over a duration of 18 months. Evaluation for ED was performed using the International Index of Erectile Function 5 (IIEF-5)., Results: A total of 139 participants were enrolled in the present study. The severity of ED was categorized as follows: 32.4% of participants had mild ED, 11.5% had mild-to-moderate ED, and 6.5% had moderate ED. A positive association was found between prediabetes-related parameters and ED., Conclusion: There is a significant prevalence of ED in prediabetic males compared to the general population, as found in our study. Overall, 50.3% of participants were found to have ED. We recommend exploring this subject with more structured and larger studies., (© Journal of the Association of Physicians of India 2024.)

Background: The cancer registry provides reliable data from the population. In this article, we provide cancer burden and its patterns from the Varanasi district., Methods: The method adopted by the Varanasi cancer registry is community interaction along with regular visits to more than 60 sources to collect data on cancer patients. The cancer registry was established by the Tata Memorial Centre, Mumbai, in 2017 covering 4 million population (57% rural and 43% urban population)., Results: The registry has recorded 1,907 incidence cases (1,058 male and 849 female). The age-adjusted incidence rate per 100,000 population in male and female of Varanasi district is 59.2 and 52.1, respectively. One in 15 male and one in 17 female are at risk of developing the disease. Mouth and tongue cancers are the predominant cancers in male, whereas breast, cervix uteri, and gallbladder are the leading cancer sites among the female. In female, cervix uteri cancer is significantly higher (double) in rural areas when compared with urban areas (rate ratio [RR] 0.5, 95% confidence interval [CI; 0.36, 0.72]), whereas in male, mouth cancer is higher in urban areas when compared with rural areas (RR 1.4, 95% CI [1.11, 1.72]). More than 50% of cancer cases in male are due to tobacco consumption. There may be underreporting of the cases., Conclusion: The results of the registry warrant policies and activities related to early detection services for the mouth, cervix uteri, and breast cancers. The Varanasi cancer registry is the foundation for cancer control and will play an important role in the evaluation of the interventions., (Copyright © 2024 Copyright: © 2024 Indian Journal of Cancer.)

Cyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA polymerases (RNAPs) with characteristics that distinguish them from canonical bacterial RNAPs. The largest subunit of cyanobacterial RNAP (cyRNAP) is divided into two polypeptides, β'1 and β'2, and contains the largest known lineage-specific insertion domain, Si3, located in the middle of the trigger loop and spanning approximately half of the β'2 subunit. In this study, we present the X-ray crystal structure of Si3 and the cryo-EM structures of the cyRNAP transcription elongation complex plus the NusG factor with and without incoming nucleoside triphosphate (iNTP) bound at the active site. Si3 has a well-ordered and elongated shape that exceeds the length of the main body of cyRNAP, fits into cavities of cyRNAP in the absence of iNTP bound at the active site and shields the binding site of secondary channel-binding proteins such as Gre and DksA. A small transition from the trigger loop to the trigger helix upon iNTP binding results in a large swing motion of Si3; however, this transition does not affect the catalytic activity of cyRNAP due to its minimal contact with cyRNAP, NusG, or DNA. This study provides a structural framework for understanding the evolutionary significance of these features unique to cyRNAP and chloroplast RNAP and may provide insights into the molecular mechanism of transcription in specific environment of photosynthetic organisms and organelle., Competing Interests: Competing interests statement:The authors declare no competing interest.

An easy access to functionalized alkenes has been developed by the C-H functionalization of anilides with Morita-Baylis-Hillman (MBH) bromides in the presence of copper chloride, TBHP and acetic acid. Unsubstituted as well as ortho / meta -substituted anilides exclusively give rise to the para -allylated products, whereas para -substitution brings about the formation of ortho -allylated anilides.

NusG is a transcription elongation factor that stimulates transcription pausing in Gram+ bacteria including B. subtilis by sequence-specific interaction with a conserved pause-inducing - 11 TTNTTT -6 motif found in the non-template DNA (ntDNA) strand within the transcription bubble. To reveal the structural basis of NusG-dependent pausing, we determined a cryo-EM structure of a paused transcription complex (PTC) containing RNA polymerase (RNAP), NusG, and the TTNTTT motif in the ntDNA strand. The interaction of NusG with the ntDNA strand rearranges the transcription bubble by positioning three consecutive T residues in a cleft between NusG and the β-lobe domain of RNAP. We revealed that the RNAP swivel module rotation (swiveling), which widens (swiveled state) and narrows (non-swiveled state) a cleft between NusG and the β-lobe, is an intrinsic motion of RNAP and is directly linked to trigger loop (TL) folding, an essential conformational change of all cellular RNAPs for the RNA synthesis reaction. We also determined cryo-EM structures of RNAP escaping from the paused transcription state. These structures revealed the NusG-dependent pausing mechanism by which NusG-ntDNA interaction inhibits the transition from swiveled to non-swiveled states, thereby preventing TL folding and RNA synthesis allosterically. This motion is also reduced by the formation of an RNA hairpin within the RNA exit channel. Thus, the pause half-life can be modulated by the strength of the NusG-ntDNA interaction and/or the stability of the RNA hairpin. NusG residues that interact with the TTNTTT motif are widely conserved in bacteria, suggesting that NusG-dependent pausing is widespread.

This prospective quasi-experimental study from the NASAM (National Approach to Standardize and Improve Mechanical Ventilation) collaborative assessed the impact of evidence-based practices including subglottic suctioning, daily assessment for spontaneous awakening trial (SAT), spontaneous breathing trial (SBT), head of bed elevation, and avoidance of neuromuscular blockers unless otherwise indicated. The study outcomes included VAE (primary) and intensive care unit (ICU) mortality. Changes in daily care process measures and outcomes were evaluated using repeated measures mixed modeling. The results were reported as incident rate ratio (IRR) for each additional month with 95% confidence interval (CI). A comprehensive program that included education on evidence-based practices for optimal care of mechanically ventilated patients with real-time benchmarking of daily care process measures to drive improvement in forty-two ICUs from 26 hospitals in Saudi Arabia (>27,000 days of observation). Compliance with subglottic suctioning, SAT and SBT increased monthly during the project by 3.5%, 2.1% and 1.9%, respectively (IRR 1.035, 95%CI 1.007-1.064, p = 0.0148; 1.021, 95% CI 1.010-1.032, p = 0.0003; and 1.019, 95%CI 1.009-1.029, p = 0.0001, respectively). The use of neuromuscular blockers decreased monthly by 2.5% (IRR 0.975, 95%CI 0.953-0.998, p = 0.0341). The compliance with head of bed elevation was high at baseline and did not change over time. Based on data for 83153 ventilator days, VAE rate was 15.2/1000 ventilator day (95%CI 12.6-18.1) at baseline and did not change during the project (IRR 1.019, 95%CI 0.985-1.053, p = 0.2812). Based on data for 8523 patients; the mortality was 30.4% (95%CI 27.4-33.6) at baseline, and decreased monthly during the project by 1.6% (IRR 0.984, 95%CI 0.973-0.996, p = 0.0067). A national quality improvement collaborative was associated with improvements in daily care processes. These changes were associated with a reduction in mortality but not VAEs. Registration The study is registered in clinicaltrials.gov (NCT03790150)., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Arabi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Self-assembly of macromolecules into higher-order symmetric structures is fundamental for the regulation of biological processes. Higher-order symmetric structure self-assembly by the gene expression machinery, such as bacterial DNA-dependent RNA polymerase (RNAP), has never been reported before. Here, we show that the stress-response σ B factor from the human pathogen, Mycobacterium tuberculosis, induces the RNAP holoenzyme oligomerization into a supramolecular complex composed of eight RNAP units. Cryo-electron microscopy revealed a pseudo-symmetric structure of the RNAP octamer in which RNAP protomers are captured in an auto-inhibited state and display an open-clamp conformation. The structure shows that σ B is sequestered by the RNAP flap and clamp domains. The transcriptional activator RbpA prevented octamer formation by promoting the initiation-competent RNAP conformation. Our results reveal that a non-conserved region of σ is an allosteric controller of transcription initiation and demonstrate how basal transcription factors can regulate gene expression by modulating the RNAP holoenzyme assembly and hibernation., (© 2023. The Author(s).)

