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Funder: Wellcome Clinical PhD fellowship, Funder: Wellcome PhD Studentship, Funder: Jean Shank/Pathological Society Intermediate Fellowship, Mutation accumulation in somatic cells contributes to cancer development and is proposed as a cause of aging. DNA polymerases Pol ε and Pol δ replicate DNA during cell division. However, in some cancers, defective proofreading due to acquired POLE/POLD1 exonuclease domain mutations causes markedly elevated somatic mutation burdens with distinctive mutational signatures. Germline POLE/POLD1 mutations cause familial cancer predisposition. Here, we sequenced normal tissue and tumor DNA from individuals with germline POLE/POLD1 mutations. Increased mutation burdens with characteristic mutational signatures were found in normal adult somatic cell types, during early embryogenesis and in sperm. Thus human physiology can tolerate ubiquitously elevated mutation burdens. Except for increased cancer risk, individuals with germline POLE/POLD1 mutations do not exhibit overt features of premature aging. These results do not support a model in which all features of aging are attributable to widespread cell malfunction directly resulting from somatic mutation burdens accrued during life.

Gene therapy (GT) has rapidly attracted renewed interest as a treatment for otherwise incurable diseases, with several GT products already on the market and many more entering clinical testing for selected indications. Clonal tracking techniques based on vector integration enable monitoring of the fate of engineered cells in the blood of patients receiving GT and allow assessment of the safety and efficacy of these procedures. However, owing to the limited number of cells that can be tested and the impracticality of studying cells residing in peripheral organs without performing invasive biopsies, this approach provides only a partial snapshot of the clonal repertoire and dynamics of genetically modified cells and reduces the predictive power as a safety readout. In this study, we developed liquid biopsy integration site sequencing, or LiBIS-seq, a polymerase chain reaction technique optimized to quantitatively retrieve vector integration sites from cell-free DNA released into the bloodstream by dying cells residing in several tissues. This approach enabled longitudinal monitoring of in vivo liver-directed GT and clonal tracking in patients receiving hematopoietic stem cell GT, improving our understanding of the clonal composition and turnover of genetically modified cells in solid tissues and, in contrast to conventional analyses based only on circulating blood cells, enabling earlier detection of vector-marked clones that are aberrantly expanding in peripheral tissues.

Objective The Covid‐19 pandemic is rapidly spreading worldwide, notably in Europe and North America, where obesity is highly prevalent. The relation between obesity and severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) has not been fully documented. Methods In this retrospective cohort study we analyzed the relationship between clinical characteristics, including body mass index (BMI), and the requirement for invasive mechanical ventilation (IMV) in 124 consecutive patients admitted in intensive care for SARS‐CoV‐2, in a single French center. Results Obesity (BMI >30 kg/m2) and severe obesity (BMI >35 kg/m2) were present in 47.6% and 28.2% of cases, respectively. Overall, 85 patients (68.6%) required IMV. The proportion of patients who required IMV increased with BMI categories (p35 kg/m2 (85.7%). In multivariate logistic regression, the need for IMV was significantly associated with male sex (p35 kg/m2 vs patients with BMI

The use of chimeric antigen receptor (CAR)-engineered regulatory T cells (Tregs) has emerged as a promising strategy to promote immune tolerance. However, in conventional T cells (Tconvs), CAR expression is often associated with tonic signaling, which can induce CAR-T cell dysfunction. The extent and effects of CAR tonic signaling vary greatly according to the expression intensity and intrinsic properties of the CAR. Here, we show that the 4-1BB CSD-associated tonic signal yields a more dramatic effect in CAR-Tregs than in CAR-Tconvs with respect to activation and proliferation. Compared to CD28 CAR-Tregs, 4-1BB CAR-Tregs exhibit decreased lineage stability and reduced in vivo suppressive capacities. Transient exposure of 4-1BB CAR-Tregs to a Treg stabilizing cocktail, including an mTOR inhibitor and vitamin C, during ex vivo expansion sharply improves their in vivo function and expansion after adoptive transfer. This study demonstrates that the negative effects of 4-1BB tonic signaling in Tregs can be mitigated by transient mTOR inhibition., Chimeric antigen receptor engineering in T cells has been shown to be of great potential therapeutic benefit in a range of immune pathologies, although the functionality of such cell therapies can be limited due to tonic signalling and the induction of dysfunction. Here the authors show transient inhibition of mTOR can rescue their 41-BB-CAR-Tregs from tonic signalling-induced dysfunction.

Motor sequence learning in persons with multiple sclerosis (pwMS) and healthy controls (HC) under implicit or explicit learning conditions has not yet been investigated in a stepping task. Given the prevalent cognitive and mobility impairments in pwMS, this is important in order to understand motor learning processes and optimize rehabilitation strategies. Nineteen pwMS (the Expanded Disability Status Scale = 3.4 ± 1.2) and 18 HC performed a modified serial reaction time task by stepping as fast as possible on a stepping tile when it lit up, either with (explicit) or without (implicit) knowledge of the presence of a sequence beforehand. Motor sequence learning was studied by examining response time changes and differences between sequence and random blocks during the learning session (acquisition), 24 h later (retention), and in three dual-task (DT) conditions at baseline and retention (automaticity) using subtracting sevens, verbal fluency, and vigilance as concurrent cognitive DTs. Response times improved and were lower for the sequenced compared with the random blocks at the post- and retention tests (P's < 0.001). Response times during DT conditions improved after learning, but DT cost improved only for the subtracting sevens DT condition. No differences in learning were observed between learning conditions or groups. This study showed motor sequence learning, by acquisition and retention, in a stepping task in pwMS with motor impairments, to a similar degree as HC and regardless of learning conditions. Whether automaticity increased remains unclear.

We studied COVID-19 associated mucormycosis based on 17 cases reported nationwide and assessed the differences with India. They differed by frequencies of diabetes mellitus (47% in France versus up to 95% in India), hematological malignancies (35% versus 1%), anatomical sites (12% versus >80% rhino-orbito-cerebral) and prognosis (88% mortality versus, We reported 17 cases of COVID-19 associated mucormycosis in France. Compared to India, they differed by frequencies of diabetes mellitus (47% versus up to 95% in India), hematological malignancies (35% versus 1%), anatomical sites and mortality (88% versus

Feeling threatened by terrorism can be associated with mental health problems and behavioural changes. However, few studies look at the association in the long-term. Using a survey, the population in Brussels, Belgium was studied using a representative database delivered by the national post service. The Patient Health Questionnaire-4 (PHQ-4) assessed mental health, and self-made questions avoidance behaviour. 170 people answered: 60% women and 50% higher educated, 28.2% between 56 and 65 years and 62.4% had a partner. 43.5% felt threatened by the terrorist attacks and 45.9% experienced no mental health problems. Both terrorist threat (p < 0.001) and avoidance behaviour (p < 0.001) significantly predicted PHQ-4 scores, while controlling for gender, age, social support, education level, and traumatic events. There is a relation between terrorist threat and anxiety/depressive symptoms 2.5 years after the last study on terrorist threat in Brussels, but it has weakened. Avoidance behaviour seems to be more present than threat.

