Cause of In-Hospital Death After Weaning from Venoarterial-Extracorporeal Membrane Oxygenation.

PURPOSE: A survival gap between weaning from venoarterial-extracorporeal membrane oxygenation (VA-ECMO) and the hospital discharge has been consistently reported. The aim of this study is to investigate the clinical features of patients who underwent successful VA-ECMO decannulation at our institution and to identify the major contributors responsible for adverse outcomes. METHODS: We retrospectively reviewed all patients supported with VA-ECMO in our institution between January 2013 and June 2020. Only patients that survived VA-ECMO and underwent successful decannulation were included and dichotomized based on survival to hospital discharge: non-survivors versus survivors. The primary study outcome was the cause of death after successful VA-ECMO decannulation. RESULTS: Of the 262 adult patients who underwent VA-ECMO decannulation, 72 (27.5%) patients did not survive to hospital discharge. Non-survivors were older (62 vs. 54 years, p < 0.001) and suffering from many pre-existing comorbidities. Pneumonia and sepsis were the most frequent infectious complication and almost twice as likely in non-survivors. Major causes of death were: cardiovascular (31.9%), infections (25.0%) and neurological (20.8%). The survival curve demonstrated that 51.4% of our patients died within 8 days after decannulation. Multivariate analysis identified older age, central venous cannulation, pulmonary bleeding and infection, dialysis after VA-ECMO, sepsis, and ischemic stroke (OR = 7.86, 95% CI: 2.76-2.43, p < 0.001) as factors significantly predisposing to patients' death. CONCLUSION: In our study, one-third of patients decannulated off VA-ECMO did not survive to hospital discharge due to end-stage heart failure, infections or neurological injury. The first 8 post-decannulation days were recognized as a critical period where thorough strategies to prevent acquired infections and cautious support of end-organ function should be warranted. Future large-scale trials are needed to confirm our results.

Non-phosphorylated Tyr-1248 form of human epidermal growth factor receptor 2 (HER2) predicts resistance to trastuzumab therapy and poor disease-free survival of HER2-positive breast cancer patients.

AIM: To determine the predictive value of phosphorylated human epidermal growth factor receptor 2 (pHER2Y1248) status in breast cancer (BC) patients undergoing trastuzumab-based adjuvant therapy. METHODS: Immunohistochemical status of pHER2Y1248, EGFR/HER1, HER3, and HER4 was determined in 124 consecutive HER2-positive BC patients (median age [range]=57 years [49.0-64.0]) treated at the University Hospital for Tumors, Zagreb, between 2008 and 2011. The median follow-up was 84 months (60.0-84.0). Prognostic factors of disease free survival (DFS) rate were evaluated with Kaplan-Meier/log-rank test and Cox regression analysis. RESULTS: pHER2Y1248, HER1, HER3, and HER4 were expressed in 66.1%, 9.7%, 70.2%, and 71.0% of patients, respectively. Disease progression (DP) was observed in 17.1% of pHER2Y1248-positive and 47.6% of pHER2Y1248-negative BCs (P=0.001). Kaplan-Meier analysis showed a worse five-year DFS in pHER2Y1248-negative patients who were older than 60 years (P<0.001) and had positive lymph node status (P<0.001); tumor size >2.0 cm (P<0.001); higher histological grade (P<0.001); HER2E intrinsic subtype (P<0.001), negative hormone receptors (P<0.001); negative HER1 status (P<0.001), positive HER3 (P=0.002); and/or positive HER4 (P=0.002) status. The only negative prognostic factor for five-year DFS in multivariate Cox regression analysis was pHER2Y1248-negative (hazard ratio [HR] 3.6, 95% confidence interval [CI] 1.8-7.2, P<0.001) and lymph node-positive status (HR 3.6, 95% CI 1.3-9.8, P=0.014). CONCLUSION: pHER2Y1248 predicts sensitivity to trastuzumab and a better five-year DFS regardless of any other prognostic parameter. In HER2-positive BC patients. Non-phosphorylated HER2Y1248 is a strong predictor of trastuzumab resistance and a poor DFS.

