Emerging roles of DNA repair factors in the stability of centromeres.

Satellite DNA sequences are an integral part of centromeres, regions critical for faithful segregation of chromosomes during cell division. Because of their complex repetitive structure, satellite DNA may act as a barrier to DNA replication and other DNA based transactions ultimately resulting in chromosome breakage. Over the past two decades, several DNA repair proteins have been shown to bind and function at centromeres. While the importance of these repair factors is highlighted by various structural and numerical chromosome aberrations resulting from their inactivation, their roles in helping to maintain genome stability by solving the intrinsic difficulties of satellite DNA replication or promoting their repair are just starting to emerge. In this review, we summarize the current knowledge on the role of DNA repair and DNA damage response proteins in maintaining the structure and function of centromeres in different contexts. We also report the recent connection between the roles of specific DNA repair factors at these genomic loci with age-related increase of chromosomal instability under physiological and pathological conditions.

De Novo VPS4A Mutations Cause Multisystem Disease with Abnormal Neurodevelopment.

The endosomal sorting complexes required for transport (ESCRTs) are essential for multiple membrane modeling and membrane-independent cellular processes. Here we describe six unrelated individuals with de novo missense variants affecting the ATPase domain of VPS4A, a critical enzyme regulating ESCRT function. Probands had structural brain abnormalities, severe neurodevelopmental delay, cataracts, growth impairment, and anemia. In cultured cells, overexpression of VPS4A mutants caused enlarged endosomal vacuoles resembling those induced by expression of known dominant-negative ATPase-defective forms of VPS4A. Proband-derived fibroblasts had enlarged endosomal structures with abnormal accumulation of the ESCRT protein IST1 on the limiting membrane. VPS4A function was also required for normal endosomal morphology and IST1 localization in iPSC-derived human neurons. Mutations affected other ESCRT-dependent cellular processes, including regulation of centrosome number, primary cilium morphology, nuclear membrane morphology, chromosome segregation, mitotic spindle formation, and cell cycle progression. We thus characterize a distinct multisystem disorder caused by mutations affecting VPS4A and demonstrate that its normal function is required for multiple human developmental and cellular processes.

The mitochondrial poison carbonyl cyanide 3-chlorophenyl hydrazone (CCCP) induces aneugenic effects in primary human fibroblasts: a possible link between mitochondrial dysfunction and chromosomal loss.

An association between proper chromosome segregation and intact mitochondria has been extensively reported. This could be related to the effects on the progression of cell division of altered energy production, increased oxidative stress, and deregulated calcium homeostasis. However, evidence for a direct relationship is still lacking. The present study was aimed at investigating the possible effect of mitochondrial dysfunction on chromosomal instability as detected in primary human cells treated with the mitochondrial poison carbonyl cyanide 3-chlorophenyl hydrazone (CCCP). Chromosome instability was analyzed in anaphase and interphase cells to follow the fate of chromosome damage during the progression of mitosis and the subsequent cell cycle. Through the combination of cytogenetic approaches and molecular analyses, i.e. morphological cell analysis, formation and characterization of micronucleus content, Comet assay, and gene expression, it was demonstrated that the prevalent DNA damage associated with CCCP treatment was the induction of chromosome loss, while primary DNA damage was not detected. No alterations in the shape of anaphase cells were observed nor induction of multipolar spindles. The proper activation of mitotic checkpoint was maintained. A linear dose-response curve characterizing the CCCP effects suggested that multiple cellular targets could be affected by the CCCP-induced mitochondrial dysfunctions triggering aneuploidy. Conversely, a steep increase was induced by the positive control vinblastine, known to have tubulin as a unique target. In addition, the effect of CCCP on mitochondrial function was demonstrated by changes in mitochondrial DNA copy number and in the expression of genes involved in mitochondrial maintenance. Overall, these results indicate that the mitochondrial poison CCCP may induce aneugenic effects.

An integrated approach for chemical water quality assessment of an urban river stretch through Effect-Based Methods and emerging pollutants analysis with a focus on genotoxicity.

