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ABSTRACT: Mountain dairy cattle farming systems are pivotal for the economy, as well as for social and environmental aspects. They significantly contribute to rural development, which is currently strongly prioritized in the common European Union agricultural policy; at the same time, they are also increasingly criticized for having a relatively high environmental impact (such as greenhouse gas emissions) per kilogram of product. Consequently, the aim of this study was to assess and compare the environmental efficiency of 2 common alpine dairy farming systems, with a focus on the effects of grazing, considering the seasonal variability in feeding at the individual cow level and farm management over a 3-yr period. This study focuses on alpine farming systems, but can also be considered to effectively represent other topographically disadvantaged mountain areas. We compared an intensively managed and globally dominating production system (high-input) aimed at high milk yield through relatively intensive feeding and the use of the high-yielding dual-purpose Simmental cattle permanently confined in stables, with a forage-based production system (low-input) based on seasonal grazing and the use of the autochthonous dual-purpose breed Tyrolean Grey. For the present analysis, we used a dataset with information on feed intake and diet composition, as well as animal productivity at the individual cow level and farm management data based on multiyear data recording. We quantified 4 impact categories for 3 consecutive years: global warming potential (GWP100), acidification potential (AP), marine eutrophication potential (MEP), and land use (LU; in square meters per year and eco points [Pt], with the latter additionally considering the soil quality index). In addition to being attributed to 1 kg of fat- and protein-corrected milk (FPCM), these impact categories were also related to 1 m2 of on-farm area. Due to limited agronomic options beyond forage production and pasture use in alpine regions, net provision of protein was calculated for both farming systems to assess food supply and quantify the respective food-feed competition. Overall, the low-input farming system had greater environmental efficiency in terms of MEP per kilogram of FPCM, as well as MEP and AP per square meter than the high-input system. Land use was found to be consistently higher for the high-input than for the low-input system, the GWP100 per kilogram of FPCM was lower for the high-input system. Additionally, pasture access had a significant effect on the reduction of environmental impacts. Lastly, the net protein provision was slightly negative for the high-input system and marginally positive for the low-input system, indicating a lower food-feed competition for the latter. Future studies should also address the social and economic aspects of the farming systems in order to offer a comprehensive overview of the 3 key factors necessary for achieving more sustainable farming systems, particularly in disadvantaged marginal regions such as mountain areas.

ABSTRACT: Cow milk microbiota has received increased attention in recent years, not only because of its importance for human health but also because of its effect on the quality and technological properties of milk. Several studies, therefore, have investigated the effect of various production factors on the microbial composition of milk. However, most of the previous studies considered a limited number of animals from experimental or single farm, which could have biased the results. Therefore, this study aimed to understand the effect of different alpine production systems in Italy on the compositional and microbiological quality of milk, considering commercial herds with different feeding intensities and cattle breeds. The results obtained in this work indicated that the month and season of sampling (July for summer or February for winter) more than farm, breed, and cow diet exerted significant effects on cow milk parameters and microbiota. In particular, significant differences were observed for urea content in milk between sampling seasons. Differences in milk fat were mainly related to breed specific effects. From a microbiological point of view, statistically significant differences were found in presumptive lactic acid bacteria counts. Based on a culture-independent method, milk obtained in February harbored the highest number of Firmicutes (e.g., Lactobacillus) and the lowest number of Actinobacteria (e.g., Corynebacterium). Moreover, bacterial richness and diversity were higher in July during alpine pasture season indicating a significant effect of pasture feed on the growth of bacterial communities. The results of this study highlighted the effect of month or season mainly related to differences in feeding management (e.g., access to pasture during vegetation period, concentrates supplementation) on composition and microbiota in milk.

Abstract BackgroundDepression and chronic pain are commonly comorbid, mutually reinforcing, and debilitating. Emerging approaches to mobile behavioral health care (mHealth) promise to improve outcomes for patients with comorbid depression and chronic pain by integrating with existing care models to bolster support and continuity between clinical visits; however, the evidence base supporting the use of mHealth to augment care for this patient population is limited. ObjectiveTo develop an evidence base that sets the stage for future research, we aimed to explore the associations between changes in depression severity and various integrated care models, with and without mHealth augmentation, among patients with comorbid depression and nonmalignant chronic pain. MethodsOur team leveraged retrospective, real-world data from 3837 patients with comorbid depression and nonmalignant chronic pain who received integrated behavioral health care (IBH) at a subspecialty pain clinic. We analyzed one IBH-only, non-mHealth cohort (n=2765), an mHealth-augmented cohort (n=844), and a collaborative care (CoCM)+mHealth cohort (n=136), which were supported by the NeuroFlow mHealth platform, and a pre-CoCM mHealth cohort (n=92), which was supported by the mHealth platform for 3 months prior to beginning the chronic pain treatment. We evaluated changes in depression severity between treatment cohorts via longitudinal analyses of both clinician- and mHealth-administered Patient Health Questionnaire-9 (PHQ-9) assessments. ResultsmHealth-augmented integrated care led to significantly greater proportions of patients reaching clinical benchmarks for reduction (725/844, 86% vs 2112/2765, 76%), response (689/844, 82% vs 2027/2765, 73%), and remission (629/844, 75% vs 1919/2765, 69%) compared with integrated care alone. Furthermore, hierarchical regression modeling revealed that patients who received mHealth-augmented psychiatric CoCM experienced the greatest sustained reductions in on-average depression severity compared with other cohorts, irrespective of clinical benchmarks. In addition, patients who engaged with an mHealth platform before entering CoCM experienced a 7.2% reduction in average depression severity before starting CoCM treatment. ConclusionsOur findings suggest that mHealth platforms have the potential to improve treatment outcomes for patients with comorbid chronic pain and depression by providing remote measurement–based care, tailored interventions, and improved continuity between appointments. Moreover, our study set the stage for further research, including randomized controlled trials to evaluate causal relationships between mHealth engagement and treatment outcomes in integrated care settings.

