N-cadherin: A diagnostic marker to help discriminate primary liver carcinomas from extrahepatic carcinomas.

Distinguishing primary liver cancer (PLC), namely hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), from liver metastases is of crucial clinical importance. Histopathology remains the gold standard, but differential diagnosis may be challenging. While absent in most epithelial, the expression of the adherens junction glycoprotein N-cadherin is commonly restricted to neural and mesenchymal cells, or carcinoma cells that undergo the phenomenon of epithelial-to-mesenchymal transition (EMT). However, we recently established N- and E-cadherin expression as hallmarks of normal hepatocytes and cholangiocytes, which are also preserved in HCC and iCCA. Therefore, we hypothesized that E- and/or N-cadherin may distinguish between carcinoma derived from the liver vs carcinoma of other origins. We comprehensively evaluated E- and N-cadherin in 3359 different tumors in a multicenter study using immunohistochemistry and compared our results with previously published 882 cases of PLC, including 570 HCC and 312 iCCA. Most carcinomas showed strong positivity for E-cadherin. Strong N-cadherin positivity was present in HCC and iCCA. However, except for clear cell renal cell carcinoma (23.6% of cases) and thyroid cancer (29.2%), N-cadherin was only in some instances faintly expressed in adenocarcinomas of the gastrointestinal tract (0%-0.5%), lung (7.1%), pancreas (3.9%), gynecological organs (0%-7.4%), breast (2.2%) as well as in urothelial (9.4%) and squamous cell carcinoma (0%-5.6%). As expected, N-cadherin was detected in neuroendocrine tumors (25%-75%), malignant melanoma (46.2%) and malignant mesothelioma (41%). In conclusion, N-cadherin is a useful marker for the distinction of PLC vs liver metastases of extrahepatic carcinomas (P < .01).

Comparison of standard mismatch repair deficiency and microsatellite instability tests in a large cancer series.

BACKGROUND: The tumor-agnostic indication of immune checkpoint inhibitors to treat cancers with mismatch repair deficiency (dMMR)/microsatellite instability (MSI) increased the demand for such tests beyond Lynch syndrome. International guideline recommendations accept immunohistochemistry (IHC) for dMMR or molecular techniques (PCR or NGS) for MSI status determinations considering the two tests are equal, although there are scattered reports contradicting to this presumption. MATERIALS AND METHODS: Here we have directly compared four protein MMR immunohistochemistry (IHC) to MSI Pentaplex PCR test in a large cancer patient cohort (n = 1306) of our diagnostic center where the two tests have been run parallel in 703 cases. RESULTS: In this study we have found a high discrepancy rate (19.3%) of the two tests which was independent of the tumor types. The MSI PCR sensitivity for MMR IHC status was found to be very low resulting in a relatively low positive and negative predicting values. As a consequence, the correlation of the two tests was low (kappa < 0.7). During analysis of the possible contributing factors of this poor performance, we have excluded low tumor percentage of the samples, but identified dMMR phenotypes (classic versus non-classic or unusual) as possible contributors. CONCLUSION: Although our cohort did not include samples with identified technical errors, our data strongly support previous reports that unidentified preanalytical factors might have the major influence on the poor performance of the MSI PCR and MMR IHC. Furthermore, the case is open whether the two test types are equally powerful predictive markers of immunotherapies.

Proteome alterations in human autopsy tissues in relation to time after death.

Protein expression is a primary area of interest for routine histological diagnostics and tissue-based research projects, but the limitations of its post-mortem applicability remain largely unclear. On the other hand, tissue specimens obtained during autopsies can provide unique insight into advanced disease states, especially in cancer research. Therefore, we aimed to identify the maximum post-mortem interval (PMI) which is still suitable for characterizing protein expression patterns, to explore organ-specific differences in protein degradation, and to investigate whether certain proteins follow specific degradation kinetics. Therefore, the proteome of human tissue samples obtained during routine autopsies of deceased patients with accurate PMI (6, 12, 18, 24, 48, 72, 96 h) and without specific diseases that significantly affect tissue preservation, from lungs, kidneys and livers, was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For the kidney and liver, significant protein degradation became apparent at 48 h. For the lung, the proteome composition was rather static for up to 48 h and substantial protein degradation was detected only at 72 h suggesting that degradation kinetics appear to be organ specific. More detailed analyses suggested that proteins with similar post-mortem kinetics are not primarily shared in their biological functions. The overrepresentation of protein families with analogous structural motifs in the kidney indicates that structural features may be a common factor in determining similar postmortem stability. Our study demonstrates that a longer post-mortem period may have a significant impact on proteome composition, but sampling within 24 h may be appropriate, as degradation is within acceptable limits even in organs with faster autolysis.

