Oxime blot: A novel method for reliable and sensitive detection of carbonylated proteins in diverse biological systems.

Oxidative stress and oxidative protein damage occur in various biological processes and diseases. The carbonyl group on amino acid side chains is the most widely used protein oxidation biomarker. Carbonyl groups are commonly detected indirectly through their reaction with 2,4-dinitrophenylhydrazine (DNPH) and subsequent labeling with an anti-DNP antibody. However, the DNPH immunoblotting method lacks protocol standardization, exhibits technical bias, and has low reliability. To overcome these shortcomings, we have developed a new blotting method in which the carbonyl group reacts with the biotin-aminooxy probe to form a chemically stable oxime bond. The reaction speed and the extent of the carbonyl group derivatization are increased by adding a p-phenylenediamine (pPDA) catalyst under neutral pH conditions. These improvements are crucial since they ensure that the carbonyl derivatization reaction reaches a plateau within hours and increases the sensitivity and robustness of protein carbonyl detection. Furthermore, derivatization under pH-neutral conditions facilitates a good SDS-PAGE protein migration pattern, avoids protein loss by acidic precipitation, and is directly compatible with protein immunoprecipitation. This work describes the new Oxime blot method and demonstrates its use in detecting protein carbonylation in complex matrices from diverse biological samples.

Role of TLRs in HIV-1 Infection and Potential of TLR Agonists in HIV-1 Vaccine Development and Treatment Strategies.

Toll-like receptors (TLRs), as a family of pattern recognition receptors, play an important role in the recognition of HIV-1 molecular structures by various cells of the innate immune system, but also provide a functional association with subsequent mechanisms of adaptive immunity. TLR7 and TLR8 play a particularly important role in the innate immune response to RNA viruses due to their ability to recognise GU-rich single-stranded RNA molecules and subsequently activate intracellular signalling pathways resulting in expression of genes coding for various biological response modifiers (interferons, proinflammatory cytokines, chemokines). The aim of this review is to summarise the most recent knowledge on the role of TLRs in the innate immune response to HIV-1 and the role of TLR gene polymorphisms in the biology and in the clinical aspects of HIV infections. In addition, the role of TLR agonists as latency reversing agents in research to treat HIV infections and as immunomodulators in HIV vaccine research will be discussed.

CDC25c expression in patients with myelofibrosis is associated with stronger myeloproliferation and shorter overall survival.

BACKGROUND: Cell division cycle 25c (CDC25c) is a gene coding a phosphatase controlling entry into mitosis upon activation through Polo-like kinase 1 (PLK1) and serves as a key regulator of cell division. The CDC25c was reported to be dysregulated in some malignant diseases, but its role in myelofibrosis has not yet been elucidated. METHODS: We have retrospectively investigated CDC25c mRNA expression in bone marrow aspirates of 43 patients with myelofibrosis (28 primary [PMF] and 15 secondary myelofibrosis [SMF]) and 12 controls. RESULTS: CDC25c mRNA expression did not significantly differ between PMF, SMF and controls (median ∆CT 3.08 vs 2.86 vs 2.29 for PMF, SMF and controls, respectively; P = 0.162). Patients presenting with higher CDC25c mRNA expression were of older age (P = 0.037), had statistically significantly higher white-blood-cells (P = 0.017), larger liver size (P = 0.022), higher absolute neutrophil (P = 0.010), monocyte (P = 0.050), basophil (P = 0.012), and eosinophil counts (P = 0.013). Patients presenting with high CDC25c mRNA expression had statistically significantly inferior overall survival compared to low CDC25c expression group (HR = 2.99; P = 0.049). Median overall survival was not reached in patients with low and was 44 months in patients with high CDC25c expression. CONCLUSION: Our data suggest that higher CDC25c expression is associated with more proliferative phenotype of myelofibrosis and is prognostic of worse survival. Future studies investigating these interesting associations are warranted.

Cancer-associated mutations in the ribosomal protein L5 gene dysregulate the HDM2/p53-mediated ribosome biogenesis checkpoint.

