Local occurrence and fast spread of B.1.1.7 lineage: A glimpse into Friuli Venezia Giulia

In-depth study of the entire SARS-CoV-2 genome has uncovered many mutations, which have replaced the lineage that characterized the first wave of infections all around the world. In December 2020, the outbreak of variant of concern (VOC) 202012/01 (lineage B.1.1.7) in the United Kingdom defined a turning point during the pandemic, immediately posing a worldwide threat on the Covid-19 vaccination campaign. Here, we reported the evolution of B.1.1.7 lineage-related infections, analyzing samples collected from January 1st 2021, until April 15th 2021, in Friuli Venezia Giulia, a northeastern region of Italy. A cohort of 1508 nasopharyngeal swabs was analyzed by High Resolution Melting (HRM) and 479 randomly selected samples underwent Next Generation Sequencing analysis (NGS), uncovering a steady and continuous accumulation of B.1.1.7 lineage-related specimens, joined by sporadic cases of other known lineages (i.e. harboring the Spike glycoprotein p.E484K mutation). All the SARS-CoV-2 genome has been analyzed in order to highlight all the rare mutations that may eventually result in a new variant of interest. This work suggests that a thorough monitoring of the SARS-CoV-2 genome by NGS is essential to contain any new variant that could jeopardize all the efforts that have been made so far to resolve the emergence of the pandemic.

Carcinosarcoma, a Rare Malignant Neoplasm of the Pancreas

Carcinosarcoma of the pancreas is a rare entity with poor prognosis. Here, we report a case of pancreatic carcinosarcoma in a 68-year-old male patient who underwent a pancreatoduodenectomy for a unilocular cystic mass in the head of the pancreas. Histologically, the lesion showed a biphasic tumor with a carcinoma component and a spindle cell sarcomatous component, which were intimately intermingled. Most of the carcinoma components are well-differentiated ductal adenocarcinoma with small areas of moderately to poorly differentiated ductal adenocarcinoma. The sarcomatous component is a high-grade highly cellular spindle cell tumor with frequent mitosis and apoptosis. Immunohistochemical studies demonstrated that the carcinomatous component was positive for epithelial markers and cyclin D1, and the sarcomatous component was negative for these markers while positive for vimentin, p16, and DOG1 with patchy positivity for S100. Other markers, including SOX10, CD117, Melan A, HMB45, actin, desmin, myogenin, beta-catenin, TLE1, and p53, were negative in both components. Molecular studies demonstrated that the tumor was microsatellite stable. Whole exome next generation sequencing analysis was performed and no pathogenic alterations in the genes were identified.

Analysis of 29 Targeted Genes for Non-obstructive Azoospermia: the Relationship Between Genetic Testing and Testicular Histology

BACKGROUND. Few studies have evaluated the relationship between testicular histology and pathogenic variations of genes regulating spermatogenesis.AIM. To analyze the presence of potentially pathogenic variants of 29 candidate genes known to cause spermatogenic failure (SPGF) in patients with non-obstructive azoospermia (NOA) who underwent testicular histology.PATIENTS AND METHODS. Sixty patients with NOA referred to the Department of Transfusion Medicine and Transplantation Biology, University Hospital Center Zagreb, Croatia, for testicular biopsy were consecutively assessed for eligibility. Twelve patients were excluded from the study because they had Klinefelter syndrome (n=1), Yq microdeletions (n=6), testicular trauma (n=2), or in-situ germ cell neoplasia (n=3). Therefore, 48 patients were considered eligible and included in this study. They were divided into three groups: those who had cryptorchidism (n=9), those with varicocele (n=14), and those with idiopathic NOA (n=25). All included patients underwent blood withdrawal for next-generation sequencing analysis and gene sequencing.RESULTS. We found a possible genetic cause in 4 patients with idiopathic NOA (16%) and in 2 with cryptorchidism (22%). No pathogenic or possibly pathogenic mutations were identified in patients with varicocele. Variants of undetermined significance (VUS) were found in 11 patients with idiopathic NOA (44%), 3 with cryptorchidism (33%), and 8 patients with varicocele (57%). VUSs of the USP9Y gene were the most frequently as they were found in 14 out of 48 patients (29%). In particular, the VUS USP9Y c.7434+14del was found in 11 patients. They showed varied histological pictures, including Sertoli cell-only syndrome, mixed atrophy, and hypospermatogenesis, regardless of cryptorchidism or varicocele. No direct correlation was found between the gene mutation/variant and the testicular histological picture. CONCLUSION. Different mutations of the same gene cause various testicular histological pictures. These results suggest that it is not the gene itself but the type of mutation/variation that determines the testicular histology picture. Based on the data presented above, it remains challenging to design a genetic panel with prognostic value for the outcome of testicular sperm extraction in patients with NOA.