Originating in 19th-century Japan, judo blends traditional martial arts with modern educational principles. As a martial art rooted in the Budo tradition, it embodies a philosophy focused on the harmony of mind and body. In the West, however, the discipline evolved with an emphasis on competition and athleticism, which has led to increased competitive pressures among athletes. We report here the case studies of two Italian judokas who retired due to the development of unhealthy eating habits, extreme exercise and other unwanted psychological distress. It will be argued that the prevalent practice of weight cutting in combat sports poses substantial physical and psychological risks, particularly for young athletes. Despite regulatory efforts to mitigate extreme weight loss methods, there remains a critical need for improved education on safe practices. It sheds light on the complex interplay between athletic success, physical and mental health, and cultural perceptions of Judo in contemporary society.

Abstract This study estimates the association between the level of biosecurity, animal welfare, milk quality, and economic performance on 2291 mountain dairy farms which largely differs in management and structure from large dairy operations in the lowlands mainly due to climatic and topographic constraints in mountain areas. The dairy industry’s increasing emphasis on biosecurity is crucial for ensuring animal health, productivity, and disease mitigation. Therefore, in the present study the biosecurity and animal welfare status of mountain dairy farms were assessed considering the official welfare protocol for dairy cows of the Italian ClassyFarm system. Our findings reveal a suboptimal adoption of biosecurity measures, attributed to structural limitations in mountain farms and farmers’ awareness gaps. Despite these challenges, the economic significance of biosecurity adoption is evident, emphasizing potential benefits for farm viability and animal health. Conversely, the study indicates a moderate to good welfare status in assessed farms, reflecting farmers’ awareness of the importance of high welfare standards for dairy cows. Improved welfare positively correlates with milk sales and productivity, highlighting the economic advantages of prioritizing animal well-being. Challenges persist, particularly regarding animal housing deficiencies and related consumer concerns about animal welfare in dairy production. Therefore, targeted interventions and educational initiatives are crucial to empower farmers and promote best practices in biosecurity and animal welfare management. However, addressing resultant production cost increases necessitates society’s willingness to pay more for animal-based food, emphasizing the importance of aligning economic incentives with sustainable farming practices.

Abstract Esophageal adenocarcinoma (EAC) is one of the deadliest tumor entities worldwide, with a 5-year survival rate of less than 25%. Unlike other tumor entities, personalized therapy options are rare, partly due to the lack of knowledge about specific subgroups. In this publication, we demonstrate a subgroup of patients with EAC in a large screening cohort of 826 patients, characterized by specific morphological and immunohistochemical features. This subgroup represents approximately 0.7% (6/826) of the total cohort. Morphological features of this subgroup show a striking clear cytoplasm of the tumour cells and the parallel existence of rare growth patterns like yolk sac-like differentiation and enteroblastic differentiation. Immunohistochemistry reveals expression of the fetal gut cell-like proteins Sal-like protein 4 (SALL4), claudin-6, and glypican 3. Interestingly, we find a correlation with alterations of SWI/SNF-complex associated genes, which are supposed to serve as tumor suppressor genes in various tumour entities. Our results suggest a possible implication of rare tumour subtypes in the WHO classification for EACs according to the classification for gastric cancer. Furthermore, claudin-6 positive tumors have shown promising efficacy of CAR T cell therapy in the recently published BNT-211-01 trial (NCT04503278). This represents a personalized therapeutic option for this tumor subtype.

This study investigates the determinants of entrepreneurial intentions among university students in the rural region of Comarca Sierra Sur, Andalusia, Spain. Using the Theory of Planned Behavior, the analysis reveals that attitudes toward entrepreneurship and perceived behavioral control are significant predictors of entrepreneurial intentions. Positive attitudes and a strong sense of control are closely associated with entrepreneurial intentions, while subjective norms show no significant effect, suggesting that social expectations may function differently in rural areas. Additionally, personal values such as openness to change and self-enhancement positively influence entrepreneurial intentions, whereas conservatism is negatively associated. This research fills a gap in the literature by extending the Theory of Planned Behavior to a rural context, where prior studies have been limited. The findings highlight the need to tailor entrepreneurship policies to the unique characteristics of rural areas and emphasize the importance of personal values and perceived control in fostering entrepreneurship. These insights are particularly relevant for policymakers aiming to stimulate economic development and entrepreneurship in rural regions.

The Cancer of Unknown Primary (CUP) syndrome is characterized by identifiable metastases while the primary tumor remains hidden. In recent years, various data-driven approaches have been suggested to predict the location of the primary tumor (LOP) in CUP patients promising improved diagnosis and outcome. These LOP prediction approaches use high-dimensional input data like images or genetic data. However, leveraging such data is challenging, resource-intensive and therefore a potential translational barrier. Instead of using high-dimensional data, we analyzed the LOP prediction performance of low-dimensional data from routine medical care. With our findings, we show that such low-dimensional routine clinical information suffices as input data for tree-based LOP prediction models. The best model reached a mean Accuracy of 94% and a mean Matthews correlation coefficient (MCC) score of 0.92 in 10-fold nested cross-validation (NCV) when distinguishing four types of cancer. When considering eight types of cancer, this model achieved a mean Accuracy of 85% and a mean MCC score of 0.81. This is comparable to the performance achieved by approaches using high-dimensional input data. Additionally, the distribution pattern of metastases appears to be important information in predicting the LOP.

