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Multicellular tumour spheroid (MCTS) cultures are excellent model systems for simulating the development and microenvironmental conditions of in vivo tumour growth. Many documented cell lines can generate differentiated MCTS when cultured in suspension or in a non-adhesive environment. While physiological and biochemical properties of MCTS have been extensively characterized, insight into the events and conditions responsible for initiation of these structures is lacking. MCTS are formed by only a small subpopulation of cells during surface-associated growth but the processes responsible for this differentiation are poorly understood and have not been previously studied experimentally. Analysis of gene expression within spheroids has provided clues but to date it is not known if the observed differences are a cause or consequence of MCTS growth. One mechanism linked to tumourigenesis in a number of cancers is genetic instability arising from impaired DNA mismatch repair (MMR). This study aimed to determine the role of MMR in MCTS initiation and development. Using surface-associated N2a and CHLA-02-ATRT culture systems we have investigated the impact of impaired MMR on MCTS growth. Analysis of the DNA MMR genes MLH1 and PMS2 revealed both to be significantly down-regulated at the mRNA level compared with non-spheroid-forming cells. By using small interfering RNA (siRNA) against these genes we show that silencing of MLH1 and PMS2 enhances both MCTS initiation and subsequent expansion. This effect was prolonged over several passages following siRNA transfection. Down-regulation of DNA MMR can contribute to tumour initiation and progression in N2a and CHLA-02-ATRT MCTS models. Studies of surface-associated MCTS differentiation may have broader applications in studying events in the initiation of cancer foci.

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) has had a tremendous impact on humanity. Prevention of transmission by disinfection of surfaces and aerosols through a chemical-free method is highly desirable. Ultraviolet C (UVC) light is uniquely positioned to achieve inactivation of pathogens. We report the inactivation of SARS-CoV-2 virus by UVC radiation and explore its mechanisms. A dose of 50mJ/cm2 using a UVC laser at 266nm achieved an inactivation efficiency of 99.89%, whilst infectious virions were undetectable at 75mJ/cm2 indicating >99.99% inactivation. Infection by SARS-CoV-2 involves viral entry mediated by the spike glycoprotein (S), and viral reproduction, reliant on translation of its genome. We demonstrate that UVC radiation damages ribonucleic acid (RNA) and provide in-depth characterisation of UVC-induced damage of the S protein. We find that UVC severely impacts the ability of the SARS-CoV- 2 spike protein to bind human angiotensin-converting enzyme 2 (hACE2) and this correlates with loss of native protein conformation and aromatic amino acid integrity. This report has important implications for the design and development of rapid and effective disinfection systems against the SARS-CoV-2 virus and other pathogens.Competing Interest StatementThe authors have declared no competing interest.

Research Summary In this study, we replicate and expand Hawn et al.’s study (Strategic Management Journal, 2018, 39, 949–976) that used Dow Jones Sustainability World Index (DJSI) events to measure variations in firms' Corporate Social Responsibility (CSR)‐activism and examined their effect on a firm's stock price. We use DJSI events to capture variations in firms' CSR visibility, holding CSR activism constant by restricting our analyses to CSR‐equivalent firms. First, we find similar results on stock price (i.e., no impact) and on trading volumes. Second, because professional market participants pay more attention to CSR‐oriented firms and use visible cues such as DJSI events, we study and find that additions to DJSI lead to more analysts following a firm, and that continuations on the DJSI lead to an increase in equity being held by long‐term investors. Managerial Summary We replicate and complement a recent study that showed that the events of a firm's addition, continuation, or deletion in a major sustainability index (DJSI) had little impact on stock market reactions (Hawn et al. Strategic Management Journal, 2018, 39, 949–976). We redefine the comparison set that was previously used and compare across observationally equivalent firms in terms of CSR orientation in order to isolate the visibility effect of DJSI events. We find that these events do not significantly impact stock price and trading volumes. However, we expand the analysis and find that sustainability events attract more attention from financial analysts and lead to an increase in the percentage of shares held by long‐term investors indicative of a trend that professional investors pay more attention to CSR‐visible firms over time.