Enhanced Recovery After Surgery in Patients Implanted with Left Ventricular Assist Device.

INTRODUCTION: We sought to develop and implement a comprehensive enhanced recovery after surgery (ERAS) protocol for patients implanted with a left ventricular assist device (LVAD). METHODS AND RESULTS: In this article, we describe our approach to the development and phased implementation of the protocol. Additionally, we reviewed prospectively collected data for patients who underwent LVAD implantation at our institution from February 2019 to August 2020. To compare early outcomes in our patients before and after protocol implementation, we dichotomized patients into two 6-month cohorts (the pre-ERAS and ERAS cohorts) separated from each other by 6 months to allow for staff adoption of the protocol. Of the 115 LVAD implants, 38 patients were implanted in the pre-ERAS period and 46 patients in the ERAS period. Preoperatively, the patients` characteristics were similar between the cohorts. Postoperatively, we observed a decrease in bleeding (chest tube output of 1006 vs 647.5 mL, P < .001) and blood transfusions (fresh frozen plasma 31.6% vs 6.7%, P = .04; platelets 42.1% vs 8.7%, P = .001). Opioid prescription at discharge were 5-fold lower with the ERAS approach (P < .01). Furthermore, the number of patients discharged to a rehabilitation facility decreased significantly (20.0% vs 2.4%, P = .02). The index hospitalization length of stay and survival were similar between the groups. CONCLUSIONS: ERAS for patients undergoing LVAD implantation is a novel, evidence-based, interdisciplinary approach to care with multiple potential benefits. In this article, we describe the details of the protocol and early positive changes in clinical outcomes. Further studies are needed to evaluate benefits of an ERAS protocol in an LVAD population.Lay Summary: Enhanced recovery after surgery (ERAS) is the implementation of standardized clinical pathways that ensures the use of best practices and decreased variation in perioperative care. Multidisciplinary teams work together on ERAS, thereby enhancing communication among health care silos. ERAS has been used for more than 30 years by other surgical services and has been shown to lead to a decreased length of stay, fewer complications, lower mortality, fewer readmissions, greater job satisfaction, and lower costs. Our goal was to translate these benefits to the perioperative care of complex patients with a left ventricular assist device. Early results suggest that this goal is possible; we have observed a decrease in transfusions, discharge on opioids, and discharge to a rehabilitation facility.

THE FREQUENCY OF FOLLICLE-STIMULATING HORMONE RECEPTOR 2039A>G GENE POLYMORPHISM AND THE RISK OF MALE INFERTILITY IN ALBANIAN POPULATION.

The purpose of this study was to determine the prevalence of allele and genotype variants of the follicle-stimulating hormone receptor (FSHR) gene polymorphic region at position Asn680Ser in the Albanian male population and associate them with the clinical parameters of infertility. The study included 114 infertile men (mean age 35.04±5.85 years) stratified according to the level of spermatogenetic impairment (oligoasthenozoospermia, asthenozoospermia and normospermia) and 112 fertile men (mean age 36.44±7.05 years) with normal semen parameters. Genotyping of the FSHR gene at position 680 was performed by TaqMan genotyping assay. All the participants underwent semen analysis, and serum reproductive hormones (FSH, luteinizing hormone, prolactin and testosterone) were also measured. The FSHR Asn680Ser genotype frequencies were as follows: Asn/Ser 42%, Ser/Ser 33.9% and Asn/Asn 24.1% in the control group, and Asn/Ser 56.1%, Ser/Ser 22.8% and Asn/Asn 21.1% in the whole group of infertile men (χ2-test: P=0.08). There was no statistically significant correlation between serum hormone levels and semen characteristics or between fertility status and FSHR Asn680Ser gene variants in the control group and the group of infertile men. However, adjusted logistic regression analysis (age, body mass index, smoking and alcohol as covariates) revealed increased odds ratio for male infertility among heterozygous Asn/Ser genotype carriers associated with lower values of semen parameters (normal morphology, concentration, total sperm count and motility). In conclusion, our case-control study further confirmed previous reports on no significant association between the FSHR Asn680Ser polymorphisms and male infertility. Nevertheless, the data presented herein indicate that the Asn/Ser genotype may increase the risk of male infertility in Albanian population.

