Calcium activated proteolysis of fibrous proteins in central nervous system

A Ca²⁺ activated protease(s) capable of hydrolyzing several polypeptides at neutral pH including cytoskeletal proteins, actin, myosin, tubulin and neurofilament triplet was identified in calf brain cortex. The enzyme activity precipitates at 75 mM KCl, pH 6.5 – 7.0 and is inhibited by the sulfhydryl inhibitors, N-ethylmaleimide and para-chloromercuribenzoate and the protease inhibitors, antipain, pepstatin and leupeptin, leupeptin being the most effective.     

Improved sensitivity of glutamine synthetase assay and of acyl hydroxamates

Sensitivity of glutamine synthetase assay by the hydroxamate method can be doubled by replacing ferric chloride with ferric nitrate or ferric perchlorate, each with the appropriate acid.     

Predicting major complications in patients undergoing laparoscopic and open hysterectomy for benign indications

Background:Hysterectomy, the most common gynecological operation, requires surgeons to counsel women about their operative risks. We aimed to develop and validate multivariable logistic regression models to predict major complications of laparoscopic or abdominal hysterectomy for benign conditions.Methods:We obtained routinely collected health administrative data from the English National Health Service (NHS) from 2011 to 2018. We defined major complications based on core outcomes for postoperative complications including ureteric, gastrointestinal and vascular injury, and wound complications. We specified 11 predictors a priori. We used internal–external cross-validation to evaluate discrimination and calibration across 7 NHS regions in the development cohort. We validated the final models using data from an additional NHS region.Results:We found that major complications occurred in 4.4% (3037/68 599) of laparoscopic and 4.9% (6201/125 971) of abdominal hysterectomies. Our models showed consistent discrimination in the development cohort (laparoscopic, C-statistic 0.61, 95% confidence interval [CI] 0.60 to 0.62; abdominal, C-statistic 0.67, 95% CI 0.64 to 0.70) and similar or better discrimination in the validation cohort (laparoscopic, C-statistic 0.67, 95% CI 0.65 to 0.69; abdominal, C-statistic 0.67, 95% CI 0.65 to 0.69). Adhesions were most predictive of complications in both models (laparoscopic, odds ratio [OR] 1.92, 95% CI 1.73 to 2.13; abdominal, OR 2.46, 95% CI 2.27 to 2.66). Other factors predictive of complications included adenomyosis in the laparoscopic model, and Asian ethnicity and diabetes in the abdominal model. Protective factors included age and diagnoses of menstrual disorders or benign adnexal mass in both models and diagnosis of fibroids in the abdominal model.Interpretation:Personalized risk estimates from these models, which showed moderate discrimination, can inform clinical decision-making for people with benign conditions who may require hysterectomy.

Hysterectomy is one of the most frequently performed surgical procedures. Canada has one of the highest rates of hysterectomy globally, with one-third of women undergoing this procedure before 60 years of age.¹ Minimal access approaches are favoured by both clinicians and patients,² and the proportion of hysterectomies being undertaken by a laparoscopic approach has increased substantially in many countries over the last 10 years.^3–7 The evidence-based medicine paradigm for surgical approaches to hysterectomy for benign disease advocates that the chosen surgical approach should be discussed with the patient by their surgeon and decided in light of the relative benefits and risks.² This advice is echoed by national guidelines.^8,9Most clinicians undertaking hysterectomy will intuitively identify patient characteristics that have the potential to increase the complexity and complications of surgery. A 2016 systematic review of studies that reported significant associations between patient characteristics and surgical outcomes for laparoscopic hysterectomy and a 2020 population-based prospective cohort study using data from the Danish hysterectomy database have suggested that older age, race, raised body mass index (BMI), diabetes mellitus, increased uterine weight, fibroids, endometriosis and adhesions are predictors of complications in patients undergoing hysterectomy for benign indications.^10,11 However, assimilating this information to individualize and anticipate the precise risk for each patient if there are multiple factors present can be challenging. A 2020 systematic review reported that surgeons in other specialties were outperformed by risk prediction models in estimating postoperative risk and outcomes; their discriminatory ability showed greater variation (C-statistic 0.51–0.75) than other risk prediction tools.¹²Patients should be given information about potential risks before surgery to manage expectations.¹³ This is especially important when surgery is considered for benign disease because nonsurgical options are often available.Our aim was to generate prediction models that can be used in conjunction with a surgeon’s intuition to enhance preoperative patient counselling and match the advances made in the technical aspects of surgery. We sought to quantify the proportion of patients who underwent hysterectomy for benign disease and will have a major complication, and to develop and validate prognostic models to individualize this risk, using a national data set.     

