The Prediction Analysis of Microarray 50 (PAM50) Gene Expression Classifier Utilized in Indeterminate-Risk Breast Cancer Patients in Hungary: A Consecutive 5-Year Experience.

BACKGROUND: Breast cancer has been categorized into molecular subtypes using immunohistochemical staining (IHC) and fluorescence in situ hybridization (FISH) since the early 2000s. However, recent research suggests that gene expression testing, specifically Prosigna® Prediction Analysis of Microarray 50 (PAM50), provides more accurate classification methods. In this retrospective study, we compared the results of IHC/FISH and PAM50 testing. We also examined the impact of various PAM50 parameters on overall survival (OS) and progression-free survival (PFS). RESULTS: We analyzed 42 unilateral breast cancer samples, with 18 classified as luminal A, 10 as luminal B, 8 as Human epidermal growth factor receptor 2 (HER2)-positive, and 6 as basal-like using PAM50. Interestingly, 17 out of the 42 samples (40.47%) showed discordant results between histopathological assessment and the PAM50 classifier. While routine IHC/FISH resulted in classification differences for a quarter to a third of samples within each subtype, all basal-like tumors were misclassified. Hormone receptor-positive tumors (hazard rate: 8.7803; p = 0.0085) and patients who had higher 10-year recurrence risk scores (hazard rate: 1.0539; p = 0.0201) had shorter OS and PFS. CONCLUSIONS: Our study supports the existing understanding of molecular subtypes in breast cancer and emphasizes the overlap between clinical characteristics and molecular subtyping. These findings underscore the value of gene expression profiling, such as PAM50, in improving treatment decisions for breast cancer patients.

The Medicago truncatula nodule-specific cysteine-rich peptides, NCR343 and NCR-new35 are required for the maintenance of rhizobia in nitrogen-fixing nodules.

In the nodules of IRLC legumes, including Medicago truncatula, nitrogen-fixing rhizobia undergo terminal differentiation resulting in elongated and endoreduplicated bacteroids specialized for nitrogen fixation. This irreversible transition of rhizobia is mediated by host produced nodule-specific cysteine-rich (NCR) peptides, of which c. 700 are encoded in the M. truncatula genome but only few of them have been proved to be essential for nitrogen fixation. We carried out the characterization of the nodulation phenotype of three ineffective nitrogen-fixing M. truncatula mutants using confocal and electron microscopy, monitored the expression of defence and senescence-related marker genes, and analysed the bacteroid differentiation with flow cytometry. Genetic mapping combined with microarray- or transcriptome-based cloning was used to identify the impaired genes. Mtsym19 and Mtsym20 mutants are defective in the same peptide NCR-new35 and the lack of NCR343 is responsible for the ineffective symbiosis of NF-FN9363. We found that the expression of NCR-new35 is significantly lower and limited to the transition zone of the nodule compared with other crucial NCRs. The fluorescent protein-tagged version of NCR343 and NCR-new35 localized to the symbiotic compartment. Our discovery added two additional members to the group of NCR genes essential for nitrogen-fixing symbiosis in M. truncatula.

Short-term effects of a novel bronchial drainage device: A pilot cohort study in subjects with cystic fibrosis.

Background and Aims: In cystic fibrosis (CF) airways, impaired airway mucociliary clearance and mucus accumulation due to cystic fibrosis transmembrane conductance regulator defects contribute to inflammation, progressive structural lung damage, and decline of lung function. Physiotherapy is essential to promote mucus mobilization and removal in CF and is a key element of rehabilitation measures, but conventional techniques may be suboptimal to mobilize viscous mucus. This study aimed to test the specific effects of a novel bronchial drainage device (BDD) (Simeox®; PhysioAssist) in subjects with CF and evaluate lung function, diaphragm mobility, and sputum properties. Methods: This prospective monocentric clinical cohort study in the setting of outpatient physiotherapy of CF patients (n = 21) with stable CF lung disease collected pulmonary lung function tests (PFT), diaphragm mobility, and sputum properties before and after two physiotherapy sessions using the novel BDD. PFT was assessed using spirometry and diaphragm mobility using m-mode ultrasound analysis. Spontaneous sputum samples were collected before and after using the BDD and analyzed for microstructure and DNA concentrations. Results: PFT parameters (FEV1, FVC, MEF25/50/75) were not affected by the use of the BDD. Ultrasound analysis of diaphragm mobility revealed an increase in maximum diaphragm excursion upon the intervention. Mucus analysis demonstrated altered microstructure and higher DNA concentrations collected after using the BDD compared to samples collected before. Pearson correlation analysis showed significant correlations between changes in mucus properties and DNA levels in respective mucus samples. Conclusion: Our results demonstrate that the novel BDD improves diaphragm mobility and alters sputum properties in subjects with CF. The novel BDD with unique properties may be further studied as a device in CF-specific physiotherapy to facilitate sputum mobilization of CF patients.