Association between Circulatory and Plaque Resistin Levels with Carotid Plaque Instability and Ischemic Stroke Events.

ses of ischemic stroke. The risk of ischemic stroke increases with the degree of carotid stenosis and plaque vulnerability. The aim of this study was to investigate the association of circulating and plaque resistin levels with plaque vulnerability and ischemic stroke events in patients with moderate- to high-grade carotid artery stenosis. METHODS: 40 patients with ischemic stroke events and 38 neurologically asymptomatic patients scheduled for carotid endarterectomy were recruited for this study. Fasting blood samples for laboratory analysis were collected preoperatively and serum resistin levels were measured by enzyme-linked immunosorbent assays. Carotid endarterectomy specimens were analyzed according to the gold-standard procedure of histological classification. Plaque resistin expression was determined by standard immunohistochemical procedure. RESULTS: Serum resistin levels and resistin plaque expression were found to be significantly higher in subjects with unstable carotid plaque (P < .001) while significantly higher serum resistin levels were also present in patients with ischemic stroke events (P < .001). In univariate stepwise logistic regression analysis, higher serum resistin levels were significantly associated with plaque instability (OR 2.223, 95% CI 1.488-3.320, P < .0001) and ischemic stroke events (OR 1.237, 95% CI 1.079-1.420, P = .002). There was also a significant association between higher serum and plaque resistin expression (OR 1.663, 95% CI1.332-2.077, P < .0001). These associations remained significant in all models of multivariate logistic regression analysis. High serum and plaque resistin levels were also significantly associated with specific histological features of plaque instability. CONCLUSION: The results suggests that serum resistin levels may be used as a potential biomarker of plaque vulnerability and ischemic stroke events in patients with moderate- to high-grade carotid artery stenosis and highlight the possible relationship that plaque resistin expression has with histological features of plaque vulnerability.

Association of polymorphic variants in serotonin re-uptake transporter gene with Crohn's disease: a retrospective case-control study.

AIM: To analyze the distribution of SLC6A4 gene polymorphisms in Crohn's disease (CD) patients and their association with the disease. METHODS: We evaluated the presence/absence of promoter (5-HTTLPR, rs25531) and intron 2 (STin2 VNTR) polymorphic variants of SLC6A4 gene in a retrospective case-control study including 192 CD patients and 157 healthy controls (HC). Genotyping was performed by polymerase chain reaction. The association of polymorphisms with CD and its clinical subtypes was analyzed using χ2 and Fisher exact test, binary logistic regression, and haplotype analysis. RESULTS: CD patients and healthy controls had similar sex (88 [45.8%] vs 84 [53.5%] women, respectively; P=0.154) and age (41.3±12.8 years vs 41.7±8.8 years, respectively, P=0.091) distribution. Significant differences were observed in the STin2 genotype and allele distribution between CD patients and healthy controls (P=0.003 and P=0.002, respectively) and between the corresponding female subgroups (P=0.004 and P=0.007, respectively), with a significant negative association of biallelic ss (STin2.9 and Stin2.10) STin2 genotype with CD (P=0.013, age- and sex-adjusted odds ratio [OR] 0.5, 95% confidence interval [CI] 0.29-0.86; women: P=0.006, age-adjusted OR 0.32, 95% CI 0.14-0.72) and a significantly higher S-STin2.12 (5-HTTLPR/rs25531: S-STin2: STin2.12) haplotype distribution in CD patients (P=0.004, OR 1.62, 95% CI 1.16-2.26). There was no significant association between 5-HTTLRP and rs25531 genotype or allele frequencies and CD and between any SLC6A4 polymorphic loci with clinical CD subtypes. CONCLUSION: STin2 VNTR polymorphism of SLC6A4 gene may contribute to CD pathogenesis.

Comparison of vitamin K and non-vitamin K oral anticoagulants and the bleeding frequency in the emergency department.