Metagenomics analysis of the fecal microbiota in Ring-necked pheasants (Phasianus colchicus) and Green pheasants (Phasianus versicolor) using next generation sequencing

Pheasant reintroduction and conservation efforts have been in place in Pakistan since the 1980 s, yet there is still a scarcity of data on pheasant microbiome and zoonosis. Instead of growing vast numbers of bacteria in the laboratory, to investigate the fecal microbiome, pheasants (green and ring neck pheasant) were analyzed using 16S rRNA metagenomics and using IonS5TMXL sequencing from two flocks more than 10 birds. Operational taxonomic unit (OTU) cluster analysis and phylogenetic tree analysis was performed using Mothur software against the SSUrRNA database of SILVA and the MUSCLE (Version 3.8.31) software. Results of the analysis showed that firmicutes were the most abundant phylum among the top ten phyla, in both pheasant species, followed by other phyla such as actinobacteria and proteobacteria in ring necked pheasant and bacteroidetes in green necked pheasant. Bacillus was the most relatively abundant genus in both pheasants followed by Oceanobacillus and Teribacillus for ring necked pheasant and Lactobacillus for green necked pheasant. Because of their well-known beneficial characteristics, these genus warrants special attention. Bird droppings comprise germs from the urinary system, gut, and reproductive sites, making it difficult to research each anatomical site at the same time. We conclude that metagenomic analysis and classification provides baseline information of the pheasant fecal microbiome that plays a role in disease and health.     

PKA modulates GSK-3beta- and cdk5-catalyzed phosphorylation of tau in site- and kinase-specific manners

Phosphorylation of tau protein is regulated by several kinases, especially glycogen synthase kinase 3beta (GSK-3beta), cyclin-dependent protein kinase 5 (cdk5) and cAMP-dependent protein kinase (PKA). Phosphorylation of tau by PKA primes it for phosphorylation by GSK-3beta, but the site-specific modulation of GSK-3beta-catalyzed tau phosphorylation by the prephosphorylation has not been well investigated. Here, we found that prephosphorylation by PKA promotes GSK-3beta-catalyzed tau phosphorylation at Thr181, Ser199, Ser202, Thr205, Thr217, Thr231, Ser396 and Ser422, but inhibits its phosphorylation at Thr212 and Ser404. In contrast, the prephosphorylation had no significant effect on its subsequent phosphorylation by cdk5 at Thr181, Ser199, Thr205, Thr231 and Ser422; inhibited it at Ser202, Thr212, Thr217 and Ser404; and slightly promoted it at Ser396. These studies reveal the nature of the inter-regulation of tau phosphorylation by the three major tau kinases.     

Promotion of hyperphosphorylation by frontotemporal dementia tau mutations

Mutations in the tau gene are known to cosegregate with the disease in frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). However, the molecular mechanism by which these mutations might lead to the disease is not understood. Here, we show that four of the FTDP-17 tau mutations, R406W, V337M, G272V, and P301L, result in tau proteins that are more favorable substrates for phosphorylation by brain protein kinases than the wild-type, largest four-repeat protein tau4L and tau4L more than tau3L. In general, at all the sites studied, mutant tau proteins were phosphorylated faster and to a higher extent than tau4L and tau4L > tau3L. The most dramatic difference found was in the rate and level of phosphorylation of tau4L(R406W) at positions Ser-396, Ser-400, Thr-403, and Ser-404. Phosphorylation of this mutant tau was 12 times faster and 400% greater at Ser-396 and less than 30% at Ser-400, Thr-403, and Ser-404 than phosphorylation of tau4L. The mutated tau proteins polymerized into filaments when 4-6 mol of phosphate per mol of tau were incorporated, whereas wild-type tau required approximately 10 mol of phosphate per mol of protein to self-assemble. Mutated and wild-type tau proteins were able to sequester normal tau upon incorporation of approximately 4 mol of phosphate per mol of protein, which was achieved at as early as 30 min of phosphorylation in the case of mutant tau proteins. These findings taken together suggest that the mutations in tau might cause neurodegeneration by making the protein a more favorable substrate for hyperphosphorylation.     

