Progression of herpesvirus infection remodels mitochondrial organization and metabolism.

Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.

Phenotyping the Responses to Systemic Corticosteroids in the Management of Asthma Attacks (PRISMA): protocol for an observational and translational pilot study.

INTRODUCTION: Asthma and its associated exacerbation are heterogeneous. Although severe asthma attacks are systematically prescribed corticosteroids and often antibiotics, little is known about the variability of response to these therapies. Blood eosinophils and fractional exhaled nitric oxide (FeNO) are type 2 inflammation biomarkers that have established mechanistic, prognostic and theragnostic values in chronic asthma, but their utility in acute asthma is unclear. We speculate that the clinical and biological response to those treatments varies according to inflammometry and microbiological test results. METHODS AND ANALYSIS: An observational longitudinal pilot study with multimodal clinical and translational assessments will be performed on 50 physician-diagnosed ≥12-year-old asthmatics presenting with an asthma attack and 12 healthy controls, including blood eosinophil count (venous and point-of-care (POC) capillary blood), FeNO and testing for airway infection (sputum cultures and POC nasopharyngeal swabs). People with asthma will be assessed on day 0 and after a 7-day corticosteroid course, with home monitoring performed in between. The primary analysis will be the change in the forced expiratory volume in 1 s according to type 2 inflammatory status (blood eosinophils ≥0.15×109/L and/or FeNO ≥25 ppb) after treatment. Key secondary analyses will compare changes in symptom scores and the proportion of patients achieving a minimal clinically important difference. Exploratory analyses will assess the relationship between clinical, lung function, inflammatory and microbiome parameters; satisfaction plus reliability indices of POC tests; and sex-gender variability in treatment response. Ultimately, this pilot study will serve to plan a larger trial comparing the clinical and biological response to systemic corticosteroids according to inflammatory biomarkers, offering valuable guidance for more personalised therapeutic strategies in asthma attacks. ETHICS AND DISSEMINATION: The protocol has been approved by the Research Ethics Committee of the CIUSSS de l'Estrie-CHUS, Sherbrooke, Quebec, Canada (#2023-4687). Results will be communicated in an international meeting and submitted to a peer-reviewed journal. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registry (NCT05870215).

G2/M checkpoint regulation and apoptosis facilitate the nuclear egress of parvoviral capsids.

The nuclear export factor CRM1-mediated pathway is known to be important for the nuclear egress of progeny parvovirus capsids in the host cells with virus-mediated cell cycle arrest at G2/M. However, it is still unclear whether this is the only pathway by which capsids exit the nucleus. Our studies show that the nuclear egress of DNA-containing full canine parvovirus. capsids was reduced but not fully inhibited when CRM1-mediated nuclear export was prevented by leptomycin B. This suggests that canine parvovirus capsids might use additional routes for nuclear escape. This hypothesis was further supported by our findings that nuclear envelope (NE) permeability was increased at the late stages of infection. Inhibitors of cell cycle regulatory protein cyclin-dependent kinase 1 (Cdk1) and pro-apoptotic caspase 3 prevented the NE leakage. The change in NE permeability could be explained by the regulation of the G2/M checkpoint which is accompanied by early mitotic and apoptotic events. The model of G2/M checkpoint activation was supported by infection-induced nuclear accumulation of cyclin B1 and Cdk1. Both NE permeability and nuclear egress of capsids were reduced by the inhibition of Cdk1. Additional proof of checkpoint function regulation and promotion of apoptotic events was the nucleocytoplasmic redistribution of nuclear transport factors, importins, and Ran, in late infection. Consistent with our findings, post-translational histone acetylation that promotes the regulation of several genes related to cell cycle transition and arrest was detected. In conclusion, the model we propose implies that parvoviral capsid egress partially depends on infection-induced G2/M checkpoint regulation involving early mitotic and apoptotic events.

Bridging Gaps in Diabetic Nephropathy Care: A Narrative Review Guided by the Lived Experiences of Patient Partners.

