Potential roles of serum ATPase and AMPase in predicting diagnosis of colorectal cancer patients.

Colorectal cancer (CRC) is a common gastrointestinal cancer with high incidence and mortality rates. CRC may be associated with regulation of circulating nucleotides. This study aimed to evaluate the serum levels of nucleotide-metabolizing enzymes (ATPase and AMPase) in patients with CRC and to explore the clinical diagnostic value of these enzymes. The gene set variation analysis (GSVA) score of the ATP-adenosine signature was calculated using tumor samples from The Cancer Genome Atlas (TCGA). ATP-adenosine signaling plays a central role in CRC progression. A total of 135 subjects, including 87 patients with CRC and 48 healthy controls, were included. The serum levels of ATPase and AMPase in the CRC group were significantly higher than those in the control group (P < 0.05). Furthermore, ATP and AMP hydrolysis levels significantly increased in the advanced CRC group (P < 0.05). ATP and AMP hydrolysis was decreased by the ENTPDase inhibitors (POM-1 and ARL67156) and CD73 inhibitor (APCP). The sensitivities of ATPase and AMPase were 95.4% and 75.9%, respectively, which were higher than those of CEA (67.8%) and CA19-9 (72.4%). The specificities of ATPase and AMPase were 69.9% and 73.9%, respectively, which were higher than that of CA19-9 (47.8%). The combination of CEA, ATPase, and AMPase demonstrated high sensitivity (92.0%) and specificity (87.0%). Collectively, ATPase and AMPase activities are upregulated in CRC with considerable diagnostic significance. The combination of CEA, ATPase, and AMPase may provide a novel approach for CRC screening.

Single-Cell RNA Sequencing Analysis of the Immunometabolic Rewiring and Immunopathogenesis of Coronavirus Disease 2019.

Although immune dysfunction is a key feature of coronavirus disease 2019 (COVID-19), the metabolism-related mechanisms remain elusive. Here, by reanalyzing single-cell RNA sequencing data, we delineated metabolic remodeling in peripheral blood mononuclear cells (PBMCs) to elucidate the metabolic mechanisms that may lead to the progression of severe COVID-19. After scoring the metabolism-related biological processes and signaling pathways, we found that mono-CD14+ cells expressed higher levels of glycolysis-related genes (PKM, LDHA and PKM) and PPP-related genes (PGD and TKT) in severe patients than in mild patients. These genes may contribute to the hyperinflammation in mono-CD14+ cells of patients with severe COVID-19. The mono-CD16+ cell population in COVID-19 patients showed reduced transcription levels of genes related to lysine degradation (NSD1, KMT2E, and SETD2) and elevated transcription levels of genes involved in OXPHOS (ATP6V1B2, ATP5A1, ATP5E, and ATP5B), which may inhibit M2-like polarization. Plasma cells also expressed higher levels of the OXPHOS gene ATP13A3 in COVID-19 patients, which was positively associated with antibody secretion and survival of PCs. Moreover, enhanced glycolysis or OXPHOS was positively associated with the differentiation of memory B cells into plasmablasts or plasma cells. This study comprehensively investigated the metabolic features of peripheral immune cells and revealed that metabolic changes exacerbated inflammation in monocytes and promoted antibody secretion and cell survival in PCs in COVID-19 patients, especially those with severe disease.

Serum from patients with hypertension promotes endothelial dysfunction to induce trophoblast invasion through the miR­27b­3p/ATPase plasma membrane Ca2+ transporting 1 axis.

