A streamlined workflow for a fast and cost-effective count of tyndallized probiotics using flow cytometry.

The use of dead probiotics and their cellular metabolites seems to exhibit immunomodulatory and anti-inflammatory properties, providing protection against pathogens. These inanimate microorganisms, often referred to as tyndallized or heat-killed bacteria, are a new class of probiotics employed in clinical practice. Safety concerns regarding the extensive use of live microbial cells have increased interest in inactivated bacteria, as they could eliminate shelf-life problems and reduce the risks of microbial translocation and infection. Culture-dependent methods are not suitable for the quality assessment of these products, and alternative methods are needed for their quantification. To date, bacterial counting chambers and microscopy have been used for tyndallized bacteria enumeration, but no alternative validated methods are now available for commercial release. The aim of the present study is to design a new method for the qualitative and quantitative determination of tyndallized bacterial cells using flow cytometric technology. Using a live/dead viability assay based on two nucleic acid stains, thiazole orange (TO) and propidium iodide (PI), we optimized a workflow to evaluate bacterial viability beyond the reproduction capacity that provides information about the structural properties and metabolic activities of probiotics on FACSVerse without using beads as a reference. The data obtained in this study represent the first analytical application that works effectively both on viable and non-viable cells. The results provided consistent evidence, and different samples were analyzed using the same staining protocol and acquisition settings. No significant discrepancies were highlighted between the declared specification of commercial strain and the analytical data obtained. For the first time, flow cytometry was used for counting tyndallized bacterial cells as a quality control assessment in probiotic production. This aspect becomes important if applied to medical devices where we cannot boast metabolic but only mechanical activities.

Verification of a Rapid Analytical Method for the Qualitative Detection of Listeria spp. and Listeria monocytogenes by a Real-Time PCR Assay according to EN UNI ISO 16140-3:2021.

Microbial contamination and foodborne infections are a significant global public health concern. For this reason, the detection, monitoring, and characterization of pathogens represent a significant challenge in quality control settings. Standard approaches, such as culture methods and biochemical tests, are known to be very time-consuming and intensive. Conversely, molecular technologies based on the genomic identification of bacteria are quick and low-cost. Listeria monocytogenes is an opportunistic pathogen and a major concern especially in food industries. It is important to understand and implement multiple quality control measures to control Listeria infection risk and prevent the contamination of products. Standardized detection and confirmation tests such as the API Listeria test, MALDI-TOF MS, and PCR analysis are available. The aim of our work is to provide a specific molecular method, designed according to the EN UNI ISO 16140-3:2021, for the specific detection, monitoring, and characterization of Listeria spp. and Listeria monocytogenes contamination. The verification of this new rapid approach by real-time PCR (qPCR) overcomes the limitations of culture-based techniques, meeting all the verification criteria required by ISO guidelines, including implementation and item confirmation. This system offers a powerful approach to the real-time assessment of food safety, useful for industry self-monitoring and regulatory inspection.

COVID-19-Related Myocarditis: Are We There Yet? A Case Report of COVID-19-Related Fulminant Myocarditis.

There is increasing evidence of cardiac involvement in COVID-19 cases, with a broad range of clinical manifestations spanning from acute life-threatening conditions such as ventricular dysrhythmias, myocarditis, acute myocardial ischemia and pulmonary thromboembolism to long-term cardiovascular sequelae. In particular, acute myocarditis represents an uncommon but frightening complication of SARS-CoV-2 infection. Even if many reports of SARS CoV-2 myocarditis are present in the literature, the majority of them lacks histological confirmation of cardiac injury. Here, we report a case of a young lady, who died suddenly a few days after testing positive for SARS-CoV-2, whose microscopic and genetics features suggested a direct cardiac involvement compatible with fulminant myocarditis.

An Integrated Analytical Approach for the Characterization of Probiotic Strains in Food Supplements.