Several risk factors have been established for colorectal cancer, yet their direct mutagenic effects in patients' tumors remain to be elucidated. Here, we leveraged whole-exome sequencing data from 900 colorectal cancer cases that had occurred in three U.S.-wide prospective studies with extensive dietary and lifestyle information. We found an alkylating signature that was previously undescribed in colorectal cancer and then showed the existence of a similar mutational process in normal colonic crypts. This alkylating signature is associated with high intakes of processed and unprocessed red meat prior to diagnosis. In addition, this signature was more abundant in the distal colorectum, predicted to target cancer driver mutations KRAS p.G12D, KRAS p.G13D, and PIK3CA p.E545K, and associated with poor survival. Together, these results link for the first time a colorectal mutational signature to a component of diet and further implicate the role of red meat in colorectal cancer initiation and progression. Significance: Colorectal cancer has several lifestyle risk factors, but the underlying mutations for most have not been observed directly in tumors. Analysis of 900 colorectal cancers with whole-exome sequencing and epidemiologic annotations revealed an alkylating mutational signature that was associated with red meat consumption and distal tumor location, as well as predicted to target KRAS p.G12D/p.G13D. This article is highlighted in the In This Issue feature, p. 2355

In clinically suspected acute full-thickness proximal hamstring tendon avulsions, MRI is the gold standard for evaluating the extent of the injury. MRI variables such as full-thickness free tendon discontinuity, extent of tendon retraction (>20 mm), and continuity of the sacrotuberous ligament with the conjoint tendon (STL-CT) are used in treatment decision-making. The objective was to assess the intra- and inter-rater reliability of these relevant MRI variables after acute full-thickness proximal hamstring tendon avulsion. Three musculoskeletal radiologists assessed MRIs of 40 patients with an acute full-thickness proximal hamstring tendon avulsion. MRI variables included assessment of free tendon discontinuity and continuity of the STL-CT and extent of tendon retraction. Absolute and relative intra- and inter-rater reliability were calculated. Intra- and inter-rater reliability for the assessment of tendon discontinuity was substantial (Kappa [ĸ]=0.78;0.77). For the retraction measurement of the conjoint and semimembranosus tendons, intra-rater reliability was moderate and poor (Intraclass correlation coefficient (ICC)=0.74;0.45), inter-rater reliability was moderate (ICC=0.73;0.57). Intra- and inter-rater reliability of the STL-CT continuity assessment was substantial and fair (ĸ=0.74;0.31). In conclusion, MRI assessment for full-thickness free tendon discontinuity is reliable. However, assessment of extent of tendon retraction and STL-CT continuity is not reliable enough to guide the treatment decision-making process.

Background: Proximal full-thickness free hamstring tendon injury (ie, tendon avulsion or rupture) is a severe injury. Treatment decision making relies on clinical factors and magnetic resonance imaging (MRI) variables; it specifically relies on which tendons are injured as well as the extent of tendon retraction. According to a worldwide evaluation of current practice, discontinuity of both proximal tendons and retraction of >2 cm are used as surgical indications. However, both the diagnosis and the use of MRI variables in decision making may be fraught with uncertainty in clinical practice. A reliable standardized MRI assessment is required. Purpose: To propose an MRI assessment for acute proximal full-thickness free hamstring tendon injury and to evaluate its interater reliability. Study Design: Cohort study (diagnosis); Level of evidence, 2. Methods: We included 40 MRI scans of patients with acute (≤4 weeks of injury) proximal full-thickness free hamstring tendon injury. Three musculoskeletal radiologists assessed proximal full-thickness free hamstring tendon discontinuity using the novel “dropped ice cream sign” and tendon retraction (in mm). Quantification of tendon retraction (in mm) was performed using 2 different methods: (1) a direct (ie, shortest distance between the center of the hamstring origin and the tendon stump) method and (2) a combined craniocaudal/mediolateral measurement method. Absolute and relative interrater reliability were calculated. Results: We found an almost perfect interrater agreement (kappa = 0.87) for assessment of full-thickness tendon discontinuity using the dropped ice cream sign. Interrater agreement for the direct and craniocaudal retraction measurements was good for both the conjoint (intraclass correlation coefficient [ICC], 0.88 and 0.83) and the semimembranosus tendons (ICC, 0.81 and 0.79). The mediolateral retraction measurement yielded only moderate to poor reliability for the conjoint (ICC, 0.53) and semimembranosus tendons (ICC, 0.41). Conclusion: The standardized MRI assessment to identify proximal hamstring tendon discontinuity and quantify tendon retraction is reliable. We recommend using the novel dropped ice cream sign and the direct retraction measurement in clinical practice and research.

Background: Late-onset retinal degeneration (L-ORD) is a rare autosomal dominant retinal dystrophy related to C1QTNF5 gene variants.Materials and methods: Twenty-six patients (21–81 years) with L-O...

Background Systemic reactivation of herpesviruses may occur in intensive care unit (ICU) patients and is associated with morbidity and mortality. Data on severe Coronavirus disease-19 (COVID-19) and concomitant reactivation of herpesviruses are lacking. Methods We selected patients admitted to ICU for confirmed COVID-19 who underwent systematic testing for Epstein–Barr virus (EBV), cytomegalovirus (CMV) and human-herpes virus-6 (HHV-6) DNAemia while in the ICU. We retrospectively analysed frequency, timing, duration and co-occurrence of viral DNAemia. Results Thirty-four patients were included. Viremia with EBV, CMV, and HHV-6 was detected in 28 (82%), 5 (15%), and 7 (22%) patients, respectively. EBV reactivation occurred early after ICU admission and was associated with longer ICU length-of-stay. Conclusions While in the ICU, critically ill patients with COVID-19 are prone to develop reactivations due to various types of herpesviruses.

T-cell acute lymphoblastic leukemia (T-ALL) represents the malignant expansion of immature T cells blocked in their differentiation. T-ALL is still associated with a poor prognosis, mainly related to occurrence of relapse or refractory disease. A critical medical need therefore exists for new therapies to improve the disease prognosis. Adenylate kinase 2 (AK2) is a mitochondrial kinase involved in adenine nucleotide homeostasis recently reported as essential in normal T-cell development, as defective AK2 signaling pathway results in a severe combined immunodeficiency with a complete absence of T-cell differentiation. In this study, we show that AK2 is constitutively expressed in T-ALL to varying levels, irrespective of the stage of maturation arrest or the underlying oncogenetic features. T-ALL cell lines and patient T-ALL–derived xenografts present addiction to AK2, whereas B-cell precursor ALL cells do not. Indeed, AK2 knockdown leads to early and massive apoptosis of T-ALL cells that could not be rescued by the cytosolic isoform AK1. Mechanistically, AK2 depletion results in mitochondrial dysfunction marked by early mitochondrial depolarization and reactive oxygen species production, together with the depletion of antiapoptotic molecules (BCL-2 and BCL-XL). Finally, T-ALL exposure to a BCL-2 inhibitor (ABT-199 [venetoclax]) significantly enhances the cytotoxic effects of AK2 depletion. We also show that AK2 depletion disrupts the oxidative phosphorylation pathway. Combined with pharmaceutical inhibition of glycolysis, AK2 silencing prevents T-ALL metabolic adaptation, resulting in dramatic apoptosis. Altogether, we pinpoint AK2 as a genuine and promising therapeutic target in T-ALL.

Somatic mutations accumulate in healthy tissues as we age, giving rise to cancer and potentially contributing to ageing. To study somatic mutations in non-neoplastic tissues, we developed a series of protocols to sequence the genomes of small populations of cells isolated from histological sections. Here, we describe a complete workflow that combines laser-capture microdissection (LCM) with low-input genome sequencing, while circumventing the use of whole-genome amplification (WGA). The protocol is subdivided broadly into four steps: tissue processing, LCM, low-input library generation and mutation calling and filtering. The tissue processing and LCM steps are provided as general guidelines that might require tailoring based on the specific requirements of the study at hand. Our protocol for low-input library generation uses enzymatic rather than acoustic fragmentation to generate WGA-free whole-genome libraries. Finally, the mutation calling and filtering strategy has been adapted from previously published protocols to account for artifacts introduced via library creation. To date, we have used this workflow to perform targeted and whole-genome sequencing of small populations of cells (typically 100–1,000 cells) in thousands of microbiopsies from a wide range of human tissues. The low-input DNA protocol is designed to be compatible with liquid handling platforms and make use of equipment and expertise standard to any core sequencing facility. However, obtaining low-input DNA material via LCM requires specialized equipment and expertise. The entire protocol from tissue reception through whole-genome library generation can be accomplished in as little as 1 week, although 2–3 weeks would be a more typical turnaround time.

