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Sea cucumbers are deposit feeding members of marine benthic communities. The over-exploitation of sea cucumber natural stocks, especially in the Mediterranean basin, is having negative impacts on the marine ecosystem. This concerns had led the Italian government to take legal actions to preserve these important marine resources. The aim of this study was to evaluate the level of genetic diversity and population structures within two sympatric Holothuria sea cucumber species, H. polii and H. tubulosa, across ten Mediterranean areas along Italy. A 323 bp portion of the COI mitochondrial gene was sequenced in a total of 441 holothurians (251 H. polii, 177 H. tubulosa and 13 from other species). Genetic diversity analyses and analysis of molecular variance (AMOVA) suggest that H. polii and H. tubulosa species are distinct, but within and among populations are homogeneous, indicating active gene flows across the Mediterranean areas investigated. H. polii showed a lower genetic diversity than H. tubulosa, probably related to differences in life history traits. Phylogenetic analyses showed a clear differentiation between the two species, even if six specimens morphologically assigned to a species clustered within the other species, indicating a possible occurrence of hybridization events. These data will be useful in implementing conservation actions for these holothurian genetic resources., (© 2024. The Author(s).)

Efficient long-range axonal transport is essential for maintaining neuronal function, and perturbations in this process underlie severe neurological diseases. Nodes of Ranvier (NoR) are short, specialized unmyelinated axonal domains with a unique molecular and structural composition. Currently, it remains unresolved how the distinct molecular structures of the NoR impact axonal transport dynamics. Using intravital time-lapse microscopy of sciatic nerves in live, anesthetized mice, we reveal (1) similar morphologies of the NoR in fast and slow motor axons, (2) signaling endosomes and mitochondria accumulate specifically at the distal node, and (3) unique axonal transport profiles of signaling endosomes and mitochondria transiting through the NoR. Collectively, these findings provide important insights into the fundamental physiology of peripheral nerve axons, motor neuron subtypes, and diverse organelle dynamics at the NoR. Furthermore, this work has relevance for several pathologies affecting peripheral nerves and the NoR., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Mitochondria are dynamic bioenergetic hubs that become compromised with age. In neurons, declining mitochondrial axonal transport has been associated with reduced cellular health. However, it is still unclear to what extent the decline of mitochondrial transport and function observed during ageing are coupled, and if somal and axonal mitochondria display compartment-specific features that make them more susceptible to the ageing process. It is also not known whether the biophysical state of the cytoplasm, thought to affect many cellular functions, changes with age to impact mitochondrial trafficking and homeostasis. Focusing on the mouse peripheral nervous system, we show that age-dependent decline in mitochondrial trafficking is accompanied by reduction of mitochondrial membrane potential and intramitochondrial viscosity, but not calcium buffering, in both somal and axonal mitochondria. Intriguingly, we observe a specific increase in cytoplasmic viscosity in the neuronal cell body, where mitochondria are most polarised, which correlates with decreased cytoplasmic diffusiveness. Increasing cytoplasmic crowding in the somatic compartment of DRG neurons grown in microfluidic chambers reduces mitochondrial axonal trafficking, suggesting a mechanistic link between the regulation of cytoplasmic viscosity and mitochondrial dynamics. Our work provides a reference for studying the relationship between neuronal mitochondrial homeostasis and the viscoelasticity of the cytoplasm in a compartment-dependent manner during ageing., (© 2024 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Background: The integration of nuclear mitochondrial DNA (mtDNA) into the mammalian genomes is an ongoing, yet rare evolutionary process that produces nuclear sequences of mitochondrial origin (NUMT). In this study, we identified and analysed NUMT inserted into the pig (Sus scrofa) genome and in the genomes of a few other Suinae species. First, we constructed a comparative distribution map of NUMT in the Sscrofa11.1 reference genome and in 22 other assembled S. scrofa genomes (from Asian and European pig breeds and populations), as well as the assembled genomes of the Visayan warty pig (Sus cebifrons) and warthog (Phacochoerus africanus). We then analysed a total of 485 whole genome sequencing datasets, from different breeds, populations, or Sus species, to discover polymorphic NUMT (inserted/deleted in the pig genome). The insertion age was inferred based on the presence or absence of orthologous NUMT in the genomes of different species, taking into account their evolutionary divergence. Additionally, the age of the NUMT was calculated based on sequence degradation compared to the authentic mtDNA sequence. We also validated a selected set of representative NUMT via PCR amplification., Results: We have constructed an atlas of 418 NUMT regions, 70 of which were not present in any assembled genomes. We identified ancient NUMT regions (older than 55 million years ago, Mya) and NUMT that appeared at different time points along the Suinae evolutionary lineage. We identified very recent polymorphic NUMT (private to S. scrofa, with < 1 Mya), and more ancient polymorphic NUMT (3.5-10 Mya) present in various Sus species. These latest polymorphic NUMT regions, which segregate in European and Asian pig breeds and populations, are likely the results of interspecies admixture within the Sus genus., Conclusions: This study provided a first comprehensive analysis of NUMT present in the Sus scrofa genome, comparing them to NUMT found in other species within the order Cetartiodactyla. The NUMT-based evolutionary window that we reconstructed from NUMT integration ages could be useful to better understand the micro-evolutionary events that shaped the modern pig genome and enriched the genetic diversity of this species., (© 2024. The Author(s).)