Research surrounding health benefits from probiotics is becoming popular because of the increasing demand for safer products with protective and therapeutic effects. Proven benefits are species- or genus-specific; however, no certified assays are available for their characterization and quantification at the strain level in the food supplement industry. The objective of this study was to develop a strain-specific Real-time quantitative polymerase chain reaction (RT-qPCR)-based method to be implemented in routine tests for the identification and quantification of Bifidobacterium longum, Bifidobacterium animalis spp. lactis, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus helveticus, starting from a powder mixture of food supplements. The method optimization was carried out in combination with flow cytometry to compare results between the two strategies and implement the analytical workflow with the information also regarding cell viability. These assays were validated in accordance with the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) criteria using the plate count enumeration as the gold standard reference. Briefly, probiotic DNAs were extracted from two powder food supplements. Strain-specific primers targeting unique sequence regions of 16S RNA were identified and amplified by RT-qPCR. Primers were tested for specificity, sensitivity, and efficiency. Both RT-qPCR and flow-cytometry methods described in our work for the quantification and identification of Lactobacillus and Bifidobacterium strains were specific, sensitive, and precise, showing better performances with respect to the morphological colony identification. This work demonstrated that RT-qPCR can be implemented in the quality control workflow of commercial probiotic products giving more standardized and effective results regarding species discrimination.

Intrauterine fetal death in a COVID positive pregnant woman: a case report and literature review.

SARS-CoV-2 disease mechanisms are not yet fully understood, especially in cases affecting pregnant women. In fact, although they suffer from the same symptoms as non-pregnant women, they are more susceptible to adverse outcomes of COVID-19 as well as pregnancy complications leading to stillbirth, premature rupture of membrane, or intrauterine fetal demise (IUFD). The consequences on pregnancy are not clearly correlated with the severity of the infection. We present a case of fetal death in a 30-year-old woman with a confirmed SARS-CoV-2 infection without any other significant clinical or obstetric disorders. A histopathological examination of the placenta indicated massive and diffuse intervillar fibrinoid deposits associated with chronic histiocytic intervillositis that can be associated with SARS-CoV-2 placentitis. Given the high rates of SARS-CoV-2 infections in pregnancy, it is important to understand the factors that determine negative pregnancy outcomes, regardless of the severity of the patient's symptoms.

Clinical relevance of the combined analysis of circulating tumor cells and anti-tumor T-cell immunity in metastatic breast cancer patients.

Background: Metastatic breast cancer (mBC) is a heterogeneous disease with varying responses to treatments and clinical outcomes, still requiring the identification of reliable predictive biomarkers. In this context, liquid biopsy has emerged as a powerful tool to assess in real-time the evolving landscape of cancer, which is both orchestrated by the metastatic process and immune-surveillance mechanisms. Thus, we investigated circulating tumor cells (CTCs) coupled with peripheral T-cell immunity to uncover their potential clinical relevance in mBC. Methods: A cohort of 20 mBC patients was evaluated, before and one month after starting therapy, through the following liquid biopsy approaches: CTCs enumerated by a metabolism-based assay, T-cell responses against tumor-associated antigens (TAA) characterized by interferon-γ enzyme-linked immunosorbent spot (ELISpot), and the T-cell receptor (TCR) repertoire investigated by a targeted next-generation sequencing technique. TCR repertoire features were characterized by the Morisita's overlap and the Productive Simpson Clonality indexes, and the TCR richness. Differences between groups were calculated by Fisher's, Mann-Whitney or Kruskal-Wallis test, as appropriate. Prognostic data analysis was estimated by Kaplan-Meier method. Results: Stratifying patients for their prognostic level of 6 CTCs before therapy, TAA specific T-cell responses were detected only in patients with a low CTC level. By analyzing the TCR repertoire, the highest TCR clonality was observed in the case of CTCs under the cut-off and a positive ELISpot response (p=0.03). Whereas, at follow-up, patients showing a good clinical response coupled with a low number of CTCs were characterized by the most elevated TCR clonality (p<0.05). The detection of CTCs≥6 in at least one time-point was associated with a lower TCR clonality (p=0.02). Intriguingly, by combining overall survival analysis with TCR repertoire, we highlighted a potential prognostic role of the TCR clonality measured at follow-up (p=0.03). Conclusion: These data, whether validated in a larger cohort of patients, suggest that the combined analysis of CTCs and circulating anti-tumor T-cell immunity could represent a valuable immune-oncological biomarker for the liquid biopsy field. The clinical application of this promising tool could improve the management of mBC patients, especially in the setting of immunotherapy, a rising approach for BC treatment requiring reliable predictive biomarkers.

One-Step Nucleic Acid Amplification System in Comparison to the Intraoperative Frozen Section and Definitive Histological Examination Among Breast Cancer Patients: A Retrospective Survival Study.

