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Rabies virus (RABV) transcription and replication take place within viral factories having liquid properties, called Negri bodies (NBs), that are formed by liquid-liquid phase separation (LLPS). The co-expression of RABV nucleoprotein (N) and phosphoprotein (P) in mammalian cells is sufficient to induce the formation of cytoplasmic biocondensates having properties that are like those of NBs. This cellular minimal system was previously used to identify P domains that are essential for biocondensates formation. Here, we constructed fluorescent versions of N and analyzed by FRAP their dynamics inside the biocondensates formed in this minimal system as well as in NBs of RABV-infected cells using FRAP. The behavior of N appears to be different of P as there was no fluorescence recovery of N proteins after photobleaching. We also identified arginine residues as well as two exposed loops of N involved in condensates formation. Corresponding N mutants exhibited distinct phenotypes in infected cells ranging from co-localization with NBs to exclusion from them associated with a dominant-negative effect on infection. We also demonstrated that in vitro , in crowded environments, purified P as well as purified N0-P complex (in which N is RNA-free) form liquid condensates. We identified P domains required for LLPS in this acellular system. P condensates were shown to associate with liposomes, concentrate RNA, and undergo a liquid-gel transition upon ageing. Conversely, N0-P droplets were disrupted upon incubation with RNA. Taken together, our data emphasize the central role of P in NBs formation and reveal some physicochemical features of P and N0-P droplets relevant for explaining NBs properties such as their envelopment by cellular membranes at late stages of infection and nucleocapsids ejections from the viral factories.

PurposeDespite the importance of follow-up and multidisciplinary care after bariatric surgery, many patients do not attend postoperative appointments, particularly those with the medical team. The present study aimed to identify factors associated with loss to follow-up after bariatric surgery.Materials and MethodsWe recruited patients who underwent bariatric surgery between 01/01/2012 and 31/12/2013. Data were collected on demographic and socioeconomic information and comorbidities. Ten baseline psychological evaluations were blindly reviewed to evaluate the relationship between emotions and compliance with follow-up. During the 3-year postoperative period, we defined frequent attendees as those who attended at least two visits, whereas non-attendees were those who attended one visit or none. We evaluated baseline variables associated with non-adherence with follow-up schedules.ResultsAmong 92 patients, 41 patients (44.6%) attended at least two postoperative appointments, while 51 (55.4%) were classified as non-attendees. Among the non-attendees, significantly more were younger than 45 years compared with attendees. No other statistically significant differences were found in terms of socioeconomic variables. Multivariate logistic regression revealed male gender and psychological issued related to obesity to be independent predictors of poor compliance with follow-up. Blinded psychological evaluation of ten patients did not suggest that psychological factors are predictive of follow-up attendance.ConclusionIdentifying factors associated with loss to follow-up after bariatric surgery is challenging. However, this is important in order to enable the design of personalized follow-up plans, especially for younger patients and those with psychological issues.

BackgroundMost studies on bariatric patients to date have only examined mortality and morbidities in terms of surgery or no surgery. Few have investigated loss to follow-up in post-surgery patients.PurposeThis study aimed to describe the dynamics behind non-adherence to follow-up in bariatric patients postsurgery.DesignUsing semi-structured interviews, we performed a qualitative study. Using a thematic analysis, we described themes involved in patient adherence to postsurgery follow-up.SettingParticipants were recruited from a university hospital near Paris and via social networks.Participants17 patients who had undergone surgery, some of whom were lost to follow-up, 15 women and 2 men, were interviewed, during a mean time of 90 min. 10 were adherent, and 7 were lost to follow-up.ResultsFollow-up was seen as a support in which the care provider–patient relationship can act on the four following themes: (1) regaining control, (2) knowledge acquisition, (3) management of fears and (4) overall restructuring of one’s life postsurgery.ConclusionsPatients’ experiences and representations of postsurgery follow-up should be documented in detail in order to define the specific roles of the various care providers offering support to this population, and to strengthen the coordination of care pathways between these actors. In addition, improving the quality of communication could improve adherence to follow-up after bariatric surgery.

miRNAs, small non-coding RNAs that regulate gene expression, are involved in various pathological processes, including viral infections. Virus infections may interfere with the miRNA pathway through the inhibition of genes involved in miRNA biogenesis. A reduction in the number and the levels of miRNAs expressed in nasopharyngeal swabs of patients with severe COVID-19 was lately observed by us, pointing towards the potential of miRNAs as possible diagnostic or prognostic biomarkers for predicting outcomes among patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The objective of the present study was to investigate whether SARS-CoV-2 infection influences the expression levels of messenger RNAs (mRNAs) of key genes involved in miRNA biogenesis. mRNA levels of AGO2, DICER1, DGCR8, DROSHA, and Exportin-5 (XPO5) were measured by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) in nasopharyngeal swab specimens from patients with COVID-19 and controls, as well as in cells infected with SARS-CoV-2 in vitro. Our data showed that the mRNA expression levels of AGO2, DICER1, DGCR8, DROSHA, and XPO5 were not significantly different in patients with severe COVID-19 when compared to patients with non-severe COVID-19 and controls. Similarly, the mRNA expression of these genes was not affected by SARS-CoV-2 infection in NHBE and Calu-3 cells. However, in Vero E6 cells, AGO2, DICER1, DGCR8, and XPO5 mRNA levels were slightly upregulated 24 h after infection with SARS-CoV-2. In conclusion, we did not find evidence for downregulation of mRNA levels of miRNA biogenesis genes during SARS-CoV-2 infection, neither ex vivo nor in vitro.