The conservation of the genetic integrity of Apis mellifera subspecies has emerged as an important objective. In 2019, the Emilia-Romagna region became the first Italian regional authority to issue a law specifically addressing the protection of the native Apis mellifera ligustica subspecies. In this study we analysed a highly informative portion of the mitochondrial DNA (mtDNA), widely used for assessing genetic diversity of honey bee populations. By analysing 1143 honey bees sampled after the introduction of this law, we provided a snapshot of the distribution of mtDNA haplotypes in this region. The two most frequent mtDNA haplotypes were C1 (characteristic of A. m. ligustica) and C2 (characteristic of A. m. carnica), reported in 86.5% and 11.0% of the analysed bees, respectively. About 1.3% and 1.1% of the analysed bees carried mtDNA haplotypes of the A and M lineages (haplotypes A1a, A1e, A4, A26, A65 and two novel ones, A2w and A6a; M3, M3a, M4 and M79). Continued genetic monitoring will be important to assess the impact of this regional law over the coming years. Based on the obtained results, we recommend a more stringent policy to prevent the erosion of the genetic integrity of the native subspecies A. m. ligustica., (© 2024. The Author(s).)

Local tetanus develops when limited amounts of tetanus neurotoxin (TeNT) are released by Clostridium tetani generated from spores inside a necrotic wound. Within days, a spastic paralysis restricted to the muscles of the affected anatomical area develops. This paralysis follows the retrograde transport of TeNT inside the axons of motoneurons and its uptake by inhibitory interneurons with cleavage of a vesicle-associated membrane protein required for neurotransmitter release. Consequently, incontrollable excitation of motoneurons causes contractures of innervated muscles and leads to local spastic paralysis. Here, the initial events occurring close to the site of TeNT release were investigated in a mouse model of local tetanus. A peripheral flaccid paralysis was found to occur, before or concurrent to the spastic paralysis. At variance from the confined TeNT proteolytic activity taking place within motor neuron terminals, central protein cleavage was detected within inhibitory interneurons controlling motor neuron efferents innervating muscle groups distant from the site of TeNT release. These results indicate peripheral activity of TeNT in tetanus and explains why the spastic paralysis observed in local tetanus, although confined to single limbs, generally affects multiple muscles. The initial TeNT neuroparalytic activity can be detected by measuring the compound muscle action potential, providing a very early diagnosis and therapy, thus preventing the ensuing life-threatening generalized tetanus., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Tetanus neurotoxin (TeNT) causes spastic paralysis by inhibiting neurotransmission in spinal inhibitory interneurons. TeNT binds to the neuromuscular junction, leading to its internalisation into motor neurons and subsequent transcytosis into interneurons. While the extracellular matrix proteins nidogens are essential for TeNT binding, the molecular composition of its receptor complex remains unclear. Here, we show that the receptor-type protein tyrosine phosphatases LAR and PTPRδ interact with the nidogen-TeNT complex, enabling its neuronal uptake. Binding of LAR and PTPRδ to the toxin complex is mediated by their immunoglobulin and fibronectin III domains, which we harnessed to inhibit TeNT entry into motor neurons and protect mice from TeNT-induced paralysis. This function of LAR is independent of its role in regulating TrkB receptor activity, which augments axonal transport of TeNT. These findings reveal a multi-subunit receptor complex for TeNT and demonstrate a novel trafficking route for extracellular matrix proteins. Our study offers potential new avenues for developing therapeutics to prevent tetanus and dissecting the mechanisms controlling the targeting of physiological ligands to long-distance axonal transport in the nervous system., (© 2024. The Author(s).)

Background: There is no gold-standard trigger for detecting drug-induced respiratory disorders, a type of Adverse Drug Event (ADE) with high morbimortality, particularly in older people., Objective: To propose and evaluate the performance of triggers for detecting hospitalizations related to drug-induced respiratory disorders in older people., Methods: A pilot cross-sectional study was conducted with older people (age ≥ 60) admitted to a Brazilian hospital. Electronic chart documentation was screened using ICD-10 codes; Global Trigger Tool (GTT); and drugs potentially associated with respiratory disorders. A chart and medication review were conducted to perform the causality assessment using the instrument developed by the World Health Organization. The performance of triggers was evaluated by the Positive Predictive Value (PPV), with values ≥ 0.20 indicating good performance., Results: Among 221 older people, 72 were eligible. Potential drug-induced dyspnea and/or cough were detected in six older people (6/72), corresponding to a prevalence of 8.3 %. The overall PPV of the triggers was 0.14, with abrupt medication stop (PPV = 1.00), codeine (PPV = 1.00), captopril (PPV = 0.33), and carvedilol (PPV = 0.33) showing good performance. Two triggers were proposed for detecting therapeutic ineffectiveness associated with respiratory disorders: furosemide (PPV = 0.23) and prednisone (PPV = 0.20)., Conclusion: The triggers enabled the identification that one in 12 hospitalizations was related to drug-induced respiratory. Although good performance was observed in the application of triggers, additional investigations are needed to assess the feasibility of incorporating them into clinical practice for the screening, detection, management, and reporting of these ADEs, which are considered to be underreported and difficult to detect., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 HCFMUSP. Published by Elsevier España, S.L.U. All rights reserved.)

