Metal regulation of Mycobacterium tuberculosis SufB intein splicing at the host-pathogen crossroad.

Intein sequences self-excise from precursor proteins to generate functional proteins in various organisms. Thus, regulation of intein splicing at the host-pathogen interface can determine the fate of infection by controlling generation of essential proteins in microbes. For instance, Mycobacterium tuberculosis (Mtu) SufB intein splicing is crucial for the functionality of SUF complex. This multiprotein system is the sole pathway for [Fe-S] cluster biogenesis in mycobacteria during oxidative stress and Fe starvation. Although metal toxicity and metal starvation are components of host immunity, correlation of metal stress to Mtu SufB intein splicing is missing till date. Current study examines the splicing and N-terminal cleavage reactions of Mtu SufB precursor protein in presence of micronutrient metal ions like Zn+2, Cu+2, and Fe+3/+2. A known intein splicing inhibitor Pt+4 was also tested to support its proposed role as an anti-TB agent. Mtu SufB precursor protein exhibited significant attenuation of splicing and N-terminal cleavage reactions across different concentration ranges for Pt+4, Cu+2, Zn+2, while Fe+3 interaction resulted in precursor accumulation. UV-Vis spectroscopy, inductively coupled plasma-optical emission spectroscopy (ICP-OES), Tryptophan fluorescence assay, and dynamic light scattering (DLS) techniques analyzed metal-protein interaction. Mutagenesis experiments and Ellman's assay identified plausible metal co-ordination sites within Mtu SufB protein. Analyzing the metal effect on Mtu SufB splicing may provide elemental information about the fate of mycobacterial infection, and a probable mechanism to attenuate intracellular survival of Mtu. Current research hints at the host regulatory mechanism on SufB splicing in its native environment and a likely target for developing next-generation anti-TB drugs.

Audit of operation theater time utilization with perspective to optimize turnaround times and theater output.

Background and Aims: Operation theater (OT) complex is an important area for a hospital as it needs expensive infrastructure, disposable, and reusable resources and a multidisciplinary highly qualified and efficient team, the metrics of which are key in generating revenue, and improved productivity. The efficient utilization of OT ensures maximum output in view of the investment of highly qualified doctors, equipment, and outcomes. Our study aimed to evaluate the utilization of OT functioning stepwise, reasons for delays, case cancellations, and areas of improvement if any. Material and Methods: This prospective observational study was planned in three phases; in phase 1 audit of OT functioning was carried out for 1 month and based on data analysis recommendations were given for improvement. In phase 2, the recommendations would be implemented over 3 months and in phase 3 re-audit will be carried out for 1 month. Data analysis was done on IBM SPSS version 26 software. Descriptive statistics measures were calculated by the mean and standard deviation. Results: The total available resource time was 52920 min and the total time utilized was 37740 min. Overall, raw utilization was 71.31%. OT was started late 63.50% times. Case cancellation occurred on 8.99% occasions. Conclusion: We conclude that utilization of operating room time can be maximized by proper planning and realistic scheduling of elective lists, communication among team members, and resource management. Audit of OT utilization is an important tool to identify problem areas and formulate protocols accordingly.

The effect of different pressures of pneumoperitoneum on the dimensions of internal jugular vein - A prospective double-blind, randomised study.

Background and Aims: During laparoscopic surgeries, pneumoperitoneum increases intraabdominal pressure (IAP) which can increase the central venous pressure (CVP), and significant haemodynamic changes. In this study, we evaluated the effect of two different pressures of pneumoperitoneum, standard (13-15 mmHg), and low (6-8 mmHg) on the cross-sectional area (CSA) of the internal jugular vein (IJV) using ultrasonography, haemodynamic changes and duration of surgery. Surgeon's comfort and feasibility of performing laparoscopic surgeries with low pressure pneumoperitoneum was also studied. Methods: This prospective, double-blind, randomised study included 148 patients of American Society of Anesthesiologists physical status class I and II undergoing laparoscopic surgeries. They were allocated into two groups: group S (standard) (number (n) = 73) had the IAP maintained between 13-15 mmHg; group L (low) had an IAP of 6-8 mmHg (n = 75). CSA of right IJV was measured before induction of anaesthesia (T1), 5 min after intubation (T2), 5 min after pneumoperitoneum (T3), before desufflation (T4) and 5 min prior to extubation (T5). Chi-square test, and Student's paired and unpaired t test were used for statistical analysis. Results: The increase in IJV CSA at T3 when compared to T2 was statistically significant in both the groups (P < 0.001). On desufflation, the change in IJV CSA showed significant decrease in T5 value than T4 value in both the groups (P < 0.001). However, the percentage change in the IJV CSA was more in group S (35.4%) than group L (21.2%). Conclusion: CSA of IJV increased significantly even with lower IAP of 6-8 mmHg. Laparoscopic surgery can be performed conveniently even at low IAP.