Simple Summary: Implementing intraoperative assessment of sentinel lymph nodes by one-step nucleic acid amplification in early breast cancer can reduce the surgical burden to the patient and the costs to the health system. However, only limited data are available in terms of long-term disease-free survival and overall survival. Therefore, this study aims to compare disease-free survival and overall survival between one-step nucleic acid amplification, frozen section, and definitive histology. These results could impact the healthcare community, adding further proof to the body of evidence supporting the broader adoption of this innovative technology that enables a safe reduction in patient surgical burden and healthcare costs. Background: The one-step nucleic acid amplification (OSNA) system is a novel molecular technique, which consents to quick intraoperative detection of sentinel lymph node metastases by the amplification of cytokeratin 19 mRNA. Our study aims to evaluate the OSNA method in comparison with frozen section (FS) and definitive histological examination of the sentinel lymph node biopsy among early breast cancer patients considering disease-free survival (DFS) and overall survival (OS). Methods: In this study, we included all women who underwent sentinel lymph node biopsy (SLNB) for breast cancers classified as TNM stage I and II in our center between January 2005 and January 2017, and the follow-up was collected up to January 2019. We divided patients among three groups based on SLNB evaluation: definitive histological examination, intra-operative FS, or OSNA. Results: We included 2412 SLNBs: 727 by definitive histological examination, 697 by FS, and 988 by OSNA. Isolated tumor cells were found in 2.32% of cases, micrometastasis in 9.12%, and macrometastases in 13.64%. Surgical procedure duration was significantly shorter in OSNA than in FS (42.1 minutes ±5.1 vs. 70.1 minutes ±10.5, p <0.05). No significant differences have been observed among the three groups regarding OS, DSF, cumulative local, or distant metastases. In particular 5-year DFS was 96.38% in definitive histology (95% C.I. 95.02-97.75%), 96.37% in FS (95% C.I. 94.98-97.78%), and 96.51% in OSNA group (95% C.I. 95.32-97.72%). Conclusions: No difference in OS and DFS was found comparing OSNA, FS, and definitive histology. Furthermore, reduced operative time was found in the OSNA group.

Reinfection of Transplanted Livers in HCV- and HCV/HIV-Infected Patients Is Characterized by a Different MicroRNA Expression Profile.

BACKGROUND: After liver transplantation, HCV/HIV co-infected patients present, compared to the HCV mono-infected ones, increased HCV viral load, rapid progression to liver fibrosis and higher mortality. Liver biopsies (LB), obtained routinely 6 months after transplantation, represent a unique model to assess the early events related to graft re-infection. Here, we used miRNA sequencing of LB obtained from both HCV-and HCV/HIV-infected recipients, to identify transcriptional profiles able to explain the more severe outcome of these latter. METHODS: miRNAs of 3 healthy livers, 3 HCV-LB and 3 HCV/HIV-LB were sequenced by Illumina HiSeq2500 platform. The DIANA-miRPath v3.0 webserver and DIANA-microT-CDS algorithm (v5.0) were used to characterize the functions of differentially expressed (DE-) miRNAs, querying the KEGG and Gene Ontology-Biological Process databases. RESULTS: LB obtained from infected patients were characterized, with respect to controls, by a miRNA profile related to viral infection, immune system signaling and DNA damage in HCV-induced carcinogenesis. Instead, HCV-LB and HCV/HIV-LB differed in the expression of miRNAs involved in immunological and apoptotic processes and in extracellular matrix remodeling. CONCLUSIONS: liver reinfection processes are associated with early miRNA changes. Further studies are necessary to establish their prognostic role and possible actionability.

Incidence and Risk Factors for Venous Thromboembolism in Female Patients Undergoing Breast Surgery.

Albeit it does not have the highest venous thromboembolism (VTE) incidence compared to other neoplasms, breast cancer contributes to many VTE events because it is the most diagnosed tumor in women. We aim to analyze the occurrence and timing of VTE during the follow-up of patients who underwent breast surgery, the possible correlated factors, and the overall survival. This retrospective study included all female patients diagnosed with mammary pathology and surgically treated in our clinic between January 2002 and January 2012. Of 5039 women who underwent breast surgery, 1056 were found to have no evidence of malignancy, whereas 3983 were diagnosed with breast cancer. VTE rate resulted significantly higher in patients with invasive breast cancer than in women with benign breast disease or carcinoma in situ. Invasive cancers other than lobular or ductal were associated with a higher VTE rate. In addition, chronic hypertension, high BMI, cancer type, and evidence of metastasis turned out to be the most significant risk factors for VTE in women who underwent breast surgery. Moreover, VTE occurrence significantly impacted survival in invasive breast cancer patients. Compared to women with benign mammary pathology, VTE prevalence in women with breast cancer is significantly higher. The knowledge about the risk factors of VTE could be helpful as prognostic information, but also to eventually target preventive treatment strategies for VTE, as far as the co-existence of invasive breast cancer and VTE has a significantly negative impact on survival.

