Antitumour effects of SFX-01 molecule in combination with ionizing radiation in preclinical and in vivo models of rhabdomyosarcoma.

BACKGROUND: Despite a multimodal approach including surgery, chemo- and radiotherapy, the 5-year event-free survival rate for rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in childhood, remains very poor for metastatic patients, mainly due to the selection and proliferation of tumour cells driving resistance mechanisms. Personalised medicine-based protocols using new drugs or targeted therapies in combination with conventional treatments have the potential to enhance the therapeutic effects, while minimizing damage to healthy tissues in a wide range of human malignancies, with several clinical trials being started. In this study, we analysed, for the first time, the antitumour activity of SFX-01, a complex of synthetic d, l-sulforaphane stabilised in alpha-cyclodextrin (Evgen Pharma plc, UK), used as single agent and in combination with irradiation, in four preclinical models of alveolar and embryonal RMS. Indeed, SFX-01 has shown promise in preclinical studies for its ability to modulate cellular pathways involved in inflammation and oxidative stress that are essential to be controlled in cancer treatment. METHODS: RH30, RH4 (alveolar RMS), RD and JR1 (embryonal RMS) cell lines as well as mouse xenograft models of RMS were used to evaluate the biological and molecular effects induced by SFX-01 treatment. Flow cytometry and the modulation of key markers analysed by q-PCR and Western blot were used to assess cell proliferation, apoptosis, autophagy and production of intracellular reactive oxygen species (ROS) in RMS cells exposed to SFX-01. The ability to migrate and invade was also investigated with specific assays. The possible synergistic effects between SFX-01 and ionising radiation (IR) was studied in both the in vitro and in vivo studies. Student's t-test or two-way ANOVA were used to test the statistical significance of two or more comparisons, respectively. RESULTS: SFX-01 treatment exhibited cytostatic and cytotoxic effects, mediated by G2 cell cycle arrest, apoptosis induction and suppression of autophagy. Moreover, SFX-01 was able to inhibit the formation and the proliferation of 3D tumorspheres as monotherapy and in combination with IR. Finally, SFX-01, when orally administered as single agent, displayed a pattern of efficacy at reducing the growth of tumour masses in RMS xenograft mouse models; when combined with a radiotherapy regime, it was observed to act synergistically, resulting in a more positive outcome than would be expected by adding each exposure alone. CONCLUSIONS: In summary, our results provide evidence for the antitumour properties of SFX-01 in preclinical models of RMS tumours, both as a standalone treatment and in combination with irradiation. These forthcoming findings are crucial for deeper investigations of SFX-01 molecular mechanisms against RMS and for setting up clinical trials in RMS patients in order to use the SFX-01/IR co-treatment as a promising therapeutic approach, particularly in the clinical management of aggressive RMS disease.

PANC-1 Cell Line as an Experimental Model for Characterizing PIVKA-II Production, Distribution, and Molecular Mechanisms Leading to Protein Release in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) represents a highly aggressive malignancy with a lack of reliable diagnostic biomarkers. Protein induced by vitamin K absence (PIVKA-II) is a protein increased in various cancers (particularly in hepatocellular carcinoma), and it has recently exhibited superior diagnostic performance in PDAC detection compared to other biomarkers. The aim of our research was to identify an in vitro model to study PIVKA-II production, distribution, and release in PDAC. We examined the presence of PIVKA-II protein in a panel of stabilized pancreatic cancer cell lines by Western blot analysis and indirect immunofluorescence (IFA). After quantitative evaluation of PIVKA-II in PaCa 44, H-Paf II, Capan-1, and PANC-1, we adopted the latter as a reference model. Subsequently, we analyzed the effect of glucose addiction on PIVKA-II production in a PANC-1 cell line in vitro; PIVKA-II production seems to be directly related to an increase in glucose concentration in the culture medium. Finally, we evaluated if PIVKA-II released in the presence of increasing doses of glucose is concomitant with the expression of two well-acknowledged epithelial-mesenchymal transition (EMT) markers (Vimentin and Snail). According to our experimental model, we can speculate that PIVKA-II release by PANC-1 cells is glucose-dependent and occurs jointly with EMT activation.

DNA Sequence Variations Affecting Serotonin Transporter Transcriptional Regulation and Activity: Do They Impact Alcohol Addiction?