International audience; In tetrahydrofuran, the electrochemical reduction of Cp2TiIVCl2 (2 mM) generated three titanium(III) complexes which were in equilibrium: [Cp2TiCl2]•−, [Cp2TiCl]• and (Cp2TiCl)2. Although the anion radical [Cp2TiCl2]•− was the main species produced under these conditions, cyclic voltammetry investigations clearly showed that the proportion of the three electrogenerated TiIII complexes can be modified as a function of the amounts of chloride ion present in the solution. Accordingly, the presence of Mg2+ ions, which led to the consumption of chloride ions through the formation of MgCl2, favoured the formation of [Cp2TiCl]• and, consequently, of the corresponding dimer (Cp2TiCl)2. The electrochemical behaviours of Cp2TiIVCl2 and of the electrogenerated low-valent Ti complexes were also investigated in the presence of amide and alkyne derivatives. Under these conditions, titanium complexes could not only interact with the amide carbonyl group, but also with the alkyne triple bond, provided the latter was not sterically hindered. Interestingly, the carbonyl group and the triple bond had antagonist effects on redox properties of titanium(III) complexes.

The function of the uncoupling protein 2 (UCP2) is different for each cancer cell. However, the mechanism of expression is still unclear. DNA methylation affects protein expression and is one factor that transforms normal cells into cancer cells. In this study, the hepatocellular carcinoma Hep3B and HepG2 cells and colorectal cancer HT-29 cells were treated with 5-azacytidine (5-aza), a DNA demethylation agent, to observe the modification of UCP2 expression and the methylation degree in the UCP2 promoter region. Promoter basal activity and degree of UCP2 expression were measured in Hep3B, HepG2, and HT-29 cells. In addition, methylation-specific PCR (MSP) was performed to investigate the degree of methylation in the UCP2 promoter region. The methylation region in the UCP2 promoter was confirmed based on bisulfite sequencing. In Hep3B cells in which UCP2 mRNA was not transcribed, the promoter basal activity was significantly higher than in HT-29 or HepG2 cells in which UCP2 mRNA was transcribed. Treatment with 5-aza increased UCP2 expression in Hep3B and HT-29 cells; however, the expression in HepG2 cells was unchanged. The UCP2 promoter in Hep3B cells has numerous methylated regions compared with HT-29 and HepG2 cells. The results of the present study revealed that inhibition of UCP2 expression in Hep3B cells was due to methylation of the promoter region. Investigating the mechanism that induces UCP2 expression in cancer cells is important to understand the function of UCP2, which could aid in cancer treatment.

Introduction The number of patients with hip or knee joint replacements is increasing. Some of these patients want to practice sport, including in high-impact sports such as ultra-trails. Is the proportion of drop-out higher among runners after a hip or knee replacement? What are the symptoms of these patients? Hypothesis Hip or knee joint replacements are associated with a higher drop-out rate during an ultra-trail. Material and methods We conducted a monocentric retrospective epidemiological study. A questionnaire translated into French, English and Spanish were sent to all runners registered for one or more of the 5 races of the 2015 to 2017 Ultra-Trail du Mont-Blanc® (UTMB®) editions. There were no exclusion criteria. The drop-out rate of patients who responded to the questionnaire was calculated among patients with and without hip or knee joint replacement. Specific symptoms related to these joint replacements were identified before, during and after the race. Results Out of the 24,855 participants having run at least one of the 3 editions, 2,469 responded. Some of them ran several races: these 2469 runners were on 3171 start lines and 2548 finish lines (drop-out rate of 19.6%). Among these 2469 runners, 18 had hip and/or knee replacement and were on 27 start lines and 17 finish lines (drop-out rate of 37%). The race was finished by 3 of the 6 runners with a total hip arthroplasty (THA), all of the 8 runners with a hip resurfacing arthroplasty (HRA) and 3 of the 4 runners with a total knee arthroplasty (TKA). Among runners with THA, pubalgia or groin pain was described by one of them before the race, 3 during the race and 2 after the race. Among runners with HRA, pygalgia or groin pain was described by 2 of them during the race. As for the TKA, there was no specific symptom. No answering runner had a unicompartmental knee prosthesis. Discussion Running ultra-trail is possible after a hip or knee joint replacement. All of the 8 runners with HRA finished the race. We may remain cautious about the long-term survival of prostheses. Level of proof IV; monocentric retrospective epidemiological study.

Gram-negative bacteria possess an asymmetric outer membrane (OM) composed primarily of lipopolysaccharides (LPSs) on the outer leaflet and phospholipids (PLs) on the inner leaflet. The loss of this asymmetry due to mutations in the LPS biosynthesis or transport pathways causes the externalization of PLs to the outer leaflet of the OM and leads to OM permeability defects. Here, we used metabolic labeling to detect a compromised OM in intact bacteria. Phosphatidylcholine synthase expression in Escherichia coli allowed for the incorporation of exogenous propargylcholine into phosphatidyl(propargyl)choline and exogenous 1-azidoethyl-choline (AECho) into phosphatidyl(azidoethyl)choline (AEPC), as confirmed by LC/MS analyses. A fluorescent copper-free click reagent poorly labeled AEPC in intact wild-type cells but readily labeled AEPC from lysed cells. Fluorescence microscopy and flow cytometry analyses confirmed the absence of significant AEPC labeling from intact wild-type E. coli strains and revealed significant AEPC labeling in an E. coli LPS transport mutant (lptD4213) and an LPS biosynthesis mutant (E. coli lpxC101). Our results suggest that metabolic PL labeling with AECho is a promising tool for detecting a compromised bacterial OM, revealing aberrant PL externalization, and identifying or characterizing novel cell-active inhibitors of LPS biosynthesis or transport.­

DNA vaccines expressing codon-optimized Venezuelan equine encephalitis virus (VEEV) and Ebola virus (EBOV) glycoprotein genes provide protective immunity to mice and nonhuman primates when delivered by intramuscular (IM) electroporation (EP). To achieve equivalent protective efficacy in the absence of EP, we evaluated VEEV and EBOV DNA vaccines constructed using minimalized Nanoplasmid expression vectors that are smaller than conventional plasmids used for DNA vaccination. These vectors may also be designed to co-express type I interferon inducing innate immune agonist genes that have an adjuvant effect. Nanoplasmid vaccinated mice had increased antibody responses as compared to those receiving our conventional pWRG7077-based vaccines when delivered by IM injection, and these responses were further enhanced by the inclusion of the innate immune agonist genes. The Nanoplasmid VEEV DNA vaccines also significantly increased protection against aerosol VEEV challenge as compared to the pWRG7077 VEEV DNA vaccine. Although all mice receiving the pWRG7077 and Nanoplasmid EBOV DNA vaccines at the dose tested survived EBOV challenge, only mice receiving the Nanoplasmid EBOV DNA vaccine that co-expresses the innate immune agonist genes failed to lose weight after challenge. Our results suggest that Nanoplasmid vectors can improve the immunogenicity and protective efficacy of alphavirus and filovirus DNA vaccines.

Highlights • Using acquired somatic mutations as a genetic barcode allows reconstruction of a stem cell “family tree” of relatedness. • Understanding HSC clonal dynamics is central to understanding how clones emerge to drive leukemias and other diseases. • Many HSC clones, most with multilineage output, make blood. • HSCs acquire mutations linearly over life, but more slowly than other tissues. • There is now a vast potential for understanding the relatedness and origin of various cell types across multiple adult tissues., Despite decades of progress in our understanding of hematopoiesis through the study of animal models and transplantation in humans, investigating physiological human hematopoiesis directly has remained challenging. Questions on the clonal structure of the human hematopoietic stem cell (HSC) pool, such as “how many HSCs are there?” and “do all HSC clones actively produce all blood cell types in equal proportions?” remain open. These questions have inherent value for understanding normal human physiology, but also directly inform our comprehension of the process by which the system is subverted to drive diseases of the blood, in particular blood cancers and bone marrow failure syndromes. The critical link between normal and abnormal hematopoiesis is perhaps best illustrated by the recent discovery of clonal hematopoiesis in healthy people with no abnormal blood parameters. In such individuals, large clones derived from single cells are present and are dominant relative to their normal counterparts, but their presence does not necessitate abnormal blood cell production. Intriguingly, however, these individuals are also at a significantly greater risk of developing leukemias and of cardiovascular events, underscoring the importance of understanding how blood stem cell clones compete against each other.

