Highly efficient and sensitive membrane-based concentration process allows quantification, surveillance, and sequencing of viruses in large volumes of wastewater.

Wastewater-based epidemiology is experiencing exponential development. Despite undeniable advantages compared to patient-centered approaches (cost, anonymity, survey of large populations without bias, detection of asymptomatic infected peoples…), major technical limitations persist. Among them is the low sensitivity of the current methods used for quantifying and sequencing viral genomes from wastewater. In situations of low viral circulation, during initial stages of viral emergences, or in areas experiencing heavy rains, the extremely low concentrations of viruses in wastewater may fall below the limit of detection of the current methods. The availability during crisis and the cost of the commercial kits, as well as the requirement of expensive materials such as high-speed centrifuge, can also present major blocks to the development of wastewater-based epidemiological survey, specifically in low-income countries. Thereby, highly sensitive, low cost and standardized methods are still needed, to increase the predictability of the viral emergences, to survey low-circulating viruses and to make the results from different labs comparable. Here, we outline and characterize new protocols for concentrating and quantifying SARS-CoV-2 from large volumes (500 mL-1 L) of untreated wastewater. In addition, we report that the methods are applicable for monitoring and sequencing. Our nucleic acid extraction technique (the routine C: 5 mL method) does not require sophisticated equipment such as automatons and is not reliant on commercial kits, making it readily available to a broader range of laboratories for routine epidemiological survey. Furthermore, we demonstrate the efficiency, the repeatability, and the high sensitivity of a new membrane-based concentration method (MBC: 500 mL method) for enveloped (SARS-CoV-2) and non-enveloped (F-specific RNA phages of genogroup II / FRNAPH GGII) viruses. We show that the MBC method allows the quantification and the monitoring of viruses in wastewater with a significantly improved sensitivity compared to the routine C method. In contexts of low viral circulation, we report quantifications of SARS-CoV-2 in wastewater at concentrations as low as 40 genome copies per liter. In highly diluted samples collected in wastewater treatment plants of French Guiana, we confirmed the accuracy of the MBC method compared to the estimations done with the routine C method. Finally, we demonstrate that both the routine C method processing 5 mL and the MBC method processing 500 mL of untreated wastewater are both compatible with SARS-CoV-2 sequencing. We show that the quality of the sequence is correlated with the concentration of the extracted viral genome. Of note, the quality of the sequences obtained with some MBC processed wastewater was improved by dilutions or enzyme substitutions suggesting the presence of specific enzyme inhibitors in some wastewater. To the best of our knowledge, our MBC method is one of the first efficient, sensitive, and repeatable method characterized for SARS-CoV-2 quantification and sequencing from large volumes of wastewater.

Doublecortin mutation leads to persistent defects in the Golgi apparatus and mitochondria in adult hippocampal pyramidal cells.

Human doublecortin (DCX) mutations are associated with severe brain malformations leading to aberrant neuron positioning (heterotopia), intellectual disability and epilepsy. DCX is a microtubule-associated protein which plays a key role during neurodevelopment in neuronal migration and differentiation. Dcx knockout (KO) mice show disorganized hippocampal pyramidal neurons. The CA2/CA3 pyramidal cell layer is present as two abnormal layers and disorganized CA3 KO pyramidal neurons are also more excitable than wild-type (WT) cells. To further identify abnormalities, we characterized Dcx KO hippocampal neurons at subcellular, molecular and ultrastructural levels. Severe defects were observed in mitochondria, affecting number and distribution. Also, the Golgi apparatus was visibly abnormal, increased in volume and abnormally organized. Transcriptome analyses from laser microdissected hippocampal tissue at postnatal day 60 (P60) highlighted organelle abnormalities. Ultrastructural studies of CA3 cells performed in P60 (young adult) and > 9 months (mature) tissue showed that organelle defects are persistent throughout life. Locomotor activity and fear memory of young and mature adults were also abnormal: Dcx KO mice consistently performed less well than WT littermates, with defects becoming more severe with age. Thus, we show that disruption of a neurodevelopmentally-regulated gene can lead to permanent organelle anomalies contributing to abnormal adult behavior.