Purpose of review: Diabetes affects almost a 10th of the Canadian population, and diabetic nephropathy is one of its main complications. It remains a leading cause of kidney failure despite the availability of effective treatments. Sources of information: The sources of information are iterative discussions between health care professionals and patient partners and literature collected through the search of multiple databases. Methods: Major pitfalls related to optimal diabetic nephropathy care were identified through discussions between patient partners and clinician researchers. We identified underlying factors that were common between pitfalls. We then conducted a narrative review of strategies to overcome them, with a focus on Canadian initiatives. Key findings: We identified 5 pitfalls along the diabetic nephropathy trajectory, including a delay in diabetes diagnosis, suboptimal glycemic control, delay in the detection of kidney involvement, suboptimal kidney protection, and deficient management of advanced chronic kidney disease. Several innovative care models and approaches have been proposed to address these pitfalls; however, they are not consistently applied. To improve diabetic nephropathy care in Canada, we recommend focusing initiatives on improving awareness of diabetic nephropathy, improving access to timely evidence-based care, fostering inclusive patient-centered care environment, and generating new evidence that supports complex disease management. It is imperative that patients and their families are included at the center of these initiatives. Limitations: This review was limited to research published in peer-reviewed journals. We did not perform a systematic review of the literature; we included articles that were relevant to the major pitfalls identified by our patient partners. Study quality was also not formally assessed. The combination of these factors limits the scope of our conclusions.

Motif de la revue: Le diabète touche près d'un dixième de la population canadienne et la néphropathie diabétique est l'une de ses principales complications. Le diabète demeure une cause principale d'insuffisance rénale malgré la disponibilité de traitements efficaces. Sources: Discussions itératives entre des professionnels de la santé et des patients partenaires, ainsi que la documentation recueillie à la suite d'une recherche dans plusieurs bases de données. Méthodologie: Les principaux obstacles liés aux soins optimaux en néphropathie diabétique ont été définis grâce à des discussions entre des patients partenaires et des cliniciens-chercheurs. Des facteurs sous-jacents, communs à tous ces obstacles, ont été dégagés, puis nous avons procédé à un examen narratif des stratégies visant à surmonter ces obstacles, en privilégiant les initiatives canadiennes. Principaux résultats: Cinq obstacles jalonnant la trajectoire de la néphropathie diabétique ont été identifiés, soit un retard dans le diagnostic du diabète, une régulation glycémique sous-optimale, un retard dans la détection de l'atteinte rénale, une protection rénale sous-optimale et une gestion déficiente de l'insuffisance rénale chronique de stade avancé. Plusieurs approches et modèles de soins novateurs ont été proposés pour remédier à ces obstacles, mais ils ne sont pas appliqués de façon uniforme. Pour améliorer les soins de néphropathie diabétique au Canada, nous recommandons de concentrer les initiatives visant la sensibilisation à la néphropathie diabétique, l'amélioration de l'accès en temps opportun à des soins fondés sur des données probantes, la promotion d'un environnement de soins inclusif axé sur le patient et la production de données probantes appuyant la gestion complexe de la maladie. Il est impératif que les patients et leurs familles soient au cœur de ces initiatives. Limites: Notre revue s'est limitée aux articles publiés dans des revues examinées par des pairs. Nous n'avons pas procédé à une revue systématique de la littérature; nous avons inclus des articles pertinents pour les principaux obstacles identifiés par nos patients partenaires. La qualité des études n'a pas été évaluée officiellement. La combinaison de ces facteurs limite la portée de nos conclusions.

A Novel Mouse Model of Idiopathic Nephrotic Syndrome Induced by Immunization with the Podocyte Protein Crb2.

