Proteomics and metabolic burden analysis to understand the impact of recombinant protein production in E. coli.

The impact of recombinant protein production (RPP) on host cells and the metabolic burden associated with it undermine the efficiency of the production system. This study utilized proteomics to investigate the dynamics of parent and recombinant cells induced at different time points for RPP. The results revealed significant changes in both transcriptional and translational machinery that may have impacted the metabolic burden, growth rate of the culture and the RPP. The timing of protein synthesis induction also played a critical role in the fate of the recombinant protein within the host cell, affecting protein and product yield. The study identified significant differences in the expression of proteins involved in fatty acid and lipid biosynthesis pathways between two E. coli host strains (M15 and DH5⍺), with the E. coli M15 strain demonstrating superior expression characteristics for the recombinant protein. Overall, these findings contribute to the knowledge base for rational strain engineering for optimized recombinant protein production.

Investigations on physiochemical and biomedical properties of Aloe vera - Sterculia gum copolymeric dressings impregnated with antibiotic-anesthetic drugs to enhance wound healing.

Recently, various innovative advancements have been made in carbohydrate research to design versatile materials for biomedical applications. The current research focuses on the development of copolymeric hydrogel wound dressings (HWD) using a combination of aloe vera (AV) - sterculia gum (SG) - poly (vinylsulfonic acid) (VSA)-based with the aim to enhancing their efficacy in drug delivery (DD) applications. These hydrogel dressings were encapsulated with levofloxacin and lidocaine to address both microbial infection and pain. Copolymers were characterized by FESEM, SEM, EDS, AFM, 13C NMR, FTIR, XRD, and TGA-DTG analysis. Hydrogel exhibited a fluid absorption capacity of 4.52 ± 0.12 g per gram of polymeric dressing in simulated wound conditions. The hydrogels displayed a sustained release of drugs, demonstrating a non-Fickian diffusion mechanism. Polymer dressings revealed antibacterial, mucoadhesive, antioxidant, biocompatible and non-cytotoxic properties. Additionally, HWD displayed permeability to O2 and water vapour, yet was impermeable to microbial penetration. Overall, the findings of physiological, biochemical and drug delivery properties demonstrated the suitability of materials for wound dressing applications.

Maintaining independence in individuals with dementia at home after a fall: a protocol for the UK pilot cluster randomised controlled trial MAINTAIN.

INTRODUCTION: Individuals with dementia face an increased risk of falls. Falls can cause a decline in the individual's overall functionality. All types of falls, including those that do not result in injury, can lead to psychosocial consequences, such as diminished confidence and a fear of falling. Projections indicate a rising trend in dementia diagnoses, implying an increase in fall incidents. Yet, there is a lack of evidence to support interventions for people living with dementia who have fallen. Our objective is to test the feasibility of a falls intervention trial for people with dementia. METHOD AND ANALYSIS: This is a UK-based two-arm pilot cluster randomised controlled trial. In this study, six collaborating sites, which form the clusters, will be randomly allocated to either the intervention arm or the control arm (receiving treatment as usual) at a 1:1 ratio. During the 6 month recruitment phase, each cluster will enrol 10 dyads, comprising 10 individuals with dementia and their respective carers, leading to a total sample size of 60 dyads. The primary outcomes are the feasibility parameters for a full trial (ie, percentage consented, follow-up rate and cost framework). Secondary outcomes include activities of daily living, quality of life, fall efficacy, mobility, goal attainment, cognitive status, occurrence of falls, carer burden and healthcare service utilisation. Outcome measures will be collected at baseline and 28 weeks, with an additional assessment scheduled at 12 weeks for the healthcare service utilisation questionnaire. An embedded process evaluation, consisting of interviews and observations with participants and healthcare professionals, will explore how the intervention operates and the fidelity of study processes. ETHICS AND DISSEMINATION: The study was approved by the NHS and local authority research governance and research ethics committees (NHS REC reference: 23/WA/0126). The results will be shared at meetings and conferences and will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: ISRCTN16413728.

Development of novel bosentan analogues as endothelin receptor antagonists for pulmonary arterial hypertension.