Benjamini-Hochberg false discovery rate correction Of the miRNAs found of interest to discriminate between severe and non-severe COVID-19 in our study, some were reported to be implicated in viral and other infections: miR-455-5p was up-regulated in rabies virus infection in vitro, decreased suppressor of cytokine signaling 3 (SOCS3) expression and increased signal transducer and activator of transcription 3 (STAT3) activity, resulting in enhanced viral replication and the production of IL-6.6 Furthermore, this miRNA targeted the C-C motif chemokine receptor 5 (CCR5).7 Of interest, CCR5 is involved in severe COVID-19 and has been proposed as anti-inflammatory treatment target.8 Taken together, this underlines the role of hsa-miR-455-5p in viral infections and the inflammatory response and its potential as target of therapeutic interventions. hsa-miR-532-3p diminished the levels of ASK1 and downstream phosphorylation/translocation of p38 MAPK during lipopolysaccharide (LPS)/ tumor necrosis factor-α (TNF-α)-induced inflammation in macrophages and reduced the release of various pro-inflammatory cytokines and chemokines, including IL-6 and TNF-alpha.9 IL-6 is a proinflammatory cytokine that has been reported to be involved in the cytokine storm observed in severe COVID-19.10 The down-regulation of anti-inflammatory miRNAs, such as hsa-miR-532-3p, hsa-miR-340-5p and hsa-miR-455-5p, in severe COVID-19 (Table 1) is in line with a hyperinflammatory state in severe COVID-19. Recently, direct and indirect miRNA interactions with other miRNAs have been described (reviewed11). [...]it cannot be excluded that the miRNAs identified in this study directly or indirectly influence expression of other miRNAs and thus have a broad impact on miRNA and gene expression. [...]our analysis of miRNA expression in nasopharyngeal swabs revealed a general reduction of miRNA expression in severe COVID-19 patients.

(1) Background: The aim of the present study was to compare oxygen gradient ektacytometry parameters between sickle cell patients of different genotypes (SS, SC, and S/β+) or under different treatments (hydroxyurea or chronic red blood cell exchange). (2) Methods: Oxygen gradient ektacytometry was performed in 167 adults and children at steady state. In addition, five SS patients had oxygenscan measurements at steady state and during an acute complication requiring hospitalization. (3) Results: Red blood cell (RBC) deformability upon deoxygenation (EImin) and in normoxia (EImax) was increased, and the susceptibility of RBC to sickle upon deoxygenation was decreased in SC patients when compared to untreated SS patients older than 5 years old. SS patients under chronic red blood cell exchange had higher EImin and EImax and lower susceptibility of RBC to sickle upon deoxygenation compared to untreated SS patients, SS patients younger than 5 years old, and hydroxyurea-treated SS and SC patients. The susceptibility of RBC to sickle upon deoxygenation was increased in the five SS patients during acute complication compared to steady state, although the difference between steady state and acute complication was variable from one patient to another. (4) Conclusions: The present study demonstrates that oxygen gradient ektacytometry parameters are affected by sickle cell disease (SCD) genotype and treatment.

Rabies virus (RABV) transcription and replication take place within viral factories having liquid properties, called Negri bodies (NBs), that are formed by liquid-liquid phase separation (LLPS). The co-expression of RABV nucleoprotein (N) and phosphoprotein (P) in mammalian cells is sufficient to induce the formation of cytoplasmic biocondensates having properties that are like those of NBs. This cellular minimal system was previously used to identify P domains that are essential for biocondensates formation. Here, we constructed fluorescent versions of N and analyzed by FRAP their dynamics inside the biocondensates formed in this minimal system as well as in NBs of RABV-infected cells using FRAP. The behavior of N appears to be different of P as there was no fluorescence recovery of N proteins after photobleaching. We also identified arginine residues as well as two exposed loops of N involved in condensates formation. Corresponding N mutants exhibited distinct phenotypes in infected cells ranging from co-localization with NBs to exclusion from them associated with a dominant-negative effect on infection. We also demonstrated that in vitro , in crowded environments, purified P as well as purified N0-P complex (in which N is RNA-free) form liquid condensates. We identified P domains required for LLPS in this acellular system. P condensates were shown to associate with liposomes, concentrate RNA, and undergo a liquid-gel transition upon ageing. Conversely, N0-P droplets were disrupted upon incubation with RNA. Taken together, our data emphasize the central role of P in NBs formation and reveal some physicochemical features of P and N0-P droplets relevant for explaining NBs properties such as their envelopment by cellular membranes at late stages of infection and nucleocapsids ejections from the viral factories.