BACKGROUND: The cause of podocyte injury in idiopathic nephrotic syndrome (INS) remains unknown. Although recent evidence points to the role of B cells and autoimmunity, the lack of animal models mediated by autoimmunity limits further research. We aimed to establish a mouse model mimicking human INS by immunizing mice with Crb2, a transmembrane protein expressed at the podocyte foot process. METHODS: C3H/HeN mice were immunized with the recombinant extracellular domain of mouse Crb2. Serum anti-Crb2 antibody, urine protein-to-creatinine ratio, and kidney histology were studied. For signaling studies, a Crb2-expressing mouse podocyte line was incubated with anti-Crb2 antibody. RESULTS: Serum anti-Crb2 autoantibodies and significant proteinuria were detected 4 weeks after the first immunization. The proteinuria reached nephrotic range at 9-13 weeks and persisted up to 29 weeks. Initial kidney histology resembled minimal change disease in humans, and immunofluorescence staining showed delicate punctate IgG staining in the glomerulus, which colocalized with Crb2 at the podocyte foot process. A subset of mice developed features resembling FSGS after 18 weeks. In glomeruli of immunized mice and in Crb2-expressing podocytes incubated with anti-Crb2 antibody, phosphorylation of ezrin, which connects Crb2 to the cytoskeleton, increased, accompanied by altered Crb2 localization and actin distribution. CONCLUSION: The results highlight the causative role of anti-Crb2 autoantibody in podocyte injury in mice. Crb2 immunization could be a useful model to study the immunologic pathogenesis of human INS, and may support the role of autoimmunity against podocyte proteins in INS.

Qualitative and Quantitative Dosage of the Anti M-Type Phospholipase A2 Receptor Autoantibody: One-Year Experience in Quebec's Reference Center.

BACKGROUND: Quantification of the M-type phospholipase A2 receptor antibodies (anti-PLA2R) is now an essential tool for diagnosis and management of primary membranous nephropathy (MN). Since October 2018, Hôpital Maisonneuve-Rosemont (HMR) has been designated as Quebec's reference center for serum anti-PLA2R antibody testing by the Institut National d'Excellence en Santé et Services Sociaux (INESSS), the regulatory body on drugs and tests usage in Quebec. OBJECTIVES: To describe the 2-step method of serum qualitative and quantitative anti-PLA2R antibody testing during its first year of use in Quebec and analyze its diagnostic value in the province's population. DESIGN: Retrospective cohort study. SETTING: Single-center academic teaching hospital in Quebec, Canada. PATIENTS: All patients who had a serum anti-PLA2R antibody test analyzed at HMR from October 1, 2018, to October 1, 2019, were included in the study. MEASUREMENTS: Serum anti-PLA2R antibodies were screened by indirect immunofluorescence tests. If results were positive or undetermined, it was followed by a quantitative enzyme-linked immunosorbent assay (ELISA) test. Both tests were based on a commercial kit developed by the same company. METHODS: We calculated sensitivity, specificity, predictive value, and likelihood ratio for both tests, using kidney biopsy findings performed at HMR as the gold standard. RESULTS: In Quebec, a total of 1690 tests were performed among 1025 patients during the study year. A small proportion of these patients (8%) were followed at HMR. Patients tested at HMR and in the rest of Quebec had similar characteristics. Test validity was only characterized for patients tested at HMR. Sensitivity and specificity were, respectively, 58% and 100% for the qualitative test, and 71% and 100% for the quantitative test. The combined net sensitivity was 42% and the net specificity 100%. The net positive and negative predictive value were 100% and 84% respectively, whereas the net negative likelihood ratio was 0.58. LIMITATIONS: As the detailed analysis was only possible in the small proportion of patients clinically followed at HMR, there is a possible selection bias. Another potential selection bias was the focus on patients who were selected to have a kidney biopsy, probably because of more severe disease, higher probability of glomerulonephritis, or lesser number of comorbidities. Given the retrospective nature of this study, there was no systematic kidney biopsy or serum PLA2R antibody testing performed. Finally, we were unable to provide detailed information on the timing between immunosuppressive therapy and anti-PLA2R results. CONCLUSIONS: Serum anti-PLA2R antibody testing was widely used in Quebec during its first year of availability. A 2-step approach, using a qualitative test first, followed by a quantitative test if the results are positive or undetermined, appears efficient to avoid useless quantitative testing in negative patients and to better characterize undetermined results on immunofluorescence. TRIAL REGISTRATION: Due to the retrospective nature of this study, no trial registration was performed.

