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The aim of this cross-sectional study was to determine the body fluid volume in patients diagnosed with both chronic kidney disease (CKD) and arterial hypertension (AH), and to investigate the relationship between fluid overload (FO), nutritional status and arterial stiffness in this specific patient population. A total of 169 participants with CKD and AH were enrolled in the study, and data on general parameters, comorbidities, medication use, and laboratory parameters were collected. Body composition was assessed with a Tanita MC 780 device, and data on the central and peripheral systolic and diastolic blood pressure, as well as pulse wave velocity (PWV) and the augmentation index (AIx) were collected with an IEM Mobil-O-Graph 24 h ambulatory blood pressure monitor, which was based on oscillometry. The Mediterranean Diet Serving Score (MDSS) questionnaire was used to determine the adherence to the Mediterranean diet (MeDi). Our results showed that the significant positive predictors of hydration status were the use of diuretics and oral hypoglycemic agents, whereas the negative predictors were female sex, higher body mass index level and use of two or more antihypertensives in the form of a single-pill combination. We also found differences in blood pressure and arterial stiffness parameters in relation to volume status, along with differences based on the presence of diabetes mellitus (DM). In conclusion, these results call for a higher awareness of volume status in the care of CKD patients with AH, especially in those with diabetes mellitus.

Infectious diseases still threaten global human health, and host genetic factors have been indicated as determining risk factors for observed variations in disease susceptibility, severity, and outcome. We performed a genome-wide meta-analysis on 4624 subjects from the 10,001 Dalmatians cohort, with 14 infection-related traits. Despite a rather small number of cases in some instances, we detected 29 infection-related genetic associations, mostly belonging to rare variants. Notably, the list included the genes CD28, INPP5D, ITPKB, MACROD2, and RSF1, all of which have known roles in the immune response. Expanding our knowledge on rare variants could contribute to the development of genetic panels that could assist in predicting an individual’s life-long susceptibility to major infectious diseases. In addition, longitudinal biobanks are an interesting source of information for identifying the host genetic variants involved in infectious disease susceptibility and severity. Since infectious diseases continue to act as a selective pressure on our genomes, there is a constant need for a large consortium of biobanks with access to genetic and environmental data to further elucidate the complex mechanisms behind host–pathogen interactions and infectious disease susceptibility.

Background: Kidney transplant recipients (KTRs) represent a vulnerable group of patients who develop a number of comorbidities. Severe periodontitis (SP) is associated with the most common chronic systemic diseases including kidney diseases. The objective of this study was to explore the risk factors for SP in KTRs. Methods: In this study, KTRs were divided into those with or without periodontitis and in relation to the severity of periodontitis. A comprehensive medical and periodontal examination was performed and evaluated. Multivariate logistic regression was performed to examine possible risk factors for SP among KTRs. Results: A total of 100 KTRs was included in the analysis, of which 87% had periodontitis. Significant predictors of periodontitis were older age (OR (95% CI) = 1.07 (1.01-1.13), P = 0.016) and lower skeletal muscle mass (OR (95% CI) = 0.88 (0.78-0.99), P = 0.035). When examining periodontitis severity, predictors of SP (n = 21, 24%) were increased levels of uric acid (OR (95% CI) = 1.01 (1.00-1.02), P = 0.022) and dental plaque (OR (95% CI) = 1.04 (1.01-1.07), P = 0.013). In the subset analysis that included only KTRs with measured advanced glycation end products (AGE) (n = 47), 34% (n = 16) had SP. The predictors of SP were AGE (OR (95% CI) = 3.89 (1.28-11.82), P = 0.017) and dental plaque (OR (95% CI) = 1.07 (1.01-1.13), P = 0.028). Conclusions: KTRs with SP had significantly higher uric acid level and AGE, which may contribute to the systemic health status of this patient population.

Modern teaching and learning in biomedical education require introducing new learning methods with students in focus. Student-oriented pedagogies represent new approaches in the European higher education area, in particular for biomedical studies. Their introduction strives to modernize biomedical education and increase its quality. Personalized, predictive, participatory, and preventive (P4) medicine is revolutionizing medical care in all its aspects. However, the appropriate training that develops the P4 medicine understanding and the skills to implement this new approach needs to be improved in biomedical studies. Various initiatives are promoting new innovative pedagogies in biomedical education, striving to develop research, knowledge, and transversal skills, crucial for the students of the 21st century. Erasmus+ project “Personalized Medicine Inquiry-Based Education - PROMISE” in partnership with five European countries Mediterranean Institute for Life Sciences (Croatia), University of Split (Croatia), University of Zagreb (Croatia), University Pompeu Fabra (Spain), European Alliance for Personalized Medicine (Belgium) and Tamara Milosevic (France) tried to achieve “P4 Education” in biomedical studies. The target group of the project was students from Croatia and Spain. The main objective of the PROMISE project was to prepare students in different biomedical fields, such as biology, biotechnology, pharmacy, and medicine, for the rapidly changing landscape of medical practice and research by creating innovative inquiry-based teaching tools in an online format on the principles of P4 Medicine and Responsible Research and Innovation (RRI). During the project, the partnership developed and implemented the PROMISE program with four online modules, each dealing with one element of P4 Medicine, supplemented with in-person learning expeditions held in Paris, Split, and Zagreb where students deepened their knowledge and skills crucial for their future careers in the biomedical field. Students, after finishing the course, identified several key elements as high-quality content: 1) relevant topics for their future career, 2) raised awareness about the importance of P4 Medicine, 3) relevance of integration with different disciplines, and 4) opportunity to develop transversal skills. Students evaluated the PROMISE program as excellent regarding the methodology used, acquired knowledge, and skills. As a result of their satisfaction with the project activities, students created their youth association through which they are now raising awareness and educating others about P4 Medicine and RRI principles. The project results were evaluated by external evaluators and achieved a high score of 94/100 for project implementation.