MicroRNA Profiling in Papillary Thyroid Cancer.

Genetic alterations are well known to be related to the pathogenesis and prognosis of papillary thyroid carcinoma (PTC). Some miRNA expression dysregulations have previously been described in the context of cancer development including thyroid carcinoma. In our study, we performed original molecular diagnostics on tissue samples related to our own patients. We aimed to identify all dysregulated miRNAs in potential association with PTC development via sequencing much higher numbers of control-matched PTC tissue samples and analyzing a wider variety of miRNA types than previous studies. We analyzed the expression levels of 2656 different human miRNAs in the context of 236 thyroid tissue samples (118 tumor and control pairs) related to anonymized PTC cases. Also, KEGG pathway enrichment analysis and GO framework analysis were used to establish the links between miRNA dysregulation and certain biological processes, pathways of signaling, molecular functions, and cellular components. A total of 30 significant differential miRNA expressions with at least ±1 log2 fold change were found related to PTC including, e.g., miR-551b, miR-146b, miR-221, miR-222, and miR-375, among others, being highly upregulated, as well as miR-873 and miR-204 being downregulated. In addition, we identified miRNA patterns in vast databases (KEGG and GO) closely similar to that of PTC including, e.g., miRNA patterns of prostate cancer, HTLV infection, HIF-1 signaling, cellular responses to growth factor stimulus and organic substance, and negative regulation of gene expression. We also found 352 potential associations between certain miRNA expressions and states of clinicopathological variables. Our findings-supported by the largest case number of original matched-control PTC-miRNA relation research-suggest a distinct miRNA expression profile in PTC that could contribute to a deeper understanding of the underlying molecular mechanisms promoting the pathogenesis of the disease. Moreover, significant miRNA expression deviations and their signaling pathways in PTC presented in our study may serve as potential biomarkers for PTC diagnosis and prognosis or even therapeutic targets in the future.

Descriptive Analysis of Common Fusion Mutations in Papillary Thyroid Carcinoma in Hungary.

Thyroid cancer is the most common type of endocrine malignancy. Papillary thyroid carcinoma (PTC) is its predominant subtype, which is responsible for the vast majority of cases. It is true that PTC is a malignant tumor with a very good prognosis due to effective primary therapeutic approaches such as thyroidectomy and radioiodine (RAI) therapy. However, we are often required to indicate second-line treatments to eradicate the tumor properly. In these scenarios, molecular therapies are promising alternatives, especially if specifically targetable mutations are present. Many of these targetable gene alterations originate from gene fusions, which can be found using molecular diagnostics like next-generation sequencing (NGS). Nonetheless, molecular profiling is far from being a routine procedure in the initial phase of PTC diagnostics. As a result, the mutation status, except for BRAF V600E mutation, is not included in risk classification algorithms either. This study aims to provide a comprehensive analysis of fusion mutations in PTC and their associations with clinicopathological variables in order to underscore certain clinical settings when molecular diagnostics should be considered earlier, and to demonstrate yet unknown molecular-clinicopathological connections. We conducted a retrospective fusion mutation screening in formalin-fixed paraffin-embedded (FFPE) PTC tissue samples of 100 patients. After quality evaluation by an expert pathologist, RNA isolation was performed, and then NGS was applied to detect 23 relevant gene fusions in the tumor samples. Clinicopathological data were collected from medical and histological records. To obtain the most associations from the multivariate dataset, we used the d-correlation method for our principal component analysis (PCA). Further statistical analyses, including Chi-square tests and logistic regressions, were performed to identify additional significant correlations within certain subsets of the data. Fusion mutations were identified in 27% of the PTC samples, involving nine distinct genes: RET, NTRK3, CCDC6, ETV6, MET, ALK, NCOA4, EML4, and SQSTM1. RET and CCDC6 fusions were associated with type of thyroidectomy, RAI therapy, smaller tumor size, and history of Hashimoto's disease. NCOA4 fusion correlated with sex, multifocality, microcarcinoma character, history of goiter, and obstructive pulmonary disease. EML4 fusion was also linked with surgical procedure type and smaller tumor size, as well as the history of hypothyroidism. SQSTM1 fusion was associated with multifocality and a medical history of thyroid/parathyroid adenoma. NTRK3 and ETV6 fusions showed significant associations with Hashimoto's disease, and ETV6, also with endometriosis. Moreover, fusion mutations were linked to younger age at the time of diagnosis, particularly the fusion of ETV6. The frequent occurrence of fusion mutations and their associations with certain clinicopathological metrics highlight the importance of integrating molecular profiling into routine PTC management. Early detection of fusion mutations can inform surgical decisions and therapeutic strategies, potentially improving clinical outcomes.

