Metaviromic Characterization of Betaflexivirus Populations Associated with a Vitis cultivar Collection in South Africa.

South Africa is associated with a centuries-old viticultural industry, accompanied by a diverse range of wine and table grape cultivars and an extensive history of pervasive introductions of vine material and associated viruses. The Vitis D2 collection in Stellenbosch represents the most comprehensive collection of Vitis species, hybrids, and cultivars in South Africa. We collected leaf petiole material from 229 accessions from this collection. Our metaviromic analyses revealed a total of 406 complete/near complete genomes of various betaflexiviruses. Among these, we identified the presence of grapevine rupestris stem pitting-associated virus and grapevine viruses A, B, E, F, H (GVH), I (GVI), and M (GVM). Notably, this study marks the first report of GVH, GVI, and GVM in South Africa, which were confirmed via RT-PCR. This research significantly contributes to our understanding of viral diversity and introductions in South African viticulture and emphasizes the need for vigilant monitoring and management of viral infections. Our findings lay the groundwork for strategies that mitigate the impact of viruses on South Africa's wine industry, which generates an annual revenue of approximately 500 million USD.

Cost of exome analysis in patients with intellectual disability: a micro-costing study in a French setting.

BACKGROUND: With the development of next generation sequencing technologies in France, exome sequencing (ES) has recently emerged as an opportunity to improve the diagnosis rate of patients presenting an intellectual disability (ID). To help French policy makers determine an adequate tariff for ES, we aimed to assess the unit cost per ES diagnostic test for ID from the preparation of the pre-analytical step until the report writing step and to identify its main cost drivers. METHODS: A micro-costing bottom-up approach was conducted for the year 2018 in a French setting as part of the DISSEQ study, a cost-effectiveness study funded by the Ministry of Health and performed in collaboration with the GAD (Génétique des Anomalies du Développement), a genetic team from the Dijon University Hospital, and a public sequencing platform, the Centre National de Recherche en Génomique Humaine (CNRGH). The analysis was conducted from the point of view of these two ES stakeholders. All of the resources (labor, equipment, disposables and reagents, reusable material) required to analyze blood samples were identified, collected and valued. Several sensitivity analyses were performed. RESULTS: The unit nominal cost per ES diagnostic test for ID was estimated to be €2,019.39. Labor represented 50.7% of the total cost. The analytical step (from the preparation of libraries to the analysis of sequences) represented 88% of the total cost. Sensitivity analyses suggested that a simultaneous price decrease of 20% for the capture kit and 50% for the sequencing support kit led to an estimation of €1,769 per ES diagnostic test for ID. CONCLUSION: This is the first estimation of ES cost to be done in the French setting of ID diagnosis. The estimation is especially influenced by the price of equipment kits, but more generally by the organization of the centers involved in the different steps of the analysis and the time period in which the study was conducted. This information can now be used to define an adequate tariff and assess the efficiency of ES. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT03287206 on September 19, 2017.

Effect of repeat refresher courses on neonatal resuscitation skill decay: an experimental comparative study of in-person and video-based simulation training.

Neonatal deaths are a major contributor to global under-5-year-old mortality. Training birth attendants can improve perinatal outcomes, but skills may fade over time. In this pilot study, we assessed skill decay of nursing students after remote video versus in-person resuscitation training in a low-resource setting. Filipino nursing students (n = 49) underwent traditional, in-person simulation-based Helping Babies Breathe (HBB) training in Mindanao, Philippines. Participants were then assigned to receive refresher training at 2-month intervals either in-person or via tele-simulation beginning at 2 months, 4 months, or 6 months after initial training. A knowledge examination and practical examination, also known as objective structured clinical examination B in the HBB curriculum, were administered before retraining to assess knowledge and skill retention at time of scheduled follow-up. Time to initiation of bag-mask ventilation (BMV) in seconds during simulated birth asphyxia was the primary outcome. Skill decay was evident at first follow-up, with average time to BMV increasing from 56.9 (range 15-87) s at initial post-training to 83.8 (range 32-128) s at 2 months and 90.2 (range 51-180) s at 4 months. At second follow-up of the 2-month group, students showed improved pre-training time to BMV (average 70.4; range 46-97 s). No statistical difference was observed between in-person and video-trained students in time to BMV. Because of COVID-19 restrictions, the 6-month follow-up was not completed. We conclude that remote video refresher training is a reasonable alternative to traditional in-person HBB training. Our study also suggests that refreshers may be needed more frequently than every 2 months to mitigate skill decay. Additional studies are necessary to assess the longitudinal impact of tele-simulation on clinical outcomes.