Abstract Background Fibroblast activation protein (FAP) is expressed in the tumor microenvironment (TME) of various cancers. In our analysis, we describe the impact of dual-tracer imaging with Gallium-68-radiolabeled inhibitors of FAP (FAPI-46-PET/CT) and fluorodeoxy-D-glucose (FDG-PET/CT) on the radiotherapeutic management of primary esophageal cancer (EC). Methods 32 patients with EC, who are scheduled for chemoradiation, received FDG and FAPI-46 PET/CT on the same day (dual-tracer protocol, 71%) or on two separate days (29%) We compared functional tumor volumes (FTVs), gross tumor volumes (GTVs) and tumor stages before and after PET-imaging. Changes in treatment were categorized as “minor” (adaption of radiation field) or “major” (change of treatment regimen). Immunohistochemistry (IHC) staining for FAP was performed in all patients with available tissue. Results Primary tumor was detected in all FAPI-46/dual-tracer scans and in 30/32 (93%) of FDG scans. Compared to the initial staging CT scan, 12/32 patients (38%) were upstaged in nodal status after the combination of FDG and FAPI-46 PET scans. Two lymph node metastases were only visible in FAPI-46/dual-tracer. New distant metastasis was observed in 2/32 (6%) patients following FAPI-4 -PET/CT. Our findings led to larger RT fields (“minor change”) in 5/32 patients (16%) and changed treatment regimen (“major change”) in 3/32 patients after FAPI-46/dual-tracer PET/CT. GTVs were larger in FAPI-46/dual-tracer scans compared to FDG-PET/CT (mean 99.0 vs. 80.3 ml, respectively (p

Abstract:: The aim of the present study was to assess the impact of altitude on the quality of milk from dairy cows housed in small-scale farms in alpine areas. Therefore, a dataset comprising 5,680 bulk milk samples from 32 farms located at different geographical altitudes was considered. Milk traits such as fat, protein, lactose, free fatty acids, casein, milk urea nitrogen content, pH value, and somatic cell score were examined in the laboratories of the South Tyrolean Dairy Association using mid-infrared spectroscopy. The data were analyzed using a statistical model that considered altitude category, grazing practice, housing system, and season of milk analysis as fixed effects. The findings revealed a positive association between altitude and milk fat, free fatty acids, and somatic cell count. Conversely, lactose content, milk urea nitrogen, and pH value in milk from cows kept on farms at higher altitude (>1,200 m above sea level) showed a negative relationship with altitude. Farms located at altitudes above 1,200 m exhibited higher fat, protein, urea, and somatic cell content than farms located at lower levels. The results provide new insights into a production effect that has so far received little attention and should be considered in farm management (e.g., feeding management, breed decision) to ensure animal health and the associated animal welfare as well as the productivity of dairy cows reared in traditional small-scale mountain dairy farms.

Abstract Background Colorectal cancer (CRC) frequently involves mutations in the KRAS gene, impacting therapeutic strategies and prognosis. The occurrence of KRAS mutations typically precludes the presence of RET fusions, with current medical literature suggesting a mutual exclusivity between these two genetic alterations. We present a unique case that challenges this notion. Case Presentation An 85-year-old female with metastatic CRC was found to have a combination of genetic anomalies that is to the best of our knowledge not yet described in the medical literature: a KRAS p.G12C mutation, associated with oncogenesis and treatment resistance, and an ANK3::RET fusion, an infrequent but targetable mutation in CRC. This molecular profile was uncovered through comprehensive genomic sequencing after the patient experienced metachronous tumor dissemination. The presence of both genetic events complicates the treatment approach. Conclusions The identification of both a KRAS p.G12C mutation and an ANK3::RET fusion in the same CRC patient adds a new layer to the oncogenic landscape and treatment considerations for CRC. It highlights the intricate decision-making required in the era of precision medicine, where targeted therapies must be carefully chosen and potentially combined to combat complex genetic profiles. The case emphasizes the urgency of investigating the clinical effects of concurrent or sequential use of KRAS p.G12C and RET inhibitors to inform future therapeutic guidelines and improve patient outcomes in similar cases.

Abstract One factor for the lacking integration of the middle ear stapes footplate prosthesis or the missing healing of stapes footplate fractures could be the known osteogenic inactivity. In contrast, it was recently demonstrated that titanium prostheses with an applied collagen matrix and immobilised growth factors stimulate osteoblastic activation and differentiation on the stapes footplate. Regarding those findings, the aim of this study was to evaluate the potential of bone regeneration including bone remodeling in the middle ear. Ten one-year-old female merino sheep underwent a middle ear surgery without implantation of middle ear prostheses or any other component for activating bone formation. Post-operatively, four fluorochromes (tetracycline, alizarin complexion, calcein green and xylenol orange) were administered by subcutaneous injection at different time points after surgery (1 day: tetracycline, 7 days: alizarin, 14 days: calcein, 28 days: xylenol). After 12 weeks, the temporal bones including the lateral skull base were extracted and histologically analyzed. Fluorescence microscopy analysis of the entire stapes with the oval niche, but in particular stapes footplate and the Crura stapedis revealed evidence of new bone formation. Calcein was detected in all and xylenol in 60% of the animals. In contrast, tetracycline and alizarin could only be verified in two animals. The authors were able to demonstrate the osseoregenerative potential of the middle ear, in particular of the stapes footplate, using fluorescence sequence labelling.

