Role of prophylactic central compartment lymph node dissection in clinically N0 differentiated thyroid cancer patients: Analysis of risk factors and review of modern trends

In the last years, especially thanks to a large diffusion of ultrasound-guided FNBs, a surprising increased incidence of differentiated thyroid cancer (DTC), "small" tumors and microcarcinomas have been reported in the international series. This led endocrinologists and surgeons to search for "tailored" and "less aggressive" therapeutic protocols avoiding risky morbidity and useless "overtreatment". Considering the most recent guidelines of referral endocrine societies, we analyzed the role of routine or so-called prophylactic central compartment lymph node dissection (RCLD), also considering its benefits and risks. Literature data showed that the debate is still open and the surgeons are divided between proponents and opponents of its use. Even if lymph node metastases are commonly observed, and in up to 90 % of DTC cases micrometastases are reported, the impact of lymphatic involvement on long-term survival is subject to intensive research and the best indications of lymph node dissection are still controversial. Identification of prognostic factors for central compartment metastases could assist surgeons in determining whether to perform RLCD. Considering available evidence, a general agreement to definitely reserve RCLD to "high-risk" cases was observed. More clinical researches, in order to identify risk factors of meaningful predictive power and prospective long-term randomized trials, should be useful to validate this selective approach

The Streptococcus pneumoniae Pilus-1 Displays a Biphasic Expression Pattern

The Streptococcus pneumoniae pilus-1 is encoded by pilus islet 1 (PI-1), which has three clonal variants (clade I, II and III) and is present in about 30% of clinical pneumococcal isolates. In vitro and in vivo assays have demonstrated that pilus-1 is involved in attachment to epithelial cells and virulence, as well as protection in mouse models of infection. Several reports suggest that pilus-1 expression is tightly regulated and involves the interplay of numerous genetic regulators, including the PI-1 positive regulator RlrA. In this report we provide evidence that pilus expression, when analyzed at the single-cell level in PI-1 positive strains, is biphasic. In fact, the strains present two phenotypically different sub-populations of bacteria, one that expresses the pilus, while the other does not. The proportions of these two phenotypes are variable among the strains tested and are not influenced by genotype, serotype, growth conditions, colony morphology or by the presence of antibodies directed toward the pilus components. Two sub-populations, enriched in pilus expressing or not expressing bacteria were obtained by means of colony selection and immuno-detection methods for five strains. PI-1 sequencing in the two sub-populations revealed the absence of mutations, thus indicating that the biphasic expression observed is not due to a genetic modification within PI-1. Microarray expression profile and western blot analyses on whole bacterial lysates performed comparing the two enriched sub-populations, revealed that pilus expression is regulated at the transcriptional level (on/off regulation), and that there are no other genes, in addition to those encoded by PI-1, concurrently regulated across the strains tested. Finally, we provide evidence that the over-expression of the RrlA positive regulator is sufficient to induce pilus expression in pilus-1 negative bacteria. Overall, the data presented here suggest that the observed biphasic pilus expression phenotype could be an example of bistability in pneumococcus

Increased serum miR-193a-5p during non-alcoholic fatty liver disease progression: diagnostic and mechanistic relevance

Background & Aims: Serum microRNAs (miRNAs) levels are known to change in non-alcoholic fatty liver disease (NAFLD) and may serve as useful biomarkers. This study aimed to profile miRNAs comprehensively at all NAFLD stages.Methods: We profiled 2,083 serum miRNAs in a discovery cohort (183 NAFLD cases representing the complete NAFLD spectrum and 10 population controls). MiRNA libraries generated by HTG EdgeSeq were sequenced by Illumina NextSeq. Selected serum miRNAs were profiled in 372 additional NAFLD cases and 15 population controls by quantitative reverse transcriptase-polymerase chain reaction.Results: Levels of 275 miRNAs differed between cases and population controls. Fewer differences were seen within individual NAFLD stages but miR-193a-5p consistently the showed increased levels in all comparisons. Relative to NAFL/NASH with mild fibrosis (stage 0/1), three miRNAs (miR-193a-5p, miR-378d and miR378d) were increased in cases with NASH and clinically significant fibrosis (stage 2-4), seven (miR193a-5p, miR-378d, miR-378e, miR-320b, c, d & e) increased in cases with NAFLD Activity Score (NAS) 5-8 compared with lower NAS, and three (miR-193a-5p, miR-378d, miR-378e) increased but one (miR-19b-3p) decreased in steatosis, activity, and fibrosis "activity" (SAF-A) score 2-4 compared with lower SAF-A. The significant findings for miR-193a-5p were replicated in the additional NAFLD cohort. Studies in Hep G2 cells showed that following palmitic acid treatment, miR-193a-5p expression decreased significantly. Gene targets for miR-193a-5p were investigated in liver RNAseq data for a case subgroup (n=80); liver GPX8 levels correlated positively with serum miR-193a-5p. Conclusions: Serum miR-193a-5p levels correlate strongly with NAFLD activity grade and fibrosis stage. MiR-193a-5p may have a role in the hepatic response to oxidative stress and is a potential clinically tractable circulating biomarker for progressive NAFLD

