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Abstract Background The epigenetic status of patients 6-month post-COVID-19 infection remains largely unexplored. The existence of long-COVID, or post-acute sequelae of SARS-CoV-2 infection (PASC), suggests potential long-term changes. Long-COVID includes symptoms like fatigue, neurological issues, and organ-related problems, regardless of initial infection severity. The mechanisms behind long-COVID are unclear, but virus-induced epigenetic changes could play a role. Methods and results Our study explores the lasting epigenetic impacts of SARS-CoV-2 infection. We analyzed genome-wide DNA methylation patterns in an Italian cohort of 96 patients 6 months after COVID-19 exposure, comparing them to 191 healthy controls. We identified 42 CpG sites with significant methylation differences (FDR

Platelet lysate, derived from platelets, are valuable biological products rich in bioactive molecules. Their use promotes tissue healing and modulates inflammation. However, maintaining the stability and bioactivity of platelet lysate is challenging since they degrade rapidly at room temperature. This study focused on the possibility to confer enhanced stability to freeze-dried equine platelet lysate as an alternative to platelet-rich plasma (PRP). Platelet lysate (PL) was derived from PRP and freeze-dried either as such or using various adjuvants. Primary cell cultures of porcine Vascular Wall-Mesenchymal Stem Cells were treated with different PL formulations, and cell viability was assessed using an MTT assay. Overall, the addition of PL significantly improved cell viability as compared to controls without growth factor supplementation or with foetal bovine serum. Notably, the freeze-drying process maintained the effectiveness of the PL for at least a week. Furthermore, the study revealed that varying the horse as the source of PL could yield varying effects on cell viability. Detailed freeze-drying protocols were established, including freezing, primary drying and secondary drying phases, and the type of adjuvant. This study demonstrated the potential of freeze-dried equine PL as a viable alternative to PRP and highlighted the importance of precise freeze-drying protocols and adjuvants for standardization. Equine PL showed promise for medical treatment in horses, offering advantages such as extended shelf life, ease of handling, and reduced transportation costs, with the potential for broadened therapeutic usage.

Abstract Background Individuals with type 2 diabetes (T2D) face an increased mortality risk, not fully captured by canonical risk factors. Biological age estimation through DNA methylation (DNAm), i.e. the epigenetic clocks, is emerging as a possible tool to improve risk stratification for multiple outcomes. However, whether these tools predict mortality independently of canonical risk factors in subjects with T2D is unknown. Methods Among a cohort of 568 T2D patients followed for 16.8 years, we selected a subgroup of 50 subjects, 27 survived and 23 deceased at present, passing the quality check and balanced for all risk factors after propensity score matching. We analyzed DNAm from peripheral blood leukocytes using the Infinium Human MethylationEPIC BeadChip (Illumina) to evaluate biological aging through previously validated epigenetic clocks and assess the DNAm-estimated levels of selected inflammatory proteins and blood cell counts. We tested the associations of these estimates with mortality using two-stage residual-outcome regression analysis, creating a reference model on data from the group of survived patients. Results Deceased subjects had higher median epigenetic age expressed with DNAmPhenoAge algorithm (57.49 [54.72; 60.58] years. vs. 53.40 [49.73; 56.75] years; p = 0.012), and accelerated DunedinPoAm pace of aging (1.05 [1.02; 1.11] vs. 1.02 [0.98; 1.06]; p = 0.012). DNAm PhenoAge (HR 1.16, 95% CI 1.05–1.28; p = 0.004) and DunedinPoAm (HR 3.65, 95% CI 1.43–9.35; p = 0.007) showed an association with mortality independently of canonical risk factors. The epigenetic predictors of 3 chronic inflammation-related proteins, i.e. CXCL10, CXCL11 and enRAGE, C-reactive protein methylation risk score and DNAm-based estimates of exhausted CD8 + T cell counts were higher in deceased subjects when compared to survived. Conclusions These findings suggest that biological aging, as estimated through existing epigenetic tools, is associated with mortality risk in individuals with T2D, independently of common risk factors and that increased DNAm-surrogates of inflammatory protein levels characterize deceased T2D patients. Replication in larger cohorts is needed to assess the potential of this approach to refine mortality risk in T2D.

Abstract Autism spectrum disorder (ASD) is a complex neurodevelopmental condition with a strong genetic component in which rare variants contribute significantly to risk. We performed whole genome and/or exome sequencing (WGS and WES) and SNP-array analysis to identify both rare sequence and copy number variants (SNVs and CNVs) in 435 individuals from 116 ASD families. We identified 37 rare potentially damaging de novo SNVs (pdSNVs) in the cases (n = 144). Interestingly, two of them (one stop-gain and one missense variant) occurred in the same gene, BRSK2. Moreover, the identification of 8 severe de novo pdSNVs in genes not previously implicated in ASD (AGPAT3, IRX5, MGAT5B, RAB8B, RAP1A, RASAL2, SLC9A1, YME1L1) highlighted promising candidates. Potentially damaging CNVs (pdCNVs) provided support to the involvement of inherited variants in PHF3, NEGR1, TIAM1 and HOMER1 in neurodevelopmental disorders (NDD), although mostly acting as susceptibility factors with incomplete penetrance. Interpretation of identified pdSNVs/pdCNVs according to the ACMG guidelines led to a molecular diagnosis in 19/144 cases, although this figure represents a lower limit and is expected to increase thanks to further clarification of the role of likely pathogenic variants in ASD/NDD candidate genes not yet established. In conclusion, our study highlights promising ASD candidate genes and contributes to characterize the allelic diversity, mode of inheritance and phenotypic impact of de novo and inherited risk variants in ASD/NDD genes.