Genetic features of alcohol dependence have been extensively investigated in recent years. A large body of studies has underlined the important role of genetic variants not only in metabolic pathways but also in the neurobiology of alcohol dependence, mediated by the neuronal circuits regulating reward and craving. Serotonin transporter (5-HTT), encoded by the SLC6A4 gene (Solute carrier family 6-neurotransmitter transporter-member 4), is targeted by antidepressant drugs such as selective serotonin reuptake inhibitors (SSRIs) and plays a pivotal role in serotoninergic transmission; it has been associated with psychiatric diseases and alcohol dependence. Transcriptional regulation and expression of 5-HTT depend not only on epigenetic modifications, among which DNA methylation (CpG and non-CpG) is primarily involved, but also on sequence variations occurring in intron/exon regions and in untranslated regions in 5' and 3', being the first sequences important for the splicing machinery and the last for the binding of transcription factors and micro RNAs. This work intends to shed light on the role of sequence variations known to affect the expression or function of 5-HTT in alcohol-dependent individuals. We found a statistically significant difference in the allelic (p = 0.0083) and genotypic (p = 0.0151) frequencies of the tri-allelic polymorphism, with higher function alleles and genotypes more represented in the control population. Furthermore, we identified three haplotypes more frequent in subjects with AUD (p < 0.0001) and one more frequent in the control population (p < 0.0001). The results obtained for the tri-allelic polymorphism in alcohol dependence confirm what is already present in part of the literature. The role of haplotypes requires further studies to be clarified.

Systems Biology and Inborn Error of Metabolism: Analytical Strategy in Investigating Different Biochemical/Genetic Parameters.

Inborn errors of metabolism (IEM) are a group of about 500 rare genetic diseases with large diversity and complexity due to number of metabolic pathways involved in. Establishing a correct diagnosis and identifying the specific clinical phenotype is consequently a difficult task. However, an inclusive diagnosis able in capturing the different clinical phenotypes is mandatory for successful treatment. However, in contrast with Garrod's basic assumption "one-gene one-disease," no "simple" correlation between genotype-phenotype can be vindicated in IEMs. An illustrative example of IEM is Phenylketonuria (PKU), an autosomal recessive inborn error of L-phenylalanine (Phe) metabolism, ascribed to variants of the phenylalanine hydroxylase (PAH) gene encoding for the enzyme complex phenylalanine-hydroxylase. Blood values of Phe allow classifying PKU into different clinical phenotypes, albeit the participation of other genetic/biochemical pathways in the pathogenetic mechanisms remains elusive. Indeed, it has been shown that the most serious complications, such as cognitive impairment, are not only related to the gene dysfunction but also to the patient's background and the participation of several nongenetic factors.Therefore, a Systems Biology-based strategy is required in addressing IEM complexity, and in identifying the interplay between different pathways in shaping the clinical phenotype. Such an approach should entail the concerted investigation of genomic, transcriptomics, proteomics, metabolomics profiles altogether with phenylalanine and amino acids metabolism. Noticeably, this "omic" perspective could be instrumental in planning personalized treatment, tailored accordingly to the disease profile and prognosis.

Combined PIVKA II and Vimentin-Guided EMT Tracking in Pancreatic Adenocarcinoma Combined Biomarker-Guided EMT Tracking in PDAC.

"Background/Aim": the current inability to diagnose Pancreatic Cancer Adenocarcinoma (PDAC) at an early stage strongly influences therapeutic strategies. Protein Induced by Vitamin K Absence (PIVKA II) showed an accurate diagnostic performance for PDAC. Since circulating PIVKA II has been recently associated with pancreatic origin cells with Vimentin, an epithelial-to-mesenchymal transition (EMT) early activation marker, the aim of this study was to investigate in vivo the combination between the two proteins. "Materials and Methods": we assayed the presence of PIVKA II and Vimentin proteins by using different diagnostic methods. A total of 20 PDAC patients and 10 healthy donors were tested by Western Blot analysis; 74 PDAC patient and 46 healthy donors were assayed by ECLIA and Elisa. "Results": Western Blot analysis showed the concomitant expression of PIVKA II and Vimentin in PDAC patient sera. Immunometric assay performed on a larger cohort of patients demonstrated that 72% of PIVKA II-positive PDAC patients were Vimentin-positive. Additionally, in a group of PDAC patients with PIVKA II levels ≥2070 ng/mL, the percentage of Vimentin-positive subjects reached 84%. "Conclusion": the association between PIVKA II protein and the EMT suggests that this molecule could be considered a marker of the acquisition of an aggressive phenotype.

Association between serum levels of GDF-15, suPAR, PIVKA-II, sdLDL and clinical outcomes in hospitalized COVID-19 patients.