Perturbations in ribosome biogenesis have been associated with cancer. Such aberrations activate p53 through the RPL5/RPL11/5S rRNA complex-mediated inhibition of HDM2. Studies using animal models have suggested that this signaling pathway might constitute an important anticancer barrier. To gain a deeper insight into this issue in humans, here we analyze somatic mutations in RPL5 and RPL11 coding regions, reported in The Cancer Genome Atlas and International Cancer Genome Consortium databases. Using a combined computational and statistical approach, complemented by a range of biochemical and functional analyses in human cancer cell models, we demonstrate the existence of several mechanisms by which RPL5 mutations may impair wild-type p53 upregulation and ribosome biogenesis. Unexpectedly, the same approach provides only modest evidence for a similar role of RPL11, suggesting that RPL5 represents a preferred target during human tumorigenesis in cancers with wild-type p53. Furthermore, we find that several functional cancer-associated RPL5 somatic mutations occur as rare germline variants in general population. Our results shed light on the so-far enigmatic role of cancer-associated mutations in genes encoding ribosomal proteins, with implications for our understanding of the tumor suppressive role of the RPL5/RPL11/5S rRNA complex in human malignancies.

Higher AURKA and PLK1 expression are associated with inferior overall survival in patients with myelofibrosis.

Aurora-kinase-A (AURKA), BORA and Polo-like-kinase-1 (PLK1) are regulating cell-cycle control and promotion of mitosis entry. AURKA contributes to Janus-kinase-2 (JAK2) activation and increased AURKA protein levels were reported in CD34+ and CD41+ cells of myeloproliferative neoplasm patients, leading to aneuploidy and aberrant megakaryopoiesis. We aimed to investigate AURKA, BORA and PLK1 mRNA expression in unfractionated bone-marrow aspirates of 43 patients with myelofibrosis (28 primary-/PMF, 15 secondary-myelofibrosis/SMF) and 12 controls and to assess their clinical correlations. AURKA expression did not significantly differ between myelofibrosis and controls (P = 0.466). Higher AURKA expression was significantly associated with higher absolute monocyte-count (P = 0.024) and shorter overall survival (HR = 3.77; P = 0.012). Patients with both PMF and SMF had lower BORA expression than controls (P = 0.009). Higher BORA expression was significantly associated with absence of constitutional symptoms (P = 0.049), absence of circulatory blasts (P = 0.047), higher monocyte- (P = 0.040) and higher eosinophil-counts (P = 0.016) and had neutral effect on survival (P > 0.05). PLK1 expression did not significantly differ between myelofibrosis and controls (P = 0.103). Higher PLK1 expression was significantly associated with higher white-blood-cell-count (P = 0.042) and inferior overall survival (HR = 5.87; P = 0.003). In conclusion, AURKA, BORA and PLK1 are involved in pathogenesis of myelofibrosis and may affect survival. Future studies investigating these interesting associations are warranted.

Weather conditions: a neglected factor in human salivary cortisol research?

There is ample evidence that environmental stressors such as extreme weather conditions affect animal behavior and that this process is in part mediated through the elevated activity of the hypothalamic pituitary adrenal axis which results in an increase in cortisol secretion. This relationship has not been extensively researched in humans, and weather conditions have not been analyzed as a potential confounder in human studies of stress. Consequently, the goal of this paper was to assess the relationship between salivary cortisol and weather conditions in the course of everyday life and to test a possible moderating effect of two weather-related variables, the climate region and timing of exposure to outdoors conditions. The sample consisted of 903 secondary school students aged 18 to 21 years from Mediterranean and Continental regions. Cortisol from saliva was sampled in naturalistic settings at three time points over the course of a single day. We found that weather conditions are related to salivary cortisol concentration and that this relationship may be moderated by both the specific climate and the anticipation of immediate exposure to outdoors conditions. Unpleasant weather conditions are predictive for the level of salivary cortisol, but only among individuals who anticipate being exposed to it in the immediate future (e.g., in students attending school in the morning shift). We also demonstrated that isolated weather conditions or their patterns may be relevant in one climate area (e.g., Continental) while less relevant in the other (e.g., Mediterranean). Results of this study draw attention to the importance of controlling weather conditions in human salivary cortisol research.

