Improved Coinfection with Amphotropic Pseudotyped Retroviral Vectors

Amphotropic pseudotyped retroviral vectors have typically been used to infect target cells without prior concentration. Although this can yield high rates of infection, higher rates may be needed where highly efficient coinfection of two or more vectors is needed. In this investigation we used amphotropic retroviral vectors produced by the Plat-A cell line and studied coinfection rates using green and red fluorescent proteins (EGFP and dsRed2). Target cells were primary human fibroblasts (PHF) and 3T3 cells. Unconcentrated vector preparations produced a coinfection rate of ∼4% (defined as cells that are both red and green as a percentage of all cells infected). Optimized spinoculation, comprising centrifugation at 1200 g for 2 hours at 15°C, increased the coinfection rate to ∼10%. Concentration by centrifugation at 10,000 g or by flocculation using Polybrene increased the coinfection rate to ∼25%. Combining the two processes, concentration by Polybrene flocculation and optimized spinoculation, increased the coinfection rate to 35% (3T3) or >50% (PHF). Improved coinfection should be valuable in protocols that require high transduction by combinations of two or more retroviral vectors

Nonhuman Primate Induced Pluripotent Stem Cells in Regenerative Medicine

Among the various species from which induced pluripotent stem cells have been derived, nonhuman primates (NHPs) have a unique role as preclinical models. Their relatedness to humans and similar physiology, including central nervous system, make them ideal for translational studies. We review here the progress made in deriving and characterizing iPS cell lines from different NHP species. We focus on iPS cell lines from the marmoset, a small NHP in which several human disease states can be modeled. The marmoset can serve as a model for the implementation of patient-specific autologous cell therapy in regenerative medicine

Enhanced Protein Homeostasis Mechanisms in Naked Mole Rats Cells

Phylogenic studies suggest that cells from long-lived species are more resistant to a variety of stressors than short-lived species. However, there is little information on the cellular mechanisms that give rise to increased resistance to stress. Our previous studies have shown that liver proteins from a long-lived species have lower levels of protein ubiquitination which is associated with increased proteasome activity, suggesting that mechanisms of protein quality control could play a critical role in assuring longevity of long-lived species. In this study, we evaluated whether autophagy and heat shock chaperones proteins (HSPs) are associated with longevity in rodents using skin fibroblasts isolated from mice and naked mole rats (NMR); two species that are similar in body size but differ almost 10 fold in longevity. Our results indicate that macroautophagy induced by serum-starvation is significantly enhanced in NMR compared to mouse which correlates with an inhibition of the mTOR pathway and increased LC3 conversion. We also found that several HSPs (e.g., Hsp90, Hsp70, Hsp40, Hsp 27) were significantly higher at both basal and after heat shock conditions. These observations suggest that NMR, a long-lived species, has increased mechanisms to ensure protein quality (autophagy and HSPs) and support the idea that protein homeostasis could play an important role in promoting longevity

Migratory shorebird adheres to Bergmann’s Rule by responding to environmental conditions through the annual lifecycle

The inverse relationship between body size and environmental temperature is a widespread ecogeographic pattern. However, the underlying forces that produce this pattern are unclear in many taxa. Expectations are particularly unclear for migratory species, as individuals may escape environmental extremes and reorient themselves along the environmental gradient. In addition, some aspects of body size are largely fixed while others are environmentally flexible and may vary seasonally. Here, we used a long-term dataset that tracked multiple populations of the migratory piping plover Charadrius melodus across their breeding and non-breeding ranges to investigate ecogeographic patterns of phenotypically flexible (body mass) and fixed (wing length) size traits in relation to latitude (Bergmann’s Rule), environmental temperature (heat conservation hypothesis), and migratory distance. We found that body mass was correlated with both latitude and temperature across the breeding and non-breeding ranges, which is consistent with predictions of Bergmann’s Rule and heat conservation. However, wing length was correlated with latitude and temperature only on the breeding range. This discrepancy resulted from low migratory connectivity across seasons and the tendency for individuals with longer wings to migrate farther than those with shorter wings. Ultimately, these results suggest that wing length may be driven more by conditions experienced during the breeding season or tradeoffs related to migration, whereas body mass is modified by environmental conditions experienced throughout the annual lifecycle

Migratory shorebird adheres to Bergmann’s Rule by responding to environmental conditions through the annual lifecycle

The inverse relationship between body size and environmental temperature is a widespread ecogeographic pattern. However, the underlying forces that produce this pattern are unclear in many taxa. Expectations are particularly unclear for migratory species, as individuals may escape environmental extremes and reorient themselves along the environmental gradient. In addition, some aspects of body size are largely fixed while others are environmentally flexible and may vary seasonally. Here, we used a long-term dataset that tracked multiple populations of the migratory piping plover Charadrius melodus across their breeding and non-breeding ranges to investigate ecogeographic patterns of phenotypically flexible (body mass) and fixed (wing length) size traits in relation to latitude (Bergmann’s Rule), environmental temperature (heat conservation hypothesis), and migratory distance. We found that body mass was correlated with both latitude and temperature across the breeding and non-breeding ranges, which is consistent with predictions of Bergmann’s Rule and heat conservation. However, wing length was correlated with latitude and temperature only on the breeding range. This discrepancy resulted from low migratory connectivity across seasons and the tendency for individuals with longer wings to migrate farther than those with shorter wings. Ultimately, these results suggest that wing length may be driven more by conditions experienced during the breeding season or tradeoffs related to migration, whereas body mass is modified by environmental conditions experienced throughout the annual lifecycle