The impact of emerging chemical pollutants, on both status and functionality of aquatic ecosystems is worldwide recognized as a relevant issue of concern that should be assessed and managed by researchers, policymakers, and all relevant stakeholders. In Europe, the Reach Regulation has registered more than 100.000 chemical substances daily released in the environment. Furthermore, the effects related to the mixture of substances present in aquatic ecosystems may not be predictable on the basis of chemical analyses alone. This evidence, coupled with the dramatic effects of climate changes on water resources through water scarcity and flooding, makes urgent the application of innovative, fast and reliable monitoring methods. In this context, Effect-Based Methods (EBMs) have been applied in the urban stretch of the Tiber River (Central Italy) with the aim of understanding if detrimental pressures affect aquatic environmental health. In particular, different eco-genotoxicological assays have been used in order to detect genotoxic activity of chemicals present in the river, concurrently characterized by chemical analysis. Teratogenicity and embryo-toxicity have been studied in order to cover additional endpoints. The EBMs have highlighted the presence of diffuse chemical pollution and ecotoxicological effects in the three sampling stations, genotoxicological effects have been also detected through the use of different tests and organisms. The chemical analyses confirmed that in the aquatic ecosystems there is a diffuse presence, even at low concentrations, of emerging contaminants such as pharmaceuticals, not routinely monitored pesticides, personal care products, PFAS. The results of this study can help to identify an appropriate battery of EBMs for future studies and the application of more appropriate measures in order to monitor, mitigate or eliminate chemical contamination and remediate its adverse/detrimental effects on the ecosystem health.

Recurrence patterns of pancreatic cancer after pancreatoduodenectomy: systematic review and a single-centre retrospective study.

BACKGROUND: Positive margins in pancreatoduodenectomy (PD) for pancreatic cancer, specifically the superior mesenteric artery (SMA) margin, are associated with worse outcomes. Local therapies targeting these margins could impact on recurrence. This study analysed recurrence-patterns to identify whether strategies to control local disease could have a meaningful impact. METHODS: (I) Systematic review to define recurrence patterns and resection margin status. (II) Additional retrospective study of PD performed at our centre. RESULTS: In the systematic review, 23/617 evaluated studies were included (n = 3815). Local recurrence was observed in 7-69%. SMA margin (6 studies) was positive in 15-35%. In the retrospective study (n = 204), local recurrence was more frequently observed with a positive SMA margin (66 vs.45%; p = 0.005). Furthermore, in a multivariate cox-proportional hazard model, only a positive SMA margin was associated with disease recurrence (HR 1.615; 95%CI 1.127-2.315; p = 0.009). Interestingly, median overall survival was 20 months and similar for patients who developed local only, metastases only or simultaneous recurrence (p = 0.124). CONCLUSION: Local recurrence of pancreatic cancer is common and associated with similar mortality rates as those who present with simultaneous or metastatic recurrence. Involvement of the SMA margin is an independent predictor for disease progression and should be the target of future adjuvant local therapies.

Risk adjusted assessment of individual surgeon's pancreatic fistula outcomes.

BACKGROUND: Post-operative pancreatic fistula (POPF) is a major cause of morbidity following pancreatoduodenectomy. The risk of POPF varies between individuals and thus assessment without risk adjustment is crude. However, despite the availability of numerous scores to determine risk, no study has provided a risk adjusted assessment of POPF outcomes. METHODS: The observed and risk adjusted occurrence of POPF from consecutive patients operated upon by eight surgeons were recorded. Surgeons varied in experience from newly appointed (n = 5) to established (n = 3). CUSUM (cumulative sum) analysis was used to assess performance. RESULTS: 104 POPF occurred among 519 patients (20.0%). The occurrence of POPF was significantly lower among experienced surgeons (20/186, 10.7% vs 84/333, 25.2%; p < 0.001). Following risk adjustment surgeons observed 16.6 fewer to 6.5 excess POPF per 100 patients than predicted. Analysis of the CUSUM plots demonstrated the experienced surgeons performed steadily with a gradual reduction in observed POPF compared to what was predicted. The new surgeon's performance was less consistent and evidence of a learning curve was observed with steady improvement occurring after 50-70 patients. CONCLUSIONS: Risk adjusted assessment of POPF demonstrates differences between experienced and less experienced surgeons. This method could be used to audit practice and observe effects of changes to technique.

