CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy–associated TCF4 triplet repeat

PURPOSE: To demonstrate the utility of an amplification-free long-read sequencing method to characterize the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (CTG18.1). METHODS: We applied an amplification-free method, utilizing the CRISPR/Cas9 system, in combination with PacBio single-molecule real-time (SMRT) long-read sequencing, to study CTG18.1. FECD patient samples displaying a diverse range of CTG18.1 allele lengths and zygosity status (n = 11) were analyzed. A robust data analysis pipeline was developed to effectively filter, align, and interrogate CTG18.1-specific reads. All results were compared with conventional polymerase chain reaction (PCR)-based fragment analysis. RESULTS: CRISPR-guided SMRT sequencing of CTG18.1 provided accurate genotyping information for all samples and phasing was possible for 18/22 alleles sequenced. Repeat length instability was observed for all expanded (≥50 repeats) phased CTG18.1 alleles analyzed. Furthermore, higher levels of repeat instability were associated with increased CTG18.1 allele length (mode length ≥91 repeats) indicating that expanded alleles behave dynamically. CONCLUSION: CRISPR-guided SMRT sequencing of CTG18.1 has revealed novel insights into CTG18.1 length instability. Furthermore, this study provides a framework to improve the molecular diagnostic accuracy for CTG18.1-mediated FECD, which we anticipate will become increasingly important as gene-directed therapies are developed for this common age-related and sight threatening disease

Atividade Antimicrobiana de Peptídeos Obtidos de Caseína Bovina

<p class="MsoNormal" style="margin: 0cm 0cm 0pt; line-height: normal; text-align: justify; mso-layout-grid-align: none;"> As proteínas de leite são precursoras de vários peptídeos biologicamente ativos. Estes, inativos dentro da seqüência da proteína original, podem ser liberados pela proteólise enzimática. Após a hidrólise, algumas proteínas apresentam atividade antibacteriana. Caseinofosfopeptídeos (CPP) podem ser liberados in vivo na digestão normal da caseína ou formados in vitro pela hidrólise enzimática. O objetivo do trabalho foi a investigação do potencial de peptídeos obtidos pela hidrólise tríptica da caseína em inibir o crescimento de bactérias. Os peptídeos foram testados contra as bactérias Gram-positivas Listeria monocytogenes e Staphylococcus aureus e as bactérias Gram-negativas, Escherichia coli e Salmonella typhimurium. Foram testadas diferentes formas de produção de CPP. O peptídeo denominado CPP-A apresentou atividade bacteriostática contra Escherichia coli a 1 mg/mL e 0,2 mg/mL. O CPP-C inibiu o crescimento de Staphylococcus aureus, Escherichia coli e Salmonella typhimurium em quase todas as concentrações testadas. A Listeria monocytogenes foi inibida por todos os peptídeos testados

Minimally invasive approach to incisional hernia in elective and emergency surgery: a SICE (Italian Society of Endoscopic Surgery and new technologies) and ISHAWS (Italian Society of Hernia and Abdominal Wall Surgery) online survey

Minimally invasive abdominal wall surgery is growing worldwide, with a constant and fast improvement of surgical techniques and surgeons’ confidence in treating both primary and incisional hernias (IH). The Italian Society of Endoscopic Surgery and new technologies (SICE) and the ISHAWS (Italian Society of Hernia and Abdominal Wall Surgery) worked together to investigate state of the art in IH treatment in elective and emergency settings in Italy. An online open survey was designed, and Italian surgeons interested in abdominal wall surgery were invited to fill out a 20-point questionnaire on IH surgical procedures performed in their departments. Surgeons were asked to express their points of view on specific questions about technical and clinical variables in IH treatment. Preferred approach in elective IH surgery was minimally invasive (59.7%). Open surgery was the preferred approach in 40.3% of the responses. In emergency settings, open surgery was the preferred approach (65.4%); however, 34.5% of the involved surgeons declare to prefer the laparoscopic/endoscopic approach. Most respondents opted for conversion to open surgery in case of relevant surgical field contamination, with a non-mesh repair of abdominal wall defects. Among those that used the laparoscopic approach in the emergent setting, the majority (74%) used the size of the defect of 5 cm as a decisional cut-off. The spread of minimally invasive approaches to IH repair in emergency surgery in Italy is gaining relevance. Code-sharing through scientific societies can improve clinical practice in different departments and promote a tailored approach to IH surgery