Objective: To identify trigger tools applied to detect adverse drug events (ADEs) in older people and describe their utility and performance., Methods: A systematic review was conducted in the PubMed, Lilacs, and Scopus databases (January 2024). Studies that developed, applied, or validated trigger tools and evaluated their utility and/or performance for detecting ADEs in older people were considered. Direct proportion meta-analyses using the inverse-variance method were performed for prevalence of ADEs and positive predictive value (PPV)., Results: Twenty-four studies (25 publications) were included. Twelve trigger tools were identified, of which six were developed for detecting ADEs in older population, four developed for general population and modified for older people, and two developed for general population. No tools for detecting ADEs in older people receiving palliative care or hospitalized in intensive or surgical care units were found. The performance of triggers was presented through PPV (11.5-71%), negative predictive values (83.3%), and sensitivity (30-94.8%). The overall PPV was 33.3% (95%CI: 32.5-34.2%). Triggers with good performance were changes in plasma levels of digoxin, glucose, and potassium; changes in international normalized ratio; abrupt medication stop; hypotension; and constipation. The prevalence of ADEs ranged from 2.8 to 66%, with overall prevalence of ADEs of 20% (95%CI: 19.3-20.8%). Preventability ranged from 8.4 to 94.4%. Metabolic or electrolyte disturbances induced by diuretics, constipation induced by opioids, and falls and delirium induced by benzodiazepines were the most prevalent ADEs., Conclusion: The trigger tools are flexible and easy to apply, and they can contribute to the detection of ADEs, their associated risk factors, the level of harm, and preventability in different health settings. However, there is no consensus on good or poor values of PPV, which indicate the performance of triggers. Furthermore, there is limited evidence regarding the evaluation of performance through negative predictive value, sensitivity, and specificity., Prospero: CRD42022379893., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

TDP-43 loss of function induces multiple splicing changes, including a cryptic exon in the amyotrophic lateral sclerosis and fronto-temporal lobar degeneration risk gene UNC13A , leading to nonsense-mediated decay of UNC13A transcripts and loss of protein. UNC13A is an active zone protein with an integral role in coordinating pre-synaptic function. Here, we show TDP-43 depletion induces a severe reduction in synaptic transmission, leading to an asynchronous pattern of network activity. We demonstrate that these deficits are largely driven by a single cryptic exon in UNC13A . Antisense oligonucleotides targeting the UNC13A cryptic exon robustly rescue UNC13A protein levels and restore normal synaptic function, providing a potential new therapeutic approach for ALS and other TDP-43-related disorders., Competing Interests: Competing interests: PF, MJK and OGW have filed a patent application relating to the use of antisense oligonucleotides for the correction of cryptic splicing in UNC13A. PF is founder, advisor, and holds shares in Trace Neuroscience Inc. MJK performs consulting for and holds shares in Trace Neuroscience Inc. PH performs consulting for Trace Neuroscience Inc.

Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes, which transfer amino acids to partner tRNAs for protein synthesis, represent the largest protein family genetically linked to CMT aetiology, suggesting pathomechanistic commonalities. Dominant intermediate CMT type C (DI-CMTC) is caused by YARS1 mutations driving a toxic gain-of-function in the encoded tyrosyl-tRNA synthetase (TyrRS), which is mediated by exposure of consensus neomorphic surfaces through conformational changes of the mutant protein. In this study, we first showed that human DI-CMTC-causing TyrRS E196K mis-interacts with the extracellular domain of the BDNF receptor TrkB, an aberrant association we have previously characterised for several mutant glycyl-tRNA synthetases linked to CMT type 2D (CMT2D). We then performed temporal neuromuscular assessments of Yars E196K mice modelling DI-CMT. We determined that Yars E196K homozygotes display a selective, age-dependent impairment in in vivo axonal transport of neurotrophin-containing signalling endosomes, phenocopying CMT2D mice. This impairment is replicated by injection of recombinant TyrRS E196K , but not TyrRS WT , into muscles of wild-type mice. Augmenting BDNF in DI-CMTC muscles, through injection of recombinant protein or muscle-specific gene therapy, resulted in complete axonal transport correction. Therefore, this work identifies a non-cell autonomous pathomechanism common to ARS-related neuropathies, and highlights the potential of boosting BDNF levels in muscles as a therapeutic strategy., Competing Interests: Declaration of competing interest The technology described in this work has been protected in the patent GB2303495.2 (patent applicant, UCL Business Ltd., status pending), in which GS and JNS are named as inventors. The other authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Selection and breeding strategies to improve resistance to enteropathies are essential to reaching the sustainability of the rabbit production systems. However, disease heterogeneity (having only as major visible symptom diarrhoea) and low disease heritability are two barriers for the implementation of these strategies. Diarrhoea condition can affect rabbits at different life stages, starting from the suckling period, with large negative economic impacts. In this study, from a commercial population of suckling rabbits (derived from 133 litters) that experienced an outbreak of enteropathy, we first selected a few animals that died with severe symptoms of diarrhoea and characterized their microbiota, using 16S rRNA gene sequencing data. Clostridium genus was consistently present in all affected specimens. In addition, with the aim to identify genetic markers in the rabbit genome that could be used as selection tools, we performed genome-wide association studies for symptoms of diarrhoea in the same commercial rabbit population. These studies were also complemented with F ST analyses between the same groups of rabbits. A total of 332 suckling rabbits (151 with severe symptoms of diarrhoea, 42 with mild symptoms and 129 without any symptoms till the weaning period), derived from 45 different litters (a subset of the 133 litters) were genotyped with the Affymetrix Axiom OrcunSNP Array. In both genomic approaches, rabbits within litters were paired to constitute two groups (susceptible and resistant, including the mildly affected in one or the other group) and run case and control genome-wide association analyses. Genomic heritability estimated in the designed experimental structure integrated in a commercial breeding scheme was 0.19-0.21 (s.e. 0.09-0.10). A total of eight genomic regions on rabbit chromosome 2 (OCU2), OCU3, OCU7, OCU12, OCU13, OCU16 and in an unassembled scaffold had significant single nucleotide polymorphisms (SNPs) and/or markers that trespassed the F ST percentile distribution. Among these regions, three main peaks of SNPs were identified on OCU12, OCU13 and OCU16. The QTL region on OCU13 encompasses several genes that encode members of a family of immunoglobulin Fc receptors (FCER1G, FCRLA, FCRLB and FCGR2A) involved in the immune innate system, which might be important candidate genes for this pathogenic condition. The results obtained in this study demonstrated that resistance to an enteropathy occurring in suckling rabbits is in part genetically determined and can be dissected at the genomic level, providing DNA markers that could be used in breeding programmes to increase resistance to enteropathies in meat rabbits., (© 2023 The Authors. Journal of Animal Breeding and Genetics published by John Wiley & Sons Ltd.)