Background: This systematic review and meta-analysis aimed to summarize current evidence on vitamin D status in patients with psoriatic arthritis (PsA) with a particular focus on disease activity. Methods: PubMed, Web of Science, Scopus and Cochrane Library databases were searched for studies that investigated vitamin D levels in PsA. The search was conducted on 12th October 2022. Included studies were cohorts, RCTs or observational studies, those assessing the level of 25(OH)D3 with control group consisting of healthy or psoriasis (Pso) patients. Nottingham-Ottawa Quality Scale was used to assess methodological quality. Random effects meta-analysis model was applied with inverse variance weighting and mean difference with 95% CI was calculated. Results: Of 356 retrieved studies, 76 duplicates and 270 studies were excluded according to the exclusion criteria with one study unavailable. Four studies including 264 PsA patients and 287 healthy controls and five studies including 225 PsA patients and 391 Pso patients assessing vitamin D levels were eligible for meta-analysis. Vitamin D levels were lower in PsA patients compared to the healthy group (MD = -6.42 ; 95 % CI -8.31, -4.53 ; P < 0.01), while higher compared to Pso patients (MD = 2.37 ; 95 % CI 0.97, 3.78 ; P < 0.01). Included studies had moderate to low risk of bias. Conclusion: In conclusion, PsA patients have lower vitamin D levels than the general population. However, further studies are essential to understand the role of vitamin D in the development and treatment of PsA and the differences in vitamin D metabolism in PsA and Pso.

There is limited evidence on the associations between dietary patterns, body composition, and nonclassical predictors of worse outcomes such as advanced glycation end products (AGE) in kidney transplant recipients (KTRs). The aim of this cross-sectional study was to determine the level of AGE-determined cardiovascular (CV) risk in Dalmatian KTRs and possible associations between AGE, adherence to the Mediterranean diet (MeDi), and nutritional status. Eighty-five (85) KTRs were enrolled in this study. For each study participant, data were collected on the level of AGE, as measured by skin autofluorescence (SAF), Mediterranean Diet Serving Score (MDSS), body mass composition, anthropometric parameters, and clinical and laboratory parameters. Only 11.76% of the participants were adherent to the MeDi. Sixty-nine percent (69%) of KTRs had severe CV risk based on AGE, while 31% of KTRs had mild to moderate CV risk. The results of the LASSO regression analysis showed that age, dialysis type, dialysis vintage, presence of CV and chronic kidney disease, C- reactive protein level, urate level, percentage of muscle mass, and adherence to recommendations for nuts, meat, and sweets were identified as positive predictors of AGE. The negative predictors for AGE were calcium, phosphate, cereal adherence according to the MeDi, and trunk fat mass. These results demonstrate extremely low adherence to the MeDi and high AGE levels related CV risk in Dalmatian KTRs. Lifestyle interventions in terms of CV risk management and adherence to the MeDi of KTRs should be taken into consideration when taking care of this patient population.

Canine babesiosis is a tick-borne disease caused by Babesia canis, with acute kidney injury as one of the common complications. In the study 8 healthy control dogs and 22 dogs with naturally occurring babesiosis were enrolled, with the aim to analyse differences in serum and urinary proteomes between healthy dogs and dogs with different degree of renal dysfunction in babesiosis using a label-based high-throughput quantitative proteomic approach. In serum, 58 proteins were found differentially abundant between healthy controls and groups of dogs with different degrees of renal dysfunction in babesiosis, while in urine there were 259 differentially abundant proteins. In addition, altered biological pathways were detected in the diseased dogs using bioinformatics tools and validation of several candidate biomarkers was performed. SIGNIFICANCE: The main aim of this comprehensive study was to perform analyses of serum and urinary proteomes of dogs with renal dysfunction in babesiosis compared to healthy dogs using, for the first time, a high-throughput proteomic method and functional enrichment analyses. Serum and urine samples of the same dogs were investigated in order to gain a more complete picture of pathologic changes taking place in renal dysfunction in babesiosis. We highlighted two putative biomarkers validated herein which could be of importance for early diagnosis of renal dysfunction in canine babesiosis, as they are easily accessible from urine and their concentration rises before the appearance of azotaemia: urinary neutrophil gelatinase-associated lipocalin (NGAL) and urinary liver-type fatty acid-binding protein (L-FABP).

Canine chronic enteropathy (CCE) is a collective term used to describe a group of idiopathic enteropathies of dogs that result in a variety of clinical manifestations of intestinal dysfunction. Clinical stratification into food-responsive enteropathy (FRE) or non-food responsive chronic inflammatory enteropathy (CIE), is made retrospectively based on response to treatments. Faecal extracts from those with a FRE (n = 5) and those with non-food responsive chronic inflammatory enteropathies (CIE) (n = 6) were compared to a healthy control group (n = 14) by applying TMT-based quantitative proteomic approach. Many of the proteins with significant differential abundance between groups were pancreatic or intestinal enzymes with pancreatitis-associated protein (identified as REG3α) and pancreatic M14 metallocarboxypeptidase proteins carboxypeptidase A1 and B identified as being of significantly increased abundance in the CCE group. The reactome analysis revealed the recycling of bile acids and salts and their metabolism to be present in the FRE group, suggesting a possible dysbiotic aetiology. Several acute phase proteins were significantly more abundant in the CCE group with the significant increase in haptoglobin in the CIE group especially notable. Further research of these proteins is needed to fully assess their clinical utility as faecal biomarkers for differentiating CCE cases. SIGNIFICANCE: The identification and characterisation of biomarkers that differentiate FRE from other forms of CIE would prove invaluable in streamlining clinical decision-making and would avoid costly and invasive investigations and delays in implementing effective treatment. Many of the proteins described here, as canine faecal proteins for the first time, have been highlighted in previous human and murine inflammatory bowl disease (IBD) studies initiating a new chapter in canine faecal biomarker research, where early and non-invasive biomarkers for early clinical stratification of CCE cases are needed. Pancreatitis-associated protein, pancreatic M14 metallocarboxypeptidase along with carboxypeptidase A1 and B are identified as being of significantly increased abundance in the CCE groups. Several acute phase proteins, were significantly more abundant in the CCE group notably haptoglobin in dogs with inflammatory enteropathy. The recognition of altered bile acid metabolism in the reactome analysis in the FRE group is significant in CCE which is a complex condition incorporating of immunological, dysbiotic and faecal bile acid dysmetabolism. Both proteomics and immunoassays will enable the characterisation of faecal APPs as well as other inflammatory and immune mediators, and the utilisation of assays, validated for use in analysis of faeces of veterinary species will enable clinical utilisation of faecal matrix to be fully realised.