Genomic and phylogenetic characterization of wheat yellows virus, a novel tenuivirus infecting wheat in South Africa.

During the 2019 winter wheat season, symptoms of severe chlorosis and stunting were observed on wheat in the irrigation production areas of South Africa. RNAtag-seq data were generated for seven samples from KwaZulu-Natal province and one from Limpopo. Analysis of assembled contigs indicated the presence of a putatively novel member of the genus Tenuivirus, tentatively named "wheat yellows virus" (WhYV). The genome is made up of four segments, which are 8952, 3451, 2338, and 2045 nucleotides in length and code for a total of seven ORFs. Phylogenies of each segment (nucleotide) and the polymerase gene (amino acid), as well as amino acid sequence comparisons of each gene product, showed that WhYV is most closely related to rice stripe virus.

Genomic characterization of grapevine viruses N and O: novel vitiviruses from South Africa.

A survey was performed on a Vitis cultivar collection in Stellenbosch, South Africa. Metaviromes were generated for each cultivar, using an RNAtag-seq workflow. Analysis of assembled contigs indicated the presence of two putatively novel members of the genus Vitivirus, provisionally named "grapevine virus N" (GVN) and "grapevine virus O" (GVO). Comparisons of amino acid sequences showed that GVN and GVO are most closely related to grapevine virus G and grapevine virus E, respectively. The incidence of these novel viruses within the sampling site was low, with GVO and GVN associated with only five and two cultivars, respectively, of the 229 sampled.

Novel viruses associated with plants of the family Amaryllidaceae in South Africa.

Nineteen samples from members of the plant genera Agapanthus, Clivia, Hippeastrum, and Scadoxus were collected from gardens in the Gauteng and Western Cape provinces of South Africa. The plants displayed highly variable symptoms of viral disease, including chlorosis, necrosis, streaking, and ringspot. RNAtag-seq was used to characterize the associated viral populations. Plants of the genus Agapanthus were found to be associated with three novel viruses from the families Caulimoviridae, Closteroviridae, and Betaflexiviridae; plants of the genus Clivia were associated with novel members of the families Potyviridae and Betaflexiviridae; and plants of the genus Scadoxus were associated with a novel member of the family Tospoviridae. Nerine latent virus was associated with plants of the genera Agapanthus, Clivia, and Hippeastrum, while hippeastrum mosaic virus was associated exclusively with a Hippeastrum cultivar.

Quantification of 1.5 T T1 and T2* Relaxation Times of Fetal Tissues in Uncomplicated Pregnancies.

BACKGROUND: Despite its many advantages, experience with fetal magnetic resonance imaging (MRI) is limited, as is knowledge of how fetal tissue relaxation times change with gestational age (GA). Quantification of fetal tissue relaxation times as a function of GA provides insight into tissue changes during fetal development and facilitates comparison of images across time and subjects. This, therefore, can allow the determination of biophysical tissue parameters that may have clinical utility. PURPOSE: To demonstrate the feasibility of quantifying previously unknown T1 and T2* relaxation times of fetal tissues in uncomplicated pregnancies as a function of GA at 1.5 T. STUDY TYPE: Pilot. POPULATION: Nine women with singleton, uncomplicated pregnancies (28-38 weeks GA). FIELD STRENGTH/SEQUENCE: All participants underwent two iterative decomposition of water and fat with echo asymmetry and least-squares estimation (IDEAL-IQ) acquisitions at different flip angles (6° and 20°) at 1.5 T. ASSESSMENT: Segmentations of the lungs, liver, spleen, kidneys, muscle, and adipose tissue (AT) were conducted using water-only images and proton density fat fraction maps. Driven equilibrium single pulse observation of T1 (DESPOT1 ) was used to quantify the mean water T1 of the lungs, intraabdominal organs, and muscle, and the mean water and lipid T1 of AT. IDEAL T2* maps were used to quantify the T2* values of the lungs, intraabdominal organs, and muscle. STATISTICAL TESTS: F-tests were performed to assess the T1 and T2* changes of each analyzed tissue as a function of GA. RESULTS: No tissue demonstrated a significant change in T1 as a function of GA (lungs [P = 0.89]; liver [P = 0.14]; spleen [P = 0.59]; kidneys [P = 0.97]; muscle [P = 0.22]; AT: water [P = 0.36] and lipid [P = 0.14]). Only the spleen and muscle T2* showed a significant decrease as a function of GA (lungs [P = 0.67); liver [P = 0.05]; spleen [P < 0.05]; kidneys [P = 0.70]; muscle [P < 0.05]). DATA CONCLUSION: These preliminary data suggest that the T1 of the investigated tissues is relatively stable over 28-38 weeks GA, while the T2* change in spleen and muscle decreases significantly in that period. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY STAGE: 2.

