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.

Everolimus in combination with vandetanib in children, adolescents, and young adults: a phase I study.

BACKGROUND: Combined use of inhibitors of mammalian target of rapamycin (mTOR) and vascular endothelial growth factor (VEGF-2) receptors is a potential strategy to overcome resistance to either class of drugs when used alone. PATIENTS AND METHODS: We designed a phase 1 trial to test the drug combination of a multikinase VEGF receptor 2 inhibitor, vandetanib, and an mTOR inhibitor, everolimus, in a pediatric and young adult patient cohort with advanced cancers. Exceptional responders were probed for tumor mutational profile to explore possible molecular mechanisms of response. RESULTS: Among 21 enrolled patients, clinical benefit was observed in 38% (one patient with partial response and eight patients with stable disease) with a median progression-free survival of 3.3 months. The most common treatment-related adverse event was rash (n = 13). Other treatment-related toxicities included diarrhea, fatigue, hypertension, QT prolongation, hypertriglyceridemia/hypercholesterolemia, transaminitis, thrombocytopenia, and weight loss. None of the patients experienced dose-limiting toxicities. Three exceptional responders were analyzed and were found to harbor genetic alterations including kinase insert domain receptor (KDR) Q472H mutation, EWSR1-CREB3L1, CDKN2A/B loss, and ASPL/ASPSCR1-TFE3 fusion. CONCLUSIONS: The combination of vandetanib and everolimus showed early activity and tolerable toxicity profile in pediatric patients with advanced cancers.

Long-term follow-up of patients with extensive segmental infantile hemangioma of the cervical or facial region: A French single-center prospective study.

BACKGROUND: Infantile hemangiomas (IHs) can be part of PHACE (posterior fossa anomalies, hemangioma, arterial anomalies, cardiac anomalies, eye anomalies) syndrome when they are segmental, extensive, and located on the face or neck. The initial assessment is codified and well known, but there are no recommendations for the follow-up of these patients. The aim of this study was to assess the long-term prevalence of different associated abnormalities. METHODS: Patients with a history of large segmental IHs of the face or neck. diagnosed between 2011 and 2016 were included in the study. Each patient underwent an ophthalmological, dental, ENT (ear, nose, and throat), dermatological, neuro-pediatric, and radiological assessment at inclusion. Eight patients including five with PHACE syndrome were prospectively evaluated. RESULTS: After a mean follow-up of 8.5 years, three patients presented with an angiomatous aspect of the oral mucosa, two with hearing loss, and two with otoscopic abnormalities. No patients developed ophthalmological abnormalities. The neurological examination was altered in three cases. Brain magnetic resonance imaging follow-up was unchanged in three out four patients and revealed atrophy of the cerebellar vermis in 1 patient. Neurodevelopmental disorders were found in five of the patients and learning difficulties were observed in five patients. The S1 location appears to be associated with a higher risk of neurodevelopmental disorders and cerebellar malformations, while the S3 location was associated with more progressive complications, including neurovascular, cardiovascular, and ENT abnormalities. CONCLUSION: Our study reported late complications in patients with a large segmental IH of the face or neck, whether associated with PHACE syndrome or not, and we proposed an algorithm to optimize the long-term follow-up.

Break on through: The role of innate immunity and barrier defence in atopic dermatitis and psoriasis.

The human skin can be affected by a multitude of diseases including inflammatory conditions such as atopic dermatitis and psoriasis. Here, we describe how skin barrier integrity and immunity become dysregulated during these two most common inflammatory skin conditions. We summarise recent advances made in the field of the skin innate immune system and its interaction with adaptive immunity. We review gene variants associated with atopic dermatitis and psoriasis that affect innate immune mechanisms and skin barrier integrity. Finally, we discuss how current and future therapies may affect innate immune responses and skin barrier integrity in a generalized or more targeted approach in order to ameliorate disease in patients.

Small molecule allosteric inhibitors of RORγt block Th17-dependent inflammation and associated gene expression in vivo.