The aim of the study was to monitor and analyse injured and diseased loggerhead sea turtles (Caretta caretta) plasma proteome profiles and biochemistry parameters during their recovery period in rescue centre within different age and recovery period groups, and determine the potential biomarkers that can be used in diagnostics. The plasma biochemical parameters of total protein and glucose content, accompanied by aspartate aminotransferase (AST) and N-acetyl-cystein-activated creatinine kinase (CK-NAC) are highlighted as valuable and potential biomarkers of turtle's health status and condition. Using high throughput tandem mass tag (TMT)-based proteomic approach we identified 913 plasma proteins, 12 of which shown to be modulated in loggerheads age groups, and identified as a part of (i) platelet degranulation, (ii) neutrophil degranulation, and (iii) innate immune system pathways. The neurofascin (NFASC) is shown to be differentially abundant among all the age groups, and alpha-1-acid glycoprotein 2-like (ORM2) and alpha-1-antitrypsin-like (SERPINA1) proteins were recognized as members of all three above mentioned REACTOME pathways. Furthermore, 29 of plasma proteins were significantly differentially abundant in loggerheads age and recovery period groups. Out of 15 recognized pathways, those proteins were mostly included in three specific REACTOME pathways: (i) post-translational phosphorylation, (ii) regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs), and (iii) platelet degranulation. The alpha-fetoprotein (AFP) was the only protein which showed statistically significant up-regulation patterns in all loggerhead age groups before release from the rescue centre, and the complement component 3 (C3) protein was the only protein modulated in all recovery period groups. Furthermore, C3 protein takes part in 9; and followed up with apolipoprotein A-I (APOA1) in 7; complement component 4 (C4), complement component 5 (C5) and kininogen-1 (KNG1) in 6 REACTOME pathways. Thereby, those proteins are highlighted and recommended as potential biomarkers of turtle's health status. Data are available via ProteomeXchange with identifier PXD029569. Finally, based on our results, we believe that comprehensive omics approach and routine plasma biochemical analysis, accompanied by proteins of acute phase, acid-base status and immune-response indicator analysis may significantly and reliably improve assessment of captive loggerheads rehabilitation and medication. SIGNIFICANCE: Monitoring and comparison of loggerhead sea turtles (C. caretta) blood plasma biochemistry parameters and plasma proteome profiles in relation to the age, and recovery period pointed out significantly differentially abundant proteins, along with certain biochemical parameter contents as potential biomarkers of turtle's fitness, health status and physiology.

Depression has been addressed as a predictor of worse outcomes in kidney transplant recipients (KTRs). Nutritional status plays a great role in treatment of this population. The Mediterranean diet (MeDi) has been associated with lower levels of depressive symptoms. The aim of this cross-sectional study was to determine the rate of depression and its correlations to nutritional status and dietary habits according to the MeDi in Dalmatian KTRs. We included 115 KTRs, and data about body composition and anthropometric, laboratory and clinical parameters were obtained for each study participant. The Beck Depression Inventory-II (BDI-II) questionnaire was used to assess depressive symptoms and the Mediterranean Diet Serving Score (MDSS) was used to assess adherence to the MeDi. We found the presence of depressive symptoms in 21.73% of the Dalmatian KTRs. BDI-II score was reciprocally associated with fat mass, trunk visceral fat, anthropometric parameters of obesity, triglyceride levels and olive oil consumption. Inverse associations were found between BDI-II score and skeletal muscle mass, handgrip strength, MCV, hemoglobin levels and consumption of fish and white meat, as suggested by the MeDi. Our results showed the interconnections between nutritional status, dietary habits and depression in Dalmatian KTRs.

DNA-protein crosslinks (DPCs), formed by the covalent conjugation of proteins to DNA, are toxic lesions that interfere with DNA metabolic processing and transcription. The development of an accurate biochemical assay for DPC isolation is a priority for the mechanistic understanding of their repair. Here, we propose the STAR assay for the direct quantification of DPCs, sensitive to physiologically relevant treatment conditions. Implementing the STAR assay revealed the formation of small cross-linked peptides on DNA, created by the proteolytic degradation of DPCs by SPRTN. The initial proteolytic degradation of DPCs is required for the downstream activation of DNA repair, which is mediated through the phosphorylation of H2Ax. This leads to the accumulation of DNA repair factors on chromatin and the subsequent complete removal of the cross-linked peptides. These results confirmed that the repair of DPCs is a two-step process, starting with proteolytic resection by SPRTN, followed by the repair of the underlying damage to the DNA.

Background: The aim of the present review was to summarize the current evidence about the impact of vitamin D deficiency on pathology and clinical manifestations of Sjögren’s disease (SD). Methods: Databases PubMed, Web of Science, Scopus, and Cochrane library were searched for studies assessing the levels of vitamin D in SD patients using the following keywords: (vitamin D OR calciferol OR cholecalciferol OR 25-hydroxyvitamin D OR 25-hydroxycholecalciferol OR calcidiol OR calcitriol OR 1,25-dihydroxycholecalciferol) AND (Sjögren’s Syndrome OR Sjögren’s disease) accessed on 20 September 2022. Out of 248 retrieved studies, following the systematic review methodology and defined inclusion and exclusion criteria, 9 clinical studies were eligible to be included in the present review: 4 of them case-control, 4 cross-sectional, and 1 cohort study. Results: Nine studies totaling 670 SD patients and 857 healthy controls were eligible for meta-analysis with moderate to high methodological quality as determined by the Newcastle–Ottawa Quality Scale (NOS). According to the obtained results, a high prevalence of hypovitaminosis D was observed in SD patients when compared to healthy controls (95% CI −10.43, −2.39; p < 0.01). Conclusion: Available evidence points to lower levels of vitamin D in patients with SD in comparison to healthy controls. However, further studies are necessary to understand the underlying mechanisms associated with the role of vitamin D in the development and disease severity of SD.