Large genotyping datasets, obtained from high-density single nucleotide polymorphism (SNP) arrays, developed for different livestock species, can be used to describe and differentiate breeds or populations. To identify the most discriminating genetic markers among thousands of genotyped SNPs, a few statistical approaches have been proposed. In this study, we applied the Boruta algorithm, a wrapper of the machine learning random forest algorithm, on a database of 23 European pig breeds (20 autochthonous and three cosmopolitan breeds) genotyped with a 70k SNP chip, to pre-select informative SNPs. To identify different sets of SNPs, these pre-selected markers were then ranked with random forest based on their mean decrease accuracy and mean decrease gene indexes. We evaluated the efficiency of these subsets for breed classification and the usefulness of this approach to detect candidate genes affecting breed-specific phenotypes and relevant production traits that might differ among breeds. The lowest overall classification error (2.3%) was reached with a subpanel including only 398 SNPs (ranked based on their mean decrease accuracy), with no classification error in seven breeds using up to 49 SNPs. Several SNPs of these selected subpanels were in genomic regions in which previous studies had identified signatures of selection or genes associated with morphological or production traits that distinguish the analysed breeds. Therefore, even if these approaches have not been originally designed to identify signatures of selection, the obtained results showed that they could potentially be useful for this purpose., (© 2024 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics.)

Crossbreeding might be a valid strategy to valorize local pig breeds. Crossbreeding should reduce homozygosity and, as a consequence, yield hybrid vigor for fitness and production traits. This study aimed to quantify the persistence of autozygosity in terminal crossbred pigs compared with purebreds and, in turn, identify genomic regions where autozygosity's persistence would not be found. The study was based on genotyping data from 20 European local pig breeds and three cosmopolitan pig breeds used to simulate crossbred offspring. This study consisted of two steps. First, one hundred matings were simulated for each pairwise combination of the 23 considered breeds (for a total of 276 combinations), ignoring the sex of the parent individuals in order to generate purebred and crossbred matings leveraging all the germplasm available. Second, a few preselected terminal-maternal breed pairs were used to mimic a realistic terminal crossbreeding system: (i) Mora Romagnola (boars) or Cinta Senese (boars) crossed with Large White (sows) or Landrace (sows); (ii) Duroc (boars) crossed with Mora Romagnola (sows) or Cinta Senese (sows). Runs of homozygosity was used to estimate genome-wide autozygosity (F ROH ). Observed F ROH was higher in purebreds than in crossbreds, although some crossbred combinations showed higher F ROH than other purebred combinations. Among the purebreds, the highest F ROH values were observed in Mora Romagnola and Turopolje (0.50 and 0.46, respectively). F ROH ranged from 0.04 to 0.16 in the crossbreds Alentejana × Large White and Alentejana × Iberian, respectively. Persistence of autozygosity was found in several genomic segments harboring regions where quantitative trait loci (QTLs) were found in the literature. The regions were enriched in QTLs involved in fatty acid metabolism and associated with performance traits. This simulation shows that autozygosity persists in most breed combinations of terminal crosses. Results suggest that a strategy for crossbreeding is implemented when leveraging autochthonous and cosmopolitan breeds to obtain most of the hybrid vigor., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Polymorphisms in the human ABO gene determine the major blood classification system based on the three well-known forms: A ; B ; and O . In pigs that carry only two main alleles in this gene ( A and O ), we still need to obtain a more comprehensive distribution of variants, which could also impact its function. In this study, we mined more than 500 whole-genome sequencing datasets to obtain information on the ABO gene in different Suidae species, pig breeds, and populations and provide (i) a comprehensive distribution of the A and O alleles, (ii) evolutionary relationships of ABO gene sequences across Suidae species, and (iii) an exploratory evaluation of the effect of the different ABO gene variants on production traits and blood-related parameters in Italian Large White pigs. We confirmed that allele O is likely under balancing selection, present in all Sus species investigated, without being fixed in any of them. We reported a novel structural variant in perfect linkage disequilibrium with allele O that made it possible to estimate the evolutionary time window of occurrence of this functional allele. We also identified two single nucleotide polymorphisms that were suggestively associated with plasma magnesium levels in pigs. Other studies can also be constructed over our results to further evaluate the effect of this gene on economically relevant traits and basic biological functions.