Can sustained exposure to PFAS trigger a genotoxic response? A comprehensive genotoxicity assessment in mice after subacute oral administration of PFOA and PFBA.

PFAS (perfluoroalkyl substances) are considered non-genotoxic. However, PFAS exposure has been associated with the induction of oxidative stress in vitro and in vivo, and the possible induction of indirect genotoxic effects under sustained PFAS exposure has not been investigated. In order to shed light on this aspect, in this study a comprehensive assessment of genotoxicity was carried out in mice administered with perfluorooctanoic acid (PFOA, 0.1, 1 and 5 mg/kg body weight) and its C4 analogue perfluorobutyric acid (PFBA, 5 mg/kg body weight) for five weeks through drinking water. Markers of cell toxicity, oxidative stress and DNA strand breaks were measured in liver, the main target of toxicity of PFOA in rodents; systemic genotoxicity was also assessed by the analysis of micronuclei in reticulocytes and spleen lymphocytes, and germ cell effects by the Comet assay on testis cells. PFOA administration at the highest dose (5 mg/kg body weight) induced marked liver hypertrophy with signs of cell injury (elevated ALT and AST), with no concurrent evidence of lipid peroxidation and oxidative stress (decreased antioxidant capacity). Only mild liver hypertrophy, with no other signs of toxicity, was determined by PFBA administration. No evidence of treatment related genotoxicity was observed in any experimental group. Overall, data indicate that under the experimental conditions of this study, severe liver toxicity induced by PFOA administration is not associated with oxidative stress. Accordingly, no genotoxic effect is observed in liver and in the other tissues examined. Milder evidence of liver toxicity, with no genotoxicity, and a lower tendency to bioaccumulation were observed in PFBA treated mice.

Defining Benchmarks in Liver Transplantation: A Multicenter Outcome Analysis Determining Best Achievable Results.

: This multicentric study of 17 high-volume centers presents 12 benchmark values for liver transplantation. Those values, mostly targeting markers of morbidity, were gathered from 2024 "low risk" cases, and may serve as reference to assess outcome of single or any groups of patients. OBJECTIVE: To propose benchmark outcome values in liver transplantation, serving as reference for assessing individual patients or any other patient groups. BACKGROUND: Best achievable results in liver transplantation, that is, benchmarks, are unknown. Consequently, outcome comparisons within or across centers over time remain speculative. METHODS: Out of 7492 liver transplantation performed in 17 international centers from 3 continents, we identified 2024 low risk adult cases with a laboratory model for end-stage liver disease score ≤20 points, a balance of risk score ≤9, and receiving a primary graft by donation after brain death. We chose clinically relevant endpoints covering intra- and postoperative course, with a focus on complications graded by severity including the complication comprehensive index (CCI). Respective benchmarks were derived from the median value in each center, and the 75 percentile was considered the benchmark cutoff. RESULTS: Benchmark cases represented 8% to 49% of cases per center. One-year patient-survival was 91.6% with 3.5% retransplantations. Eighty-two percent of patients developed at least 1 complication during 1-year follow-up. Biliary complications occurred in one-fifth of the patients up to 6 months after surgery. Benchmark cutoffs were ≤4 days for ICU stay, ≤18 days for hospital stay, ≤59% for patients with severe complications (≥ Grade III) and ≤42.1 for 1-year CCI. Comparisons with the next higher risk group (model for end stage liver disease 21-30) disclosed an increase in morbidity but within benchmark cutoffs for most, but not all indicators, while in patients receiving a second graft from 1 center (n = 50) outcome values were all outside of benchmark values. CONCLUSIONS: Despite excellent 1-year survival, morbidity in benchmark cases remains high with half of patients developing severe complications during 1-year follow-up. Benchmark cutoffs targeting morbidity parameters offer a valid tool to assess higher risk groups.