Retinoic acid receptor-related orphan nuclear receptor (ROR) γt is a member of the RORC nuclear hormone receptor family of transcription factors. RORγt functions as a critical regulator of thymopoiesis and immune responses. RORγt is expressed in multiple immune cell populations including Th17 cells, where its primary function is regulation of immune responses to bacteria and fungi through IL-17A production. However, excessive IL-17A production has been linked to numerous autoimmune diseases. Moreover, Th17 cells have been shown to elicit both pro- and anti-tumor effects. Thus, modulation of the RORγt/IL-17A axis may represent an attractive therapeutic target for the treatment of autoimmune disorders and some cancers. Herein we report the design, synthesis and characterization of three selective allosteric RORγt inhibitors in preclinical models of inflammation and tumor growth. We demonstrate that these compounds can inhibit Th17 differentiation and maintenance in vitro and Th17-dependent inflammation and associated gene expression in vivo, in a dose-dependent manner. Finally, RORγt inhibitors were assessed for efficacy against tumor formation. While, RORγt inhibitors were shown to inhibit tumor formation in pancreatic ductal adenocarcinoma (PDAC) organoids in vitro and modulate RORγt target genes in vivo, this activity was not sufficient to delay tumor volume in a KP/C human tumor mouse model of pancreatic cancer.

A new Cannabis genome assembly associates elevated cannabidiol (CBD) with hemp introgressed into marijuana.

Demand for cannabidiol (CBD), the predominant cannabinoid in hemp (Cannabis sativa), has favored cultivars producing unprecedented quantities of CBD. We investigated the ancestry of a new cultivar and cannabinoid synthase genes in relation to cannabinoid inheritance. A nanopore-based assembly anchored to a high-resolution linkage map provided a chromosome-resolved genome for CBDRx, a potent CBD-type cultivar. We measured cannabinoid synthase expression by cDNA sequencing and conducted a population genetic analysis of diverse Cannabis accessions. Quantitative trait locus mapping of cannabinoids in a hemp × marijuana segregating population was also performed. Cannabinoid synthase paralogs are arranged in tandem arrays embedded in long terminal repeat retrotransposons on chromosome 7. Although CBDRx is predominantly of marijuana ancestry, the genome has cannabidiolic acid synthase (CBDAS) introgressed from hemp and lacks a complete sequence for tetrahydrocannabinolic acid synthase (THCAS). Three additional genomes, including one with complete THCAS, confirmed this genomic structure. Only cannabidiolic acid synthase (CBDAS) was expressed in CBD-type Cannabis, while both CBDAS and THCAS were expressed in a cultivar with an intermediate tetrahydrocannabinol (THC) : CBD ratio. Although variation among cannabinoid synthase loci might affect the THC : CBD ratio, variability among cultivars in overall cannabinoid content (potency) was also associated with other chromosomes.

DNA methylation epi-signature is associated with two molecularly and phenotypically distinct clinical subtypes of Phelan-McDermid syndrome.

BACKGROUND: Phelan-McDermid syndrome is characterized by a range of neurodevelopmental phenotypes with incomplete penetrance and variable expressivity. It is caused by a variable size and breakpoint microdeletions in the distal long arm of chromosome 22, referred to as 22q13.3 deletion syndrome, including the SHANK3 gene. Genetic defects in a growing number of neurodevelopmental genes have been shown to cause genome-wide disruptions in epigenomic profiles referred to as epi-signatures in affected individuals. RESULTS: In this study we assessed genome-wide DNA methylation profiles in a cohort of 22 individuals with Phelan-McDermid syndrome, including 11 individuals with large (2 to 5.8 Mb) 22q13.3 deletions, 10 with small deletions (< 1 Mb) or intragenic variants in SHANK3 and one mosaic case. We describe a novel genome-wide DNA methylation epi-signature in a subset of individuals with Phelan-McDermid syndrome. CONCLUSION: We identified the critical region including the BRD1 gene as responsible for the Phelan-McDermid syndrome epi-signature. Metabolomic profiles of individuals with the DNA methylation epi-signature showed significantly different metabolomic profiles indicating evidence of two molecularly and phenotypically distinct clinical subtypes of Phelan-McDermid syndrome.

Fostering infant food texture acceptance: A pilot intervention promoting food texture introduction between 8 and 15 months.