Image Analysis of Circulating Tumor Cells and Leukocytes Predicts Survival and Metastatic Pattern in Breast Cancer Patients.

BACKGROUND: The purpose of the present work was to test whether quantitative image analysis of circulating cells can provide useful clinical information targeting bone metastasis (BM) and overall survival (OS >30 months) in metastatic breast cancer (MBC). METHODS: Starting from cell images of epithelial circulating tumor cells (eCTC) and leukocytes (CD45pos) obtained with DEPArray, we identified the most significant features and applied single-variable and multi-variable methods, screening all combinations of four machine-learning approaches (Naïve Bayes, Logistic regression, Decision Trees, Random Forest). RESULTS: Best predictive features were circularity (OS) and diameter (BM), in both eCTC and CD45pos. Median difference in OS was 15 vs. 43 (months), p = 0.03 for eCTC and 19 vs. 36, p = 0.16 for CD45pos. Prediction for BM showed low accuracy (64%, 53%) but strong positive predictive value PPV (79%, 91%) for eCTC and CD45, respectively. Best machine learning model was Naïve Bayes, showing 46 vs 11 (months), p <0.0001 for eCTC; 12.5 vs. 45, p = 0.0004 for CD45pos and 11 vs. 45, p = 0.0003 for eCTC + CD45pos. BM prediction reached 91% accuracy with eCTC, 84% with CD45pos and 91% with combined model. CONCLUSIONS: Quantitative image analysis and machine learning models were effective methods to predict survival and metastatic pattern, with both eCTC and CD45pos containing significant and complementary information.

The Longevity-Associated Variant of BPIFB4 Reduces Senescence in Glioma Cells and in Patients' Lymphocytes Favoring Chemotherapy Efficacy.

Glioblastoma (GBM) is the most common primary brain cancer with the median age at diagnosis around 64 years, thus pointing to aging as an important risk factor. Indeed, aging, by increasing the senescence burden, is configured as a negative prognostic factor for GBM stage. Furthermore, several anti-GBM therapies exist, such as temozolomide (TMZ) and etoposide (ETP), that unfortunately trigger senescence and the secretion of proinflammatory senescence-associated secretory phenotype (SASP) factors that are responsible for the improper burst of (i) tumorigenesis, (ii) cancer metastasis, (iii) immunosuppression, and (iv) tissue dysfunction. Thus, adjuvant therapies that limit senescence are urgently needed. The longevity-associated variant (LAV) of the bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4) gene previously demonstrated a modulatory activity in restoring age-related immune dysfunction and in balancing the low-grade inflammatory status of elderly people. Based on the above findings, we tested LAV-BPIFB4 senotherapeutic effects on senescent glioblastoma U87-MG cells and on T cells from GBM patients. We interrogated SA-ß-gal and HLA-E senescence markers, SASP factors, and proliferation and apoptosis assays. The results highlighted a LAV-BPIFB4 remodeling of the senescent phenotype of GBM cells, enhancement of their sensitivity to temozolomide and a selective reduction of the T cells' senescence from GBM patients. Overall, these findings candidate LAV-BPIFB4 as an adjuvant therapy for GBM.

Validation of a Saliva-Based Test for the Molecular Diagnosis of SARS-CoV-2 Infection.