Mapping solar magnetic fields from the photosphere to the base of the corona.

Routine ultraviolet imaging of the Sun's upper atmosphere shows the spectacular manifestation of solar activity; yet, we remain blind to its main driver, the magnetic field. Here, we report unprecedented spectropolarimetric observations of an active region plage and its surrounding enhanced network, showing circular polarization in ultraviolet (Mg ii h & k and Mn i) and visible (Fe i) lines. We infer the longitudinal magnetic field from the photosphere to the very upper chromosphere. At the top of the plage chromosphere, the field strengths reach more than 300 G, strongly correlated with the Mg ii k line core intensity and the electron pressure. This unique mapping shows how the magnetic field couples the different atmospheric layers and reveals the magnetic origin of the heating in the plage chromosphere.

Water-fat magnetic resonance imaging of adipose tissue compartments in the normal third trimester fetus.

BACKGROUND: Assessment of fetal adipose tissue gives information about the future metabolic health of an individual, with evidence that the development of this tissue has regional heterogeneity. OBJECTIVE: To assess differences in the proton density fat fraction (PDFF) between fetal adipose tissue compartments in the third trimester using water-fat magnetic resonance imaging (MRI). MATERIALS AND METHODS: Water-fat MRI was performed in a 1.5-T scanner. Fetal adipose tissue was segmented into cheeks, thorax, abdomen, upper arms, forearms, thighs and lower legs. PDFF and R2* values were measured in each compartment. RESULTS: Twenty-eight women with singleton pregnancies were imaged between 28 and 38 weeks of gestation. At 30 weeks' gestation (n=22), the PDFF was statistically different between the compartments (P<0.0001), with the highest PDFF in cheeks, followed by upper arms, thorax, thighs, forearms, lower legs and abdomen. There were no statistical differences in the rate of PDFF change with gestational age between the white adipose tissue compartments (P=0.97). Perirenal brown adipose tissue had a different PDFF and R2* compared to white adipose tissue, while the rate of R2* change did not significantly change with gestational age between white adipose tissue compartments (P=0.96). CONCLUSION: Fetal adipose tissue accumulates lipids at a similar rate in all white adipose tissue compartments. PDFF variances between the compartments suggest that accumulation begins at different gestational ages, starting with cheeks, followed by extremities, trunk and abdomen. Additionally, MRI was able to detect differences in the PDFF between fetal brown adipose tissue and white adipose tissue.

Measuring fetal adipose tissue using 3D water-fat magnetic resonance imaging: a feasibility study.

Purpose: Analysis of fetal adipose tissue volumes may provide useful insight towards assessment of overall fetal health, especially in cases with abnormal fetal growth. Here, we assess whether fetal adipose tissue volume can be reliably measured using 3D water-fat MRI, using a quantitative assessment of the lipid content of tissues.Materials and methods: Seventeen women with singleton pregnancies underwent a fetal MRI and water-only and fat-only images were acquired (modified 2-point Dixon technique). Water and fat images were used to generate a fat signal fraction (fat/(water + fat)) from which subcutaneous adipose tissue was segmented along the fetal trunk. Inter-rater (three readers) and intrarater reliability was assessed using intraclass-correlation coefficients (ICC) for 10 image sets. Relationships between adipose tissue measurements and gestational age and estimated fetal weight percentiles were examined.Results: The ICC of the inter-rater reliability was 0.936 (p < .001), and the ICC of the intrarater reliability was 0.992 (p < .001). Strong positive correlations were found between adipose tissue measurements (lipid volume, lipid volume/total fetal volume, mean fat signal fraction) and gestational age.Conclusions: 3D water-fat MRI can reliably measure volume and quantify lipid content of fetal subcutaneous adipose tissues.