INTRODUCTION: Safety studies of anticoagulant therapy have so far been conducted on many subjects in controlled conditions (i.e., clinically monitored) and demonstrated the noninferiority of new ones over old anticoagulant drugs. Data on the propositions for the presence of symptoms and signs of bleeding among various anticoagulants in the emergency department indicate that these data do not match the data published so far. AIM: The aim of the study was to investigate the differences in the frequency of bleeding and bleeding-related symptoms as a reason for emergency department attendance in patients on anticoagulant therapy. METHODS: The study included patients from the emergency department of University Hospital for one year, who were on anticoagulant therapy and who met the inclusion criteria. Out of a total of 595 patients, 409 were on warfarin (68.74%), and the rest were taking direct oral anticoagulants (DOAC): dabigatran 71 (11.93%), rivaroxaban 66 (11.09%) and apixaban 49 (8.23%). RESULTS: Out of 409 patients taking warfarin, 34.4% were adequately anticoagulated with the frequency of bleeding 13.7%, while in 57.2% of patients, PT INR was higher than the reference values with the frequency of bleeding 15.0%. A comparison between all DOAC groups and adequately anticoagulated warfarin patients in the frequency of bleeding and bleeding-related symptoms as a reason for emergency attendance yielded a difference that was marginally statistically significant (Pearson Chi-Square = 7.554, p = 0.052). CONCLUSION: Monitoring the frequency of bleeding and bleeding-related symptoms in patients on oral anticoagulant therapy as a reason for emergency department attendance may be a new safety and efficacy factor in real-life patient scenarios.

WNT5A, ß­catenin and SUFU expression patterns, and the significance of microRNA deregulation in placentas with intrauterine growth restriction.

Placental insufficiency is a common cause of intrauterine growth restriction (IUGR). It affects ~10% of pregnancies and increases fetal and neonatal morbidity and mortality. Although Wnt and Hh pathways are crucial for embryonic development and placentation, their role in the pathology of IUGR is still not sufficiently explored. The present study analyzed the expression of positive regulators of the Wnt pathway, WNT5A and ß­catenin, and the expression of the Hh pathway negative regulator suppressor of fused (SUFU). Immunohistochemical and reverse transcription­quantitative PCR (RT­qPCR) assays were performed on 34 IUGR and 18 placental tissue samples from physiologic singleton­term pregnancies. Epigenetic mechanisms of SUFU gene regulation were also investigated by methylation­specific PCR analysis of its promoter and RT­qPCR analysis of miR­214­3p and miR­378a­5p expression. WNT5A protein expression was higher in endothelial cells of placental villi from IUGR compared with control tissues. That was also the case for ß­catenin protein expression in trophoblasts and endothelial cells and SUFU protein expression in trophoblasts from IUGR placentas. The SUFU gene promoter remained unmethylated in all tissue samples, while miR­214­3p and miR­378a­5p were downregulated in IUGR. The present results suggested altered Wnt and Hh signaling in IUGR. DNA methylation did not appear to be a mechanism of SUFU regulation in the pathogenesis of IUGR, but its expression could be regulated by miRNA targeting.

Clinical and histopathological characteristics of COL4A3 c.2881+1G>A variant causing Alport spectrum disorders in Croatian population.

Alport syndrome (AS) and thin basement membrane nephropathy (TBMN) are part of the spectrum of kidney disorders caused by pathogenic variants in α3, α4, or α5 chains of the collagen type IV, the major structural component of the glomerular basement membrane (GBM). Using targeted next-generation sequencing (NGS), 34 AS/TBMN patients (58.8% male) from 12 unrelated families were found positive for heterozygous c.2881+1G>A variant of the COL4A3gene, that is considered disease-causing. All patients were from the continental or island part of Croatia. Clinical, laboratory, and histopathological data collected from the medical records were analyzed and compared to understand the clinical course and prognosis of the affected patients. At the time of biopsy or first clinical evaluation, the mean age was 31 years (median: 35 years; range: 1 - 72 years). Hematuria was present in 33 patients (97.1%) and 19 (55.9%) patients had proteinuria. There were 6 (17.6%) patients with hearing loss, 4 (11.8%) with ocular lesions, and 11 (32.4%) with hypertension. Twenty-three (67.6%) patients had proteinuria at follow-up, and 5 (14.7%) patients with the median age of 48 years (range: 27-55) progressed to kidney failure, started dialysis, or underwent kidney transplantation. Of the 13 patients who underwent kidney biopsy, 4 (30.8%) developed focal segmental glomerulosclerosis (FSGS), and 8 (66.7%) showed lamellation of the GBM, including all patients with FSGS. It is essential to conduct a detailed analysis of each collagen type IV genetic variant to optimize the prognosis and therapeutic approach for affected patients.