This study examines whether maternal low ω6:ω3 ratio diet and offspring SW supplementation can improve offspring immunity and performance by elucidating the effects on piglet serum proteome. A total of 16 sows were given either a standard (CR, 13:1) or low ω6:ω3 ratio diet (LR, 4:1) during pregnancy and lactation and their male weaned piglets were supplemented with SW powder (4 g/kg, SW) or not (CT) in a 21-day post-weaning (PW) diet. Four PW piglet groups were then identified based on dam and piglet treatment, namely CRCT, CRSW, LRCT, and LRSW (n = 10 each). Piglet serum collected at weaning and d21 PW were analysed (n = 5 each) using TMT-based quantitative proteomics and validated by appropriate assays. The differentially abundant proteins (n = 122) displayed positive effects of maternal LR diet on anti-inflammatory properties and innate immune stimulation. Progeny SW diet activated the innate immunity and enhance the host defence during inflammation. These data demonstrate the value of decreasing ω6:ω3 ratio in maternal diet and SW supplementation in PW piglet's diet to boost their immunity and anti-inflammation properties. SIGNIFICANCE: This novel proteomic study in post-weaned piglets addresses the interplay between maternal and offspring nutritional interventions in a context of rapid and dynamic alterations in piglet metabolic status around weaning. Decreasing ω6:ω3 ratio in maternal diet and SW supplementation in PW piglet's diet can boost their immunity and anti-inflammation properties. This study also provides new insights into piglet serum proteome regulation during post-weaning, a critical development period in swine.

Herniation of the intervertebral disc (IVDH) is the most common cause of neurological\ud and intervertebral disc degeneration-related diseases. Since the disc starts to degenerate before\ud it can be observed by currently available diagnostic methods, there is an urgent need for novel\ud diagnostic approaches. To identify molecular networks and pathways which may play important\ud roles in intervertebral disc herniation, as well as to reveal the potential features which could be\ud useful for monitoring disease progression and prognosis, multi-omics profiling, including highresolution liquid chromatography-mass spectrometry (LC-MS)-based metabolomics and tandem\ud mass tag (TMT)-based proteomics was performed. Cerebrospinal fluid of nine dogs with IVDH\ud and six healthy controls were used for the analyses, and an additional five IVDH samples were\ud used for proteomic data validation. Furthermore, multi-omics data were integrated to decipher a\ud complex interaction between individual omics layers, leading to an improved prediction model.\ud Together with metabolic pathways related to amino acids and lipid metabolism and coagulation\ud cascades, our integromics prediction model identified the key features in IVDH, namely the proteins\ud follistatin Like 1 (FSTL1), secretogranin V (SCG5), nucleobindin 1 (NUCB1), calcitonin re-ceptorstimulating peptide 2 precursor (CRSP2) and the metabolites N-acetyl-D-glucosamine and adenine,\ud involved in neuropathic pain, myelination, and neurotransmission and inflammatory response,\ud respectively. Their clinical application is to be further investigated. The utilization of a novel\ud integrative interdisciplinary approach may provide new opportunities to apply innovative diagnostic\ud and monitoring methods as well as improve treatment strategies and personalized care for patients\ud with degenerative spinal disorders.

Liver fluke, Fascioloides magna, is an important non-native parasite introduced to Europe, posing a threat to survival of local wildlife populations. The aim of this study was to assess the serum and liver protein profile of control and F. magna infected wild boars, by means of shotgun tandem mass tag - based quantitative high resolution proteomics approach. In serum, 4 differentially abundant proteins were found out of total 1073 identified, while in liver from 3520 identified proteins, 116 were differentially abundant between healthy and F. magna infected wild boars. Pathway analysis revealed that most of the proteins differing in abundance are involved in metabolism, biological oxidations, cellular responses to stimuli, fatty acid metabolism, and others. Validation of proteomic results was performed for paraoxonase-1, ceruloplasmin, glutathione S-transferase and liver enzymes by ELISA and automated assays. Complementary analysis of liver and serum in F. magna infection enabled insight into changes of proteome profile of the host at local and sistemic level. Our findings showed that chronic infection with F. magna is associated with immune response in host, oxidative stress and metabolomic changes in liver. SIGNIFICANCE: Liver fluke infections are recognised as worldwide neglected diseases with considerable veterinary and public health importance. Pathological changes, clinical signs and outcome of F. magna infection are strongly related to the type of final hosts and their different tolerance to infection. In order to gain insight into host-parasite interactions in wild boars, dead-end host for F. magna, we assessed proteomics profile of serum and liver of control animals and those infected with F. magna. Proteomics analysis of serum and liver in parallel showed as advantageous and beneficial, demonstrating protein alterations mainly at local level. Bioinformatics analysis enabled elucidation of molecular pathways associated with F. magna infection. Identification and validation of proteins associated with infection may have added value to current tools for efficient liver fluke control.

Although kidney transplantation significantly improves the quality of life of patients with end-stage renal disease (ESRD), the prevalence of cardiovascular disease (CVD) in kidney transplant recipients (KTRs) remains high. Atherosclerosis, post-transplantation metabolic changes, immunosuppressive therapy, and periodontitis contribute to elevated cardiovascular risk in this population. The aim of the study was to evaluate carotid intima-media thickness (IMT) as a surrogate marker of atherosclerosis and to analyze the possible risk factors for IMT in Dalmatian KTRs. Ninety-three KTRs were included in this study. Data on clinical and laboratory parameters, body composition, anthropometry, advanced glycation end-product (AGE) measurements, blood pressure, and arterial stiffness were collected. All participants underwent ultrasound examination of IMT and evaluation of periodontal status. KTRs with carotid IMT ≥ 0.9 were significantly older, had a lower level of total cholesterol, fat mass, end-diastolic velocity (EDV), and had fewer teeth. They also had significantly higher values of pulse wave velocity (PWV) and resistive index (RI). We found positive correlations between carotid IMT and duration of dialysis, age, PWV, AGE, RI, and average total clinical attachment level (CAL). The regression model showed that IMT in KTRs is associated with higher PWV, lower fat mass, and fewer teeth. The results of our study suggest that nutritional and periodontal status are associated with carotid IMT in KTRs.