Analysis of SIRT1 Gene SNPs and Clinical Characteristics in Medication-Related Osteonecrosis of the Jaw.

Certain genetic factors, including single-nucleotide polymorphisms (SNPs) in the SIRT1 gene, have been linked to medication-related osteonecrosis of the jaw (MRONJ). This study examined four SNPs in the SIRT1 gene and implemented multivariate statistical analysis to analyze genetic and clinical factors in MRONJ patients. Genomic DNA was isolated from peripheral blood samples of 63 patients of European origin treated for MRONJ, and four SNP genotypes in the gene encoding the SIRT-1 protein were determined by Sanger sequencing. The allele frequencies measured in the MRONJ population were compared with allele frequencies measured in the European population in the National Center for Biotechnology Information Allele Frequency Aggregator (NCBI ALFA) database. Genetic and clinical factors were examined with multivariate statistical analysis. A C:A allele distribution ratio of 77.8:22.2 was measured in the rs932658 SNP. In the ALFA project, a C:A allele distribution ratio of 59.9:40.1 was detected in the European population, which was found to be a significant difference (p = 4.5 × 10-5). Multivariate statistical analysis revealed a positive correlation (0.275) between the genotype of SNP rs932658 and the number of stages improved during appropriate MRONJ therapy. It is concluded that allele A in SNP rs932658 in the SIRT1 gene acts as a protective factor in MRONJ.

Comparison of surgical strategies in the treatment of low-risk differentiated thyroid cancer.

CONTEXT: Increasing diagnostic sensitivity in the detection of thyroid cancer has led to uncertainties in the optimal surgical approach of the smaller, low risk tumors. Current ATA guidelines consider lobectomy safe between 1 and 4 cm, while ETA advocates for primary total thyroidectomy to avoid reoperation, as final risk stratification is based on the histological results. OBJECTIVE: Our aim was to compare the differences in outcomes that are potentially achievable with adherence to the different guidelines, and also to examine the predictive value of clinical parameters on the incidence of postoperative risk factors. METHODS: We performed a retrospective cohort database analysis to identify the different surgical outcomes (based on postoperative risk factors) using ATA and ETA guidelines; the hypothetical rate of completion thyroidectomy when ATA or ETA recommends lobectomy; the accuracy of our preoperative evaluation; the utility of preoperative findings in predicting the optimal surgical strategy using binary logistic regression. RESULTS: Out of 248 patients, 152 (ATA) and 23 (ETA) cases would have been recommended for initial lobectomy. Following the guidelines, a postoperative risk factor would have been present in 61.8, and 65.2% of the cases, respectively. Except for angioinvasion, tumor size was not a significant predictor for the presence of postoperative risk factors. CONCLUSION: Current pre-operative criteria are inadequate to accurately determine the extent of initial surgery and our postoperative findings verify the frequent need for completion thyroidectomy using both guidelines. As a consequence, in the absence of effective pre-operative set of criteria, we advocate primary total thyroidectomy in most cases.

Dynamic Interplay in Tumor Ecosystems: Communication between Hepatoma Cells and Fibroblasts.