Pregnancy­induced hypertension is often accompanied by preeclampsia. The present study investigated whether microRNA (miR)­27b­3p affected the occurrence of preeclampsia by regulating the function of endothelial cells. Expressions levels of miR­27b­3p and ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) were determined using reverse­transcription quantitative PCR. miR­27b­3p targeting ATP2B1 was predicted using bioinformatics and further confirmed by dual­luciferase reporter assays. Cell Counting Kit­8, Transwell and Matrigel tube formation assays were performed to detect the effects of miR­27b­3p on proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs), respectively. Moreover, HTR8/SVneos cells were co­cultured with HUVECs to detect the invasion of trophoblast cells, and the expression levels of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)­2 and MMP­9 of HUVECs and HTR8/SVneos were detected by western blotting. Expression levels of miR­27b­3p were upregulated in the serum of patients with hypertension and preeclampsia, which could target and regulate the expression of ATP2B1. The expression levels of miR­27b­3p were increased and those of ATP2B1 were reduced in HUVECs from hypertensive serums. Moreover, miR­27b­3p mimics reduced the expression level of ATP2B1, and miR­27b­3p inhibitor reversed the effect of hypertensive serum on ATP2B1 expression. Furthermore, patients with hypertension showed increased endothelial dysfunction, reduced trophoblastic invasion and the expressions of VEGF, MMP­2 and MMP­9, and miR­27b­3p mimics and silencing of ATP2B1 produced similar results to HUVECs. The miR­27b­3p inhibitor reversed the effect of hypertensive serum, and silencing of ATP2B1 inhibited the improvement of miR­27b­3p inhibitor to HUVECs and HTR­8/SVneo cells in proliferation, migration and tube formation. The current findings suggested that miR­27b­3p promoted proliferation, migration and tube formation of HUVECs and enhanced invasion of trophoblast cells, via regulation of ATP2B1. Thus, miR­27b­3p could be considered as a molecular risk factor in the pathogenesis and development of preeclampsia.

The levels of follicular fluid cell-free mitochondrial DNA could serve as a biomarker for pregnancy success in patients with small ovarian endometriosis cysts: A case-control study.

Ovarian endometriosis cyst (OEC) is caused by the growth of ectopic endometrium into the ovarian cortex, leading to disrupted ovarian cortical structures and infertility. Large OECs are usually surgically removed, and assisted reproductive technology (ART) is required for future pregnancy. The oocyte reserve and development of patients with small non-surgical OECs are unknown. In this study, we compared mitochondrial abnormality, ATPase and IF1 mRNA expression levels, and OXPHO complex proteins between OEC vs control mural granulosa cells (mGCs).OEC mGCs show fewer mitochondria per cell, a higher proportion of aberrant morphology, lower ATPase mRNA levels, higher IF1 mRNA levels, and impaired expression of 3 of the 5 critical proteins involved in the OXPHOS complex, compared with control mGCs. Cell-free mitochondrial DNA (cfmtDNA) levels are higher in the follicular fluid of patients with OEC and were inversely associated with the expression of mtDNA in mGCs and cumulus granulosa cells (cGCs).Taken together, this study indicates that small non-surgical OECs lead to poor quality of oocytes and subsequent embryos during ART compared with control, which was accompanied by mGC mitochondrial dysfunction. mGC and cGC mtDNA and FF cfmtDNA might serve as efficient biomarkers for the non-invasive prediction of pregnancy outcomes in patients with OEC undergoing ART.

Calpain cleaves phospholipid flippase ATP8A1 during apoptosis in platelets.

The asymmetric distribution of phospholipids in the plasma/organellar membranes is generated and maintained through phospholipid flippases in resting cells, but becomes disrupted in apoptotic cells and activated platelets, resulting in phosphatidylserine (PS) exposure on the cell surface. Stable PS exposure during apoptosis requires inactivation of flippases to prevent PS from being reinternalized. Here we show that flippase ATP8A1 is highly expressed in both murine and human platelets, but is not present in the plasma membrane. ATP8A1 is cleaved by the cysteine protease calpain during apoptosis, and the cleavage is prevented indirectly by caspase inhibition, involving blockage of calcium influx into platelets and subsequent calpain activation. In contrast, in platelets activated with thrombin and collagen and exposing PS, ATP8A1 remains intact. These data reveal a novel mechanism of flippase cleavage and suggest that flippase activity in intracellular membranes differs between platelets undergoing apoptosis and activation.