BACKGROUND: Since the beginning of the pandemic, clinicians and researchers have been searching for alternative tests to improve the screening and diagnosis of the SARS-CoV-2 infection. Currently, the gold standard for virus identification is the nasopharyngeal (NP) swab. Saliva samples, however, offer clear, practical, and logistical advantages but due to a lack of collection, transport, and storage solutions, high-throughput saliva-based laboratory tests are difficult to scale up as a screening or diagnostic tool. With this study, we aimed to validate an intralaboratory molecular detection method for SARS-CoV-2 on saliva samples collected in a new storage saline solution, comparing the results to NP swabs to determine the difference in sensitivity between the two tests. METHODS: In this study, 156 patients (cases) and 1005 asymptomatic subjects (controls) were enrolled and tested simultaneously for the detection of the SARS-CoV-2 viral genome by RT-PCR on both NP swab and saliva samples. Saliva samples were collected in a preservative and inhibiting saline solution (Biofarma Srl). Internal method validation was performed to standardize the entire workflow for saliva samples. RESULTS: The identification of SARS-CoV-2 conducted on saliva samples showed a clinical sensitivity of 95.1% and specificity of 97.8% compared to NP swabs. The positive predictive value (PPV) was 81% while the negative predictive value (NPV) was 99.5%. Test concordance was 97.6% (Cohen's Kappa = 0.86; 95% CI 0.81-0.91). The LoD of the test was 5 viral copies for both samples. CONCLUSIONS: RT-PCR assays conducted on a stored saliva sample achieved similar performance to those on NP swabs, and this may provide a very effective tool for population screening and diagnosis. Collection of saliva in a stabilizing solution makes the test more convenient and widely available; furthermore, the denaturing properties of the solution reduce the infective risks belonging to sample manipulation.

STRA6 and Placental Retinoid Metabolism in Gestational Diabetes Mellitus.

BACKGROUND: Recent reports indicate the potential role of the stimulated by retinoic acid 6 (STRA6) protein in developing insulin resistance. The study's objective was to assess placental STRA6 expression and staining pattern in human pregnancy complicated by gestational diabetes mellitus (GDM). The expression pattern of further relevant genes involved in retinoid metabolism was also evaluated. METHODS: A retrospective case-control study on paraffin-embedded placental tissue. Twenty-two human pregnancies affected by GDM, namely, 11 insulin-treated (iGDM) and 11 diet-controlled (dGDM), were compared with 22 normal-developed pregnancies (controls). An RT-PCR was performed in a random sample of 18 patients (six iGDM, six dGDM, and six controls) to assess RNA expression of STRA6 and further markers of retinoid metabolism. A semi-quantitative intensity evaluation at immunohistochemistry was performed for STRA6 in all 44 recruited patients. RESULTS: STRA6 showed a decreased placental staining (9.09% vs. 68.18% positively stained samples, p < 0.05) and augmented RNA expression in dGDM patients than controls (ΔCT expression 0.473, IQR 0.403-0.566 vs. 0.149, IQR 0.092-0.276, p < 0.05). The protein staining pattern in patients affected by iGDM was comparable to controls. A reduced RNA expression of LPL, LRP1, VLDLR, and MTTP besides an augmented expression of LDLR was found in dGDM, while overexpression of LRP1 and LPL was found in iGDM patients. Unlike in the control group, significant positive correlations were found between RXRα and the proteins involved in the intracellular uptake of ROH, such as STRA6, LRP1, LRP2, and VLDLR. CONCLUSIONS: An altered placental expression and staining pattern of STRA6 were found in pregnancies complicated by GDM compared to the controls. These changes were coupled to an altered expression pattern of several other genes involved in the retinoid metabolism.

High Viral Load in Upper Respiratory Specimens of Persons With SARS-CoV-2 Infection in a Northern Italian Area.

BACKGROUND: An assessment of viral load in biologic specimens of subjects with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may have important implications for public health planning. The aim of this study was to estimate the prevalence of high viral load in upper respiratory specimens of patients with SARS-CoV-2 infection during the first Italian wave (spring) and at the beginning of the second wave (summer) of the COVID-19 epidemic, through the measurement of cycle threshold (Ct) values from real-time reverse transcription-polymerase chain reaction tests conducted at the University Hospital of Udine, Italy, serving 530,000 inhabitants. METHODS: We compared the prevalence of high viral load, defined as Ct ≤ 20 at the first positive test result, in 262 subjects from the spring and 453 from the summer period. Logistic regression was used to account for potential confounding due to sex, age, and severity of infection. RESULTS: In the spring, 9.2% of subjects had Ct ≤ 20 versus 21.4% in the summer. After adjusting for confounders, the likelihood of having high viral load was 2.9 times higher in the summer than in the spring (95% confidence interval, 1.7-5.0). CONCLUSIONS: In this Italian area, more COVID-19 patients had high viral load in the spring epidemic wave than at the beginning of the second, during the summer. Cycle threshold values may represent useful information to monitor viral load at a population level in subjects with SARS-CoV-2 infection.

