KCNA2 IgG autoimmunity in neuropsychiatric diseases.

BACKGROUND: Autoantibodies against the potassium voltage-gated channel subfamily A member 2 (KCNA2) have been described in a few cases of neuropsychiatric disorders, but their diagnostic and pathophysiological role is currently unknown, imposing challenges to medical practice. DESIGN / METHODS: We retrospectively collected comprehensive clinical and paraclinical data of 35 patients with KCNA2 IgG autoantibodies detected in cell-based and tissue-based assays. Patients' sera and cerebrospinal fluid (CSF) were used for characterization of the antigen, clinical-serological correlations, and determination of IgG subclasses. RESULTS: KCNA2 autoantibody-positive patients (n = 35, median age at disease onset of 65 years, range of 16-83 years, 74 % male) mostly presented with cognitive impairment and/or epileptic seizures but also ataxia, gait disorder and personality changes. Serum autoantibodies belonged to IgG3 and IgG1 subclasses and titers ranged from 1:32 to 1:10,000. KCNA2 IgG was found in the CSF of 8/21 (38 %) patients and in the serum of 4/96 (4.2 %) healthy blood donors. KCNA2 autoantibodies bound to characteristic anatomical areas in the cerebellum and hippocampus of mammalian brain and juxtaparanodal regions of peripheral nerves but reacted exclusively with intracellular epitopes. A subset of four KCNA2 autoantibody-positive patients responded markedly to immunotherapy alongside with conversion to seronegativity, in particular those presenting an autoimmune encephalitis phenotype and receiving early immunotherapy. An available brain biopsy showed strong immune cell invasion. KCNA2 autoantibodies occurred in less than 10 % in association with an underlying tumor. CONCLUSION: Our data suggest that KCNA2 autoimmunity is clinically heterogeneous. Future studies should determine whether KCNA2 autoantibodies are directly pathogenic or develop secondarily. Early immunotherapy should be considered, in particular if autoantibodies occur in CSF or if clinical or diagnostic findings suggest ongoing inflammation. Suspicious clinical phenotypes include autoimmune encephalitis, atypical dementia, new-onset epilepsy and unexplained epileptic seizures.

The Association of Severe Coronary Tortuosity and Non-Obstructive Coronary Artery Disease.

Background and Objectives: There is an increasing interest in the coronary tortuosity as a novel pathophysiological mechanism of ischemia in coronary artery disease without significant obstruction, but there are a lack of studies to confirm this relationship in the clinical setting. The aim of our study was to evaluate the association of severe coronary tortuosity and the potential role of coronary blood supply dominance in the appearance of myocardial ischemia in patients with non-obstructive coronary artery disease (non-CAD), compared to patients with obstructive coronary artery disease (CAD). Materials and Methods: The study enrolled 131 participants (71 male and 60 female), recruited among patients referred to cardiologists due to angina symptoms with ischemic alterations established by cardiac stress tests, as well as those admitted to the hospital for acute coronary syndrome. Results: Mean age of recruited patients was 61.6 (±10.1) years. According to the coronary angiography, they were divided into two groups: non-obstructive and obstructive CAD (77 and 54, respectively). There were significantly more women (61% vs. 24%, p < 0.001) in the non-CAD group. Both tortuous coronary arteries (50.6% vs. 14.8%, p < 0.001) and left coronary dominance (37.7% vs. 16.7%, p = 0.006) were more frequent in the non-CAD group compared to the CAD group. Female sex (OR = 17.516, p = 0.001), tortuous coronary arteries (OR = 7.962, p = 0.006) and left dominance of blood supply were significant predictors for non-CAD. Conclusions: Non-obstructive CAD is common among patients, especially women, who are referred for coronary angiography. Severe coronary artery tortuosity is the strongest independent predictor of non-obstructive CAD, followed by female gender and left coronary dominance.

THE PREVALENCE OF SUBCLINICAL HYPOTHYROIDISM IN THE POPULATION OF ELDERLY NURSING HOME RESIDENTS IN ZAGREB.