Tumors are intricate ecosystems where cancer cells and non-malignant stromal cells, including cancer-associated fibroblasts (CAFs), engage in complex communication. In this study, we investigated the interaction between poorly (HLE) and well-differentiated (HuH7) hepatoma cells and LX2 fibroblasts. We explored various communication channels, including soluble factors, metabolites, extracellular vesicles (EVs), and miRNAs. Co-culture with HLE cells induced LX2 to produce higher levels of laminin ß1, type IV collagen, and CD44, with pronounced syndecan-1 shedding. Conversely, in HuH7/LX2 co-culture, fibronectin, thrombospondin-1, type IV collagen, and cell surface syndecan-1 were dominant matrix components. Integrins α6ß4 and α6ß1 were upregulated in HLE, while α5ß1 and αVß1 were increased in HuH7. HLE-stimulated LX2 produced excess MMP-2 and 9, whereas HuH7-stimulated LX2 produced excess MMP-1. LX2 activated MAPK and Wnt signaling in hepatoma cells, and conversely, hepatoma-derived EVs upregulated MAPK and Wnt in LX2 cells. LX2-derived EVs induced over tenfold upregulation of SPOCK1/testican-1 in hepatoma EV cargo. We also identified liver cancer-specific miRNAs in hepatoma EVs, with potential implications for early diagnosis. In summary, our study reveals tumor type-dependent communication between hepatoma cells and fibroblasts, shedding light on potential implications for tumor progression. However, the clinical relevance of liver cancer-specific miRNAs requires further investigation.

Surface Analysis by Secondary Ion Mass Spectrometry (SIMS): Principles and Applications from Swiss laboratories.

Secondary Ion Mass Spectrometry (SIMS) extracts chemical, elemental, or isotopic information about a localized area of a solid target by performing mass spectrometry on secondary ions sputtered from its surface by the impact of a beam of charged particles. This primary beam sputters ionized atoms and small molecules (as well as many neutral particles) from the upper few nanometers of the sample surface. The physical basis of SIMS has been applied to a large range of applications utilizing instruments optimized with different types of mass analyzer, either dynamic SIMS with a double focusing mass spectrometer or static SIMS with a Time of Flight (TOF) analyzer. Here, we present a short review of the principles and major applications of three different SIMS instruments located in Switzerland.

The Hippo Effector Transcriptional Coactivator with PDZ-Binding Motif Cooperates with Oncogenic ß-Catenin to Induce Hepatoblastoma Development in Mice and Humans.

Hepatoblastoma (HB) is the most common pediatric liver tumor. Though Wnt/ß-catenin and Hippo cascades are implicated in HB development, studies on crosstalk between ß-catenin and Hippo downstream effector transcriptional coactivator with PDZ-binding motif (TAZ) in HB are lacking. Expression levels of TAZ and ß-catenin in human HB specimens were assessed by immunohistochemistry. Functional interplay between TAZ and ß-catenin was determined by overexpression of an activated form of TAZ (TAZS89A), either alone or combined with an oncogenic form of ß-catenin (ΔN90-ß-catenin), in mouse liver via hydrodynamic transfection. Activation of TAZ often co-occurred with that of ß-catenin in clinical specimens. Although the overexpression of TAZS89A alone did not induce hepatocarcinogenesis, concomitant overexpression of TAZS89A and ΔN90-ß-catenin triggered the development of HB lesions exhibiting both epithelial and mesenchymal features. Mechanistically, TAZ/ß-catenin-driven HB development required TAZ interaction with transcriptional enhanced associate domain factors. Blockade of the Notch cascade did not inhibit TAZ/ß-catenin-dependent HB formation in mice but suppressed the mesenchymal phenotype. Neither Yes-associated protein nor heat shock factor 1 depletion affected HB development in TAZ/ß-catenin mice. In human HB cell lines, silencing of TAZ resulted in decreased cell growth, which was further reduced when TAZ knockdown was associated with suppression of either ß-catenin or Yes-associated protein. Overall, our study identified TAZ as a crucial oncogene in HB development and progression.

Co-occurrence of thyroid and breast cancer is associated with an increased oncogenic SNP burden.

