Case report: Tissue positivity for SARS-CoV-2 in a preterm born infant death of thrombosis: possible intrauterine transmission

Intrauterine transmission of SARS-CoV-2 (Severe Acute Respiratory Syndrome Corona Virus 2) is still matter of debate among scientists and there is limited information concerning this aspect of research. This could lead to severe complications of the growing fetus and, theoretically, of the newborn as well. We report the case of a male infant of 1,100 grams, born at 27th week of gestation to a SARS-CoV-2 mother, tested negative for viral detection at delivery. He was immediately admitted to neonatal Intensive Care Unit (ICU) for severe complications, where he died after 37 days by pulmonary embolism and thrombosis of the superior vena cava. After autopsy, SARS-CoV-2 N-protein and Spike RBD were detected in several tissues, particularly in the esophagus, stomach, spleen, and heart, with a significantly higher H-Score than the placenta. In conclusion, immunohistochemical analysis demonstrated SARS-CoV-2 NP and Spike RBD positivity in different tissues suggesting a possible intrauterine transmission. Newborn thrombo-embolism could be a complication of SARS-CoV-2 infection as observed in adult patients

A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field

Genetic markers (DNA barcodes) are often used to support and confirm species identification. Barcode sequences can be generated in the field using portable systems based on the Oxford Nanopore Technologies (ONT) MinION sequencer. However, to achieve a broader application, current proof-of-principle workflows for on-site barcoding analysis must be standardized to ensure a reliable and robust performance under suboptimal field conditions without increasing costs. Here, we demonstrate the implementation of a new on-site workflow for DNA extraction, PCR-based barcoding, and the generation of consensus sequences. The portable laboratory features inexpensive instruments that can be carried as hand luggage and uses standard molecular biology protocols and reagents that tolerate adverse environmental conditions. Barcodes are sequenced using MinION technology and analyzed with ONTrack, an original de novo assembly pipeline that requires as few as 1000 reads per sample. ONTrack-derived consensus barcodes have a high accuracy, ranging from 99.8 to 100%, despite the presence of homopolymer runs. The ONTrack pipeline has a user-friendly interface and returns consensus sequences in minutes. The remarkable accuracy and low computational demand of the ONTrack pipeline, together with the inexpensive equipment and simple protocols, make the proposed workflow particularly suitable for tracking species under field conditions

Hydraena (s.str.) dinarica, new species (Coleoptera: Hydraenidae) along with further records of Hydraena spp. from Durmitor National Park, Montenegro and comments on the DNA barcoding problem with the genus

Background Long-palped Water Beetles were collected during a taxon expedition in Montenegro which involved citizen scientists, students and taxonomists. The material was collected from springs, brooks, fens and the Tara River, at altitudes between 600 m and 1450 m above sea level, using fine-meshed hand-nets and by manual checking of submerged substrates. The morphological species delimitation was supplemented and congruent with mtDNA sequences mainly obtained in the field using the newly-developed MinION-based ONTrack pipeline. New information The new species Hydraena dinarica Freitag & de Vries, sp. n. from Durmitor Mt. is described, illustrated and compared in detail to closely-related congeners of the H. saga d\u27Orchymont, 1930/H. emarginata Rey, 1885 species complex. Five additional species and female specimens of two unidentified morphospecies of the genus were also recorded in the vicinity of Durmitor National Park. New records and the first DNA barcodes for Hydraena biltoni Jäch & Díaz, 2012 (endemic to Montenegro) and H. morio Kiesenwetter, 1849 are provided. Further records of H. nigrita Germar, 1824, H. minutissima Stephens, 1829, H. subintegra Ganglbauer, 1901 and females of two unidentified morphospecies are commented upon. The resulting inter- and intraspecific genetic distances and some observations of low or zero sequence divergence between recently-diverged species of Hydraena Kugelann, 1794 are briefly discussed

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

An Altered Splicing Registry Explains the Differential ExSpeU1-Mediated Rescue of Splicing Mutations Causing Haemophilia A

The exon recognition and removal of introns (splicing) from pre-mRNA is a crucial step in the gene expression flow. The process is very complex and therefore susceptible to derangements. Not surprisingly, a significant and still underestimated proportion of disease-causing mutations affects splicing, with those occurring at the 5' splice site (5'ss) being the most severe ones. This led to the development of a correction approach based on variants of the spliceosomal U1snRNA, which has been proven on splicing mutations in several cellular and mouse models of human disease. Since the alternative splicing mechanisms are strictly related to the sequence context of the exon, we challenged the U1snRNA-mediated strategy in the singular model of the exon 5 of coagulation factor (F)VIII gene (F8) in which the authentic 5'ss is surrounded by various cryptic 5'ss. This scenario is further complicated in the presence of nucleotide changes associated with FVIII deficiency (Haemophilia A), which weaken the authentic 5'ss and create/strengthen cryptic 5'ss. We focused on the splicing mutations (c.602-32A > G, c.602-10T > G, c.602G > A, c.655G > A, c.667G > A, c.669A > G, c.669A > T, c.670G > T, c.670+1G > T, c.670+1G > A, c.670+2T > G, c.670+5G > A, and c.670+6T > C) found in patients with severe to mild Haemophilia A. Minigenes expression studies demonstrated that all mutations occurring within the 5'ss, both intronic or exonic, lead to aberrant transcripts arising from the usage of two cryptic intronic 5'ss at positions c.670+64 and c.670+176. For most of them, the observed proportion of correct transcripts is in accordance with the coagulation phenotype of patients. In co-transfection experiments, we identified a U1snRNA variant targeting an intronic region downstream of the defective exon (Exon Specific U1snRNA, U1sh7) capable to re-direct usage of the proper 5'ss (similar to 80%) for several mutations. However, deep investigation of rescued transcripts from +1 and +2 variants revealed only the usage of adjacent cryptic 5'ss, leading to frameshifted transcript forms. These data demonstrate that a single ExSpeU1 can efficiently rescue different mutations in the F8 exon 5, and provide the first evidence of the applicability of the U1snRNA-based approach to Haemophilia A