CONTEXTE: La quantification des anticorps des récepteurs de la phospholipase A2 de type M (anti-PLA2R) est désormais un outil essentiel pour le diagnostic et la prise en charge de la glomérulonéphrite extra-membraneuse primaire (GEMp). Depuis octobre 2018, l'Hôpital Maisonneuve-Rosemont (HMR) a été désigné par l'Institut National d'Excellence en Santé et Services Sociaux (INESSS)­l'organisme règlementant l'usage des médicaments et des tests au Québec­comme le centre hospitalier de référence dans la province pour le dépistage des anticorps sériques anti-PLA2R. OBJECTIFS: Décrire la méthode en deux étapes du test qualitatif et quantitatif des anticorps anti-PLA2R sériques au cours de sa première année d'utilisation au Québec et évaluer sa valeur diagnostique dans la population de la province. TYPE D'ÉTUDE: Étude de cohorte rétrospective. CADRE: Un centre hospitalier universitaire du Québec (Canada). SUJETS: Ont été inclus tous les patients dont le test des anticorps sériques anti-PLA2R a été analysé à HMR entre le 1er octobre 2018 et le 1er octobre 2019. MESURES: Les anticorps sériques anti-PLA2R ont été détectés par immunofluorescence indirecte. Les résultats positifs ou indéterminés ont été suivis d'un test ELISA quantitatif. Les deux tests ont été réalisés à l'aide de trousses commerciales développées par la même entreprise. MÉTHODOLOGIE: Nous avons analysé la sensibilité, la spécificité, la valeur prédictive et le rapport de vraisemblance des deux tests avec comme référence des résultats de biopsie rénale obtenus à HMR. RÉSULTATS: Au Québec, au cours de l'année de l'étude, 1 690 tests ont été effectués sur 1 025 patients; une faible proportion de ces patients (8 %) étaient suivis à HMR. Les patients, qu'ils aient été testés à HMR et ailleurs au Québec, présentaient des caractéristiques semblables. La validité du test n'a été caractérisée que pour les patients testés à HMR. La sensibilité et la spécificité s'établissaient respectivement à 58 % et à 100 % pour le test qualitatif, et à 71 % et 100 % pour le test quantitatif. La sensibilité nette combinée était de 42 % et la spécificité nette, de 100 %. Les valeurs prédictives nettes, positive et négative, étaient respectivement de 100 % et de 84 %, alors que le ratio net de probabilité négative était de 0,58. LIMITES: L'étude présente un possible biais de sélection puisque l'analyse détaillée n'était possible que pour la faible proportion de patients suivis à HMR. L'accent mis sur les patients sélectionnés pour une biopsie rénale, probablement en raison d'une maladie plus grave, d'une probabilité plus élevée de glomérulonéphrite ou d'un moins grand nombre de comorbidités, constitue un autre possible biais de sélection. Aucune biopsie rénale ou test d'anticorps de PLA2R sérique systématique n'a été effectué puisque l'étude est rétrospective. Enfin, il n'a pas été possible de fournir des informations détaillées sur le temps écoulé entre le traitement immunosuppresseur et les résultats du test d'anticorps anti-PLA2R. CONCLUSION: Le test d'anticorps sériques anti-PLA2R a été largement utilisé au Québec au cours de sa première année de disponibilité. Une approche en deux étapes, constituée d'un test qualitatif suivi d'un test quantitatif si le résultat est positif ou indéterminé, semble efficace pour éviter de procéder inutilement à des tests quantitatifs chez les patients négatifs et pour caractériser plus précisément les résultats indéterminés par immunofluorescence. ENREGISTREMENT DE L'ESSAI: L'essai n'a pas été enregistré puisqu'il s'agit d'une étude rétrospective.

Cellular therapies in kidney transplantation.