Abstract: The prevalent manual destalking of dried red chillies is a labour-intensive, tedious, and unhealthy practice that can cause severe health ailments to the labourers. This study focuses on the design and fabrication of a mechanized system for removing stalks from dried chillies to reduce the drudgery involved in manual operation. The machine's primary components including hopper, rotating cylinder, triangular bridges, cutting blades, destalked chilli outlet, and stalk outlet were fabricated based on the design considerations and ease of operation. The machine was evaluated considering the independent parameters viz. feed rate, cylinder speed, and cylinder inclination for optimization of its performance using one-factor-at-a-time statistical design. Physical properties of chillies, destalking efficiency of the machine, and the effect of destalking on powder characteristics were determined. Optimal performance of the machine was achieved with the chillies having 8-10% (moisture), 6.5 kg/h (feed rate), 20 rpm (cylinder's speed), and 3° (cylinder's inclination) with destalking efficiency of 85-87%. The evaluation of powder characteristics also revealed the prominence of destalking with enhanced colour attributes. The developed machine is compact, efficient, economical, requires less labour and thereby it may encourage its adoption at the farmer's level and strengthening of the value chain of dried chillies., Competing Interests: Conflict of interestThe authors declare that they don’t have any conflict of interest., (© Association of Food Scientists & Technologists (India) 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

The transcriptome-wide contributions of Rho-dependent and intrinsic (Rho-independent) transcription termination mechanisms in bacteria are unclear. By sequencing released transcripts in a wild-type strain and strains containing deficiencies in NusA, NusG and/or Rho (10 strains), we produced an atlas of terminators for the model Gram-positive bacterium Bacillus subtilis. We found that NusA and NusG stimulate 77% and 19% of all intrinsic terminators, respectively, and that both proteins participate in Rho-dependent termination. We also show that Rho stimulates termination at 10% of the intrinsic terminators in vivo. We recapitulated Rho-stimulated intrinsic termination at 5 terminators in vitro and found that Rho requires the KOW domain of NusG to stimulate this process at one of these terminators. Computational analyses of our atlas using RNAstructure, MEME suite and DiffLogo, combined with in vitro transcription experiments, revealed that Rho stimulates intrinsic terminators with weak hairpins and/or U-rich tracts by remodelling the RNA upstream of the intrinsic terminator to prevent the formation of RNA structures that could otherwise compete with the terminator hairpin. We also identified 56 putative examples of 'hybrid Rho-dependent termination', wherein classical Rho-dependent termination occurs after readthrough of a Rho-stimulated intrinsic terminator., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Objective: To examine the accuracy of our national Life-Saving Protocol (LSP). To the best of our knowledge, this is the first study addressing this issue in Saudi Arabia., Background: LSP was created to facilitate triaging patients with LIFE or LIMB threatening conditions in peripheral hospitals with limited services to large regional hospitals to receive definitive care., Method: This is a retrospective single-center observational study over 12 months studying the patients who arrived via LSP to our Emergency room (ED), at the only regional pediatric hospital. For the subgroup of patients who were admitted to PICU through LSP, we further assessed their outcomes like mortality and length of stay (LOS) through a matched case-control study of 1:1 with similar patients who were admitted to our PICU via other routes rather than LSP. The primary outcome is to assess the accuracy of the LSP in triaging pediatric patients with LIFE of LIMB conditions. Secondary outcomes include assessing the association between LSP and (mortality, LOS) for those who were admitted to the regional PICU via LSP compared to patients admitted to PICU via other sources of admission., Results: During the study period, 118 patients arrived at our ED via LSP. Only 43 patients (36 %) were admitted to the PICU with LIFE or LIMB conditions. A total of 64 patients (54%) of the patients were admitted directly to the general pediatric ward from ED level due to absence of LIFE of LIMB threatening condition and 8% (n=9) were discharged immediately home from the ED level due to lack of any significant illness. One patient died at ED level, and one was referred to another hospital with a minor orthopedic injury. For those who were admitted to the PICU via LSP, the mortality rate was (13.9%) (6/43), and the control group was (4.6%) (2/43) with a p-value of 0.08., Conclusion: LSP is an excellent initiative and essential tool in our healthcare system; however, our study showed huge variation in the ability of the system to recognize true pediatric patients with LIFE or LIMB conditions. Our study might form a stepping-stone in future studies assessing the LSP at the national level.

Background: It is unclear whether screening for sepsis using an electronic alert in hospitalized ward patients improves outcomes. The objective of the Stepped-wedge Cluster Randomized Trial of Electronic Early Notification of Sepsis in Hospitalized Ward Patients (SCREEN) trial is to evaluate whether an electronic screening for sepsis compared to no screening among hospitalized ward patients reduces all-cause 90-day in-hospital mortality., Methods and Design: This study is designed as a stepped-wedge cluster randomized trial in which the unit of randomization or cluster is the hospital ward. An electronic alert for sepsis was developed in the electronic medical record (EMR), with the feature of being active (visible to treating team) or masked (inactive in EMR frontend for the treating team but active in the backend of the EMR). Forty-five clusters in 5 hospitals are randomized into 9 sequences of 5 clusters each to receive the intervention (active alert) over 10 periods, 2 months each, the first being the baseline period. Data are extracted from EMR and are compared between the intervention (active alert) and control group (masked alert). During the study period, some of the hospital wards were allocated to manage patients with COVID-19. The primary outcome of all-cause hospital mortality by day 90 will be compared using a generalized linear mixed model with a binary distribution and a log-link function to estimate the relative risk as a measure of effect. We will include two levels of random effects to account for nested clustering within wards and periods and two levels of fixed effects: hospitals and COVID-19 ward status in addition to the intervention. Results will be expressed as relative risk with a 95% confidence interval., Conclusion: The SCREEN trial provides an opportunity for a novel trial design and analysis of routinely collected and entered data to evaluate the effectiveness of an intervention (alert) for a common medical problem (sepsis in ward patients). In this statistical analysis plan, we outline details of the planned analyses in advance of trial completion. Prior specification of the statistical methods and outcome analysis will facilitate unbiased analyses of these important clinical data., Trial Registration: ClinicalTrials.gov NCT04078594 . Registered on September 6, 2019., (© 2021. The Author(s).)

Graphene oxide-strontium (GO-Sr) composites prepared under different ambient conditions were characterized using morphological and spectroscopic techniques to optimize the uptake of Sr from aqueous solutions. These studies indicated that interactions among GO and Sr 2+ ions are highly sensitive to size and aging of GO sheets, as well as pH of the ambience. Further, the extent of Sr uptake on GO sheets was found to be largely influenced by relative fractions of the associated -COOH, -OH, C-O-C functional groups and sp 2 -C domains. Membranes prepared using various forms of GO were evaluated for their Sr separation ability and, a window of parameters for optimum separation of Sr has been proposed. Among the variety of membranes studied, those made up of fresh and large GO sheets were found to exhibit superior Sr adsorption capacity (~296 mg/g) at limited GO mass. Further, adsorption efficiency of these GO membranes was observed to deteriorate with aging of GO sheets and rise of GO mass on membrane. The membrane based filtration procedure introduced in present work facilitates to provide a lamellar structure of GO sheets with abundant surface area, diverse and accessible sites for Sr 2+ ion uptake and offer high Sr adsorption efficiencies., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: To evaluate the effect of screening for sepsis using an electronic sepsis alert vs. no alert in hospitalized ward patients on 90-day in-hospital mortality., Methods: The SCREEN trial is designed as a stepped-wedge cluster randomized controlled trial. Hospital wards (total of 45 wards, constituting clusters in this design) are randomized to have active alert vs. masked alert, 5 wards at a time, with each 5 wards constituting a sequence. The study consists of ten 2-month periods with a phased introduction of the intervention. In the first period, all wards have a masked alert for 2 months. Afterwards the intervention (alert system) is implemented in a new sequence every 2-month period until the intervention is implemented in all sequences. The intervention includes the implementation of an electronic alert system developed in the hospital electronic medical records based on the quick sequential organ failure assessment (qSOFA). The alert system sends notifications of "possible sepsis alert" to the bedside nurse, charge nurse, and primary medical team and requires an acknowledgment in the health information system from the bedside nurse and physician. The calculated sample size is 65,250. The primary endpoint is in-hospital mortality by 90 days., Discussion: The trial started on October 1, 2019, and is expected to complete patient follow-up by the end of October 2021., Trial Registration: ClinicalTrials.gov NCT04078594 . Registered on September 6, 2019., (© 2021. The Author(s).)