Since decades, bosentan has been in use for the treatment of pulmonary arterial hypertension (PAH). However, chronic exposure to bosentan leads to the development of resistance, tolerance, and serious adverse effects that have restricted its usage in clinical practices. To surmount these limitations, some new bosentan derivatives have been synthesized and evaluated for their therapeutic efficacy in PAH. Molecular docking analyses of all the synthesized derivatives were carried out using the endothelin (ET) receptor. In addition, the inhibitory ability of synthesized derivatives was determined in in vitro assay employing an ET-1 human ELISA kit. Among the synthesized derivatives, three derivatives namely 17d, 16j, and 16h with higher docking scores and lower IC50 values were selected for determination of the magnitude of the binding force between the derivative and ET receptor using molecular dynamics (MD) simulations study. Further, these derivatives were subjected to in vivo studies using monocrotaline (MCT) induced PAH in rat model. Results of in vivo studies inferred that the derivatives exhibit impressive ability to reduce PAH. Besides, its protective role was also evidenced in hemodynamic and right ventricular hypertrophy analyses, histological analysis, cardiac biomarkers, hypoxia-inducible factor 1 alpha (HIF1α) levels, and biochemical studies. Furthermore, gene quantification by quantitative RT-PCR and Western blot analysis was also performed to examine its effect on the expression of key proteins in PAH. Notably, amongst three, derivative 16h exhibited the most encouraging results in molecular docking analysis, in vitro, in vivo, histopathological, biochemical, protein expression, and MD studies. Besides, derivative 16h also showed impressive pharmacokinetic features in ADMET analysis. In conclusion, derivative 16 h could act as a reliable ET receptor antagonist and requires further exploration to attain its therapeutic utility in PAH management.

Monkeypox epidemiology, clinical presentation, and transmission: a systematic review.

BACKGROUND: The new zoonotic viral infection, monkeypox, is a global health issue. Our study aimed at studying the epidemiology, clinical presentation, complications, case fatality rate, and transmission among the present cases of monkeypox infection. METHODS: Articles were searched in PubMed, Google Scholar, and Science Direct databases using the keywords "Monkeypox" [MeSH] or "Monkeypox virus" (MeSH). Narrative reviews, conference abstracts, commentaries, and articles in languages other than English were excluded. RESULTS: From three databases, 1442 studies were identified. Seven hundred ten articles were excluded because they included data before 2022, leaving 732 items for screening. After filtering 320 data due to data duplication, 412 remained. Due to the inclusion of systematic reviews, meta-analyses, reviews, comments, and articles in languages other than English, 257 were excluded. Eligibility based on full-text review was applied to the remaining 155, excluding 129. So, the study covered a total of remaining 26 articles. We studied 2352 confirmed cases from published literature, accounting for approximately 4% of infected cases worldwide. Around 81.71% of patients have a bisexual or men having sex with men (MSM) preference. Approximately 30.18% of confirmed cases were HIV positive. Male sex was also identified as a risk factor in our review. CONCLUSION: Monkeypox human-to-human and human-to-animal transmission are rising. Thus, it is essential to do research on the prevention, clinicodemographic trends, and treatment of monkeypox. Understanding this will enable us to treat monkeypox patients with a targeted and focused approach.

Improved Interobserver Reliability in Diagnosing and Staging Lesions of COVID-19 Between Radiologist and Emergency Medicine Physicians After an Online Course.

BACKGROUND: Chest radiographs are the most basic and readily available imaging modality for visualizing the lungs and are potentially useful for describing the disease severity in patients showing respiratory symptoms in COVID-19 patients. The early diagnosis of COVID-19 features on radiography helps in triaging and starting treatment. MATERIAL AND METHODS: Our study consisted of 145 radiographs, and these were reported by two radiologists, two emergency physicians and one intern working in the Emergency department. The scores given by them were correlated. A targeted short lecture for the scoring was imparted and after a sufficient latent period the scoring of chest radiographs was done again, and the scores correlated and compared. RESULTS: We observed agreement between radiologists with emergency medicine physicians was "none to slight" to "fair," before the dedicated online teaching course. Following the meeting, there was an increase in interobserver agreement in-between radiologists and between radiologists and emergency medicine physicians. CONCLUSIONS: We propose a focused online meeting, targeted at explaining radiological features of a specific pathology, in a pandemic situation like COVID, to our clinical counterparts in the emergency medicine department can help in increasing their interpretation skills. This can directly benefit triaging, admission/discharge and monitoring of the status of patients, in intensive care units and emergency medicine. This also helps in allaying the anxiety, while waiting for a final report from the Radiologist.