PURPOSE OF REVIEW: Current immunosuppressive regimens used in kidney transplantation are sometimes ineffective and carry significant risks of morbidity and mortality. Cellular therapies are a promising alternative to prolong graft survival while minimizing treatment toxicity. We review the recently published breakthrough studies using cell therapies in kidney transplantation. RECENT FINDINGS: The reviewed phase I and II trials showed that cell therapies are feasible and safe in kidney transplantation, sometimes associated with less infectious complications than traditional regimens. Regulatory T cells and macrophages were added to the induction regimen, allowing for lower immunosuppressive drug doses without higher rejection risk. Regulatory T cells are also a treatment for subclinical rejection on the 6 months biopsy. Other strategies, like bone marrow-derived mesenchymal cells, genetically modified regulatory T cells, and chimerism-based tolerance are also really promising. In addition, to improve graft tolerance, cell therapy could be used to prevent or treat viral infection after transplantation. SUMMARY: Emerging data underline that cell therapy is a feasible and safe treatment in kidney transplantation. Although the evidence points to a benefit for transplant recipients, studies with standardized protocols, representative control groups, and longer follow-up are needed to answer the question definitively and guide future research.

Minimal Change Disease With Severe Acute Kidney Injury Following the Oxford-AstraZeneca COVID-19 Vaccine: A Case Report.

We report a case of minimal change disease (MCD) with severe acute kidney injury (AKI) following the first injection of the ChAdOx1 nCoV-19 (AZD1222) vaccine from Oxford-AstraZeneca against coronavirus disease 2019 (COVID-19). A 71-year-old man with a history of dyslipidemia and a baseline serum creatinine of 0.7mg/dL presented with nephrotic syndrome, AKI, and severe hypertension 13 days after receiving the Oxford-AstraZeneca vaccine. Refractory hyperkalemia and hypervolemia with oligoanuria prompted initiation of hemodialysis. His serum albumin was 2.6g/dL and his urinary protein-creatinine ratio was 2,321mg/mmol. Given a high suspicion for rapidly progressive glomerulonephritis, empirical glucocorticoid treatment was initiated (3 methylprednisolone pulses followed by high-dose prednisone). A kidney biopsy showed MCD and acute tubular injury. Kidney function and proteinuria subsequently improved, and hemodialysis was discontinued 38 days after the start of therapy. This case describes de novo MCD after the Oxford-AstraZeneca vaccine. It adds to the few published case reports of MCD after the Pfizer-BioNTech COVID-19 vaccine. Further reports and studies will be needed to elucidate whether MCD is truly associated with COVID-19 vaccination.

The Role of Human Centromeric RNA in Chromosome Stability.

Chromosome instability is a hallmark of cancer and is caused by inaccurate segregation of chromosomes. One cellular structure used to avoid this fate is the kinetochore, which binds to the centromere on the chromosome. Human centromeres are poorly understood, since sequencing and analyzing repeated alpha-satellite DNA regions, which can span a few megabases at the centromere, are particularly difficult. However, recent analyses revealed that these regions are actively transcribed and that transcription levels are tightly regulated, unveiling a possible role of RNA at the centromere. In this short review, we focus on the recent discovery of the function of human centromeric RNA in the regulation and structure of the centromere, and discuss the consequences of dysregulation of centromeric RNA in cancer.

Use of Desmopressin Prior to Kidney Biopsy in Patients With High Bleeding Risk.

INTRODUCTION: To prevent bleeding after native kidney biopsy (NKB), nephrologists often prescribe desmopressin, especially for patients with reduced estimated glomerular filtration rate (eGFR) at risk of uremia-related platelet dysfunction. However, only 1 randomized study has suggested a beneficial effect for desmopressin in patients with eGFR ≥60 ml/min per 1.73 m2. This retrospective cohort study aimed to evaluate desmopressin effect on postbiopsy bleeding in all patients, regardless of eGFR and other comorbidities. METHODS: In this retrospective cohort study, all adult patients who underwent an NKB from April 1, 2013, to April 30, 2018, in a tertiary hospital were identified. The association between desmopressin use and bleeding complications, including hemoglobin fall, transfusion, hematoma, symptomatic hematoma, urgent radiologic study, and hypotension, was analyzed using multivariable logistic regression models. RESULTS: A total of 413 native kidney biopsies were studied, 79% of which were performed after receiving desmopressin. Patients receiving desmopressin had worse chronic kidney disease (eGFR 28 vs. 45 ml/min per 1.73 m2; P < 0.001) and were more often hospitalized (48% vs. 32%; P = 0.009). Despite higher bleeding risk, patients using desmopressin had a similar likelihood of symptomatic hematomas (odds ratio [OR], 0.39; 95% confidence interval [CI], 0.13-1.14) and a lower need for urgent radiologic studies (OR, 0.33; 95% CI, 0.11-0.98). CONCLUSION: Patients at higher risk of bleeding using desmopressin before kidney biopsy had bleeding complications similar to those not using desmopressin. These results highlight potential important clinical and financial benefits of desmopressin use before kidney biopsy.