A facile photocatalyzed strategy for difunctionalization of styrenes in the presence of CS 2 and amines providing β-keto dithiocarbamates has been developed. In the case of 4-nitrostyrene and 2-vinylpyridine, however, only 2-arylethylthiocarbamates are interestingly formed without the aid of photoredox catalysis/TBHP.

Background: Coronavirus disease-19 (COVID-19) manifested by a broad spectrum of symptoms, ranging from asymptomatic manifestations to severe illness and death. The purpose of the study was to extensively describe the clinical features and outcomes in critically ill patients with COVID-19 in Saudi Arabia., Method: This was a multicenter, non-interventional cohort study for all critically ill patients aged 18 years or older, admitted to intensive care units (ICUs) between March 1 to August 31, 2020, with an objectively confirmed diagnosis of COVID-19. The diagnosis of COVID-19 was confirmed by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) on nasopharyngeal and/or throat swabs. Multivariate logistic regression and generalized linear regression were used. We considered a P value of <0.05 statistically significant., Results: A total of 560 patients met the inclusion criteria. An extensive list of clinical features was associated with higher 30-day ICU mortality rates, such as requiring mechanical ventilation (MV) or developing acute kidney injury within 24 hours of ICU admission, higher body temperature, white blood cells, blood glucose level, serum creatinine, fibrinogen, procalcitonin, creatine phosphokinase, aspartate aminotransferase, and total iron-binding capacity. During ICU stay, the most common complication was respiratory failure that required MV (71.4%), followed by acute kidney injury (AKI) and thrombosis with a proportion of 46.8% and 11.4%, respectively., Conclusion: Among patients with COVID-19 who were admitted to the ICU, several variables were associated with an increased risk of ICU mortality at 30 days. Respiratory failure that required MV, AKI, and thrombosis were the most common complications during ICU stay., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: In this study, we evaluated the inflammatory response in patients with severe acute respiratory infection due to the Middle East respiratory syndrome and non-Middle East respiratory syndrome and assessed the presence of distinct inflammatory subphenotypes using latent class analysis., Design: Prospective cohort study., Setting: A tertiary care ICU in Riyadh, Saudi Arabia., Patients: Consecutive critically ill patients with laboratory-confirmed Middle East respiratory syndrome severe acute respiratory infection and non-Middle East respiratory syndrome severe acute respiratory infection., Intervention: None., Measurements and Main Results: We measured cytokines on days 1, 3, 7, and 14 of ICU stay. We included 116 patients (40 with Middle East respiratory syndrome severe acute respiratory infection and 76 with non-Middle East respiratory syndrome severe acute respiratory infection). On ICU day 1, both patients with Middle East respiratory syndrome severe acute respiratory infection and non-Middle East respiratory syndrome severe acute respiratory infection had higher levels of interleukin-3, interleukin-4, interleukin-6, interleukin-8, interleukin-17A, eotaxin, and epidermal growth factor compared with healthy controls. There were no differences in cytokines over time between patients with Middle East respiratory syndrome severe acute respiratory infection and non-Middle East respiratory syndrome severe acute respiratory infection. Using day 1 cytokine levels, latent class analysis categorized patients into two subphenotypes: subphenotype 1 (n = 74 [64%]) and subphenotype 2 (n = 42 [36%]); the latter had significantly higher levels of interleukin-1β, interleukin-1ra, interleukin-2, interleukin-6, interleukin-7, interleukin-8, interleukin-10, interleukin-12p70, interleukin-15, interleukin-17A, inducible protein-10, monocyte chemoattractant protein-1, macrophage inflammatory protein-1α, macrophage inflammatory protein-1β, tumor necrosis factor-α, granulocyte-macrophage colony-stimulating factor, granulocyte-colony stimulating factor, interferon-α, and interferon-γ. Although baseline characteristics were not different between the two subphenotypes, patients in the subphenotype 2 had higher ICU mortality compared with the subphenotype 1 (18/42 [43%] vs 17/74 [23%]; p = 0.03)., Conclusions: One third of critically ill patients with Middle East respiratory syndrome severe acute respiratory infection and non-Middle East respiratory syndrome severe acute respiratory infection demonstrated a subphenotype characterized by increased proinflammatory cytokines, consistent with cytokine storm. Further research is needed to examine whether immunomodulators have differential effects based on inflammatory subphenotypes., Competing Interests: Dr. Arabi is the principal investigator on a clinical trial for lopinavir/ritonavir and interferon in Middle East respiratory syndrome; he is an investigator on A Randomised, Embedded, Multi-factorial, Adaptive Platform Trial for Community-Acquired Pneumonia (REMAP-CAP) and Board Members of the International Severe Acute Respiratory and Emerging Infection Consortium. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2020 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.)

Rationale, Aims, and Objectives: The recent outbreak of coronavirus (COVID-19) has infected around 1 560 000 individuals till 10 April 2020, which has resulted in 95 000 deaths globally. While no vaccine or anti-viral drugs for COVID-19 are available, lockdown acts as a protective public health measures to reduce human interaction and lower transmission. The study aims to explore the impact of delayed planning or lack of planning for the lockdown and inadequate implementation of the lockdown, on the transmission rate of COVID-19., Method: Epidemiological data on the incidence and mortality of COVID-19 cases as reported by public health authorities were accessed from six countries based on total number of infected cases, namely, United States and Italy (more than 100 000 cases); United Kingdom, and France (50 000-100 000 cases), and India and Russia (6000-10 000 cases). The Bayesian inferential technique was used to observe the changes (three points) in pattern of number of cases on different duration of exposure (in days) in these selected countries 1 month after World Health Organization (WHO) declaration about COVID-19 as a global pandemic., Results: On comparing the pattern of transmission rates observed in these six countries at posterior estimated change points, it is found that partial implementation of lockdown (in the United States), delayed planning in lockdown (Russia, United Kingdom, and France), and inadequate implementation of the lockdown (in India and Italy) were responsible to the spread of infections., Conclusions: In order to control the spreading of COVID-19, like other national and international laws, lockdown must be implemented and enforced. It is suggested that on-time or adequate implementation of lockdown is a step towards social distancing and to control the spread of this pandemic., (© 2020 John Wiley & Sons Ltd.)

Outdoor and early mosquito biters challenge the efficacy of bed-nets and indoor residual spraying on the Thailand-Myanmar border. Outdoor residual spraying is proposed for the control of exophilic mosquito species. The objective of this study was to assess the impact of outdoor residual spraying on the biting rate of malaria vectors in Kayin state, Myanmar. Outdoor residual spraying using lambda-cyhalothrin was carried out in two villages in December 2016 (beginning of the dry season) and two villages were used as a control. Malaria mosquitoes were captured at baseline and monthly for four months after the intervention using human-landing catch and cow-baited trap collection methods. The impact of outdoor residual spraying on human-biting rate was estimated with propensity score adjusted generalized linear mixed-effect regressions. At baseline, mean indoor and outdoor human-biting rate estimates ranged between 2.12 and 29.16 bites /person /night, and between 0.20 and 1.72 bites /person /night in the intervention and control villages respectively. Using model output, we estimated that human-biting rate was reduced by 91% (95%CI = 88-96, P <0.0001) immediately after outdoor residual spraying. Human-biting rate remained low in all sprayed villages for 3 months after the intervention. Malaria vector populations rose at month 4 in the intervention villages but not in the controls. This coincided with the expected end of insecticide mist residual effects, thereby suggesting that residual effects are important determinants of intervention outcome. We conclude that outdoor residual spraying with a capsule suspension of lambda-cyhalothrin rapidly reduced the biting rate malaria vectors in this area where pyrethroid resistance has been documented., Competing Interests: The authors have declared that no competing interests exist.

The 2019-Coronavirus (COVID-19) pandemic has had a global impact. The effect of environmental temperature on transmissibility and fatality rate of COVID-19 and protective efficacy of Bacillus Calmette-Guérin (BCG) vaccination towards COVID-19 remains ambiguous. Therefore, we explored the global impact of environmental temperature and neonatal BCG vaccination coverage on transmissibility and fatality rate of COVID-19. The COVID-19 data for reported cases, deaths and global temperature were collected from 31st December 2020 to 3rd April 2020 for 67 countries. Temperature data were split into quartiles for all three categories (minimum temperature, maximum temperature and mean temperature). The impact of three types of temperature data and policy of BCG vaccination on COVID-19 infection was determined by applying the multivariable two-level negative binomial regression analysis keeping daily new cases and daily mortality as outcome. The highest number of cases fell in the temperature categories as following: mean temperature in the second quartile (6°C to 10.5°C), median 26, interquartile range (IQR) 237; minimum temperature in the first quartile (-26°C to 1°C), median 23, IQR 173; maximum temperature in the second quartile (10°C to 16°C), median 27.5, IQR 219. For the minimum temperature category, 28% statistically significant lower incidence was noted for new cases from the countries falling in the second quartile (2°C to 6°C) compared with countries falling in the first quartile (-26°C to 1°C) (incidence rate ratio [IRR] 0.72, 95% confidence interval [CI] 0.57 to 0.93). However, no statistically significant difference in incidence rate was observed for mean temperature categories in comparison to the first quartile. Countries with BCG vaccination policy had 58% less mortality as compared with countries without BCG coverage (IRR 0.42; 95% CI 0.18 to 0.95). Our exploratory study provides evidence that high temperature might not be associated with low transmissibility and countries having neonatal BCG vaccination policy had a low fatality rate of COVID-19., Competing Interests: The authors have declared that no competing interests exist.

Objective: To investigate the association between antibiotics administration timing with morbidity and mortality in children with severe sepsis and septic shock, presenting to a tertiary care center in a developing country. Methods: This is a retrospective study of children aged 14 years or younger diagnosed with severe sepsis or septic shock at a free-standing tertiary children's hospital in Saudi Arabia between April 2015 and February 2018. We investigated the association between antibiotic administration timing and pediatric intensive care unit (PICU) mortality, PICU length of stay (LOS), hospital LOS, and ventilation-free days after adjusting for confounders. Results: Among the 189 admissions, 77 patients were admitted with septic shock and 112 with severe sepsis. Overall, the mortality rate was 16.9%. The overall median time from sepsis recognition to antibiotic administration was 105 min (IQR: 65-185.5 min); for septic shock patients, it was 85 min (IQR: 55-148 min), and for severe sepsis, 130 min (IQR: 75.5-199 min). Delayed antibiotic administration (> 3 h) was associated with 3.85 times higher PICU mortality (95% confidence intervals 1.032-14.374) in children with septic shock than in children who receive antibiotics within 3 h, after controlling for severity of illness, age, comorbidities, and volume resuscitation. However, delayed antibiotics administration was not significantly associated with higher PICU mortality in children diagnosed with severe sepsis. Conclusions: Delayed antibiotics administration in children with septic shock admitted to a free-standing children's hospital in a developing country was associated with PICU mortality., (Copyright © 2020 Alsadoon, Alhamwah, Alomar, Alsubaiel, Almutairi, Vishwakarma, Alharthy and Kazzaz.)

Transcription initiation is a key checkpoint and highly regulated step of gene expression. The sigma (σ) subunit of RNA polymerase (RNAP) controls all transcription initiation steps, from recognition of the -10/-35 promoter elements, upon formation of the closed promoter complex (RPc), to stabilization of the open promoter complex (RPo) and stimulation of the primary steps in RNA synthesis. The canonical mechanism to regulate σ activity upon transcription initiation relies on activators that recognize specific DNA motifs and recruit RNAP to promoters. This mini-review describes an emerging group of transcriptional regulators that form a complex with σ or/and RNAP prior to promoter binding, remodel the σ subunit conformation, and thus modify RNAP activity. Such strategy is widely used by bacteriophages to appropriate the host RNAP. Recent findings on RNAP-binding protein A (RbpA) from Mycobacterium tuberculosis and Crl from Escherichia coli suggest that activator-driven changes in σ conformation can be a widespread regulatory mechanism in bacteria., (Copyright © 2020 Vishwakarma and Brodolin.)

Background: The outbreak of coronavirus disease, 2019 (COVID-19), which started from Wuhan, China, in late 2019, have spread worldwide. A total of 5,91,971 cases and 2,70,90 deaths were registered till 28th March, 2020. We aimed to predict the impact of duration of exposure to COVID-19 on the mortality rates increment., Methods: In the present study, data on COVID-19 infected top seven countries viz., Germany, China, France, United Kingdom, Iran, Italy and Spain, and World as a whole, were used for modeling. The analytical procedure of generalized linear model followed by Gompertz link function was used to predict the impact lethal duration of exposure on the mortality rates., Findings: Of the selected countries and World as whole, the projection based on 21st March, 2020 cases, suggest that a total (95% Cl) of 76 (65-151) days of exposure in Germany, mortality rate will increase by 5 times to 1%. In countries like France and United Kingdom, our projection suggests that additional exposure of 48 days and 7 days, respectively, will raise the mortality rates to10%. Regarding Iran, Italy and Spain, mortality rate will rise to 10% with an additional 3-10 days of exposure. World's mortality rates will continue increase by 1% in every three weeks. The predicted interval of lethal duration corresponding to each country has found to be consistent with the mortality rates observed on 28th March, 2020., Conclusion: The prediction of lethal duration was found to have apparently effective in predicting mortality, and shows concordance with prevailing rates. In absence of any vaccine against COVID-19 infection, the present study adds information about the quantum of the severity and time elapsed to death will help the Government to take necessary and appropriate steps to control this pandemic., Competing Interests: The authors have declared that no competing interests exist.

Background: Caesarean section is considered a relatively preferable and safe method of delivery as compared to normal delivery. Since the last decade, its prevalence has increased in both developed and developing countries. In the context of developing countries viz., South Asia (the highest populated region) and South-East Asia (the third-highest populated region), where a significant proportion of home deliveries were reported,however, the preference for, caesarean delivery and its associated factors are not well understood., Objective: To study the caesarean delivery in the South and South-East Asian countries and to determine the factors associated with the preference for caesarean delivery., Methodology: Demographic and Health Survey Data on from ever-married women of nine developing countries of South and South-East Asia viz., Vietnam, India, Maldives, Timor-Leste, Nepal, Indonesia, Pakistan, Bangladesh, and Cambodia have been considered. Both bivariate and binary logistic regression models were used to estimate the propensity of a woman undergoing for caesarean delivery and to assess the influence of maternal socioeconomic characteristics towards the preference for caesarean delivery., Results: Obtained results have shown an inclination of caesarean delivery among urban than rural women and are quite conspicuous, but is found to be underestimated mostly among rural women. Caesarean delivery in general is mostly predisposed among women whose baby sizes are either very large or smaller than average, have a higher level of education and place of delivery is private medical institutions. The logistic regression also revealed the influence of maternal socioeconomic characteristics towards the preference for caesarean delivery. Based on nine South and South-East Asian countries an overall C-section prevalence of 13%, but based on institutional births its increase to 19%. The forest plot demonstrated that a significant inclination of C-section among urban than rural regions. In Meta-Analysis, very high and significant heterogeneity among countries is observed, but confirms that in terms of prevalence of C-section all of the countries follow independent pattern., Conclusion: Study of seven urban and four rural regions of nine South and South- East Asian countries showed, a significant inclination towards the caesarean delivery above the more recent outdated WHO recommended an optimal range of 10-15%and are associated maternal socioeconomic characteristics. In order to control unwanted caesarean delivery, the government needs to develop better healthcare infrastructure and along with more antenatal care related schemes to reduce the risks associated with increased caesarean delivery., Competing Interests: The authors have read the journal's policy and the authors of this study have the following competing interests: RP is a paid employee of Eurofins Limited (https://www.eurofins.com/agroscience-services/about-us/latest-news/eurofins-scientific-to-acquire-advinus-therapeutics/). There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Fungal diseases result in significant losses of fruits and vegetables during handling, transportation and storage. At present, post-production fungal spoilage is predominantly controlled by using synthetic fungicides. Under the global climate change scenario and with the need for sustainable agriculture, biological control methods of fungal diseases, using antagonistic microorganisms, are emerging as ecofriendly alternatives to the use of fungicides. The potential of microbial antagonists, isolated from a diversity of natural habitats, for postharvest disease suppression has been investigated. Postharvest biocontrol systems involve tripartite interaction between microbial antagonists, the pathogen and the host, affected by environmental conditions. Several modes for fungistatic activities of microbial antagonists have been suggested, including competition for nutrients and space, mycoparasitism, secretion of antifungal antibiotics and volatile metabolites and induction of host resistance. Postharvest application of microbial antagonists is more successful for efficient disease control in comparison to pre-harvest application. Attempts have also been made to improve the overall efficacy of antagonists by combining them with different physical and chemical substances and methods. Globally, many microbe-based biocontrol products have been developed and registered for commercial use. The present review provides a brief overview on the use of microbial antagonists as postharvest biocontrol agents and summarises information on their isolation, mechanisms of action, application methods, efficacy enhancement, product formulation and commercialisation.

The transcriptional activator RbpA associates with Mycobacterium tuberculosis RNA polymerase (MtbRNAP) during transcription initiation, and stimulates formation of the MtbRNAP-promoter open complex (RPo). Here, we explored the influence of promoter motifs on RbpA-mediated activation of MtbRNAP containing the stress-response σB subunit. We show that both the 'extended -10' promoter motif (T-17G-16T-15G-14) and RbpA stabilized RPo and allowed promoter opening at suboptimal temperatures. Furthermore, in the presence of the T-17G-16T-15G-14 motif, RbpA was dispensable for RNA synthesis initiation, while exerting a stabilization effect on RPo. On the other hand, RbpA compensated for the lack of sequence-specific interactions of domains 3 and 4 of σB with the extended -10 and the -35 motifs, respectively. Mutations of the positively charged residues K73, K74 and R79 in RbpA basic linker (BL) had little effect on RPo formation, but affected MtbRNAP capacity for de novo transcription initiation. We propose that RbpA stimulates transcription by strengthening the non-specific interaction of the σ subunit with promoter DNA upstream of the -10 element, and by indirectly optimizing MtbRNAP interaction with initiation substrates. Consequently, RbpA renders MtbRNAP promiscuous in promoter selection, thus compensating for the weak conservation of the -35 motif in mycobacteria.

This work used an approach of enzyme engineering towards the improved production of baicalin as well as alteration of acceptor and donor substrate preferences in UGT73A16. The 3D model of Withania somnifera family-1 glycosyltransferase (UGT73A16) was constructed based on the known crystal structures of plant UGTs. Structural and functional properties of UGT73A16 were investigated using docking and mutagenesis. The docking studies were performed to understand the key residues involved in substrate recognition. In the molecular model of UGT73A16, substrates binding pockets are located between N- and C-terminal domains. Modeled UGT73A16 was docked with UDP-glucose, UDP-glucuronic acid (UDPGA), kaempferol, isorhamnetin, 3-hydroxy flavones, naringenin, genistein and baicalein. The protein-ligand interactions showed that His 16, Asp 246, Lys 255, Ala 337, Gln 339, Val 340, Asn 358 and Glu 362 amino acid residues may be important for catalytic activity. The kinetic parameters indicated that mutants A337C and Q339A exhibited 2-3 fold and 6-7 fold more catalytic efficiency, respectively than wild type, and shifted the sugar donor specificity from UDP-glucose to UDPGA. The mutant Q379H displayed large loss of activity with UDP-glucose and UDPGA strongly suggested that last amino acid residue of PSPG box is important for glucuronosylation and glucosylation and highly specific to sugar binding sites. The information obtained from docking and mutational studies could be beneficial in future to engineer this biocatalyst for development of better ones.

Pulses are essential component of the human diet in the underdeveloped and the developing countries. Pulse milling mainly focuses on complete removal of the hull with minimum generation of powder, broken, and in certain cases dehulled split formation. Physical and mechanical properties of different pulses are described and their role in dehulling is discussed in this review. The review discusses nature of gums that binds hull and cotyledons together and their effect on milling characteristics. Pitting operation is performed for all pulses prior to the pretreatment for better dehulling. Various pretreatment processes (soaking, edible oils, chemicals, enzymes, hydrothermal) developed to loosen the bond are examined for their dehulling efficacy from commercial application viewpoint. Dependence of dehulling characteristics of different pulses on size, shape, variety, grain hardness, and moisture content are discussed. Most of the machines developed for pulses dehulling in India are abrasion based emery-cylinder concave system. The studies done for optimization of different pretreatment process conditions are also reviewed. The losses taking place in the form of broken and powder during pulse milling are described. Methods for performance evaluation of the pulse dehulling system are reviewed and expressions are suggested for proper evaluation. This review gives complete overview of the processes and technological status of pulse milling in present context.

The σ subunit of bacterial RNA polymerase (RNAP) controls recognition of the -10 and -35 promoter elements during transcription initiation. Free σ adopts a "closed," or inactive, conformation incompatible with promoter binding. The conventional two-state model of σ activation proposes that binding to core RNAP induces formation of an "open," active, σ conformation, which is optimal for promoter recognition. Using single-molecule Förster resonance energy transfer, we demonstrate that vegetative-type σ subunits exist in open and closed states even after binding to the RNAP core. As an extreme case, RNAP from Mycobacterium tuberculosis preferentially retains σ in the closed conformation, which is converted to the open conformation only upon binding by the activator protein RbpA and interaction with promoter DNA. These findings reveal that the conformational dynamics of the σ subunit in the RNAP holoenzyme is a target for regulation by transcription factors and plays a critical role in promoter recognition.

Fruit drinks contain negligible amount of protein as nutritional component. Fortification of fruit drinks with protein is a challenge due to protein stability in acidic and ionic environment. Mango ready-to-serve (RTS) beverage was fortified with modified whey protein and its rheological properties were studied. Whey protein was hydrolysed with papain to improve its stability in acidic medium. The water holding capacity of whey protein increased about two times after hydrolysis. Hydrolysed and native whey protein was used at 2, 3 and 4% levels for fortification of mango based RTS beverage. Addition of hydrolysed whey protein at all the three levels did not significantly change the flow behaviour of the beverage. Native whey protein fortification resulted in precipitation; however, addition of hydrolysed whey protein led to stable beverage formulation at all the three levels. Hydrolysed whey protein imparted slight bitter taste to the RTS beverage, which was masked by β-cyclodextrin @ 0.15% of total protein. The mango RTS beverage with 3.0% hydrolysed whey protein was found acceptable with good sensory appeal and stability during thermal processing as well storage in glass bottles.