Performance of non-invasive tests and histology for the prediction of clinical outcomes in patients with non-alcoholic fatty liver disease: an individual participant data meta-analysis

BackgroundHistologically assessed liver fibrosis stage has prognostic significance in patients with non-alcoholic fatty liver disease (NAFLD) and is accepted as a surrogate endpoint in clinical trials for non-cirrhotic NAFLD. Our aim was to compare the prognostic performance of non-invasive tests with liver histology in patients with NAFLD.MethodsThis was an individual participant data meta-analysis of the prognostic performance of histologically assessed fibrosis stage (F0–4), liver stiffness measured by vibration-controlled transient elastography (LSM-VCTE), fibrosis-4 index (FIB-4), and NAFLD fibrosis score (NFS) in patients with NAFLD. The literature was searched for a previously published systematic review on the diagnostic accuracy of imaging and simple non-invasive tests and updated to Jan 12, 2022 for this study. Studies were identified through PubMed/MEDLINE, EMBASE, and CENTRAL, and authors were contacted for individual participant data, including outcome data, with a minimum of 12 months of follow-up. The primary outcome was a composite endpoint of all-cause mortality, hepatocellular carcinoma, liver transplantation, or cirrhosis complications (ie, ascites, variceal bleeding, hepatic encephalopathy, or progression to a MELD score ≥15). We calculated aggregated survival curves for trichotomised groups and compared them using stratified log-rank tests (histology: F0–2 vs F3 vs F4; LSM: 2·67; NFS: 0·676), calculated areas under the time-dependent receiver operating characteristic curves (tAUC), and performed Cox proportional-hazards regression to adjust for confounding. This study was registered with PROSPERO, CRD42022312226.FindingsOf 65 eligible studies, we included data on 2518 patients with biopsy-proven NAFLD from 25 studies (1126 [44·7%] were female, median age was 54 years [IQR 44–63), and 1161 [46·1%] had type 2 diabetes). After a median follow-up of 57 months [IQR 33–91], the composite endpoint was observed in 145 (5·8%) patients. Stratified log-rank tests showed significant differences between the trichotomised patient groups (p<0·0001 for all comparisons). The tAUC at 5 years were 0·72 (95% CI 0·62–0·81) for histology, 0·76 (0·70–0·83) for LSM-VCTE, 0·74 (0·64–0·82) for FIB-4, and 0·70 (0·63–0·80) for NFS. All index tests were significant predictors of the primary outcome after adjustment for confounders in the Cox regression.InterpretationSimple non-invasive tests performed as well as histologically assessed fibrosis in predicting clinical outcomes in patients with NAFLD and could be considered as alternatives to liver biopsy in some cases

Discovering Sortases: From pilus biogenesis in Streptococcus pneumoniae to application in biotecnology