Less Invasive Left Ventricular Assist Device Implantation Is Safe and Feasible in Patients With Smaller Body Surface Area.

OBJECTIVE: Smaller body surface area (BSA) frequently precludes patients from left ventricular assist device (LVAD) therapy. We sought to investigate the clinical outcomes in patients with small BSA undergoing less invasive LVAD implantation. METHODS: We conducted a retrospective review of 216 patients implanted with HeartMate 3 LVAD (Abbott, Chicago, IL) via less invasive surgery at our institution. Patients were dichotomized based on their preimplant BSA for comparison between small BSA (≤1.8 m2) and normal/large BSA (>1.8 m2). We analyzed patient perioperative characteristics and outcomes. RESULTS: In our study, small BSA was found in 32 patients (14.8%), while 184 patients (85.2%) had normal/large BSA. Women were more prevalent in the small BSA group (50.0% vs 13.0%, P < 0.001). Preoperative and intraoperative data showed comparable results. Major complications and hospital length of stay did not differ by BSA group. Patients with smaller BSA had significantly decreased pump parameters at discharge, including LVAD flow (4.11 ± 0.49 vs 4.60 ± 0.54 L/min, P < 0.001) and pump speed (5,200 vs 5,400 rpm, P < 0.001). Survival to discharge and within 6 months after implantation were similar between the groups. CONCLUSIONS: Our study results suggest that less invasive HeartMate 3 implantation can be safely performed and demonstrates equivalent outcomes in patients with smaller body habitus. Randomized trials are required to confirm our data.

Dishevelled family proteins (DVL1-3) expression in intrauterine growth restriction (IUGR) placentas.

Dishevelled family proteins (DVL1, DVL2, and DVL3) are cytoplasmic proteins that are involved in canonical and non-canonical Wnt signaling pathway during embryonic development. The role of DVL proteins in the placental tissue remains mostly unknown. In the current study, we explored the role of Dishevelled proteins in naturally invasive tissue, trophoblast. Formalin-fixed paraffin-embedded samples of 15 term placentas from physiologic term pregnancies and 15 term placentas from pregnancies complicated with intrauterine growth restrictions (IUGR) were used for the study. Expression levels of mRNA for DVL1, DVL2, and DVL3 in placentas were analyzed by quantitative real-time PCR (qRTPCR). DVL1, DVL2, and DVL3 protein expression were semi-quantitatively analyzed using immunohistochemistry. The expression of DVL2 and DVL3 proteins was significantly higher in trophoblasts in placental villi from IUGR pregnancies compared with the control group of term placentas. In contrast, DVL3 protein expression was significantly higher in endothelial cells in placental villi from IUGR pregnancies compared with normal term placentas. The observed differences at protein levels between normal and IUGR placentas were not confirmed at the mRNA levels of DVL genes. Our data indicate the active involvement of DVL proteins in IUGR-related placentas. No significant changes were observed in DVL mRNA levels between the two groups of placentas. Further studies are required to explore the clinical relevance of these observations.

Aberrant expression of SFRP1, SFRP3, DVL2 and DVL3 Wnt signaling pathway components in diffuse gastric carcinoma.