Background: Intense selection of modern pig breeds has resulted in genetic improvement of production traits while the performance of local pig breeds has remained lower. As local pig breeds have been bred in extensive systems, they have adapted to specific environmental conditions, resulting in a rich genotypic and phenotypic diversity. This study is based on European local pig breeds that have been genetically characterized using DNA-pool sequencing data and phenotypically characterized using breed level phenotypes related to stature, fatness, growth, and reproductive performance traits. These data were analyzed using a dedicated approach to detect signatures of selection linked to phenotypic traits in order to uncover potential candidate genes that may underlie adaptation to specific environments., Results: Analysis of the genetic data of European pig breeds revealed four main axes of genetic variation represented by the Iberian and three modern breeds (i.e. Large White, Landrace, and Duroc). In addition, breeds clustered according to their geographical origin, for example French Gascon and Basque breeds, Italian Apulo Calabrese and Casertana breeds, Spanish Iberian, and Portuguese Alentejano breeds. Principal component analysis of the phenotypic data distinguished the larger and leaner breeds with better growth potential and reproductive performance from the smaller and fatter breeds with low growth and reproductive efficiency. Linking the signatures of selection with phenotype identified 16 significant genomic regions associated with stature, 24 with fatness, 2 with growth, and 192 with reproduction. Among them, several regions contained candidate genes with possible biological effects on stature, fatness, growth, and reproductive performance traits. For example, strong associations were found for stature in two regions containing, respectively, the ANXA4 and ANTXR1 genes, for fatness in a region containing the DNMT3A and POMC genes and for reproductive performance in a region containing the HSD17B7 gene., Conclusions: In this study on European local pig breeds, we used a dedicated approach for detecting signatures of selection that were supported by phenotypic data at the breed level to identify potential candidate genes that may have adapted to different living environments and production systems., (© 2023. The Author(s).)

Metabolomics has been used to characterise many biological matrices and obtain detailed pictures of biological systems based on many metabolites. Plasma and serum are two blood-derived biofluids commonly used to assess and monitor the organismal metabolism and obtain information on the physiological and health conditions of an animal. Plasma is the supernatant that is separated from the cellular components after centrifugation of the blood that is first added with an anticoagulant. Serum is obtained after centrifugation of the blood that has been coagulated. The choice of one or the other biofluid for metabolomic analyses is related to specific analytical needs and technical issues, to problems derived by the collection and preparation steps, in particular when specimens are sampled from animals involved in field studies. Thus far, most of the metabolomic studies that compared plasma and serum have been carried out in humans and very little is known on the pigs. In this study, we used a targeted metabolomic platform that can detect about 180 metabolites of five biochemical classes to compare plasma and serum profiles of samples collected from 24 pigs. To also obtain a cross-species comparative metabolomic analysis, information for human plasma and serum derived from the same platform was retrieved from previous studies. Statistical analyses included univariate and multivariate approaches aimed at identifying stable and/or differentially abundant metabolites between the two porcine biofluids. A total of 154 (∼83%) metabolites passed the initial quality control, indicating a good repeatability of the analytical platform in pigs. Discarded metabolites included aspartate and biogenic amines that were already reported to be unstable in human studies. More than 80% of the metabolites had similar profiles in both porcine biofluids (average correlation was 0.75). Concentrations were usually higher in serum than in plasma, in agreement with what was already reported in humans. The univariate analysis identified 44 metabolites that had statistically different concentrations between porcine plasma and serum, of which 28 metabolites were also confirmed by the multivariate analysis. The obtained picture described similarities and differences between these two biofluids in pigs and the related human-pig comparisons. The obtained information can be useful for the choice of one or the other matrix for the implementation of metabolomic studies in this livestock species. The results can also provide useful hints to valuing the pig as animal model, in particular when metabolite-derived physiological states are relevant., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Objectives: To assess the survival predictivity of baseline blood cell differential count (BCDC), discretised according to two different methods, in adults visiting an emergency room (ER) for illness or trauma over 1 year., Design: Retrospective cohort study of hospital records., Setting: Tertiary care public hospital in northern Italy., Participants: 11 052 patients aged >18 years, consecutively admitted to the ER in 1 year, and for whom BCDC collection was indicated by ER medical staff at first presentation., Primary Outcome: Survival was the referral outcome for explorative model development. Automated BCDC analysis at baseline assessed haemoglobin, mean cell volume (MCV), red cell distribution width (RDW), platelet distribution width (PDW), platelet haematocrit (PCT), absolute red blood cells, white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils and platelets. Discretisation cut-offs were defined by benchmark and tailored methods. Benchmark cut-offs were stated based on laboratory reference values (Clinical and Laboratory Standards Institute). Tailored cut-offs for linear, sigmoid-shaped and U-shaped distributed variables were discretised by maximally selected rank statistics and by optimal-equal HR, respectively. Explanatory variables (age, gender, ER admission during SARS-CoV2 surges and in-hospital admission) were analysed using Cox multivariable regression. Receiver operating curves were drawn by summing the Cox-significant variables for each method., Results: Of 11 052 patients (median age 67 years, IQR 51-81, 48% female), 59% (n=6489) were discharged and 41% (n=4563) were admitted to the hospital. After a 306-day median follow-up (IQR 208-417 days), 9455 (86%) patients were alive and 1597 (14%) deceased. Increased HRs were associated with age >73 years (HR=4.6, 95% CI=4.0 to 5.2), in-hospital admission (HR=2.2, 95% CI=1.9 to 2.4), ER admission during SARS-CoV2 surges (Wave I: HR=1.7, 95% CI=1.5 to 1.9; Wave II: HR=1.2, 95% CI=1.0 to 1.3). Gender, haemoglobin, MCV, RDW, PDW, neutrophils, lymphocytes and eosinophil counts were significant overall. Benchmark-BCDC model included basophils and platelet count (area under the ROC (AUROC) 0.74). Tailored-BCDC model included monocyte counts and PCT (AUROC 0.79)., Conclusions: Baseline discretised BCDC provides meaningful insight regarding ER patients' survival., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Following the recent domestication process of the European rabbit (Oryctolagus cuniculus), many different breeds and lines, distinguished primarily by exterior traits such as coat colour, fur structure and body size and shape, have been constituted. In this study, we genotyped, with a high-density single-nucleotide polymorphism panel, a total of 645 rabbits from 10 fancy breeds (Belgian Hare, Champagne d'Argent, Checkered Giant, Coloured Dwarf, Dwarf Lop, Ermine, Giant Grey, Giant White, Rex and Rhinelander) and three meat breeds (Italian White, Italian Spotted and Italian Silver). ADMIXTURE analysis indicated that breeds with similar phenotypic traits (e.g. coat colour and body size) shared common ancestries. Signatures of selection using two haplotype-based approaches (iHS and XP-EHH), combined with the results obtained with other methods previously reported that we applied to the same breeds, we identified a total of 5079 independent genomic regions with some signatures of selection, covering about 1777 Mb of the rabbit genome. These regions consistently encompassed many genes involved in pigmentation processes (ASIP, EDNRA, EDNRB, KIT, KITLG, MITF, OCA2, TYR and TYRP1), coat structure (LIPH) and body size, including two major genes (LCORL and HMGA2) among many others. This study revealed novel genomic regions under signatures of selection and further demonstrated that population structures and signatures of selection, left into the genome of these rabbit breeds, may contribute to understanding the genetic events that led to their constitution and the complex genetic mechanisms determining the broad phenotypic variability present in these untapped rabbit genetic resources., (© 2023 The Authors. Journal of Animal Breeding and Genetics published by John Wiley & Sons Ltd.)