An Observational Crossover Study of N95 Respirator with Surgical Mask and Visor in Various Combinations on Healthy Volunteers and Their Impact on Physiological Variables.

Background and Aim: The COVID pandemic necessitated the use of masks to reduce the propagation of coronavirus by airborne transmission. This research was conducted in healthy volunteers to assess the changes in noninvasive measurable physiological variables over 45 min at rest. Methods: This was a prospective randomized controlled crossover trial. Twenty-one healthy volunteers were monitored for pulse rate (PR), peripheral oxygen saturation (SpO2), systolic blood pressure (SBP), diastolic blood pressure (DBP), respiratory rate (RR), inspired carbon dioxide and expired carbon dioxide (ECO2), inspired (FiO2) and expired oxygen (FeO2), every 15 min for 45 minute (min) with N95 respirator, N95 respirator with surgical mask (SM), N95 with SM and visor (V), SM with N95, and N95 respirator with visor. Results: Repeated measures analysis of variance (ANOVA) of PR, RR, SpO2, SBP, and DBP over time within the group and intragroup was calculated and found statistically insignificant. P value for comparison of mean value within the group was calculated by paired t-test with Bonferroni correction. There was a significant rise in ECO2 in the N95 group over time, and repeated measures ANOVA showed P = 0.04 at 30 min between the N95 + V group and the N95 + SM + V group. Inspired CO2 was statistically significant over time in the N95 + SM + V with P = 0.02. Conclusion: N95 alone or in combination with a SM and visor does not cause any clinically significant measurable physiological derangements. The inspired CO2 may be implicated in the symptoms manifested by individuals.

SufB intein splicing in Mycobacterium tuberculosis is influenced by two remote conserved N-extein histidines.

Inteins are auto-processing domains that implement a multistep biochemical reaction termed protein splicing, marked by cleavage and formation of peptide bonds. They excise from a precursor protein, generating a functional protein via covalent bonding of flanking exteins. We report the kinetic study of splicing and cleavage reaction in [Fe-S] cluster assembly protein SufB from Mycobacterium tuberculosis (Mtu). Although it follows a canonical intein splicing pathway, distinct features are added by extein residues present in the active site. Sequence analysis identified two conserved histidines in the N-extein region; His-5 and His-38. Kinetic analyses of His-5Ala and His-38Ala SufB mutants exhibited significant reductions in splicing and cleavage rates relative to the SufB wildtype (WT) precursor protein. Structural analysis and molecular dynamics (MD) simulations suggested that Mtu SufB displays a unique mechanism where two remote histidines work concurrently to facilitate N-terminal cleavage reaction. His-38 is stabilized by the solvent-exposed His-5, and can impact N-S acyl shift by direct interaction with the catalytic Cys1. Development of inteins as biotechnological tools or as pathogen-specific novel antimicrobial targets requires a more complete understanding of such unexpected roles of conserved extein residues in protein splicing.

Epidemiological Analysis of SARS-CoV-2 Transmission Dynamics in the State of Odisha, India: A Yearlong Exploratory Data Analysis.