Reference intervals for six salivary cortisol measures based on the Croatian Late Adolescence Stress Study (CLASS).

INTRODUCTION: The aim of this nested study is to provide the reference intervals for already published measurements of salivary cortisol from the Croatian Adolescence Stress Study (CLASS). MATERIAL AND METHODS: A total of 969 individuals (372 males and 597 females) were included in the reference sample (age range: 18-21 years). Salivary cortisol concentrations were determined by the enzyme immunoassay (LUCIO-Medical ELISA Salivary Cortisol Kit, Nal von Minden, Germany) in the Department of Medical Laboratory Diagnostics, University Hospital Split. Nonparametric statistics were used for calculating the reference intervals (RIs) and 90% confidence intervals (90% CIs). RESULTS: The lower limits of RIs determined by the direct method were higher in females (> 10%) than in males for the cortisol concentrations at awakening (SCC0), 30 to 45 after awakening (SCC30-45) and at bedtime (SCCbedtime). The upper limits of RIs for the SCCbedtime were higher (> 10%) in males than in females. Females also had higher upper limits of RIs for the cortisol awakening response (CAR) and the diurnal cortisol slope (DCS) and higher lower limits of RIs for the CAR and the area under the curve with respect to ground (AUCG). The lower limits of RIs for the DCS were higher in males than in females. CONCLUSIONS: Obtained reference values open the arena for introducing salivary bioscience in Croatian clinical laboratory practice and provide important data for better understanding of gender differences in adaptation to stress during late adolescence.

Characterization of blood flow through intrapulmonary arteriovenous anastomoses and patent foramen ovale at rest and during exercise in stroke and transient ischemic attack patients.

OBJECTIVES: We determined whether stroke and/or TIA subjects have exercise-induced blood flow through intrapulmonary arteriovenous anastomoses (QIPAVA ) and/or patent foramen ovale (QPFO ) and a genetic predisposition for ischemic stroke. METHODS: Twenty-eight stroke and/or TIA subjects (33-63 years old) underwent transthoracic saline contrast echocardiography concomitant with transcranial Doppler to detect QIPAVA and QPFO at rest and during supine exercise with and without breathing 100% O2 . We also examined genetic polymorphisms in FV Leiden (G1691A; rs6025), factor II (FII) prothrombin (G20210A; rs1799963), methylene tetrahydfropholate reductase (C677T, rs1801133), and plasminogen-activator inhibitor-1 (PAI-1) (4G/5G; rs1799889) and angiotensin-converting enzyme (ACE; I/D, rs4646994) in 24/28 subjects. RESULTS: No subject without PFO had QIPAVA at rest (n=17), but 12/17 did with exercise. All PFO subjects had QPFO at rest (n=11) and 7/11 had either QIPAVA or QPFO with exercise. Breathing 100% O2 during exercise reduced or eliminated left heart contrast in all subjects. Gene analyses revealed that 15/24 patients were either heterozygous or homozygous for methylenetetrahydrofolate reductase gene polymorphism; 4G/4G and 4G/5G genotypes of plasminogen-activator inhibitor-1 were present in 7/24 and 13/24 patients, respectively; polymorphisms of ACE D/D genotype were present in 6/24 and I/D in 14/24 patients. Having both I/D and 4G/4G genotypes was more prevalent in PFO+ subjects (P=.03), and there was a trend (P=.06) for PFO- subjects to have a greater D/D genotype prevalence. CONCLUSIONS: Novel genetic predispositions reported here in PFO subjects should be investigated further in larger stroke and/or TIA patient datasets.

Protective effect of urapidil on testicular torsion-detorsion injury in rats.