Valutazione di parametri prognostici nel carcinoma del colon-retto. II. Ploidia mediante citometria a flusso.

none4noValutazione di parametri prognostici nel carcinoma del colon-retto. Valutazione del contenuto nucleare di DNA ( ploidia) mediante citometria a flusso.noneG. LANZA; MAESTRI I.; DUBINI A.; GAFA' R.Lanza, Giovanni; Maestri, Iva; Dubini, A.; Gafa', Robert

Exon-Specific U1snRNA-Mediated Rescue of Splicing and Missense Changes in Hemophilia A

Background. Splicing mutations account for 8-10% of Hemophilia A (HA)-causing defects, a highly underestimated proportion since even exonic variants, besides acting on protein biology, can affect splicing regulatory elements. In this context, splicing-rescuing approaches might represent innovative personalized therapies. Over years we demonstrated that engineered variants of the spliceosomal U1snRNA, named exon-specific U1snRNA (ExSpeU1), can correct multiple splicing mutations for therapeutic purposes. Aims. To elucidate the molecular mechanisms underlying all HA-causing mutations on exon 19 and to test ExSpeU1s as a correction strategy. Methods. In vitro expression of F8 exon 19 minigenes to assess splicing pattern. Expression via lentiviral vectors (LV) of FVIII missense variants and evaluation of FVIII antigen (ELISA) and activity (chromogenic assays) levels. Results. Highly variable degree of aberrant splicing was observed, ranging from complete exon 19 skipping for all changes at the 5’ splice site (5’ss) to different proportions of exon 19 inclusion for exonic changes (p.Gly2000Ala, p.Arg2016Gly and p.Tyr2036Tyr). FVIII protein expression studies demonstrated that the p.Arg2016Gly change leads to reduced antigen and activity levels (8.3±1.6% and 10.7±1.0% of wild-type, respectively). Differently, the p.Gly2000Ala change did not affect FVIII antigen nor activity, indicating a major effect on splicing for this variant. Co-transfection experiments led to the identification of a single ExSpeU1, designed to minimize potential off-target effects, able to properly restore splicing. In particular, the ExSpeU1 was able to completely rescue (>90%) splicing variants (c.6115+3G>T, c.6115+4A>G and c.6115+6T>A) as well as exonic changes (p.Gly2000Ala and p.Tyr2036Tyr). Conclusions. Overall, we provided insights into the molecular mechanisms underlying HA caused by all splicing and exonic changes in exon 19, strengthening the notion that also exonic mutations can impair splicing process by affecting splicing regulatory elements. Moreover, we provided evidence of the ability of a single ExSpeU1 in rescuing multiple HA-causing mutations, thus expanding the therapeutic potential of this approach

Evaluation of FV mRNA to define the residual FV expression levels in severe FV deficiency

We evaluated FV mRNA in severe factor V deficiency caused by the -12T/A IVS18 mutation, activating a cryptic splice site and leading to premature translation termination. Quantitative evaluation of factor V cDNA from homozygous and heterozygous subjects, and correction for nonsense mediated decay, suggested the presence of 0.1% of normal factor V mRNA

Next-generation sequencing and recombinant expression characterized aberrant splicing mechanisms and provided correction strategies in factor VII deficiency

Despite the exhaustive screening of F7 gene exons and exon-intron boundaries and promoter region, a significant proportion of mutated alleles remains unidentified in patients with coagulation factor VII deficiency. Here, we applied next-generation sequencing to thirteen FVII-deficient patients displaying genotype-phenotype discrepancies upon conventional sequencing, and identified six rare intronic variants. Computational analysis predicted splicing effects for three of them, which would strengthen (c.571+78G>A; c.806-329G>A) or create (c.572-392C>G) intronic 5' splice sites (5'ss). In F7 minigenes assays the c.806-329G>A was ineffective while the c.571+78G>A change led to usage of the +79 cryptic 5'ss with only trace levels of correct transcripts (3% of wild-type), in accordance with factor VII activity levels in homozygotes (1-3% of normal). The c.572-392C>G change led to pseudo-exonization and frame-shift, but also substantial levels of correct transcripts (~70%). However, this variant was associated with the common F7 polymorphic haplotype predicted to further decrease factor VII levels, thus roughly explaining the factor VII levels of 10% in the homozygous patient. Intriguingly, the effect of the c.571+78G>A and c.572-392C>G changes, and particularly of the former, the severest and well-represented in our cohort, was counteracted by antisense U7snRNA variants targeting the intronic 5'ss, thus demonstrating their pathogenic role. In conclusion, the combination of next-generation sequencing of the entire F7 gene with the minigene expression studies elucidated the molecular bases of factor VII deficiency in ten out of thirteen patients, thus improving diagnosis and genetic counselling, and provided a potential therapeutic approach based on antisense molecules, successfully exploited in other disorders