Integrative and comparative genomic analyses identify clinically relevant pulmonary carcinoid groups and unveil the supra-carcinoids.

The worldwide incidence of pulmonary carcinoids is increasing, but little is known about their molecular characteristics. Through machine learning and multi-omics factor analysis, we compare and contrast the genomic profiles of 116 pulmonary carcinoids (including 35 atypical), 75 large-cell neuroendocrine carcinomas (LCNEC), and 66 small-cell lung cancers. Here we report that the integrative analyses on 257 lung neuroendocrine neoplasms stratify atypical carcinoids into two prognostic groups with a 10-year overall survival of 88% and 27%, respectively. We identify therapeutically relevant molecular groups of pulmonary carcinoids, suggesting DLL3 and the immune system as candidate therapeutic targets; we confirm the value of OTP expression levels for the prognosis and diagnosis of these diseases, and we unveil the group of supra-carcinoids. This group comprises samples with carcinoid-like morphology yet the molecular and clinical features of the deadly LCNEC, further supporting the previously proposed molecular link between the low- and high-grade lung neuroendocrine neoplasms.

Minor allele of the factor V K858R variant protects from venous thrombosis only in non-carriers of factor V Leiden mutation.

Factor V serves an important role in the regulation of blood coagulation. The rs6025 (R534Q) and rs4524 (K858R) polymorphisms in the F5 gene, are known to influence the risk of venous thrombosis. While the rare Q534 (factor V Leiden) allele is associated with an increased risk of venous thrombosis, the minor R858 allele is associated with a lower risk of disease. However, no study has deeply examined the cumulative impact of these two variations on venous thrombosis risk. We study the association of these polymorphisms with the risk of venous thrombosis in 4 French case-control populations comprising 3719 patients and 4086 controls. We demonstrate that the Q534 allele has a dominant effect over R858. Besides, we show that in individuals not carrying the Q534 allele, the protective effect of the R858 allele acts in a dominant mode. Thrombin generation-based normalized activated protein C sensitivity ratio was lower in the 858R/R homozygotes than in the 858K/K homozygotes (1.92 ± 1.61 vs 2.81 ± 1.57, p = 0.025). We demonstrate that the R858 allele of the F5 rs4524 variant protects from venous thrombosis only in non-carriers of the Q534 allele of the F5 rs6025. Its protective effect is mediated by reduced factor VIII levels and reduced activated protein C resistance.

17q21.31 duplication causes prominent tau-related dementia with increased MAPT expression.

To assess the role of rare copy number variations in Alzheimer's disease (AD), we conducted a case-control study using whole-exome sequencing data from 522 early-onset cases and 584 controls. The most recurrent rearrangement was a 17q21.31 microduplication, overlapping the CRHR1, MAPT, STH and KANSL1 genes that was found in four cases, including one de novo rearrangement, and was absent in controls. The increased MAPT gene dosage led to a 1.6-1.9-fold expression of the MAPT messenger RNA. Clinical signs, neuroimaging and cerebrospinal fluid biomarker profiles were consistent with an AD diagnosis in MAPT duplication carriers. However, amyloid positon emission tomography (PET) imaging, performed in three patients, was negative. Analysis of an additional case with neuropathological examination confirmed that the MAPT duplication causes a complex tauopathy, including prominent neurofibrillary tangle pathology in the medial temporal lobe without amyloid-ß deposits. 17q21.31 duplication is the genetic basis of a novel entity marked by prominent tauopathy, leading to early-onset dementia with an AD clinical phenotype. This entity could account for a proportion of probable AD cases with negative amyloid PET imaging recently identified in large clinical series.

Is there still room for additional common susceptibility alleles for venous thromboembolism?