TFEB, SIRT1, CARM1, Beclin-1 expression and PITX2 methylation in breast cancer chemoresistance: a retrospective study.

BACKGROUND: Breast cancer chemoresistance is attributed to a wide variety of mechanisms, including autophagy. Transcription factor EB (TFEB) has been recently identified and characterized as one major regulator of autophagy and lysosomal genesis. OBJECTIVE: This study aims to evaluate the prognostic impact of TFEB and its pathway in breast cancer chemoresistance. METHODS: This retrospective study analyzes the expression of TFEB, CARM1, SIRT1, and Beclin-1 and the methylation of PITX2 in breast carcinoma. A group of breast cancer patients treated with chemotherapy, who relapsed within 12 months from treatment initiation, were compared to a sub-cohort of chemo-treated patients who did not recur within 12 months of follow-up. The expression of TFEB, CARM1, SIRT1, and Belcin-1 was analyzed using immunohistochemistry or RT-PCR on formalin-fixed paraffin-embedded samples. PITX2 methylation was tested with the diagnostic CE-marked kit Therascreen PITX2 RGQ PCR. In the final model, 136 cases of chemo-treated breast cancer were included. RESULTS: A higher TFEB and Beclin-1 expression correlate with shorter survival in patients with chemo-treated invasive breast cancer (respectively HR 3.46, CI.95 1.27-9.47, p < 0.05 and 7.11, CI.95 2.54-19.9). TFEB, CARM1, and SIRT1 are positively correlated with Beclin-1. The protein expression of SIRT1 is significantly associated with TFEB and CARM1 so that a very low SIRT1 expression (lower than the first quartile of the H-score distribution) correlates with a low expression of TFEB and CARM1 and with longer survival. SIRT1 seems to have a lower H-score in the basal-like and HER2-enriched tumors than the luminal subtypes. Beclin-1 and TFEB seem to have a higher H-score in the basal-like and HER2-enriched tumors than the luminal subtypes. PITX2 methylation analysis was feasible only in 65% of the selected samples, but no significant differences between cases and controls were found, and there was also no correlation with the expression of the TFEB pathway. CONCLUSIONS: TFEB, SIRT1, and Beclin-1 seem to have a potential prognostic significance in patients with chemo-treated breast cancer, likely because of their role in the regulation of autophagy. In addition, no correlation between TFEB and PITX2 methylation was found, likely because they perform two different roles within the autophagy process.

Intra-Laboratory Validation of Alpha-Galactosidase Activity Measurement in Dietary Supplements.

INTRODUCTION: Alpha-galactosidase (α-Gal) is an enzyme responsible for the hydrolyzation of glycolipids and glycoprotein commonly found in dietary sources. More than 20% of the general population suffers from abdominal pain or discomfort caused by intestinal gas and by indigested or partially digested food residuals. Therefore, α-Gal is used in dietary supplements to reduce intestinal gases and help complex food digestion. Marketed enzyme-containing dietary supplements must be produced in accordance with the Food and Drug Administration (FDA) regulations for Current Good Manufacturing Practice (cGMPs). AIM: in this work we illustrated the process used to develop and validate a spectrophotometric enzymatic assay for α-Gal activity quantification in dietary supplements. METHODS: The validation workflow included an initial statistical-phase optimization of materials, reagents, and conditions, and subsequently a comparative study with another fluorimetric assay. A final validation of method performance in terms of specificity, linearity, accuracy, intermediate-precision repeatability, and system precision was then executed. RESULTS AND CONCLUSIONS: The proven method achieved good performance in the quantitative determination of α-Gal activity in commercial food supplements in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals (ICH) guidelines and is suitable as a rapid in-house quality control test.

miRNA expression profiles in liver grafts of HCV and HIV/HCV-infected recipients, 6 months after liver transplantation.