BHi-Cect: a top-down algorithm for identifying the multi-scale hierarchical structure of chromosomes.

High-throughput chromosome conformation capture (Hi-C) technology enables the investigation of genome-wide interactions among chromosome loci. Current algorithms focus on topologically associating domains (TADs), that are contiguous clusters along the genome coordinate, to describe the hierarchical structure of chromosomes. However, high resolution Hi-C displays a variety of interaction patterns beyond what current TAD detection methods can capture. Here, we present BHi-Cect, a novel top-down algorithm that finds clusters by considering every locus with no assumption of genomic contiguity using spectral clustering. Our results reveal that the hierarchical structure of chromosome is organized as 'enclaves', which are complex interwoven clusters at both local and global scales. We show that the nesting of local clusters within global clusters characterizing enclaves, is associated with the epigenomic activity found on the underlying DNA. Furthermore, we show that the hierarchical nesting that links different enclaves integrates their respective function. BHi-Cect provides means to uncover the general principles guiding chromatin architecture.

Pharmacodynamics of 250 mg and 500 mg oral furosemide in peritoneal dialysis
.

Residual renal function and diuresis preservation are associated with improved volume control and lower mortality in peritoneal dialysis (PD). Loop diuretics are used to maintain diuresis, although their optimal dosage remains unclear. This study aimed to compare the pharmacodynamics of a 250-mg and a 500-mg dose of oral furosemide in PD patients. 12 patients with a diuresis > 100 mL per day were randomized in a crossover pattern to successively receive an oral dose of 250 mg and 500 mg of furosemide. Twelve-hour natriuresis and diuresis were measured before and after each furosemide dose. Fractional excretion of sodium (FENa) and absolute sodium excretion increased after each dose, although these rises were not statistically significantly different (5.8% (250 mg) vs. 6.9% (500 mg), p = 0.57 for FENa and 42.6 mmol/12h (250 mg) vs. 70.8 mmol/12h (500 mg), p = 0.07 for absolute sodium excretion). Urinary volume was significantly increased after the 500-mg dose, whilst the difference did not reach statistical significance after the 250-mg dose. Furthermore, the higher dose was associated with a greater increase in diuresis than the lower dose (226 mL (250 mg) vs. 522 mL (500 mg), p = 0.04). Furosemide could be used at oral single doses reaching 500 mg in PD patients requiring greater volume control.

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed 'labeling occupancy' (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Prehospital analysis of northern trauma outcome measures: the PHANTOM study.

OBJECTIVE: To compare the mortality and morbidity of traumatically injured patients who received additional prehospital care by a doctor and critical care paramedic enhanced care team (ECT), with those solely treated by a paramedic non-ECT. METHODS: A retrospective analysis of Trauma Audit and Research Network (TARN) data and case note review of all severe trauma cases (Injury Severity Score ≥9) in North East England from 1 January 2014 to 1 December 2017 who were treated by the North East Ambulance Service, the Great North Air Ambulance Service or both. TARN methods were used to calculate the number of unexpected survivors or deaths in each group (W score (Ws)). The Glasgow Outcome Scores were contrasted to evaluate morbidity. RESULTS: The ECT group treated 531 patients: there were 17 unexpected survivors and no unexpected deaths. The non-ECT group treated 1202 patients independently: there were no unexpected survivors and 31 unexpected deaths. The proportion of patients requiring critical care interventions differed between the two groups 49% versus 33% (CI for difference 12% to 20%). In the ECT group, the Ws was 3.22 (95% CI 0.79 to 5.64). In the non-ECT group, the Ws was -2.97 (95% CI -1.22 to -4.71). The difference between the Ws was 6.18 (95% CI 3.19 to 9.17). There was no evidence of worse morbidity in the ECT group. CONCLUSION: This is the first UK ECT service to demonstrate a risk-adjusted mortality benefit in trauma patients with no detriment in morbidity: our results demonstrate an additional 3.22 survivors per 100 severe trauma casualties when treated by an ECT. The authors encourage other ECT services to conduct similar research.