A rare combination of methemoglobinemia and carboxyhemoglobinemia in pesticide poisoning.

Dyshemoglobinemias are disorders in which the haemoglobin is functionally altered and prevented from carrying oxygen. They include carboxyhemoglobin, methemoglobin, and sulfhemoglobin. This increase in abnormal haemoglobin has reduced oxygen binding capacity, which leads to decrease in total oxygen content in the blood causing anaemic-hypoxia. The anaemic-hypoxia which is present in these disorders are refractory to the oxygen supplementation and cause many systemic and life threatening complications.Many cases are reported in literature with either of haemoglobin. It is very rare to have two abnormal haemoglobin levels in the same patient. Here we discuss an uncommon case which presented to our tertiary care hospital after consuming pesticide with suicidal intention. The patient was very pale, had peripheral cyanosis,tachypnea and tachycardia and dizziness on presentation. The SpO2 of 85% was our clue to suspect methemoglobinemia which was confirmed along with carboxyhemoglobinemia on arterial blood gas saving result. Despite that patient being very unstable, she was successfully managed with 100% oxygen through High flow nasal cannula (HFNC), methylene blue and blood transfusion. The patient's signs and symptoms gradually reduced in a few days and got discharged after 2 weeks without any neurological and cardiorespiratory sequelae. An early suspicion and personalized emergency management was the key to success. As in all fields of Medicine, Emergency Medicine is also witnessing a change towards precision and personalized Medicine practice.

Veterans Health Administration COVID-19 Index to Risk Stratify the Geriatric Population with COVID-19 Infection.

The risk for severe illness with coronavirus disease-2019 (COVID-19) increases with age, with older adults at highest risk. Adults 65-years-old and older who were fully vaccinated with an mRNA COVID-19 vaccine had a 94% reduction in risk of COVID-19 hospitalizations, and vaccination was 64% effective among those who were partially vaccinated. How to cite this article: Sharma A, Ranjan A. Veterans Health Administration COVID-19 Index to Risk Stratify the Geriatric Population with COVID-19 Infection. Indian J Crit Care Med 2022;26(5):632-633.

Biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase.

Acyl-ACP reductase (AAR) is one of the two key cyanobacterial enzymes along with aldehyde deformylating oxygenase (ADO) involved in the synthesis of long-chain alkanes, a drop-in biofuel. The enzyme is prone to aggregation when expressed in Escherichia coli, leading to varying alkane levels. The present work attempts to investigate the crucial structural aspects of AAR protein associated with its stability and folding. Characterization by dynamic light scattering experiment and intact mass spectrometry revealed that recombinantly expressed AAR in E. coli existed in multiple-sized protein particles due to diverse lipidation. Interestingly, while thermal- and urea-based denaturation of AAR showed 2-state unfolding transition in circular dichroism and intrinsic fluorescent spectroscopy, the unfolding process of AAR was a 3-state pathway in GdnHCl solution suggesting that the protein milieu plays a significant role in dictating its folding. Apparent standard free energy [Formula: see text] of ~ 4.5 kcal/mol for the steady-state unfolding of AAR indicated borderline stability of the protein. Based on these evidences, we propose that the marginal stability of AAR are plausible contributing reasons for aggregation propensity and hence the low catalytic activity of the enzyme when expressed in E. coli for biofuel production. Our results show a path for building superior biocatalyst for higher biofuel production.

Short research paper: Personal protective equipment for the care of suspected and confirmed COVID-19 patients - Modelling requirements and burn rate.