The European epidemic monophasic variant of Salmonella enterica serovar Typhimurium (S. 1,4,[5],12:i:-) characterized by the multi locus sequence type ST34 and the antimicrobial resistance ASSuT profile has become one of the most common serovars in Europe (EU) and the United States (US). In this study, we reconstructed the time-scaled phylogeny and evolution of this Salmonella in Europe. The epidemic S. 1,4,[5],12:i:- ST34 emerged in the 1980s by an acquisition of the Salmonella Genomic Island (SGI)-4 at the 3′ end of the phenylalanine phe tRNA locus conferring resistance to copper and arsenic toxicity. Subsequent integration of the Tn21 transposon into the fljAB locus gave resistance to mercury toxicity and several classes of antibiotics used in food-producing animals (ASSuT profile). The second step of the evolution occurred in the 1990s, with the integration of mTmV and mTmV-like prophages carrying the perC and/or sopE genes involved in the ability to reduce nitrates in intestinal contents and facilitate the disruption of the junctions of the host intestinal epithelial cells. Heavy metals are largely used as food supplements or pesticide for cultivation of seeds intended for animal feed so the expansion of the epidemic S. 1,4,[5],12:i:- ST34 was strongly related to the multiple-heavy metal resistance acquired by transposons, integrative and conjugative elements and facilitated by the escape until 2011 from the regulatory actions applied in the control of S. Typhimurium in Europe. The genomic plasticity of the epidemic S. 1,4,[5],12:i:- was demonstrated in our study by the analysis of the plasmidome. We were able to identify plasmids harboring genes mediating resistance to phenicols, colistin, and fluoroquinolone and also describe for the first time in six of the analyzed genomes the presence of two plasmids (pERR1744967-1 and pERR2174855-2) previously described only in strains of enterotoxigenic Escherichia coli and E. fergusonii.

FOXP3 deficiency in mice and in patients with immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome results in fatal autoimmunity by altering regulatory T (T-reg) cells. CD4(+) T cells in patients with IPEX syndrome and Foxp3-deficient mice were analyzed by single-cell cytometry and RNA-sequencing, revealing heterogeneous T-reg-like cells, some very similar to normal T-reg cells, others more distant. Conventional T cells showed no widespread activation or helper T cell bias, but a monomorphic disease signature affected all CD4(+) T cells. This signature proved to be cell extrinsic since it was extinguished in mixed bone marrow chimeric mice and heterozygous mothers of patients with IPEX syndrome. Normal T-reg cells exerted dominant suppression, quenching the disease signature and revealing in mutant T-reg-like cells a small cluster of genes regulated cell-intrinsically by FOXP3, including key homeostatic regulators. We propose a two-step pathogenesis model: cell-intrinsic downregulation of core FOXP3-dependent genes destabilizes T-reg cells, de-repressing systemic mediators that imprint the disease signature on all T cells, furthering T-reg cell dysfunction. Accordingly, interleukin-2 treatment improved the T-reg-like compartment and survival. FOXP3 deficiency leads to dramatic loss of immune homeostasis. This multicenter collaborative group finds that loss of FOXP3 function only disrupts a few core genes, but this unmasks a degree of systemic inflammation, and it is this environment that then strongly perturbs T-reg cells.

Somatic mutations drive the development of cancer and may contribute to ageing and other diseases1,2. Despite their importance, the difficulty of detecting mutations that are only present in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. Here, to overcome these limitations, we developed nanorate sequencing (NanoSeq), a duplex sequencing protocol with error rates of less than five errors per billion base pairs in single DNA molecules from cell populations. This rate is two orders of magnitude lower than typical somatic mutation loads, enabling the study of somatic mutations in any tissue independently of clonality. We used this single-molecule sensitivity to study somatic mutations in non-dividing cells across several tissues, comparing stem cells to differentiated cells and studying mutagenesis in the absence of cell division. Differentiated cells in blood and colon displayed remarkably similar mutation loads and signatures to their corresponding stem cells, despite mature blood cells having undergone considerably more divisions. We then characterized the mutational landscape of post-mitotic neurons and polyclonal smooth muscle, confirming that neurons accumulate somatic mutations at a constant rate throughout life without cell division, with similar rates to mitotically active tissues. Together, our results suggest that mutational processes that are independent of cell division are important contributors to somatic mutagenesis. We anticipate that the ability to reliably detect mutations in single DNA molecules could transform our understanding of somatic mutagenesis and enable non-invasive studies on large-scale cohorts. NanoSeq is used to detect mutations in single DNA molecules and analyses show that mutational processes that are independent of cell division are important contributors to somatic mutagenesis.

Background: Palliative sedation requires no formal registration in Belgium. For euthanasia, however, there are clear guidelines in certain countries, including mandatory registration and evaluation of cases. Official guidelines have been developed for palliative sedation, but research still shows unskilled use of sedation and unclear demarcation between palliative sedation and euthanasia. Registration could be used to avoid unskilled use of sedation and to avoid use or abuse of sedation to hasten the patient's death (described as "slow/passive euthanasia"). Objective: To evaluate the current practice of palliative sedation and use of opioids and sedatives at the end of life by using a registration document. Design: Retrospective observational study. Setting/Subjects: We included all in-hospital deaths at an academic hospital in Belgium of patients (age ≥18 years) who had received parenteral opioids, benzodiazepines, barbiturates, or other anesthetics during the last 24 hours. Measurements: We investigated indications for palliative sedation, patients' and physicians' characteristics, types of medication used, and the decision-making process with the patients and family. The questionnaires were collected between July 9 and November 25, 2016. The study was approved by the Biomedical Ethics Committee of UZ Brussel (B.U.N. 1432016293). Results: In a population of 124 patients, refractory symptoms were reported in 94.4%. All patients received parenteral opioids (intravenously). Benzodiazepines were used in 51.6%. In 75.8%, physicians reported no change in treatment plan during end of life. Hastening death by increasing analgesics and/or sedatives was mentioned in 19.3%. The treatment plan was discussed with patients in 26.6% of cases. In 6 cases, an explicit intention to hasten death was mentioned; in 36 cases, doses of opioids/sedatives may not have been strictly proportional to symptoms. Conclusion: This Belgian study shows that objective reporting of palliative sedation can be used as a tool to ensure good clinicalpractice where patients receive the most appropriate end-of-life care, avoiding abusive and injudicious or unskilled use of sedation.