BackgroundImage and Performance-Enhancing Drugs (IPEDs) can enhance mental and physical capabilities and impact one’s overall health. Initially confined in sport environments, IPEDs use has become increasingly widespread in a high-performing society. The present study was aimed at profiling IPEDs use during the COVID-19 lockdown among an international sample of young adults.MethodsA cross-sectional observational study was carried out in eight countries (United Kingdom, Italy, Lithuania, Hungary, Portugal, Spain, Brazil, and Japan) between April and May 2020. The survey questionnaire included validated measurements such as Exercise Addiction Inventory (EAI), Appearance Anxiety Inventory (AAI), and Self-Compassion Scale (SCS) as well as questions about the type of IPEDs, purchasing methods and socio-demographic information.ResultsA total of 736 IPEDs users were included in the survey. Their mean age was 33.05 years (±SD = 10.06), and 64.2% were female participants. Overall, 6.8% were found at risk of exercise addiction (EAI >24), 27.6% presented high levels of appearance anxiety, and 24.9% revealed low levels of emotional regulation’s self-compassion. Most participants (55.6%) purchased IPEDs through pharmacies/specialized shops, while 41.3% purchased IPEDs on the Internet. Online IPEDs buyers were mainly men who had higher scores on the Exercise Addiction Inventory. One or more IPEDs classifiable as “potentially risky” were used by 66.3% of the sample. Users of “potentially risky IPEDs” were younger and primarily men. They showed higher scores both on the Exercise Addiction Inventory and Appearance Anxiety Inventory.ConclusionThis study profiled users of IPEDs when the most restrictive COVID-19 lockdown policies were implemented in all the participating countries. More targeted post-COVID 19 prevention strategies should be implemented according to the emerged socio-demographic and psychopathological traits and cross-cultural differences emerged. Longitudinal studies will also be needed to determine the long-term effect of the COVID-19 lockdown on IPEDs consumption.

Abstract Benzodiazepine (BDZ) addiction is a widespread and multifaceted phenomenon. For many patients, especially females, the concomitant use of other drugs also increases their risk of QTc prolongation, possibly leading to complications such as seizures and even sudden death. However, the relationship between BDZ use and QTc prolongation is currently unclear. The present study aims to examine patterns of polysubstance use among a sample of Italian adults with BDZ dependence in relation with their QTc prolongation risk. We used Latent Class Analysis (LCA) on data collected from 251 inpatients of the Addiction Medicine Unit in Verona to group patients into three classes according to their substance use and their QTc prolongation risk. Results showed no significant relationship between QTc prolongation and BDZ use in any of the classes considered. We conclude that BDZs, even if used long-term and at high dosages, can be considered safe in terms of cardiovascular complications for patients.

The main aim of the study was to assess the recent evolution of cattle breeding in Alpine areas based on the number, quality and price of calves sold at local auctions in the South Tyrol region over the last decade, as case study. In addition, the effect of grazing during dam’s late gestation on calves’ auction quality was investigated to assess whether the inclusion of pasture in Alpine production system has an impact on auction parameters of calves beside its well-known positive effects on cattle welfare and the environment. Similarly, to other geographical areas, the study revealed that the numbers of crossbred calves (especially from specialised dairy breeds) has increased over the last years also in the Alpine area, because of more targeted breeding plans with an intensive use of beef bulls’ semen. Further, purebred calves from dual-purpose breeds (Simmental, Alpine Grey, Pinzgauer) received a higher remuneration in terms of price and market value at auction than purebred calves from specialised Holstein Friesian and Brown Swiss cows. A part of these animals could be available to support an innovative alpine meat supply chain which, if grass-fed, could further strengthen the current landscape maintenance service provided by the mountain livestock husbandry. Dams grazing during the last 90 days of gestation had a limited negative effect on calves’ liveweight and quality traits at auctions. All these results provide useful technical insights to support the switch from dairy to suckler herd system as future productive alternative for some small-scale mountain cattle farms whose viability is essential for the provision of several ecosystem services in the fragile Alpine environment.

Literature provides indications that the personality and temperament of a horse are sufficiently genetically determined. This background was the impetus to continue collecting phenotypic information on the temperament in the South Tyrolean Haflinger horses, not only to have marketing tools but also to be able to estimate genetic parameters for a future genetic evaluation. The latter should open the possibility to include temperament traits in the breeding goal. Therefore, the aim of the present study was to consider for the very first-time phenotypic information from character testing for estimating genetic parameters for temperament in Haflinger horses. For the present study, data from routine character testing of 210 South Tyrolean Haflinger mares between 2019 and 2021 were considered. A multivariate estimation of heritability (h2) and genetic correlations (r) was carried out for the criteria ‘Interest’, ‘Activity’ and ‘Excitement’. In addition, univariate and bivariate estimation runs were also carried out. The pedigree-based heritability for the behavioural trait Interest was lowest with 0.07 and a standard error of ±0.04. For the other two criteria, Activity and Excitement, h2 was 0.19 ± 0.05 and 0.21 ± 0.04, respectively. The genetic correlation between Interest and Activity and between Interest and Excitement was in both cases negative with −0.52 and −0.61, respectively while the genetic correlation between Excitement and Activity was positive (0.99). Results of the present study highlight the potential of considering temperament traits in breeding of Haflinger horses.

The challenges facing the health sector and the agri-food industry have become increasingly complex over the years. Animal welfare, drug consumption, the use of the slaughterhouse as an epidemiological observatory and biosecurity, as well as the health risks associated with it, are increasingly interconnected, making an integrated approach necessary. Hence the European Commission developed regulations to ensure a certain standard for animal welfare in livestock husbandry, which led the Animal Health and Veterinary Drugs Directorate of the Italian Ministry of Health to design an integrated system called Classyfarm, aimed at categorising farming according to risk. The aim of our study was to consider the protocol of the Classyfarm system for evaluating the present situation in South Tyrolean dairy farming (1) and discuss the practical implementation of this new system in small-scale mountain dairy farming (2) which largely differs from large dairy enterprises in the lowlands. The results provide important insights about farm structure and management. However, in terms of animal welfare the questionnaire of Classyfarm is not able to properly differentiate and therefore results need to be considered with caution. Similarly, the section about biosafety is hardly applicable for small-scale mountain farms due to limited herd size and building capacity. Therefore, authors underline the necessity of developing a further differentiated questionnaire for the Classyfarm system, which considers the peculiarities of small-scale mountain dairy farming and allow proper conclusions regarding sanitary risk for public health unless the long-term aim is that such structure will stop producing which will have significant effects on many other sectors in the region, e.g. tourism.

