Droplet digital PCR (ddPCR) is a sensitive and reproducible technology widely used for quantitation of several viruses. The aim of this study was to evaluate the 2019-nCoV CDC ddPCR Triplex Probe Assay (BioRad) performance, comparing the direct quantitation of SARS-CoV-2 on nasopharyngeal swab with the procedure applied to the extracted RNA. Moreover, two widely used swab types were compared (UTM 3 mL and ESwab 1 mL, COPAN). A total of 50 nasopharyngeal swabs (n = 25 UTM 3 mL and n = 25 ESwab 1 mL) from SARS-CoV-2 patients, collected during the pandemic at IRCCS Sacro Cuore Don Calabria Hospital (Veneto Region, North-East Italy), were used for our purpose. After heat inactivation, an aliquot of swab medium was used for the direct quantitation. Then, we compared the direct method with the quantitation performed on the RNA purified from nasopharyngeal swab by automated extraction. We observed that the direct approach achieved generally equal RNA copies compared to the extracted RNA. The results with the direct quantitation were more accurate on ESwab with a sensitivity of 93.33% [95% CI, 68.05 to 99.83] and specificity of 100.00% for both N1 and N2. On the other hand, on UTM we observed a higher rate of discordant results for N1 and N2. The human internal amplification control (RPP30) showed 100% of both sensitivity and specificity independent of swabs and approaches. In conclusion, we described a direct quantitation of SARS-CoV-2 in nasopharyngeal swab. Our approach resulted in an efficient quantitation, without automated RNA extraction and purification. However, special care needs to be taken on the potential bias due to the conservation of samples and to the heating treatment, as we used thawed and heat inactivated material. Further studies on a larger cohort of samples are warranted to evaluate the clinical value of this direct approach.

Background: We assessed the sensitivity, specificity and positive and negative predictive value (PPV and NPV) of molecular and serological tests for the diagnosis of SARS-CoV-2 infection. Methods: A total of 346 patients were enrolled in the emergency room. We evaluated three Reverse Transcriptase-real time PCRs (RT-PCRs) including six different gene targets, five serologic rapid diagnostic tests (RDT) and one ELISA. The final classification of infected/non-infected patients was performed using Latent Class Analysis combined with clinical re-assessment of incongruous cases. Results: Out of these, 24.6% of patients were classified as infected. The molecular test RQ-SARS-nCoV-2 showed the highest performance with 91.8% sensitivity, 100% specificity, 100.0% PPV and 97.4% NPV respectively. Considering the single gene targets, S and RdRp of RQ-SARS-nCoV-2 had the highest sensitivity (94.1%). The in-house RdRp presented the lowest sensitivity (62.4%). The specificity ranged from 99.2% for in-house RdRp and N2 to 95.0% for E. The PPV ranged from 97.1% of N2 to 85.4% of E and the NPV from 98.1% of S to 89.0% of in-house RdRp. All serological tests had < 50% sensitivity and low PPV and NPV. VivaDiag IgM (RDT) had 98.5% specificity, with 84.0% PPV, but 24.7% sensitivity. Conclusion: Molecular tests for SARS-CoV-2 infection showed excellent specificity, but significant differences in sensitivity. Serological tests have limited utility in a clinical context.

Objectives We assessed the antibody response to BNT162b2 mRNA COVID-19 vaccine in a cohort of health care workers (HCW), comparing subjects with previous SARS-CoV-2 infection and naïve subjects. Methods HCW were tested at T0 (day of first dose), T1 (day of second dose), and T2 (2-3 weeks after) for IgG anti nucleocapside protein, IgM anti spike protein and IgG anti receptor binding domain (IgG-RBD-S). The antibody response was compared between 4 main groups: A) Subjects with previous infection and positive antibodies at baseline; B) subjects with same history but negative antibodies; C) subjects with no infection history but positive antibodies; D) naïve subjects. Repeated measures analysis was used to compare results over time points. Results 1,935 HCW were included. Median IgG-RBD-S titre was significantly higher for group A (232 subjects) than for group B (56 subjects) both at T1 (A: 22,763 AU/mL, IQR 14,222-37,204; B: 1,373 AU/mL, IQR 783-3,078, p=0.0003) and T2 (A: 30,765 AU/mL, IQR 19,841-42,813; B:13,171 AU/mL, IQR 2,324-22,688, p=0.0038) and for group D (1563 subjects): 796 AU/mL, IQR 379-1,510 at T1; 15,494 AU/mL, IQR 9,122-23,916 at T2, p<0.0001 both timepoints. T1 values of group A were also significantly higher than T2 values of group D (p<0.0001). Presence of symptoms, younger age and female gender were associated with stronger antibody response. HCW infected in March showed a significantly stronger response (T1: 35,324 AU/mL, IQR 22,003-44,531; T2: 37,648 AU/mL, IQR 27,088-50,451) than those infected in November (T1: 18,499 AU/mL, IQR 11,492-27,283; T2: 23,210 AU/mL, IQR 18,074-36,086): p<0.0001, both timepoints. Conclusions Subjects with past COVID-19 infection had a strong antibody response after one single vaccine shot. A single dose might be sufficient for this group, regardless the time elapsed since infection, however the clinical correlation with antibody response needs to be studied.