COVID-19 remains a matter of global public health concern. Previous research suggested the association between local environmental factors and viral transmission. We present a multivariate observational analysis of SARS-CoV-2 transmission in the state of Odisha, India, hinting at a seasonal activity. We aim to investigate the demographic characteristics of COVID-19 in the Indian state of Odisha for two specific timelines in 2020 and 2021. For a comparative outlook, we chose similar datasets from the state of New York, USA. Further, we present a critical analysis pertaining to the effects of environmental factors and the emergence of variants on SARS-CoV-2 transmission and persistence. We assessed the datasets for confirmed cases, death, age, and gender for 29 February 2020 to 31 May 2020, and 1 March 2021 to 31 May 2021. We determined the case fatalities, crude death rates, sex ratio, and incidence rates for both states along with monthly average temperature analysis. A yearlong epi-curve analysis was conducted to depict the coronavirus infection spread pattern in the respective states. The Indian state of Odisha reported a massive 436,455 confirmed cases and 875 deaths during the 2021 timeline as compared to a mere 2223 cases and 7 deaths during the 2020 timeline. We further discuss the demographic and temperature association of SARS-CoV-2 transmission during early 2020 and additionally comment on the variant-associated massive rise in cases during 2021. Along with the rapid rise of variants, the high population density and population behavior seem to be leading causes for the 2021 pandemic, whereas factors such as age group, gender, and average local temperature were prominent during the 2020 spread. A seasonal occurrence of SARS-CoV-2 transmission is also observed from the yearlong epidemiological plot. The recent second wave of COVID-19 is a lesson that emphasizes the significance of continuous epidemiological surveillance to predict the relative risk of viral transmission for a specific region.

Metal effect on intein splicing: A review.

Inteins are intervening polypeptides that interrupt the functional domains of several important proteins across the three domains of life. Inteins excise themselves from the precursor protein, ligating concomitant extein residues in a process called protein splicing. Post-translational auto-removal of inteins remain critical for the generation of active proteins. The perspective of inteins in science is a robust field of research, however fundamental studies centralized upon splicing regulatory mechanism are imperative for addressing more intricate issues. Controlled engineering of intein splicing has many applications; intein inhibition can facilitate novel drug design, while activation of intein splicing is exploited in protein purification. This paper provides a comprehensive review of the past and recent advances in the splicing regulation via metal-intein interaction. We compare the behavior of different metal ions on diverse intein systems. Though metals such as Zn, Cu, Pt, Cd, Co, Ni exhibit intein inhibitory effect heterogeneously on different inteins, divalent metal ions such as Ca and Mg fail to do so. The observed diversity in the metal-intein interaction arises mostly due to intein polymorphism and variations in atomic structure of metals. A mechanistic understanding of intein regulation by metals in native as well as synthetically engineered intein systems may yield potent intein inhibitors via direct or indirect approach.

Gold Nanoparticles Augment N-Terminal Cleavage and Splicing Reactions in Mycobacterium tuberculosis SufB.

Protein splicing is a self-catalyzed event where the intervening sequence intein cleaves off, joining the flanking exteins together to generate a functional protein. Attempts have been made to regulate the splicing rate through variations in temperature, pH, and metals. Although metal-regulated protein splicing has been more captivating to researchers, metals were shown to only inhibit splicing reactions that confine their application. This is the first study to show the effect of nanoparticles (NPs) on protein splicing. We found that gold nanoparticles (AuNPs) of various sizes can increase the splicing efficiency by more than 50% and the N-terminal cleavage efficiency by more than 45% in Mycobacterium tuberculosis SufB precursor protein. This study provides an effective strategy for engineering splicing-enhanced intein platforms. UV-vis absorption spectroscopy, isothermal titration calorimetry (ITC), and transmission electron microscopy (TEM) confirmed AuNP interaction with the native protein. Quantum mechanics/molecular mechanics (QM/MM) analysis suggested a significant reduction in the energy barrier at the N-terminal cleavage site in the presence of gold atom, strengthening our experimental evidence on heightened the N-terminal cleavage reaction. The encouraging observation of enhanced N-terminal cleavage and splicing reaction can have potential implementations from developing a rapid drug delivery system to designing a contemporary protein purification system.

Inteins in Science: Evolution to Application.

Inteins are mobile genetic elements that apply standard enzymatic strategies to excise themselves post-translationally from the precursor protein via protein splicing. Since their discovery in the 1990s, recent advances in intein technology allow for them to be implemented as a modern biotechnological contrivance. Radical improvement in the structure and catalytic framework of cis- and trans-splicing inteins devised the development of engineered inteins that contribute to various efficient downstream techniques. Previous literature indicates that implementation of intein-mediated splicing has been extended to in vivo systems. Besides, the homing endonuclease domain also acts as a versatile biotechnological tool involving genetic manipulation and control of monogenic diseases. This review orients the understanding of inteins by sequentially studying the distribution and evolution pattern of intein, thereby highlighting a role in genetic mobility. Further, we include an in-depth summary of specific applications branching from protein purification using self-cleaving tags to protein modification, post-translational processing and labelling, followed by the development of intein-based biosensors. These engineered inteins offer a disruptive approach towards research avenues like biomaterial construction, metabolic engineering and synthetic biology. Therefore, this linear perspective allows for a more comprehensive understanding of intein function and its diverse applications.