The use of substances to improve sports performance is a widespread phenomenon among adolescents. Several anti-doping programs have been developed, mainly based on knowledge-based evidence. The main aim of the present study was to implement an anti-doping intervention in sports high school students, based on a psychological framework, such as Socio-Cognitive Theory, through the development of a Serious Game (SG), i.e., digital learning based on the game. The experimental design included an intervention group (n = 167; F = 37.7%; Meanage = 17.5 years; SD = 0.58) and a control group (n = 112; F = 42%; Meanage = 17.6; SD = 1). Both of the groups completed the same questionnaire on two different occasions (i.e., time 1 and time 2) for measuring doping intention, self-regulatory efficacy to resist social pressure for the use of substances, moral disengagement, and doping knowledge. Data were analyzed through repeated measures of Group X Time ANOVA, demonstrating some degree of efficacy of the intervention, in particular in terms of the decrease in doping intention and the strengthening of doping knowledge. Moreover, the study demonstrated that the score obtained during the implementation of the SG could partially represent a coherent measure of the participants’ beliefs regarding doping. These results could be considered a starting point for future research to better develop technological anti-doping interventions.

Abstract Background Anti-Hu antibody neuropathy is considered a rare acquired peripheral neuropathy, but common among paraneoplastic syndromes. Typically, is described as subacute sensory neuronopathy and electrophysiological findings are usually suggestive of a sensory axonal neuropathy. Case presentation We report the case of a 67-year-old man referred to our clinic with a 4-month history of progressive pain and paresthesias of distal lower limbs. He had a 30-year history of alcohol abuse and smoking. Alcoholic neuropathy was considered the most likely diagnosis, considering his history and evaluation. The patient’s neurological examination revealed symmetric bilateral superficial and deep sensory loss in the lower extremities, reduced Achilles tendon reflexes and wide based gait. Electrophysiological testing was suggestive of axonal sensory-motor polyneuropathy and small fiber involvement. Even though alcohol consumption was discontinued, symptoms gradually worsened. Further testing was performed and the patient was found seropositive for anti-Hu antibody. Small-cell lung cancer was detected later, but patient passed away before treatment for cancer was administrated. Conclusions The aim of our paper is to report a case of a rare paraneoplastic syndrome that can cause progressive sensory-motor neuropathy with large and small fiber involvement, which should be rapidly differentially diagnosed from other neuropathies, so that the underlying cause can be identified and, potentially, treated.

Background/Objective: There are several questionnaires for the challenge of anticipating opioid use disorder (OUD). However, many are not specific for chronic non-cancer pain (CNCP) or have been developed in the American population, whose sociodemographic factors are very different from the Spanish population, leading to scarce translation into clinical practice. Thus, the aim of this study is to prospectively validate a predictive model for OUD in Spanish patients under long-term opioids. Methods: An innovative two-stage predictive model was developed from retrospective (n = 129) and non-overlapping prospective (n = 100) cohorts of real-world CNCP outpatients. All subjects used prescribed opioids for 6 or more months. Sociodemographic, clinical and pharmacological covariates were registered. Mu-opioid receptor 1 (OPRM1, A118G, rs1799971) and catechol-O-methyltransferase (COMT, G472A, rs4680) genetic variants plus cytochrome P450 2D6 (CYP2D6) liver enzyme phenotypes were also analyzed. The model performance and diagnostic accuracy were calculated. Results: The two-stage model comprised risk factors related to OUD (younger age, work disability and high daily opioid dose) and provided new useful information about other risk factors (low quality of life, OPRM-G allele and CYP2D6 extreme phenotypes). The validation showed a satisfactory accuracy (70% specificity and 75% sensitivity) for our predictive model with acceptable discrimination and goodness of fit. Conclusions: Our study presents the results of an innovative model for predicting OUD in our setting. After external validation, it could represent a change in the paradigm of opioid treatment, helping clinicians to better identify and manage the risks and reduce the side effects and complications.

The application of venovenous (VV) extracorporeal membrane oxygenation (ECMO) has gained wide acceptance for the treatment of acute severe respiratory failure. Since no rat model of VV ECMO therapy with femoral drainage has yet been described, although this cannulation strategy is commonly used in humans, this study aimed to establish such a model. Twenty male Lewis rats were randomly assigned to receive a sham procedure or VV ECMO therapy. After the inhalative induction of anesthesia, animals were intubated and the vascular accesses were placed surgically. While venous drainage was achieved through a modified multi-orifice 18 G cannula that was placed in the inferior vena cava through the femoral vein over a guide wire with an ultra-flexible tip, the venous return was realized via a shortened 20 G cannula into the jugular vein. Hemodynamic data were obtained from a tail artery and left ventricular pressure–volume catheter. Repetitive blood gas analyses were carried out, and systemic inflammation was measured using an enzyme-linked immunosorbent assay. While animals in the ECMO group showed adequate oxygenation and decarboxylation, there was no evidence of recirculation. VV ECMO therapy increased stroke volume (SV), cardiac output (CO), and left ventricular end-diastolic volume (LVEDV). ECMO-induced inflammation was reflected in increased levels of tumor necrosis factor alpha. However, no differences in interleukins 6 and 10 were seen. This study describes a frequently used cannulation strategy in humans for a rat model of VV ECMO. Despite successful oxygenation and decarboxylation, the oxygenated blood may reduce pulmonary vascular resistance and lead to an increased LVEDV, which is associated with increased SV and CO. This model allows us to answer research questions about topics such as intestinal microcirculation in further studies.