BACKGROUND: The COVID-19 pandemic has caused unprecedented global demand for personal protective equipment (PPE). A paucity of data on PPE burn rate (PPE consumption over time) in pandemic situations exacerbated these issues as there was little historic research to indicate volumes of PPE required to care for surges in infective patients and thus plan procurement requirements. METHODS: To better understand PPE requirements for care of suspected or confirmed COVID-19 patients in our Australian quaternary referral hospital, the number of staff-to-patient interactions in a 24-h period for three patient groups (ward-based COVID suspect, ward-based COVID confirmed, intensive care COVID confirmed) was audited prospectively from 1st to 30th April 2020. RESULTS: The average number of staff-to-patient interactions in a 24-h period was: 13.1 ± 5.0 (mean ± SD) for stable ward-managed COVID-19 suspect patients; 11.9 ± 3.8 for stable ward-managed confirmed COVID-19 patients; and 30.0 ± 5.3 for stable, mechanically ventilated, ICU-managed COVID-19 patients. This data can be used in PPE demand simulation modelling for COVID-19 and potentially other respiratory illnesses. CONCLUSION: Data on the average number of staff-to-patient interactions needed for the care of COVID-19 patients is presented. This data can be used for PPE demand simulation modelling.

Microbial engineering to produce fatty alcohols and alkanes.

Owing to their high energy density and composition, fatty acid-derived chemicals possess a wide range of applications such as biofuels, biomaterials, and other biochemical, and as a consequence, the global annual demand for products has surpassed 2 million tons. With the exhausting petroleum reservoirs and emerging environmental concerns on using petroleum feedstock, it has become indispensable to shift to a renewable-based industry. With the advancement in the field of synthetic biology and metabolic engineering, the use of microbes as factories for the production of fatty acid-derived chemicals is becoming a promising alternative approach for the production of these derivatives. Numerous metabolic approaches have been developed for conditioning the microbes to improve existing or develop new methodologies capable of efficient oleochemical production. However, there still exist several limitations that need to be addressed for the commercial viability of the microbial cell factory production. Though substantial advancement has been made toward successfully producing these fatty acids derived chemicals, a considerable amount of work needs to be done for improving the titers. In the present review, we aim to address the roadblocks impeding the heterologous production, the engineering pathway strategies implemented across the range of microbes in a detailed manner, and the commercial readiness of these molecules of immense application.

Selective Fluorometric Analysis of Hg(II) in Industrial Waste Water Samples.

The highly selective and sensitive fluorometric method has been developed for trace level determination of Hg(II) is based on photo-induced electron transfer between rhodamine-6G dye and metal complex. Quenching in fluorescence intensity by fluorescence resonance energy transfer (FRET) is due to interaction between metal ion complex and dye. The fluorescence emitted was measured at 510 and 550 nm, for excitation and emission wavelengths respectively. Possible interferences present in water samples, which could affect the analytical response are studied and determined. The calibration graph was dynamically linear from 0.002 to 0.05 mgL-1 of Hg(II) with limit of detection 7 × 10-4 mgL-1 and limit of quantitation 1.9 × 10-3 mgL-1. The Stern-Volmer constant (KSV) calculated for the quenching of R-6G with Hg (II) was 8.47 Lmg-1 s-1 at optimized reaction conditions. The proposed FRET based fluorometric method was applied successfully in different industrial wastewater samples with satisfactory outcome.

Building cell factories for the production of advanced fuels.