Bovine mastitis causes changes in the milk and serum proteomes. Here changes in both proteomes caused by naturally occurring subclinical and clinical mastitis have been characterised and quantified. Milk and serum samples from healthy dairy cows (n = 10) were compared to those of cows with subclinical (n = 12) and clinical mastitis (n = 10) using tandem mass tag (TMT) proteomics. Proteins that significantly increased or decreased in milk (n = 237) or serum (n = 117) were quantified and classified by the type of change in subclinical and clinical mastitis. A group of the proteins (n = 38) showed changes in both milk and serum a number of which decreased in the serum but increased in milk, suggesting a particular role in host defence for maintaining and restoring homeostasis during the disease. Proteins affected by bovine mastitis included proteins in host defence and coagulation pathways. Investigation of the modified proteomes in milk and serum was assessed by assays for haptoglobin, serum amyloid A and α1 acid glycoprotein validating the results obtained by quantitative proteomics. Alteration of abundance patterns of milk and serum proteins, together with pathway analysis reveal multiple interactions related to proteins affected by mastitis. Data are available via ProteomeXchange with identifier PXD022595 . Significance Mastitis is the most serious condition to affect dairy cows and leads to reduced animal welfare as well as having a negative economic effect for the dairy industry. Proteomics has previously identified changes in abundance of milk proteins during mastitis, but there have been few investigations addressing changes that may affect proteins in the blood during the infection. In this study, changes in the abundance of proteins of milk and serum, caused by naturally occurring mastitis have been characterised by proteomics using a quantitative approach and both subclinical and clinical cases of mastitis have been investigated. In both milk and serum, change in individual proteins was determined and classified into varying types of altering abundance, such as increasing in subclinical mastitis, but showing no further increase in clinical mastitis. Of special interest were the proteins that altered in abundance in both milk and serum which either showed similar trends - increasing or decreasing in both biological fluids or showed reciprocal change decreasing in serum but increasing in milk. As well as characterising proteins as potential markers of mastitis and the severity of the disease, these results provide insight into the pathophysiology of the host response to bovine mastitis.

Objective: The balance between oxidative stress (OS) and antioxidants is speculated to be associated with occurrence, severity, and progression of periodontal disease. Previous studies suggested that OS regulates the relationship between periodontitis and endothelial dysfunction. The increased arterial stiffness is a major pathophysiological player involved in the adverse cardiovascular profi le of renal transplant recipients (RTRs) and graft survival due to the occurrence of cardiovascular disease. The aim of this study was to evaluate the incidence and severity of periodontitis among RTRs suffering from arterial hypertension (AH) and to investigate possible correlation with OS arterial stiffness and blood pressure parameters regards severity of periodontitis. Design and method: A comprehensive medical and periodontal examination was performed and for each RTR and data were collected. To measure arterial pressure (both peripheral and central, systolic and diastolic) and parameters of arterial stiffness (pulse wave velocity (PWV) and augmentation Index (AIx)) Agedio B900 device was used. Oral periodontal status included full mouth plaque score (FMPS), full mouth bleeding score (FMBS), periodontal probing depth (PPD) and clinical attachment loss (CAL), all measured at six sites of each tooth. Also, to measure Advanced Glycation End products (AGE) proteins in tissue the AGE Reader was used. Results: Out of 55 RTRs suffering from periodontitis, 45 (82%) had AH, out of which 9 (20%) were classifi ed as periodontitis stage I (mild), 14 (31%) as stage Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved. e158 Journal of Hypertension Vol 39, e-Supplement 1, March 2021 II (moderate) and 22 (49%) as stage III and IV (severe). With the severity of periodontitis increasing, statistically signifi cant increase was observed in parameters CAL and AGE (P

The aim of this study was to evaluate adherence to Mediterranean diet (MeDi) and possible correlation of MeDi adherence and nutritional status parameters in Dalmatian kidney transplant recipients (KTRs). One hundred and sixteen KTRs were included in this study. Data about Mediterranean Diet Serving Score (MDSS), body mass composition, anthropometric parameters, clinical and laboratory parameters were collected for each study participant. The results showed 25% adherence to the MeDi in Dalmatian KTRs. MDSS showed association with higher serum albumin and phosphorus level and higher skeletal muscle mass. Also, significant association between diabetic status and MDSS was found. Adherence to olive oil intake suggested by the MeDi showed significant association with lower level of triglycerides and adherence to nuts suggestions was associated with lower level of fat mass. Following MeDi recommendations for consumption of other foods (cereals, potato, eggs, vegetables, fruits and dairy) were also associated with body mass composition parameters and laboratory findings. In conclusion, low adherence to the MeDi in Dalmatian KTRs raises high concerns. The results showed that MeDi can have favorable effects on nutritional status in KTRs. A structured nutritional approach is needed to enhance adherence to the MeDi and prevent possible adverse effects in this patient population.

