Thermal Sensitivity of the Enzymatic Activity of ß-Glucosidase: Simulations Lend Mechanistic Insights into Experimental Observations.

A crucial prerequisite for industrial applications of enzymes is the maintenance of specific activity across wide thermal ranges. ß-Glucosidase (EC 3.2.1.21) is an essential enzyme for converting cellulose in biomass to glucose. While the reaction mechanisms of ß-glucosidases from various thermal ranges (hyperthermophilic, thermophilic, and mesophilic) are similar, the factors underlying their thermal sensitivity remain obscure. The work presented here aims to unravel the molecular mechanisms underlying the thermal sensitivity of the enzymatic activity of the ß-glucosidase BglB from the bacterium Paenibacillus polymyxa. Experiments reveal a maximum enzymatic activity at 315 K, with a marked decrease in the activity below and above this temperature. Employing in silico simulations, we identified the crucial role of the active site tunnel residues in the thermal sensitivity. Specific tunnel residues were identified via energetic decomposition and protein-substrate hydrogen bond analyses. The experimentally observed trends in specific activity with temperature coincide with variations in overall binding free energy changes, showcasing a predominantly electrostatic effect that is consistent with enhanced catalytic pocket-substrate hydrogen bonding (HB) at Topt. The entropic advantage owing to the HB substate reorganization was found to facilitate better substrate binding at 315 K. This study elicits molecular-level insights into the associative mechanisms between thermally enabled fluctuations and enzymatic activity. Crucial differences emerge between molecular mechanisms involving the actual substrate (cellobiose) and a commonly employed chemical analogue. We posit that leveraging the role of fluctuations may reveal unexpected insights into enzyme behavior and offer novel paradigms for enzyme engineering.

Genomic Variability of Canine Parvoviruses from a Selected Population of Dogs and Cats in Sri Lanka.

The aim of the study was to identify canine parvovirus type 2 (CPV-2) subtypes circulating among a selected population of domestic dogs and cats in Sri Lanka and to investigate the evolutionary patterns among Sri Lankan viruses in the context of contemporary global CPV-2 sequences. Altogether, 40/61 (65.6%) samples tested were positive for CPV-2 DNA, including 31/48 (64.6%) dogs and 9/13 (69%) cats. All three subtypes (CPV-2a, CPV-2b and CPV-2c) were detected, with CPV-2a being most common. International median joining haplotype network of 291 CPV-2 sequences suggested that there was little barrier for CPV-2 moving between different geographical regions worldwide, including Sri Lanka, and that there was no correlation between the genetic structure within the molecular network and the decade of sample collection. By contrast, there was correlation between CPV-2 subtype and genetic structure, both within the international network and within the network built from 31 Sri Lankan CPV-2 sequences only. The structure within the latter was not correlated with the location of the veterinary clinic where the samples were submitted, the age or species of the host. Altogether, we have shown that there is considerable variability of CPV-2 genotypes circulating in Sri Lanka, which is likely driven by both local evolution and introduction from other countries. The similarity of CPV-2 obtained from cats and dogs suggests that cats may play a role in the epidemiology of CPV-2 in Sri Lanka.

Virucidal Efficacy of Blue LED and Far-UVC Light Disinfection against Feline Infectious Peritonitis Virus as a Model for SARS-CoV-2.

Transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurs through respiratory droplets passed directly from person to person or indirectly through fomites, such as common use surfaces or objects. The aim of this study was to determine the virucidal efficacy of blue LED (405 nm) and far-UVC (222 nm) light in comparison to standard UVC (254 nm) irradiation for the inactivation of feline infectious peritonitis virus (FIPV) on different matrices as a model for SARS-CoV-2. Wet or dried FIPV on stainless steel, plastic, or paper discs, in the presence or absence of artificial saliva, were exposed to various wavelengths of light for different time periods (1-90 min). Dual activity of blue LED and far-UVC lights were virucidal for most wet and dried FIPV within 4 to 16 min on all matrices. Individual action of blue LED and far-UVC lights were virucidal for wet FIPV but required longer irradiation times (8-90 min) to reach a 4-log reduction. In comparison, LED (265 nm) and germicidal UVC (254 nm) were virucidal on almost all matrices for both wet and dried FIPV within 1 min exposure. UVC was more effective for the disinfection of surfaces as compared to blue LED and far-UVC individually or together. However, dual action of blue LED and far-UVC was virucidal. This combination of lights could be used as a safer alternative to traditional UVC.