Chronic wound is characterized by slow healing time, persistence, and abnormal healing progress. Therefore, serious complications can lead at worst to the tissue removal. In this scenario, there is an urgent need for an ideal dressing capable of high absorbency, moisture retention and antimicrobial properties. Herein we investigate the technical properties of a novel advanced non-woven triple layer gauze imbibed with a cream containing Rigenase, an aqueous extract of Triticum vulgare used for the treatment of skin injuries. To assess the applicability of this system we analyzed the dressing properties by wettability, dehydration, absorbency, Water Vapor Transmission Rate (WVTR), lateral diffusion and microbiological tests. The dressing showed an exudate absorption up to 50%. It created a most environment allowing a proper gaseous exchange as attested by the WVTR and a controlled dehydration rate. The results candidate the new dressing as an ideal medical device for the treatment of the chronic wound repairing process. It acts as a mechanical barrier providing a good management of the bacterial load and proper absorption of abundant wound exudate. Finally, its vertical transmission minimizes horizontal diffusion and side effects on perilesional skin as maceration and bacterial infection.

In the adult mammalian brain, neural stem cells (NSCs) located in highly restricted niches sustain the generation of new neurons that integrate into existing circuits. A reduction in adult neurogenesis is linked to ageing and neurodegeneration, whereas dysregulation of proliferation and survival of NSCs have been hypothesized to be at the origin of glioma. Thus, unravelling the molecular underpinnings of the regulated activation that NSCs must undergo to proliferate and generate new progeny is of considerable relevance. Current research has identified cues promoting or restraining NSCs activation. Yet, whether NSCs depend on external signals to survive or if intrinsic factors establish a threshold for sustaining their viability remains elusive, even if this knowledge could involve potential for devising novel therapeutic strategies. Kidins220 (Kinase D-interacting substrate of 220 kDa) is an essential effector of crucial pathways for neuronal survival and differentiation. It is dramatically altered in cancer and in neurological and neurodegenerative disorders, emerging as a regulatory molecule with important functions in human disease. Herein, we discover severe neurogenic deficits and hippocampal-based spatial memory defects accompanied by increased neuroblast death and high loss of newly formed neurons in Kidins220 deficient mice. Mechanistically, we demonstrate that Kidins220-dependent activation of AKT in response to EGF restraints GSK3 activity preventing NSCs apoptosis. We also show that NSCs with Kidins220 can survive with lower concentrations of EGF than the ones lacking this molecule. Hence, Kidins220 levels set a molecular threshold for survival in response to mitogens, allowing adult NSCs growth and expansion. Our study identifies Kidins220 as a key player for sensing the availability of growth factors to sustain adult neurogenesis, uncovering a molecular link that may help paving the way towards neurorepair., (© 2023. The Author(s).)