Lymphoma is the most common spontaneous malignant tumor in dogs. As a heterogeneous disease, canine lymphoma represents malignant transformation of lymphocyte subtypes at different stages of development. Different biomarkers of canine lymphoma for diagnosis, prognosis, and therapy monitoring have been of clinical interest for more than a decade. Moreover, canine lymphoma is similar to human non-Hodgkin's lymphoma and represents an excellent model as the most widely investigated tumor both in veterinary and comparative oncology. This study employed quantitative label-based proteomic and bioinformatic approaches to identify potential serum biomarkers in dogs with lymphoma. Ten dogs with multicentric lymphoma were followed in 3 time points: at the presentation to the Clinic for internal diseases, during the treatment and after the treatment. The results were compared with samples obtained from 10 healthy dogs. Results summary based on analysis of 378 proteins and four different groups of dogs: 50 proteins were significantly different between the four groups after FDR correction (all FDR < 0.05). In post-hoc tests, 152 proteins were significantly differentially abundant with adjusted P-value (FDR < 0.05) according to the group combinations (10 unique genes). Involved proteins had a role in different biological processes: response to stress, blood coagulation, regulation of biological quality, defence response, adaptive and innate immune response and acute-phase response. Differentially expressed REACTOME pathways are: complement cascade, common pathway of fibrin clot formation, signaling for integrins, innate immune system, thrombin signalling through proteinase activated receptors (PARs), platelet activation, signaling and aggregation, regulation of complement cascade and cell surface interactions at the vascular wall. Overexpressed subunit of fibrinogen - fibrinogen gamma chain isoform X2, different isforms of fibronectin and rearrangement of genes for immunoglobulin are previously connected with human cancers and non-Hodgkin's lymphoma, so they deserve further validation. Spontaneously occurring malignancies in dogs provide a tool for translational research for human oncology. Complete characterization of canine proteome in lymphoma, as well as biomarker validation with detailed explanation of the role of involved proteins becomes useful for diagnosis and treatment monitoring.

Background and Aims While kidney transplantation (Ktx) offers several advantages in terms of improved clinical outcomes and quality of life compared to dialysis modalities, depressive symptoms are still present in approximately 25% of patients, rates comparable to that of the hemodialysis population. Correlates of depressive symptoms include marital status, income, and kidney function, history of affective illness, malnutrition, and inflammation. The aim of this study was to evaluate possible correlations between nutritional status evaluated through body mass composition parameters, muscle strength and depression in kidney transplant recipients (KtR). Method 115 KtR, 61 (53 %) men and 54 (47 %) women, aged 59.3 (range 25 - 83) years were included. For each patient data about duration after Ktx, duration of dialysis before Ktx, age, gender, body weight and height were collected and body mass index (BMI) was calculated. Tanita MC780 Multi Frequency segmental body composition analyser was used to measure content of body fat, muscle mass, and visceral fat each study subject measured in kilograms (kg) and percentage (%.) Peripheral muscle strength of KtR was measured by using a standardized handgrip dynamometry protocol. The Beck Depression Inventory-Second Edition (BDI-II) was used as an indicator of the severity of depression. Results Out of all 115 KtR patients, according BDI-II score 25 (21.7 %) were depressed; 15 (13 %) had mild, 6 (5.2 %) moderate and 4 (3.5 %) severe depression. Significantly negative correlations between BDI-II scores (higher score – more depressive symptoms) and muscle mass (kg and %) was found (R=-0.205, P=0.032), (R=-0.278, P=0.003), respectively. Therefore, significantly positive correlations between BDI-II scores (higher score – more depressive symptoms), fat mass (%) and age was found (R=-0.24, P=0.012), (R=-0.231, P=0.013), respectively. Those KtR patients with more depressive symptoms (higher BDI-II score) had statistically lower muscle strength (R=-0.205, P=0.032). Significantly correlation between duration after Ktx and duration of dialysis before Ktx with BDI-II score was not found. Conclusion Prevalence rates for depression in this study is similar with previous reports and results have shown that depressive KtR patients were older and had significantly lower muscle mass and muscle strength and significantly higher fat mass content. Possible explanation for this findings might be that depression in KtR is associated with unhealthy behaviours including lower activity levels and worse dietary habits, which may in turn impact body mass composition parameters and upon transplant-related outcomes. Efforts to detect and treat depression should be a priority if one is to improve treatment and dietary adherence, quality of life, and outcomes in KtR.

Background and Aims Cognitive impairment is correlated with increased mortality of chronic kidney disease patients and hypertension is a well known comorbidity. While transplantation offers various general health and cognitive function improvement over dialysis, kidney transplant recipients (KTRs) still show greater cognitive impairment than healthy control. Researched pathophysiological mechanisms in KTR population include infection and immunosuppressive induced neurotoxicity. The aim of this study was to determine what impact do body composition, dietary habits and muscular strength have on cognitive impairment of hypertensive KTRs. Method 48 (52.17 %) men and 44 (47.83%) women, aged 59.7 (range25-83) years were included. For each patient data about duration after Ktx, duration of dialysis before Ktx, age, gender, body weight and height were collected and BMI and waist-to-height ratio (WHtR) were calculated. Tanita MC780 Multi Frequency segmental body composition analyser was used to measure content of body fat, muscle mass, and visceral fat each study subject measured in kilograms (kg) and percentage (%.) Peripheral muscle strength of KtR was measured by using a standardized handgrip dynamometry protocol. The Mini Mental State Exam (MMSE) was used as an indicator of cognitive impairment. Mediterranean Diet Serving Score (MDSS) questionnaire was used to determine adherence to Mediterranean Diet (MeDi) considering the consumption of foods and food groups per meal, day or week. Results Out of 92 KTR patients with hypertension, 5 of them scored ≤ 24 points on MMSE scale implying serious cognitive dysfunction. Mean score of MMSE was 28.2, (range 22-30) Regarding MDSS score, 11 patients scored ≥ 16 meaning highest adherence, while 67 KTRs scored < 16 meaning low adherence to MeDi, (results range 2-18) . Positive correlations were found between MMSE score (higher score meaning better cognitive functioning) and MDSS score ( higher score implying better adherence to MeDi)( R=0,282, p=0,012), consuming 1-2 portions of fruits( R=0,266,p=0,016) or ≥ 2 portions of vegetables ( R=0,241,p=0,03) per main meal, skeletal muscle mass (%) (R=0,278, p=0,008), peripheral muscular strength( R=0,325,p=0,003) and fat free mass (kg)(R=0,212,p=0,045). On the other hand we found statistically significant negative correlation between MMSE score and WHtR ( R=-0,255, p= 0,023) . as well as PWV(R=-0,244, p=0,022). Conclusion Cognitive impairment correlations in this study are similar to ones in general population specially regarding Mediterrranean diet and muscle mass . Possible explanation of positive MeDi impact on cognitive function in KTRs could be its low phosphate intake which prevents vascular calcification and negative effects of hyperparatiroidism. Although benefits of MeDi in CKD patients can be argued due to high pottasium levels, after kidney Tx it should not be an obstacle anymore. These results suggest that lifestyle interventions such as following MeDi, building more muscle and reducing fat could provide hypertensive KTR patients with longer preservation of cognitive function.