Association between activities of SOD, MDA and Na+-K+-ATPase in peripheral blood of patients with acute myocardial infarction and the complication of varying degrees of arrhythmia.

OBJECTIVE: To investigate the changes of ambulatory electrocardiography and peripheral blood SOD, MDA and Na+-K+-ATP enzymes in patients of acute myocardial infarction (AMI) complicated with arrhythmia. METHODS: From January 2012 to March 2015, 135 cases AMI complicated with arrhythmia in our hospital were divided into 2 groups: 70 cases in the AMI uncomplicated with arrhythmia and 65 cases in the AMI complicated with arrhythmia. 62 cases volunteers accepted physical examination in our hospital in the same period were collected as the control group. 24 hour-electrocardiogram detected by ambulatory electrocardiogram (AECG), SOD and MDA in peripheral blood detected by diagnostic reagent kit and Na+-K+-ATP enzymes in peripheral blood detected by malachite green Kit Method phosphate determination method were collected. ROC curve was used to evaluate the prognostic value of SOD, MDA and Na+-K+-ATP enzymes in AMI patients. RESULTS: Compared with the control group, the patients had unusual ambulatory electrocardiography had increased (P <0.05), peripheral blood SOD and Na+-K+-ATP enzymes had decreased, peripheral blood MDA had increased in 2 AMI groups (P <0.05). Compared with AMI uncomplicated with arrhythmia group, the patients had unusual ambulatory electrocardiography had increased (P <0.05), peripheral blood SOD and Na+-K+-ATP enzymes had decreased, peripheral blood MDA had increased in AMI complicated with arrhythmia group (P <0.05). Among 135 cases AMI patients, 120 (88.9%) survived and 15 (11.1%) died, of whom 11 cases were AMI complicated with arrhythmia group, 4 cases were AMI uncomplicated with arrhythmia group. Compared with the AMI uncomplicated with arrhythmia group, the dead patients were more in the AMI complicated with arrhythmia group (c2 = 4.287, P = 0.038). Compared with the survival group, the SOD and Na+-K+-ATP enzymes were significantly lower (P <0.05) and MDA significantly higher (P <0.05) in the death group. The area under the ROC curve of SOD, MDA and Na+-K+-ATP enzymes were 0.958, 0.954 and 0.993 respectively, and the cut-off values were 30.66 ng/ml, 576.70 nmol/ml and 57.42 nmol/mgh, respectively. CONCLUSION: Ambulatory electrocardiography has a close relationship with the peripheral blood SOD, MDA and Na+-K+-ATP enzymes in AMI patients complicated with arrhythmia, which might predict AMI condition.

Involvement of circular RNA SMARCA5/microRNA-620 axis in the regulation of cervical cancer cell proliferation, invasion and migration.

OBJECTIVE: Circular RNAs (circRNAs) have emerged as crucial regulators for tumor progression. However, the effects of circRNAs are not entirely clear in cervical cancer (CC). The objective of this study was to investigate the function and regulation mechanism of circular RNA SMARCA5 (circSMARCA5, also named hsa_circ_0001445) in cervical cancer. PATIENTS AND METHODS: circSMARCA5 and miR-620 expressions were analyzed by qRT-PCR assay. The cell proliferation, cell cycle, migration, invasion abilities were measured by CCK-8 and colony formation, Flow cytometry, Wound-healing and transwell assays. In addition, the interaction between circSMARCA5 and miR-620 was accessed by luciferase reporter assay. RESULTS: The results indicated that circSMARCA5 was downregulated in CC; overexpression of circSMARCA5 inhibited CC cell proliferation, migration and invasion, and induced cell cycle arrest. In addition, we found that circSMARCA5 could bind to miR-620 and significantly downregulated its expression. Furthermore, the results revealed that circSMARCA5 suppressed proliferation and invasion by miR-620 in CC. CONCLUSIONS: We suggested that circSMARCA5/miR-620 regulatory axis is involved in the development of CC and may serve as a potential therapy target.