Purpose: To investigate the association between the use of antidepressants and the risk of upper gastrointestinal tract bleeding (UGIB)., Methods: A Case-control study was conducted in a Brazilian hospital complex. Cases were defined as patients with a diagnosis of UGIB and controls as patients admitted for reasons unrelated to gastrointestinal bleeding, gastric concerns, or complications associated with low-dose aspirin (LDA) or nonsteroidal anti-inflammatory drugs (NSAIDs) use. Sociodemographic and clinical data, comorbidities, drug therapy in use (long-term use and self-medication), and lifestyle habits were recorded through face-to-face interviews. Two groups were defined: use of antidepressants in general and use of antidepressants according to their affinity for serotonin transporters. The presence of synergism between the concomitant use of antidepressants and LDA or NSAIDs on the risk of UGIB was also explored., Findings: A total of 906 participants were recruited (200 in the case group and 706 in the control group). The use of antidepressants was not associated with the risk of UGIB (odds ratio [OR] = 1.503; 95% CI, 0.78-2.88) or the use of antidepressants with high affinity for serotonin receptors (OR = 1.983; 95% CI, 0.81-4.85). An increased risk of UGIB was observed in concomitant users of antidepressants and LDA (OR = 5.489; 95% CI, 1.60-18.81) or NSAIDs (OR = 18.286; 95% CI, 3.18-105.29). Despite the lack of significance, the use of antidepressants appears to be a positive modifier of UGIB risk in LDA and NSAID users., Implications: These findings indicate an increased risk of UGIB in concomitant users of antidepressants and LDA or NSAIDs, suggesting the need to monitor antidepressant users, especially those most likely to develop UGIB. In addition, further studies with larger sample sizes are needed to confirm these findings., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Pathological tau aggregates propagate across functionally connected neuronal networks in human neurodegenerative pathologies, such as Alzheimer's disease. However, the mechanism underlying this process is poorly understood. Several studies have showed that tau release is dependent on neuronal activity and that pathological tau is found in the extracellular space in free form, as well as in the lumen of extracellular vesicles. We recently showed that metabotropic glutamate receptor activity and SNAP25 integrity modulate the release of pathological tau from human and mouse synaptosomes. Here, we have leveraged botulinum neurotoxins (BoNTs), which impair neurotransmitter release by cleaving specific synaptic SNARE proteins, to dissect molecular mechanisms related to tau release at synapses. In particular, we have tested the effect of botulinum neurotoxin A (BoNT/A) on the synaptic release of tau in primary mouse neurons. Hippocampal neurons were grown in microfluidic chambers and transduced with lentiviruses expressing human tau (hTau). We found that neuronal stimulation significantly increases the release of mutant hTau, whereas wild-type hTau is unaffected. Importantly, BoNT/A blocks mutant hTau release, indicating that this process is controlled by SNAP25, a component of the SNARE complex, in intact neurons. These results suggest that BoNTs are potent tools to study the spreading of pathological proteins in neurodegenerative diseases and could play a central role in identifying novel molecular targets for the development of therapeutic interventions to treat tauopathies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Giampietro Schiavo reports a relationship with Eli Lilly and Company that includes: funding grants/fellowship. GS was recipient of a grant (LRAP program) from Eli Lilly and Company that supported a previously published work (Mazzo et al., 2022)., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The Greek Black Pig (or Greek Pig) is the only recognized autochthonous pig breed raised in Greece, usually in extensive or semi-extensive production systems. According to its name, the characteristic breed coat color is solid black. In this study, with the aim to start a systematic genetic characterization of the Greek Black Pig breed, we investigated polymorphisms in major genes well known to affect exterior and production traits ( MC1R , KIT , NR6A1 , VRTN and IGF2 ) and compared these data with population genetic information available in other Mediterranean and Western Balkan pig breeds and wild boars. None of the investigated gene markers were fixed for one allele, suggesting that, in the past, this breed experienced introgression from wild boars and admixture from cosmopolitan pig breeds, enriching the breed genetic pool that should be further investigated to design appropriate conservation genetic strategies. We identified a new MC1R allele, containing two missense mutations already reported in two other independent alleles, but here present in the same haplotype. This allele might be useful to disclose biological information that can lead to better understanding the cascade transmission of signals to produce melanin pigments. This study demonstrated that autochthonous genetic resources can be an interesting reservoir of unexpected genetic variants.

Hearing loss and peripheral neuropathy are two clinical entities that are genetically and phenotypically heterogeneous and sometimes co-occurring. Using exome sequencing and targeted segregation analysis, we investigated the genetic etiology of peripheral neuropathy and hearing loss in a large Ashkenazi Jewish family. Moreover, we assessed the production of the candidate protein via western blotting of lysates from fibroblasts from an affected individual and an unaffected control. Pathogenic variants in known disease genes associated with hearing loss and peripheral neuropathy were excluded. A homozygous frameshift variant in the BICD1 gene, c.1683dup (p.(Arg562Thrfs*18)), was identified in the proband and segregated with hearing loss and peripheral neuropathy in the family. The BIDC1 RNA analysis from patient fibroblasts showed a modest reduction in gene transcripts compared to the controls. In contrast, protein could not be detected in fibroblasts from a homozygous c.1683dup individual, whereas BICD1 was detected in an unaffected individual. Our findings indicate that bi-allelic loss-of-function variants in BICD1 are associated with hearing loss and peripheral neuropathy. Definitive evidence that bi-allelic loss-of-function variants in BICD1 cause peripheral neuropathy and hearing loss will require the identification of other families and individuals with similar variants with the same phenotype.