UNLABELLED: Essentials Genetic architecture of venous thromboembolism (VTE) remains to be fully disentangled. 11 newly discovered candidate polymorphisms were genotyped in 3019 VTE cases and 2605 controls. None of the 11 polymorphisms were significantly associated with VTE risk. Additional major efforts are needed to identify VTE-associated genetic variants. SUMMARY: Background Through a meta-analysis of 12 genome-wide association studies, the International Network against VENous Thrombosis (INVENT) consortium identified two novel susceptibility loci for venous thromboembolism (VTE). This project has also generated other candidates that need to be confirmed. Objectives To assess the association with VTE of common single-nucleotide polymorphisms (SNPs) that demonstrated strong statistical, but not genome-wide, significance in the INVENT cohorts. Patients/methods Eleven SNPs were genotyped and tested for association with VTE in three case-control studies totaling 3019 patients and 2605 healthy individuals. Results and conclusions None of the tested SNPs showed evidence for association with VTE. Different strategies are needed to decipher the whole spectrum of common and rare genetic variations associated with VTE risk.

SORL1 rare variants: a major risk factor for familial early-onset Alzheimer's disease.

The SORL1 protein plays a protective role against the secretion of the amyloid ß peptide, a key event in the pathogeny of Alzheimer's disease. We assessed the impact of SORL1 rare variants in early-onset Alzheimer's disease (EOAD) in a case-control setting. We conducted a whole exome analysis among 484 French EOAD patients and 498 ethnically matched controls. After collapsing rare variants (minor allele frequency ≤1%), we detected an enrichment of disruptive and predicted damaging missense SORL1 variants in cases (odds radio (OR)=5.03, 95% confidence interval (CI)=(2.02-14.99), P=7.49.10(-5)). This enrichment was even stronger when restricting the analysis to the 205 cases with a positive family history (OR=8.86, 95% CI=(3.35-27.31), P=3.82.10(-7)). We conclude that predicted damaging rare SORL1 variants are a strong risk factor for EOAD and that the association signal is mainly driven by cases with positive family history.

Development of the foetal and neonatal testis.

The foetal testis originates from a proliferation of the mesonephric and the coelomic epithelia which are colonized by the primordial germ cells. In the foetal testis, the development and functions of the three main cell type precursors (Leydig, Sertoli and germ cells) do not depend upon gonadotropins. Numerous intra- and extra-testicular factors are candidates for the control of its development and functions. To study the potential involvement of these factors, we developed an organotypic culture system. In absence of any growth factors or hormone, this system allows a development of the three main cell types which mimics that observed in vivo. The effects of different regulators (gonadotropin-releasing hormone, follicle-stimulating hormone, transforming growth factor-beta, insulin-like growth factor-I, anti-Mullerian hormone, retinoic acid, oestrogens) were tested in this system. Whether or not some of the effects observed in vitro have a physiological relevance was evaluated using appropriate transgenic mice. It is concluded that the foetal testis cannot be considered as an adult mini-testis since it has a specific physiology which largely differs from that of the immature or adult testis.

The serine/threonine transmembrane receptor ALK2 mediates Müllerian inhibiting substance signaling.

Müllerian inhibiting substance (MIS or anti-Müllerian hormone) is a member of the transforming growth factor-beta family and plays a pivotal role in proper male sexual differentiation. Members of this family signal by the assembly of two related serine/threonine kinase receptors, referred to as type I or type II receptors, and downstream cytoplasmic Smad effector proteins. Although the MIS type II receptor (MISRII) has been identified, the identity of the type I receptor is unclear. Here we report that MIS activates a bone morphogenetic protein-like signaling pathway, which is solely dependent on the presence of the MISRII and bioactive MIS ligand. Among the multiple type I candidates tested, only ALK2 resulted in significant enhancement of the MIS signaling response. Furthermore, dominant-negative and antisense strategies showed that ALK2 is essential for MIS-induced signaling in two independent assays, the cellular Tlx-2 reporter gene assay and the Müllerian duct regression organ culture assay. In contrast, ALK6, the other candidate MIS type I receptor, was not required. Expression analyses revealed that ALK2 is present in all MIS target tissues including the mesenchyme surrounding the epithelial Müllerian duct. Collectively, we conclude that MIS employs a bone morphogenetic protein-like signaling pathway and uses ALK2 as its type I receptor. The use of this ubiquitously expressed type I receptor underscores the role of the MIS ligand and the MIS type II receptor in establishing the specificity of the MIS signaling cascade.