APOBEC3 (A3) enzymes are best known for their role as antiviral restriction factors and as mutagens in cancer. Although four of them, A3A, A3B, A3F and A3G, are induced by type-1-interferon (IFN-I), their role in inflammatory conditions is unknown. We thus investigated the expression of A3 , and particularly A3A and A3B because of their ability to edit cellular DNA, in Systemic Lupus Erythematosus (SLE), a chronic inflammatory disease characterized by high IFN-α serum levels. In a cohort of 57 SLE patients, A3A and A3B, but also A3C and A3G, were upregulated ~ 10 to 15-fold (> 1000-fold for A3B ) compared to healthy controls, particularly in patients with flares and elevated serum IFN-α levels. Hydroxychloroquine, corticosteroids and immunosuppressive treatment did not reverse A3 levels. The A3AΔ3B polymorphism, which potentiates A3A , was detected in 14.9% of patients and in 10% of controls, and was associated with higher A3A mRNA expression. A3A and A3B mRNA levels, but not A3C or A3G , were correlated positively with dsDNA breaks and negatively with lymphopenia. Exposure of SLE PBMCs to IFN-α in culture induced massive and sustained A3A levels by 4 h and led to massive cell death. Furthermore, the rs2853669 A > G polymorphism in the telomerase reverse transcriptase (TERT) promoter, which disrupts an Ets-TCF-binding site and influences certain cancers, was highly prevalent in SLE patients, possibly contributing to lymphopenia. Taken together, these findings suggest that high baseline A3A and A3B levels may contribute to cell frailty, lymphopenia and to the generation of neoantigens in SLE patients. Targeting A3 expression could be a strategy to reverse cell death and the generation of neoantigens.

Long-term follow up studies from Ebola virus disease (EVD) survivors (EBOV_S) are lacking. Here, we evaluate immune and gene expression profiles in 35 Guinean EBOV_S from the last West African outbreak, a median of 23 months (IQR [18–25]) after discharge from treatment center. Compared with healthy donors, EBOV_S exhibit increases of blood markers of inflammation, intestinal tissue damage, T cell and B cell activation and a depletion of circulating dendritic cells. All survivors have EBOV-specific IgG antibodies and robust and polyfunctional EBOV-specific memory T-cell responses. Deep sequencing of the genes expressed in blood reveals an enrichment in ‘inflammation’ and ‘antiviral’ pathways. Integrated analyses identify specific immune markers associated with the persistence of clinical symptoms. This study identifies a set of biological and genetic markers that could be used to define a signature of “chronic Ebola virus disease (CEVD)”. Patients who have recovered from Ebola virus can have ongoing health problems. Here, the authors show that 35 Guinean survivors of the last West African Ebola epidemic have a chronic disease with high inflammatory cytokine expression and other markers of immune activation as well as evidence of intestinal tissue damage nearly two years after their release from hospital.