The aim of the study was to investigate the prevalence of thyroid dysfunction, positive thyroid peroxidase antibodies (TPOAb) and hypercholesterolemia in elderly and younger subjects, and the association of subclinical hypothyroidism with hypercholesterolemia. The study included 204 elderly (136 females and 68 males, age median 71, range 60-92 years), and 83 younger control subjects (63 females and 20 males, age median 45, range 19-55 years). Subjects with prior thyroid dysfunction were excluded. Serum thyrotropin (TSH), free triiodothyronine (FT3), free thyroxine (FT4), TPOAb, total cholesterol, height and weight were measured. Mann-Whitney, χ2-test and Student's t-test were used on statistical analysis. The prevalence of subclinical hypothyroidism (TSH >5 mU/L) in elderly was 7.4% vs. 3.6% in younger subjects, with the highest prevalence of 8.8% in elderly women vs. 4.8% in younger women, and 4.4% in elderly men. The prevalence of hypothyroidism and subclinical hyperthyroidism in elderly subjects was 0.5% and 1.5%, respectively. In women with subclinical hypothyroidism, the prevalence of TPOAb was 77% in elderly women and 67% in younger women (overall 19.9% in elderly and 14.3% in younger women). The mean FT3 level was lower in elderly women as compared with elderly men (p<0.01) and younger women (p<0.05). The mean cholesterol level was higher in elderly subjects in comparison with younger ones (p<0.01), and in elderly women vs. elderly men (p<0.01), but without difference between subclinical hypothyroidism and euthyroid subjects (6.0 mmol/L). In conclusion, subclinical hypothyroidism is the most prevalent thyroid dysfunction in elderly, with the highest prevalence in elderly women, and autoimmune thyroiditis is the most common etiology. Hypercholesterolemia was more related to older age, especially elderly females, but not influenced by subclinical hypothyroidism.

Thyroglobulin Measurement in Needle Aspiration for Detection of Recurrences and Neck Metastases in Patients with Differentiated Thyroid Carcinoma: Significance of Anti-Tg Antibodies.

The worldwide incidence of differentiated thyroid cancer (DTC) has increased in recent decades, likely due to frequent use of cervical ultrasonography (US) and US-guided fine needle aspiration biopsy (FNA)., US is performed during follow-up after thyroidectomy, and US-guided FNA with cytology is used if suspicious cervical lymph nodes (LN) or thyroid bed masses are detected. Knowing that serum anti-Tg antibodies (sTgAb) affect the use of serum Tg (sTg) as a tumor marker, the aim of our study was to assess the usefulness of Tg determination in needle aspirates (FNA-Tg) in presence of sTgAb. This retrospective study included 149 patients with DTC and 159 aspirations of suspicious LN and thyroid bed masses. As expected, there was a negative correlation between sTg and sTgAb levels (p<0.05), while FNA-Tg levels had a positive correlation with FNA-TgAb levels (p<0.05). Furthermore, we found a positive correlation between sTg and FNA-Tg levels (p<0.05), but not between sTgAb and FNA-TgAb or sTgAb and FNA-Tg. In conclusion, these results show that FNA-Tg values were not affected by sTgAb and that FNA-Tg measurement were highly effective in detecting cervical DTC metastases. However, combined use with cytology is suggested for neck evaluation because cytology could reveal metastases from other tumor sites.

LIFESTYLE RISK FACTORS AND BONE MASS IN RECURRENT STONE-FORMING PATIENTS: A CROSS-SECTIONAL STUDY IN 144 SUBJECTS.

Patients with urolithiasis, particularly hypercalciuria, may have reduced bone mineral density (BMD). There are numerous risk factors contributing to reduction of BMD such as advanced age, sedentary lifestyle, smoking, low calcium intake, etc. The aim of our study was to investigate the association of lifestyle risk factors and daily intake of milk and dairy products with determinants of BMD in a group of recurrent calcium stone formers (RSF) compared with healthy subjects (HS). The study was carried out at the Department of Mineral Research, Faculty of Medicine in Osijek, Croatia. The study included 144 subjects, i.e. 56 RSF and 78 HS. BMD was assessed by dual-energy x-ray absorptiometry. A standard self-reported questionnaire was used to collect data on lifestyle risk factors. Current dietary intake was assessed by personal interview that included questions about milk and dairy product intake. Low BMD was observed in 44.64% of RSF and 35.90% of HS. RSF consumed significantly less milk and dairy products than HS. Calcium restriction in dietary recommendations might be unnecessary due to the impact on bone mineral loss in RSF and dual-energy x-ray absorptiometry should be included in the routine evaluation of RSF.