Lack of physical exercise is considered an important risk factor for chronic diseases. On the contrary, physical exercise reduces the morbidity rates of obesity, diabetes, bone disease, and hypertension. In order to gain novel molecular and cellular clues, we analyzed the effects of physical exercise on differentiation of mesenchymal circulating progenitor cells (M-CPCs) obtained from runners. We also investigated autophagy and telomerase-related gene expression to evaluate the involvement of specific cellular functions in the differentiation process. We performed cellular and molecular analyses in M-CPCs, obtained by a depletion method, of 22 subjects before (PRE RUN) and after (POST RUN) a half marathon performance. In order to prove our findings, we performed also in vitro analyses by testing the effects of runners' sera on a human bone marrow-derived mesenchymal stem (hBM-MSC) cell line. PCR array analyses of PRE RUN versus POST RUN M-CPC total RNAs put in evidence several genes which appeared to be modulated by physical activity. Our results showed that physical exercise promotes differentiation. Osteogenesis-related genes as RUNX2, MSX1, and SPP1 appeared to be upregulated after the run; data showed also increased levels of BMP2 and BMP6 expressions. SOX9, COL2A1, and COMP gene enhanced expression suggested the induction of chondrocytic differentiation as well. The expression of telomerase-associated genes and of two autophagy-related genes, ATG3 and ULK1, was also affected and correlated positively with MSC differentiation. These data highlight an attractive cellular scenario, outlining the role of autophagic response to physical exercise and suggesting new insights into the benefits of physical exercise in counteracting chronic degenerative conditions.

The mortality rate for malignant melanoma (MM) is very high, since it is highly invasive and resistant to chemotherapeutic treatments. The modulation of some transcription factors affects cellular processes in MM. In particular, a higher expression of the osteogenic master gene RUNX2 has been reported in melanoma cells, compared to normal melanocytes. By analyzing public databases for recurrent RUNX2 genetic and epigenetic modifications in melanoma, we found that the most common RUNX2 genetic alteration that exists in transcription upregulation is, followed by genomic amplification, nucleotide substitution and multiple changes. Additionally, altered RUNX2 is involved in unchecked pathways promoting tumor progression, Epithelial Mesenchymal Transition (EMT), and metastasis. In order to investigate further the role of RUNX2 in melanoma development and to identify a therapeutic target, we applied the CRISPR/Cas9 technique to explore the role of the RUNT domain of RUNX2 in a melanoma cell line. RUNT-deleted cells showed reduced proliferation, increased apoptosis, and reduced EMT features, suggesting the involvement of the RUNT domain in different pathways. In addition, del-RUNT cells showed a downregulation of genes involved in migration ability. In an in vivo zebrafish model, we observed that wild-type melanoma cells migrated in 81% of transplanted fishes, while del-RUNT cells migrated in 58%. All these findings strongly suggest the involvement of the RUNT domain in melanoma metastasis and cell migration and indicate RUNX2 as a prospective target in MM therapy.

Physical exercise is known to promote beneficial effects on overall health, counteracting risks related to degenerative diseases. MicroRNAs (miRNAs), short non-coding RNAs affecting the expression of a cell’s transcriptome, can be modulated by different stimuli. Yet, the molecular effects on osteogenic differentiation triggered by miRNAs upon physical exercise are not completely understood. In this study, we recruited 20 male amateur runners participating in a half marathon. Runners’ sera, collected before (PRE RUN) and after (POST RUN) the run, were added to cultured human mesenchymal stromal cells. We then investigated their effects on the modulation of selected miRNAs and the consequential effects on osteogenic differentiation. Our results showed an increased expression of miRNAs promoting osteogenic differentiation (miR-21-5p, miR-129-5p, and miR-378-5p) and a reduced expression of miRNAs involved in the adipogenic differentiation of progenitor cells (miR-188-5p). In addition, we observed the downregulation of PTEN and SMAD7 expression along with increased AKT/pAKT and SMAD4 protein levels in MSCs treated with POST RUN sera. The consequent upregulation of RUNX2 expression was also proven, highlighting the molecular mechanisms by which miR-21-5p promotes osteogenic differentiation. In conclusion, our work proposes novel data, which demonstrate how miRNAs may regulate the osteogenic commitment of progenitor cells in response to physical exercise.

High concentrations of ivermectin demonstrated antiviral activity against SARS-CoV-2 in vitro . Aim of this study was to assess safety and efficacy of high-dose ivermectin in reducing viral load in individuals with early SARS-CoV-2 infection. Randomised, double-blind, multicentre, phase II, dose-finding, proof-of-concept clinical trial. Participants: adults recently diagnosed with asymptomatic/oligosymptomatic SARS-CoV-2 infection, providing informed consent. Exclusion criteria: pregnant or lactating women; CNS diseases; dialysis; severe medical condition with prognosis < 6 months; warfarin treatment; antiviral/chloroquine phosphate/hydroxychloroquine treatment. Participants were assigned according to a randomized permuted block procedure to one of the following arms with allocation ratio 1:1:1: placebo (arm A); single dose ivermectin 600 μg/kg plus placebo for 5 days (arm B); single dose ivermectin 1200 μg/kg for 5 days (arm C). Primary outcomes: serious adverse drug reactions (SADR) and change of viral load at Day 7. From 31 th July, 2020 to 26 th May, 2021, 32 participants were randomized to arm A, 29 to arm B and 32 to arm C. The recruitment was stopped on 10 th June, because of a dramatic drop of cases. Eighty-nine participants were included in the safety analysis set, the change in viral load was calculated on 87 participants. No SADR were registered. The mean log10 viral load reduction was 2.9 in arm C (SD 1.6), 2.5 (2.2) in arm B and 2.0 (2.1) in arm A, with no significant differences (p=0.099 and 0.122 for C versus A and B versus A, respectively). High-dose ivermectin was safe, but did not prove efficacy to reduce viral load.

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