Herpes simplex virus 1 (HSV-1) impairs the mitochondrial network through the viral protein UL12.5. This leads to the fusion of mitochondria and simultaneous release of mitochondrial DNA (mtDNA) in a mouse model. Viruses have evolved a plethora of mechanisms to impair host innate immune responses. Herpes simplex virus type 1 (HSV-1), a double-stranded linear DNA virus, impairs the mitochondrial network and dynamics predominantly through the UL12.5 gene. We demonstrated that HSV-1 infection induced a remodeling of mitochondrial shape, resulting in a fragmentation of the mitochondria associated with a decrease in their volume and an increase in their sphericity. This damage leads to the release of mitochondrial DNA (mtDNA) to the cytosol. By generating a stable THP-1 cell line expressing the DNase I-mCherry fusion protein and a THP-1 cell line specifically depleted of mtDNA upon ethidium bromide treatment, we showed that cytosolic mtDNA contributes to type I interferon and APOBEC3A upregulation. This was confirmed by using an HSV-1 strain (KOS37 UL98-SPA) with a deletion of the UL12.5 gene that impaired its ability to induce mtDNA stress. Furthermore, by using an inhibitor of RNA polymerase III, we demonstrated that upon HSV-1 infection, cytosolic mtDNA enhanced type I interferon induction through the RNA polymerase III/RIG-I pathway. APOBEC3A was in turn induced by interferon. Deep sequencing analyses of cytosolic mtDNA mutations revealed an APOBEC3A signature predominantly in the 5′TpCpG context. These data demonstrate that upon HSV-1 infection, the mitochondrial network is disrupted, leading to the release of mtDNA and ultimately to its catabolism through APOBEC3-induced mutations. IMPORTANCE Herpes simplex virus 1 (HSV-1) impairs the mitochondrial network through the viral protein UL12.5. This leads to the fusion of mitochondria and simultaneous release of mitochondrial DNA (mtDNA) in a mouse model. We have shown that released mtDNA is recognized as a danger signal, capable of stimulating signaling pathways and inducing the production of proinflammatory cytokines. The expression of the human cytidine deaminase APOBEC3A is highly upregulated by interferon responses. This enzyme catalyzes the deamination of cytidine to uridine in single-stranded DNA substrates, resulting in the catabolism of edited DNA. Using human cell lines deprived of mtDNA and viral strains deficient in UL12 , we demonstrated the implication of mtDNA in the production of interferon and APOBEC3A expression during viral infection. We have shown that HSV-1 induces mitochondrial network fragmentation in a human model and confirmed the implication of RNA polymerase III/RIG-I signaling in the capture of cytosolic mtDNA.

Ostreopsis cf. ovata produces palytoxin analogues including ovatoxins (OVTXs) and a putative palytoxin (p-PLTX), which can accumulate in marine organisms and may possibly lead to food intoxication. However, purified ovatoxins are not widely available and their toxicities are still unknown. The aim of this study was to improve understanding of the ecophysiology of Ostreopsis cf. ovata and its toxin production as well as to optimize the purification process for ovatoxin. During Ostreopsis blooms in 2011 and 2012 in Villefranche-sur-Mer (France, NW Mediterranean Sea), microalgae epiphytic cells and marine organisms were collected and analyzed both by LC-MS/MS and hemolysis assay. Results obtained with these two methods were comparable, suggesting ovatoxins have hemolytic properties. An average of 223 μg·kg−1 of palytoxin equivalent of whole flesh was found, thus exceeding the threshold of 30 μg·kg−1 in shellfish recommended by the European Food Safety Authority (EFSA). Ostreopsis cells showed the same toxin profile both in situ and in laboratory culture, with ovatoxin-a (OVTX-a) being the most abundant analogue (~50%), followed by OVTX-b (~15%), p-PLTX (12%), OVTX-d (8%), OVTX-c (5%) and OVTX-e (4%). Ostreopsis cf. ovata produced up to 2 g of biomass per L of culture, with a maximum concentration of 300 pg PLTX equivalent cell−1. Thus, an approximate amount of 10 mg of PLTX-group toxins may be produced with 10 L of this strain. Toxin extracts obtained from collected biomass were purified using different techniques such as liquid-liquid partition or size exclusion. Among these methods, open-column chromatography with Sephadex LH20 phase yielded the best results with a cleanup efficiency of 93% and recovery of about 85%, representing an increase of toxin percentage by 13 fold. Hence, this purification step should be incorporated into future isolation exercises.