A reduced time to surgery within a 'fast track' pathway for periampullary malignancy is associated with an increased rate of pancreatoduodenectomy.

INTRODUCTION: Pancreatoduodenectomy (PD) typically follows preoperative biliary drainage (PBD) despite PBD being potentially harmful. This study evaluated a pathway to avoid PBD within the framework of the UK's NHS. METHOD: A prospective observational study of jaundiced patients undergoing PD for periampullary cancer. A pathway to provide early surgery without PBD was introduced at the start of the study period. RESULTS: Over 12 months 61 and 32 patients underwent surgery with and without PBD respectively; 95% of patients in the PBD group had been stented before referral. The time from CT scan to surgery was shorter in the no PBD group (16 vs 65 days, p < 0.0001). Significantly more patients underwent PD in the no PBD group (31/32 vs 46/61, p = 0.009) and venous resection (10/31 vs 4/46, p = 0.014). The sensitivity of initial CT scan to define borderline resectable disease was worse in the PBD group (91 vs 50%, p = 0.042). CONCLUSIONS: Early surgery to avoid PBD is possible within the NHS. By reducing the time to surgery it appears that more patients undergo potentially curative resection. It is desirable to understand why surgery without PBD is not performed routinely as are the development of strategies to support its more widespread practice.

Risk assessment of feed components of botanical origin - Approaches taken in the European Union.

In view of a continuous trend in replacing synthetic feed additives and especially flavouring compounds by botanical preparations, different aspects of the safety evaluations of plants and plant-derived preparations and components in feed are discussed. This includes risk assessment approaches developed by the European Food Safety Authority (EFSA) for phytotoxins regarding unintentional exposure of target animals and of consumers to animal derived food via carry-over from feed. Relevant regulatory frameworks for feed additives and feed contaminants in the European Union are summarised and the essentials of existing guidelines used in the safety evaluation of botanicals and their preparations and components in feed are outlined. The examples presented illustrate how the safety of the botanicals, their preparations and components present in feed is assessed. An outlook on possible future developments in risk assessment by applying new in vitro and in silico methodologies is given.

Efficacy of a feed additive consisting of carvacrol (Nimicoat®) for weaned piglets (Techna France Nutrition).

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the efficacy of a feed additive consisting of carvacrol (Nimicoat®) as a zootechnical feed additive for weaned piglets at the recommended use level of 250 mg/kg complete feed. In a previous assessment, three efficacy trials and one tolerance-efficacy trial were assessed. Only one of the efficacy trials was considered to support the efficacy at the recommended use level. The applicant provided amendments to two previously submitted studies and a new trial. The amendments to the previously submitted studies did not change the conclusions from the previous assessment. The new efficacy study showed a significant improvement of the zootechnical parameters. Two studies showed positive and significant effects on the performance of the weaned piglets when the additive was administered at 250 mg/kg feed. Due to the lack of sufficient data, the FEEDAP Panel is not in the position to conclude on the efficacy of the additive for the target species.

Assessment of the feed additive consisting of Bacillus velezensisATCC PTA-6737 (PB6) for turkeys for fattening and turkeys reared for breeding for the renewal of its authorisation and the modification of the conditions of the authorisation for other growing poultry species (Kemin Europe N.V).

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on Bacillus velezensis ATCC PTA-6737 as a zootechnical additive (functional group: gut-flora stabiliser) in the context of the renewal of the authorisation for turkeys for fattening and turkeys reared for breeding. The applicant is also requesting to modify the target species in the current authorisations to 'all growing poultry', the increase of the recommended use level in chickens for fattening, chickens reared for laying and minor poultry species except minor poultry for laying from 1 × 107 to 1 × 108 CFU/kg complete feed and the compatibility of the additive with halofuginone. The applicant provided evidence that the additive currently in the market complies with the conditions of the authorisation. There was no new evidence that would lead to reconsider previous conclusions. Therefore, the FEEDAP Panel concluded that the additive remains safe for all poultry species for fattening and reared for laying/breeding, the consumers and the environment under the current authorised conditions of use. The additive is not irritant to the skin and eyes, but it should be considered a respiratory sensitiser. The Panel could not conclude on the skin sensitisation potential of the additive. The Panel concluded that the additive has a potential to be efficacious as a zootechnical additive for poultry for fattening and reared for laying/breeding under the proposed conditions of use.