ObjectivesCoronavirus-disease-19 (COVID-19) continues to affect millions of individuals worldwide. Antiviral activity of mouthrinses remains an important research area as the oral cavity is a site of SARS-CoV-2 initial replication. The aim of this study was to assess the effectiveness of three different mouthrinses in reducing the oral/oropharyngeal SARS-CoV-2 viral load.MethodsAdult patients, hospitalized with confirmed COVID-19 were recruited for the study. Oral/oropharyngeal baseline SARS-CoV-2 samples were collected and analyzed by Real-Time-PCR. Subsequently, patients were instructed to rinse with 1 % hydrogen peroxide (H2O2), 0.12 % chlorhexidine (CHX), 1 % povidone‑iodine (PVP-I) or Sodium Chloride 0.9 % (placebo). Viral loads were measured right after (T1), and at 45 min (T2) from the rinse.ResultsIn the PVP-I 1 % group, 5/8 (62.5 %) patients at T1, and 3/8 (37.5 %) patients at T2, SARS-CoV-2 was not detectable in the swab specimens. In the H2O2 1 % group, 2/11 (18.2 %) patients at T1, and 2/11 (18.2 %) other patients at T2 showed no SARS-CoV-2 loads. One (12.5 %) patient in the CHX 0.12 % group showed SARS-CoV-2 negativity at T2. One (9.1 %) patient at T1, and another (9.1 %) patient at T2 showed no SARS-CoV-2 loads in the placebo group.ConclusionsOral SARS-CoV-2 loads were reduced at T1 in the PVP-I 1 % and H2O2 1 % groups.Clinical relevancePVP-I 1 % was the most effective rinse especially in patients with low viral copy numbers at baseline.

Abstract Translational research for the evaluation of physical activity habits and lifestyle modifications based on nutrition and exercise has recently gained attention. In this study, we evaluated the effects of serum samples obtained before and after a 12-week home-based lifestyle intervention based on nutrition and exercise in breast cancer survivors in terms of modulation of the tumorigenic potential of breast cancer cells. The home-based lifestyle intervention proposed in this work consisted of educational counselling on exercise and nutritional behaviors and in 12 weeks of structured home-based exercise. Triple-negative breast cancer cell line MDA-MB-231 was cultured in semi-solid medium (3D culture) with sera collected before (PRE) and after (POST) the lifestyle intervention program. Spheroid formation was evaluated by counting cell colonies after 3 weeks of incubation. Results show a slight but significant reduction of spheroid formation induced by serum collected POST in comparison to those obtained PRE. Moreover, statistical analyses aimed to find physiologic and metabolic parameters associated with 3D cell proliferation revealed the proliferative inducer IGF-1 as the only predictor of cell tumorigenic potential. These results highlight the importance of lifestyle changes for cancer progression control in a tertiary prevention context. Translational research could offer a useful tool to identify metabolic and physiological changes induced by exercise and nutritional behaviors associated with cancer progression and recurrence risk.

Healthcare workers (HCWs) are a high-risk group for hepatitis B virus (HBV) infection. Notably, about 5–10% of the general population does not respond to the HBV vaccination. In this study, we aimed to investigate DNA methylation (DNAm) in order to estimate the biological age of B cells from HCW of both sexes, either responder (R) or non-responder (NR), to HBV vaccination. We used genome-wide DNA methylation data to calculate a set of biomarkers in B cells collected from 41 Rs and 30 NRs between 22 and 62 years old. Unresponsiveness to HBV vaccination was associated with accelerated epigenetic aging (DNAmAge, AltumAge, DunedinPoAm) and was accompanied by epigenetic drift. Female non-responders had higher estimates of telomere length and lower CRP inflammation risk score when compared to responders. Overall, epigenetic differences between responders and non-responders were more evident in females than males. In this study we demonstrated that several methylation DNAm-based clocks and biomarkers are associated with an increased risk of non-response to HBV vaccination, particularly in females. Based on these results, we propose that accelerated epigenetic age could contribute to vaccine unresponsiveness. These insights may help improve the evaluation of the effectiveness of vaccination strategies, especially among HCWs and vulnerable patients.

This narrative work highlights a selection of published work from 2023 with potential implications for breast cancer practice. We feature publications that have provided new knowledge immediately relevant to patient care or for future research. We also highlight guidelines that have reported evidence-based or consensus recommendations to support practice and evaluation in breast cancer diagnosis and treatment. The scope of selected highlights represents various domains and disciplines in cancer control, from prevention to treatment of early and advanced breast cancer.