Wiskott Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency associated with thrombocytopenia, eczema, infectious, autoimmune complications, and lymphomas. Patients lacking an HLA-matched donor may benefit from an alternative therapeutic approach based on the infusion of autologous gene corrected CD34+ cells. We previously reported a non-randomised, open-label, phase 1/2 clinical study applying a lentiviral vector based gene therapy (GT) protocol in 7 paediatric patients with severe WAS (score ≥ 3/5) (S. Hacein-Bey Abina et al, JAMA 2015). One patient died 7 months after GT because of pre-existing severe opportunistic infections, as reported. Two additional patients have been treated since that initial report, with a follow-up of at least 4 years. We here present a comprehensive long-term study on 8 patients with a follow-up from 4 to 9 years (median 7.6). The safety and efficacy of the approach is thoroughly investigated, with a particular focus on the correction of thrombocytopenia and auto-immunity. A stable engraftment of genetically and functionally corrected lymphoid and myeloid cells was reached in all patients, with no serious treatment-associated adverse events or concerning clonal expansion. Corrected lymphoid cells displayed a selective advantage over time with increasing vector copy number (VCN) level. In turn, this led to (i) sustained expression of WAS protein (WASp) in the patients' cells and (ii) clinical resolution of severe eczema and susceptibility to recurrent infections. In line with these results, T-cell function was restored after GT, as shown by the recovery of immune synapse assembly and the normalization of naïve T cell numbers. The T-cell compartment was also reconstituted in the patient treated at the age of 30 years, suggesting that GT for WAS is a treatment option in adult patients. In parallel with the robustness of T-cell reconstitution a normalized B-cell compartment was observed after GT, as shown in particular by increasing levels of WASp + switched memory B cells over time and the age-matched levels of KRECs. Five patients out of 8 were able to discontinue Ig replacement therapy while achieving normal post-vaccination antibody titers. Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. After GT, a few autoimmune manifestations were observed: the persistence of lower extremity vasculitis (P2, very severe prior to GT), the new occurrence of nephrotic syndrome (P9), and the presence of anti-platelet antibodies (P2, P4, P7). The levels of circulating autoantibodies detected before GT (including ANA and vasculitis-related autoantibodies) normalized after treatment. Following GT, platelets were found to express sub-normal levels of WASp and to only partially augment their size. Platelet function studies indicated a partial correction of the platelet compartment achieved by GT, which may be sufficient to prevent occurrence of the hemorrhagic symptoms typical of WAS. Our results suggest that lentiviral GT provides sustained clinical benefits for patients with WAS. Overall clinical remission was observed in our patients despite very severe disease scores before GT. More efficacious and more reliable transduction protocols and conditioning regimen are likely to further improve outcomes, particularly with regard to platelet recovery, where the advantages of intrinsic correction are less apparent. Disclosures Booth: Orchard Therapeutics: Consultancy, Honoraria; SOBI: Consultancy, Honoraria; Takeda: Honoraria; GSK: Honoraria; Rocket Pharmaceuticals, Inc.: Consultancy. Thrasher: Orchard Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Cavazzana: Smart Immune: Other: co-founder.

Rainfall seasonality and timing are key climatic features affecting crop yield in rainfed agriculture (RFA). We evaluated the dynamics of temporal rainfall attributes – the strength of seasonality, the date of onset, cessation, and duration of the rainy season – over RFA areas across Ethiopia for the period 1981-2010 and explored their impacts on cereal crop production (including maize, teff, sorghum, wheat, barley, millet, oats and rice) between 1995 and 2010. First, we quantified the rainfall seasonality using an entropy-based seasonality index, defined the onset and cessation dates of rainy seasons, computed the rainy season duration, and analyzed their interannual variability and trends. Second, we correlated de-trended total cereal production during the Meher (i.e., long rainy) season (April to September) with the anomalies of the temporal rainfall attributes, and we used a univariate linear regression model to estimate the influence of changes in these attributes on crop production. We show that RFA areas in northern Ethiopia are characterized by a highly seasonal and unimodal rainfall regime. The southern parts of the RFA areas are characterized by less seasonal rainfall of bimodal and erratic nature. Cereal crop production during the Meher season, especially in teff and maize-dominated regions, is found to be correlated to both the onset (median ρ=-0.32 and -0.37, respectively) and duration (ρ=0.34 and 0.19) of the rainy season in the unimodal rainfall regime, whereas it is correlated with the rainfall seasonality (ρ=0.21) in regions with a bimodal rainfall. We estimate that on average over RFA areas, a late-onset and shorter rainy season lead to ∼1.5% and 1.1% crop production losses per pentad (5-day period), respectively., Agricultural and Forest Meteorology, 310, ISSN:0168-1923, ISSN:1873-2240

Cellular DNA damage caused by reactive oxygen species is repaired by the base excision repair (BER) pathway which includes the DNA glycosylase MUTYH. Inherited biallelic MUTYH mutations cause predisposition to colorectal adenomas and carcinoma. However, the mechanistic progression from germline MUTYH mutations to MUTYH-Associated Polyposis (MAP) is incompletely understood. Here, we sequenced normal tissue DNAs from 10 individuals with MAP. Somatic base substitution mutation rates in intestinal epithelial cells were elevated 2 to 5-fold in all individuals, except for one showing a 33-fold increase, and were also increased in other tissues. The increased mutation burdens were of multiple mutational signatures characterised by C>A changes. Different mutation rates and signatures between individuals were likely due to different MUTYH mutations or additional inherited mutations in other BER pathway genes. The elevated base substitution rate in normal cells likely accounts for the predisposition to neoplasia in MAP. Despite ubiquitously elevated mutation rates, individuals with MAP do not display overt evidence of premature ageing. Thus, accumulation of somatic mutations may not be sufficient to cause the global organismal functional decline of ageing.Summary

Gram-negative bacteria producing carbapenemases are resistant to a variety of β-lactam antibiotics and pose a significant health risk. Given the dearth of new antibiotics, combinations of new broad-spectrum β-lactamase inhibitors (BLIs) with approved β-lactams have provided treatment options for resistant bacterial infections. Taniborbactam (formerly VNRX-5133) is an investigational BLI that is effective against both serine- and metallo-β-lactamases, including the serine carbapenemase KPC. In the current study, we assessed the effectiveness of taniborbactam to restore antibacterial activity of cefepime against KPC-3-producing Escherichia coli by inhibiting the KPC-3-dependent hydrolysis of cefepime. Time-lapse microscopy revealed that cells treated with greater than 1× MIC of cefepime (128 μg/ml) and cefepime-taniborbactam (4 μg/ml cefepime and 4 μg/ml taniborbactam) exhibited significant elongation, whereas cells treated with taniborbactam alone did not owing to a lack of standalone antibacterial activity of the BLI. The elongated cells also had frequent cellular voids thought to be formed by attempted cell divisions and pinching of the cytoplasmic membrane. Additionally, the effect of taniborbactam continued even after its removal from the growth medium. Pretreatment with 4 μg/ml taniborbactam helped to restore the antibacterial action of cefepime by neutralizing the effect of the KPC-3 β-lactamase. IMPORTANCE β-lactam (BL) antibiotics are the most prescribed antimicrobial class. The efficacy of β-lactams is threatened by the production of β-lactamase enzymes, the predominant resistance mechanism impacting these agents in Gram-negative bacterial pathogens. This study visualizes the effects of a combination treatment of taniborbactam, a broad spectrum β-lactamase inhibitor (BLI), and the BL antibiotic cefepime on a carbapenemase-producing E. coli strain. While this treatment has been described in the context of other cephalosporin-resistant bacteria, this is the first description of a microscopic evaluation of a KPC-3-producing strain of E. coli challenged by this BL-BLI combination. Live-cell microscopy analysis of cells treated with taniborbactam and cefepime demonstrated the antimicrobial effects on cellular morphology and highlighted the long-lasting inhibition of β-lactamases by taniborbactam even after it was removed from the medium. This research speaks to the importance of taniborbactam in fighting BL-mediated antibiotic resistance.

Background Bloodstream infections with antibiotic-resistant Pseudomonas aeruginosa are common and increasingly difficult to treat. Pyocins are naturally occurring protein antibiotics produced by P. aeruginosa that have potential for human use. Objectives To determine if pyocin treatment is effective in a murine model of sepsis with P. aeruginosa. Methods Recombinant pyocins S5 and AP41 were purified and tested for efficacy in a Galleria mellonella infection model and a murine model of P. aeruginosa sepsis. Results Both pyocins produced no adverse effects when injected alone into mice and showed good in vitro antipseudomonal activity. In an invertebrate model of sepsis using G. mellonella, both pyocins significantly prolonged survival from 1/10 (10%) survival in controls to 80%–100% survival among groups of 10 pyocin-treated larvae. Following injection into mice, both showed extensive distribution into different organs. When administered 5 h after infection, pyocin S5 significantly increased survival from 33% (2/6) to 83% (5/6) in a murine model of sepsis (difference significant by log-rank test, P Conclusions Pyocins S5 and AP41 show in vivo biological activity and can improve survival in two models of P. aeruginosa infection. They hold promise as novel antimicrobial agents for treatment of MDR infections with this microbe.

