Quantitative evaluation of RASSF1A methylation in the non-lesional, regenerative and neoplastic liver

BACKGROUND: Epigenetic changes during ageing and their relationship with cancer are under the focus of intense research. RASSF1A and NORE1A are novel genes acting in concert in the proapoptotic pathway of the RAS signalling. While NORE1A has not been previously investigated in the human liver, recent reports have suggested that RASSF1A is frequently epigenetically methylated not only in HCC but also in the cirrhotic liver. METHODS: To address whether epigenetic changes take place in connection to age and/or to the underlying disease, we investigated RASSF1A and NORE1A gene promoter methylation by conventional methylation specific PCR and Real-Time MSP in a series of hepatitic and non-hepatitic livers harboring regenerative/hyperplastic (cirrhosis/focal nodular hyperplasia), dysplastic (large regenerative, low and high grade dysplastic nodules) and neoplastic (hepatocellular adenoma and carcinoma) growths. RESULTS: In the hepatitic liver (chronic hepatitic/cirrhosis, hepatocellular nodules and HCC) we found widespread RASSF1A gene promoter methylation with a methylation index that increased from regenerative conditions (cirrhosis) to hepatocellular nodules (p < 0.01) to HCC (p < 0.001). In the non-hepatitic liver a consistent pattern of gene methylation was also found in both lesional (focal nodular hyperplasia and hepatocellular adenoma) and non-lesional tissue. Specifically, hepatocellular adenomas (HA) showed a methylation index significantly higher than that detected in focal nodular hyperplasia (FNH) (p < 0.01) and in non-lesional tissue (p < 0.001). In non-lesional liver also the methylation index gradually increased by ageing (p = 0.002), suggesting a progressive spreading of methylated cells over time. As opposed to RASSF1A gene promoter methylation, NORE1A gene was never found epigenetically alterated in both hepatitic and non-hepatitic liver. CONCLUSION: We have shown that in non-lesional, regenerative and neoplastic liver the RASSF1A gene is increasingly methylated, that this condition takes place as an age-related phenomenon and that the early setting and spreading over time of an epigenetically methylated hepatocyte subpopulation, might be related to liver tumorigenesis

Global disparities in surgeons’ workloads, academic engagement and rest periods: the on-calL shIft fOr geNEral SurgeonS (LIONESS) study

: The workload of general surgeons is multifaceted, encompassing not only surgical procedures but also a myriad of other responsibilities. From April to May 2023, we conducted a CHERRIES-compliant internet-based survey analyzing clinical practice, academic engagement, and post-on-call rest. The questionnaire featured six sections with 35 questions. Statistical analysis used Chi-square tests, ANOVA, and logistic regression (SPSS® v. 28). The survey received a total of 1.046 responses (65.4%). Over 78.0% of responders came from Europe, 65.1% came from a general surgery unit; 92.8% of European and 87.5% of North American respondents were involved in research, compared to 71.7% in Africa. Europe led in publishing research studies (6.6 ± 8.6 yearly). Teaching involvement was high in North America (100%) and Africa (91.7%). Surgeons reported an average of 6.7 ± 4.9 on-call shifts per month, with European and North American surgeons experiencing 6.5 ± 4.9 and 7.8 ± 4.1 on-calls monthly, respectively. African surgeons had the highest on-call frequency (8.7 ± 6.1). Post-on-call, only 35.1% of respondents received a day off. Europeans were most likely (40%) to have a day off, while African surgeons were least likely (6.7%). On the adjusted multivariable analysis HDI (Human Development Index) (aOR 1.993) hospital capacity &gt; 400 beds (aOR 2.423), working in a specialty surgery unit (aOR 2.087), and making the on-call in-house (aOR 5.446), significantly predicted the likelihood of having a day off after an on-call shift. Our study revealed critical insights into the disparities in workload, access to research, and professional opportunities for surgeons across different continents, underscored by the HDI

