Recommendations for accurate genotyping of SARS-CoV-2 using amplicon-based sequencing of clinical samples

Objectives: Genotyping of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in monitoring viral evolution and transmission during the pandemic. The quality of the sequence data obtained from these genotyping efforts depends on several factors, including the quantity/integrity of the input material, the technology, and laboratory-specific implementation. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in genomic epidemiology studies. We aimed to establish clear and broadly applicable recommendations for reliable virus genotyping. Methods: We established and used a sequencing data analysis workflow that reliably identifies and removes technical artefacts; such artefacts can result in miscalls when using alternative pipelines to process clinical samples and synthetic viral genomes with an amplicon-based genotyping approach. We evaluated the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination. Results: We found that at least 1000 viral genomes are necessary to confidently detect variants in the SARS-CoV-2 genome at frequencies of ≥10%. The broad applicability of our recommendations was validated in over 200 clinical samples from six independent laboratories. The genotypes we determined for clinical isolates with sufficient quality cluster by sampling location and period. Our analysis also supports the rise in frequencies of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was facilitated by travel during the summer of 2020. Conclusions: We present much-needed recommendations for the reliable determination of SARS-CoV-2 genome sequences and demonstrate their broad applicability in a large cohort of clinical samples

Face masks and nanotechnology: Keep the blue side up

Coronavirus Disease 2019 (COVID-19) is one of the biggest challenges of the 21st century. While researchers are working on vaccine development and elucidating the mechanism of action and evolution of the harmful SARS-CoV-2, the current most important public health measure, second only to social distancing, is the obligatory wearing of facial protection. The Centers for Disease Control and Prevention recommended in April 2020 that the public wear face coverings in areas with high rates of transmission based on epidemiological evidence on the strong relationship between mask wearing and pandemic control. This protection against SARS-CoV-2 and other airborne pathogens, boost the design and production of innovative solutions by industry stakeholders. Nanoparticles, nanofibers, and other pioneering technologies based on nanomaterials have been introduced in mask production chains to improve performance and confer antiviral properties. During an emergency like COVID-19, these products directly available to the public should be carefully analyzed in terms of efficacy and possible long-term effects on the wearers’ skin and lungs as well as on the environment. This opinion paper provides a wealth of information on the role of nanotechnologies in improving the performance of facial masks and on possible future consequences caused by a poorly regulated use of nanotechnology in textiles

PE_PGRS proteins of Mycobacterium tuberculosis: A specialized molecular task force at the forefront of host–pathogen interaction

To the PE_PGRS protein subfamily belongs a group of surface-exposed mycobacterial antigens that in Mycobacterium tuberculosis (Mtb) H37Rv accounts to more than 65 genes, 51 of which are thought to express a functional protein. PE_PGRS proteins share a conserved structural architecture with three main domains: the N-terminal PE domain; the PGRS domain, that can vary in sequence and size and is characterized by the presence of multiple GGA-GGX amino acid repeats; the highly conserved sequence containing the GRPLI motif that links the PE and PGRS domains; the unique C-terminus end that can vary in size from few to up to ≈ 300 amino acids. pe_pgrs genes emerged in slow-growing mycobacteria and expanded and diversified in MTBC and few other pathogenic mycobacteria. Interestingly, despite sequence homology and apparent redundancy, PE_PGRS proteins seem to have evolved a peculiar function. In this review, we summarize the actual knowledge on this elusive protein family in terms of evolution, structure, and function, focusing on the role of PE_PGRS in TB pathogenesis. We provide an original hypothesis on the role of the PE domain and propose a structural model for the polymorphic PGRS domain that might explain how so similar proteins can have different physiological functions

Structural Basis of PE_PGRS Polymorphism, a Tool for Functional Modulation

Background: The mycobacterial PE_PGRS protein family is present only in pathogenic strains of the genus mycobacterium, such as Mtb and members of the MTB complex, suggesting a likely important role of this family in pathogenesis. Their PGRS domains are highly polymorphic and have been suggested to cause antigenic variations and facilitate pathogen survival. The availability of AlphaFold2.0 offered us a unique opportunity to better understand structural and functional properties of these domains and a role of polymorphism in Mtb evolution and dissemination. Methods: We made extensive use of AlphaFold2.0 computations and coupled them with sequence distribution phylogenetic and frequency analyses, and antigenic predictions. Results: Modeling of several polymorphic forms of PE_PGRS33, the prototype of the PE_PGRS family and sequence analyses allowed us to predict the structural impact of mutations/deletions/insertions present in the most frequent variants. These analyses well correlate with the observed frequency and with the phenotypic features of the described variants. Conclusions: Here, we provide a thorough description of structural impacts of the observed polymorphism of PE_PGRS33 protein and we correlate predicted structures to the known fitness of strains containing specific variants. Finally, we also identify protein variants associated with bacterial evolution, showing sophisticated modifications likely endowed with a gain-of-function role during bacterial evolution