Objective To assess intertester reliability of isometric knee flexor strength testing in high-level rugby players with testers of different physical capacity and different methods of dynamometer fixation. Design Reliability study. Patients Thirty noninjured high-level (Tegner Activity Score ≥9) rugby players, free from hamstring injury in the previous 2 months. Assessment Isometric knee flexor strength (in N) in prone 0/15 degrees (hip/knee flexion) and supine 90/90 degrees position. Tests were performed by 1 female and 2 male testers whose upper-body strength was measured with a 6-repetition maximum bench press test. The prone 0/15 degrees measurement was performed with manual and external belt fixation of the dynamometer. Main outcome measures Absolute and relative intertester reliability were calculated using intraclass correlation coefficient (ICC) and minimal detectable change. Paired t-tests were used to identify systematic measurement error between testers and to test for a difference in recorded knee flexor strength between methods of dynamometer fixation. Methods Isometric knee flexor strength was measured in prone 0/15 degrees (hip/knee flexion) and supine 90/90 degrees position. Results Good intertester reliability was found for all pairwise comparisons (ICC 0.80-0.87). MDCs (as percentage of mean strength) ranged from 15.2% to 25.4%. For tester couples where systematic error was identified, Bland-Altman plots and Pearson correlation coefficients demonstrated no statistically significant correlation between mean knee flexor strength and between-tester difference. There was no significant difference in isometric knee flexor strength between manual and belt fixation of the dynamometer. Conclusions In strong high-level rugby players, hand-held dynamometry for isometric knee flexor strength assessment in prone 0/15 degrees and supine 90/90 degrees position is intertester reliable.

International audience; Objectives and method.-We conducted a prospective study in 2013 to compare the whole-cell versus acellular pertussis vaccines effectiveness and duration of protection, following the occurrence of pertussis clusters. Results.-During seven school outbreaks, we identified 102 clinical pertussis cases, including 10 cases biologically confirmed by Bordetella pertussis specific PCR, among a cohort of 305 children in 2nd to 6th grade. The risk of pertussis when vaccinated with an acellular vaccine alone was 1.6 (RR = 1.6; 95% CI = 1.1-2.5) times higher than when vaccinated with a whole-cell vaccine or using a combined schedule. Conclusions.-The limited duration of protection conferred by the acellular vaccine reinforces the 2013 introduction of the pertussis booster at six years old.

Tobacco smoking causes lung cancer1–3, a process that is driven by more than 60 carcinogens in cigarette smoke that directly damage and mutate DNA4,5. The profound effects of tobacco on the genome of lung cancer cells are well-documented6–10, but equivalent data for normal bronchial cells are lacking. Here we sequenced whole genomes of 632 colonies derived from single bronchial epithelial cells across 16 subjects. Tobacco smoking was the major influence on mutational burden, typically adding from 1,000 to 10,000 mutations per cell; massively increasing the variance both within and between subjects; and generating several distinct mutational signatures of substitutions and of insertions and deletions. A population of cells in individuals with a history of smoking had mutational burdens that were equivalent to those expected for people who had never smoked: these cells had less damage from tobacco-specific mutational processes, were fourfold more frequent in ex-smokers than current smokers and had considerably longer telomeres than their more-mutated counterparts. Driver mutations increased in frequency with age, affecting 4–14% of cells in middle-aged subjects who had never smoked. In current smokers, at least 25% of cells carried driver mutations and 0–6% of cells had two or even three drivers. Thus, tobacco smoking increases mutational burden, cell-to-cell heterogeneity and driver mutations, but quitting promotes replenishment of the bronchial epithelium from mitotically quiescent cells that have avoided tobacco mutagenesis. Whole-genome sequencing of normal bronchial epithelium from 16 individuals shows that tobacco smoking increases genomic heterogeneity, mutational burden and driver mutations, whereas stopping smoking promotes replenishment of the epithelium with near-normal cells.

Cancer develops through a process of somatic evolution1,2. Sequencing data from a single biopsy represent a snapshot of this process that can reveal the timing of specific genomic aberrations and the changing influence of mutational processes3. Here, by whole-genome sequencing analysis of 2,658 cancers as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA)4, we reconstruct the life history and evolution of mutational processes and driver mutation sequences of 38 types of cancer. Early oncogenesis is characterized by mutations in a constrained set of driver genes, and specific copy number gains, such as trisomy 7 in glioblastoma and isochromosome 17q in medulloblastoma. The mutational spectrum changes significantly throughout tumour evolution in 40% of samples. A nearly fourfold diversification of driver genes and increased genomic instability are features of later stages. Copy number alterations often occur in mitotic crises, and lead to simultaneous gains of chromosomal segments. Timing analyses suggest that driver mutations often precede diagnosis by many years, if not decades. Together, these results determine the evolutionary trajectories of cancer, and highlight opportunities for early cancer detection., Whole-genome sequencing data for 2,778 cancer samples from 2,658 unique donors across 38 cancer types is used to reconstruct the evolutionary history of cancer, revealing that driver mutations can precede diagnosis by several years to decades.

This study aimed to extend the shelf-life of coffee-containing milk beverage by adding Theobroma cacao (cacao nibs) extract. To prepare the beverage sample containing cacao nibs extract, 0.8% cacao nibs hydrothermal extract was aseptically injected. Qualitative changes in the beverage samples, including antioxidant effect, peroxide value (POV), caffeine content, and sensory parameters were monitored regularly during storage at 10°C, 20°C, and 30°C for 4 wk. The inclusion of cacao nibs extract produced higher antioxidant activity compared to the control. As the storage temperature increased, the POV of all samples increased. Samples with cacao nibs extract generally displayed lower POV than the control. The caffeine content of all samples tended to decrease during storage, with the decrease accentuated by higher storage temperatures. In the shelf-life prediction using the Arrhenius model, the kinetic regressions of the cacao nibs extract-added sample and control were YPOV=1.2212X–2.1141 (r2=0.9713) and YPOV=1.8075X–2.0189 (r2=0.9883), respectively. Finally, the predicted shelf-life of cacao nibs-added group and control to reach the quality limit (20 meq/kg POV) were approximately 18.11 and 12.18 wk, respectively. The results collectively indicate that the addition of cacao nibs extract extends the shelf-life of the coffee-containing milk beverage and heightened the antioxidant effect.

Background The southern African yellow dog tick, Haemaphysalis elliptica, occurs in eastern and southern Africa and adults infest domestic and wild carnivores. This tick species is also a vector of the highly virulent Babesia rossi pathogen, the causative agent of canine babesiosis in sub-Saharan Africa. Sustained high levels of efficacy of a parasiticide are not only important in protecting dogs against the direct effects of tick infestation, but also in reducing the risk of tick-borne diseases. Sarolaner (Simparica™ chewable tablets) has been reported to be effective against the major tick species infesting dogs in Europe and the USA, including representatives from the genera Amblyomma, Ixodes, Rhipicephalus and Dermacentor. Until now no efficacy evaluations have been reported against species of the genus Haemaphysalis. The objective of the study was to confirm the efficacy of a single 2 mg sarolaner/kg oral dose of Simparica™ against induced infestations with H. (R.) elliptica, an important parasite of dogs in southern Africa. Methods This blinded, randomised, single centre, placebo controlled efficacy study followed a parallel group design and was conducted on two groups consisting of eight purpose-bred dogs each. Animals were treated orally, once on Day 0, with either a placebo compound (Group 1) or Simparica™ (Group 2). Simparica™ was administered orally at a dose rate of 2 mg sarolaner/kg body weight. The dogs were infested with ticks on Days − 7, − 2, 5, 12, 19, 26 and 33, with removal counts conducted on Days − 5, 2, 7, 14, 21, 28 and 35. Results A single oral administration of Simparica™ (sarolaner) at a minimum dose of 2 mg/kg resulted in a 100% efficacy against existing infestations of H. (R.) elliptica on dogs and a 100% reduction in live ticks following weekly re-infestations for 35 days. Moreover, the immediate and persistent high levels of efficacy observed in this study for 35 days is consistent with those observed in previous studies against ticks in other genera. Conclusions The efficacy of sarolaner (Simparica™), administered orally to dogs at the minimum label dose of 2.0 mg/kg, was demonstrated against existing and weekly re-infestations of H. (R.) elliptica for at least 5 weeks. Efficacy of 100% was achieved against existing infestations as well as weekly re-infestations.