To quantify the circulating levels of novel serum biomarkers including GDF-15, PIVKA-II, sdLDL, suPAR, and of CRP in hospitalized COVID-19 patients compared with healthy subjects, and to evaluate their association(s) with outcomes in COVID-19. We considered patients with confirmed COVID-19, hospitalized in an Internal Medicine ward. The clinical characteristics were collected, including the number and type of comorbidities. Serum levels of GDF-15, PIVKA-II, suPAR, sdLDL, as well as CRP were measured. As outcomes, we considered Intensive Care Unit (ICU) transfer or death, as well as the length of stay (days) and in-hospital complications. Data were statistically analyzed, as appropriate, and a p value < 0.05 was considered significant. Ninety-three patients and 20 healthy controls were enrolled. COVID-19 patients vs. controls showed higher median levels of GDF-15 (p < 0.0001), PIVKA-II (p < 0.0001) and sdLDL (p = 0.0002), whereas no difference was observed for suPAR. In COVID-19 patients, the most frequent comorbidities were arterial hypertension (62.4%) and cardiovascular disease (30.1%). GDF-15 levels positively correlated with age (r = 0.433, p < 0.0001), and this correlation was confirmed for suPAR (r = 0.308, p = 0.003) and CRP (Rho = 0.40 p < 0.0001), but not for PIVKA-II and sdLDL. Higher GDF-15 levels were associated with a higher number of comorbidities (p = 0.021). The median length of stay was 22 (15; 30) days. During hospitalization, 15 patients (16%) were ICU transferred, and 6 (6.45%) died. GDF-15 serum levels correlated with the length of stay (rho = 0.27 p = 0.010), and were associated with ICU transfer or death (p = 0.003), as well as PIVKA-II (p = 0.038) and CRP (p < 0.001). Moreover, higher GDF-15 and PIVKA-II serum levels were associated with infectious complications (p = 0.008 and p = 0.017, respectively). In this cohort of hospitalized COVID-19 patients, novel inflammatory biomarkers, including GDF-15, suPAR and PIVKA II were associated with some patient's clinical characteristics, complications, and poor outcomes.

Effect of 2 Years of Monthly Calcifediol Administration in Postmenopausal Women with Vitamin D Insufficiency.

BACKGROUND: We assessed the long-term (24 months) efficacy and safety of monthly calcifediol (0.266 mg) in the correction and maintenance of total 25(OH)D levels in postmenopausal women with basal values <30 ng/mL. METHODS: We initially enrolled 45 consecutive patients during the period September 2019-September 2020. After an initial visit, patients were instructed to return at 3, 6, 9, 12 and 24 months for measuring serum total 25(OH)D, ionised calcium, creatinine and isoenzyme of alkaline phosphatase (bALP). Here, we report only the per-protocol analysis, because the COVID-19 pandemic precluded adherence to the scheduled visits for some patients. RESULTS: The patients' mean age was 62.4 ± 9.0 years. Mean basal 25(OH)D levels were 20.5 ± 5.3 ng/mL. There was a continuous increase of mean 25(OH)D values (p for trend < 0.001). However, mean values at month 24 (36.7 ± 15.9) were not significantly different in respect to values at month 12 (41.2 ± 11.18). At 24 months, only 1 out 19 patients had a value <20 ng/mL. There was a significant decrease with time of mean values of bALP (p < 0.0216), with no significant changes between 12 and 24 months. No significant changes were observed as far as ionised calcium or creatinine were concerned. CONCLUSIONS: The long-term administration of calcifediol maintains stable and sustained 25(OH)D concentrations, with no safety concerns.

Keeping university open did not increase the risk of SARS-CoV-2 acquisition: A test negative case-control study among students.

BACKGROUND: During the SARS-CoV-2 testing program offered through the RT-PCR test by Sapienza University of Rome, we conducted a test-negative case-control study to identify risk factors for acquiring SARS-CoV-2 infection among university students. METHODS: Each SARS-CoV-2-positive case detected was matched to two controls randomly selected from students who tested negative on the same day. 122 positive students and 244 negative students were enrolled in the study. Multivariable conditional logistic regression models were built. Adjusted odds ratios (aORs) and 95% confidence intervals (CIs) were calculated. A second model was limited to students who had attended campus. RESULTS: Out of 8223 tests for SARS-CoV-2, 173 students tested positive (2.1%), of whom 122 (71.5%) were included in the case-control study. In the first analysis, being a non-Italian student (aOR: 8.93, 95% CI: 2.71-29.41), having received only the primary vaccination course (aOR: 2.94, 95% CI: 1.24-6.96) compared to the booster dose, known exposure to a COVID-19 case or someone with signs/symptoms suggestive of COVID-19 (aOR: 6.51, 95% CI: 3.48-12.18), and visiting discos (aOR: 4.07, 95% CI: 1.52-10.90) in the two weeks before testing increased the likelihood of SARS-CoV-2 infection. Conversely, students attending in-person lectures on campus seemed less likely to become infected (aOR: 0.34, 95% CI: 0.15-0.77). No association was found with other variables. The results of the second model were comparable to the first analysis. CONCLUSIONS: This study indicates that if universities adopt strict prevention measures, it is safe for students to attend, even in the case of an infectious disease epidemic.