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Metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) is an agnostic method for broad-based diagnosis of central nervous system (CNS) infections. Here we analyzed the 7-year performance of clinical CSF mNGS testing of 4,828 samples from June 2016 to April 2023 performed by the University of California, San Francisco (UCSF) clinical microbiology laboratory. Overall, mNGS testing detected 797 organisms from 697 (14.4%) of 4,828 samples, consisting of 363 (45.5%) DNA viruses, 211 (26.4%) RNA viruses, 132 (16.6%) bacteria, 68 (8.5%) fungi and 23 (2.9%) parasites. We also extracted clinical and laboratory metadata from a subset of the samples (n = 1,164) from 1,053 UCSF patients. Among the 220 infectious diagnoses in this subset, 48 (21.8%) were identified by mNGS alone. The sensitivity, specificity and accuracy of mNGS testing for CNS infections were 63.1%, 99.6% and 92.9%, respectively. mNGS testing exhibited higher sensitivity (63.1%) than indirect serologic testing (28.8%) and direct detection testing from both CSF (45.9%) and non-CSF (15.0%) samples (P < 0.001 for all three comparisons). When only considering diagnoses made by CSF direct detection testing, the sensitivity of mNGS testing increased to 86%. These results justify the routine use of diagnostic mNGS testing for hospitalized patients with suspected CNS infection., Competing Interests: Competing interests: A.P.W., B.O., T.B., B.M. and S.M. are employed by and own equity in Delve Bio. C.Y.C. is a co-founder of Delve Bio and on the scientific advisory board for Delve Bio, Flightpath Biosciences, Biomeme, Mammoth Biosciences, BiomeSense and Poppy Health. He is also an inventor on US patent 11380421, ‘Pathogen detection using next-generation sequencing’, under which algorithms for taxonomic classification, filtering and pathogen detection are used by SURPI+ software. C.Y.C. receives research support from Delve Bio and Abbott Laboratories, Inc. M.R.W. is a co-founder and on the scientific advisory board for Delve Bio. The other authors declare no competing interests., (© 2024. The Author(s).)

Background: Bacillus cereus ( B. cereus ) is known to cause 2 types of foodborne diseases; the diarrheal and emetic syndromes. They are largely underreported due to their usually self-limiting course. Rare and sometimes fatal cases of liver failure, pulmonary hemorrhage and cerebral oedema have been reported mainly in children and young adults. We present here a case of liver failure associated with B. cereus food poisoning in a middle-aged patient., Case Summary: A 48-year-old female patient presented to the emergency department for emesis, diarrhea, chills without fever, asthenia and diffuse abdominal cramps that started less than 30 minutes after eating a rice salad. Her past medical history was relevant for cholecystectomy and a cured Hashimoto's disease. She did not take any medication, drugs and declared a consumption of one glass of wine per week. In the emergency department, she was treated with acetaminophen, metoclopramide, ondansetron, and an intravenous normal saline infusion. Blood gas analysis revealed a metabolic acidosis with hyperlactatemia, coagulation revealed a low prothrombin activity [32 %; normal values (N): 70-140] and a low Factor V activity (15%; N: > 70). Transaminases were elevated with hyperbilirubinemia, elevated lipase and rhabdomyolysis. N-acetylcysteine treatment was introduced. Abdominal echography revealed no signs of chronic hepatopathy or hepatomegaly. Day after the admission, psychomotor activity improved, transaminases and lipase started decreasing. Rhabdomyolysis gradually worsened to peak on day 3. Screening tests for liver disease were negative for viral and autoimmune cause of liver failure. Stools cultures were positive for colonies of the B. cereus group which were also identified in the rice salad samples processed whereas blood cultures were negative. The patient's condition improved gradually including her liver function parameters and psychomotor activity which allowed her discharged home on day 9., Conclusion: We describe a rare case of hepatocellular dysfunction due to a foodborne B. cereus intoxication in an adult patient. Even if it is uncommon, the severity of liver dysfunction reported and mechanism of the cereulide toxin toxicity on liver suggest that acetaminophen should be avoided in case of a foodborne intoxication and n-acetylcysteine could be a potential therapy helping to prevent hepatocytes necrosis due to the oxidative stress induced by mitochondrial dysfunction., Competing Interests: Conflict-of-interest statement: The authors declare that they have no conflict of interest., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

Tools for rapid identification of novel and/or emerging viruses are urgently needed for clinical diagnosis of unexplained infections and pandemic preparedness. Here we developed and clinically validated a largely automated metagenomic next-generation sequencing (mNGS) assay for agnostic detection of respiratory viral pathogens from upper respiratory swab and bronchoalveolar lavage samples in <24 h. The mNGS assay achieved mean limits of detection of 543 copies/mL, viral load quantification with 100% linearity, and 93.6% sensitivity, 93.8% specificity, and 93.7% accuracy compared to gold-standard clinical multiplex RT-PCR testing. Performance increased to 97.9% overall predictive agreement after discrepancy testing and clinical adjudication, which was superior to that of RT-PCR (95.0% agreement). To enable discovery of novel, sequence-divergent human viruses with pandemic potential, de novo assembly and translated nucleotide algorithms were incorporated into the automated SURPI+ computational pipeline used by the mNGS assay for pathogen detection. Using in silico analysis, we showed that after removal of all human viral sequences from the reference database, 70 (100%) of 70 representative human viral pathogens could still be identified based on homology to related animal or plant viruses. Our assay, which was granted breakthrough device designation from the US Food and Drug Administration (FDA) in August of 2023, demonstrates the feasibility of routine mNGS testing in clinical and public health laboratories, thus facilitating a robust and rapid response to the next viral pandemic., Competing Interests: Competing interests C.Y.C. is a founder of Delve Bio and on the scientific advisory board for Delve Bio, Flightpath Biosciences, Biomeme, Mammoth Biosciences, BiomeSense and Poppy Health. He is also an inventor on US patent 11380421, “Pathogen detection using next generation sequencing”, under which algorithms for taxonomic classification, filtering, and pathogen detection are used by SURPI+ software. C.Y.C. receives research support from Delve Bio and Abbott Laboratories, Inc. The other authors declare no competing interests., (© 2024. The Author(s).)

Merkel cell polyomavirus (MCPyV) is the foremost causative factor of Merkel cell carcinoma (MCC), a rare yet highly aggressive skin cancer. Although the evaluation of circulating IgG antibodies against Merkel cell polyomavirus (MCPyV) LT/sT oncoproteins is clinically useful for MCC diagnosis/prognosis, a limited number of assays for identifying such antibodies have been developed. Herein, a novel indirect immunoassay with synthetic epitopes/mimotopes of MCPyV oncoproteins was computationally designed and experimentally validated on control sera and sera from healthy individuals and MCC patients. Upon computational design of five synthetic peptides, the performance of the immunoassay in detecting anti-oncoprotein IgGs in MCPyV-positive and -negative control sera was evaluated. The immunoassay was afterwards extended on sera from healthy individuals, and, for longitudinal analysis, MCC patients. Performance properties such as sensitivity and specificity and positive/negative predictive values were adequate. Receiver-operating characteristic (ROC) curves indicated that the areas under the curves (AUCs) were within the low/moderately accurate ranges. Immunoassay was repeatable, reproducible and accurate. As expected, the serum anti-oncoprotein IgG prevalence in healthy individuals was low (2%-5%). Anti-oncoprotein IgGs slightly increased when MCC patients experienced partial tumour remission and/or stable disease, compared to baseline. Our data indicate that the newly developed immunoassay is reliable for detecting circulating anti-oncoprotein IgGs both in healthy individuals and MCC patients., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Background: The limited therapies available for treating Merkel cell carcinoma (MCC), a highly aggressive skin neoplasm, still pose clinical challenges, and novel treatments are required. Targeting retinoid signalling with retinoids, such as all-trans retinoic acid (ATRA), is a promising and clinically useful antitumor approach. ATRA drives tumour cell differentiation by modulating retinoid signalling, leading to anti-proliferative and pro-apoptotic effects. Although retinoid signalling is dysregulated in MCC, ATRA activity in this tumour is unknown. This study aimed to evaluate the impact of ATRA on the pathological phenotype of MCC cells., Methods: The effect of ATRA was tested in various Merkel cell polyomavirus-positive and polyomavirus-negative MCC cell lines in terms of cell proliferation, viability, migration and clonogenic abilities. In addition, cell cycle, apoptosis/cell death and the retinoid gene signature were evaluated upon ATRA treatments., Results: ATRA efficiently impaired MCC cell proliferation and viability in MCC cells. A strong effect in reducing cell migration and clonogenicity was determined in ATRA-treated cells. Moreover, ATRA resulted as strongly effective in arresting cell cycle and inducing apoptosis/cell death in all tested MCC cells. Enrichment analyses indicated that ATRA was effective in modulating the retinoid gene signature in MCC cells to promote cell differentiation pathways, which led to anti-proliferative and pro-apoptotic/cell death effects., Conclusions: These results underline the potential of retinoid-based therapy for MCC management and might open the way to novel experimental approaches with other retinoids and/or combinatorial treatments., (© 2024 European Academy of Dermatology and Venereology.)

CRISPR base editing screens enable analysis of disease-associated variants at scale; however, variable efficiency and precision confounds the assessment of variant-induced phenotypes. Here, we provide an integrated experimental and computational pipeline that improves estimation of variant effects in base editing screens. We use a reporter construct to measure guide RNA (gRNA) editing outcomes alongside their phenotypic consequences and introduce base editor screen analysis with activity normalization (BEAN), a Bayesian network that uses per-guide editing outcomes provided by the reporter and target site chromatin accessibility to estimate variant impacts. BEAN outperforms existing tools in variant effect quantification. We use BEAN to pinpoint common regulatory variants that alter low-density lipoprotein (LDL) uptake, implicating previously unreported genes. Additionally, through saturation base editing of LDLR, we accurately quantify missense variant pathogenicity that is consistent with measurements in UK Biobank patients and identify underlying structural mechanisms. This work provides a widely applicable approach to improve the power of base editing screens for disease-associated variant characterization., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Antimicrobial/antibiotic de-escalation (ADE) is a key feature of antimicrobial stewardship programs (ASP) that relies mainly on individual panels for determining ADE events based on subjective ranking of antibiotics' spectrum activity. The lack of consensus among ASP experts leads to reproducibility issues in the measure of this clinical outcome, making difficult to assess its real impact on patient care. The S 3 score (Simplified Spectrum Score) app was developed to allow an objective ranking of antibiotics. Ranking was achieved by developing a database harboring pairs of bacteria-antibiotics for which each molecule was assigned a score based on published and clinically validated data from a recognized international committee. S 3 score shows a strong correlation relationship and substantial agreement to a clinically validated spectrum score, and its framework enables any person to use it for ADE detection without assuming prior knowledge or training. In addition, its design enables regular updates and sustainability., (© 2024. The Author(s).)

Introduction: Human polyomaviruses (HPyVs) cause persistent/latent infections in a large fraction of the population. HPyV infections may cause severe diseases in immunocompromised patients. Malawi polyomavirus (MWPyV) is the 10th discovered human polyomavirus (HPyV 10). MWPyV was found in stool samples of healthy children. So far, the few investigations carried out on HPyV 10 did not find an association with human disease., Methods: In this study, to verify the putative association between MWPyV and human diseases, MWPyV seroprevalence was investigated in patients affected by i) lymphoproliferative disorders (LPDs) and ii) immune system disorders, i.e., autoimmune diseases (ADs), and in iii) healthy subjects. An indirect ELISA, employing virus-like particles (VLPs) to detect serum IgG antibodies against MWPyV/HPyV 10, was carried out. The study also revealed the prevalence of another polyomavirus, Merkel cell polyomavirus (MCPyV)., Results: Sera from patients with distinct autoimmune diseases ( n = 44; mean age 20 years) had a prevalence of MWPyV antibodies of 68%, while in patients with lymphoproliferative disorders ( n = 15; mean age 14 years), subjected to bone marrow transplantation, the prevalence was 47%. In healthy subjects ( n = 66; mean age 13 years), the prevalence of MWPyV antibodies was 67%. Our immunological investigation indicates that MWPyV/HPyV 10 seroconversion occurs early in life and MWPyV/HPyV 10 appears to be another polyomavirus ubiquitous in the human population. A significantly lower MWPyV antibody reactivity together with a lower immunological profile was detected in the sera of LPD patients compared with HS2 (* p < 0.05) (Fisher's exact test). LPD and AD patients have a similar MCPyV seroprevalence compared with healthy subjects., Discussion: MWPyV seroprevalence indicates that this HPyV is not associated with lymphoproliferative and autoimmune diseases. However, the ability to produce high levels of antibodies against MWPyV appears to be impaired in patients with lymphoproliferative disorders. Immunological investigations indicate that MWPyV seroconversion occurs early in life. MCPyV appears to be a ubiquitous polyomavirus, like other HPyVs, in the human population., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Nicol, Mazzoni, Iaquinta, De Pace, Gaboriaud, Maximova, Cason, De Martino, Mazziotta, Coursaget, Touzé, Boz, Comar, Tognon and Martini.)

Human osteogenic differentiation is a complex and well-orchestrated process which involves a plethora of molecular players and cellular processes. A growing number of studies have underlined that circular RNAs (circRNAs) play an important regulatory role during human osteogenic differentiation. CircRNAs are single-stranded, covalently closed non-coding RNA molecules that are acquiring increased attention as epigenetic regulators of gene expression. Given their intrinsic high conformational stability, abundance, and specificity, circRNAs can undertake various biological activities in order to regulate multiple cellular processes, including osteogenic differentiation. The most recent evidence indicates that circRNAs control human osteogenesis by preventing the inhibitory activity of miRNAs on their downstream target genes, using a competitive endogenous RNA mechanism. The aim of this review is to draw attention to the currently known regulatory mechanisms of circRNAs during human osteogenic differentiation. Specifically, we provide an understanding of recent advances in research conducted on various human mesenchymal stem cell types that underlined the importance of circRNAs in regulating osteogenesis. A comprehensive understanding of the underlying regulatory mechanisms of circRNA in osteogenesis will improve knowledge on the molecular processes of bone growth, resulting in the potential development of novel preclinical and clinical studies and the discovery of novel diagnostic and therapeutic tools for bone disorders., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Intercellular adhesion is a key function for epithelial cells. The fundamental mechanisms relying on epithelial cell adhesion have been partially uncovered. Hsa-microRNA-1249-3p (hsa-miR-1249-3p) plays a role in the epithelial mesenchymal transition in carcinoma cells, but its physiological function in epithelial cells is unknown. We aimed to investigate the role and molecular mechanisms of hsa-miR-1249-3p on epithelial cell functions. Hsa-miR-1249-3p was overexpressed in human epithelial cells and uterine cervical tissues, compared to cervical carcinoma cells and precancerous tissues, respectively. Hsa-miR-1249-3p was analyzed to verify its regulatory function on Homeobox A13 (HOXA13) target gene and its downstream cell adhesion gene β-catenin. Functional experiments indicated that hsa-miR-1249-3p inhibition prompted the mRNA and protein overexpression of HOXA13 which, in turn, led to the β-catenin protein expression. Moreover, hsa-miR-1249-3p inhibition induced a strong colony forming ability in epithelial cells, suggesting the miR involvement in cell adhesion machinery. These data indicate that hsa-miR-1249-3p regulates the expression of HOXA13 and its downstream cell adhesion gene β-catenin, possible resulting in cell adhesion modification in epithelial cells. This study will allow the set-up of further investigations aimed at exploring the relationship between the hsa-miR-1249-3p/HOXA13 axis and downstream cell adhesion genes., (© 2023. The Author(s).)

Stem cell-based therapy is a significant topic in regenerative medicine, with a predominant role being played by human mesenchymal stem cells (hMSCs). The hMSCs have been shown to be suitable in regenerative medicine for the treatment of bone tissue. In the last few years, the average lifespan of our population has gradually increased. The need of biocompatible materials, which exhibit high performances, such as efficiency in bone regeneration, has been highlighted by aging. Current studies emphasize the benefit of using biomimetic biomaterials, also known as scaffolds, for bone grafts to speed up bone repair at the fracture site. For the healing of injured bone and bone regeneration, regenerative medicine techniques utilizing a combination of these biomaterials, together with cells and bioactive substances, have drawn a great interest. Cell therapy, based on the use of hMSCs, alongside materials for the healing of damaged bone, has obtained promising results. In this work, several aspects of cell biology, tissue engineering, and biomaterials applied to bone healing/regrowth will be considered. In addition, the role of hMSCs in these fields and recent progress in clinical applications are discussed. Impact Statement The restoration of large bone defects is both a challenging clinical issue and a socioeconomic problem on a global scale. Different therapeutic approaches have been proposed for human mesenchymal stem cells (hMSCs), considering their paracrine effect and potential differentiation into osteoblasts. However, different limitations are still to be overcome in using hMSCs as a therapeutic opportunity in bone fracture repair, including hMSC administration methods. To identify a suitable hMSC delivery system, new strategies have been proposed using innovative biomaterials. This review provides an update of the literature on hMSC/scaffold clinical applications for the management of bone fractures.

CRISPR base editing screens are powerful tools for studying disease-associated variants at scale. However, the efficiency and precision of base editing perturbations vary, confounding the assessment of variant-induced phenotypic effects. Here, we provide an integrated pipeline that improves the estimation of variant impact in base editing screens. We perform high-throughput ABE8e-SpRY base editing screens with an integrated reporter construct to measure the editing efficiency and outcomes of each gRNA alongside their phenotypic consequences. We introduce BEAN, a Bayesian network that accounts for per-guide editing outcomes and target site chromatin accessibility to estimate variant impacts. We show this pipeline attains superior performance compared to existing tools in variant classification and effect size quantification. We use BEAN to pinpoint common variants that alter LDL uptake, implicating novel genes. Additionally, through saturation base editing of LDLR , we enable accurate quantitative prediction of the effects of missense variants on LDL-C levels, which aligns with measurements in UK Biobank individuals, and identify structural mechanisms underlying variant pathogenicity. This work provides a widely applicable approach to improve the power of base editor screens for disease-associated variant characterization., Competing Interests: Competing interests L.P. has financial interests in Edilytics, Inc., Excelsior Genomics, and SeQure Dx, Inc. L.P.’s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. The remaining authors declare no competing interests.

Background: Genetic and metabolic heterogeneity are well-known features of cancer and tumors can be viewed as an evolving mix of subclonal populations, subjected to selection driven by microenvironmental pressures or drug treatment. In previous studies, anti-VEGF therapy was found to elicit rewiring of tumor metabolism, causing marked alterations in glucose, lactate ad ATP levels in tumors. The aim of this study was to evaluate whether differences in the sensitivity to glucose starvation existed at the clonal level in ovarian cancer cells and to investigate the effects induced by anti-VEGF therapy on this phenotype by multi-omics analysis., Methods: Clonal populations, obtained from both ovarian cancer cell lines (IGROV-1 and SKOV3) and tumor xenografts upon glucose deprivation, were defined as glucose deprivation resistant (GDR) or glucose deprivation sensitive (GDS) clones based on their in vitro behaviour. GDR and GDS clones were characterized using a multi-omics approach, including genetic, transcriptomic and metabolic analysis, and tested for their tumorigenic potential and reaction to anti-angiogenic therapy., Results: Two clonal populations, GDR and GDS, with strikingly different viability following in vitro glucose starvation, were identified in ovarian cancer cell lines. GDR clones survived and overcame glucose starvation-induced stress by enhancing mitochondrial oxidative phosphorylation (OXPHOS) and both pyruvate and lipids uptake, whereas GDS clones were less able to adapt and died. Treatment of ovarian cancer xenografts with the anti-VEGF drug bevacizumab positively selected for GDR clones that disclosed increased tumorigenic properties in NOD/SCID mice. Remarkably, GDR clones were more sensitive than GDS clones to the mitochondrial respiratory chain complex I inhibitor metformin, thus suggesting a potential therapeutic strategy to target the OXPHOS-metabolic dependency of this subpopulation., Conclusion: A glucose-deprivation resistant population of ovarian cancer cells showing druggable OXPHOS-dependent metabolic traits is enriched in experimental tumors treated by anti-VEGF therapy., (© 2023. Italian National Cancer Institute ‘Regina Elena’.)

Limited molecular knowledge of Merkel cell polyomavirus (MCPyV)-positive and -negative Merkel cell carcinoma (MCC) subsets (MCCP/MCCN) has prevented so far the identification of the MCC origin cell type and, therefore, the development of effective therapies. The retinoic gene signature was investigated in various MCCP, MCCN, and control fibroblast/epithelial cell lines to elucidate the heterogeneous nature of MCC. Hierarchical clustering and principal component analysis indicated that MCCP and MCCN cells were clusterizable from each other and control cells, according to their retinoic gene signature. MCCP versus MCCN differentially expressed genes (n = 43) were identified. Protein-protein interaction network indicated SOX2, ISL1, PAX6, FGF8, ASCL1, OLIG2, SHH, and GLI1 as upregulated hub genes and JAG1 and MYC as downregulated hub genes in MCCP compared to MCCN. Numerous MCCP-associated hub genes were DNA-binding/-transcription factors involved in neurological and Merkel cell development and stemness. Enrichment analyses indicated that MCCP versus MCCN differentially expressed genes predominantly encode for to DNA-binding/-transcription factors involved in development, stemness, invasiveness, and cancer. Our findings suggest the neuroendocrine origin of MCCP, by which neuronal precursor cells could undergo an MCPyV-driven transformation. These overarching results might open the way to novel retinoid-based MCC therapies., (© 2023 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

The microbiota represents all the microorganisms including viruses, bacteria, fungi, and parasites, that have a symbiotic relationship with their host and that are present in a particular system (or niche) of the human body such as the skin, the respiratory tract, the urogenital tract or the digestive tract. This paper is a narrative review of all talks given at the 8th edition of the « Feeding the Microbiota » symposium organized at the Geneva University Hospitals. The symposium gathered 346 participants, both onsite and online, from 23 countries all-around the world. The main thematic of this edition focused on the composition of the gut microbiota as affected by prebiotics and postbiotics and their effects on various diseases., Competing Interests: Les auteurs n’ont déclaré aucun conflit d’intérêts en relation avec cet article.

Background: Blood transfusions represent common medical procedures, which provide essential supportive therapy. However, these procedures are notoriously expensive for healthcare services and not without risk. The potential threat of transfusion-related complications, such as the development of pathogenic infections and the occurring of alloimmunization events, alongside the donor's dependence, strongly limits the availability of transfusion units and represents significant concerns in transfusion medicine. Moreover, a further increase in the demand for donated blood and blood transfusion, combined with a reduction in blood donors, is expected as a consequence of the decrease in birth rates and increase in life expectancy in industrialized countries., Main Body: An emerging and alternative strategy preferred over blood transfusion is the in vitro production of blood cells from immortalized erythroid cells. The high survival capacity alongside the stable and longest proliferation time of immortalized erythroid cells could allow the generation of a large number of cells over time, which are able to differentiate into blood cells. However, a large-scale, cost-effective production of blood cells is not yet a routine clinical procedure, as being dependent on the optimization of culture conditions of immortalized erythroid cells., Conclusion: In our review, we provide an overview of the most recent erythroid cell immortalization approaches, while also describing and discussing related advancements of establishing immortalized erythroid cell lines., (© 2023. The Author(s).)

Transcription factors (TFs) are key regulatory proteins that control the transcriptional rate of cells by binding short DNA sequences called transcription factor binding sites (TFBS) or motifs. Identifying and characterizing TFBS is fundamental to understanding the regulatory mechanisms governing the transcriptional state of cells. During the last decades, several experimental methods have been developed to recover DNA sequences containing TFBS. In parallel, computational methods have been proposed to discover and identify TFBS motifs based on these DNA sequences. This is one of the most widely investigated problems in bioinformatics and is referred to as the motif discovery problem. In this manuscript, we review classical and novel experimental and computational methods developed to discover and characterize TFBS motifs in DNA sequences, highlighting their advantages and drawbacks. We also discuss open challenges and future perspectives that could fill the remaining gaps in the field., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The epigenetic role of microRNAs is established at both physiological and pathological levels. Dysregulated miRNAs and their targets appear to be a promising approach for innovative anticancer therapies. In our previous study, circulating miR-197-3p tested dysregulated in workers ex-exposed to asbestos (WEA). Herein, an epigenetic investigation on this circulating miRNA was carried out in sera from malignant pleural mesothelioma (MPM) patients. MiR-197-3p was quantified in MPM (n = 75) sera and comparatively analyzed to WEA (n = 75) and healthy subject (n = 75) sera, using ddPCR and RT-qPCR techniques. Clinicopathological characteristics, occupational, non-occupational information and overall survival (OS) were evaluated in correlation studies. MiR-197-3p levels, analyzed by ddPCR, were significantly higher in MPM than in WEA cohort, with a mean copies/µl of 981.7 and 525.01, respectively. Consistently, RT-qPCR showed higher miR-197-3p levels in sera from MPM with a mean copies/µl of 603.7, compared to WEA with 336.1 copies/µl. OS data were significantly associated with histologic subtype and pleurectomy. Circulating miR-197-3p is proposed as a new potential biomarker for an early diagnosis of the MPM onset. Indeed, miR-197-3p epigenetic investigations along with chest X-ray, computed tomography scan and spirometry could provide relevant information useful to reach an early and effective diagnosis for MPM., (© 2023. The Author(s).)

Oncogenic Merkel cell polyomavirus (MCPyV) provokes a widespread and asymptomatic infection in humans. Herein, sera from healthy children and young adults (HC, n = 344) aged 0-20 years old were evaluated for anti-MCPyV immunoglobulin G (IgG) and IgM antibodies employing a recently developed immunoassay. Serum MCPyV IgG data from healthy subjects (HS, n = 510) and elderlies (ES, n = 226), aged 21-65/66-100 years old, from our previous studies, were included. The anti-MCPyV IgG and IgM rates in HC sera were 40.7% and 29.7%, respectively. A lower prevalence of anti-MCPyV IgGs was found in HC aged 0-5 years old (13%) compared to 6-10 (52.3%), 11-15 (60.5%) and 16-20 years old (61.6%) cohorts. Age-stratified HCs exhibited similar anti-MCPyV IgM rates (27.9%-32.9%). Serological profiles indicated that anti-MCPyV IgGs and IgMs had low optical densities (ODs) during the first years of life, while IgM ODs appeared to decrease throughout young adulthood. A lower anti-MCPyV IgGs rate was found in HC (40.7%) than HS (61.8%) and ES (63.7%). Upon the 5-years range age-stratification, a lower anti-MCPyV IgGs rate was found in the younger HC cohort aged 0-5 years old compared to the remaining older HC/HS/ES cohorts (52.3%-72%). The younger HC cohort exhibited the lowest anti-MCPyV IgG ODs than the older cohorts. Low anti-MCPyV IgMs rates and ODs were found in the 21-25 (17.5%) and 26-30 (7.7%) years old cohorts. Our data indicate that, upon an early-in-life seroconversion, the seropositivity for oncogenic MCPyV peaks in late childhood/young adulthood and remains at high prevalence and relatively stable throughout life., (© 2022 John Wiley & Sons Ltd.)

Immune cells and commensal microbes in the human intestine constantly communicate with and react to each other in a stable environment in order to maintain healthy immune activities. Immune system-microbiota cross-talk relies on a complex network of pathways that sustain the balance between immune tolerance and immunogenicity. Probiotic bacteria can interact and stimulate intestinal immune cells and commensal microflora to modulate specific immune functions and immune homeostasis. Growing evidence shows that probiotic bacteria present important health-promoting and immunomodulatory properties. Thus, the use of probiotics might represent a promising approach for improving immune system activities. So far, few studies have been reported on the beneficial immune modulatory effect of probiotics. However, many others, which are mainly focused on their metabolic/nutritional properties, have been published. Therefore, the mechanisms behind the interaction between host immune cells and probiotics have only been partially described. The present review aims to collect and summarize the most recent scientific results and the resulting implications of how probiotic bacteria and immune cells interact to improve immune functions. Hence, a description of the currently known immunomodulatory mechanisms of probiotic bacteria in improving the host immune system is provided.

Merkel cell carcinoma (MCC) is an aggressive skin malignancy with two distinct etiologies. The first, which accounts for the highest proportion, is caused by Merkel cell polyomavirus (MCPyV), a DNA tumor virus. A second, UV-induced, MCC form has also been identified. Few MCC diagnostic, prognostic, and therapeutic options are available. MicroRNAs (miRNAs) are small noncoding RNA molecules, which play a key role in regulating various physiologic cellular functions including cell cycling, proliferation, differentiation, and apoptosis. Numerous miRNAs are dysregulated in cancer, by acting as either tumor suppressors or oncomiRs. The aim of this review is to collect, summarize, and discuss recent findings on miRNAs whose dysregulation has been assumed to play a role in MCC. The potential clinical application of miRNAs as diagnostic and prognostic biomarkers in MCC is also described. In the future, miRNAs will potentially gain clinical significance for the improvement of MCC diagnostic, prognostic, and therapeutic options., (© 2022 Wiley Periodicals LLC.)

CRISPR gene editing holds great promise to modify DNA sequences in somatic cells to treat disease. However, standard computational and biochemical methods to predict off-target potential focus on reference genomes. We developed an efficient tool called CRISPRme that considers single-nucleotide polymorphism (SNP) and indel genetic variants to nominate and prioritize off-target sites. We tested the software with a BCL11A enhancer targeting guide RNA (gRNA) showing promise in clinical trials for sickle cell disease and β-thalassemia and found that the top candidate off-target is produced by an allele common in African-ancestry populations (MAF 4.5%) that introduces a protospacer adjacent motif (PAM) sequence. We validated that SpCas9 generates strictly allele-specific indels and pericentric inversions in CD34 + hematopoietic stem and progenitor cells (HSPCs), although high-fidelity Cas9 mitigates this off-target. This report illustrates how genetic variants should be considered as modifiers of gene editing outcomes. We expect that variant-aware off-target assessment will become integral to therapeutic genome editing evaluation and provide a powerful approach for comprehensive off-target nomination., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The purpose of this study is to identify predictive factors associated with missed diagnosis of B. pertussis-B. holmesii co-infection by assessing the analytical performance of a commercially available multiplexed PCR assay and by building a prediction model based on clinical signs and symptoms for detecting co-infections. This is a retrospective study on the electronic health records of all clinical samples that tested positive to either B. pertussis or B. holmesii from January 2015 to January 2018 at Geneva University Hospitals. Multivariate logistic regression was used to build a model for co-infection prediction based on the electronic health record chart review. Limit of detection was determined for all targets of the commercial multiplexed PCR assay used on respiratory samples. A regression model, developed from clinical symptoms and signs, predicted B. pertussis and B. holmesii co-infection with an accuracy of 82.9% (95% CI 67.9-92.8%, p value = .012), for respiratory samples positive with any of the two tested Bordetella species. We found that the LOD of the PCR reaction targeting ptxS1 is higher than that reported by the manufacturer by a factor 10. The current testing strategy misses B. pertussis and B. holmesii co-infections by reporting only B. holmesii infections. Thus, we advocate to perform serological testing for detecting a response against pertussis toxin whenever a sample is found positive for B. holmesii. These findings are important, both from a clinical and epidemiological point of view, as the former impacts the choice of antimicrobial drugs and the latter biases surveillance data, by underestimating B. pertussis infections during co-infections., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Multiple sclerosis (MS) is a neurological disease characterized by immune dysregulations. Different viruses may act as MS triggering agents. MS patients respond differently to distinct viruses. The aim of our study is to verify the association between the polyomavirus BKPyV and MS, together with other neurological diseases, through the investigation of serum IgG antibodies against the virus. Sera were from patients affected by MS and other neurologic diseases, both inflammatory (OIND) and noninflammatory (NIND). Control sera were from healthy subjects (HS). Samples were analyzed for IgG antibodies against BKPyV with an indirect ELISA with synthetic peptides mimicking the viral capsid protein 1 (VP1) antigens. As control, ELISAs were carried out to verify the immune response against the Epstein-Barr virus (EBV) of patients and controls. In addition, we assessed values for total IgG in each experimental groups. A significant lower prevalence of IgG antibodies against BKPyV VP 1 epitopes, together with a low titer, was detected in sera from MS patients and other inflammatory neurologic diseases than HS. In MS patients and OIND and NIND groups, the EBV-antibody values and total IgG did not differ from HS. Experimental data indicate that patients affected by neurological diseases, including MS, are poor responders to BKPyV VP 1 antigens, thus suggesting specific immunologic dysfunctions for this polyomavirus. Our findings are relevant in understanding the immune reactions implicated in neurological disorders., Competing Interests: Data of this work were enclosed, in part, in the Italian patent application number I0167478/BRE-EC/rp. In addition to this, the authors declare that there is no other conflict of interest regarding the publication of this paper., (Copyright © 2022 Ilaria Bononi et al.)

Malignant pleural mesothelioma (MPM), a fatal tumor, is mainly linked to the asbestos exposure. It has been reported that together with the inhalation of asbestos fibers, other factors are involved in the MPM onset, including simian virus 40 (SV40). SV40, a polyomavirus with oncogenic potential, induces (i) in vitro the mesenchymal cell transformation, whereas (ii) in vivo the MPM onset in experimental animals. The association between MPM and SV40 in humans remains to be elucidated. Sera (n = 415) from MPM-affected patients (MPM cohort 1; n = 152) and healthy subjects (HSs, n = 263) were investigated for their immunoglobulin G (IgG) against simian virus 40 large tumor antigen (Tag), which is the transforming protein. Sera were investigated with an indirect enzyme-linked immunosorbent assay (ELISA) using two synthetic peptides from SV40 Tag protein. SV40 Tag protein was evaluated by immunohistochemical (IHC) staining on MPM samples (MPM cohort 2; n = 20). Formalin-fixed and paraffin-embedded (FFPE) samples were obtained from MPM patients unrelated to MPM serum donors. The proportion of sera, from MPM patients, showing antibodies against SV40 Tag (34%) was significantly higher compared to HSs (20%) (odds ratio 2.049, CI 95% 1.32-3.224; p =0.0026). Immunohistochemical staining (IHS) assays showed SV40 Tag expression in 8/20, 40% of MPM specimens. These results indicate that SV40 is linked to a large fraction of MPM. It is worth noting that the prevalence of SV40 Tag antibodies detected in sera from cohort 1 of MPM patients is similar to the prevalence of SV40 Tag found to be expressed in FFPE tissues from MPM cohort 2., Competing Interests: The authors report no conflicts of interest for this work., (Copyright © 2022 Elisa Mazzoni et al.)

Competing Interests: Les auteurs n’ont déclaré aucun conflit d’intérêts en relation avec cet article.

Bone defects in maxillofacial regions lead to noticeable deformity and dysfunctions. Therefore, the use of biomaterials/scaffolds for maxillofacial bone regrowth has been attracting great interest from many surgical specialties and experts. Many approaches have been devised in order to create an optimal bone scaffold capable of achieving desirable degrees of bone integration and osteogenesis. Osteogenesis represents a complex physiological process involving multiple cooperating systems. A tight relationship between the immune and skeletal systems has lately been established using the concept of "osteoimmunology," since various molecules, particularly those regulating immunological and inflammatory processes, are shared. Inflammatory mediators are now being implicated in bone remodeling, according to new scientific data. In this study, a profiler PCR array was employed to evaluate the expression of cytokines and chemokines in human adipose derived-mesenchymal stem cells (hASCs) cultured on porous hydroxylapatite (HA)/Collagen derived Bio-Oss ® /Avitene scaffolds, up to day 21. In hASCs grown on the Bio-Oss ® /Avitene biomaterial, 12 differentially expressed genes (DEGs) were found to be up-regulated, together with 12 DEG down-regulated. Chemokine CCL2, which affects bone metabolism, tested down-regulated. Interestingly, the Bio-Oss ® /Avitene induced the down-regulation of pro-inflammatory inter-leukin IL-6. In conclusion, our investigation carried out on the Bio-Oss ® /Avitene scaffold indicates that it could be successfully employed in maxillofacial surgery. Indeed, this composite material has the advantage of being customized on the basis of the individual patients favoring a novel personalized medicine approach., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Iaquinta, Martini, D’Agostino, Trevisiol, Bersani, Torreggiani, Tognon, Rotondo and Mazzoni.)

COVID-19 emerged in late 2019 in China and quickly spread across the globe, causing over 521 million cases of infection and 6.26 million deaths to date. After 2 years, numerous advances have been made. First of all, the preventive vaccine, which has been implemented in record time, is effective in more than 95% of cases. Additionally, in the diagnostic field, there are numerous molecular and antigenic diagnostic kits that are equipped with high sensitivity and specificity. Real Time-PCR-based assays for the detection of viral RNA are currently considered the gold-standard method for SARS-CoV-2 diagnosis and can be used efficiently on pooled nasopharyngeal, or oropharyngeal samples for widespread screening. Moreover, additional, and more advanced molecular methods such as droplet-digital PCR (ddPCR), clustered regularly interspaced short palindromic repeats (CRISPR) and next-generation sequencing (NGS), are currently under development to detect the SARS-CoV-2 RNA. However, as the number of subjects infected with SARS-CoV-2 continuously increases globally, health care systems are being placed under increased stress. Thus, the clinical laboratory plays an important role, helping to select especially asymptomatic individuals who are actively carrying the live replicating virus, with fast and non-invasive molecular technologies. Recent diagnostic strategies, other than molecular methods, have been adopted to either detect viral antigens, i.e., antigen-based immunoassays, or human anti-SARS-CoV-2 antibodies, i.e., antibody-based immunoassays, in nasal or oropharyngeal swabs, as well as in blood or saliva samples. However, the role of mucosal sIgAs, which are essential in the control of viruses entering the body through mucosal surfaces, remains to be elucidated, and in particular the role of the immune response in counteracting SARS-CoV-2 infection, primarily at the site(s) of virus entry that appears to be promising.