NONOBSTRUCTIVE CORONARY ARTERY DISEASE - CLINICAL RELEVANCE, DIAGNOSIS, MANAGEMENT AND PROPOSAL OF NEW PATHOPHYSIOLOGICAL CLASSIFICATION.

- New data gathered from large clinical trials indicate that nonobstructive coronary artery disease (non-CAD) is a clinical entity that should not be ignored. It is estimated that 50% of female population undergoing coronarography are diagnosed with non-CAD. There is also an increase in the prevalence of non-CAD in both genders, which is probably due to gradual expanding of clinical indications for angiography in patients with angina. Furthermore, considering the increased mortality risk established recently, a prognosis of non-CAD is not benign as previously thought. However, the concept and definition of non-CAD remains elusive causing difficulties in diagnosis and treatment. One of the major shortcomings is the exclusion-based diagnosis of non-CAD. Furthermore, treatment of non-CAD still presents a great challenge and optimal therapy is yet to be determined. There are two major hypotheses explaining the pathophysiological mechanisms of non-CAD, i.e. ischemic hypothesis based on abnormal microvascular dysfunction and non-ischemic one based on altered pain perception. This review encompasses a broader spectrum of pathophysiological mechanisms of non-CAD, and proposes a new way of classification based on the major disorder involved: type I (ischemic mechanisms) and type II (non-ischemic mechanisms), depending on which mechanism predominates. Hopefully, this would provide new insights in the understanding of this disorder, thus leading to accurate and early diagnosis and successful treatment, especially considering the increased mortality risk in these patients.

Bone Mineral Density in Relation to Metabolic Syndrome Components in Postmenopausal Women With Diabetes Mellitus Type 2

Diabetes mellitus type 2 is associated with greater bone mineral density (BMD) due to obesity, although rapid bone loss observed over time could be explained by elevated chronic inflammation. The objective of this study was to investigate the relationship between central adiposity and hyperinsulinemia, as well as inflammation markers with vertebral and femoral BMD and bone turnover markers in postmenopausal women with type 2 diabetes. Femoral and vertebral BMD, osteocalcin, pyrilinks D, beta-CrossLaps (B-CTx), insulin, C-reactive protein (CRP), fibrinogen and plasminogen activator inhibitor-1 (PAI-1) were measured in 114 postmenopausal female patients with diabetes type 2. The patients of similar age, HbA1c levels and diabetes duration were divided into 2 groups based on their body mass index (BMI) values: lower or equal to 27 kg/m(2) (31 patients) and higher than 27 kg/m(2) (83 patients). Lower levels of osteocalcin (p=0.001), B-CTx (p=0.000007) and pyrilinks D (p=0.0365), and higher femoral BMD (p=0.00006), insulin level (p=0.0002), PAI-1 (p=0.00000) and CRP (p=0.002) were found in the overweight group. There were no signifi cant differences in vertebral BMD and fibrinogen. Osteocalcin and B-CTx showed inverse correlation, and femoral BMD positive correlation with waist circumference, insulin level and PAI-1. This suggests that abdominal obesity and hyperinsulinemia as components of the metabolic syndrome could increase femoral BMD by lowering bone rate. In addition, the only inflammation marker linked with femoral BMD was PAI-1, which is associated with increased mineralization of cortical bone in mouse.

Phylogenetic, syntenic, and tissue expression analysis of slc22 genes in zebrafish (Danio rerio).