Mevalonate diphosphate decarboxylase (MDD; EC 4.1.1.33) is an important enzyme in the mevalonic acid pathway catalyzing the Mg(2+)-ATP dependant decarboxylation of mevalonate 5-diphosphate (MVAPP) to isopentenyl diphosphate (IPP). Bacopa monniera recombinant MDD (BmMDD) protein was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Km and Vmax for MVAPP were 144 μM and 52 U mg(-1) respectively. The values of turnover (kcat) and kcat/Km for mevalonate 5-diphosphate were determined to be 40s(-1) and 2.77×10(5) M(-1) s(-1) and kcat and kcat/Km values for ATP were found to be 30 s(-1) and 2.20×10(4) M(-1) s(-1), respectively. pH activity profile indicated the involvement of carboxylate ion, lysine and arginine for the activity of enzyme. The apparent activation energy for the BmMDD catalyzed reaction was 12.7 kJ mol(-1). Optimum pH and temperature for the forward reaction was found to be 8.0 and 45 °C. The enzyme was most stable at pH 7 at 20 °C with the deactivation rate constant (Kd(*)) of 1.69×10(-4) and half life (t1/2) of 68 h. The cation studies suggested that BmMDD is a cation dependant enzyme and optimum activity was achieved in the presence of Mg(2+)., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Bacopa monniera is an important source of metabolites with pharmaceutical value. It has been regarded as a valuable medicinal plant and its entire commercial requirement is met from wild natural population. Recently, metabolic engineering has emerged as an important solution for sustained supply of assured and quality raw material for the production of active principles. Present report describes efficient in vitro multiplication and transformation method for genetic manipulation of this species. MS medium supplemented with 2 mgl(-1) BA and 0.2 mgl(-1) IAA was found optimum for maximum shoot regeneration (98.33 %) from in vitro leaves with 2-3 longitudinal cuts. Agrobacterium tumefaciens-mediated transformation method was used for generating transgenic B. monniera plants. Putative transformants were confirmed by GUS assay and PCR based confirmation of hptII gene. DNA blot analysis showed single copy insertion of transgene cassette. An average of 87.5 % of the regenerated shoots were found PCR positive for hptII gene and GUS activity was detected in leaves of transgenic shoots at a frequency of 82.5 % The efficient multiple shoots regeneration system described herein may help in mass production of B. monniera plant. Also, the high frequency transformation protocol described here can be used for genetic engineering of B. monniera for enhancement of its pharmaceutically important metabolites.

Aboitic stress such as drought and salinity are class of major threats, which plants undergo through their lifetime. Lignin deposition is one of the responses to such abiotic stresses. The gene encoding Cinnamoyl CoA Reductase (CCR) is a key gene for lignin biosynthesis, which has been shown to be over-expressed under stress conditions. In the present study, developing seedlings of Leucaena leucocephala (Vernacular name: Subabul, White popinac) were treated with 1 % mannitol and 200 mM NaCl to mimic drought and salinity stress conditions, respectively. Enzyme linked immunosorbant assay (ELISA) based expression pattern of CCR protein was monitored coupled with Phlorogucinol/HCl activity staining of lignin in transverse sections of developing L. leucocephala seedlings under stress. Our result suggests a differential lignification pattern in developing root and stem under stress conditions. Increase in lignification was observed in mannitol treated stems and corresponding CCR protein accumulation was also higher than control and salt stress treated samples. On the contrary CCR protein was lower in NaCl treated stems and corresponding lignin deposition was also low. Developing root tissue showed a high level of CCR content and lignin deposition than stem samples under all conditions tested. Overall result suggested that lignin accumulation was not affected much in case of developing root however developing stems were significantly affected under drought and salinity stress condition.

Mevalonate kinase (MK; ATP: mevalonate 5-phosphotransferase; EC 2.7.1.36) plays a key role in isoprenoid biosynthetic pathway in plants. MK catalyzes the phosphorylation of mevalonate to form mevalonate-5-phosphate. The recombinant BmMK was cloned and over-expressed in E. coli BL21 (DE3), and purified to homogeneity by affinity chromatography followed by gel filtration. Optimum pH and temperature for forward reaction was found to be 7.0 and 30 °C, respectively. The enzyme was most stable at pH 8 at 25 °C with deactivation rate constant (Kd*) 1.398 × 10(-4) and half life (t1/2) 49 h. pH activity profile of BmMK indicates the involvement of carboxylate ion, histidine, lysine, arginine or aspartic acid at the active site of enzyme. Activity of recombinant BmMK was confirmed by phosphorylation of RS-mevalonate in the presence of Mg(2+), having Km and Vmax 331.9 μM and 719.1 pKat μg(-1), respectively. The values of kcat and kcat/Km for RS-mevalonate were determined to be 143.82 s(-1) and 0.43332 M(-1) s(-1) and kcat and kcat/Km values for ATP were found 150.9 s(-1) and 1.023 M(-1) s(-1). The metal ion studies suggested that BmMK is a metal dependent enzyme and highly active in the presence of MgCl2., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Gentiana scabra is commonly known as 'Longdan' is an important herb in traditional Chinese medicines, commonly used for the treatment of inflammation, anorexia, indigestion and gastric infections. Iridoids and secoiridoids are main bioactive compounds which attributed to the pharmacological properties of this plant. The use of hairy root cultures as an excellent alternative for the production of pharmaceutically important metabolites in less time period with ensured quality of raw materials., Results: An efficient hairy root culture system of Gentiana scabra and influence of different plant growth regulators (PGRs) on the production of gentiopicroside, swertiamarin and loganic acid constituents were described. Leaf explants were infected with Agrobacterium rhizogenes, which induced hairy roots up to 21%. The transformed hairy root lines were confirmed by PCR using rolB and rolC gene-specific primers. Among various solid and liquid media, B5 liquid medium resulted maximum root biomass (36- fold higher) in 4-weeks. Quantitative analysis showed loganic acid was 6.6- fold higher in the presence of zeatin (1 mg/l) and gentiopicroside accumulation was 1.8- fold higher in the presence of naphthaleneacetic acid (NAA, 1 mg/l), as compared to the roots of plants grown in greenhouse. On the other hand, 1.4- and 2.5- fold higher gentiopicroside and swertiamarin were observed in the presence of 1.0 mg/l NAA as compared to commercial Gentiana herb No. 2. The result also showed iridoid and secoiridoid contents affected greatly by age, physiology and growing environment of the plant., Conclusions: The use of hairy root cultures is an excellent alternative to harvesting natural or in vitro grown plants to produce pharmaceutically important metabolites in less time with ensured quality.

Health related benefits of isoflavones such as genistein are well known. Glycosylation of genistein yields different glycosides like genistein 7-O-glycoside (genistin) and genistein 4'-O-glycoside (sophoricoside). This is the first report on isolation, cloning and functional characterization of a glycosyltransferase specific for genistein 4'-O-glucoside from Bacopa monniera, an important Indian medicinal herb. The glycosyltransferase from B. monniera (UGT74W1) showed 49% identity at amino acid level with the glycosyltransferases from Lycium barbarum. The UGT74W1 sequence contained all the conserved motifs present in plant glycosyltransferases. UGT74W1 was cloned in pET-30b (+) expression vector and transformed into E. coli. The molecular mass of over expressed protein was found to be around 52 kDa. Functional characterization of the enzyme was performed using different substrates. Product analysis was done using LC-MS and HPLC, which confirmed its specificity for genistein 4'-O-glucoside. Immuno-localization studies of the UGT74W1 showed its localization in the vascular bundle. Spatio-temporal expression studies under normal and stressed conditions were also performed. The control B. monniera plant showed maximum expression of UGT74W1 in leaves followed by roots and stem. Salicylic acid treatment causes almost tenfold increase in UGT74W1 expression in roots, while leaves and stem showed decrease in expression. Since salicylic acid is generated at the time of injury or wound caused by pathogens, this increase in UGT74W1 expression under salicylic acid stress might point towards its role in defense mechanism.

The effect of hot water blanching treatment on thin layer drying kinetics of aonla shreds was studied at drying air temperatures of 50, 55 and 60 °C with the air velocity of 1.2 m/s. The drying time decreased with the increase in air temperature and blanching. The drying process was observed in falling rate. Drying after blanching reduced the vitamin C content of aonla shreds by 69.36% whereas it decreased by 27.78% in unblanched shreds. Eight commonly used mathematical models were evaluated to predict the drying behavior of aonla shreds. The Midilli model described the drying behaviour of unblanched aonla shreds at all temperatures better than other models whereas two-term model described the drying kinetics of blanched aonla shreds satisfactorily. The effective diffusivities of the unblanched and blanched aonla shreds were determined using Fick's law of diffusion. The activation energy was found to be 47.21 kJ/mol for unblanched and 43.98 kJ/mol for blanched aonla shreds.