BACKGROUND: Epidemiological evidence suggests that synchronous or metachronous presentation of breast and thyroid cancers exceeds that predicted by chance alone. The following potential explanations have been hypothesized: common environmental or hormonal factors, oncogenic effect of the treatment for the first cancer, closer follow-up of cancer survivors, shared underlying genetic risk factors. While some cases were found to be related to monogenic disorders with autosomal inheritance, the genetic background of most cases of co-occurring breast and thyroid cancer is thought to be polygenic. METHODS: In this retrospective case-control study we compared the genetic profile of patients with a history of breast cancer (n = 15) to patients with co-occurring breast and thyroid cancer (n = 19) using next generation sequencing of 112 hereditary cancer risk genes. Identified variants were categorized based on their known association with breast cancer and oncogenesis in general. RESULTS: No difference between patients with breast and double cancers was observed in clinical and pathological characteristics or the number of neutral SNPs. The unweighted and weighted number of SNPs with an established or potential association with breast cancer was significantly lower in the group with breast cancer only (mean difference - 0.58, BCa 95% CI [- 1.09, - 0.06], p = 0.029, and mean difference - 0.36, BCa 95% CI [- 0.70, - 0.02], p = 0.039, respectively). The difference was also significant when we compared the number of SNPs with potential or known association with any malignancy (mean difference - 1.19, BCa 95% CI [- 2.27, - 0.11], p = 0.032 for unweighted, and mean difference - 0.73, BCa 95% CI [- 1.32, - 0.14], p = 0.017 for weighted scores). CONCLUSION: Our findings are compatible with the hypothesis of genetic predisposition in the co-occurrence of breast and thyroid cancer. Further exploration of the underlying genetic mechanisms may help in the identification of patients with an elevated risk for a second cancer at the diagnosis of the first cancer.

TEA Domain Transcription Factor 4 Is the Major Mediator of Yes-Associated Protein Oncogenic Activity in Mouse and Human Hepatoblastoma.

Hepatoblastoma (HB) is the most common type of pediatric liver cancer. Activation of yes-associated protein (YAP) has been implicated in HB molecular pathogenesis. The transcriptional co-activator Yap regulates downstream gene expression through interaction with the TEA domain (TEAD) proteins. Nonetheless, YAP also displays functions that are independent of its transcriptional activity. The underlying molecular mechanisms by which Yap promotes HB development remain elusive. In the current study, we demonstrated that blocking TEAD function via the dominant-negative form of TEAD2 abolishes Yap-driven HB formation in mice and restrains human HB growth in vitro. When TEAD2 DNA-binding domain was fused with virus protein 16 transcriptional activation domain, it synergized with activated ß-catenin to promote HB formation in vivo. Among TEAD genes, silencing of TEAD4 consistently inhibited tumor growth and Yap target gene expression in HB cell lines. Furthermore, TEAD4 mRNA expression was significantly higher in human HB lesions when compared with corresponding nontumorous liver tissues. Human HB specimens also exhibited strong nuclear immunoreactivity for TEAD4. Altogether, data demonstrate that TEAD-mediated transcriptional activity is both sufficient and necessary for Yap-driven HB development. TEAD4 is the major TEAD isoform and Yap partner in human HB. Targeting TEAD4 may represent an effective treatment option for human HB.

Helicobacter pylori heteroresistance to clarithromycin in adults-New data by in situ detection and improved concept.

BACKGROUND: Clarithromycin (Cla) heteroresistance of Helicobacter pylori (H pylori) infections is commonly assessed by comparing the resistance status of antrum and corpus biopsy samples and by demonstrating the discrepancy between them (interniche heteroresistance). However, fluorescence in situ hybridization (FISH) technique is capable of showing the synchronous presence of susceptible and resistant bacteria (intraniche heteroresistance), enabling the detection of heteroresistant H pylori populations within one biopsy sample. MATERIALS AND METHODS: Antrum and corpus biopsy specimens of 305 H pylori-infected patients were investigated with an rRNA-targeted Cla-resistance FISH test. Anamnestic data were collected from the institutional electronic register. Prevalence rates of susceptible, homo- and heteroresistant cases were correlated with the anamnestic and clinicopathological data. RESULTS: Overall Cla-resistance rate was 23.9% (73 cases), consisting of 35 (11.5%) homoresistant and 38 (12.5%) heteroresistant cases. Thirty-five patients had at least one biopsy site where susceptible and resistant bacteria were present simultaneously. From this subset, 20 cases demonstrated intraniche heteroresistance on both sites. Prior Cla-based eradication attempts were more frequent in homoresistant than in susceptible and heteroresistant cases (P < .001, P < .001, respectively). Cla-containing therapy eradicated heteroresistant infections at a significantly lower rate in comparison with susceptible cases (P = .0112), but more effectively than homoresistants (P = .0393). CONCLUSIONS: The most frequent type of Cla-heteroresistance is the coexistence of susceptible and resistant H pylori bacteria in the same location (intraniche heteroresistance). A previous Cla-based eradication attempt predisposes patients to homoresistant infection. Heteroresistance is characterized by a non-eradication-related background and intermediate characteristics in many respects when compared to susceptible and homoresistant cases.