Utilisation of an operative difficulty grading scale for laparoscopic cholecystectomy

Background A reliable system for grading operative difficulty of laparoscopic cholecystectomy would standardise description of findings and reporting of outcomes. The aim of this study was to validate a difficulty grading system (Nassar scale), testing its applicability and consistency in two large prospective datasets. Methods Patient and disease-related variables and 30-day outcomes were identified in two prospective cholecystectomy databases: the multi-centre prospective cohort of 8820 patients from the recent CholeS Study and the single-surgeon series containing 4089 patients. Operative data and patient outcomes were correlated with Nassar operative difficultly scale, using Kendall’s tau for dichotomous variables, or Jonckheere–Terpstra tests for continuous variables. A ROC curve analysis was performed, to quantify the predictive accuracy of the scale for each outcome, with continuous outcomes dichotomised, prior to analysis. Results A higher operative difficulty grade was consistently associated with worse outcomes for the patients in both the reference and CholeS cohorts. The median length of stay increased from 0 to 4 days, and the 30-day complication rate from 7.6 to 24.4% as the difficulty grade increased from 1 to 4/5 (both p < 0.001). In the CholeS cohort, a higher difficulty grade was found to be most strongly associated with conversion to open and 30-day mortality (AUROC = 0.903, 0.822, respectively). On multivariable analysis, the Nassar operative difficultly scale was found to be a significant independent predictor of operative duration, conversion to open surgery, 30-day complications and 30-day reintervention (all p < 0.001). Conclusion We have shown that an operative difficulty scale can standardise the description of operative findings by multiple grades of surgeons to facilitate audit, training assessment and research. It provides a tool for reporting operative findings, disease severity and technical difficulty and can be utilised in future research to reliably compare outcomes according to case mix and intra-operative difficulty

GWAS of thyroid stimulating hormone highlights pleiotropic effects and inverse association with thyroid cancer

Correction: Volume12, Issue1 Article Number7354 DOI10.1038/s41467-021-27675-w PublishedDEC 16 2021Thyroid stimulating hormone (TSH) is critical for normal development and metabolism. To better understand the genetic contribution to TSH levels, we conduct a GWAS meta-analysis at 22.4 million genetic markers in up to 119,715 individuals and identify 74 genome-wide significant loci for TSH, of which 28 are previously unreported. Functional experiments show that the thyroglobulin protein-altering variants P118L and G67S impact thyroglobulin secretion. Phenome-wide association analysis in the UK Biobank demonstrates the pleiotropic effects of TSH-associated variants and a polygenic score for higher TSH levels is associated with a reduced risk of thyroid cancer in the UK Biobank and three other independent studies. Two-sample Mendelian randomization using TSH index variants as instrumental variables suggests a protective effect of higher TSH levels (indicating lower thyroid function) on risk of thyroid cancer and goiter. Our findings highlight the pleiotropic effects of TSH-associated variants on thyroid function and growth of malignant and benign thyroid tumors. Thyroid stimulating hormone (TSH) is critical for normal development and metabolism. Here, the authors conduct a GWAS and suggest protective effect of higher TSH on risk of thyroid cancer and goitre.Peer reviewe

Author Correction:GWAS of thyroid stimulating hormone highlights the pleiotropic effects and inverse association with thyroid cancer

The original version of this article contained an error in the results, in the second paragraph of the subsection entitled “Fine-mapping for potentially causal variants among TSH loci”, in which effect sizes for two variants were incorrectly reported

Population‐based cohort study of outcomes following cholecystectomy for benign gallbladder diseases

Background The aim was to describe the management of benign gallbladder disease and identify characteristics associated with all‐cause 30‐day readmissions and complications in a prospective population‐based cohort. Methods Data were collected on consecutive patients undergoing cholecystectomy in acute UK and Irish hospitals between 1 March and 1 May 2014. Potential explanatory variables influencing all‐cause 30‐day readmissions and complications were analysed by means of multilevel, multivariable logistic regression modelling using a two‐level hierarchical structure with patients (level 1) nested within hospitals (level 2). Results Data were collected on 8909 patients undergoing cholecystectomy from 167 hospitals. Some 1451 cholecystectomies (16·3 per cent) were performed as an emergency, 4165 (46·8 per cent) as elective operations, and 3293 patients (37·0 per cent) had had at least one previous emergency admission, but had surgery on a delayed basis. The readmission and complication rates at 30 days were 7·1 per cent (633 of 8909) and 10·8 per cent (962 of 8909) respectively. Both readmissions and complications were independently associated with increasing ASA fitness grade, duration of surgery, and increasing numbers of emergency admissions with gallbladder disease before cholecystectomy. No identifiable hospital characteristics were linked to readmissions and complications. Conclusion Readmissions and complications following cholecystectomy are common and associated with patient and disease characteristics

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice