New Insights into Pediatric Kidney Transplant Rejection Biomarkers: Tissue, Plasma and Urine MicroRNAs Compared to Protocol Biopsy Histology.

The early identification of a subclinical rejection (SCR) can improve the long-term outcome of the transplanted kidney through intensified immunosuppression. However, the only approved diagnostic method is the protocol biopsy, which remains an invasive method and not without minor and/or major complications. The protocol biopsy is defined as the sampling of allograft tissue at pre-established times even in the absence of an impaired renal function; however, it does not avoid histological damage. Therefore, the discovery of new possible biomarkers useful in the prevention of SCR has gained great interest. Among all the possible candidates, there are microRNAs (miRNAs), which are short, noncoding RNA sequences, that are involved in mediating numerous post-transcriptional pathways. They can be found not only in tissues, but also in different biological fluids, both as free particles and contained in extracellular vesicles (EVs) released by different cell types. In this study, we firstly performed a retrospective miRNA screening analysis on biopsies and serum EV samples of 20 pediatric transplanted patients, followed by a second screening on another 10 pediatric transplanted patients' urine samples at one year post-transplant. In both cohorts, we divided the patients into two groups: patients with histological SCR and patients without histological SCR at one year post-transplantation. The isolated miRNAs were analyzed in an NGS platform to identify different expressions in the two allograft states. Although no statistical data were found in sera, in the tissue and urinary EVs, we highlighted signatures of miRNAs associated with the histological SCR state.

Genome-wide profiling of patient-derived glioblastoma stem-like cells reveals recurrent genetic and transcriptomic signatures associated with brain tumors.

PURPOSE: Patient-derived cancer cell lines can be very useful to investigate genetic as well as epigenetic mechanisms of transformation and to test new drugs. In this multi-centric study, we performed genomic and transcriptomic characterization of a large set of patient-derived glioblastoma (GBM) stem-like cells (GSCs). METHODS: 94 (80 I surgery/14 II surgery) and 53 (42 I surgery/11 II surgery) GSCs lines underwent whole exome and trascriptome analysis, respectively. RESULTS: Exome sequencing revealed TP53 as the main mutated gene (41/94 samples, 44%), followed by PTEN (33/94, 35%), RB1 (16/94, 17%) and NF1 (15/94, 16%), among other genes associated to brain tumors. One GSC sample bearing a BRAF p.V600E mutation showed sensitivity in vitro to a BRAF inhibitor. Gene Ontology and Reactome analysis uncovered several biological processes mostly associated to gliogenesis and glial cell differentiation, S - adenosylmethionine metabolic process, mismatch repair and methylation. Comparison of I and II surgery samples disclosed a similar distribution of mutated genes, with an overrepresentation of mutations in mismatch repair, cell cycle, p53 and methylation pathways in I surgery samples, and of mutations in receptor tyrosine kinase and MAPK signaling pathways in II surgery samples. Unsupervised hierarchical clustering of RNA-seq data produced 3 clusters characterized by distinctive sets of up-regulated genes and signaling pathways. CONCLUSION: The availability of a large set of fully molecularly characterized GCSs represents a valuable public resource to support the advancement of precision oncology for the treatment of GBM.

RNAseq analysis of olfactory neuroepithelium cytological samples in individuals with Down syndrome compared to euploid controls: a pilot study.

Down syndrome is a common genetic disorder caused by partial or complete triplication of chromosome 21. This syndrome shows an overall and progressive impairment of olfactory function, detected early in adulthood. The olfactory neuronal cells are located in the nasal olfactory mucosa and represent the first sensory neurons of the olfactory pathway. Herein, we applied the olfactory swabbing procedure to allow a gentle collection of olfactory epithelial cells in seven individuals with Down syndrome and in ten euploid controls. The aim of this research was to investigate the peripheral gene expression pattern in olfactory epithelial cells through RNAseq analysis. Validated tests (Sniffin' Sticks Extended test) were used to assess olfactory function. Olfactory scores were correlated with RNAseq results and cognitive scores (Vineland II and Leiter scales). All Down syndrome individuals showed both olfactory deficit and intellectual disability. Down syndrome individuals and euploid controls exhibited clear expression differences in genes located in and outside the chromosome 21. In addition, a significant correlation was found between olfactory test scores and gene expression, while a non-significant correlation emerged between olfactory and cognitive scores. This first preliminary step gives new insights into the Down syndrome olfactory system research, starting from the olfactory neuroepithelium, the first cellular step on the olfactory way.

Expression profiles of PRKG1, SDF2L1 and PPP1R12A are predictive and prognostic factors for therapy response and survival in high-grade serous ovarian cancer.

High-grade serous ovarian cancer (HGS-EOCs) is generally sensitive to front-line platinum (Pt)-based chemotherapy although most patients at an advanced stage relapse with progressive resistant disease. Clinical or molecular data to identify primary resistant cases at diagnosis are not yet available. HGS-EOC biopsies from 105 Pt-sensitive (Pt-s) and 89 Pt-resistant (Pt-r) patients were retrospectively selected from two independent tumor tissue collections. Pathway analysis was done integrating miRNA and mRNA expression profiles. Signatures were further validated in silico on a cohort of 838 HGS-EOC cases from a published dataset. In all, 131 mRNAs and 5 miRNAs belonging to different functionally related molecular pathways distinguish Pt-s from Pt-r cases. Then, 17 out of 23 selected elements were validated by orthogonal approaches (SI signature). As resistance to Pt is associated with a short progression-free survival (PFS) and overall survival (OS), the prognostic role of the SI signature was assessed, and 14 genes associated with PFS and OS, in multivariate analyses (SII signature). The prognostic value of the SII signature was validated in a third extensive cohort. The expression profiles of SDF2L1, PPP1R12A and PRKG1 genes (SIII signature) served as independent prognostic biomarkers of Pt-response and survival. The study identified a prognostic molecular signature based on the combined expression profile of three genes which had never been associated with the clinical outcome of HGS-EOC. This may lead to early identification, at the time of diagnosis, of patients who would not greatly benefit from standard chemotherapy and are thus eligible for novel investigational approaches.

The fecal bacterial microbiota is not useful for discriminating between lymphoplasmacytic enteritis and low-grade intestinal T-cell lymphoma in cats nor for predicting therapeutic response.

OBJECTIVE: To evaluate the fecal bacterial microbiota at the time of diagnosis (T0) and after 1 month of therapy (T1) in cats diagnosed with lymphoplasmacytic enteritis (LPE) or cats with low-grade intestinal T-cell lymphoma (LGITL) and to compare these findings with those of healthy cats. ANIMALS: 5 healthy cats, 13 cats with LPE, and 7 cats with LGITL were prospectively enrolled between June 2020 and June 2021. METHODS: Fecal samples were collected at T0 and T1, and DNA was extracted for 16S ribosomal amplicon sequencing. Alpha diversity and beta diversity were computed. The taxonomic assignment was performed using sequences from the Silva v138 formatted reference database. Differential abundant taxa were selected in each taxonomic level, with the P value adjusted < .05, as the cut-off. RESULTS: No significant differences in alpha and beta diversity were found either at T0 or T1 between healthy and diseased cats or between cats with LPE and LGITL. Beta-diversity analysis showed an increase in the Fusobacteriaceae family in cats with LGITL at T0, compared to cats with LPE. Regardless of histological diagnosis, several microbiota differences were found at T0 based on serum cobalamin levels. CLINICAL RELEVANCE: Fecal samples were successfully used to characterize the bacteriome of the intestinal tract in cats by 16S rRNA gene sequencing. However, results highlighted that the metagenomic evaluation was not useful to discriminate between LPE and LGITL nor to predict the therapeutic response in this study population.

Clinical evaluation and microbiota analysis in 9 dogs with antibiotic-responsive enteropathy: A prospective comparison study.

BACKGROUND: Antibiotic-responsive enteropathy (ARE) is diagnosed by excluding other causes of diarrhea and when there is a short-term response to administration of antibiotics. OBJECTIVES: To characterize the gut microbiota and clinical trend of dogs with suspected ARE and to evaluate the variation in microbiota before (T0), after 30 days (T30) of tylosin treatment, and 30 days after discontinuation of treatment (T60). A further objective was to evaluate whether changes in gut microbiota are related to relapses of diarrhea when the therapy is tapered. ANIMALS: Study sample (group A) was composed of 15 dogs with chronic diarrhea, group B was composed of 15 healthy dogs. Group A was given tylosin for 30 days. METHODS: A multicentric prospective study. Clinical Indexes, fecal score, and samples for microbiota analysis were collected at T0, T30, and T60 in group A and T0 and T30 in group B. The gut microbiota was analyzed via 16S ribosomal RNA gene. Qiime2 version 2020.2 was used to perform bioinformatic analyses, and Alpha- and Beta-diversity were computed. RESULTS: Diarrhea recurred after T30 in 9 of 14 dogs, which were classified as affected by ARE. At T0, a difference was noted in the beta-diversity between groups (Bray Curtis metric P = .006). A T0-T30 difference in alpha-diversity was noted in group A (Shannon index P = .001, Faith PD P = .007). CONCLUSIONS AND CLINICAL IMPORTANCE: Although tylosin influences the microbiota of dogs with ARE, we failed to find any specific characteristic in the microbiota of dogs with ARE.

Genome-scale metabolic modelling of SARS-CoV-2 in cancer cells reveals an increased shift to glycolytic energy production.

Cancer is considered a high-risk condition for severe illness resulting from COVID-19. The interaction between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and human metabolism is key to elucidating the risk posed by COVID-19 for cancer patients and identifying effective treatments, yet it is largely uncharacterised on a mechanistic level. We present a genome-scale map of short-term metabolic alterations triggered by SARS-CoV-2 infection of cancer cells. Through transcriptomic- and proteomic-informed genome-scale metabolic modelling, we characterise the role of RNA and fatty acid biosynthesis in conjunction with a rewiring in energy production pathways and enhanced cytokine secretion. These findings link together complementary aspects of viral invasion of cancer cells, while providing mechanistic insights that can inform the development of treatment strategies.

First Isolation and Molecular Typing of Pathogenic and Intermediate Leptospira Species from Urine of Symptomatic Dogs.

Aim of this study was to evaluate, the presence and diversity of Leptospira spp. in blood and urine samples collected from 175 owned-dogs from Sardinia, Italy. After determination of leptospiral infection by microscopic agglutination test (MAT), urine from MAT-positive dogs were examined by real-time polymerase chain reaction (lipL32 rt-PCR) and then isolated by culture. In order to characterize obtained serovars, positive cultures were then subjected to 16S rRNA and secY sequencing, phylogenetic analysis and Multilocus Sequence Typing (MLST). Results showed that seven dogs (4%; 95% CI: 0-55) had Leptospira DNAs in their urine and five strains were isolated from urine cultures. The three different sequence types (ST17, ST198 and ST24) belonging to Leptospira interrogans genomospecies identified by MLST analyses in this study, confirmed that the leptospiral infection was widespread in Sardinian dogs. We also reported the first characterization of a new Leptospira spp. isolated from urine of one dog living in the study area. Whole genome sequencing and phylogenetic analysis, confirmed that this genospecies was closely related to Leptospira hovindhougenii, an intermediate Leptospira spp. with unknown pathogenicity previously isolated from a rat in Denmark. Further studies are required to clarify whether healthy dogs that shed leptospires in their urine could represent a zoonotic risk for humans in this region.

MicroRNA expression profiling with a droplet digital PCR assay enables molecular diagnosis and prognosis of cancers of unknown primary.

Metastasis is responsible for the majority of cancer-related deaths. Particularly, challenging is the management of metastatic cancer of unknown primary site (CUP), whose tissue of origin (TOO) remains undetermined even after extensive investigations and whose therapy is rather unspecific and poorly effective. Molecular approaches to identify the most probable TOO of CUPs can overcome some of these issues. In this study, we applied a predetermined set of 89 microRNAs (miRNAs) to infer the TOO of 53 metastatic cancers of unknown or uncertain origin. The miRNA expression was assessed with droplet digital PCR in 159 samples, including primary tumors from 17 tumor classes (reference set) and metastases of known and unknown origin (test set). We combined two different statistical models for class prediction to obtain the most probable TOOs: the nearest shrunken centroids approach of Prediction Analysis of Microarrays (PAMR) and the least absolute shrinkage and selection operator (LASSO) models. The molecular test was successful for all formalin-fixed paraffin-embedded samples and provided a TOO identification within 1 week from the biopsy procedure. The most frequently predicted origins were gastrointestinal, pancreas, breast, lung, and bile duct. The assay was applied also to multiple metastases from the same CUP, collected from different metastatic sites: The predictions showed a strong agreement, intrinsically validating our assay. The final CUPs' TOO prediction was compared with the clinicopathological hypothesis of primary site. Moreover, a panel of 13 miRNAs proved to have prognostic value and be associated with overall survival in CUP patients. Our study demonstrated that miRNA expression profiling in CUP samples could be employed as diagnostic and prognostic test. Our molecular analysis can be performed on request, concomitantly with standard diagnostic workup and in association with genetic profiling, to offer valuable indications about the possible primary site, thereby supporting treatment decisions.

Newly-Discovered Neural Features Expand the Pathobiological Knowledge of Blastic Plasmacytoid Dendritic Cell Neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive hematologic malignancy originating from plasmacytoid dendritic cells (pDCs). The microRNA expression profile of BPDCN was compared to that of normal pDCs and the impact of miRNA dysregulation on the BPDCN transcriptional program was assessed. MiRNA and gene expression profiling data were integrated to obtain the BPDCN miRNA-regulatory network. The biological process mainly dysregulated by this network was predicted to be neurogenesis, a phenomenon raising growing interest in solid tumors. Neurogenesis was explored in BPDCN by querying different molecular sources (RNA sequencing, Chromatin immunoprecipitation-sequencing, and immunohistochemistry). It was shown that BPDCN cells upregulated neural mitogen genes possibly critical for tumor dissemination, expressed neuronal progenitor markers involved in cell migration, exchanged acetylcholine neurotransmitter, and overexpressed multiple neural receptors that may stimulate tumor proliferation, migration and cross-talk with the nervous system. Most neural genes upregulated in BPDCN are currently investigated as therapeutic targets.

Transcriptional Characterization of Stage I Epithelial Ovarian Cancer: A Multicentric Study.

Stage I epithelial ovarian cancer (EOC) represents about 10% of all EOCs. It is characterized by a complex histopathological and molecular heterogeneity, and it is composed of five main histological subtypes (mucinous, endometrioid, clear cell and high, and low grade serous), which have peculiar genetic, molecular, and clinical characteristics. As it occurs less frequently than advanced-stage EOC, its molecular features have not been thoroughly investigated. In this study, using in silico approaches and gene expression data, on a multicentric cohort composed of 208 snap-frozen tumor biopsies, we explored the subtype-specific molecular alterations that regulate tumor aggressiveness in stage I EOC. We found that single genes rather than pathways are responsible for histotype specificities and that a cAMP-PKA-CREB1 signaling axis seems to play a central role in histotype differentiation. Moreover, our results indicate that immune response seems to be, at least in part, involved in histotype differences, as a higher immune-reactive behavior of serous and mucinous samples was observed with respect to other histotypes.

Chemotherapy-related cardiotoxicity: new diagnostic and preventive strategies.

Chemotherapy is an established approach for several malignancies, but its utility may be hampered by induced cardiac toxicity possibly leading to heart failure, with a negative impact on the patient's quality of life and ultimately survival. Prospective left ventricular systolic function monitoring has demonstrated that cardiotoxicity could be subclinically present for many months or years before its overt manifestation. Although considered irreversible, some reports suggested recovery or delayed progression of cardiac dysfunction by preventive cardioactive therapies. Thus, the identification of earlier instrumental or biochemical markers of cardiac injury able to predict heart failure remains a major task. Diastolic indexes as a primary expression of hemodynamic dysfunction after cardiac damage, analyzed by means of conventional or newer Doppler technologies (tissue Doppler, color M-mode, etc.) are discussed. Moreover, brain natriuretic peptides, troponins and endothelin-1, as possible sensitive/specific markers/predictors of subclinical cardiotoxicity are reviewed in order to update and possibly improve the strategy for the detection and clinical management of chemotherapy-related cardiotoxic effects.