Synthetic biology-based engineering strategies are being extensively employed for microbial production of advanced fuels. Advanced fuels, being comparable in energy efficiency and properties to conventional fuels, have been increasingly explored as they can be directly incorporated into the current fuel infrastructure without the need for reconstructing the pre-existing set-up rendering them economically viable. Multiple metabolic engineering approaches have been used for rewiring microbes to improve existing or develop newly programmed cells capable of efficient fuel production. The primary challenge in using these approaches is improving the product yield for the feasibility of the commercial processes. Some of the common roadblocks towards enhanced fuel production include - limited availability of flux towards precursors and desired pathways due to presence of competing pathways, limited cofactor and energy supply in cells, the low catalytic activity of pathway enzymes, obstructed product transport, and poor tolerance of host cells for end products. Consequently, despite extensive studies on the engineering of microbial hosts, the costs of industrial-scale production of most of these heterologously produced fuel compounds are still too high. Though considerable progress has been made towards successfully producing some of these biofuels, a substantial amount of work needs to be done for improving the titers of others. In this review, we have summarized the different engineering strategies that have been successfully used for engineering pathways into commercial hosts for the production of advanced fuels and different approaches implemented for tuning host strains and pathway enzymes for scaling up production levels.

Feasibility of Early Noninvasive Ventilation Strategy for Patients with Acute Onset Shortness of Breath in Emergency Department - A Prospective Interventional Study.

INTRODUCTION: Noninvasive ventilation (NIV) has revolutionized the initial respiratory support provided to a patient in respiratory distress presenting to emergency department. Standardization of NIV practices and safety has always been a matter of concern and debate in emergency medicine. In this study, we tried to assess the clinical outcome of NIV in respiratory failures of varied etiologies. MATERIALS AND METHODS: This study was conducted from August 2017 to August 2018 at our emergency department which is a tertiary care teaching institute. All patients presenting to the ED with shortness of breath were screened for acute respiratory failure and enrolled after confirming the inclusion criteria. RESULTS: Out of the 236 patients presenting with acute respiratory failure, 182 fulfilled the study criteria. However, 154 patients with a mean age of 55.19 + 16.73 years were enrolled in the study. Bilevel PAP was initiated in 103 patients whereas 51 patients received CPAP. 115 (74.67%) NIV trials were successful whereas 36 (23.37%) patients had to be intubated. 32 patients died among the study group among which 3 had not consented for intubation. The in-hospital mortality has been 20.77% whereas the percentage of NIV failure with consequent intubation was 25.32%. CONCLUSION: In conclusion, our study shows that NIV is not only safe and efficacious but also significantly brings down the requirement of endotracheal intubations and its complications provided proper patient selection and close monitoring is assured. HOW TO CITE THIS ARTICLE: Nizami MI, Sharma A, Jayaram K. Feasibility of Early Noninvasive Ventilation Strategy for Patients with Acute Onset Shortness of Breath in Emergency Department - A Prospective Interventional Study. Indian J Crit Care Med 2019;23(9):400-404.

Convenient and ultra-sensitive fluorescence detection of bovine serum albumin by using Rhodamine-6G modified gold nanoparticles in biological samples.

This study comprises a convenient, rapid and very sensitive method for the determination of bovine serum albumin (BSA). The technique is based on fluorescence resonance energy transfer (FRET) between Rhodamine-6G (R6G) acting as donor and gold nanoparticles (AuNPs) acting as acceptors. This method takes advantage of AuNPs that have high quenching efficiency, therefore the absorption spectra range shifts from 521 to 635 nm when aggregation of the AuNPs takes place. Furthermore, when R6G was electrostatically self-adsorbed to the citrate-stabilized AuNPs surface the fluorescence intensity was quenched. After addition of BSA, the fluorescence intensity of the R6G recovered as BSA induced aggregation of the AuNPs and the adsorbed R6G was released to the solution. The recovery of intensity displays a linear relationship with BSA concentration over the range from 0.8 × 10-11  M to 5.6 × 10-11  M. The detection limit for BSA was found to be 4.58 × 10-11  M. The proposed method exhibited rapid analysis with high selectivity for BSA determination in human urine, blood and serum samples.

Understanding the mode of binding mechanism of doripenem to human serum albumin: Spectroscopic and molecular docking approaches.