BACKGROUND: SLC22 protein family is a member of the SLC (Solute carriers) superfamily of polyspecific membrane transporters responsible for uptake of a wide range of organic anions and cations, including numerous endo- and xenobiotics. Due to the lack of knowledge on zebrafish Slc22 family, we performed initial characterization of these transporters using a detailed phylogenetic and conserved synteny analysis followed by the tissue specific expression profiling of slc22 transcripts. RESULTS: We identified 20 zebrafish slc22 genes which are organized in the same functional subgroups as human SLC22 members. Orthologies and syntenic relations between zebrafish and other vertebrates revealed consequences of the teleost-specific whole genome duplication as shown through one-to-many orthologies for certain zebrafish slc22 genes. Tissue expression profiles of slc22 transcripts were analyzed using qRT-PCR determinations in nine zebrafish tissues: liver, kidney, intestine, gills, brain, skeletal muscle, eye, heart, and gonads. Our analysis revealed high expression of oct1 in kidney, especially in females, followed by oat3 and oat2c in females, oat2e in males and orctl4 in females. oct1 was also dominant in male liver. oat2d showed the highest expression in intestine with less noticeable gender differences. All slc22 genes showed low expression in gills, and moderate expression in heart and skeletal muscle. Dominant genes in brain were oat1 in females and oct1 in males, while the highest gender differences were determined in gonads, with dominant expression of almost all slc22 genes in testes and the highest expression of oat2a. CONCLUSIONS: Our study offers the first insight into the orthology relationships, gene expression and potential role of Slc22 membrane transporters in zebrafish. Clear orthological relationships of zebrafish slc22 and other vertebrate slc22 genes were established. slc22 members are mostly highly conserved, suggesting their physiological and toxicological importance. One-to-many orthologies and differences in tissue expression patterns of zebrafish slc22 genes in comparison to human orthologs were observed. Our expression data point to partial similarity of zebrafish versus human Slc22 members, with possible compensatory roles of certain zebrafish transporters, whereas higher number of some orthologs implies potentially more diverse and specific roles of these proteins in zebrafish.

Histopathological Changes in Tracheal Mucosa Following Total Laryngectomy.

The aim of this study was to determine the long term histopathologic changes in tracheal mucosa after a total laryngectomy , and to find out the relationship between the progression of histopathologic changes in tracheal mucosa and the duration of breathing through the tracheostomy. Tracheal mucosal biopsies were taken from a total of 35 patients, of both sexes, who underwent a total laryngectomy for laryngeal carcinoma at least one year prior. Histologic specimens of tracheal mucosa were stained with hematoxylin and eosin and examined under light microscopy. Almost all of the patients demonstrated histopathologic changes or abnormalities. Based on the results, histological findings were grouped into seven categories: normal respiratory epithelium, mild, moderate and advanced basal cell hyperplasia, squamous metaplasia, and slight and moderate and dysplasia. The time elapsed since surgery was calculated for each histopathological change separately. In laryngeal carcinoma patients, after a total laryngectomy histopathologic changes occur in tracheal mucosa. The mildest histopathological changes are found in the patients who had a longer period between the operation and the examination.

The role of cyclo-oxygenase-1 in high-salt diet-induced microvascular dysfunction in humans.