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Synthesis and characterization of novel chitosan-dopamine or chitosan-tyrosine conjugates for potential nose-to-brain delivery

This work aims to the synthesis of novel carboxylated chitosan-dopamine (DA) and -tyrosine (Tyr) conjugates as systems for improving the brain delivery of the neurotransmitter DA following nasal administration. For this purpose, ester or amide conjugates were synthesized by N,N-dicyclohexylcarbodiimide (DCC) mediated coupling reactions between the appropriate N-tert-butyloxycarbonyl (Boc) protected starting polymers N,O-carboxymethyl chitosan and 6-carboxy chitosan and DA or O-tert-Butyl-L-tyrosine-tert-butyl ester hydrochloride. The resulting conjugates were characterized by FT-IR and 1H- and 13C-NMR spectroscopies and their in vitro mucoadhesive properties in simulated nasal fluid (SNF), toxicity and uptake from Olfactory Ensheathing Cells (OECs) were assessed. Results demonstrated that N,O-carboxymethyl chitosan-DA conjugate was the most mucoadhesive polymer in the series examined and, together with the 6-carboxy chitosan-DA-conjugate were able to release the neurotransmitter in SNF. The MTT assay showed that the starting polymers as well as all the prepared conjugates in OECs resulted not toxic at any concentration tested. Likewise, the three synthesized conjugates were not cytotoxic as well. Cytofluorimetric analysis revealed that the N,O-carboxymethyl chitosan DA conjugate was internalized by OECs in a superior manner at 24 h as compared with the starting polymer. Overall, the N,O-CMCS-DA conjugate seems promising for improving the delivery of DA by nose-to-brain administration

Multifunctional Membranes Based on β-Glucans and Chitosan Useful in Wound Treatment

In this work, bio-based membranes prepared using a crosslinked β-glucans–chitosan dispersed in the chitosan matrix useful in promoting wound healing were studied for the first-time. Wound healing is a process that includes sequential steps designed to restore the structure and function of damaged cells and tissue. To minimize damage and the risk of infection during the healing process and to promote restoration of the integrity of damaged tissue, the wound should be dressed. Generally, according to their function in the wound, dressings are classified on the basis of type of material and physical form. The substances used to make a dressing are generally natural polymers such as hydrocolloids, alginates, polyurethane, collagen, chitosan, pectin and hyaluronic acid. The combination of polymeric substances, with antibacterial and antioxidant properties, could be exploited in the biomedical field for the development of biocompatible materials able to act as a barrier between the wound and the external environment, protecting the site from bacterial contamination and promoting healing. To this aim, bio-based membranes were prepared by the phase inversion induced by solvent evaporation, using the crosslinked β-glucans–chitosan obtained by esterification reactions as a functional additive in the chitosan membrane. The reaction intermediates and the final products were characterized by Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) while the morphological properties of membranes were analyzed using electronic scanning microscopy (SEM). The chemical bonding between chitosan and β-glucans allowed for the obtainment of a better dispersion of the combined new material into the membrane’s matrix and as a consequence, an enhanced antibacterial property evaluated through in vitro tests, with respect to the starting materials

Nose-to-brain delivery: a comparative study between carboxymethyl chitosan based conjugates of dopamine

Herein, the synthesis of a novel polymeric conjugate N,O-CMCS-Dopamine (DA) based on an amide linkage is reported. The performances of this conjugate were compared with those of an analogous N,O-CMCS-DA ester conjugate previously studied (Cassano et al., 2020) to gain insight into their potential utility for Parkinson's disease treatment. The new amide conjugate was synthesized by standard carbodiimide coupling procedure and characterized by FT-IR, 1H-NMR spectroscopies and thermal analysis (Differential Scanning Calorimetry). In vitro mucoadhesive studies in simulated nasal fluid (SNF) evidenced high adhesive effect of both ester and amide conjugates. Results demonstrated that the amide conjugate exerted an important role to prevent DA spontaneous autoxidation both under stressed conditions and physiological mimicking ones. MTT test indicated cytocompatibility of the amide conjugate with Olfactory Ensheating Cells (OECs), which were shown by cytofluorimetry to internalize efficiently the conjugate. Overall, among the two conjugates herein studied, the N,O-CMCS-DA amide conjugate seems a promising candidate for improving the delivery of DA by nose-to-brain administration