Genetic and cellular analysis of male germ cell development.

The evolution of the germline has been studied for many decades. Although the pathway of germ cell primordial migration and the kinetic evolution of the gonocytes are well known, their genetic and cellular controls are poorly understood. Recently, a genetic approach using gene knockout and a cellular investigation using several germ cell culture models has allowed a better understanding of the involvement of several genes and factors in the development of germ cells during fetal and neonatal life. Because of the obvious importance of the development of primordial germ cells and gonocytes in adult fertility and its eventual alteration by toxins or xenobiotics, a better knowledge of these interactions appears as an important research area.

Transforming growth factor beta3 in the fetal and neonatal rat testis: immunolocalization and effect on fetal Leydig cell function.

The localization of transforming growth factor beta3 (TGFbeta3) in the fetal and neonatal testis (from fetal day 13.5 to postnatal day 6) was investigated by immunohistochemical staining with a specific polyclonal antibody raised against a synthetic peptide corresponding to residues 50-75 of TGFbeta3. This antibody recognized 0.5 ng TGFbeta3 in western blot analysis, but did not detect 25 ng TGFbeta1 or TGFbeta2. The immunolocalization of TGFbeta3 in the fetal and neonatal testis changed throughout development. Immunostaining was present in the gonocytes by fetal day 13.5, persisted until postnatal day 3, and was heterogeneous in spermatogonia on postnatal day 6. The Sertoli cells contained no immunoreactivity at any age. The fetal-type Leydig cells were first immunostained for TGFbeta3 on day 16.5 and staining became very intense from day 18.5 onward. Staining disappeared when the antibody was presaturated with the synthetic peptide, but persisted when the antibody was presaturated with a tenfold excess of the corresponding peptide from TGFbeta2. Furthermore, we researched whether TGFbeta3 could act as a local regulator of fetal Leydig cell function. In a dispersed fetal testicular cell system, TGFbeta3 inhibited the LH-stimulated testosterone production by Leydig cells from 20.5-day-old fetuses. The inhibitory effect of TGFbeta3 was equal to that observed with TGFbeta1 or TGFbeta2. When compared with our previous studies showing the immunolocalization of TGFbeta1 and TGFbeta2, the present study shows that TGFbeta3 may have a specific role in the developing rat testis, but may also overlap the action of TGFbeta1 and TGFbeta2.

Apoptosis and mitosis in gonocytes of the rat testis during foetal and neonatal development.

This study was undertaken to determine the extent of apoptosis and mitosis in the various testicular cell types throughout rat development from foetal day 14.5 to postnatal days 9-10. Apoptotic activity was studied by detecting DNA fragmentation (TUNEL method) in situ. A TUNEL-positive reaction was detected in gonocytes, while none of the other testicular cells were labelled. The morphology of the TUNEL-positive gonocytes was characteristic of apoptotic cells and was different from that observed in experimentally induced necrosis. The percentage of stained gonocytes peaked on day 15.5-16.5 post-conception (dpc), decreased thereafter and no TUNEL-positive gonocytes were found from foetal day 18.5 onwards. On postnatal day 2, apoptosis resumed and increased to reach a maximum on day 7. Mitosis in the gonocytes, as evaluated by the immunodetection of 5-bromo-2'-deoxyuridine (BrdU) incorporation, was present during the same developmental periods but the ratio of BrdU-positive/TUNEL-positive gonocytes was much greater in the foetal period than in the neonatal period. In an organotypic culture system, the changes in the apoptotic and mitotic activities of the gonocytes in testicular explants from foetuses on days 18.5 and 20.5 or from neonates on day 3, cultured for two days were similar to those observed in vivo. Addition of LH or FSH did not influence either apoptosis or mitosis in the germ cells. These results suggest that both apoptosis and mitosis of gonocytes are independent of gonadotrophins and are mainly controlled by intratesticular factors.