Evaluation of the accuracy of exchangeable copper and relative exchangeable copper (REC) in a mouse model of Wilson's disease.

Wilson's disease (WD) is caused by mutations in the ATP7B gene responsible for a toxic copper overload mainly in the liver and the central nervous system. Phenotypic heterogeneity may challenge the diagnostic confirmation. Exchangeable copper (CuEXC) has recently been proposed as a new marker of WD, and its ratio to the total serum copper (Cus), Relative Exchangeable Copper (REC = CuEXC/Cus), as a diagnostic marker. This study aimed to investigate whether this could be confirmed in Atp7b-/- mice, an engineered WD animal model. Atp7b-/- (n = 137) and wild type (WT; n = 101) mice were investigated under the same conditions at 6-8, 20, 39, or 50 weeks of age. Twenty-four Atp7b-/- mice received D-penicillamine treatment from 39 to 50 weeks of age. Serum and liver [histology and intrahepatic copper (IHCu)] data were evaluated. In the WT group, all serum and liver data were normal. Atp7b-/- livers developed a chronic injury from isolated moderate inflammation (6-8 weeks: 16/33 = 48%) to inflammatory fibrosis with cirrhosis (50 weeks: 25/25 = 100% and 16/25 = 64% respectively). Cus and CuEXC increased until week 39, whereas IHCu and REC were stable with increasing age and much higher than in WT mice (mean ±â€¯SD: 669 ±â€¯269 vs. 13 ±â€¯3 µg/g dry liver and 39 ±â€¯12 vs. 11 ±â€¯3%, respectively). A threshold value of 20% for REC provided a diagnostic sensitivity and specificity of 100%, regardless of sex, age, or the use of D-penicillamine. Eleven weeks of 100 mg/kg D-penicillamine reduced liver fibrosis (p = 0.001), IHCu (p = 0.026) and CuEXC (p = 0.175). In conclusion, this study confirms REC as a WD diagnostic marker in a mouse model of chronic liver disease caused by copper overload. Further studies are needed to assess the usefulness of CuEXC to monitor the evolution of WD, particularly during treatment.

[Effects of soybean isoflavones on the energy metabolism of swimming mice].

OBJECTIVE: To establish an animal model for loaded swimming, so as to investigate the energy metabolism effects of soybean isoflavones (SI) on swimming mice. METHODS: Thirty male Kunming mice were randomly divided into three groups:normal control, swimming group, and swimming+SI group. The normal control group mice were fed a basic AIN-93M diet, the SI groups were supplied with soybean isoflavones(4 g/kg).Two weeks later, the mice were forced to swim for an hour,and then all the mice were killed, the samples of blood, liver and muscles of hind were collected.The serum contents of lactic acid(Lac), the activities of lactic dehydrogenase (LDH), succinate dehydrogenase (SDH), creatine kinase (CK) and ATPase were measured. RESULTS: Compared with normal control,the serum content of Lac was significantly improved in the group of the swimming control and SI(P<0.05),the activity of LDH in the serum was obviously improved in the group of the swimming control and SI, and the activity of CK and SDH were both significantly improved in the group of the swimming control and SI except the activity of SDH in the liver of the group SI; compared with the swimming control,the serum contents of Lac,the activities of LDH, ATPase, SDH, CK were obviously improved(P<0.05). CONCLUSIONS: Soybean isoflavones can improve the energy metabolism,antioxidant capacity of the swimming mice.

Creatine kinase and ATPase activities in piglets fed a fungal mycotoxin co-contaminated diet: Consequences in the pathogenesis of subclinical intoxication.