Abstract Background In humans, there is a growing body of evidence that neoangiogenesis is crucial for tumour growth and progression in urothelial carcinomas (UC) which also typically exhibit overactivation of the RAS‐MAPK pathway. In canine UC (cUC), the same pathway has been aberrantly activated due to V595E BRAF variant and BRAF inhibitors has been evaluated as more effective treatment. However, BRAF inhibition is hampered in humans by rapidly occurring of chemoresistance. Targeting angiogenesis has been speculated to increase the effectiveness of BRAF inhibitors and to delay the development of chemoresistance. Objectives This study aimed to investigate the level of angiogenic markers in urine samples of UC affected dogs (n = 15) in comparison to an unmatched control group (n = 16) along with the clinical, morphological and molecular features. Methods In urine, both vascular endothelial growth factor (VEGF) concentration, using an ELISA assay, and MMP2 and 9 activities, using the zymographic assay, were measured. BRAF analysis was carried out using a digital PCR method. Results Urinary VEGF concentration (mean pg/g_uCrea 6.9 ± 27.7 vs. 1074 ± 1797, p

Introduction The empty pelvis syndrome is a significant source of morbidity following pelvic exenteration surgery. It remains poorly defined with research in this field being heterogeneous and of low quality. Furthermore, there has been minimal engagement with patient representatives following pelvic exenteration with respect to the empty pelvic syndrome. ‘PelvEx—Beating the empty pelvis syndrome’ aims to engage both patient representatives and healthcare professionals to achieve an international consensus on a core outcome set, pathophysiology and mitigation of the empty pelvis syndrome.Methods and analysis A modified-Delphi approach will be followed with a three-stage study design. First, statements will be longlisted using a recent systematic review, healthcare professional event, patient and public engagement, and Delphi piloting. Second, statements will be shortlisted using up to three rounds of online modified Delphi. Third, statements will be confirmed and instruments for measurable statements selected using a virtual patient-representative consensus meeting, and finally a face-to-face healthcare professional consensus meeting.Ethics and dissemination The University of Southampton Faculty of Medicine ethics committee has approved this protocol, which is registered as a study with the Core Outcome Measures in Effectiveness Trials Initiative. Publication of this study will increase the potential for comparative research to further understanding and prevent the empty pelvis syndrome.Trial registration number NCT05683795.

Abstract Introduction Type 2 diabetes mellitus (T2DM) is a non-communicable disease representing one of the most serious public health challenges of the twenty-first century. Its incidence continues to rise in both developed and developing countries, causing the death of 1.5 million people every year. The use of technology (e.g. smartphone application—App) in the health field has progressively increased as it has been proved to be effective in helping individuals manage their long-term diseases. Therefore, it has the potential to reduce the use of health service and its related costs. The objective of this study is to evaluate the impact of using a digital platform called “TreC Diabete” embedded into a novel organisational asset targeting poorly controlled T2DM individuals in the Autonomous Province of Trento (PAT), Italy. Methods This trial was designed as a multi-centre, open-label, randomised, superiority study with two parallel groups and a 1:1 allocation ratio. Individuals regularly attending outpatient diabetes clinics, providing informed consent, are randomised to be prescribed TreC Diabete platform as part of their personalised care plan. Healthcare staff members will remotely assess the data shared by the participants through the App by using a dedicated online medical dashboard. The primary end-point is the evaluation of the Hb1Ac level at 12-month post-randomisation. Data will be analysed on an intention-to-treat (ITT) basis. Discussion This trial is the first conducted in the PAT area for the use of an App specifically designed for individuals with poorly controlled T2DM. If the effects of introducing this specific App within a new organisational asset are positive, the digital platform will represent a possible way for people diagnosed with T2DM to better manage their health in the future. Results will be disseminated through conferences and peer-reviewed journals once the study is completed. Trial registration ClinicalTrials.gov NCT05629221. Registered on November 29, 2022, prior start of inclusion.

Day 3 embryo quality is a predictor of in vitro fertilization (IVF) success rates in cleavage-stage embryo transfer. However, the association between day 3 embryo quality and clinical outcomes in blastocyst transfer policy is largely unknown. This retrospective study included 1074 frozen–thawed single day 5/6 blastocyst transfers between January 2019 and December 2022. Three groups were assessed depending on whether the transferred blastocyst derived from a top-quality, good-quality, or poor-quality embryo at day 3. The analysis was conducted independently for each blastocyst quality group (top, good, and poor) using multivariable logistic regression. We applied a Factorial Analysis of Mixed Data (FAMD) to reduce the potential collinearity between the covariates used in the model. All the blastocysts included in this study were obtained from the first ICSI freeze-all cycles. The cleavage and blastocysts stages were assessed between 67 ± 0.5 (day 3), 115 ± 0.5 (day 5), and 139 ± 0.5 (day 6) hours post-insemination (hpi), respectively. After adjusting for the day of transfer (day 5 or day 6) and FAMD dimensions, no statistical differences in a β-HCG, clinical pregnancy, and live birth were observed among the same-quality blastocysts derived from different day 3 embryo quality groups (top = A, good = B, and poor = C). Our findings showed that a day 3 embryo quality assessment may be unnecessary in planned freeze-all blastocyst cycles.

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