SUMMARY Gram-positive bacteria display various surface proteins on their cell wall, many of them performing virulence related functions, such as host surface attachment and immune system escape. Several of these exposed proteins are covalently linked to the peptidoglycan layer through the sortase-mediated transpeptidation mechanism. Sortases recognize protein substrates with a C-terminal LPXTG motif, cleaving the peptide bond between the motif’s threonine and glycine residues, forming a thioacyl intermediate. Transpeptidation proceeds by the nucleophilic attack by the N-terminus of the peptidyl side chain of a lipid II peptidoglycan precursor resulting in the formation of a protein-lipid II adduct (Ton-That and Schneewind, 1999). Incorporation of the protein-loaded lipid II into the peptidoglycan layer finally leads to covalent attachment of the sortase substrate to the cell wall. Sortases are also responsible for the polymerization of new virulence factors recently identified in Gram-positive bacteria known as Pili. Pili consist of covalently linked subunits assembled by specialized pilus-related sortases, phylogenetically and mechanisti¬cally related to housekeeping sortases that anchor proteins to the cell wall (Telford et al., 2006). Several studies describe pilus-like surface structures as eligible components for vaccine development; however, understanding the mechanism of pilus assembly in Gram positive bacteria is still rudimentary. To elucidate the role of pilus-related sortases, the human pathogen Streptococcus pneumoniae was used as a model system. This thesis describes the role of pilus-related sortases in biogenesis of pili encoded by a region known as Pilus islet 1 (PI-1). PI-1 related sortases of S. pneumoniae were functionally characterized using various biochemical methods. FRET (Fluorescent Resonance Energy Transfer) and HPLC-based sortase activity assays were developed allowing kinetic and mechanistic analysis and studying substrate specificity of PI-1 related sortases. In vivo studies were performed to confirm in vitro data. Furthermore, this thesis explores possible applications of sortases in biotechnology. Discovery of sortase-catalyzed transpeptidation lead to development of the Sortase tagging technology (Sortagging), a powerful tool to create functionalized protein-conjugates. Sortases may exert their activity in vitro in the presence of an LPXTG tagged protein and derivatized glycine compounds that act as nucleophiles to yield a functionalized transpeptidation product. Depending on the function carried by the glycine derivative, it is possible to obtain fluorescent labeled proteins, circularized proteins, covalently linked proteins or proteins that are covalently attached to a solid support. The housekeeping sortase A from S. aureus is extensively applied for these biotechnological purposes and it was also used for the novel biotechnological application described here. This thesis explores the application of sortase technology for the creation of protein arrays to be used in high throughput screening of protein-protein interactions. In this context, an Influenza virus protein array was developed to screen sera against the H1N1 virus. Bacterial crude extracts were applied to immobilize expressed LPETG-tagged Influenza virus proteins at their carboxyl-termini on pre-activated amine-glass slides derivatized with Gly3 peptide. The specificity of the Sortase A reaction permits avoiding purification steps in array building and allows for immobilization of proteins in an oriented fashion. This section of the work describes how, for the first time, sortagging was implemented to covalently link proteins of a viral genome to a solid support. This approach allows for scaling-up to proteins of larger genomes in order to develop a high density protein array to be used in high throughput screening.SOMMARIO I batteri Gram positivi espongono sulla loro parete cellulare proteine di superficie, molte di queste hanno una funzione relativa alla virulenza del batterio stesso, tra gli esempi del loro ruolo ci sono l’evasione dal sistema immunitario o l’iterazione con la superficie dell’ospite. Diverse proteine di superficie sono legate covalentemente allo strato di peptidoglicano dei Gram positivi attraverso un meccanismo di transpeptidazione, enzimi responsabili di quenza reazione sono stati denominati sortasi. Le sortasi sono in grado di riconoscere un motivo C-terminal LPxTG, effettuando un taglio tra i residui aminoacidici di Thr e di Gly, in maniera tale da avere in un primo step di reazione la formazione di un intermedio che viene poi risolto in un secondo momento da un attacco nucleofilo mediato dall’N-terminal del peptydil side chain del Lipid II precursore del peptidoglicano (Ton-That et al., 1999). Oltre all’ancoraggio di proteine di superficie le sortasi sono responsabili della polimerizzazione di nuovi fattori di virulenza recentemente identificati in batteri Gram positivi e noti come Pili. I Pili consistono in subunità covalentemente legate e assemblate grazie alle sortasi associate al pilo, le quali sono filogeneticamente e maccanicisticamente vicine alle housekeeping sortasi il cui ruolo è quello di ancorare le proteine alla parete cellulare (Telford et al., 2006). Diversi studi descrivono queste strutture come componenti elegibili per lo sviluppo di un vaccino, sebbene il meccanismo di assemblaggio risulta ancora rudimentale. Per meglio chiarire il ruolo delle sortasi associate al pilo in questo lavoro di tesi si è preso come modello lo studio delle sortasi in S. pneumoniae. In questo studio viene descritto infatti il ruolo delle sortasi nella biogenesi del pilo di S. pneumonaie, codificato da una regione nota come Pilus islet 1 (PI-1). Le sortasi relative a questa regione sono state caratterizzate usando diversi metodi biochimici, tra cui saggi di FRET ( Fluorescent Resonance Energy Transfer) e saggi di attività basati su analisi HPLC. Questi studi hanno permesso di fare analisi cinetiche e di studiare la specificità di substrato delle diverse sortasi relativa alle subunità che compongono il pilo, ossia RrgB, RrgA e RrgC. Analisi in vivo hanno poi permesso di confermare i dati ottenuti dallo studio in vitro. Dopo questa prima sessione, in questo lavoro si sono anche valutate le possibili applicazioni biotecnologiche capaci di sfruttare l’attività biologica delle sortasi. Recentemente svariate applicazioni biotecnologiche hanno permesso di sfruttare la transpeptidazione per di funzionalizzare proteine, processo noto col nome di Sortagging. Infatti le sortasi esercitano la loro attività anche in vitro in presenza di una proteina substrato ricombinante, contenente un motivo LPxTG e composti derivatizzati con oligoglicine che agendo da nucleofili risolvono la reazione di transpeptidazione in un prodotto finito modificato e funzionalizzato. Attraverso il Sortagging è possibile ottenere proteine fluorescenti, circolarizzarle o covalentemente unirle a supporto solido o banalmente è possibile ricreare un legame peptidico tra proteine con prodotti ricombinanti opportunamente modificati col motivo LPxTG e con oligoglicine (e.Gly3). Per le suddette applicazioni è stata ampliamente utilizzata l’housekeeping sortase A di S. aureus che è anche stata impiegata in questa tesi. Obiettivo è quello di creare un protein array per high troughput screening capace di rilevare iterazioni proteina-proteina. In questo contesto un protein array del Virus Influenza è stato sviluppato per lo screening di siero commerciale H1N1. Lisati batterici che esprimono proteine dell’Influenza virus modificate al C-terminal con il mofivo sortagging sono state immobilizate su glass-slide opportunamente derivatizzate con Gly3. Grazie alla specificità della sortasi A è possibile utilizzare estratti crudi evitandodi step di purificazione e un’immobilizzazione orientata. É stato applicato per la prima volta il processo di Sortagging per legare proteine di un genoma intero su supporto solido. L’idea è quella di poter applpicare la tecnologia a un ampio proteoma in grado di sviluppare un protein microarray ad alta densità