PURPOSE: The aim of this study was to investigate the effect of urapidil and low-molecular weight heparin (LMWH) on testicular torsion-detorsion injury in rats. METHODS: Thirty-two male Sprague-Dawley rats were used. In the torsion-detorsion (T/D) group, the left testis was twisted at 720° for 3 h. After 3 h of reperfusion, the testis was removed. Urapidil or LMWH was administered intraperitoneally 30 min before detorsion in the treatment groups. RESULTS: Unilateral testicular torsion-detorsion caused significant increases in the malondialdehyde level and apoptosis and significant decreases in the superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in ipsilateral testes (p < 0.001). The rats treated with urapidil had a significant decrease in the malondialdehyde level and apoptosis and significant increases in the SOD and GPx activities in ipsilateral testes compared to the T/D group (p < 0.001). Animals treated with LMWH showed non-significant reductions in malondialdehyde levels and apoptosis compared to the T/D group. In addition, no significant difference in the SOD activities (p = 0.52) between the groups was found. The increase in the GPx activities was significant in the LMWH group compared to the T/D group (p < 0.001). CONCLUSION: The administration of urapidil before detorsion prevents ischemia/reperfusion cellular damage in testicular tissue. LMWH was not found to have a beneficial effect on testicular T/D injury in rats.

A case report of Anisakis pegreffii (Nematoda, Anisakidae) identified from archival paraffin sections of a Croatian patient.

BACKGROUND: This is the first report of anisakiasis in a Croatian patient, evidenced from an archival paraffin-embedded and hematoxylin-eosin stained tissue section. Anisakiasis has been only suspected in the country based on previously detected anti-Anisakis IgE seroprevalence in the healthy coastal population, as well as an acute case where pathohistological and serological findings suggested the diseases, but the migrating larva has not been retrieved. CASE PRESENTATION: Seventy years-old female, operated in 1998 for pulmonary carcinoma, was admitted to the General hospital Sibenik, Croatia in 2003, because of gastric pain and nausea that lasted for couple of days. She was showing good general condition, full mobility and lucidity, subfebrile status. Abdominal palpation inferred acute pain in paraumbilical and ileocecal region. Exploratory right pararectal laparotomy revealed a hardened, 5 cm-long structure, located intraluminally in the sigmoid colon, not perforating colon serosa. The process has been dissected and sent for patohistological diagnosis. Results showed a 2 mm-long whitish nematode spiralised in muscular layer of colon mucosa surrounded by granulomatous inflammation. CONCLUSION: After genomic DNA isolation of the nematode from the histological section, and amplification at the mitochondrial cytochrome oxidase 2 locus, etiological agent has been identified as Anisakis pegreffii. Used methodology suggests that screening of archival suspicious sections is feasible in order to study epidemiology of this zoonotic disease poorly recognised in Croatia.

Low molecular weight heparin treatment and impact of inherited thrombophilia type in pregnancies with previous adverse outcome.

OBJECTIVE: To assess the impact of low molecular weight heparin (LMWH) treatment in 50 pregnancies of women with inherited thrombophilia and adverse pregnancy outcome (APO) in previous untreated pregnancies. The impact of "Conventional" (FVL, PT, AT, PC, PS) and "Novel" (MTHFR, PAI-1, ACE) thrombophilias on APO was investigated. METHODS: The primary outcomes (PO) were: early and late pregnancy loss (EPL, LPL), preterm birth (PTB) or term birth (TB) compared to the last untreated pregnancies of the same women. Secondary outcomes (SO) were APO in LMWH treated and last untreated pregnancies ended with birth. PO and SO were compared in relation to the thrombophilia type. RESULTS: LMWH decreased EPL and LPL rate and improved TB rate compared with last untreated pregnancies (p < 0.001). There were less PTB (p = 0.019) and no cases of intrauterine fetal death (IUFD) (p = 0.0019) in LWMH-treated pregnancies. The division to Conventional and Novel thrombophilias showed: (a) difference between pregnancy losses and birth rate (p = 0.0069) and (b) no difference in the prevalence of APO in untreated pregnancies ended with birth. CONCLUSIONS: LMWH treatment improves pregnancy outcome in women with inherited thrombophilia and APO in previous pregnancies. Novel thrombophilias have the equal impact on the pregnancy outcome compared to the Conventional thrombophilias.

Beneficial effect of nifedipine on testicular torsion-detorsion injury in rats.