Impact of Structured Clinical Handover Protocol on Communication and Patient Satisfaction.

The Handover process is an essential aspect of patient care in daily clinical practice to ensure continuity of patient care. Standardization of clinical handover may reduce sentinel events due to inaccurate and ineffective communication. Single arm experimental trial was conducted to assess the effect of standard Situation, Background, Assessment, Recommendation (SBAR) protocol implementation in overall bedside nursing handover process, patient satisfaction, and nurses' acceptance. As a sample, all nursing staff of specified unit, all handover process performed by them, and patients admitted during study the period were included. Initially, the prevailing handover process and patient satisfaction regarding nursing handover was assessed using a structured observation checklist. During the implementation phase, nurses were trained on an SBAR handover protocol. After implementation, nursing handovers were again assessed and data regarding patient satisfaction and nurses' acceptance were collected. There was a statistically significant difference (P < .05) in median scores between the pre and post-intervention group on overall nursing handover and patient satisfaction regarding nursing handover. Standardization of patient's handover process is effective in terms of improving nursing handover process, patient satisfaction, and health professionals' acceptance.

In Vitro Effects of Doxycycline on Replication of Feline Coronavirus.

Feline infectious peritonitis (FIP) is a sporadic fatal disease of cats caused by a virulent variant of feline coronavirus (FCoV), referred to as FIP virus (FIPV). Treatment options are limited, and most of the affected cats die or are euthanized. Anecdotally, doxycycline has been used to treat FIP-affected cats, but there are currently no data to support or discourage such treatment. The aim of this study was to establish whether doxycycline inhibits replication of FIPV in vitro. The virus was cultured in Crandell-Rees feline kidney cells with various concentrations of doxycycline (0 to 50 µg/mL). The level of FIPV in cultures was determined by virus titration and FCoV-specific reverse-transcription quantitative PCR. Cell viability was also monitored. There was no difference in the level of infectious virus or viral RNA between doxycycline-treated and untreated cultures at 3, 12- and 18-hours post-infection. However, at 24 h, the growth of FIPV was inhibited by approximately two logs in cultures with >10 µg/mL doxycycline. This inhibition was dose-dependent, with inhibitory concentration 50% (IC50) 4.1 µg/mL and IC90 5.4 µg/mL. Our data suggest that doxycycline has some inhibitory effect on FIPV replication in vitro, which supports future clinical trials of its use for the treatment of FIP-affected cats.

Similar immune mechanisms control experimental airway eosinophilia elicited by different allergens and treatment protocols.

BACKGROUND: Mouse models have been extremely valuable in identifying the fundamental mechanisms of airway inflammation that underlie human allergic asthma. Several models are commonly used, employing different methods and routes of sensitisation, and allergens of varying clinical relevance. Although all models elicit similar hallmarks of allergic airway inflammation, including airway eosinophilia, goblet cell hyperplasia and cellular infiltration in lung, it is not established whether they do so by involving the same mechanisms. RESULTS: We compared the impact of inactivation of various innate or adaptive immune genes, as well as sex, in different models of allergic airway inflammation in mice of C57BL/6 background. Chicken ovalbumin (OVA) and house dust mite (HDM) were used as allergens in settings of single or multiple intranasal (i.n.) challenges, after sensitisation in adjuvant or in adjuvant-free conditions. Eosinophil numbers in the broncho-alveolar lavage and lung histopathology were assessed in each model. We found that Major Histocompatibility Complex Class II (MHCII) deficiency and lack of conventional CD4+ T cells had the most profound effect, essentially ablating airway eosinophilia and goblet cell hyperplasia in all models. In contrast, Thymic stromal lymphopoietin receptor (TSLPR) deficiency greatly reduced eosinophilia but had a variable effect on goblet cells. CD1d deficiency and lack of Natural Killer T (NKT) cells moderately impaired inflammation in OVA models but not HDM, whereas sex affected the response to HDM but not OVA. Lastly, defective Toll-like receptor (TLR)4 expression had only a relatively modest overall impact on inflammation. CONCLUSION: All the models studied were comparably dependent on adaptive CD4+ T cell responses and TSLP. In contrast, sex, NKT cells and TLR4 appeared to play subtler and more variable roles that were dependent on the type of allergen and mode of immunization and challenge. These results are consistent with clinical data suggesting a key role of CD4+ T cells and TSLP in patients with allergic asthma.