Comparative Evaluation of Efficacy of Preventive Analgesia with Diclofenac and Buprenorphine Patch versus Single Diclofenac Patch for Postoperative Pain following General Anesthesia for Laparoscopic Cholecystectomy.

BACKGROUND: Acute postoperative pain is managed with parenteral and oral medications requiring skilled personnel for administration and vigilance. Previous studies have been done either with transdermal buprenorphine or diclofenac patch separately in mitigating postoperative pain. AIMS: The primary aim was to compare pain scores and rescue analgesia requirement. The secondary aims were comparison of side effects, time to mobilization and oral intake start, sedation scores, and satisfaction scores of surgeons and patients receiving transdermal patches of buprenorphine and diclofenac versus placebo and diclofenac patches following laparoscopic cholecystectomy. SETTINGS AND DESIGN: This is a randomized double-blinded (1:1), case-control study. MATERIALS AND METHODS: One hundred patients undergoing laparoscopic cholecystectomies were enrolled for the study. Patients were allocated into two groups by computer-generated randomization: those receiving dual patch of buprenorphine and diclofenac (DP) and those receiving patches of diclofenac and placebo (SP). Outcomes were measured after extubation and at 4, 8, 12, 24, 36, and 48 h after surgery. STATISTICS: Analysis was done using the Statistical Package for the Social Sciences version 22.0, R environment 3.2.2 for data analysis, and Microsoft Excel to generate graphs and tables. RESULTS: The pain scores and rescue analgesia requirements were significantly higher in group SP compared to group DP. Patient satisfaction scores were better with group DP. No significant difference was found in both the groups concerning sedation scores, side effects, and time to patient mobilization and start of oral intake. CONCLUSIONS: Concomitant use of transdermal patches of buprenorphine and diclofenac for postoperative pain in laparoscopic cholecystectomy provides adequate analgesia and patient satisfaction without compromising postoperative recovery.

Comparison of injection lignocaine (preservative free) 1.5 mg/kg i.v with oral pregabalin 150 mg for attenuation haemodynamic response to laryngoscopy and tracheal intubation.

Endotracheal intubation is sine quo non for safe conduct of general anaesthesia. Pregabalin, an anticonvulsant drug is being studied for control of haemodynamic response to laryngoscopy and intubation. Some authors have found that pregabalin 150 mg orally attenuates the haemodynamic response to laryngoscopy. The purpose of the present study was to compare injection lignocaine (preservative free) i.v. and oral pregabalin for attenuation of haemodynamic response to laryngoscopy and intubation. The study consisted of 60 patients of ASA class I, divided into two groups of 30 each. Group I received injection lignocaine (preservative free) 1.5 mg/kg i.v., 3 minutes prior to laryngoscopy. Group II received oral pregabalin 150 mg capsule 1 hour prior to induction. The parameters recorded were heart rate, systolic BP, diastolic BP, mean BP and rate pressure product was calculated at baseline before induction and at 1, 3 and 5 minutes following laryngoscopy. The data obtained was analysed using unpaired "t" test. Lignocaine was more effective than pregabalin in controlling the heart rate at one minute following laryngoscopy. Pregabalin was more effective than lignocaine in controlling diastolic blood pressure at 1, 3 and 5 minutes following laryngoscopy, and mean arterial pressure at land 3 minutes following laryngoscopy. There was no difference between two groups when systolic BP and rate pressure product were compared. Lignocaine (preservative free) thus, exerts better control over heart rate and pregabalin exerts better control over diastolic and mean blood pressure following laryngoscopy.

Relationship between consanguinity and depression in a south Indian population.

A Pilot study was Carried out to study the association of consanguinity marriage with depression. It was observed that the consanguinity of marriage was associated with depression. The odds ratio was 5.66 (CI: 2.42-13.54). The age and sex had an association with depression. The age and sex adjusted odds ratio of consanguinity marriage was 7.66 (CI: 3.93-19.45) indicating that it is independently associated with depression.