CDK9 is dispensable for YAP-driven hepatoblastoma development.

BACKGROUND: Hepatoblastoma (HB) is the most common pediatric liver malignancy, occurring mainly during the first 4 years of life. Recent studies unraveled the frequent, coordinated activation of Wnt/ß-catenin and YAP/Hippo (where YAP is yes-associated protein) pathways in human HB samples. Furthermore, it was found that concomitant overexpression of activated forms of ß-catenin and YAP in the mouse liver triggers HB formation in YAP/ß-catenin mice. Cyclin-dependent kinases 9 (CDK9) is an elongating kinase, which has been shown to mediate YAP-driven tumorigenesis. The role of CDK9 in HB molecular pathogenesis has not been investigated to date. METHODS: CDK9 expression was determined in human HB lesions, HB cell lines, and YAP/ß-catenin mouse livers. CDK9 was silenced in human HB cell lines and the effects on growth rate and YAP targets were analyzed. Hydrodynamic transfection of YAPS127A and ∆N90-ß-catenin together with either shCdk9 or control shLuc (where Luc is luciferase) plasmids was employed to assess the requirement of Cdk9 for HB development in vivo. RESULTS: Nuclear immunoreactivity for CDK9 protein was more pronounced in human HB samples and YAP/ß-catenin mouse HB tumor tissues than in corresponding surrounding nontumorous liver tissues. CDK9 protein was also expressed in human HB cell lines. Silencing of CDK9 in human HB cell lines did not lead to consistent effects on HB cell growth or YAP target gene expression. Surprisingly, silencing of Cdk9 led to accelerated liver tumorigenesis in YAP/ß-catenin mice. CONCLUSION: CDK9 is not a major downstream mediator of YAP oncogenic function in HB development and progression.

Claudin-18 expression in oesophagogastric adenocarcinomas: a tissue microarray study of 523 molecularly profiled cases.

BACKGROUND: Claudin-18 (CLDN18) is a highly specific tight junction protein of the gastric mucosa. An isoform of CLDN18, the Claudin 18.2, has recently emerged as an innovative drug target for metastatic gastric cancer. METHODS: We investigated the immunohistochemical profile of CLDN18, p53, p16, E-cadherin, MSH2, MSH6, MLH1, PSM2, HER2, and PDL-1 in a large series of 523 primary gastric carcinomas (GCs; n = 408) and gastro-oesophageal carcinomas (GECs; n = 115) and 135 matched and synchronous nodal metastases. The status of HER2 and EBER by means of chromogenic in situ hybridisation (CISH) was also evaluated. RESULTS: High membranous CLDN18 expression was present in 150/510 (29.4%) primary cases and in 45/132 (34.1%) metastases. An abnormal expression (i.e. nuclear and/or cytoplasmic) was observed in 115 (22.5%) primary cases and in 33 (25.0%) metastases. A 38.8% of the cases showed significant CLDN18 intratumoural variability among the different tissue microarray cores obtained from the same tumour. Positive membrane CLDN18 expression was statistically associated with non-antral GCs (p = 0.016), Lauren diffuse type (p = 0.009), and with EBV-associated cancers (p < 0.001). CONCLUSIONS: CLDN18 is frequently expressed in gastric and gastro-oesophageal cancers; further studies should investigate the prognostic significance of CLDN18 heterogeneity in order to implement its test into clinical practice.