Creatine kinase (CK) activity, through the creatine-kinase-phosphocreatine (CK/PCr) system, provides a temporal and spatial energy buffer to maintain cellular energetic homeostasis, being responsible to provide adenosine triphosphate (ATP) to the proper function of ATPases enzymes, such as the sodium-potassium (Na+, K+-ATPase) and hydrogen (H+-ATPase) pumps. Thus, the aim of this study was to evaluate the involvement of CK/PCr system in the impairment of energetic homeostasis in piglets fed with a diet co-contaminated with mycotoxins, as well as the effects on ATPases enzymes. Animals were randomly divided in two groups (eight repetitions with two animals each): CON (basal diet) and MYC (mycotoxin diet; 9300 µg/kg of aflatoxins and 8000 µg/kg of fumonisins) which were feed during 15 days. Piglets that received a diet containing 300 µg/kg of aflatoxins and 8000 µg/kg of fumonisins (MYC group) presented lower body weight on days 10 and 15 of experiment when compared to control (CON group). Serum CK activity was lower on days 5, 10 and 15 of experiment in the MYC group. The same occurred for serum Na+, K+-ATPase and H+-ATPase activities on days 10 and 15 when compared to CON group. Moreover, serum calcium levels were superior on day 15 of experiment in the MYC group, while serum potassium and sodium levels were lower in comparison to CON group. Based on these evidences, a diet co-contaminated by aflatoxins and fumonisins inhibits serum CK activity, impairing the energetic homeostasis. This inhibition alters the activities of ATPases (Na+, K+-ATPase and H+-ATPase), contributing to the imbalance of Na+, K+ and Ca+ ionic levels. In summary, the cascade of alterations contributes directly to disease pathogenesis of piglets intoxicated by mycotoxins.

The use of Escherichia coli total antigens as a complementary approach to address seropositivity to Leishmania antigens in canine leishmaniosis.

Canine leishmaniosis (CanL) is a major veterinary concern and a public health issue. Serological data are essential for disease management. Several antigens used in serological assays have specificity related problems preventing relevant seropositivity values establishment. Herein we report significant seropositivity level disparity in a study cohort with 384 dogs from eight countries, for antigens traditionally used in CanL - soluble promastigote Leishmania antigens (SPLA) and K39 recombinant protein (rK39): 43·8 and 2·9% for SPLA and rK39, respectively. To better understand the reasons for this disparity, CanL-associated serological response was characterized using, for complement serological evaluation, a ubiquitous antigen - soluble Escherichia coli antigens (SECAs). Using cohorts of CanL dogs and dogs without clinical evidences of CanL from non-endemic regions of Portugal, the serological response of CanL animals followed specific trend of seropositivity rK39 > SPLA > SECA absent in non-diseased animals. Using receiver operating characteristic curve analysis, these characteristic trends were converted in ratios, SPLA/SECA, rK39/SECA and rK39/SPLA, that presented high predictive for discriminating the CanL cohort that was potentiated when applied in a scoring system involving positivity to four out of five predictors (rK39, SPLA, SPLA/SECA, rK39/SECA and rK39/SPLA). In fact, this approach discriminated CanL with similar sensitivity/specificity as reference antigens, diminishing seropositivity in European cohort to 1·8%. Ultimately, non-related antigens like SECA and seropositivity ratios between antigens enable different perspectives into serological data focusing on the search of characteristic serological signatures and not simple absolute serology values contributing to comprehensive serological status characterization.

A predictive model for lack of partial clinical remission in new-onset pediatric type 1 diabetes.