Background and Aims Arterial stiffness is an important risk factor for cardiovascular disease (CVD) in patients with end-stage renal disease (ESRD). However, little is known about risk factors that contribute to arterial rigidity in peritoneal dialysis (PD) patients. The periodontal diseases are highly prevalent among patients with ESRD. The aim of this study was to evaluate the dental status, incidence and severity of periodontitis among Dalmatian PD patients and to investigate possible correlations with arterial stiffness. Method 24 PD patients, 12 (50 %) men and 12 (50 %) women, aged 54.9 (range 21–79) years were included. Arterial stiffening was estimated by using pulse wave velocity (PWV). To measure PWV Agedio B900 device was used. Oral periodontal status included full mouth plaque score, full mouth bleeding score, periodontal probing depth and clinical attachment loss (CAL), all measured at six sites of each tooth. Also periodontitis stage was determined for each patient. Results Out of 24 PD patients 17 (70.1 %) was suffering from periodontitis. Among those with periodontitis 1 patient (5.9 %) was classified as periodontitis stage I (mild), 6 (35.3 %) as stage II (moderate), 10 (58.8%) as stage III and IV (severe). Significantly positive correlation between PWV, average interdental CAL and average total CAL, among all PD patients were found (R= 0.764, P=0.001), (R= 0.738, P=0.001), respectively, Also, in those PD patients statistically negative correlations between PWV and number of teeth were found (R= -0.565, P=0.012). Among those PD patients with periodontitis statistically positive correlation between PWV, periodontitis stage, interdental CAL, average total CAL and dental plaque were found (R= 0.73, P=0.002),(R= 0.731, P=0.00¸5), (R= 0.72, P=0.005), (R= 0.558, P=0.047), respectively. Conclusion The incidence of peritonitis is high in Dalmatian PD patients and more than half of PD patients had severe stage of periodontitis. Despite small sample size, we showed that those PD patients with more severe periodontitis stage had significantly higher arterial stiffness. Routine dental examination should be suggested for all PD patients. A prospective cohort study needs to link the association of severe periodontitis and CVD and confirm desirable survival outcome on successful treatment of periodontal diseases.

This prospective study included four dog groups (group A: healthy dogs, groups B: dogs with idiopathic epilepsy under antiepileptic medication (AEM), C: idiopathic epilepsy dogs without AEM administration, D: dogs with structural epilepsy). The purpose of the study was to compare the proteomic profile among the four groups. Samples were analyzed by a quantitative Tandem Mass Tags approach using a Q-Exactive-Plus mass-spectrometer. Identification and relative quantification were performed using Proteome Discoverer, and data were analyzed using R. Gene ontology terms were analyzed based on Canis lupus familiaris database. Data are available via ProteomeXchange with identifier PXD018893. Eighteen proteins were statistically significant among the four groups (P < 0.05). MMP2 and EFEMP2 appeared down-regulated whereas HP and APO-A1 were up- regulated (groups B, D). CLEC3B and PEBP4 were up- regulated whereas APO-A1 was down-regulated (group C). IGLL1 was down-regulated (groups B, C) and up-regulated (group D). EFEMP2 was the only protein detected among the four groups and PEBP4 was significantly different among the epileptic dogs. Western blot and SPARCL immunoassay were used to quantify HP abundance change, validating proteomic analysis. Both, showed good correlation with HP levels identified through proteomic analysis (r = 0.712 and r = 0.703, respectively). Significance: The proteomic analysis from CSF of dogs with epileptic seizures could reflect that MMP2, HP and APO-A1 may contribute to a blood-brain barrier disruption through the seizure-induced inflammatory process in the brain. MMP2 change may indicate the activation of protective mechanisms within the brain tissue. Antiepileptic medication could influence several cellular responses and alter the CSF proteome composition.

This study aimed to investigate the association between dispositional optimism, handedness and pressure pain perception in general population and under experimental conditions. We included 2500 community-dwelling adults of both sexes from Croatia in a cross-sectional study. We measured pressure pain threshold, pressure pain tolerance, and pressure pain interval using a hand-held algometer. A detailed questionnaire was used to collect data on sociodemographic and psychological characteristics. The association between optimism and pain measures was assessed using multiple linear regression analysis, controlling for sex, age, and a number of socioeconomic and psychological confounding factors. Additionally, we tested whether handedness was associated with pain perception. Dispositional optimism was significantly and positively associated with pressure pain tolerance and interval on both hands, but not with pain threshold. Pressure pain perception did not differ substantially according to handedness, and only right-handed subjects displayed higher pain threshold, tolerance and pain interval on their dominant hand. These findings suggest that optimists can tolerate acute pressure pain better, but they are equally sensitive to pain. Further studies are needed to demonstrate causality between optimism and pain tolerance, and possibly even to translate these findings into supplementary, non-pharmaceutical optimism-based clinical pain management.

Bladder cancer (BC) is the ninth leading cause of cancer death with one of the highest recurrence rates among all cancers. One of the main risks for BC development is exposure to nitrosamines present in tobacco smoke or in other products. Aberrant epigenetic (DNA methylation) changes accompanied by deregulated gene expression are an important element of cancer pathogenesis. Therefore, we aimed to determine DNA methylation signatures and their impacts on gene expression in mice treated with N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), a carcinogen similar to compounds found in tobacco smoke. Following BBN administration mice developed non-invasive or invasive bladder cancers. Surprisingly, muscle- and neuronal-related pathways emerged as the most affected in those tumors. Hypo- and hypermethylation changes were present within non-invasive BC, across CpGs mapping to the genes involved in muscle- and neuronal-related pathways, however, methylation differences were not sufficient to affect the expression of the majority of associated genes. Conversely, invasive tumors displayed hypermethylation changes that were linked with alterations in gene expression profiles. Together, these findings indicate that bladder cancer progression could be revealed through methylation profiling at the pre-invasive cancer stage that could assist monitoring of cancer patients and guide novel therapeutic approaches.