Profiling the gut microbiota to assess infection risk in Klebsiella pneumoniae-colonized patients

Vornhagen et al. introduced a model combining gut microbiota structure and Klebsiella pneumoniae genotype to assess infection risk in K. pneumoniae-colonized patients. Building on their findings, we investigated the gut microbiota composition and K. pneumoniae genotype in 16 colonized patients, five of whom had bloodstream infections at the time of fecal sampling. Importantly, we did not apply the original machine learning model due to the small sample size of our cohort. Instead, we explored the distribution of key antimicrobial resistance and stress resistance genes and analyzed gut community structure based on amplicon sequence variants (ASVs) of the V3–V4 16S rRNA region. Notably, distinct gene profiles were observed in both infected and non-infected patients, and three patients without bloodstream infections showed no detectable Klebsiella ASVs despite microbiological confirmation of colonization. These findings highlight the need to integrate gut microbiota composition data into infection risk assessment and address limitations in taxonomic resolution and sample size. Future studies should aim to develop streamlined tools for clinical application in K. pneumoniae-colonized patients

PDZ2-conjugated-PLGA nanoparticles are tiny heroes in the battle against SARS-CoV-2

The COVID-19 pandemic caused by SARS-CoV-2 has highlighted the urgent need for innovative antiviral strategies to fight viral infections. Although a substantial part of the overall effort has been directed at the Spike protein to create an effective global vaccination strategy, other proteins have also been examined and identified as possible therapeutic targets. Among them, although initially underestimated, there is the SARS-CoV-2 E-protein, which turned out to be a key factor in viral pathogenesis due to its role in virus budding, assembly and spreading. The C-terminus of E-protein contains a PDZ-binding motif (PBM) that plays a key role in SARS-CoV-2 virulence as it is recognized and bound by the PDZ2 domain of the human tight junction protein ZO-1. The binding between the PDZ2 domain of ZO-1 and the C-terminal portion of SARS-CoV-2 E-protein has been extensively characterized. Our results prompted us to develop a possible adjuvant therapeutic strategy aimed at slowing down or inhibiting virus-mediated pathogenesis. Such innovation consists in the design and synthesis of externally PDZ2-ZO1 functionalized PLGA-based nanoparticles to be used as intracellular decoy. Contrary to conventional strategies, this innovative approach aims to capitalize on the E protein-PDZ2 interaction to prevent virus assembly and replication. In fact, the conjugation of the PDZ2 domain to polymeric nanoparticles increases the affinity toward the E protein effectively creating a “molecular sponge” able to sequester E proteins within the intracellular environment of infected cells. Our in vitro studies on selected cellular models, show that these nanodevices significantly reduce SARS-CoV-2-mediated virulence, emphasizing the importance of exploiting viral-host interactions for therapeutic benefit

Evaluation of Novel Guanidino-Containing Isonipecotamide Inhibitors of Blood Coagulation Factors against SARS-CoV-2 Virus Infection

Coagulation factor Xa (fXa) and thrombin (thr) are widely expressed in pulmonary tissues, where they may catalyze, together with the transmembrane serine protease 2 (TMPRSS2), the coronaviruses spike protein (SP) cleavage and activation, thus enhancing the SP binding to ACE2 and cell infection. In this study, we evaluate in vitro the ability of approved (i.e., dabigatran and rivaroxaban) and newly synthesized isonipecotamide-based reversible inhibitors of fXa/thr (cmpds 1-3) to hinder the SARS-CoV-2 infectivity of VERO cells. Nafamostat, which is a guanidine/amidine antithrombin and antiplasmin agent, disclosed as a covalent inhibitor of TMPRSS2, was also evaluated. While dabigatran and rivaroxaban at 100 mu M concentration did not show any effect on SARS-CoV-2 infection, the virus preincubation with new guanidino-containing fXa-selective inhibitors 1 and 3 did decrease viral infectivity of VERO cells at subtoxic doses. When the cells were pre-incubated with 3, a reversible nanomolar inhibitor of fXa (K-i = 15 nM) showing the best in silico docking score toward TMPRSS2 (pdb 7MEQ), the SARS-CoV-2 infectivity was completely inhibited at 100 mu M (p < 0.0001), where the cytopathic effect was just about 10%. The inhibitory effects of 3 on SARS-CoV-2 infection was evident (ca. 30%) at lower concentrations (3-50 mu M). The covalent TMPRSS2 and the selective inhibitor nafamostat mesylate, although showing some effect (15-20% inhibition), did not achieve statistically significant activity against SARS-CoV-2 infection in the whole range of test concentrations (3-100 mu M). These findings suggest that direct inhibitors of the main serine proteases of the blood coagulation cascade may have potential in SARS-CoV-2 drug discovery. Furthermore, they prove that basic amidino-containing fXa inhibitors with a higher docking score towards TMPRSS2 may be considered hits for optimizing novel small molecules protecting guest cells from SARS-CoV-2 infection