De novo variants in CNOT3 cause a variable neurodevelopmental disorder.

As a result of exome-based sequencing work performed by the DDD study, de novo variants in CNOT3 have emerged as a newly recognised cause of a developmental disorder. This paper describes molecular and clinical details of 16 probands with developmental disorders and de novo CNOT3 variants. It is the first such description of the developmental phenotype associated with CNOT3 variants. Eight of these cases were discovered as part of the DDD study, while the other eight were found as a result of large-scale sequencing work performed by other groups. A highly specific phenotype was not recognised in these 16 cases. The most consistent phenotypic features seen in subjects with de novo variants in CNOT3 were hypotonia, relatively small stature, developmental delay, behavioural problems and intellectual disability. There is no easily recognisable facial phenotype, but some common dysmorphic features such as anteverted nares, thin upper lip and low set eyebrows were shared among some of the probands. Haploinsufficiency appears to be the most likely mechanism of action, with eight cases found to have protein-truncating variants. Of the other eight cases (all missense variants), three share an amino acid substitution at the same position which may therefore represent an important functional domain.

Secondary findings from whole-exome/genome sequencing evaluating stakeholder perspectives. A review of the literature.

With the development of next generation sequencing, beyond identifying the cause of manifestations that justified prescription of the test, other information with potential interest for patients and their families, defined as secondary findings (SF), can be provided once patients have given informed consent, in particular when therapeutic and preventive options are available. The disclosure of such findings has caused much debate. The aim of this work was to summarize all opinion-based studies focusing on SF, so as to shed light on the concerns that this question generate. A review of the literature was performed, focusing on all PubMed articles reporting qualitative, quantitative or mixed studies that interviewed healthcare providers, participants, or society regarding this subject. The methodology was carefully analysed, in particular whether or not studies made the distinction between actionable and non-actionable SF, in a clinical or research context. From 2010 to 2016, 39 articles were compiled. A total of 14,868 people were interviewed (1259 participants, 6104 healthcare providers, 7505 representatives of society). When actionable and non-actionable SF were distinguished (20 articles), 92% of respondents were keen to have results regarding actionable SF (participants: 88%, healthcare providers: 86%, society: 97%), against 70% (participants: 83%, healthcare providers: 62%, society: 73%) for non-actionable SF. These percentages were slightly lower in the specific situation of children probands. For respondents, the notion of the «patient's choice¼ is crucial. For healthcare providers, the importance of defining policies for SF among diagnostic lab, learning societies and/or countries is outlined, in particular regarding the content and extension of the list of actionable genes to propose, the modalities of information, and the access to information about adult-onset diseases in minors. However, the existing literature should be taken with caution, since most articles lack a clear definition of SF and actionability, and referred to hypothetical scenarios with limited information to respondents. Studies conducted by multidisciplinary teams involving patients with access to results are sadly lacking, in particular in the medium term after the results have been given. Such studies would feed the debate and make it possible to measure the impact of such findings and their benefit-risk ratio.

Dermatological manifestations in cardiofaciocutaneous syndrome: a prospective multicentric study of 45 mutation-positive patients.