Healthy infant feeding practices form the basis of healthy eating behaviour later in life. The effect of providing parents with recommendations on textured food introduction between 8 and 15 months on children's experience with and acceptance of textured foods was studied. Sixty parent/child dyads were randomly assigned to a control group (CG) receiving current French recommendations and an intervention group (IG) receiving a brochure with supplementary advice, tips and monthly counselling on food texture introduction. After the intervention, parents completed self-report measures about the introduction of 188 food items, including purees, soft/small pieces, hard/large pieces and double textures. Children's acceptance of eight textured foods was assessed in a laboratory setting. Parents in the IG introduced more soft/small food pieces (P = 0.004) but not more complex textures (P = 0.15). There was no group difference in children acceptance for any of the textured foods offered in the laboratory. Independent of their study group, children's exposure to texture was associated with birth order, self-feeding with fingers, low gagging frequency and seldom use of commercial baby foods. Higher acceptance was associated with higher exposure to food pieces but not to pureed foods (either smooth or rough) and with children's eating behavioural traits (high food enjoyment, high food responsiveness and low food fussiness). This pilot intervention demonstrated that providing information can be effective in promoting the introduction of small and soft food pieces, but the most effective way to influence the introduction of more challenging textures (hard pieces and double texture) is uncertain. Further research should focus on the identification of barriers to complex texture introduction and on how building on this knowledge for a population-based public health intervention.

Validation of the Kinetik Blood Pressure Monitor-Series 1 for use in adults at home and in clinical settings, according to the 2002 European Society of Hypertension International Protocol on the validation of blood pressure devices.

The aim of this study was to assess the blood pressure (BP) measurement accuracy of the Kinetik Blood Pressure Monitor-Series 1 (BPM-1) for use in home or clinical settings according to the 2002 European Society of Hypertension International Protocol (ESH-IP). Forty-two participants were recruited to fulfil the required number of systolic and diastolic BP measurements according to the ESH-IP. Nine sequential same-arm BP readings were measured and analysed for each participant using the test device and observer mercury standard readings according to the 2002 ESH-IP. Forty one participants were used to obtain 33 sets of systolic and diastolic BP readings and were included in the analysis. Mean difference between the device measurements and the observer (mercury standard) measurements was 1.1 ± 7.2/1.1 ± 6.8 mmHg (mean ± standard deviation; systolic/diastolic). The number of systolic BP differences between the test and observer measurements that fell within 5, 10 and 15 mmHg was 65, 86 and 92. For diastolic readings, the number of test-observer measurement differences within 5, 10 and 15 mmHg was 77, 91 and 94. The number of participants with at least two out of three differences within 5 mmHg was 28 for systolic and 40 for diastolic BP readings. Three participants had no differences between the test and observer measurements within 5 mmHg in both the systolic and diastolic measurement categories. The Kinetik BPM-1 device fulfilled the requirements of the ESH-IP validation procedure and can be recommended for clinical use and self-measurement within the home.

A versatile Johansson-type tender x-ray emission spectrometer.

We present a high energy resolution x-ray spectrometer for the tender x-ray regime (1.6-5.0 keV) that was designed and operated at Stanford Synchrotron Radiation Lightsource. The instrument is developed on a Rowland geometry (500 mm of radius) using cylindrically bent Johansson analyzers and a position sensitive detector. By placing the sample inside the Rowland circle, the spectrometer operates in an energy-dispersive mode with a subnatural line-width energy resolution (∼0.32 eV at 2400 eV), even when an extended incident x-ray beam is used across a wide range of diffraction angles (∼30° to 65°). The spectrometer is enclosed in a vacuum chamber, and a sample chamber with independent ambient conditions is introduced to enable a versatile and fast-access sample environment (e.g., solid/gas/liquid samples, in situ cells, and radioactive materials). The design, capabilities, and performance are presented and discussed.

Trace Elements in Soils and Vegetables from Market Gardens of Urban Areas in Marrakech City.

The consumption of vegetables grown on soils polluted by trace elements can cause a serious threat for animal and human health and disturb the functioning of the ecosystem. The aim of this work is to determine the concentrations of As, Cd, Co, Cr, Cu, Ni, Pb, Mn, and Zn in soils and different vegetables from market gardens of urban areas in Marrakech city in order to investigate human health risk through ingestion of contaminated vegetables. Plant transfer factor (TF), daily intake of metals (DIM), and health risk index (HRI) were calculated. The concentrations of all metals studied in the garden top soils were within the threshold values. The Cu, Zn, Co, Mn, and As concentration in the edible parts of vegetables were within the safe limits. However, Cd and Pb in Malva parviflora and Cd in Brassica rapa, and Cr and Ni in Coriandrum sativum exceeded their permissible limits. The TF for these nine metals varied between 0.01 and 1.35. The classification of TEs according to their TF is as follows: Cu > Zn > Cd > Ni > Cr > Pb > Mn > Co > As. The maximum value was recorded in Coriandrum sativum while the minimum value was recorded in Cynara cardunculus. The HRI values were within the safe limit (< one) except for Pb in Malva parviflora. This can lead to risks to the health of the human population, especially children, consuming contaminated plants. Thus, the monitoring and prevention of health risks related to the consumption of plants grown in (peri)urban areas are necessary and essential to propose recommendations to both gardeners and decision-makers.