OBJECTIVE: To investigate the effect of nifedipine on testicular torsion-detorsion injury. MATERIALS AND METHODS: Twenty-four adult male Sprague-Dawley rats were randomly divided into 3 groups, each containing 8 rats. Rats in the control group underwent a sham operation of the left testis. In the torsion-detorsion (T/D) group, the left testis was twisted at 720° for 3 hours. After 3 hours of reperfusion, at the end of the experiment, the testes were removed. Rats in the treatment group received the same surgical procedure as the T/D group, but nifedipine was administered intraperitoneally (100 µg/kg) 30 minutes before the time of detorsion. RESULTS: Unilateral testicular torsion-detorsion caused a significant increase in the malondialdehyde level and apoptosis and caused significant decreases in superoxide dismutase and glutathione peroxidase activities in ipsilateral testes. The rats treated with nifedipine had a significant decrease in malondialdehyde level and apoptosis and had significant increases in superoxide dismutase and glutathione peroxidase activities in ipsilateral testes compared with those of the T/D group. CONCLUSION: These results suggest that biochemical and histological torsion-detorsion injury occurs in the ipsilateral testes after a 3-hour torsion and 3-hour detorsion and that administration of nifedipine before detorsion prevents ischemia/reperfusion cellular damage in the testicular tissue.

Skeletal remains from late Roman period: "as old as Diocletian's Palace".

In 2000, human skeletal remains were discovered in Split (Croatia). As archaeologists confirmed, it was an ancient skeleton accompanied by ceramics and bracelet characteristic for late Roman period whose possible violent death was excluded. The bone sample was radiocarbon dated by AMS to 1750 years. DNA was successfully extracted from the bone sample and subsequently typed using mt DNA and STR systems. The metal content was determined by atomic absorption spectrometry (AAS) in flame mode. Mercury concentration was determined by direct consecutive measures taken with a mercury analyzer. According to our results, we consider that the bones could belong to the one of the last citizens of the Diocletian's Palace.

The significance of immunohistochemical expression of merlin, Ki-67, and p53 in meningiomas.

Meningiomas are one of the most common CNS tumors whose appearance is closely linked to NF2 gene product merlin. Tumor markers Ki-67 and p53 play established role in tumor progression which should be analyzed in close association with merlin expression. The aim of this study was to investigate the immunohistochemical expression of merlin in meningiomas, correlation with Ki-67 and p53, and to determine the association of these results with histologic grade and subtype. The histologic sections of 170 patients with totally resected meningiomas, between January 2000 and December 2010, were classified according to WHO, immunohistochemically stained for Ki-67, p53, and merlin, and analyzed using light microscope. Ki-67 median was 5.6 times higher in group of patients with negative merlin than in those with positive merlin (P=0.05). Statistically significant correlation of merlin with p53 was found (P<0.001). Merlin expression between 2 combined groups (meningothelial/secretory and fibroblastic/transitional) was statistically significant (P=0.002). By comparing merlin expression and p53 levels, statistically significant difference was found (P=0.017). In the group with positive merlin and negative p53 as well as positive merlin and low p53, meningothelial/secretory subtypes of meningiomas were more common. In combination of negative merlin and negative p53 as well as negative merlin and high p53, there were more meningiomas of fibroblastic/transitional subtype. There was no statistically significant correlation between merlin and tumor grade (P=0.420). There is undeniable influence of merlin on the development and the proliferative ability of meningioma subtypes. Significant role of p53 pathway was confirmed.

Shiga toxin receptor Gb3Cer/CD77: tumor-association and promising therapeutic target in pancreas and colon cancer.