Efficacy of a 24-week course of PEG-Interferon a-2b monotherapy in patients with acute hepatitis C after failure of spontaneous clearance.

Abstract BACKGROUND/AIMS: Interferon (IFN) monotherapy significantly reduces the chronicity rate of acute hepatitis C (AHC) but optimal regimen and treatment timing remain undefined. The aim of this study was to assess the efficacy of a 6-month course of pegylated IFN (PEG-IFN) alpha-2b monotherapy in AHC patients and to investigate if IFN treatment initiated after 12 weeks from clinical presentation, still achieved a high response rate. METHODS: Sixteen AHC patients still viremic after 12 weeks from the onset were treated with PEG-IFN alpha-2b (1.5 mcg/kg once weekly) for 6 months and followed for at least 12 months. Response to therapy was defined as normal ALT values and undetectable HCV RNA (&lt;50 IU/ml) at the end of therapy, after 6 (sustained response) and 12 months follow-up (long-term response). RESULTS: At the end of treatment, HCV RNA was undetectable in 15/16 patients while ALT normalized in 14/16 patients. After 6 and 12 months follow-up, 15/16 patients (94%) showed virological and biochemical response. CONCLUSIONS: A 6-month course of PEG-IFN alpha-2b is effective in inducing resolution of AHC in 94% of patients. Our results provide a rationale for delaying treatment for 12 weeks, targeting only patients who fail to clear the virus spontaneously and truly requiring therapy without loss of efficacy

Sortase A Confers Protection against Streptococcus pneumoniae in Mice▿

Streptococcus pneumoniae sortase A (SrtA) is a transpeptidase that is highly conserved among pneumococcal strains, whose involvement in adhesion/colonization has been reported. We found that intraperitoneal immunization with recombinant SrtA conferred to mice protection against S. pneumoniae intraperitoneal challenge and that the passive transfer of immune serum before intraperitoneal challenge was also protective. Moreover, by using the intranasal challenge model, we observed a significant reduction of bacteremia when mice were intraperitoneally immunized with SrtA, while a moderate decrease of lung infection was achieved by intranasal immunization, even though no influence on nasopharynx colonization was seen. Taken together, our results suggest that SrtA is a good candidate for inclusion in a multicomponent, protein-based, pneumococcal vaccine

Evaluation of AIH-associated autoantigens.

<p>(A) The heatmap summarizes the recognition frequencies among different sample groups (AIH, HD and the VH) for the new four autoantigens out of the 31 candidates selected with the proposed bioinformatic strategy after DELFIA screening. Colour intensity denotes the degree of recognition frequency within the sample group. (B) Recognition frequency for two of the four autoantigens, that were statistical significant. (C) Signals distribution detected for each of the new two proteins are displayed. Statistical differences in recognition frequency (ChiSquare test) and in signal intensity (t test) are denoted as single (p<0,05), double (p<0,001) or triple stars (p <0,0001). (D), ROC curve of the biomarker candidates exhibited AUCs of 0.899 (SE = 87.5% and SP = 77.7%), 0.782 (SE = 65.0% and SP = 81.5%) for UNQ9419 and CHAD, respectively. The arrows denote best cut-off points. Combo curve represents the combination, UNQ9419+CHAD, which exhibited AUC of 0.915 (SE = 95.0% and SP = 76.2%).</p