The PARP inhibitor olaparib exerts beneficial effects in mice subjected to cecal ligature and puncture and in cells subjected to oxidative stress without impairing DNA integrity: A potential opportunity for repurposing a clinically used oncological drug for the experimental therapy of sepsis.

Poly(ADP-ribose) polymerase (PARP) is involved in the pathogenesis of cell dysfunction, inflammation and organ failure during septic shock. The goal of the current study was to investigate the efficacy and safety of the clinically approved PARP inhibitor olaparib in experimental models of oxidative stress in vitro and in sepsis in vivo. In mice subjected to cecal ligation and puncture (CLP) organ injury markers, circulating and splenic immune cell distributions, circulating mediators, DNA integrity and survival was measured. In U937 cells subjected to oxidative stress, cellular bioenergetics, viability and DNA integrity were measured. Olaparib was used to inhibit PARP. The results show that in adult male mice subjected to CLP, olaparib (1-10 mg/kg i.p.) improved multiorgan dysfunction. Olaparib treatment reduced the degree of bacterial CFUs. Olaparib attenuated the increases in the levels of several circulating mediators in the plasma. In the spleen, the number of CD4+ and CD8+ lymphocytes were reduced in response to CLP; this reduction was inhibited by olaparib treatment. Treg but not Th17 lymphocytes increased in response to CLP; these cell populations were reduced in sepsis when the animals received olaparib. The Th17/Treg ratio was lower in CLP-olaparib group than in the CLP control group. Analysis of miRNA expression identified a multitude of changes in spleen and circulating white blood cell miRNA levels after CLP; olaparib treatment selectively modulated these responses. Olaparib extended the survival rate of mice subjected to CLP. In contrast to males, in female mice olaparib did not have significant protective effects in CLP. In aged mice olaparib exerted beneficial effects that were less pronounced than the effects obtained in young adult males. In in vitro experiments in U937 cells subjected to oxidative stress, olaparib (1-100 µM) inhibited PARP activity, protected against the loss of cell viability, preserved NAD+ levels and improved cellular bioenergetics. In none of the in vivo or in vitro experiments did we observe any adverse effects of olaparib on nuclear or mitochondrial DNA integrity. In conclusion, olaparib improves organ function and extends survival in septic shock. Repurposing and eventual clinical introduction of this clinically approved PARP inhibitor may be warranted for the experimental therapy of septic shock.

Type IV Collagen Is Essential for Proper Function of Integrin-Mediated Adhesion in Drosophila Muscle Fibers.

Congenital muscular dystrophy (CMD), a subgroup of myopathies is a genetically and clinically heterogeneous group of inherited muscle disorders and is characterized by progressive muscle weakness, fiber size variability, fibrosis, clustered necrotic fibers, and central myonuclei present in regenerating muscle. Type IV collagen (COL4A1) mutations have recently been identified in patients with intracerebral, vascular, renal, ophthalmologic pathologies and congenital muscular dystrophy, consistent with diagnoses of Walker-Warburg Syndrome or Muscle-Eye-Brain disease. Morphological characteristics of muscular dystrophy have also been demonstrated Col4a1 mutant mice. Yet, several aspects of the pathomechanism of COL4A1-associated muscle defects remained largely uncharacterized. Based on the results of genetic, histological, molecular, and biochemical analyses in an allelic series of Drosophila col4a1 mutants, we provide evidence that col4a1 mutations arise by transitions in glycine triplets, associate with severely compromised muscle fibers within the single-layer striated muscle of the common oviduct, characterized by loss of sarcomere structure, disintegration and streaming of Z-discs, indicating an essential role for the COL4A1 protein. Features of altered cytoskeletal phenotype include actin bundles traversing over sarcomere units, amorphous actin aggregates, atrophy, and aberrant fiber size. The mutant COL4A1-associated defects appear to recapitulate integrin-mediated adhesion phenotypes observed in RNA-inhibitory Drosophila. Our results provide insight into the mechanistic details of COL4A1-associated muscle disorders and suggest a role for integrin-collagen interaction in the maintenance of sarcomeres.