Interplay in galectin expression predicts patient outcomes in a spatially restricted manner in PDAC.

BACKGROUND: Galectins (Gal's) are a family of carbohydrate-binding proteins that are known to support the tumour microenvironment through their immunosuppressive activity and ability to promote metastasis. As such they are attractive therapeutic targets, but little is known about the cellular expression pattern of galectins within the tumour and its neighbouring stromal microenvironment. Here we investigated the cellular expression pattern of Gals within pancreatic ductal adenocarcinoma (PDAC). METHODS: Galectin gene and protein expression were analysed by scRNAseq (n=4) and immunofluorescence imaging (n=19) in fibroblasts and epithelial cells of pancreatic biopsies from PDAC patients. Galectin surface expression was also assessed on tumour adjacent normal fibroblasts and cancer associated primary fibroblasts from PDAC biopsies using flow cytometry. RESULTS: scRNAseq revealed higher Gal-1 expression in fibroblasts and higher Gal-3 and -4 expression in epithelial cells. Both podoplanin (PDPN+, stromal/fibroblast) cells and EpCAM+ epithelial cells expressed Gal-1 protein, with highest expression seen in the stromal compartment. By contrast, significantly more Gal-3 and -4 protein was expressed in ductal cells expressing either EpCAM or PDPN, when compared to the stroma. Ductal Gal-4 cellular expression negatively correlated with ductal Gal-1, but not Gal-3 expression. Higher ductal cellular expression of Gal-1 correlated with smaller tumour size and better patient survival. CONCLUSIONS: In summary, the intricate interplay and cell-specific expression patterns of galectins within the PDAC tissue, particularly the inverse correlation between Gal-1 and Gal-4 in ducts and its significant association with patient survival, highlights the complex molecular landscape underlying PDAC and provides valuable insights for future therapeutic interventions.

Safety of a feed additive consisting of propyl gallate for all animal species (FEFANA ABL).

Following a request from the European Commission, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety of propyl gallate as a technological feed additive for all animal species. In its previous opinions on the safety and efficacy of the product, the FEEDAP Panel could not conclude on a safe level of propyl gallate for cats and on the safety for the consumer. Based on the new data provided, the FEEDAP Panel concluded that propyl gallate at a maximum concentration of 71 mg/kg complete feed is safe for cats. Propyl gallate is considered safe for the consumer when used in complete feed for all animal species at the concentrations considered safe for the target species.

Safety of a feed additive consisting of hydroxypropyl cellulose for all animal species (Association Management & Regulatory Services Ltd).

Following a request from the European Commission, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety of hydroxypropyl cellulose as a technological feed additive for all animal species. In its previous opinions on the safety and efficacy of the product, the FEEDAP Panel could not conclude on proper identification and characterisation as required for a feed additive. The occurrence of potential toxic impurities could also not be assessed. Based on the new data provided, the feed additive hydroxypropyl cellulose was properly identified and characterised and was shown to meet the specifications set for the food additive. Therefore, the conclusions of the safety assessment reached in the previous opinion for hydroxypropyl cellulose meeting the food additive specifications, apply to the hydroxypropyl cellulose under assessment as a feed additive. The feed additive is considered safe for all animal species, the consumer and the environment. In the absence of data, the FEEDAP Panel is not in the position to conclude on the safety for the user.

Safety of a feed additive consisting of ethyl cellulose for all animal species (Association Management & Regulatory Services Ltd).