The infections caused by multidrug resistant bacteria are widely treated with carabapenem antibiotics as a drug of choice, and human serum albumin (HSA) plays a vital role in binding with drugs and affecting its rate of delivery and efficacy. So, we have initiated this study to characterize the mechanism of doripenem binding and to locate its site of binding on HSA by using spectroscopic and docking approaches. The binding of doripenem leads to alteration of the environment surrounding Trp-214 residue of HSA as observed by UV spectroscopic study. Fluorescence spectroscopic study revealed considerable interaction and complex formation of doripenem and HSA as indicated by Ksv and Kq values of the order of 104  M-1 and 1012  M-1  s-1 , respectively. Furthermore, doripenem quenches the fluorescence of HSA spontaneously on a single binding site with binding constant of the order of 103  M-1 , through an exothermic process. Van der Waals forces and hydrogen bonding are the major forces operating to stabilize HSA-doripenem complex. Circular dichroism spectroscopic study showed changes in the structure of HSA upon doripenem binding. Drug displacement and molecular docking studies revealed that the binding site of doripenem on HSA is located on subdomain IB and III A. This study concludes that, due to significant interaction of doripenem on either subdomain IB or IIIA of HSA, the availability of doripenem on the target site may be compromised. Hence, there is a possibility of unavailability of threshold amount of drug to be reached to the target; consequently, resistance may develop in the bacterial population.

Physicochemical characterization of E. coli-derived human serum albumin and its comparison with the human plasma counterpart reveals it as a promising biosimilar.

Human serum albumin one of the most demanded proteins possess an array of clinical and biotechnological applications. Currently, the prime source for HSA production is the human blood which possesses the risk of pathogen contamination and is limited. Thus, there exists an indispensable need to promote non-animal derived HSA production. In the present work, we have exploited the opportunity and promoted the preparation of pathogen-free rHSA from the E. coli host which is blessed with numerous advantages like scalability, cost-effectiveness etc. Upon overcoming the difficulties to produce functional rHSA in E. coli, through engineering the biological system of protein folding in the cell, the E. coli-derived rHSA has been purified to homogeneity. Its detailed physicochemical characterization has been performed, by monitoring its conformational properties, secondary and tertiary structure elements, surface properties, ligand binding properties, stability issues etc. These parameters of the recombinant protein have been compared with the naturally occurring protein from the human source. The outcome of the comparison reveals that the recombinant protein resembles exactly the same as the natural one. Hence, we propose and promote that the E. coli-derived rHSA is an ideal biosimilar for human blood plasma-derived serum albumin.

A study of pulmonary function in end-stage renal disease patients on hemodialysis: a cross-sectional study.

BACKGROUND: The aim here was to study acute effects of hemodialysis among end-stage renal disease (ESRD) patients. DESIGN AND SETTING: Prospective study in tertiary-level care center. METHODS: Fifty ESRD patients undergoing hemodialysis were studied. Spirometric pulmonary function tests were performed before and after four-hour hemodialysis sessions. RESULTS: The patients' average age was 45.8 ± 10.0 years; 64% were males and 64% had normal body mass index. Anemia (94%) and hypoalbuminemia (72%) were common. Diabetes mellitus (68%), hypertension (34%) and coronary artery disease (18%) were major comorbidities. Forty-five patients (90%) had been on hemodialysis for six months to three years. The patients' pre-dialysis mean forced vital capacity (FVC) and forced expiratory volume in 1 second (FEV1) were below normal: 45.8 ± 24.9% and 43.5 ± 25.9% of predicted, respectively. After hemodialysis, these increased significantly, to 51.1 ± 23.4% and 49.3 ± 25.5% of predicted, respectively (P < 0.01). The increase in mean FEV1/FVC, from 97.8 ± 20.8% to 99.3 ± 20.1% of predicted, was not significant (P > 0.05). The pre-dialysis mean forced expiratory flow 25-75% was 50.1 ± 31% and increased significantly, to 56.3 ± 31.6% of predicted (P < 0.05). The mean peak expiratory flow was below normal (43.8 ± 30.7%) and increased significantly, to 49.1 ± 29.9% of predicted (P < 0.05). Males and females showed similar directions of change after hemodialysis. CONCLUSIONS: Pulmonary function abnormalities are common among ESRD patients. Comparison of pre and post-hemodialysis parameters showed significant improvements, but normal predicted values were still not achieved.