Fluorescence quenching and time resolved fluorescence studies of wild type recombinant cinnamoyl CoA reductase (Ll-CCRH1), a multitryptophan protein from Leucaena leucocephala and 10 different active site mutants were carried out to investigate tryptophan environment. The enzyme showed highest affinity for feruloyl CoA (K(a)  = 3.72 × 10(5) M(-1)) over other CoA esters and cinnamaldehydes, as determined by fluorescence spectroscopy. Quenching of the fluorescence by acrylamide for wild type and active site mutants was collisional with almost 100% of the tryptophan fluorescence accessible under native condition and remained same after denaturation of protein with 6 M GdnHCl. In wild type Ll-CCRH1, the extent of quenching achieved with iodide (f(a) = 1.0) was significantly higher than cesium ions (f(a) = 0.33) suggesting more density of positive charge around surface of trp conformers under native conditions. Denaturation of wild type protein with 6 M GdnHCl led to significant increase in the quenching with cesium (f(a) = 0.54), whereas quenching with iodide ion was decreased (f(a) = 0.78), indicating reorientation of charge density around trp from positive to negative and heterogeneity in trp environment. The Stern-Volmer plots for wild type and mutants Ll-CCRH1 under native and denatured conditions, with cesium ion yielded biphasic quenching profiles. The extent of quenching for cesium and iodide ions under native and denatured conditions observed in active site mutants was significantly different from wild type Ll-CCRH1 under the same conditions. Thus, single substitution type mutations of active site residues showed heterogeneity in tryptophan microenvironment and differential degree of conformation of protein under native or denatured conditions.

Background: Salvia miltiorrhiza Bunge (Danshen), an important herb in traditional Chinese medicine, is commonly used for treatment of cardiovascular diseases. One of the major bioactive constituents of Danshen, diterpenoid tanshinone, has been proved with pharmacological properties and have the potential to be a new drug candidate against various diseases. In our previous study, we have established an activation tagging mutagenesis (ATM) population of callus lines of S. miltiorrhiza Bunge by Agrobacterium- mediated transformation., Results: In the present study, we have identified ATM transgenic Salvia plant (SH41) with different leaf morphology and more tanshinones in its roots. The transgenic background of SH41 was identified by PCR (using hpt II primers) and Southern blots. PCR analysis showed a single band of hpt II gene and Southern blot analysis showed single insertion in SH41. External appearance of ATM transgenic SH41 was observed with broader leaves comparing to non-transformed plants. More healthy trichomes as well as bigger and wobbly guard cells and stomata were observed in SH41 by scanning electron microscopy (SEM). Quantitative analysis of active compounds in SH41 roots revealed a significant increase in tanshinone I (3.7 fold) and tanshinone IIA (2 fold) contents as compared to the wild plant., Conclusions: We have generated an activation tagged transgenic Salvia plant (SH41) with different leaf morphology and high diterpenes content in its roots. The increased amount of tanshinones in SH41 will definitely offer a route for maximizing the benefits of this plant in traditional Chinese herbal medicines. The present report may also facilitate the application of ATM for genetic manipulation of other medicinal crops and subsequent improved metabolite contents.

Triterpenoid saponins are the class of secondary metabolites, synthesized via isoprenoid pathway. Oxidosqualene cyclases (OSCs) catalyzes the cyclization of 2, 3-oxidosqualene to various triterpene skeletons, the first committed step in triterpenoid biosynthesis. A full-length oxidosqualene cyclase cDNA from Bacopa monniera (BmOSC) was isolated and characterized. The open reading frame (ORF) of BmOSC consists of 2,292 bp, encoding 764 amino acid residues with an apparent molecular mass of 87.62 kDa and theoretical pI 6.21. It contained four QxxxxxW motifs, one Asp-Cys-Thr-Ala-Glu (DCTAE) motif which is highly conserved among the triterpene synthases and another MWCYCR motif involved in the formation of triterpenoid skeletons. The deduced amino acid sequence of BmOSC shares 80.5 % & 71.8 % identity and 89.7 % & 83.5 % similarity with Olea europaea mixed amyrin synthase and Panax notoginseng dammarenediol synthase respectively. Phylogenetic analysis revealed that BmOSC is closely related with other plant OSCs. Quantitative real-time PCR (qRT-PCR) data showed that BmOSC is expressed in all tissues examined with higher expression in stem and leaves as compared to roots and floral parts.

Lack of three dimensional crystal structure of cinnamoyl CoA reductase (CCR) limits its detailed active site characterization studies. Putative active site residues involved in the substrate/NADPH binding and catalysis for Leucaena leucocephala CCR (Ll-CCRH1; GenBank: DQ986907) were identified by amino acid sequence alignment and homology modeling. Putative active site residues and proximal H215 were subjected for site directed mutagenesis, and mutated enzymes were expressed, purified and assayed to confirm their functional roles. Mutagenesis of S136, Y170 and K174 showed complete loss of activity, indicating their pivotal roles in catalysis. Mutant S212G exhibited the catalytic efficiencies less than 10% of wild type, showing its indirect involvement in substrate binding or catalysis. R51G, D77G, F30V and I31N double mutants showed significant changes in Km values, specifying their roles in substrate binding. Finally, chemical modification and substrate protection studies corroborated the presence Ser, Tyr, Lys, Arg and carboxylate group at the active site of Ll-CCRH1., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) protein from Leucaena leucocephala was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Optimum pH for forward and reverse reaction was found to be 6.5 and 7.8 respectively. The enzyme was most stable around pH 6.5 at 25°C for 90 min. The enzyme showed Kcat/Km for feruloyl, caffeoyl, sinapoyl, coumaroyl CoA, coniferaldehyde and sinapaldehyde as 4.6, 2.4, 2.3, 1.7, 1.9 and 1.2 (×10(6) M(-1) s(-1)), respectively, indicating affinity of enzyme for feruloyl CoA over other substrates and preference of reduction reaction over oxidation. Activation energy, Ea for various substrates was found to be in the range of 20-50 kJ/mol. Involvement of probable carboxylate ion, histidine, lysine or tyrosine at the active site of enzyme was predicted by pH activity profile. SAXS studies of protein showed radius 3.04 nm and volume 49.25 nm(3) with oblate ellipsoid shape. Finally, metal ion inhibition studies revealed that Ll-CCRH1 is a metal independent enzyme., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Glycosylation of flavonoids is mediated by family 1 uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs). Until date, there are few reports on functionally characterized flavonoid glycosyltransferases from Withania somnifera. In this study, we cloned the glycosyltransferase gene from W. somnifera (UGT73A16) showing 85-92 % homology with UGTs from other plants. UGT73A16 was expressed as a His(6)-tagged fusion protein in Escherichia coli. Several compounds, including flavonoids, were screened as potential substrates for UGT73A16. HPLC analysis and hypsochromic shift indicated that UGT73A16 transfers a glucose molecule to several different flavonoids. Based on kinetic parameters, UGT73A16 shows more catalytic efficiency towards naringenin. Here, we explored UGT73A16 of W. somnifera as whole cell catalyst in E. coli. We used flavonoids (genistein, apigenin, kaempferol, naringenin, biochanin A, and daidzein) as substrates for this study. More than 95 % of the glucoside products were released into the medium, facilitating their isolation. Glycosylation of substrates occurred on the 7- and 3-hydroxyl group of the aglycone. UGT73A16 also displayed regiospecific glucosyl transfer activity towards 3-hydroxy flavone compound, which is the backbone of all flavonols and also for a chemically synthesized compound, not found naturally. The present study generates essential knowledge and molecular as well as biochemical tools that allow the verification of UGT73A16 in glycosylation.