A Brønsted Acidic Deep Eutectic Solvent for <i>N</i>-Boc Deprotection

The tert-butyloxycarbonyl (Boc) group is one of the most widely used amine-protecting groups in multistep reactions in synthetic organic chemistry as well as in peptide synthesis. Traditional methods to remove the Boc group have disadvantages in terms of high acidity, the use of expensive reagents, excessive amounts of catalysts and harmful solvents as well as high temperatures, making them environmentally unsustainable. Therefore, more efforts must be stepwise tightened to make Boc removal practical, clean, and minimize any potential impact. We describe an efficient and sustainable method for N-Boc deprotection by means of a choline chloride/p-toluenesulfonic acid deep eutectic solvent (DES), which is used as a reaction medium plus catalyst. The adopted conditions allow the deprotection of a wide variety of N-Boc derivatives in excellent yields. The strategy has found advantages in greening, simplicity, and short reaction times, resulting in a useful alternative to standard methods

Novel Nanoparticles Based on N,O-Carboxymethyl Chitosan-Dopamine Amide Conjugate for Nose-to-Brain Delivery

A widely investigated approach to bypass the blood brain barrier is represented by the intranasal delivery of therapeutic agents exploiting the olfactory or trigeminal connections nosebrain. As for Parkinson’s disease (PD), characterized by dopaminergic midbrain neurons degeneration, currently there is no disease modifying therapy. Although several bio-nanomaterials have been evaluated for encapsulation of neurotransmitter dopamine (DA) or dopaminergic drugs in order to restore the DA content in parkinsonian patients, the premature leakage of the therapeutic agent limits this approach. To tackle this drawback, we undertook a study where the active was linked to the polymeric backbone by a covalent bond. Thus, novel nanoparticles (NPs) based on N,O-Carboxymethylchitosan-DA amide conjugate (N,O-CMCS-DA) were prepared by the nanoprecipitation method and characterized from a technological view point, cytotoxicity and uptake by Olfactory Ensheating Cells (OECs). Thermogravimetric analysis showed high chemical stability of N,O-CMCS-DA NPs and X-ray photoelectron spectroscopy evidenced the presence of amide linkages on the NPs surface. MTT test indicated their cytocompatibility with OECs, while cytofluorimetry and fluorescent microscopy revealed the internalization of labelled N,O-CMCS-DA NPs by OECs, that was increased by the presence of mucin. Altogether, these findings seem promising for further development of N,O-CMCS-DA NPs for nose-to-brain delivery application in PD

A Br&oslash;nsted Acidic Deep Eutectic Solvent for N-Boc Deprotection

The tert-butyloxycarbonyl (Boc) group is one of the most widely used amine-protecting groups in multistep reactions in synthetic organic chemistry as well as in peptide synthesis. Traditional methods to remove the Boc group have disadvantages in terms of high acidity, the use of expensive reagents, excessive amounts of catalysts and harmful solvents as well as high temperatures, making them environmentally unsustainable. Therefore, more efforts must be stepwise tightened to make Boc removal practical, clean, and minimize any potential impact. We describe an efficient and sustainable method for N-Boc deprotection by means of a choline chloride/p-toluenesulfonic acid deep eutectic solvent (DES), which is used as a reaction medium plus catalyst. The adopted conditions allow the deprotection of a wide variety of N-Boc derivatives in excellent yields. The strategy has found advantages in greening, simplicity, and short reaction times, resulting in a useful alternative to standard methods