KEY POINTS: Recent studies have shown that some of the deleterious effects of a high-salt (HS) diet are independent of elevated blood pressure and are associated with impaired endothelial function. Increased generation of cyclo-oxygenase (COX-1 and COX-2)-derived vasoconstrictor factors and endothelial activation may contribute to impaired vascular relaxation during HS loading. The present study aimed to assess the regulation of microvascular reactivity and to clarify the role of COX-1 and COX-2 in normotensive subjects on a short-term HS diet. The present study demonstrates the important role of COX-1 derived vasoconstrictor metabolites in regulation of microvascular blood flow during a HS diet. These results help to explain how even short-term HS diets may impact upon microvascular reactivity without changes in blood pressure and suggest that a vasoconstrictor metabolite of COX-1 could play a role in this impaired tissue blood flow. ABSTRACT: The present study aimed to assess the effect of a 1-week high-salt (HS) diet on the role of cyclo-oxygenases (COX-1 and COX-2) and the vasoconstrictor prostaglandins, thromboxane A2 (TXA2 ) and prostaglandin F2α (PGF2α ), on skin microcirculatory blood flow, as well as to detect its effect on markers of endothelial activation such as soluble cell adhesion molecules. Young women (n = 54) were assigned to either the HS diet group (N = 30) (∼14 g day(-1) NaCl ) or low-salt (LS) diet group (N = 24) (<2.3 g day(-1) NaCl ) for 7 days. Post-occlusive reactive hyperaemia (PORH) in the skin microcirculation was assessed by laser Doppler flowmetry. Plasma renin activity, plasma aldosterone, plasma and 24 h urine sodium and potassium, plasma concentrations of TXB2 (stable TXA2 metabolite) and PGF2α , soluble cell adhesion molecules and blood pressure were measured before and after the diet protocols. One HS diet group subset received 100 mg of indomethacin (non-selective COX-1 and COX-2 inhibitor), and another HS group subset received 200 mg of celecoxib (selective COX-2 inhibitor) before repeating laser Doppler flowmetry measurements. Blood pressure was unchanged after the HS diet, although it significantly reduced after the LS diet. Twenty-four hour urinary sodium was increased, and plasma renin activity and plasma aldosterone levels were decreased after the HS diet. The HS diet significantly impaired PORH and increased TXA2 but did not change PGF2α levels. Indomethacin restored microcirculatory blood flow and reduced TXA2 . By contrast, celecoxib decreased TXA2 levels but had no significant effects on blood flow. Restoration of of PORH by indomethacin during a HS diet suggests an important role of COX-1 derived vasoconstrictor metabolites in the regulation of microvascular blood flow during HS intake.

Transcranial sonography and the pocket smell test in the differential diagnosis between parkinson's disease and essential tremor.

The aim of this study was to determine the specificity and sensitivity of transcranial sonography (TCS) and the Pocket Smell Test (PST) in differing Parkinson's disease from essential tremor. The results were compared with the dopamin transporter scan (DaTSCAN) findings. Based on the DaTSCAN finding we formed a group of patients with essential tremor (51 patients) and a group with the Parkinson's disease (59 patients). The control group consisted of 26 healthy one. To evaluate the olfactory dysfunction the PST was used, whereas by TCS the substantia nigra hyperechogenicity was marked. The sensitivity and specificity of each diagnostic method was statistically calculated. In confirming Parkinson's disease the specificity of TCS was 88.2 % and the sensitivity 94.9 %. The specificity of PST was 80.4 % whereas the sensitivity was 74.6 %. TCS and PST should be performed to evaluate which patients need to be examined by DaTSCAN.

Osteoprotegerin and Vascular Calcification: Clinical and Prognostic Relevance.

Osteoprotegerin (OPG) is a key regulator in bone metabolism, that also has effect in vascular system. Studies suggest that osteoprotegerin is a critical arterial calcification inhibitor, and is released by endothelial cells as a protective mechanism for their survival in certain pathological conditions, such as diabetes mellitus, chronic kidney disease, and other metabolic disorders. That has been shown in studies in vitro and in animal models. The discovery that OPG deficient mice (OPG -/- mice) develop severe osteoporosis and arterial calcification, has led to conclusion that osteoprotegerin might be mulecule linking vascular and bone system. Paradoxically however, clinical trials have shown recently that OPG serum levels is increased in coronary artery disease and correlates with its severity, ischemic cardial decompensation, and future cardiovascular events. Therefore it is possible that osteoprotegerin could have a new function as a potential biomarker in early identification and monitoring patients with cardiovascular disease. Amongst that osteoprotegerin is in association with well known atherosclerosis risc factors: undoubtedly it is proven its relationship with age, smoking and diabetes mellitus. There is evidence regarding presence of hyperlipoproteinemia and increased serum levels of osteoprotegerin. Also the researches have been directed in genetic level, linking certain single nucleotid genetic polymorphisms of osteoprotegerin and vascular calcification appearance. This review emphasises multifactorial role of OPG, presenting numerous clinical and experimental studies regarding its role in vascular pathology, suggesting a novel biomarker in cardiovascular diseases, showing latest conclusions about this interesting topic that needs to be further explored.