IMPORTANCE: >50% of patients with new-onset type 1 diabetes (T1D) do not enter partial clinical remission (PCR); early identification of these patients may improve initial glycemic control and reduce long-term complications. AIM: To determine whether routinely obtainable clinical parameters predict non-remission in children and adolescents with new-onset T1D. SUBJECTS AND METHODS: Data on remission were collected for the first 36 months of disease in 204 subjects of ages 2-14 years with new-onset type 1 diabetes. There were 86 remitters (age 9.1±3.0y; male 57%), and 118 non-remitters (age 7.0±3.1y; male 40.7%). PCR was defined as insulin-dose adjusted hemoglobin A1c of ≤9. RESULTS: Non-remission occurred in 57.8% of subjects. Univariable analysis showed that the risk for non-remission was increased 9-fold in patients with 4 diabetes-associated auto-antibodies (OR = 9.90, p = 0.010); 5-fold in patients <5 years old (odds ratio = 5.38, p = 0.032), 3-fold in those with bicarbonate of <15 mg/dL at diagnosis (OR = 3.71, p = 0.008). Combined estimates of risk potential for HC03 and the number of autoantibodies by multivariable analysis, adjusted for BMI standard deviation score, showed HC03 <15 mg/dL with a clinically significant 10-fold risk (OR = 10.1, p = 0.074); and the number of autoantibodies with a 2-fold risk for non-remission (OR = 1.9, p = 0.105). Male sex and older age were associated with decreased risk for non-remission. A receiver-operating characteristic curve model depicting sensitivity by 1-specificity for non-remission as predicted by bicarbonate <15 mg/dL, age <5y, female sex, and >3 diabetes-associated autoantibodies had an area under the curve of 0.73. CONCLUSIONS: More than 50% of children and adolescents with new-onset T1D do not undergo partial clinical remission and are thus at an increased risk for long-term complications of diabetes mellitus. A predictive model comprising of bicarbonate <15 mg/dL, age <5y, female sex, and >3 diabetes-associated autoantibodies has 73% power for correctly predicting non-remission in children and adolescents with new-onset T1D. Early identification of these non-remitters may guide the institution of targeted therapy to limit dysglycemia and reduce the prevalence of long-term deleterious complications.

Antinuclear Matrix Protein 2 Autoantibodies and Edema, Muscle Disease, and Malignancy Risk in Dermatomyositis Patients.

OBJECTIVE: Dermatomyositis (DM) patients typically present with proximal weakness and autoantibodies that are associated with distinct clinical phenotypes. We observed that DM patients with autoantibodies recognizing the nuclear matrix protein NXP-2 often presented with especially severe weakness. The aim of this study was to characterize the clinical features associated with anti-NXP-2 autoantibodies. METHODS: There were 235 DM patients who underwent testing for anti-NXP-2 autoantibodies. Patient characteristics, including muscle strength, were compared between those with and without these autoantibodies. The number of cancer cases observed in anti-NXP-2-positive subjects was compared with the number expected in the general population. RESULTS: Of the DM patients, 56 (23.8%) were anti-NXP-2-positive. There was no significant difference in the prevalence of proximal extremity weakness in patients with and without anti-NXP-2. In contrast, anti-NXP-2-positive patients had more prevalent weakness in the distal arms (35% versus 20%; P = 0.02), distal legs (25% versus 8%; P < 0.001), and neck (48% versus 23%; P < 0.001). Anti-NXP-2-positive subjects were also more likely to have dysphagia (62% versus 35%; P < 0.001), myalgia (46% versus 25%; P = 0.002), calcinosis (30% versus 17%; P = 0.02), and subcutaneous edema (36% versus 19%; P = 0.01) than anti-NXP-2-negative patients. Five anti-NXP-2-positive subjects (9%) had cancer-associated myositis, representing a 3.68-fold increased risk (95% confidence interval 1.2-8.6) compared to the expected prevalence in the general population. CONCLUSION: In DM, anti-NXP-2 autoantibodies are associated with subcutaneous edema, calcinosis, and a muscle phenotype characterized by myalgia, proximal and distal weakness, and dysphagia. As anti-NXP-2-positive patients have an increased risk of cancer, we suggest that they undergo comprehensive cancer screening.

Changes in erythrocyte ATPase activity under different pathological conditions.