Gain-of-function mutations in the housekeeping gene GARS1, which lead to the expression of toxic versions of glycyl-tRNA synthetase (GlyRS), cause the selective motor and sensory pathology characterizing Charcot-Marie-Tooth disease (CMT). Aberrant interactions between GlyRS mutants and different proteins, including neurotrophin receptor tropomyosin receptor kinase receptor B (TrkB), underlie CMT type 2D (CMT2D); however, our pathomechanistic understanding of this untreatable peripheral neuropathy remains incomplete. Through intravital imaging of the sciatic nerve, we show that CMT2D mice displayed early and persistent disturbances in axonal transport of neurotrophin-containing signaling endosomes in vivo. We discovered that brain-derived neurotrophic factor (BDNF)/TrkB impairments correlated with transport disruption and overall CMT2D neuropathology and that inhibition of this pathway at the nerve-muscle interface perturbed endosome transport in wild-type axons. Accordingly, supplementation of muscles with BDNF, but not other neurotrophins, completely restored physiological axonal transport in neuropathic mice. Together, these findings suggest that selectively targeting muscles with BDNF-boosting therapies could represent a viable therapeutic strategy for CMT2D.

Amyotrophic lateral sclerosis is a complex disorder most of which is 'sporadic' of unknown origin but approximately 10% is familial, arising from single mutations in any of more than 30 genes. Thus, there are more than 30 familial ALS subtypes, with different, often unknown, molecular pathologies leading to a complex constellation of clinical phenotypes. We have mouse models for many genetic forms of the disorder, but these do not, on their own, necessarily show us the key pathological pathways at work in human patients. To date, we have no models for the 90% of ALS that is 'sporadic'. Potential therapies have been developed mainly using a limited set of mouse models, and through lack of alternatives, in the past these have been tested on patients regardless of aetiology. Cancer researchers have undertaken therapy development with similar challenges; they have responded by producing complex mouse models that have transformed understanding of pathological processes, and they have implemented patient stratification in multi-centre trials, leading to the effective translation of basic research findings to the clinic. ALS researchers have successfully adopted this combined approach, and now to increase our understanding of key disease pathologies, and our rate of progress for moving from mouse models to mechanism to ALS therapies we need more, innovative, complex mouse models to address specific questions., (© 2023. The Author(s).)

The prognostic value of conventional histopathological parameters in the sinonasal intestinal-type adenocarcinoma (ITAC) has been debated and novel variables should be investigated. Increasing evidence demonstrated that the evolution of cancer is strongly dependent upon the complex interactions within tumor microenvironment. The aim of this retrospective study was to assess the features of immune microenvironment in terms of CD3 + and CD8 + cells in a series of ITAC and explore their prognostic role, as well as their relations with clinicopathological variables. A computer-assisted image analysis of CD3 + and CD8 + tumor-infiltrating lymphocytes (TIL) density was conducted on surgical specimens of 51 patients with ITAC that underwent a curative treatment including surgery. ITAC displays variable TIL density, which is associated with OS. In a univariate model, the density of CD3 + TIL was significantly related to OS ( p = 0.012), whereas the association with CD8+ TIL density resulted in being non-significant ( p = 0.056). Patients with intermediate CD3 + TIL density were associated with the best outcome, whereas 5-year OS was the lowest for intermediate CD8 + TIL density. CD3 + TIL density maintained a significant association with OS in the multivariable analysis. TIL density was not significantly related to demographic and clinicopathological variables. CD3 + TIL density was independently associated with OS in a non-linear fashion and patients with intermediate CD3 + TIL density had the best outcome. Though based on a preliminary analysis on a relatively small series of patients, this finding makes TIL density a potential independent prognostic factor of ITAC.

The gilthead seabream ( Sparus aurata ) is a species of relevance for the Mediterranean aquaculture industry. Despite the advancement of genetic tools for the species, breeding programs still do not often include genomics. In this study, we designed a genomic strategy to identify signatures of selection and genomic regions of high differentiation among populations of farmed fish stocks. A comparative DNA pooling sequencing approach was applied to identify signatures of selection in gilthead seabream from the same hatchery and from different nuclei that had not been subjected to genetic selection. Identified genomic regions were further investigated to detect SNPs with predicted high impact. The analyses underlined major genomic differences in the proportion of fixed alleles among the investigated nuclei. Some of these differences highlighted genomic regions, including genes involved in general metabolism and development already detected in QTL for growth, size, skeletal deformity, and adaptation to variation of oxygen levels in other teleosts. The obtained results pointed out the need to control the genetic effect of breeding programs in this species to avoid the reduction of genetic variability within populations and the increase in inbreeding level that, in turn, might lead to an increased frequency of alleles with deleterious effects.

Neurons process real-time information from axon terminals to coordinate gene expression, growth, and plasticity. Inputs from distal axons are encoded as a stream of endocytic organelles, termed signalling endosomes, targeted to the soma. Formation of these organelles depends on target-derived molecules, such as brain-derived neurotrophic factor (BDNF), which is recognised by TrkB receptors on the plasma membrane, endocytosed, and transported to the cell body along the microtubules network. Notwithstanding its physiological and neuropathological importance, the mechanism controlling the sorting of TrkB to signalling endosomes is currently unknown. In this work, we use primary mouse neurons to uncover the small GTPase Rab10 as critical for TrkB sorting and propagation of BDNF signalling from axon terminals to the soma. Our data demonstrate that Rab10 defines a novel membrane compartment that is rapidly mobilised towards the axon terminal upon BDNF stimulation, enabling the axon to fine-tune retrograde signalling depending on BDNF availability at the synapse. These results help clarifying the neuroprotective phenotype recently associated to Rab10 polymorphisms in Alzheimer's disease and provide a new therapeutic target to halt neurodegeneration., Competing Interests: OL, GS No competing interests declared, (© 2023, Lazo and Schiavo.)