In the latest 2016 World Health Organization classification of hematological malignancies, T-cell lymphoblastic lymphoma (T-LBL) and lymphoblastic leukemia (T-ALL) are grouped together into one entity called T-cell lymphoblastic leukemia/lymphoma (T-LBLL). However, the question of whether these entities represent one or two diseases remains. Multiple studies on driver alterations in T-ALL have led to a better understanding of the disease while, so far, little data on genetic profiles in T-LBL is available. We sought to define recurrent genetic alterations in T-LBL and provide a comprehensive comparison with T-ALL. Targeted whole-exome next-generation sequencing of 105 genes, multiplex ligation-dependent probe amplification, and quantitative PCR allowed comprehensive genotype assessment in 818, consecutive, unselected, newly diagnosed patients (342 T-LBL vs. 476 T-ALL). The median age at diagnosis was similar in T-LBL and T-ALL (17 vs. 15 years old, respectively; p = 0.2). Although we found commonly altered signaling pathways and co-occurring mutations, we identified recurrent dissimilarities in actionable gene alterations in T-LBL as compared to T-ALL. HOX abnormalities (TLX1 and TLX3 overexpression) were more frequent in T-ALL (5% of T-LBL vs 13% of T-ALL had TLX1 overexpression; p = 0.04 and 6% of T-LBL vs 17% of T-ALL had TLX3 overexpression; p = 0.006). The PI3K signaling pathway was significantly more frequently altered in T-LBL as compared to T-ALL (33% vs 19%; p < 0.001), especially through PIK3CA alterations (9% vs 2%; p < 0.001) with PIK3CAH1047 as the most common hotspot. Similarly, T-LBL genotypes were significantly enriched in alterations in genes coding for the EZH2 epigenetic regulator and in TP53 mutations (respectively, 13% vs 8%; p = 0.016 and 7% vs 2%; p < 0.001). This genetic landscape of T-LBLL identifies differential involvement of recurrent alterations in T-LBL as compared to T-ALL, thus contributing to better understanding and management of this rare disease.

Despite poor survival, controversies remain in the treatment for refractory or relapsed pediatric non-Hodgkin lymphoma (r/r NHL). The current project aimed to collect international experience on the re-induction treatment of r/r NHL, hematopoietic stem cell transplantation (HSCT), risk factors associated with outcome, and to suggest treatment recommendations. Inclusion criteria were (i) refractory disease, disease progression or relapse of any NHL subtype except anaplastic large cell lymphoma, (ii) age < 18 years at initial diagnosis, (iii) diagnosis in/after January 2000. Data from 639 eligible patients were evaluable. The eight-year probability of overall survival was 34 ± 2% with highly significant differences according to NHL subtypes: 28 ± 3% for 254 Burkitt lymphoma/leukemia, 50 ± 6% for 98 diffuse large B-cell lymphomas, 57 ± 8% for 41 primary mediastinal large B-cell lymphomas, 27 ± 3% for 177 T-lymphoblastic lymphomas, 52 ± 10% for 34 precursor-B-cell lymphoblastic lymphomas and 30 ± 9% for 35 patients with rare NHL subtypes. Subtype-specific factors associated with survival and treatment recommendations are suggested. There were no survivors without HSCT, except in few very small subgroups. Conclusions: There is an urgent need to further improve survival in r/r NHL. The current study provides the largest real-world series, which underlines the role of HSCT and suggests treatment recommendations.

Documenting bacteremia at the onset of fever in immunosuppressed children is challenging; therefore, it leads to the early administration of broad-spectrum antibiotics. We aimed to analyse the evolution of antibiotic resistance profiles of bacterial bloodstream infections (BSI) and gut colonisations in a large cohort of immunocompromised children carrying a central venous catheter, in comparison with a prior, similar study conducted in our centre from 2014 to 2017. A retrospective, observational cohort study was conducted from January 2018 to December 2021, in a tertiary centre for paediatric immuno-haematology and oncology. Empirical antibiotic therapy was adapted to the immunosuppression risk group and prior bacterial colonisation. There was a mean of 6.9 BSI/1000 patient bed days. Multidrug-resistant bacteria (MDRB) associated BSI accounted for 35/273 (12.8%). The incidence of MDRB gum/gut colonisation and MDRB associated BSI increased annually and correlated with the level of immunosuppression (p = 0.024). One third (34.7%) of the BSI episodes were not associated with neutropenia. As compared to the previous study, an alarming emergence of MDRB responsible for gut colonisations and BSI in immunosuppressed children was reported over the last four years. The degree of immunosuppression directly correlates with the risk of having an MDRB gut colonisation or MDRB BSI.