Neuronal connections of the central amygdalar nucleus with refeeding-activated brain areas in rats.

Following fasting, satiety is accompanied by neuronal activation in brain areas including the central amygdalar nucleus (CEA). Since CEA is known to inhibit food intake, we hypothesized that CEA contributes to the termination of meal during refeeding. To better understand the organization of this satiety-related circuit, the interconnections of the CEA with refeeding-activated neuronal groups were elucidated using retrograde (cholera toxin-ß subunit, CTB) and anterograde (phaseolus vulgaris leucoagglutinin, PHA-L) tracers in male rats. C-Fos-immunoreactivity was used as marker of neuronal activation. The refeeding-activated input of the CEA primarily originated from the paraventricular thalamic, parasubthalamic and parabrachial nuclei. Few CTB-c-Fos double-labeled neurons were detected in the prefrontal cortex, lateral hypothalamic area, nucleus of the solitary tract (NTS) and the bed nuclei of the stria terminalis (BNST). Only few refeeding-activated proopiomelanocortin-producing neurons of the arcuate nucleus projected to the CEA. Anterograde tract tracing revealed a high density of PHAL-labeled axons contacted with refeeding-activated neurons in the BNST, lateral hypothalamic area, parasubthalamic, paraventricular thalamic and parabrachial nuclei and NTS; a low density of labeled axons was found in the paraventricular hypothalamic nucleus. Chemogenetic activation of the medial CEA (CEAm) inhibited food intake during the first hour of refeeding, while activation of lateral CEA had no effect. These data demonstrate the existence of reciprocal connections between the CEA and distinct refeeding-activated hypothalamic, thalamic and brainstem nuclei, suggesting the importance of short feedback loops in the regulation of satiety and importance of the CEAm in the regulation of food intake during refeeding.

Risk factors for multiple suicide attempts among Roma in Hungary.

In recent years, suicide rates in Hungary have been among the highest in the European Union. Attempted suicide rates in the Roma population are 2-3 times higher than in the non-Roma population. Since individuals making multiple attempts have a higher pro-bability of eventual death by suicide, and there are limited data on suicidal behaviour of the Roma population, the aim of this study was to explore the sociodemographic and psychological background factors of multiple suicide attempts in the Hungarian Roma population. Semistructured interviews were conducted with 150 individuals admitted to hospital toxicology departments, who made suicide attempts by deliberate self-poisoning, 65 of whom were multiple attempters. Detailed information regarding the current attempt and previous suicidal acts was recorded. Patients also completed the Shortened Beck Depression, the Beck Hopelessness Questionnaire, and the Social Support Questionnaire. Independent samples t-tests were used to evaluate differences in psychological variables between the Roma ( N = 90) and non-Roma ( N = 60) groups. Stepwise linear regression and odds ratios analyses were performed to identify potential background factors of multiple suicide attempts. There was a significantly higher level of previous suicidal events among the Roma in the sample population (3.53 vs. 0.84, p < .001). Roma ethnicity was found to be a strong predictor of multiple suicide attempts. Current major depression, hopelessness, and diagnosed mood disorder were identified as significant risk factors of repeated attempts. Smoking (OR = 5.4), family history of suicide (OR = 4.9), and long-term unemployment (OR = 4.6) were additional risk factors among Roma patients. A thorough understanding of the ethnicity-specific risk factors for multiple suicide attempts could facilitate the development of effective intervention and postvention programmes.

Pentoxifylline attenuates the local and systemic inflammatory response after infrarenal abdominal aortic ischemia-reperfusion.

AIMS: We studied the new anti-inflammatory effects of non-specific phosphodiesterase (PDE) inhibitor pentoxifylline (PTX) on ischaemia-reperfusion injury and postconditioning of the lower extremities. We aimed to examine the oxidative stress parameters (OSP), the inflammatory response and the changes in structure of skeletal muscle after revascularization surgery. METHODS: 50 Wistar rats in five groups underwent a 60 min infrarenal aortic cross clamping. After the ischaemia in IR+PC group ischemic postconditioning was performed, intermittent 15 seconds reperfusion, 15 seconds ischaemic periods were applied four times. The ischemic phase was followed by a 120 min of reperfusion. In IR+PTX group the animals were treated with PTX. In IR+PC+PTX group both ischemic postconditioning and PTX treatment were performed. Blood samples and biopsy from quadriceps muscle were collected. Plasma malondialdehyde, reduced glutathione, -SH-groups, TNF-alpha, IL-6 concentrations and superoxide dismutase enzyme activity were measured. RESULTS: The levels of OSP and the inflammatory proteins were significantly higher in the IR group. PTX treatment and PC could significantly decrease the levels of OSP and inflammatory proteins. When the animals were co-treated with PTX and PC the results were even better. CONCLUSIONS: Inhibition of PDE by PTX could markedly decrease the inflammatory response and moderate the ischaemia-reperfusion damages after lower limb ischemia and reperfusion. Administration of PTX could potentiate the beneficial effects of PC.