In hepatitis C virus (HCV)/human immunodeficiency virus (HIV) co-infected patients, HIV enhances HCV replication and liver damage. Several microRNAs (miRNAs), active in pro-fibrotic and inflammatory pathways, have been implicated in the pathogenesis of this phenomenon. However, these miRNAs have been tested only in explanted cirrhotic livers, when the liver damage has become chronic and irreversible. No data are available on the early phase of viral infection, such as early after liver transplantation (LT). In the present study, the expression of miR-101, miR-122, miR-155, miR-192, miR-200c, miR-338, and miR-532 was determined by quantitative real-time polymerase chain reaction in liver biopsies of HCV (n = 19) and HCV/HIV-infected (n = 20) LT recipients, as well as in a control group (n = 18) of noninfected patients, transplanted for alcoholic cirrhosis. The timing of liver biopsy was 6 months post-LT. None of the patients was treated with direct-acting anti-HCV drugs. All co-infected recipients had suppressed HIV viral load. Grading and staging were assessed according to the Ishak Classification. HCV and HIV viral load were measured in the sera. miR-101 (p = .03), miR-122 (p = .012), and miR-192 (p = .038) were significantly downregulated in HCV/HIV co-infected and HCV mono-infected recipients when compared with noninfected recipients, and such downregulation was more pronounced in co-infected ones. Moreover, in co-infected recipients but not in mono-infected ones, miR-101 inversely correlated with the peripheral HCV-RNA levels (r = .41, p = .04) and miR-122 inversely correlated with peripheral HCV-RNA levels (r = .49, p = .03) and with the histological grading (r = .51, p = .02).  In conclusion, as early as 6 months after LT, the presence of HIV-HCV co-infection enhanced a significant downregulation of certain miRNAs that showed a direct correlation with HCV viral load and liver inflammation.

Heart failure impairs the mechanotransduction properties of human cardiac pericytes.

The prominent impact that coronary microcirculation disease (CMD) exerts on heart failure symptoms and prognosis, even in the presence of macrovascular atherosclerosis, has been recently acknowledged. Experimental delivery of pericytes in non-revascularized myocardial infarction improves cardiac function by stimulating angiogenesis and myocardial perfusion. Aim of this work is to verify if pericytes (Pc) residing in ischemic failing human hearts display altered mechano-transduction properties and to assess which alterations of the mechano-sensing machinery are associated with the observed impaired response to mechanical cues. RESULTS: Microvascular rarefaction and defects of YAP/TAZ activation characterize failing human hearts. Although both donor (D-) and explanted (E-) heart derived cardiac Pc support angiogenesis, D-Pc exert this effect significantly better than E-Pc. The latter are characterized by reduced focal adhesion density, decreased activation of the focal adhesion kinase (FAK)/ Crk-associated substrate (CAS) pathway, low expression of caveolin-1, and defective transduction of extracellular stiffness into cytoskeletal stiffening, together with an impaired response to both fibronectin and lysophosphatidic acid. Importantly, Mitogen-activated protein kinase kinase inhibition restores YAP/TAZ nuclear translocation. CONCLUSION: Heart failure impairs Pc mechano-transduction properties, but this defect could be reversed pharmacologically.

Dysmetabolic Circulating Tumor Cells Are Prognostic in Metastatic Breast Cancer.

Circulating tumor cells (CTCs) belong to a heterogeneous pool of rare cells, and a unequivocal phenotypic definition of CTC is lacking. Here, we present a definition of metabolically-altered CTC (MBA-CTCs) as CD45-negative cells with an increased extracellular acidification rate, detected with a single-cell droplet microfluidic technique. We tested the prognostic value of MBA-CTCs in 31 metastatic breast cancer patients before starting a new systemic therapy (T0) and 3-4 weeks after (T1), comparing results with a parallel FDA-approved CellSearch (CS) approach. An increased level of MBA-CTCs was associated with: i) a shorter median PFS pre-therapy (123 days vs. 306; p < 0.0001) and during therapy (139 vs. 266 days; p = 0.0009); ii) a worse OS pre-therapy (p = 0.0003, 82% survival vs. 20%) and during therapy (p = 0.0301, 67% survival vs. 38%); iii) good agreement with therapy response (kappa = 0.685). The trend of MBA-CTCs over time (combining data at T0 and T1) added information with respect to separate evaluation of T0 and T1. The combined results of the two assays (MBA and CS) increased stratification accuracy, while correlation between MBA and CS was not significant, suggesting that the two assays are detecting different CTC subsets. In conclusion, this study suggests that MBA allows detection of both EpCAM-negative and EpCAM-positive, viable and label-free CTCs, which provide clinical information apparently equivalent and complementary to CS. A further validation of proposed method and cut-offs is needed in a larger, separate study.