BACKGROUND: Data on dermatological manifestations of cardiofaciocutaneous syndrome (CFCS) remain heterogeneous and almost without expert dermatological classification. OBJECTIVES: To describe the dermatological manifestations of CFCS; to compare them with the literature findings; to assess those discriminating CFCS from other RASopathies, including Noonan syndrome (NS) and Costello syndrome (CS); and to test for dermatological phenotype-genotype correlations. METHODS: We performed a 4-year, large, prospective, multicentric, collaborative dermatological and genetic study. RESULTS: Forty-five patients were enrolled. Hair abnormalities were ubiquitous, including scarcity or absence of eyebrows and wavy or curly hair in 73% and 69% of patients, respectively. Keratosis pilaris (KP), ulerythema ophryogenes (UO), palmoplantar hyperkeratosis (PPHK) and multiple melanocytic naevi (MMN; over 50 naevi) were noted in 82%, 44%, 27% and 29% of patients, respectively. Scarcity or absence of eyebrows, association of UO and PPHK, diffuse KP and MMN best differentiated CFCS from NS and CS. Oral acitretin may be highly beneficial for therapeutic management of PPHK, whereas treatment of UO by topical sirolimus 1% failed. No significant dermatological phenotype-genotype correlation was determined. CONCLUSIONS: A thorough knowledge of CFCS skin manifestations would help in making a positive diagnosis and differentiating CFCS from CS and NS.

Dermatological manifestations in Noonan syndrome: a prospective multicentric study of 129 patients positive for mutation.

BACKGROUND: Data on dermatological manifestations of Noonan syndrome (NS) remain heterogeneous and are based on limited dermatological expertise. OBJECTIVES: To describe the dermatological manifestations of NS, compare them with the literature findings, and test for dermatological phenotype-genotype correlations with or without the presence of PTPN11 mutations. METHODS: We performed a large 4-year, prospective, multicentric, collaborative dermatological and genetic study. RESULTS: Overall, 129 patients with NS were enrolled, including 65 patients with PTPN11-NS, 34 patients with PTPN11-NS with multiple lentigines (NSML), and 30 patients with NS who had a mutation other than PTPN11. Easy bruising was the most frequent dermatological finding in PTPN11-NS, present in 53·8% of patients. Multiple lentigines and café-au-lait macules (n ≥ 3) were present in 94% and 80% of cases of NSML linked to specific mutations of PTPN11, respectively. Atypical forms of NSML could be associated with NS with RAF1 or NRAS mutations. In univariate analysis, patients without a PTPN11 mutation showed (i) a significantly higher frequency of keratinization disorders (P = 0·001), including keratosis pilaris (P = 0·005), ulerythema ophryogenes (P = 0·0001) and palmar and/or plantar hyperkeratosis (P = 0·06, trend association), and (ii) a significantly higher frequency of scarce scalp hair (P = 0·035) and scarce or absent eyelashes (P = 0·06, trend association) than those with PTPN11 mutations. CONCLUSIONS: The cutaneous phenotype of NS with a PTPN11 mutation is generally mild and nonspecific, whereas the absence of a PTPN11 mutation is associated with a high frequency of keratinization disorders and hair abnormalities.

TBL1XR1 mutations in Pierpont syndrome are not restricted to the recurrent p.Tyr446Cys mutation.

Pierpont syndrome is a rare and sporadic syndrome, including developmental delay, facial characteristics, and abnormal extremities. Recently, a recurrent de novo TBL1XR1 variant (c.1337A > G; p.Tyr446Cys) has been identified in eight patients by whole-exome sequencing. A dominant-negative effect of this mutation is strongly suspected, since patients with TBL1XR1 deletion and other variants predicting loss of function do not share the same phenotype. We report two patients with typical Pierpont-like syndrome features. Exome sequencing allowed identifying a de novo heterozygous missense TBL1XR1 variant in both patients, different from those already reported: p.Cys325Tyr and p.Tyr446His. The localization of these mutations and clinical features of Pierpont-like syndrome suggest that their functional consequences are comparable with the recurrent mutation previously described, and provided additional data to understand molecular mechanisms of TBL1XR1 anomalies.

Super-resolution fluorescence microscopy by stepwise optical saturation.

Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the super-resolution microscopy is not feasible in many applications. In this paper, we propose and demonstrate a saturation-based super-resolution fluorescence microscopy technique that can be easily implemented and requires neither additional hardware nor complex post-processing. The method is based on the principle of stepwise optical saturation (SOS), where M steps of raw fluorescence images are linearly combined to generate an image with a [Formula: see text]-fold increase in resolution compared with conventional diffraction-limited images. For example, linearly combining (scaling and subtracting) two images obtained at regular powers extends the resolution by a factor of 1.4 beyond the diffraction limit. The resolution improvement in SOS microscopy is theoretically infinite but practically is limited by the signal-to-noise ratio. We perform simulations and experimentally demonstrate super-resolution microscopy with both one-photon (confocal) and multiphoton excitation fluorescence. We show that with the multiphoton modality, the SOS microscopy can provide super-resolution imaging deep in scattering samples.