Growth inhibitory effect of rapamycin in Hodgkin-lymphoma cell lines characterized by constitutive NOTCH1 activation.

Growing evidence suggests that deregulation of signalling elements of Notch and mammalian target of rapamycin (mTOR) pathways contribute to tumorigenesis. These signals play important roles in cellular functions and malignancies. Their tumorigenic role in T-cell acute lymphoblastic leukaemia (T-ALL) is well known; however, their potential interactions and functions are poorly characterized in Hodgkin lymphoma (HL). The aim of our study was to characterize mTOR and Notch signalling elements in HL cell lines (DEV, L1236, KMH2) and human biopsies and to investigate their cross-talk in the tumorous process. High mTOR activity and constitutive NOTCH1 activation was confirmed in HL cell lines, without any known oncogenic mutations in key elements, including those common to both pathways. The anti-tumour effect of Notch inhibitors are well known from several preclinical models but resistance and side effects occur in many cases. Here, we tested mTOR and Notch inhibitors and their combinations in gamma-secretase inhibitor (GSI) resistant HL cells in vitro and in vivo. mTOR inhibitor alone or in combination was able to reduce tumour growth; furthermore, it was more effective in xenograft models in vivo. Based on these results, we suggest that constitutively activated NOTCH1 may be a potential target in HL therapy; furthermore, mTOR inhibitors may be effective for decreasing tumour growth if resistance to Notch inhibitors develop.

Chronic Activation of γ2 AMPK Induces Obesity and Reduces ß Cell Function.

Despite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease.

Cytosolic localization of NADH cytochrome b5 oxidoreductase (Ncb5or).

Acyl-CoA desaturation in the endoplasmic reticulum (ER) membrane depends on cytosolic NADH or NADPH, whereas NADPH in the ER lumen is utilized by prereceptor glucocorticoid production. It was assumed that NADH cytochrome b5 oxidoreductase (Ncb5or) might connect Acyl-CoA desaturation to ER luminal redox. We aimed to clarify the ambiguous compartmentalization of Ncb5or and test the possible effect of stearoyl-CoA on microsomal NADPH level. Amino acid sequence analysis, fluorescence microscopy of GFP-tagged protein, immunocytochemistry, and western blot analysis of subcellular fractions unequivocally demonstrated that Ncb5or, either endogenous or exogenous, is localized in the cytoplasm and not in the ER lumen in cultured cells and liver tissue. Moreover, the involvement of ER-luminal reducing equivalents in stearoyl-CoA desaturation was excluded.

Elevated level of lysine 9-acetylated histone H3 at the MDR1 promoter in multidrug-resistant cells.

Failure of chemotherapy in breast cancer presents a major problem and is often due to elevated expression of ATP binding cassette (ABC)-type transporters, such as MDR1 protein. It has been shown that MDR1/ABCB1 gene expression is regulated at the chromatin level by DNA methylation and histone acetylation. However, the modified histone residues have not been identified and the role of various histone acetyl transferases (HATs) is not fully understood. By studying a breast carcinoma model cell line and its MDR1-overexpressing derivative, we show that the histone 3 lysine 9 (H3K9) acetylation level is elevated 100-fold in the promoter and first exon of the MDR1 gene in the drug-resistant cell line compared to the drug-sensitive cell line. The acetylation level of the other examined lysine residues (H3K4, H3K14, H4K8, and H4K12) is weakly or not at all elevated in the MDR1 locus, although their acetylation is generally increased genome-wide in the drug-resistant cell. Downregulation of the expression of HATs PCAF and GCN5 by RNAi effectively reduces the expression of MDR1. Unexpectedly, treatment with a p300-selective inhibitor (HAT inhibitor II) further increases MDR1 expression and drug efflux in the drug-resistant cells. Our data suggest that repeated exposure to chemotherapy may result in deregulated histone acetylation genome-wide and in the MDR1 promoter.