Expression of type I and II receptors for transforming growth factor beta in the adult rat testis.

After having established the specificity of the antibodies for the rat testis by western blot analysis, the potential target cells for transforming growth factors (TGFbetas) were identified by immunohistochemical detection of both type I (TbetaRI) and type II (TbetaRII) transducing receptors for TGFbetas in the adult rat testis in situ. Leydig cells showed a strong TbetaRII immunoreactivity whereas the TbetaRI staining was weak. Only TbetaRII was detectable in Sertoli cells. In germ cells, staining for TbetaRI was stronger than for TbetaRII and the expression of both receptors depended on the seminiferous cycle stage. TbetaRI first appeared in pachytene spermatocytes and was absent in elongated spermatids from stage XIV onwards. Labelling for TbetaRII was observed as early as the spermatogonia stage; it increased in pachytene spermatocytes at the onset of TbetaRI and disappeared in elongating spermatids from stage XI onwards. These results show that TGFbetas can affect somatic cells functions and suggest that these factors are involved in the control of meiosis and early spermiogenesis, exerting a direct effect on germ cells.

Transforming growth factor beta1 and beta2 reduce the number of gonocytes by increasing apoptosis.

Transforming growth factors beta1 and beta2 (TGFbetas) have recently been detected by immunohistochemistry in the fetal and neonatal rat testis, and the aim of the present study was to determine whether these factors can act as local regulators to control the number of gonocytes. Testes were kept in organ culture, and TGFbeta1 was found to have dose-dependent inhibitory effect on the number of gonocytes in testes explanted on fetal day 13.5. Either TGFbeta1 or beta2 at 10 ng/ml reduced the number of gonocytes by half after 2 days culture. TGFbetas did not decrease the BrdU labeling index of gonocytes or Sertoli cells, whereas these factors significantly increased the DNA fragmentation in gonocytes (TUNEL method). The other testicular cell types showed no positive TUNEL reaction. TGFbeta1 did not reduce the number of gonocytes in testes explanted on fetal day 17.5 (i.e. during the quiescent phase), but it did so in testes explanted on postnatal day 3 (i.e. stage of resumption of mitosis). To determine the potential cell type targets for TGFbetas, type I and type II TGFbeta receptors were immunolocalized in developing testis from fetal day 13.5 to postnatal day 3. Both receptors were present in the gonocytes throughout the whole period studied, and in the Leydig cells from fetal day 16.5 onward, but they were not detected in the Sertoli cells. Taken together, these results suggest that TGFbetas directly increase apoptosis in gonocytes without changing their mitotic activity during the developmental phases of proliferation.

The immunohistochemical localization of transforming growth factor-beta 2 in the fetal and neonatal rat testis.

The localization of transforming growth factor beta-2 (TGF beta 2) in the fetal and neonatal testis (from day 13.5 of fetal life to postnatal day 9) was investigated by an immunohistochemical staining method employing a specific polyclonal antibody. Immunostaining appeared on fetal day 13.5 in primitive Sertoli cells as they begin to come in contact with each other and surround the germ cells to form the seminiferous cords. Staining in Sertoli cells was still clearly observed until fetal day 16.5 and became faint or undetectable from fetal day 18.5 onwards. In fetal-type Leydig cells, a positive reaction for TGF beta 2 appeared on day 16.5 and became very intense from day 18.5 onwards. In the germ cells, immunoreactivity for TGF beta 2 appeared on fetal day 20.5, rose to a maximum on postnatal day 4 and decreased thereafter. On postnatal day 9, staining was still present in type A spermatogonia and absent in type B spermatogonia. No immunoreactivity was detected in peritubular cells on any day studied. In conclusion, our results are in favour of an autocrine/paracrine role of TGF beta 2 in the differentiation of the testis during the perinatal period. It may be involved in the organization of the seminiferous cords, the regulation of testosterone production and the regulation of the number of germ cells. When compared with the immunolocalization of TGF beta 1 that we have previously reported [1], the present study suggests that the roles of TGF beta 2 in the developing rat testis can be specific but also overlap from those of TGF beta 1.