Three-dimensional computer-assisted dissection of pancreatic lymphatic anatomy on human fetuses: a step toward automatic image alignment.

PURPOSE: Pancreatic cancer is the fourth cause of death by cancer worldwide. Lymph node (LN) involvement is known to be the main prognostic factor. However, lymphatic anatomy is complex and only partially characterized. The aim of the study was to study the pancreatic lymphatic system using computer-assisted anatomic dissection (CAAD) technique and also to update CAAD technique by automatizing slice alignment. METHODS: We dissected three human fetuses aged from 18 to 34 WA. 5-µm serial sections of duodeno-pancreas and spleen blocks were stained (hematoxylin-eosin, hematoxylin of Mayer and Masson trichrome), scanned, aligned and modeled in three dimensions. RESULTS: We observed a rich, diffuse but not systematized lymphatic network in the peri-pancreatic region. There was an equal distribution of LNs between the cephalic and body-tail portions. The lymphatic vascularization appeared in continuity from the celiac trunk to the distal ends of its hepatic and splenic arterial branches parallel to the nerve ramifications of the celiac plexus. We also observed a continuity between the drainage of the pancreatic head and the para-aortic region posteriorly. CONCLUSION: In view of the wealth of peri-pancreatic LNs, the number of LNs to harvest could be increased to improve nodal staging and prognostic evaluation. Pancreatic anatomy as described does not seem to be compatible with the sentinel LN procedure in pancreatic surgery. Finally, we are now able to offer an alternative to manual alignment with a semi-automated alignment.

MED13L-related intellectual disability: involvement of missense variants and delineation of the phenotype.

Molecular anomalies in MED13L, leading to haploinsufficiency, have been reported in patients with moderate to severe intellectual disability (ID) and distinct facial features, with or without congenital heart defects. Phenotype of the patients was referred to "MED13L haploinsufficiency syndrome." Missense variants in MED13L were already previously described to cause the MED13L-related syndrome, but only in a limited number of patients. Here we report 36 patients with MED13L molecular anomaly, recruited through an international collaboration between centers of expertise for developmental anomalies. All patients presented with intellectual disability and severe language impairment. Hypotonia, ataxia, and recognizable facial gestalt were frequent findings, but not congenital heart defects. We identified seven de novo missense variations, in addition to protein-truncating variants and intragenic deletions. Missense variants clustered in two mutation hot-spots, i.e., exons 15-17 and 25-31. We found that patients carrying missense mutations had more frequently epilepsy and showed a more severe phenotype. This study ascertains missense variations in MED13L as a cause for MED13L-related intellectual disability and improves the clinical delineation of the condition.

Wiedemann-Steiner syndrome as a major cause of syndromic intellectual disability: A study of 33 French cases.

Wiedemann-Steiner syndrome (WSS) is a rare syndromic condition in which intellectual disability (ID) is associated with hypertrichosis cubiti, short stature, and characteristic facies. Following the identification of the causative gene (KMT2A) in 2012, only 31 cases of WSS have been described precisely in the literature. We report on 33 French individuals with a KMT2A mutation confirmed by targeted gene sequencing, high-throughput sequencing or exome sequencing. Patients' molecular and clinical features were recorded and compared with the literature data. On the molecular level, we found 29 novel mutations. We observed autosomal dominant transmission of WSS in 3 families and mosaicism in one family. Clinically, we observed a broad phenotypic spectrum with regard to ID (mild to severe), the facies (typical or not of WSS) and associated malformations (bone, cerebral, renal, cardiac and ophthalmological anomalies). Hypertrichosis cubiti that was supposed to be pathognomonic in the literature was found only in 61% of our cases. This is the largest series of WSS cases yet described to date. A majority of patients exhibited suggestive features, but others were less characteristic, only identified by molecular diagnosis. The prevalence of WSS was higher than expected in patients with ID, suggesting than KMT2A is a major gene in ID.