BACKGROUND: Despite progress in adjuvant chemotherapy in the recent decades, pancreatic and colon cancers remain common causes of death worldwide. Bacterial toxins, which specifically bind to cell surface-exposed glycosphingolipids, are a potential novel therapy. We determined the expression of globotriaosylceramide (Gb3Cer/CD77), the Shiga toxin receptor, in human pancreatic and colon adenocarcinomas. METHODOLOGY/PRINCIPAL FINDINGS: Tissue lipid extracts of matched pairs of cancerous and adjacent normal tissue from 21 pancreatic and 16 colon cancer patients were investigated with thin-layer chromatography overlay assay combined with a novel mass spectrometry approach. Gb3Cer/CD77 was localized by immunofluorescence microscopy of cryosections from malignant and corresponding healthy tissue samples. 62% of pancreatic and 81% of colon adenocarcinomas showed increased Gb3Cer/CD77 expression, whereas 38% and 19% of malignant pancreas and colon tissue, respectively, did not, indicating an association of this marker with neoplastic transformation. Also, Gb3Cer/CD77 was associated with poor differentiation (G>2) in pancreatic cancer (P = 0.039). Mass spectrometric analysis evidenced enhanced expression of Gb3Cer/CD77 with long (C24) and short chain fatty acids (C16) in malignant tissues and pointed to the presence of hydroxylated fatty acid lipoforms, which are proposed to be important for receptor targeting. They could be detected in 86% of pancreatic and about 19% of colon adenocarcinomas. Immunohistology of tissue cryosections indicated tumor-association of these receptors. CONCLUSIONS/SIGNIFICANCE: Enhanced expression of Gb3Cer/CD77 in most pancreatic and colon adenocarcinomas prompts consideration of Shiga toxin, its B-subunit or B-subunit-derivatives as novel therapeutic strategies for the treatment of these challenging malignancies.

Tumor-associated CD75s- and iso-CD75s-gangliosides are potential targets for adjuvant therapy in pancreatic cancer.

Pancreatic adenocarcinoma confers one of the highest mortality rates in malignant human tumors with very poor prognosis. Because as yet no treatments are available that produce a substantial survival benefit for this fatal neoplasia, new therapeutic concepts are urgently required to support cancer standard treatment. In search of tumor-associated gangliosides with therapeutic background, we probed a random collection of cancerous and adjacent normal postoperative tissue samples from 38 patients for the expression of CD75s- and iso-CD75s-gangliosides. We exhaustively analyzed the expression of CD75s-1-ganglioside (IV(6)Neu5Ac-nLc4Cer) and structurally closely related iso-CD75s-1-ganglioside (IV(3)Neu5Ac-nLc4Cer) by means of immunohistology of cryosections and semiquantitative TLC of tissue lipid extracts combined with mass spectrometry. CD75s-1- and iso-CD75s-1-ganglioside showed an elevated expression in 42% and 66% of the tumors, respectively, indicating a significant association with neoplastic transformation (P = 0.001). Thus, increased expression of CD75s-1- and iso-CD75s-1-gangliosides renders these cell surface molecules promising candidates for oncologic applications. Further statistical analysis revealed a significant enhancement of CD75s-1-ganglioside in the group of less differentiated tumors (grade >2) suggesting this ganglioside as a potential marker for poor differentiation. The CD75s-specific antitumor drug rViscumin, which represents the recombinant counterpart of the ribosome-inactivating lectin viscumin, has successfully passed clinical phase I trials and provides an opportunity for treating pancreatic cancer. Consequently, if an enhanced expression is existent in malignant tissues, we propose the targeting of CD75s-gangliosides with rViscumin as a novel potential strategy in adjuvant treatment of pancreatic malignancies.

Anthropological analysis of neolithic and Early Bronze Age skeletons--a classical and molecular approach (East Slavonia, Croatia).

Theories about the first Indo-European migration are numerous. Significant contribution in attempt to resolve these theories is given by analysing skeletal material from two biggest prehistoric archaeological sites from N-E Croatia. Eight skeletons of Starcevo culture from sites "Nama" and "Hotel" at Vinkovci (6100-5500 BC) and seven skeletons of Vucedol culture from the site Vineyard Streim at Vucedol near Vukovar (3000-2500 BC) were analysed. Methods of classical anthropological analysis tried to distinguish the differences among members of both populations, while the methods of molecular genetics were used in defining possible genetic structure of both ancient populations. Established differences speak on the behalf of the theory of Maria Gimbutas about the first Indo-European migration with a cattle breeding population from the east around 3500 BC.