Simple Summary The present study describes for the first time the differences in the serum and saliva proteomes between healthy bitches and bitches with mammary tumors using a high-throughput proteomic approach. More than 1000 proteins were identified and 35 in serum and 49 in saliva were significantly modulated. Additionally, their related pathways were discussed in order to improve understanding of the pathophysiology of the disease and one protein, serum albumin, was further validated. The results of the present study could be a source of potential biomarkers for canine mammary tumors in saliva and serum and increase the knowledge on the pathophysiology of the disease. Abstract The aim of this study was to evaluate changes in serum and saliva proteomes in canine mammary tumors (CMT) using a high-throughput quantitative proteomic analysis in order to potentially discover possible biomarkers of this disease. Proteomes of paired serum and saliva samples from healthy controls (HC group, n = 5) and bitches with CMT (CMT group, n = 5) were analysed using a Tandem Mass Tags-based approach. Twenty-five dogs were used to validate serum albumin as a candidate biomarker in an independent sample set. The proteomic analysis quantified 379 and 730 proteins in serum and saliva, respectively. Of those, 35 proteins in serum and 49 in saliva were differentially represented. The verification of albumin in serum was in concordance with the proteomic data, showing lower levels in CMT when compared to the HC group. Some of the modulated proteins found in the present study such as haptoglobin or S100A4 have been related to CMT or human breast cancer previously, while others such as kallikrein-1 and immunoglobulin gamma-heavy chains A and D are described here for the first time. Our results indicate that saliva and serum proteomes can reflect physiopathological changes that occur in CMT in dogs and can be a potential source of biomarkers of the disease.

The response of pathophysiological research to emerging epidemics often occurs after the epidemic and, as a consequence, has little to no impact on improving patient outcomes or on developing high-quality evidence to inform clinical management strategies during the epidemic. Rapid and informed guidance of epidemic (research) responses to severe infectious disease outbreaks requires quick compilation and integration of existing pathophysiological knowledge. As a case study we chose the Zika virus (ZIKV) outbreak that started in 2015 to develop a proof-of-concept knowledge repository. To extract data from available sources and build a computationally tractable and comprehensive molecular interaction map we applied generic knowledge management software for literature mining, expert knowledge curation, data integration, reporting and visualization. A multi-disciplinary team of experts, including clinicians, virologists, bioinformaticians and knowledge management specialists, followed a pre-defined workflow for rapid integration and evaluation of available evidence. While conventional approaches usually require months to comb through the existing literature, the initial ZIKV KnowledgeBase (ZIKA KB) was completed within a few weeks. Recently we updated the ZIKA KB with additional curated data from the large amount of literature published since 2016 and made it publicly available through a web interface together with a step-by-step guide to ensure reproducibility of the described use case. In addition, a detailed online user manual is provided to enable the ZIKV research community to generate hypotheses, share knowledge, identify knowledge gaps, and interactively explore and interpret data. A workflow for rapid response during outbreaks was generated, validated and refined and is also made available. The process described here can be used for timely structuring of pathophysiological knowledge for future threats. The resulting structured biological knowledge is a helpful tool for computational data analysis and generation of predictive models and opens new avenues for infectious disease research. ZIKV Knowledgebase is available at www.zikaknowledgebase.eu., Summary: The Zika KnowledgeBase collects and integrates literature and experimental data based on Zika virus pathobiology for future emerging epidemics.

The present study evaluated for the first time changes in the saliva proteome in bitches with pyometra through a high-throughput quantitative proteomic analysis. The aims were to explore whether saliva composition could reflect the physiopathological changes occurring in canine pyometra and to identify potential biomarkers of the disease. Saliva samples from six healthy (H) and six bitches with pyometra (P) were analyzed using tandem mass tags–based approach. Additionally, 15 samples were used for the validation of changes in haptoglobin (Hp) concentration in saliva of dogs with pyometra. Proteomic analysis quantified 707 proteins in saliva. Comparison of the two groups revealed 16 unique proteins significantly modulated in saliva, with S100A calcium-binding protein 12 (S100A12), vimentin, and Hp the most up-regulated in canine pyometra. According to PANTHER (Protein Analysis Through Evolutionary Relationships) classification tool, these proteins are mainly related to proinflammatory mediators, acute-phase proteins, and sepsis. In conclusion, it can be stated that there are changes in various proteins in saliva in canine pyometra reflecting different physiopathological changes occurring in this disease. These proteins could be a source of potential non-invasive biomarkers for this disease that should be confirmed in future studies.

Colorectal cancer (CRC) is the third most frequent cancer type in both males and females, with about 35% of patients being diagnosed in stage IV metastatic disease. Despite advancements in treatment, life expectancy in patients with metastatic disease is still not satisfying. Due to frequent drug resistance during conventional and targeted cancer treatments, the development and testing of multi-target therapies is an important research field. Medicinal mushrooms specific isolated compounds as well as complex extract mixtures have been studied in depth, and many mushroom species have been proven to be non-toxic multi-target inhibitors of specific oncogenic pathways, as well as potent immunomodulators. In this study, we have performed a tandem mass tags qualitative and quantitative proteomic analyses of CT26.WT colon cancer tumor tissues from Balb/c mice treated with the studied medicinal mushroom extract mixture, with or without 5-fluorouracil. Besides significantly improved survival, obtained results reveal that Agarikon.1 alone, and in combination with 5-fluorouracil exert their anticancer effects by affecting several fundamental processes important in CRC progression. Bioinformatic analysis of up- and downregulated proteins revealed that ribosomal biogenesis and translation is downregulated in treatment groups, while the unfolded protein response (UPR), lipid metabolism and tricarboxylic acid cycle (TCA) are upregulated. Moreover, we found that many known clinical biomarkers and protein clusters important in CRC progression and prognosis are affected, which are a good basis for an expanded translational study of the herein presented treatment.