Abstract Esophageal adenocarcinoma exhibits one of the highest mortality rates among all cancer entities. Multimodal therapy strategies have improved patients’ survival significantly. However, patients in early stages are currently limited to receiving only local therapies, even though some patients within this group showcase short survival periods. Until now, there has been no widely established clinically used biomarker to detect these high-risk patients. Telomerase reverse transcriptase (TERT), a gene encoding a crucial subunit of the telomerase enzyme, plays a significant role in establishing cancer cell immortality and is under suspicion for its potential contribution to tumor progression. Therefore, we aimed to evaluate the clinical relevance of the TERT amplification status. We included 643 patients with esophageal adenocarcinoma, who underwent Ivor-Lewis esophagectomy at the University Hospital of Cologne. The TERT amplification status was characterized using fluorescence in situ hybridization. Clinicopathological values and patients’ overall survival were compared between patients with and without TERT amplification. Further sub-cohort analyses were conducted for patients with pT1N0-3 tumor stage. Eighty-One patients (12.6%) exhibited TERT amplification. Patients with amplified TERT showed significantly worse overall survival (median OS: 22.6 vs. 36.8 months, p = 0.009). Interestingly, TERT amplification could be characterized as an independent risk factor for worse overall survival in multivariate analysis in patients with pT1N0-3 tumor stage (HR = 2.440, 95% CI 1.095–5.440, p = 0.029). In this study, we describe the TERT amplification status as an independent risk factor for worse survival in patients diagnosed with esophageal adenocarcinoma at pT1N0-3 tumor stage, encompassing cases involving tumor infiltration of the lamina propria, muscularis mucosae, and/or submucosa. Based on our findings, we put forth the proposition that evaluating the TERT amplification status may serve as a valuable tool in identifying a specific subgroup of patients, namely those with TERT amplification and pT1N0-3 tumor-stage esophageal adenocarcinoma. The patients of this subgroup could potentially benefit from enhanced follow-up protocols, more aggressive treatment approaches, or possible targeted TERT inhibition therapies, all aimed at improving their overall clinical outcomes.

Abstract Mosquitoes have profoundly affected human history and continue to threaten human health through the transmission of a diverse array of pathogens. The phylogeny of mosquitoes has remained poorly characterized due to difficulty in taxonomic sampling and limited availability of genomic data beyond the most important vector species. Here, we used phylogenomic analysis of 709 single copy ortholog groups from 256 mosquito species to produce a strongly supported phylogeny that resolves the position of the major disease vector species and the major mosquito lineages. Our analyses support an origin of mosquitoes in the early Triassic (217 MYA [highest posterior density region: 188–250 MYA]), considerably older than previous estimates. Moreover, we utilize an extensive database of host associations for mosquitoes to show that mosquitoes have shifted to feeding upon the blood of mammals numerous times, and that mosquito diversification and host-use patterns within major lineages appear to coincide in earth history both with major continental drift events and with the diversification of vertebrate classes.

Abstract Guidelines regulate how many (tumour-bearing) tissue particles should be sampled during gastric cancer biopsy to obtain representative results in predictive biomarker testing. Little is known about how well these guidelines are applied, how the number of tissue particles correlates with the actual tumour-infiltrated area and how many absolute tumour cells are captured. The study included endoscopic biopsies of untreated carcinomas of the upper gastrointestinal (GI)-tract during the 2016–2020 review period. Archival (H&E)-stained histological sections were digitised and the tumour areas were manually annotated. The tumour-bearing tissue area and absolute carcinoma cell count per case were determined by image analysis and compared with a reference primary surgical specimen. Biopsies from 253 patients were analysed. The following mean values were determined: (a) tumour tissue particle number: 6.5 (range: 1–25, standard deviation (SD) = 3.33), (b) number of tumour-bearing tissue particles: 4.7 (range: 1–20, SD = 2.80), (c) tumour-infiltrated area: 7.5 mm2 (range: 0.18–59.46 mm2, SD = 6.67 mm2), (d) absolute tumour cell count: 13,492 (range: 193–92,834, SD = 14,185) and (e) tumour cell count in a primary surgical specimen (tumour size: 6.7 cm): 105,200,176. The guideline-recommended tissue particle count of 10 was not achieved in 208 patients (82.2%) and the required tumour-bearing tissue particle count of 5 was not achieved in 133 patients (52.6%). Tissue particle count, tumour-infiltrated area and tumour cell count were only weakly correlated. Most cases featured an infiltrated area ≥ 4.5 mm2 (156, 61.7%). Cases with more tissue particles showed only a moderate increase in infiltrated area and tumour cells compared to cases with fewer particles. Biopsies are often used to determine predictive biomarkers, particularly Her2/neu and PD-L1. Diagnostic standards to ensure representative material have been suggested in guidelines to reduce false-negative predictions. However, the real-world practice seems to substantially deviate from recommended standards. To the best of our knowledge, this is the first systematic study describing the relationships between endoscopic tissue fragment number, actual infiltrated tumour area and carcinoma cell number. The data question the tissue particle number as a quality assessment parameter. We advocate histopathological reports indicating on which basis statements on therapy-relevant biomarkers were made. Digital pathology has the potential to objectively quantify the tissue for documentation, quality assessment and future clinical studies.