Unlabelled: : Cinnamoyl CoA reductase (CCR) carries out the first committed step in monolignol biosynthesis and acts as a first regulatory point in lignin formation. CCR shows multiple substrate specificity towards various cinnamoyl CoA esters. Here, in Silico mutagenesis studies of active site residues of Ll-CCRH1 were carried out. Homology modeling based modeled 3D structure of Ll-CCRH1 was used as template for in Silico mutant preparations. Docking simulations of Ll-CCRH1 mutants with CoA esters by AutoDock Vina tools showed altered substrate specificity as compared to wild type. The study evidences that conformational changes, and change in geometry or architecture of active site pocket occurred following mutations. The altered substrate specificity for active site mutants suggests the possible physiological role of CCR either in lignin formation or in defense system in plants., Abbreviations: Ll-CCRH1 - Leucaena leucocephala cinnamoyl CoA reductase 1, OPLS - Optimized Potentials for Liquid Simulations, RMSD - Root Mean Square Deviation.

Unlabelled: Hertz's theory of contact stresses was applied to predict the splitting of guar seeds during uni-axial compressive loading between 2 rigid parallel plates. The apparent modulus of elasticity of guar seeds varied between 296.18 and 116.19 MPa when force was applied normal to hilum joint (horizontal position), whereas it varied between 171.86 and 54.18 MPa when force was applied in the direction of hilum joint (vertical position) with in moisture content range of 5.16% to 15.28% (d.b.). At higher moisture contents, the seeds yielded after considerable deformation, thus showing ductile nature. Distribution of stresses below the point of contact were plotted to predict the location of critical point, which was found at 0.44 to 0.64 mm and 0.37 to 0.53 mm below the contact point in vertical and horizontal loading, respectively, depending upon moisture content. The separation of cotyledons from each other initiated before yielding of cotyledons and thus splitting of seed took place. The relationships between apparent modulus of elasticity, principal stresses with moisture content were described using second-order polynomial equations and validated experimentally., Practical Application: Manufacture of guar gum powder requires dehulling and splitting of guar seeds. This article describes splitting behavior of guar seeds under compressive loading. Results of this study may be used for design of dehulling and splitting systems of guar seeds., (© 2012 Institute of Food Technologists®)

Squalene synthase (SQS: EC 2.5.1.21) is a potential branch point regulatory enzyme and represents the first committed step to diverge the carbon flux from the main isoprenoid pathway towards sterol biosynthesis. In the present study, cloning and characterization of Withania somnifera squalene synthase (WsSQS) cDNA was investigated subsequently followed by its heterologous expression and preliminary enzyme activity. Two different types of WsSQS cDNA clones (WsSQS1and WsSQS2) were identified that contained an open reading frames of 1,236 and 1,242 bp encoding polypeptides of 412 and 414 amino acids respectively. Both WsSQS isoforms share 99 % similarity and identity with each other. WsSQS deduced amino acids sequences, when compared with SQS of other plant species, showed maximum similarity and identity with Capsicum annuum followed by Solanum tuberosum and Nicotiana tabacum. To obtain soluble recombinant enzymes, 24 hydrophobic amino acids were deleted from the carboxy terminus and expressed as 6X His-Tag fusion protein in Escherichia coli. Approximately 43 kDa recombinant protein was purified using Ni-NTA affinity chromatography and checked on SDS-PAGE. Preliminary activity of the purified enzymes was determined and the products were analyzed by gas chromatograph-mass spectrometer (GC-MS). Quantitative real-time PCR (qRT-PCR) analysis showed that WsSQS expresses more in young leaves than mature leaves, stem and root.

Unlabelled: Farnesyl pyrophosphate synthase (FPS; EC 2.5.1.10) is a key enzyme in isoprenoid biosynthetic pathway and provides precursors for the biosynthesis of various pharmaceutically important metabolites. It catalyzes head to tail condensation of two isopentenyl pyrophosphate molecules with dimethylallyl pyrophosphate to form C15 compound farnesyl pyrophosphate. Recent studies have confirmed FPS as a molecular target of bisphosphonates for drug development against bone diseases as well as pathogens. Although large numbers of FPSs from different sources are known, very few protein structures have been reported till date. In the present study, FPS gene from medicinal plant Bacopa monniera (BmFPS) was characterized by comparative modeling and docking. Multiple sequence alignment showed two highly conserved aspartate rich motifs FARM and SARM (DDXXD). The 3-D model of BmFPS was generated based on structurally resolved FPS crystal information of Gallus gallus. The generated models were validated by various bioinformatics tools and the final model contained only α-helices and coils. Further, docking studies of modeled BmFPS with substrates and inhibitors were performed to understand the protein ligand interactions. The two Asp residues from FARM (Asp100 and Asp104) as well as Asp171, Lys197 and Lys262 were found to be important for catalytic activity. Interaction of nitrogen containing bisphosphonates (risedronate, alendronate, zoledronate and pamidronate) with modeled BmFPS showed competitive inhibition; where, apart from Asp (100, 104 and 171), Thr175 played an important role. The results presented here could be useful for designing of mutants for isoprenoid biosynthetic pathway engineering well as more effective drugs against osteoporosis and human pathogens., Abbreviations: IPP - Isopentenyl Pyrophosphate, DMAPP - Dimethylallyl Pyrophosphate, GPP - Geranyl Pyrophosphate, FPP - FPPFarnesyl Pyrophosphate, DOPE - Discrete Optimized Protein Energy, BmFPS - Bacopa monniera Farnesyl Pyrophosphate Synthase, RMSD - Root Mean square Deviation, OPLS-AA - Optimized Potentials for Liquid Simulations- All Atom, FARM - First Aspartate Rich Motif, SARM - Second Aspartate Rich Motif.

Removal of lignin is a major hurdle for obtaining good quality pulp. Leucaena leucocephala (subabul) is extensively used in paper industry in India; therefore, as a first step to generate transgenic plants with low lignin content, cDNA and genomic clones of CCR gene were isolated and characterized. The cDNA encoding CCR (EC 1.2.1.44) was designated as Ll-CCR; the sequence analysis revealed an Open Reading Frame (ORF) of 1005 bp. Phylogenetic analysis showed that Ll-CCR sequence is highly homologous to CCRs from other dicot plants. The 2992 bp genomic clone of Leucaena CCR consists of 5 exons and 4 introns. The haploid genome of L. leucocephala contains two copies as revealed by DNA blot hybridization. Ll-CCR gene was over-expressed in Escherichia coli, which showed a molecular mass of approximately 38 kDa. Protein blot analysis revealed that Ll-CCR protein is expressed at higher levels in root and in stem, but undetectable in leaf tissues. Expression of CCR gene in Leucaena increased up to 15 d in case of roots and stem as revealed by QRT-PCR studies in 0-15 d old seedlings. ELISA based studies of extractable CCR protein corroborated with QRT-PCR data. CCR protein was immuno-cytolocalized around xylem tissue. Lignin estimation and expression studies of 5, 10 and 15 d old stem and root suggest that CCR expression correlates with quantity of lignin produced, which makes it a good target for antisense down regulation for producing designer species for paper industry., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

Leucaena leucocephala is a fast growing multipurpose legume tree used for forage, leaf manure, paper and pulp. Lignin in Leucaena pulp adversely influences the quality of paper produced. Developing transgenic Leucaena with altered lignin by genetic engineering demands an optimized regeneration system. The present study deals with optimization of regeneration system for L. leucocephala cv. K636. Multiple shoot induction from the cotyledonary nodes of L. leucocephala was studied in response to cytokinins, thidiazuron (TDZ) and N(6)-benzyladenine (BA) supplemented in half strength MS (½-MS) medium and also their effect on in vitro rooting of the regenerated shoots. Multiple shoots were induced from cotyledonary nodes at varied frequencies depending on the type and concentration of cytokinin used in the medium. TDZ was found to induce more number of shoots per explant than BA, with a maximum of 7 shoots at an optimum concentration of 0.23 µM. Further increase in TDZ concentration resulted in reduced shoot length and fasciation of the shoots. Liquid pulse treatment of the explants with TDZ did not improve the shoot production further but improved the subsequent rooting of the shoots that regenerated. Regenerated shoots successfully rooted on ½-MS medium supplemented with 0.54 µM α-naphthaleneacetic acid (NAA). Rooted shoots of Leucaena were transferred to coco-peat and hardened plantlets showed ≥ 90 % establishment in the green house.