Following a request from the European Commission, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety of ethyl cellulose as a technological feed additive for all animal species. In its previous opinions on the safety and efficacy of the product, the FEEDAP Panel could not conclude on proper identification and characterisation as required for a feed additive. The occurrence of potential toxic impurities could also not be assessed. Based on the new data provided, the feed additive ethyl cellulose was properly identified and characterised and was shown to meet the specifications set for the food additive. Therefore, the conclusions of the safety assessment reached in the previous opinions for ethyl cellulose meeting the food additive specifications, apply to the ethyl cellulose under assessment as a feed additive. The feed additive is considered safe for all animal species, the consumer and the environment. In the absence of data, the FEEDAP Panel is not in the position to conclude on the safety for the user.

Safety of feed additives consisting of hydroxypropyl methyl cellulose (E 464) and methyl cellulose (E 461) for all animal species (International Cellulosics Association).

Following a request from the European Commission, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety of hydroxypropyl methyl cellulose and methyl cellulose as technological feed additives for all animal species. In its previous opinions on the safety and efficacy of the products, the FEEDAP Panel could not conclude on proper identification and characterisation as required for a feed additive. The occurrence of potential toxic impurities could also not be assessed. Based on the new data provided, the feed additives hydroxypropyl methyl cellulose and methyl cellulose were properly identified and characterised and were shown to meet the specifications set for the food additives. Therefore, the conclusions of the safety assessments reached in the previous opinions for hydroxypropyl methyl cellulose and methyl cellulose meeting the food additive specifications, apply to the hydroxypropyl methyl cellulose and methyl cellulose under assessment as feed additives. The additives are considered safe for all animal species, the consumer and the environment. In the absence of data, the FEEDAP Panel is not in the position to conclude on the safety for the user.

Assessment of the feed additive consisting of Lentilactobacillus buchneri (formerly Lactobacillus buchneri) DSM 22501 for all animal species for the renewal of its authorisation (Chr. Hansen A/S).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the assessment of the application for renewal of the authorisation of the additive consisting of Lentilactobacillus buchneri DSM 22501 as a technological feed additive to improve ensiling of fresh material for all animal species. The applicant has provided evidence that the additive currently on the market complies with the existing conditions of authorisation. There is no new evidence that would lead the FEEDAP Panel to reconsider its previous conclusions. Thus, the Panel concludes that the additive remains safe for all animal species, consumer and the environment under the authorised conditions of use. Regarding user safety, the additive is not irritant to skin and eye, but owing to its proteinaceous nature it should be considered a respiratory sensitiser. No conclusions could be drawn on the skin sensitisation potential of the additive. There is no need for assessing the efficacy of the additive in the context of the renewal of the authorisation.

Consumer safety of feed additives containing selenium.

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety for the consumer of products from animals fed diets with feed additives containing selenium as an active substance. Based on the limited data set available and the several uncertainties, the FEEDAP Panel concluded that the use of organic selenium at the currently maximum authorised use level of 0.2 mg supplemented selenium from organic sources/kg complete feed (within a maximum of 0.5 mg total selenium/kg complete feed) leads to an exceedance of the UL for all the population categories (except elderly and very elderly), suggesting a concern for consumer safety. It was not possible to conclude on the safety of the currently maximum use level of 0.5 mg total selenium/kg complete feed for all consumer categories. Additional data from studies specifically designed to measure deposition of selenium in tissues and products from animal origin resulting from the use of the different sources of selenium would be required to perform a proper risk assessment.

Animal dietary exposure in the risk assessment of contaminants in feed.

EFSA performs dietary exposure assessments for food-producing and non-food-producing animals to deliver risk assessment for mandates on the presence of contaminants in feed. The CONTAM and FEEDAP Panels identified the need to update the animal dietary exposure assessment model used in those assessments in CONTAM Scientific Opinions since 2011 in cases where insufficient occurrence data are available on species specific compound feeds. The Panels proposed in this statement a series of model diets based on groups of feed materials with the possibility to use different feed materials in their formulation. The Panels considered that the currently proposed model diets cover the need of the CONTAM Panel to assess the dietary exposure of animals to contaminants in feed.