Abstract Background The prognosis of esophageal adenocarcinoma (EAC) and gastric adenocarcinoma (GAC) remains poor, and new therapeutic approaches are urgently needed. Claudin 6 (CLDN6) is an oncofetal antigen that is largely absent in healthy tissues and upregulated in several cancers, making it a promising therapeutical target. In this study, the expression of CLDN6 was assessed in an large Caucasian EAC and GAC cohort. Methods RNA-Seq data from 89 EACs and 371 GACs were obtained from The Cancer Genome Atlas project and EAC/GAC cases were stratified by CLDN6 mRNA expression based on a survival-associated cutoff. For groups with CLDN6 expression above or below this cutoff, differential gene expression analyses were performed using DESeq, and dysregulated biological pathways were identified using the Enrichr tool. Additionally, CLDN6 protein expression was assessed in more than 800 EACs and almost 600 GACs using a CLDN6-specific immunohistochemical antibody (clone 58-4B-2) that is currently used in Phase I/II trials to identify patients with CLDN6-positive tumors (NCT05262530; NCT04503278). The expression of CLDN6 was also correlated with histopathological parameters and overall survival (OS). Results EACs and GACs with high CLDN6 mRNA levels displayed an overexpression of pathways regulating the cell cycle, DNA replication, and receptor / extracellular matrix interactions. CLDN6 protein expression was associated with shorter OS in EAC and GAC, both in treatment-naïve subgroups and cohorts receiving neoadjuvant therapy. In multivariate analysis, CLDN6 protein expression was an independent adverse prognostic factor in EAC associated with a shorter OS (HR: 1.75; p = 0.01) and GAC (HR: 2.74; p = 0.028). Conclusions High expression of CLDN6 mRNA is associated with the dysregulation of distinct biological pathways regulating cell growth, proliferation, and cell–matrix interactions. Clinically, the expression of CLDN6 protein is a valuable adverse prognostic marker in EAC and GAC.

Background The individual HLA-I genotype is associated with cancer, autoimmune diseases and infections. This study elucidates the role of germline homozygosity or allelic imbalance of HLA-I loci in esophago-gastric adenocarcinoma (EGA) and determines the resulting repertoires of potentially immunogenic peptides.Methods HLA genotypes and sequences of either (1) 10 relevant tumor-associated antigens (TAAs) or (2) patient-specific mutation-associated neoantigens (MANAs) were used to predict good-affinity binders using an in silico approach for MHC-binding (www.iedb.org). Imbalanced or lost expression of HLA-I-A/B/C alleles was analyzed by transcriptome sequencing. FluoroSpot assays and TCR sequencing were used to determine peptide-specific T-cell responses.Results We show that germline homozygosity of HLA-I genes is significantly enriched in EGA patients (n=80) compared with an HLA-matched reference cohort (n=7605). Whereas the overall mutational burden is similar, the repertoire of potentially immunogenic peptides derived from TAAs and MANAs was lower in homozygous patients. Promiscuity of peptides binding to different HLA-I molecules was low for most TAAs and MANAs and in silico modeling of the homozygous to a heterozygous HLA genotype revealed normalized peptide repertoires. Transcriptome sequencing showed imbalanced expression of HLA-I alleles in 75% of heterozygous patients. Out of these, 33% showed complete loss of heterozygosity, whereas 66% had altered expression of only one or two HLA-I molecules. In a FluoroSpot assay, we determined that peptide-specific T-cell responses against NY-ESO-1 are derived from multiple peptides, which often exclusively bind only one HLA-I allele.Conclusion The high frequency of germline homozygosity in EGA patients suggests reduced cancer immunosurveillance leading to an increased cancer risk. Therapeutic targeting of allelic imbalance of HLA-I molecules should be considered in EGA.

IntroductionIt is well-known that circulating microRNAs (miRNAs) play a relevant role in many kinds of diseases by regulating the expression of genes involved in various pathophysiologic processes, including erectile dysfunction (ED) and cardiovascular diseases (CVD).PurposeThis study aimed to identify the miRNA-21 profile in the blood samples of patients with ED, CVD, and the combination of both pathologies to elucidate the potential function of miRNA-21.MethodsA total of 45 patients with CVD and/or who underwent the erectile function test were included and divided into the following categories: CVD with ED (cases, n = 29) and controls (n = 16) with either ED or CVD. Real-time polymerase chain reaction analysis verified the results. miRNA-21 expression was quantified, and informatics analysis was applied to predict the functions of this differentially expressed miRNA-21.ResultsA total of 64% of cases (63 ± 9 years, 66% with severe ED, 56% with CV ejection fraction) first presented ED as the sentinel clinical manifestation. Serum miRNA-21 levels in the control ED were significant, up to 10-fold higher than in the CVD controls and cases. A significant inverse (p = 0.0368, β = −2.046) correlation was found between erectile function and miRNA-21 levels.ConclusionsOur study provides comprehensive insights into the functional interaction between miRNA-21 and ED in CVD patients. Its relevance lies in the potential of miRNA as a biomarker to be applied in the cardiovascular predictive medicine field.