BACKGROUND: Studies have shown that Na+-K+ ATPase activity was altered in disrupted red blood cell membranes and this enzyme is believed to be the site of active transport of Na+ and K+ in intact red blood cells. The enzyme is often referred to as Na+-K+ pump because it pumps Na+ out and K+ into the cell against gradients with the concomitant hydrolysis of intracellular ATP. OBJECTIVE: The aim of this study was to find out the possibility of using Na+-K+-ATPase activity as a biomarker for the diagnosis of individuals with different physiological conditions. MATERIALS AND METHODS: The activity of Na+-K+ ATPase was determined in blood samples collected from different pathological and physiological conditions such as pregnancy, smoking, diabetes and renal dysfunction compared with healthy subjects matched for age and sex. RESULTS: The Na+-K+ ATPase activity in pregnancy (0.094 ± 0.0051 µM Pi/min. mg protein), smoking (0.064 ± 0.0011 µM), diabetes (0.047 µM 0.002 µM) and kidney disease (0.069 ± 0.0014 µM) was higher compared to the measurements in healthy individuals (0.0081 ± 0.0031 µM). CONCLUSION: Na+-K+ATPase specific activity is a biomarker for the diagnosis of individuals with different physiological diseases.

Quantification of ATP7B Protein in Dried Blood Spots by Peptide Immuno-SRM as a Potential Screen for Wilson's Disease.

Wilson's Disease (WD), a copper transport disorder caused by a genetic defect in the ATP7B gene, has been a long time strong candidate for newborn screening (NBS), since early interventions can give better results by preventing irreversible neurological disability or liver cirrhosis. Several previous pilot studies measuring ceruloplasmin (CP) in infants or children showed that this marker alone was insufficient to meet the universal screening for WD. WD results from mutations that cause absent or markedly diminished levels of ATP7B. Therefore, ATP7B could serve as a marker for the screening of WD, if the protein can be detected from dried blood spots (DBS). This study demonstrates that the immuno-SRM platform can quantify ATP7B in DBS in the picomolar range, and that the assay readily distinguishes affected cases from normal controls (p < 0.0001). The assay precision was <10% CV, and the protein was stable for a week in DBS at room temperature. These promising proof-of-concept data open up the possibility of screening WD in newborns and the potential for a multiplexed assay for screening a variety of congenital disorders using proteins as biomarkers in DBS.

Clinical presentations of Wilson disease among Polish children.

INTRODUCTION: Wilson disease (WD) may present from early childhood up to the eighth decade, presenting with variable hepatic and neuropsychiatric symptoms. Establishing the diagnosis is straightforward if the major clinical and laboratory features are present. However, clinical phenotypes are highly varied and early, proper diagnosis can be challenging. AIM: The aim of our study was to analyze clinical presentations and diagnostic tests of Polish pediatric patients with WD. METHODS: We retrospectively analyzed medical history of 156 patients with confirmed diagnosis of WD treated at our Institute from 1996 till March 2016. RESULTS: The mean age at onset of symptoms was 10.15±4.23 years of age. Hepatic presentation was the most common one (94.23%) with either liver failure (16.03%) or more frequently increased transaminases (78.2%). In 90.26% cases ceruloplasmin serum concentration was ≤0,2 g/l, in 51.93% patients basal urinary copper excretion was >100 µg/24 h. Mutation analysis was performed in 155 (99.36%) cases. The most common mutation was p.H1069Q. CONCLUSIONS: Wilson disease can present with only significantly increased transaminases activity and hepatomegaly or liver failure, but neurological symptoms are very rare in children. Diagnostic approach is challenging due to wide spectrum of clinical presentations in a high variable degree of severity. Genetic screening is supportive, ceruloplasmin and urinary copper excretion are valuable tests in the majority of patients but do not allow to exclude WD.

The defining DNA methylation signature of Floating-Harbor Syndrome.