Lung infections play a critical role in cystic fibrosis (CF) pathogenesis. CF respiratory tract is now considered to be a polymicrobial niche and advances in high-throughput sequencing allowed to analyze its microbiota and mycobiota. However, no NGS studies until now have characterized both communities during CF pulmonary exacerbation (CFPE). Thirty-three sputa isolated from patients with and without CFPE were used for metagenomic high-throughput sequencing targeting 16S and ITS2 regions of bacterial and fungal rRNA. We built inter-kingdom network and adapted Phy-Lasso method to highlight correlations in compositional data. The decline in respiratory function was associated with a decrease in bacterial diversity. The inter-kingdom network revealed three main clusters organized around Aspergillus , Candida , and Scedosporium genera. Using Phy-Lasso method, we identified Aspergillus and Malassezia as relevantly associated with CFPE, and Scedosporium plus Pseudomonas with a decline in lung function. We corroborated in vitro the cross-domain interactions between Aspergillus and Streptococcus predicted by the correlation network. For the first time, we included documented mycobiome data into a version of the ecological Climax/Attack model that opens new lines of thoughts about the physiopathology of CF lung disease and future perspectives to improve its therapeutic management.

Artificial intelligence is gaining increasing interest in medical image segmentation, including liver cancer. However, the literature lacks model implementation in the setting of colorectal liver metastases for treatment planning. We collected the portal phase abdominal CT scan images from the Nice University Hospital hepatobiliary oncologic multidisciplinary discussion of 80 patients with colorectal liver metastases, before treatment. Data from 70 patients was exploited to train and test the nnU-Net model to automatically perform parenchyma, portal vein, hepatic veins, cava vein, and colorectal liver metastases segmentation. Data from the remaining 10 patients was used for external validation. The Dice score for parenchyma segmentation was 0,964 and 0,955 in the test and validation dataset, respectively. For portal vein segmentation, a centerline Dice (clDice) of 0,758 and 0,736 was highlighted, while for hepatic veins it resulted to be 0,758 and 0,577. Cava vein segmentation showed a clDice of 0,805 and 0,734. Concerning colorectal liver metastases, the Dice score was 0,693 and 0,61. The nnU-Net showed promising segmentation results, especially for liver parenchyma. Its task could be useful to help physicians decide which is the best treatment strategy based on individual anatomical characteristics and disease extension. Training the model on a larger dataset with the same characteristics could help improve segmentation performances.

Background Studies focusing on dietary pesticides in population-based samples are scarce and little is known about potential mixture effects. We aimed to assess associations between dietary pesticide exposure profiles and Type 2 Diabetes (T2D) among NutriNet-Santé cohort participants. Methods Participants completed a Food Frequency Questionnaire at baseline, assessing conventional and organic food consumption. Exposures to 25 active substances used in European Union pesticides were estimated using the Chemisches und Veterinäruntersuchungsamt Stuttgart residue database accounting for farming practices. T2D were identified through several sources. Exposure profiles were established using Non-Negative Matrix Factorization (NMF), adapted for sparse data. Cox models adjusted for known confounders were used to estimate hazard ratios (HR) and 95% confidence interval (95% CI), for the associations between four NMF components, divided into quintiles (Q) and T2D risk. Results The sample comprised 33,013 participants aged 53 years old on average, including 76% of women. During follow-up (median: 5.95 years), 340 incident T2D cases were diagnosed. Positive associations were detected between NMF component 1 (reflecting highest exposure to several synthetic pesticides) and T2D risk on the whole sample: HR Q5vsQ1  = 1.47, 95% CI (1.00, 2.18). NMF Component 3 (reflecting low exposure to several synthetic pesticides) was associated with a decrease in T2D risk, among those with high dietary quality only (high adherence to French dietary guidelines, including high plant foods consumption): HR Q5vsQ1  = 0.31, 95% CI (0.10, 0.94). Conclusions These findings suggest a role of dietary pesticide exposure in T2D risk, with different effects depending on which types of pesticide mixture participants are exposed to. These associations need to be confirmed in other types of studies and settings, and could have important implications for developing prevention strategies (regulation, dietary guidelines). Trial Registration This study is registered in ClinicalTrials.gov ( NCT03335644 ).