Sensitivity of Helicobacter pylori detection by Giemsa staining is poor in comparison with immunohistochemistry and fluorescent in situ hybridization and strongly depends on inflammatory activity.

BACKGROUND: Conventional stainings (including H&E and special stains like Giemsa) are the most widely applied histopathologic detection methods of Helicobacter pylori (HP). MATERIALS AND METHODS: We aimed to compare the diagnostic performance of Giemsa staining with immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) on a monocentric cohort of 2896 gastric biopsies and relate results to histologic alterations in order to find such histopathologic subgroups in which these methods underperform. All cases were categorized regarding presence or absence of chronic gastritis, inflammatory activity, and mucosal structural alterations. RESULTS: Giemsa revealed 687 cases (23.7%), IHC 795 cases (27.5%), and FISH 788 cases (27.2%) as being HP positive. Giemsa showed significantly lower overall sensitivity (83.3%) compared to IHC (98.8%) and FISH (98.0%). Moreover, the sensitivity of Giemsa dramatically dropped to 33.6% in the nonactive cases. We found that sensitivity of Giemsa strongly depends on HP density and, accordingly, on the presence of activity. Structural alterations (intestinal metaplasia, atrophy, etc.) had only no or weak effect on sensitivity of the three stainings. Both IHC and FISH proved to be equally reliable HP detecting techniques whose diagnostic performance is minimally influenced by mucosal inflammatory and structural alterations contrary to conventional stainings. CONCLUSIONS: We highly recommend immunohistochemistry for clinically susceptible, nonactive chronic gastritis cases, if the conventional stain-based HP detection is negative. Moreover, we recommend to use IHC more widely as basic HP stain. Helicobacter pylori FISH technique is primarily recommended to determine bacterial clarithromycin resistance. Furthermore, it is another accurate diagnostic tool for HP.

Laser-based methods for the analysis of low molecular weight compounds in biological matrices.

Laser-based desorption and/or ionization methods play an important role in the field of the analysis of low molecular-weight compounds (LMWCs) because they allow direct analysis with high-throughput capabilities. In the recent years there were several new improvements in ionization methods with the emergence of novel atmospheric ion sources such as laser ablation electrospray ionization or laser diode thermal desorption and atmospheric pressure chemical ionization and in sample preparation methods with the development of new matrix compounds for matrix-assisted laser desorption/ionization (MALDI). Also, the combination of ion mobility separation with laser-based ionization methods starts to gain popularity with access to commercial systems. These developments have been driven mainly by the emergence of new application fields such as MS imaging and non-chromatographic analytical approaches for quantification. This review aims to present these new developments in laser-based methods for the analysis of low-molecular weight compounds by MS and several potential applications.

[Vitamin D metabolism and signaling in human hepatocellular carcinoma and surrounding non-tumorous liver]. / A D-vitamin metabolizmusa humán hepatocellularis carcinomában és az azt körülvevo tumormentes májszövetben.

INTRODUCTION: 1,25-Dihydroxy vitamin D3 mediates antitumor effects in hepatocellular carcinoma. AIM: We examined mRNA and protein expression differences in 1,25-Dihydroxy vitamin D3-inactivating CYP24A1, mRNA of activating CYP27B1 enzymes, and that of VDR between human hepatocellular carcinoma and surrounding non-tumorous liver. METHODS: Snap-frozen tissues from 13 patients were studied for mRNA and protein expression of CYP24A1. Paraffin-embedded tissues from 36 patients were used to study mRNA of VDR and CYP27B1. mRNA expression was measured by RT-PCR, CYP24A1 protein was detected by immunohistochemistry. RESULTS: Expression of VDR and CYP27B1 was significantly lower in hepatocellular carcinoma compared with non-tumorous liver (p<0.05). The majority of the HCC samples expressed CYP24A1 mRNA, but neither of the non-tumorous liver. The gene activation was followed by CYP24A1 protein synthesis. CONCLUSIONS: The presence of CYP24A1 mRNA and the reduced expression of VDR and CYP27B1 mRNA in human hepatocellular carcinoma samples indicate decreased bioavailability of 1,25-Dihydroxy vitamin D3, providing an escape mechanism from the anti-tumor effect. Orv. Hetil., 2016, 157(48), 1910-1918.