Low heart rate variability (HRV) is related to health problems that are known reasons for sick-leave or early retirement. A 1-minute-protocol could allow large scale HRV measurement for screening of health problems and, potentially, sustained employability. Our objectives were to explore the association of HRV with measures of health. Cross-sectional design with 877 Dutch employees assessed during a Workers’ Health Assessment. Personal and job characteristics, workability, psychological and mental problems, and lifestyle were measured with questionnaires. Biometry was measured (BMI, waist circumference, blood pressure, glucose, cholesterol). HRV was assessed with a 1-minute paced deep-breathing protocol and expressed as mean heart rate range (MHRR). A low MHRR indicates a higher health risk. Groups were classified age adjusted for HRV and compared. Spearman correlations between raw MHRR and the other measures were calculated. Significant univariable correlations (p < 0.05) were entered in a linear regression model to explore the multivariable association with MHRR. Age, years of employment, BMI and waist circumference differed significantly between HRV groups. Significant correlations were found between MHRR and age, workability, BMI, waist circumference, cholesterol, systolic and diastolic blood-pressure and reported physical activity and alcohol consumption. In the multivariable analyses 21.1% of variance was explained: a low HRV correlates with aging, higher BMI and higher levels of reported physically activity. HRV was significantly associated with age, measures of obesity (BMI, waist circumference), and with reported physical activity, which provides a first glance of the utility of a 1-minute paced deep-breathing HRV protocol as part of a comprehensive preventive Workers’ Health Assessment.Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat ivecommons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate redit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

BACKGROUND: Pathogen inactivation and cold or cryopreservation of platelets (PLTs) both significantly affect PLT function. It is not known how PLTs function when both are combined. STUDY DESIGN AND METHODS: Standard PLT concentrates (PCs) were compared to pathogen-inactivated PCs treated with amotosalen photochemical treatment (AS-PCT) when stored at room (RT, 22 degrees C), cold (4 degrees C, n = 6), or cryopreservation (-80 degrees C, n = 8) temperatures. The impact of alternative storage methods on both arms was studied in flow cytometry, light transmittance aggregometry, and hemostasis in collagen-coated microfluidic flow chambers. RESULTS Platelet aggregation of cold-stored AS-PCT PLTs was 44% +/- 11% compared to 57% +/- 14% for cold-stored standard PLTs and 58% +/- 21% for RT-stored AS-PCT PLTs. Integrin activation of cold-stored AS-PCT PLTs was 53% +/- 9% compared to 77% +/- 6% for cold-stored standard PLTs and 69% +/- 13% for RT-stored AS-PCT PLTs. Coagulation of cold-stored AS-PCT PLTs started faster under flow (836 +/- 140 sec) compared to cold-stored standard PLTs (960 +/- 192 sec) and RT-stored AS-PCT PLTs (1134 +/- 220 sec). Fibrin formation rate under flow was also highest for cold-stored AS-PCT PLTs. This was in line with thrombin generation in static conditions because cold-stored AS-PCT PLTs generated 297 +/- 47 nmol/L thrombin compared to 159 +/- 33 nmol/L for cold-stored standard PLTs and 83 +/- 25 nmol/L for RT-stored AS-PCT PLTs. So despite decreased PLT activation and aggregation, cold storage of AS-PCT PLTs promoted coagulation. PLT aggregation of cryopreserved AS-PCT PLTs (23% +/- 10%) was not significantly different from cryopreserved standard PLTs (25% +/- 8%). CONCLUSION: This study shows that cold storage of AS-PCT PLTs further affects PLT activation and aggregation but promotes (pro)coagulation. Increased procoagulation was not observed after cryopreservation.

Venezuelan equine encephalitis virus (VEEV) is a known biological defense threat. A live-attenuated investigational vaccine, TC-83, is available, but it has a high non-response rate and can also cause severe reactogenicity. We generated two novel VEE vaccine candidates using self-amplifying mRNA (SAM). LAV-CNE is a live-attenuated VEE SAM vaccine formulated with synthetic cationic nanoemulsion (CNE) and carrying the RNA genome of TC-83. IAV-CNE is an irreversibly-attenuated VEE SAM vaccine formulated with CNE, delivering a TC-83 genome lacking the capsid gene. LAV-CNE launches a TC-83 infection cycle in vaccinated subjects but eliminates the need for live-attenuated vaccine production and potentially reduces manufacturing time and complexity. IAV-CNE produces a single cycle of RNA amplification and antigen expression without generating infectious viruses in subjects, thereby creating a potentially safer alternative to live-attenuated vaccine. Here, we demonstrated that mice vaccinated with LAV-CNE elicited immune responses similar to those of TC-83, providing 100% protection against aerosol VEEV challenge. IAV-CNE was also immunogenic, resulting in significant protection against VEEV challenge. These studies demonstrate the proof of concept for using the SAM platform to streamline the development of effective attenuated vaccines against VEEV and closely related alphavirus pathogens such as western and eastern equine encephalitis and Chikungunya viruses.

Background and objectives: Several sources of haematopoietic stem cells have been used for static culture of megakaryocytes to produce platelets in vitro. This study compares and characterizes platelets produced in shear flow using precursor cells from either umbilical (UCB) or adult peripheral blood (PB). Materials and methods: The efficiency of platelet production of the cultured cells was studied after perfusion in custom-built von Willebrand factor-coated microfluidic flow chambers. Platelet receptor expression and morphology were investigated by flow cytometry and microscopy, respectively. Results: Proliferation of stem cells isolated out of UCB was significantly higher (P < 0 center dot 0001) compared to PB. Differentiation of these cells towards megakaryocytes was significantly lower from PB compared to UCB where the fraction of CD42b/CD41 double positive events was 44 +/- 9% versus 76 +/- 11%, respectively (P < 0 center dot 0001). However, in vitro platelet production under hydrodynamic conditions was more efficient with 7 center dot 4 platelet-like particles per input cell from PB compared to 4 center dot 2 from UCB (P = 0 center dot 02). The percentage of events positive for CD42b, CD41 and CD61 was comparable between both stem cell sources. The mean number of receptors per platelet from UCB and PB was similar to that on blood bank platelets with on average 28 000 CD42b, 57 000 CD61 and 5500 CD49b receptors. Microscopy revealed platelets appearing similar to blood bank platelets in morphology, size and actin cytoskeleton, alongside smaller fragments and source megakaryocytes. Conclusion: This characterization study suggests that platelets produced in vitro under flow either from UCB or from PB share receptor expression and morphology with donor platelets stored in the blood bank.

Pioneering gene therapy trials have shown that the genetic engineering of haematopoietic stem and progenitor cells can be an alternative to allogeneic transplantation in the treatment of primary immunodeficiencies. Early trials also highlighted the risk of insertional mutagenesis and oncogene transactivation associated with the first generation of gammaretroviral vectors. These events prompted the development of safer, self-inactivating lentiviral or gammaretroviral vectors. These lentiviral vectors have been successfully used to treat over 200 patients with 10 different haematological disorders (including primary immunodeficiencies, haemoglobinopathies and metabolic disorders) and for the generation of chimeric antigen receptor-T cells for cancer therapy. However, several challenges, such as effective reconstitution during inflammation, remain if gene therapy is to be extended to more complex diseases in which haematopoietic stem and progenitor cells can be altered by the disease environment. We discuss the progress made and future challenges for gene therapy and contrast gene therapy with gene-editing strategies. Advances in the design of vectors based on retroviruses, such as lentiviruses and gammaretroviruses, have led to improvements in the safety and stability of gene therapies directed at haematopoietic stem and progenitor cells. In this Review, Cavazzana and colleagues discuss the results from recent clinical trials of retroviral vectors for the treatment of genetic disorders, including severe combined immunodeficiencies and β-haemoglobinopathies (β-thalassaemia and sickle cell disease). They highlight the progress made and the remaining challenges in applying gene therapies more broadly.