Confirmed Activity and Tolerability of Weekly Paclitaxel in the Treatment of Advanced Angiosarcoma

Background. In several prospective and retrospective studies, weekly paclitaxel showed promising activity in patients with angiosarcoma. Patients and Methods. Our study was originally designed as a prospective, phase II multicenter trial for patients younger than 75, with ECOG performance status 0–2, affected by locally advanced or metastatic angiosarcoma. Patients received paclitaxel 80 mg/m2 intravenously, at days 1, 8, and 15 every 4 weeks, until disease progression or unacceptable toxicity. Primary endpoint was objective response. Results. Eight patients were enrolled but, due to very slow accrual, the trial was prematurely stopped and further 10 patients were retrospectively included in the analysis. Out of 17 evaluable patients, 6 patients obtained an objective response (5 partial, 1 complete), with an objective response rate of 35% (95% confidence interval 17%–59%). Of note, five responses were obtained in pretreated patients. In the paper, details of overall survival, progression-free survival, and tolerability are reported. Conclusions. In this small series of patients with locally advanced or metastatic angiosarcoma, weekly paclitaxel was confirmed to be well tolerated and active even in pretreated patients

Unraveling the potential of graphene quantum dots against Mycobacterium tuberculosis infection

Introduction The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) strains has underscored the urgent need for novel therapeutic approaches. Carbon-based nanomaterials, such as graphene oxide (GO), have shown potential in anti-TB activities but suffer from significant toxicity issues. Methods This study explores the anti-TB potential of differently functionalized graphene quantum dots (GQDs) - non-functionalized, L-GQDs, aminated (NH2-GQDs), and carboxylated (COOH-GQDs) - alone and in combination with standard TB drugs (isoniazid, amikacin, and linezolid). Their effects were assessed in both axenic cultures and in vitro infection models. Results GQDs alone did not demonstrate direct mycobactericidal effects nor trapping activity. However, the combination of NH2-GQDs with amikacin significantly reduced CFUs in in vitro models. NH2-GQDs and COOH-GQDs also enhanced the antimicrobial activity of amikacin in infected macrophages, although L-GQDs and COOH-GQDs alone showed no significant activity. Discussion The results suggest that specific types of GQDs, particularly NH2-GQDs, can enhance the efficacy of existing anti-TB drugs. These nanoparticles might serve as effective adjuvants in anti-TB therapy by boosting drug performance and reducing bacterial counts in host cells, highlighting their potential as part of advanced drug delivery systems in tuberculosis treatment. Further investigations are needed to better understand their mechanisms and optimize their use in clinical settings

Pilot study on cultural and metagenomic analysis of bile and biliary stents lead to unveiling the key players in stent occlusion

: Endoscopic Retrograde Cholangio-Pancreatography (ERCP) with biliary stenting is a minimally invasive medical procedure employed to address both malignant and benign obstructions within the biliary tract. Benign biliary strictures (BBSs), typically arising from surgical interventions such as liver transplants and cholecystectomy, as well as chronic inflammatory conditions, present a common clinical challenge. The current gold standard for treating BBSs involves the periodic insertion of plastic stents at intervals of 3-4 months, spanning a course of approximately one year. Unfortunately, stent occlusion emerges as a prevalent issue within this treatment paradigm, leading to the recurrence of symptoms and necessitating repeated ERCPs. In response to this clinical concern, we initiated a pilot study, delving into the microbial composition present in bile and on the inner surfaces of plastic stents. This investigation encompassed 22 patients afflicted by BBSs who had previously undergone ERCP with plastic stent placement. Our preliminary findings offered promising insights into the microbial culprits behind stent occlusion, with Enterobacter and Lactobacillus spp. standing out as prominent bacterial species known for their biofilm-forming tendencies on stent surfaces. These revelations hold promise for potential interventions, including targeted antimicrobial therapies aimed at curtailing bacterial growth on stents and the development of advanced stent materials boasting anti-biofilm properties