Incidence of Stroke and Transient Ischemic Attack in Croatia: A Population Based Study.

The aim of this population based neuroepidemiological study was to establish the real incidence rates of acute cerebrovascular disease (CVD): stroke and transient ischemic attack (TIA) in the Republic of Croatia. Multicentric study included 89 501 persons of all ages in four regional centres in Croatia: Zagreb, Osijek + Slavonski Brod, Rijeka and Split. The following incidence rates of stroke, expressed at population of 100 000, have been established: Zagreb 290.52, Osijek + Slavonski Brod 302.14, Rijeka 219.65, Split 195.82. Incidence rate of stroke for the Republic of Croatia is 251.39. The following incidence rates of TIA, expressed at population of 100,000, have been established: Zagreb 87.15, Osijek + Slavonski Brod 156.53, Rijeka 90.11, Split 59.10. Incidence rate of TIA for the Republic of Croatia is 100.55. In the continental part of Croatia (Zagreb, Osijek + Slavonski Brod) incidence rate of stroke is higher by 45%, while incidence rate of TIA is higher by 82% than in the coastal part of Croatia, probably due to different lifestyle and environmental factors. The study has shown relatively high incidence rates of acute CVD (stroke and TIA) in the Republic of Croatia, which proves that CVD are a great public health problem.

Uptake/depuration kinetics, bioaccumulation potential and metabolic transformation of a complex pharmaceutical mixture in zebrafish (Danio rerio).

Uptake and elimination kinetics, bioconcentration factors (BCFs), and metabolic transformation of 20 different pharmaceutically active compounds (PhACs), covering a wide range of therapeutic categories and physico-chemical properties, were studied using zebrafish (Danio rerio). The fish were exposed to the mixture of the selected PhACs at environmentally relevant concentrations similar to 10 µg L-1. The experiments were performed in semi-static conditions and comprised a 7-day uptake period followed by a 7-day depuration period. Most of the PhACs reached a concentration plateau within the 7-day uptake-phase which was followed by an efficient depuration, with the observed uptake (ku) and depuration rate constants (kd,) ranging between 0.002 and 3.752 L kg-1 h-1, and 0.010 to 0.217 h-1, respectively. The investigated PhACs showed low to moderate BCFs. The highest BCFs of 47.8, 28.6 and 47.6 L kg-1 were determined for sertraline, diazepam and desloratadine, respectively. A high contribution of metabolic products to the total internal concentration was observed for some PhACs such as codeine (69%), sulfamethoxazole (51%) and verapamil (87%), which has to be taken into account when assessing the bioconcentration potential. Moreover, most of the metabolites exhibited significantly longer half-lives in zebrafish than their parent compounds and affected the overall depuration kinetics.

Usage Durability and Comfort Properties of Socks Made from Differently Spun Modal and Micro Modal Yarns.

Socks, being a necessary item of clothing, must be comfortable and maintain their quality throughout their life. Since the applicability of modal fibers and microfibers, as well as yarns produced using unconventional processes, in sock knitting has been insufficiently researched, this paper evaluated three groups of medium sized socks knitted in a plain single jersey pattern produced with the highest percentage of ring, rotor and air-jet spun modal or micro modal yarns of the same linear density in full plating with different textured polyamide 6.6 yarns compared to conventional cotton socks. The sock quality was evaluated through an investigation of the physical properties, wear resistance and dimensional stability, as well as the water vapor absorption, air permeability and thermal resistance using the thermal foot model before and after five repeated washing and drying cycles, according to the proposed methodology. The results showed that the fiber fineness, the structure of the differently spun yarns and the sock plain knits, the polyamide content and the implementation of the pretreatment of the socks had an influence on the obtained results. The socks made from modal and micro modal yarns differed in their properties. Compared to cotton socks, they have better comfort properties, a generally better pilling resistance and, after pretreatment, a comparable abrasion resistance.