Introduction: The cytochrome P450 enzyme subfamilies, including CYP3A4 and CYP1A2, have a major role in metabolism of a range of drugs including several anti-cancer treatments. Many factors including environmental exposures, diet, diseaserelated systemic inflammation and certain genetic polymorphisms can impact the activity level of these enzymes. As a result, the net activity of each enzyme subfamily can vary widely between individuals and in the same individual over time. This variability has potential major implications for treatment efficacy and risk of drug toxicity, but currently no assays are available for routine use to guide clinical decision-making.Methods: To address this, a mass spectrometry-based method to measure activities of CYP3A4, CYP1A2 was adapted and tested in free-living participants. The assay results were compared with the predicted activity of each enzyme, based on a self-report tool capturing diet, medication, chronic disease state, and tobacco usage. In addition, a feasibility test was performed using a low-volume dried blood spots (DBS) on two different filter-paper supports, to determine if the same assay could be deployed without the need for repeated standard blood tests.Results: The results confirmed the methodology is safe and feasible to perform in free-living participants using midazolam and caffeine as test substrates for CYP3A4 and CYP1A2 respectively. Furthermore, though similar methods were previously shown to be compatible with the DBS format, the assay can also be performed successfully while incorporating glucuronidase treatment into the DBS approach. The measured CYP3A4 activity score varied 2.6-fold across participants and correlated with predicted activity score obtained with the self-report tool. The measured CYP1A2 activity varied 3.5-fold between participants but no correlation with predicted activity from the self-report tool was found.Discussion: The results confirm the wide variation in CYP activity between individuals and the important role of diet and other exposures in determining CYP3A4 activity. This methodology shows great potential and future cross-sectional and longitudinal studies using DBS are warranted to determine how best to use the assay results to guide drug treatments.

ABSTRACT: Livestock production systems with ruminants play a relevant role in the emission of the greenhouse gas CH4, which is known to significantly contribute to global warming. Consequently, it is a major societal concern to develop strategies in mitigating such emissions. In addition to breeding toward low-emitting cows, management strategies could also help in reducing greenhouse gas emissions from dairy farms. However, information is required for appropriate decision making. To the best of our knowledge, this is the first study that considers different, already available equations to estimate CH4 emissions of small-scale dairy farms in the mountain region, which largely differ from large dairy farms in the lowlands concerning management and production. For this study, 2 different production systems, both typical for small-scale dairy farming in mountain regions, were simultaneously run over 3 yr at an experimental farm as follows: (1) a high-input production system, characterized by intensive feeding with high amounts of external concentrates and maize silage, year-round housing, and high yielding Simmental cattle breed, and (2) a low-input production system, characterized by prevailing hay and pasture feeding and silage ban, thus covering most of the energy requirements by forage harvested on-farm and the use of the local Tyrolean Grey cattle breed. Results reveal that feeding management has a significant effect on the amount of CH4 emissions. The low-input production system produced less CH4 per cow and per day compared with the high-input production system. However, if calculated per kilogram of milk, the high-input scenario produced proportionally less CH4 than the low-input one. Findings of this study highlight the potential to assess in a fast and cost-effective way the CH4 emission in different dairy production systems. This information contributes to the debate about the future of sustainable milk production in mountain regions, where the production of feed resources is climatically constrained, and could be useful for breeding purposes toward lower CH4-emissions.

Background and Objectives: Parathyroid adenoma is a distinct cause of primary hyperparathyroidism, with the vast majority being sporadic ones. Proteomic analysis of parathyroid adenomas has proposed a large number of related proteins. The aim of this study is to evaluate the immunohistochemical staining of ANXA2, MED12, MAPK1 and VDR in parathyroid adenoma tissue. Materials and Methods: Fifty-one parathyroid adenomas were analyzed for ANXA2, MED12, MAPK1 and VDR expressions. Tissue was extracted from formalin-fixed paraffin-embedded parathyroid adenoma specimens; an immunohistochemical study was applied, and the percentage of allocation and intensity were evaluated. Results: ANXA2 stained positively in 60.8% of all cell types, while MED12 had positive staining in 66%. MAPK1 expression was found to be negative in total, although a specific pattern for oxyphil cells was observed, as they stained positive in 17.7%. Finally, VDR staining was positive at 22.8%, based on nuclear staining. Conclusions: These immunohistochemical results could be utilized as biomarkers for the diagnosis of sporadic parathyroid adenoma. It is of great importance that a distinct immunophenotype of nodule-forming cells in a positive adenoma could suggest a specific pattern of adenoma development, as in hereditary patterns.

A class G of graphs is called hereditary if it is closed under taking induced subgraphs. We denote by G^{apex} the class of graphs G that contain a vertex v such that G − v is in G. Borowiecki, Drgas-Burchardt, and Sidorowicz proved that if a hereditary class G has finitely many forbidden induced subgraphs, then so does G^{apex}. We provide an elementary proof of this result. The hereditary class of cographs consists of all graphs G that can be generated from K_1 using complementation and disjoint union. A graph is an apex cograph if it contains a vertex whose deletion results in a cograph. Cographs are precisely the graphs that do not have the 4-vertex path as an induced subgraph. Our main result finds all such forbidden induced subgraphs for the class of apex cographs.

BackgroundStaging, especially clinical lymph node staging in esophageal adenocarcinoma has only moderate sensitivity and specificity. Therefore, we evaluated combined molecular markers to predict prognosis.Patients and methods890 tumor tissue samples were obtained from patients who underwent surgery for esophageal adenocarcinoma with curative intent. These were stained by tissue micro array for 48 markers which are associated with tumorigenesis and correlated with clinical data (TNM-staging, overall survival) by multivariate Cox regression.ResultsTwo markers (preserved Y chromosome and high grade of (CD3+) T-cell infiltration) were found to be significantly and independently associated with better overall survival. We formed a score (called CY score) from the two markers. The more markers are positive and thus the higher the score (ranging from 0 to 2), the better the overall survival, independently of UICC. Moreover, we developed a combination score of the UICC and CY score based on cluster analysis. Patients with a UICC stage of III with the presence of both traits (CY=2) can be assigned to a better prognosis group (group II), whereas patients with a UICC stage of I without both traits (CY=0) must be assigned to a worse prognosis group (group II). Therefore, patients in stage I with adverse molecular signature might benefit of multimodal therapy.ConclusionIn summary, the CY score adds prognostic information to the UICC stage based on tumor biology in esophageal adenocarcinoma and warrants further evaluations in independent clinical cohorts.