Floating-Harbor syndrome (FHS) is an autosomal dominant genetic condition characterized by short stature, delayed osseous maturation, expressive language impairment, and unique facial dysmorphology. We previously identified mutations in the chromatin remodeling protein SRCAP (SNF2-related CBP Activator Protein) as the cause of FHS. SRCAP has multiple roles in chromatin and transcriptional regulation; however, specific epigenetic consequences of SRCAP mutations remain to be described. Using high resolution genome-wide DNA methylation analysis, we identified a unique and highly specific DNA methylation "epi-signature" in the peripheral blood of individuals with FHS. Both hyper and hypomethylated loci are distributed across the genome, preferentially occurring in CpG islands. Clonal bisulfite sequencing of two hypermethylated (FIGN and STPG2) and two hypomethylated (MYO1F and RASIP1) genes confirmed these findings. The identification of a unique methylation signature in FHS provides further insight into the biological function of SRCAP and provides a unique biomarker for this disorder.

Serum Autoantibodies in Chronic Prostate Inflammation in Prostate Cancer Patients.

BACKGROUND: Chronic inflammation is frequently observed on histological analysis of malignant and non-malignant prostate specimens. It is a suspected supporting factor for prostate diseases and their progression and a main cause of false positive PSA tests in cancer screening. We hypothesized that inflammation induces autoantibodies, which may be useful biomarkers. We aimed to identify and validate prostate inflammation associated serum autoantibodies in prostate cancer patients and evaluate the expression of corresponding autoantigens. METHODS: Radical prostatectomy specimens of prostate cancer patients (N = 70) were classified into high and low inflammation groups according to the amount of tissue infiltrating lymphocytes. The corresponding pre-surgery blood serum samples were scrutinized for autoantibodies using a low-density protein array. Selected autoantigens were identified in prostate tissue and their expression pattern analyzed by immunohistochemistry and qPCR. The identified autoantibody profile was cross-checked in an independent sample set (N = 63) using the Luminex-bead protein array technology. RESULTS: Protein array screening identified 165 autoantibodies differentially abundant in the serum of high compared to low inflammation patients. The expression pattern of three corresponding antigens were established in benign and cancer tissue by immunohistochemistry and qPCR: SPAST (Spastin), STX18 (Syntaxin 18) and SPOP (speckle-type POZ protein). Of these, SPAST was significantly increased in prostate tissue with high inflammation. All three autoantigens were differentially expressed in primary and/or castration resistant prostate tumors when analyzed in an inflammation-independent tissue microarray. Cross-validation of the inflammation autoantibody profile on an independent sample set using a Luminex-bead protein array, retrieved 51 of the significantly discriminating autoantibodies. Three autoantibodies were significantly upregulated in both screens, MUT, RAB11B and CSRP2 (p>0.05), two, SPOP and ZNF671, close to statistical significance (p = 0.051 and 0.076). CONCLUSIONS: We provide evidence of an inflammation-specific autoantibody profile and confirm the expression of corresponding autoantigens in prostate tissue. This supports evaluation of autoantibodies as non-invasive markers for prostate inflammation.

ATP7B Gene Mutations in Croatian Patients with Wilson Disease.

AIMS: Wilson disease (WD) is an autosomal recessive disorder of copper metabolism, characterized by its accumulation in tissues which results in hepatic, neurological, and/or psychiatric symptoms. The aim of this study was to investigate the genetics of WD in Croatian patients. METHODS: Correlation of the clinical presentation subtype and the age at onset of the diagnosis of WD with the ATP7B genotype was investigated in a group of Croatian WD patients. DNA from peripheral blood samples was tested for the p.His1069Gln by direct mutational analysis and other polymorphisms were identified by sequence analysis of coding and flanking intronic regions of ATP7B gene. RESULTS: In the group of 75 WD patients of Croatian origin, 18 different mutations in ATP7B gene were detected, three of which were novel. The p.His1069Gln mutation was most frequent, being detected in 44 Croatian WD patients (58.7%). Most ATP7B mutations (90.4%) were located in exons 5, 8, 13, 14, and 15. CONCLUSIONS: Clinical diagnosis of WD was confirmed in 59 patients by detecting mutations on both ATP7B alleles. The age at onset of WD and the type of WD clinical presentation showed no significant correlation with the ATP7B genotype.