Extending Single Molecule Imaging to Proteome Analysis by Quantitation of Fluorescent Labeling Homogeneity in Complex Protein Samples.

Fluorescence-based electrophoresis has been widely used for proteome analysis in which every protein species in cells is labeled with a fluorescent dye, separated by electric migration, and quantified using fluorescence detection. The ultimate limit of sensitivity for this approach could be reached by single-molecule fluorescence imaging and counting individual proteins, requiring exhaustive fluorescent labeling of proteins across molecular populations and species. However, it remains unclear how homogeneous the fluorescence labeling of individual protein molecules of each species is across the proteome. To address this question, we developed a method to measure the labeling homogeneity based on a single-molecule fluorescence counting assay. Our results reveal that the proportion of proteins labeled with at least one dye, called labeling occupancy (LO), was 35% for fluorescently labeled BSA using existing protocols. We then found that the LO could be improved to 82% under high pH and surfactant-rich conditions. Furthermore, when a proteome sample from a human cell lysate was analyzed, the total LO was 71%, whereby the values varied between 50 and 90% for low and high molecular weight proteome fractions, respectively. The results support the possibility of sensitive detection of proteins using single-molecule counting with fluorescent labeling at the proteome scale.

Bioaccumulation of Nanosilver by Chlamydomonas reinhardtii-nanoparticle or the free ion?

The bioavailability of a small silver nanoparticle (nAg; nominal size of 5 nm with a polyacrylate coating) by the green alga C. reinhardtii was investigated in order to assess the contributions of Ag(+) and nAg to cellular internalization. Upon exposure to nAg, Ag biouptake exceeded what was predicted based upon measured Ag(+) concentrations. Indeed, although Ag biouptake was greatly reduced when excess cysteine was added to the nAg, it was nonetheless significantly above control levels. For both exposures to nAg and Ag(+), expression levels of the Copper Transport Protein 2 (CTR2) indicated that Ag biouptake could be attributed to the internalization of Ag(+). Exposure to Ag(+) or nAg increased CTR2 expression, even when cysteine was present with the nAg. Darkfield microscopy coupled with hyperspectral imagery showed that the presence of silver nanoparticles inside the cells was more likely due to the rereduction of Ag(+) than to the internalization of nAg. The weight of evidence indicated that nAg increased Ag biouptake by locally increasing the surface concentrations of Ag(+).

Multimethod quantification of Ag+ release from nanosilver.

There is a significant interest in determining the effects of nanomaterials on the environment and human health. Part or all of the toxicity attributed to silver nanoparticles (nAg) may be due to the release of free silver (Ag(+)). Therefore, it is necessary to have techniques that will allow the precise determination of free Ag(+) within suspensions of nAg particles. Among the different methods used for the determination of free metals in natural waters, the ion-exchange technique (IET), has promise to both distinguish Ag(+) from nAg and to attain the low detection limits required for the analysis of natural samples. In this paper, IET. centrifugal ultrafiltration and single particle inductively coupled plasma mass spectrometry (SP ICP-MS) were used to determine very low concentrations of free or dissolved Ag in commercial suspensions of nAg. Dilution of the silver nanoparticles played an important role in the measured Ag(+) concentrations. The relative release of Ag(+) from nAg increased as samples were increasingly diluted, implying that it is critical to determine Ag(+) concentrations under the precise conditions used for determinations of toxicological or environmental fate.