Recent data on iodine intake in Croatian schoolchildren: results of 2014-2019 survey.

BACKGROUND: Both insufficient and excessive iodine intake can lead to a broad range of disorders. A cross-sectional survey was conducted to assess iodine status in schoolchildren from Croatia. DESIGN: 957 healthy 6 to 12-year-olds were enrolled (381 from northwestern region, 190 from eastern region, 215 from north Adriatic, and 171 from central Dalmatia region). Urinary iodine concentration (UIC) was measured in spot urine samples. Thyroid volume (Tvol) was recorded by ultrasound device. Standard anthropometric measures were taken, and body surface area (BSA) was determined. Tvol medians were calculated as a function of age, sex and BSA and compared with reference values. RESULTS: Total sample size included 490 boys and 467 girls. Overall median UIC was 250.68 µg/L, with statistically significant variance in geographical regions (median UIC was 244.71 µg/L in northwestern, 208.02 µg/L in eastern, 216.07 µg/L in north Adriatic and 366.43 µg/L in central Dalmatia region). There were 10.08% of samples with UIC < 100 mcg/L while 38.24% of samples had UIC > 300 mcg/L. Age-matched Tvol medians in schoolchildren from all regions of Croatia were at the upper limits of reference values, but in north Adriatic and central Dalmatia exceeded the 97th percentile. BSA-matched Tvol was within the reference range in all regions. CONCLUSIONS: Our results demonstrate sufficient (more than adequate) iodine intake in schoolchildren of Croatia, and excessive iodine intake in central Dalmatia region. Total thyroid volumes in schoolchildren of Croatia were within the normal range, however borderline enlarged age-matched thyroid glands were observed in coastal areas.

Environmental contaminants modulate transport activity of zebrafish (Danio rerio) multidrug and toxin extrusion protein 3 (Mate3/Slc47a2.1).

Zebrafish Mate3 is one of six co-orthologs of human multidrug and toxin extrusion proteins. It is highly expressed in the kidneys, intestine, testes, and brain of males. Initial interaction studies showed its interaction with xenobiotic compounds, suggesting a role in the efflux of toxic compounds. In this study, we aimed to test various environmental contaminants for their interaction with zebrafish Mate3. We developed a stable zebrafish Mate3 cell line and optimized a high-throughput screening assay using DAPI and ASP+ as fluorescent model substrates. To gain insight into the structure and function of the Mate3 protein and relate these to the results of the DAPI and ASP+ transport measurements, we predicted its 3D structure using the AlphaFold2 algorithm. A 3D structure with high per residue confidence scores with 13 transmembrane segments (TMs) was obtained, with topology and mutual positioning characteristic of the Mate protein family in a shape open to the extracellular part. Molecular docking methods were used to identify DAPI and ASP+ binding sites on the surface and in the center of the protein cavity. Because our kinetics experiments combined with molecular docking indicated that there may be additional active sites in zebrafish Mate3, additional cytotoxicity experiments were performed and highly potent Mate3 interactors were identified from a set of 55 different environmental contaminants. Our results suggest that some of the identified interactors may be of environmental concern, as their interaction with Mate3 could lead to an impairment of its normal efflux function, making fish more sensitive to harmful substances commonly released into the aquatic environment. Finally, the quality of zebrafish Mate3 structures predicted by the AlphaFold2 algorithm opens up the possibility of successfully using this tool for in silico research on transport preferences of other Mate proteins.

Stage-dependent localization of F-actin and Na+ /K+ -ATPase in zebrafish embryos detected using optimized cryosectioning immunostaining protocol.