Circulating Antibodies to Skin Bacteria Detected by Serological Lateral Flow Immunoassays Differentially Correlated With Bacterial Abundance

The serological lateral flow immunoassay (LFIA) was used to detect circulating antibodies to skin bacteria. Next-generation sequencing analysis of the skin microbiome revealed a high relative abundance of Cutibacterium acnes but low abundance of Staphylococcus aureus and Corynebacterium aurimucosum on human facial samples. Yet, results from both LFIA and antibody titer quantification in 96-well microplates illustrated antibody titers that were not correspondent, and instead negatively correlated, to their respective abundance with human blood containing higher concentrations of antibodies to both S. aureus and C. aurimucosum than C. acnes. Acne vulgaris develops several unique microbial and cellular features, but its correlation with circulating antibodies to bacteria in the pilosebaceous unit remains unknown. Results here revealed that antibodies to C. acnes and S. aureus were approximately 3-fold higher and 1.5-fold lower, respectively, in acne patients than in healthy subjects. Although the results can be further validated by larger sample sizes, the proof-of-concept study demonstrates a newfound discrepancy between the abundance of skin bacteria and amounts of their corresponding antibodies. And in light of acne-correlated amplified titers of specific anticommensal antibodies, we highlight that profiling these antibodies in the pilosebaceous unit by LFIAs may provide a unique signature for monitoring acne vulgaris.

Intertumoural Heterogeneity and Branch Evolution of Synchronous Multiple Primary Lung Adenocarcinomas by Next-Generation Sequencing Analysis

ObjectivesMultiple primary lung cancers (MPLCs) are an increasingly well-known clinical phenomenon, but there is a lack of high-level evidence for their optimal clinical diagnosis and therapeutic approaches. Thus, we analysed genetic variation to determine the intertumoural heterogeneity and branch evolution of synchronous multiple primary lung adenocarcinomas.MethodsWe performed multiplex mutational sequencing on 93 synchronous multiple primary lung adenocarcinoma lesions from 42 patients who underwent surgical resection.ResultsThe high discordance rate of mutation was 92.9% (n=39) between tumours in individual patients. EGFR, TP53 and KRAS mutations were detected in 57 (61.3%), 19 (20.4%) and 11 (11.8%) of the 93 tumours, respectively. 16 cases of multiple primary lung adenocarcinomas simultaneously harboured EGFR mutations and TP53 mutations. Matching mutations between paired tumours were observed in 1 (2.4%) patient for P20. The genotypes were all EGFR L858R mutations, but the pathological type of P20T1 was lepidic predominant, and P20T2 was adenocarcinoma in situ. In the phylogenetic tree, genetic variations were divided into trunk, shared and branch subtypes. Branch mutations accounted for 91.09% of variations in sMPLA, while the ratio of trunk (4.95%) and shared (3.96%) variations was significantly lower.ConclusionsRemarkable intertumoural heterogeneity and frequent branch mutations were found in synchronous multiple primary lung adenocarcinomas.

The preoperative abundance of Prevotella associates with anastomotic healing after colorectal surgery.