Germline RUNX1 mutations underlie a syndrome, RUNX1-familial platelet disorder (RUNX1-FPD), characterized by bleeding symptoms that result from quantitative and/or qualitative defect in platelets and a significantly increased risk for developing hematologic malignancies. Myeloid neoplasms are the most commonly diagnosed hematologic malignancies, followed by lymphoid malignancies of T-cell origin. Here, we describe the first 2 cases of B-cell acute lymphoblastic leukemia (B-ALL) in patients with confirmed germline RUNX1 mutations. While 1 of the patients had a known diagnosis of RUNX1-FPD with a RUNX1 p.P240Hfs mutation, the other was the index patient of a kindred with a novel RUNX1 variant, RUNX1 c.587C>T (p.T196I), noted on a targeted genetic testing of the B-ALL diagnostic sample. We discuss the clinical course, treatment approaches, and the outcome for the 2 patients. Additionally, we describe transient resolution of the mild thrombocytopenia and bleeding symptoms during therapy, as well as the finding of clonal hematopoiesis with a TET2 mutant clone in 1 of the patients. It is critical to consider testing for germline RUNX1 mutations in patients presenting with B-ALL who have a personal or family history of thrombocytopenia, bleeding symptoms, or RUNX1 variants identified on genetic testing at diagnosis.

International audience; Herein, we report a multistep synthesis of novel (meth)acrylate monomers based on gallic acid (GA), a biosourced phenolic acid. The objective of this work was to obtain bio-based polymers exhibiting antioxidant properties provided by monomers derived from gallic acid. The phenolic groups of GA, which are responsible for antioxidant properties, need to be protected for two reasons. On the one hand, functionalization to transform GA into polymerizable monomers must not take place at the phenolic groups because they must remain free to maintain the maximum antioxidant activity in the final polymers. On the other hand, their protection is necessary to prevent radical scavenging during the radical polymerization. After synthesis of such monomers, protected GA-based polymers were thus produced through a photo-mediated RAFT polymerization at room temperature by evaluating two trithiocarbonate-type chain transfer agents (CTAs). The kinetics and molecular weight distributions were studied depending on the monomers and the CTAs. Protected polymers were then deprotected to afford polymeric chains carrying one free gallic acid moiety on each monomer unit. The antioxidant activity of these free GA-based polymers was demonstrated either through the DPPH free radical scavenging property or through the inhibition of methyl linoleate oxidation.

International audience; Recent years have witnessed an unprecedented growth in the research area of nanomedicine. There is an increasing optimism that nanotechnology applied to medicine will bring significant advances in the diagnosis and treatment of various diseases, including colorectal cancer (CRC), a type of neoplasm affecting cells in the colon or the rectum. Recent findings suggest that the role of microbiota is crucial in the development of CRC and its progression. Dysbiosis is a condition that disturbs the normal microbial environment in the gut and is often observed in CRC patients. In order to detect and treat precancerous lesions, new tools such as nanotechnology-based theranostics, provide a promising option for targeted marker detection or therapy for CRC. Because the presence of gut microbiota influences the route of biomarker detection and the route of the interaction of nanoparticle/drug complexes with target cells, the development of nanoparticles with appropriate sizes, morphologies, chemical compositions and concentrations might overcome this fundamental barrier. Metallic particles are good candidates for nanoparticle-induced intestinal dysbiosis, but this aspect has been poorly explored to date. Herein, we focus on reviewing and discussing nanotechnologies with potential applications in CRC through the involvement of gut microbiota and highlight the clinical areas that would benefit from these new medical technologies.

ObjectivesCOVIDTrach is a UK multicentre prospective cohort study project that aims to evaluate the outcomes of tracheostomy in patients with COVID-19 receiving mechanical ventilation and record the incidence of SARS-CoV-2 infection among healthcare workers involved in the procedure.DesignData on patient demographic, clinical history and outcomes were entered prospectively and updated over time via an online database (REDCap). Clinical variables were compared with outcomes, with logistic regression used to develop a model for mortality. Participants recorded whether any operators tested positive for SARS-CoV-2 within 2 weeks of the procedure.SettingUK National Health Service departments involved in treating patients with COVID-19 receiving mechanical ventilation.ParticipantsThe cohort comprised 1605 tracheostomy cases from 126 UK hospitals collected between 6 April and 26 August 2020.Main outcome measuresMortality following tracheostomy, successful wean from mechanical ventilation and length of time from tracheostomy to wean, discharge from hospital, complications from tracheostomy, reported SARS-CoV-2 infection among operators.ResultsThe median time from intubation to tracheostomy was 15 days (IQR 11, 21). 285 (18%) patients died following the procedure. 1229 (93%) of the survivors had been successfully weaned from mechanical ventilation at censoring and 1049 (81%) had been discharged from hospital. Age, inspired oxygen concentration, positive end-expiratory pressure setting, fever, number of days of ventilation before tracheostomy, C reactive protein and the use of anticoagulation and inotropic support independently predicted mortality. Six reports were received of operators testing positive for SARS-CoV-2 within 2 weeks of the procedure.ConclusionsTracheostomy appears to be safe in mechanically ventilated patients with COVID-19 and to operators performing the procedure and we identified clinical parameters that are predictive of mortality.Trial registration numberThe study is registered with ClinicalTrials.Gov (NCT04572438).

Long-term, continuous in situ observations of the near-surface atmospheric boundary layer are critical for many weather and climate applications. Although there is a proliferation of surface stations globally, especially in and around populous areas, there are notably fewer tall meteorological towers with multiple instrumented levels. This is particularly true in remote and extreme environments such as the Eastern Antarctic plateau. In the article, we present and analyze 10 years of data from 6 levels of meteorological instrumentation mounted on a 42-m tower located at Dome C, East Antarctica near the Concordia research station, producing a unique climatology of the near-surface atmospheric environment (Genthon et al., 2021,a,b). Monthly temperature and wind data demonstrate the large seasonal differences in the near-surface boundary layer dynamics, depending on the presence or absence of solar surface forcing. Strong vertical temperature gradients (inversions) frequently develop in calm, winter conditions, while vertical convective mixing occurs in the summer leading to near-uniform temperatures along the tower. Seasonal variation in wind speed is much less notable at this location than the temperature variation as the winds are less influenced by the solar cycle; there are no katabatic winds as Dome C is quite flat. Harmonic analysis confirms that most of the energy in the power spectrum is at diurnal, annual and semi-annual time scales. Analysis of observational uncertainty and comparison to reanalysis data from ERA-5 indicate that wind speed is particularly difficult to measure at this location. Data are distributed on PANGAEA data repository, see data availability section.

Free‐living red coralline algae play an important role in the carbon and carbonate cycles of coastal environments. In this study, we examined the physiology of free‐living coralline algae forming maerl beds in the Bay of Brest (Brittany, France), where Lithothamnion corallioides is the dominant maerl (i.e., rhodolith) species. Phymatolithon calcareum and Lithophyllum incrustans are also present (in lower abundances) at a specific site in the bay. We aimed to assess how maerl physiology is affected by seasonality and/or local environmental variations at the inter‐ and intraspecific levels. Physiological measurements (respiration, photosynthetic and calcification rates) were performed using incubation chambers in winter and summer to compare (1) the dominant maerl species at three sites and (2) three co‐existing maerl species at one site. Comparison of the three co‐existing maerl species suggests that L. corallioides is the best adapted to the current environmental conditions in the Bay of Brest, because this species is the most robust to dissolution in the dark in winter and has the highest calcification efficiency in the light. Comparisons of L. corallioides metabolic rates between stations showed that morphological variations within this species are the main factor affecting its photosynthetic and calcification rates. Environmental factors such as freshwater inputs also affect its calcification rates in the dark. In addition to interspecies variation in maerl physiology, there were intraspecific variations associated with direct (water physico‐chemistry) or indirect (morphology) local environmental conditions. This study demonstrates the plasticity of maerl physiology in response to environmental changes, which is fundamental for maerl persistence.