The increasing use of the zebrafish model in biomedical and (eco)toxicological studies aimed at understanding the function of various proteins highlight the importance of optimizing existing methods to study gene and protein expression and localization in this model. In this context, zebrafish cryosections are still underutilized compared with whole-mount preparations. In this study, we used zebrafish embryos (24-120 hpf) to determine key factors for the preparation of high-quality zebrafish cryosections and to determine the optimal protocol for (immuno)fluorescence analyses of Na+ /K+ -ATPase and F-actin, across developmental stages from 1 to 5 dpf. The results showed that the highest quality zebrafish cryosections were obtained after the samples were fixed in 4% paraformaldehyde (PFA) for 1 h, incubated in 2.5% bovine gelatin/25% sucrose mixture, embedded in OCT, and then sectioned to 8 µm thickness at -20°C. Fluorescence microscopy analysis of phalloidin-labeled zebrafish skeletal muscle revealed that 1-h-4% PFA-fixed samples allowed optimal binding of phalloidin to F-actin. Further immunofluorescence analyses revealed detailed localization of F-actin and Na+ /K+ -ATPase in various tissues of the zebrafish and a stage-dependent increase in their respective expression in the somitic muscles and pronephros. Finally, staining of zebrafish cryosections and whole-mount samples revealed organ-specific and zone-dependent localizations of the Na+ /K+ -ATPase α1-subunit. RESEARCH HIGHLIGHTS: This study brings optimization of existing protocols for preparation and use of zebrafish embryos cryosections in (immuno)histological analyses. It reveals stage-dependent localization/expression of F-actin and Na+ /K+ -ATPase in zebrafish embryos.

Amyloid-ß contributes to blood-brain barrier leakage in transgenic human amyloid precursor protein mice and in humans with cerebral amyloid angiopathy.

BACKGROUND AND PURPOSE: Cerebral amyloid angiopathy (CAA) is a degenerative disorder characterized by amyloid-ß (Aß) deposition in the blood-brain barrier (BBB). CAA contributes to injuries of the neurovasculature including lobar hemorrhages, cortical microbleeds, ischemia, and superficial hemosiderosis. We postulate that CAA pathology is partially due to Aß compromising the BBB. METHODS: We characterized 19 patients with acute stroke with "probable CAA" for neurovascular pathology based on MRI and clinical findings. Also, we studied the effect of Aß on the expression of tight junction proteins and matrix metalloproteases (MMPs) in isolated rat brain microvessels. RESULTS: Two of 19 patients with CAA had asymptomatic BBB leakage and posterior reversible encephalopathic syndrome indicating increased BBB permeability. In addition to white matter changes, diffusion abnormality suggesting lacunar ischemia was found in 4 of 19 patients with CAA; superficial hemosiderosis was observed in 7 of 9 patients. Aß(40) decreased expression of the tight junction proteins claudin-1 and claudin-5 and increased expression of MMP-2 and MMP-9. Analysis of brain microvessels from transgenic mice overexpressing human amyloid precursor protein revealed the same expression pattern for tight junction and MMP proteins. Consistent with reduced tight junction and increased MMP expression and activity, permeability was increased in brain microvessels from human amyloid precursor protein mice compared with microvessels from wild-type controls. CONCLUSIONS: Our findings indicate that Aß contributes to changes in brain microvessel tight junction and MMP expression, which compromises BBB integrity. We conclude that Aß causes BBB leakage and that assessing BBB permeability could potentially help characterize CAA progression and be a surrogate marker for treatment response.

Epidemiology of laryngeal cancer in Osijek-Baranja County (eastern Croatia).

The aim of this retrospective study was to provide the incidence and mortality rate for all patients with laryngeal carcinoma diagnosed in Osijek-Baranja County, during the period of 1999-2008. In this period, there were 329 registered patients, from which 301 (91.5%) males, and 28 (8.5%) females. Age-standardized rate (ASR World) laryngeal cancer incidence was 6.4/100,000 (13.4/100,000 for males and 0.9/100,000 for females). In the same period, 238 people, including 224 (94.1%) men and 14 (5.9%) women died of laryngeal carcinoma. Age-standardized rate (ASR World) laryngeal cancer mortality was 4.4/100,000 (9.8/100,000 for males and 0.5/100 000 for females). There is a significant decline in mortality in males and increased mortality in females. Laryngeal carcinoma is a significant public health problem in the Osijek-Baranja county. Although the study period shows a tendency to decrease in the number of new cases and deaths, in the times ahead it is important to focus the emphasis on prevention and early detection with the goal of reducing incidence and mortality.