Anastomotic leakage (AL) is a frequent and potentially life-threatening postoperative complication in patients undergoing colorectal surgery with a multifactorial yet unknown pathophysiology. In the present study, we aimed to unravel AL etiology and intestinal anastomotic healing (AH) physiology focusing on both host epithelial and gut microbial components. We make use of patients-related samples and two high throughput next generation sequencing analysis approaches, including anastomotic tissue-associated bulk RNA sequencing and preoperative fecal 16S rRNA gene sequencing. We find that in the absence of host epithelial gene expression differences, the preoperative fecal microbiota of patients who undergo colorectal resection with successful AH differs in the group of patients who develop AL in the abundance of Prevotella and suggests a potential beneficial role of Prevotella that may prove useful as predictive biomarker for AH.

A dual-reporter system for investigating and optimizing protein translation and folding in E. coli

Strategies for investigating and optimizing the expression and folding of proteins for biotechnological and pharmaceutical purposes are in high demand. Here, we describe a dual-reporter biosensor system that simultaneously assesses in vivo protein translation and protein folding, thereby enabling rapid screening of mutant libraries. We have validated the dual-reporter system on five different proteins and find an excellent correlation between reporter signals and the levels of protein expression and solubility of the proteins. We further demonstrate the applicability of the dual-reporter system as a screening assay for deep mutational scanning experiments. The system enables high throughput selection of protein variants with high expression levels and altered protein stability. Next generation sequencing analysis of the resulting libraries of protein variants show a good correlation between computationally predicted and experimentally determined protein stabilities. We furthermore show that the mutational experimental data obtained using this system may be useful for protein structure calculations.

Bartonella and Coxiella infections presenting as systemic vasculitis: case series and review of literature

Objectives Coxiella and Bartonella sp. display particular tropism for endothelial or endocardial tissues and an abnormal host response to infections with induced autoimmunity. We aimed, through a case series combined with a comprehensive literature review, to outline characteristics of Coxiella and Bartonella infections presenting as systemic vasculitis. Methods We retrospectively included cases of definite Coxiella and Bartonella infections presenting with vasculitis features and performed a comprehensive literature review. Results Six cases of Bartonella infections were added to 18 cases from literature review. Causative pathogens were mainly B. henselae. Bartonella infection mimicked anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis in 83% with PR3-ANCA and presented as cryoglobulinemic vasculitis in 8%. Glomerulonephritis was present in 92%, and 88% had endocarditis. Complement fractions were low in 82% and rheumatoid factor positive in 85%. Kidney biopsies showed cell proliferation, mostly crescentic, with pauci-immune glomerulonephritis in 29%. Outcome was favorable, with the use of antibiotics alone in one third. Five cases of Coxiella infections were added to 16 from literature review. Sixteen had small-vessel vasculitides, mainly cryoglobulinemia vasculitis in 75%. One patient had polyarteritis nodosa-like vasculitis and four large-vessel vasculitis. Outcome was good except for one death. A highly sensitive next generation sequencing analysis on 3 Coxiella and 2 Bartonella-related vasculitides biopsies did not find any bacterial DNA. Conclusion Coxiella and Bartonella are both able to induce vasculitis but display distinct vasculitis features. Bartonella mimics PR3-ANCA-associated vasculitis in the setting of endocarditis, whereas Coxiella may induce vasculitis involving all vessel sizes.

Next-Generation Sequencing Analysis of Root Canal Microbiota Associated with a Severe Endodontic-Periodontal Lesion

A patient with an unusual endo-periodontal lesion, without coronal decay or damage, likely caused by a deep periodontal lesion with subsequent endodontic bacterial migration, required medical care. Next-generation sequencing (NGS) was used to assess the endodontic microbiota in vestibular and palatal canals after tooth extraction, evidencing a predominant population (Fusobacterium nucleatum) in one endodontic canal, and a mixed bacterial population with six major populations almost equally distributed in the other endodontic canal (F. nucleatum, Porphyromonas gingivalis, P. endodontis, Parvimonas, Peptostreptococcus stomatis, Prevotella multiformis). These data could suggest different, separated ecologic niches in the same endodontic system, with potentially different pathogenicity levels, clinical manifestations and prognoses for every single canal of the same tooth.