Phosphatase Activity Toward Abnormally Phosphorylated τ: Decrease in Alzheimer Disease Brain

Abstract: Microtubule-associated protein τ is abnormally hyperphosphorylated and aggregated in affected neurons of Alzheimer disease brain. This hyperphosphorylated τ can be dephosphorylated at some of the abnormal phosphorylated sites by purified protein phosphatase-1, 2A, and 2B in vitro. In the present study, we have developed an assay to measure protein phosphatase activity toward τ-1 sites (Ser¹⁹⁹/Ser²⁰²) using the hyperphosphorylated τ isolated from Alzheimer disease brain as substrate. Using this assay, we have identified that in normal brain, protein phosphatase-2A and 2B and, to a lesser extent, 1 are involved in the dephosphorylation of τ. The K _m values of dephosphorylation of the hyperphosphorylated τ by protein phosphatase-2A and 2B are similar. The τ phosphatase activity is decreased by ∼30% in brain of Alzheimer disease patients compared with those of age-matched controls. These findings suggest that a defect of protein phosphatase could be the cause of the abnormal hyperphosphorylation of τ in Alzheimer disease.     

Spinach-thylakoid phosphorylation: Studies on the kinetics of changes in photosystem antenna size, spill-over and phosphorylation of light-harvesting chlorophyll a/b protein

The kinetics of LHCP phosphorylation and associated changes in photosystem cross-section and energy ‘spill-over’ from PS II to PS I have been examined in isolated spinach chloroplasts. During an initial phosphorylation period of 3–6 min, in the presence of saturating concentrations of Mg²⁺, the increase in PS I and decrease in PS II cross-section are largely completed, as judged by both measurements of the steady-state redox state of Q and fluorescence yield changes. This corresponds to a period of rapid ³²P incorporation into the low-molecular weight LHCP polypeptide. Subsequent to this initial 3–6-min period there is substantial further phosphorylation of both LHCP polypeptides, which is not accompanied by significant changes in photosystem cross-section, even after the chloroplasts had been unstacked with extensive mixing of PS I and PS II by Mg-removal. It is suggested that there exists a specific ‘mobile’ population of LHCP molecules which is rapidly phosphorylated and which may be enriched in the low-molecular-weight polypeptide. In addition, measurements of the kinetics of the ‘spill-over’ changes upon either Mg²⁺ addition or removal indicate that the continued phosphorylation of LHCP is able to increase the ‘spill-over’ process under favourable ionic conditions.     

Inter-rater reliability of physiotherapists using the Action Research Arm Test in chronic stroke

Objectives:The purpose of this study is to establish whether physiotherapists’ ratings are consistent, when using the Action Research Arm Test (ARAT) to score a chronic stroke patient.Methods:This was part of a large project establishing the reliability in chronic stroke. This study used a correlational design comparing the association between physiotherapist scores of the same patient, to establish the ARAT’s inter-rater reliability. The COSMIN checklist was followed to enhance the methodology of the study.Results:Twenty physiotherapists (8 female and 12 male) aged between 25 and 53 years were selected. There were no participant dropouts or withdrawals. The sample size was normally distributed. The physiotherapists appeared representative of the UK physiotherapy population, with the exception of gender. The distribution of scores showed a normal distribution with standard deviation of score of 1.9. The Kendall’s W test showed 0.711 of agreement between the raters. The scores achieved statistical significance showing consistency between physiotherapists’ scores with chronic stroke. Limitations of the study were the use of a small single center convenience sample that may reduce the generalizability of the findings.Conclusions:The ARAT is consistent when scored by physiotherapists in a chronic stroke population. The inter-rater reliability range was (0.70 to 0.90) which is categorized as good.