HIV-1 Vpr mediates the depletion of the cellular repressor CTIP2 to counteract viral gene silencing.

Mammals have evolved many antiviral factors impacting different steps of the viral life cycle. Associated with chromatin-modifying enzymes, the cellular cofactor CTIP2 contributes to HIV-1 gene silencing in latently infected reservoirs that constitute the major block toward an HIV cure. We report, for the first time, that the virus has developed a strategy to overcome this major transcriptional block. Productive HIV-1 infection results in a Vpr-mediated depletion of CTIP2 in microglial cells and CD4+ T cells, two of the major viral reservoirs. Associated to the Cul4A-DDB1-DCAF1 ubiquitin ligase complex, Vpr promotes CTIP2 degradation via the proteasome pathway in the nuclei of target cells and notably at the latent HIV-1 promoter. Importantly, Vpr targets CTIP2 associated with heterochromatin-promoting enzymes dedicated to HIV-1 gene silencing. Thereby, Vpr reactivates HIV-1 expression in a microglial model of HIV-1 latency. Altogether our results suggest that HIV-1 Vpr mediates the depletion of the cellular repressor CTIP2 to counteract viral gene silencing.

Targeting the DNA-PK complex: Its rationale use in cancer and HIV-1 infection.

The DNA-PK complex is the major component of the predominant mechanism of DSB repair in humans. In addition, this complex is involved in many other processes such as DNA recombination, genome maintenance, apoptosis and transcription regulation. Several studies have linked the decrease of the DNA-PK activity with cancer initiation, due to defects in the repair. On another hand, higher DNA-PK expression and activity have been observed in various other tumor cells and have been linked with a decrease of the efficiency of anti-tumor drugs. It has also been shown that DNA-PK is critical for the integration of the HIV-1 DNA into the cell host genome and promotes replication and transcription of the virus. Targeting this complex makes therefore sense to treat these two pathologies. However, according to the status of HIV-1 replication (active versus latent replication) or to the tumor grade cells (initiation versus metastasis), the way to target this DNA-PK complex might be rather different. In this review, we discuss the importance of DNA-PK complex in two major pathologies i.e. HIV-1 infection and cancer, and the rationale use of therapies aiming to target this complex.

Soft X-Ray Second Harmonic Generation as an Interfacial Probe.

Nonlinear optical processes at soft x-ray wavelengths have remained largely unexplored due to the lack of available light sources with the requisite intensity and coherence. Here we report the observation of soft x-ray second harmonic generation near the carbon K edge (∼284 eV) in graphite thin films generated by high intensity, coherent soft x-ray pulses at the FERMI free electron laser. Our experimental results and accompanying first-principles theoretical analysis highlight the effect of resonant enhancement above the carbon K edge and show the technique to be interfacially sensitive in a centrosymmetric sample with second harmonic intensity arising primarily from the first atomic layer at the open surface. This technique and the associated theoretical framework demonstrate the ability to selectively probe interfaces, including those that are buried, with elemental specificity, providing a new tool for a range of scientific problems.

Influence of a dynamic seating habit on lumbar motor control in schoolchildren.

BACKGROUND: Adequate motor control is considered important for spinal stability and the prevention of low back pain in adulthood and in childhood. OBJECTIVE: Given that the sitting position can affect proprioception, this study aimed to evaluate the influence of using at school a triangular and dynamic cushion on schoolchildren's trunk motor control. METHODS: Thirty 8-year-old schoolchildren were randomized into a control group (n= 15) and a "cushion group" (n= 15), in which the children used the cushion for one year and a half. At the end of this period, a 3D-analysis was used to assess lumbar spine proprioception by means of a trunk repositioning task performed blindfolded in a seated position in two conditions (on a stable and on an unstable surface). RESULTS: The schoolchildren in the cushion group performed better at the trunk repositioning task (p= 0.02) and hold their lumbar lordosis (p= 0.03) better than the control children, in both conditions (stable and unstable). CONCLUSIONS: This preliminary study suggests that daily use of a triangular dynamic cushion has a beneficial impact on children's lumbar proprioception. Further studies are needed to confirm these results and investigate the effectiveness of its use to prevent low back pain in adulthood.