Diffuse gastric carcinoma (DGC) is characterized by poorly cohesive cells, highly invasive growth patterns, poor prognosis and resistance to the majority of available systemic therapeutic strategies. It has been previously reported that the Wnt/ß-catenin signaling pathway serves a prominent role in the tumorigenesis of gastric carcinoma. However, the mechanism underlying the dysregulation of this pathway in DGC has not been fully elucidated. Therefore, the present study aimed to investigate the expression profiles of Wnt antagonists, secreted frizzled-related protein 1 (SFRP1) and secreted frizzled-related protein 3 (SFRP3), and dishevelled protein family members, dishevelled segment polarity protein 2 (DVL2) and dishevelled segment polarity protein 3 (DVL3), in DGC tissues. The association between the expression levels of these factors and the clinicopathological parameters of the patients was determined. Protein and mRNA expression levels in 62 DGC tumor tissues and 62 normal gastric mucosal tissues obtained from patients with non-malignant disease were measured using immunohistochemical and reverse transcription-quantitative PCR (RT-qPCR) analysis. Significantly lower protein expression levels of SFRP1 (P<0.001) and SFRP3 (P<0.001), but significantly higher protein expression levels of DVL2 (P<0.001) and DVL3 (P<0.001) were observed in DGC tissues compared with in control tissues by immunohistochemistry. In addition, significantly lower expression levels of SFRP1 (P<0.05) and higher expression levels of DVL3 (P<0.05) were found in in DGC tissues compared with those in normal gastric mucosal tissues using RT-qPCR. According to correlation analysis between the SFRP1, SFRP3, DVL2 and DVL3 protein expression levels and the clinicopathological characteristics of patients with DGC, a statistically significant correlation was found between the SFRP3 volume density and T stage (r=0.304; P=0.017) and between the SFRP3 volume density and clinical stage (r=0.336; P=0.008). In conclusion, the findings of the present study suggested that the Wnt signaling pathway components SFRP1, SFRP3, DVL2 and DVL3 may be aberrantly expressed in DGC tissues, implicating their possible role in the development of this malignant disease. The present data also revealed a positive relationship between SFRP3 protein expression and the clinical and T stage of DGC.

Study results suggest less invasive HeartMate 3 implantation is a safe and effective approach for obese patients.

BACKGROUND: Historically, obesity was considered a relative contraindication to left ventricular assist device (LVAD) implantation with less invasive surgery (LIS). The present study aimed to compare the outcomes of obese patients who underwent LVAD implantation through LIS with those who received full sternotomy (FS) implantation. METHODS: We retrospectively reviewed all patients implanted with HeartMate 3 LVAD in our institution between September 2015 and June 2020. Obese patients (BMI ≥ 30 kg/m2) were included and dichotomized based on surgical approach into the FS or LIS cohort. RESULTS: Of 231 implanted patients, 107 (46%) were obese and included in the study. FS was performed in 26 (24%) patients and LIS approach in 81 (76%) patients. Preoperative patient characteristics were similar between the cohorts. Postoperatively, patients in LIS cohort had less bleeding (p = 0.029), fewer transfusions (p = 0.042), shorter duration of inotropic support (p = 0.049), and decreased incidence of severe RV failure (11.1% vs 30.8%, p = 0.028). Survival to discharge for the obese population was 87.5% overall and did not differ based on an approach (91.4% LIS vs 76.9% FS, p = 0.079). More LIS patients were discharged home (60.0% vs 82.4%, p = 0.041) rather than to rehabilitation center. CONCLUSION: Our results showed that the LIS approach in obese patients is associated with fewer postoperative complications and a trend towards better short-term survival. These results suggest that less invasive LVAD implantation is a safe and effective approach for obese patients. Future prospective randomized trials are required to substantiate these results.

Dishevelled family proteins in serous ovarian carcinomas: a clinicopathologic and molecular study.

Dishevelled family proteins (DVL1, DVL2, and DVL3) are cytoplasmic mediators involved in canonical and non-canonical Wnt signaling that are important for embryonic development. Since Wnt signaling promotes cell proliferation and invasion, its increased activation is associated with cancer development as well. To get deeper insight into the behavior of Dishevelled proteins in cancer, we studied their expression in serous ovarian carcinomas [both low- (LGSC) and high-grade (HGSC)], and HGSC cell lines OVCAR5, OVCAR8, and OVSAHO. DVL protein expression in serous ovarian carcinomas tissues was analyzed using immunohistochemistry, while DVL protein and mRNA expressions in HGSC cell lines were analyzed using Western blot and quantitative real-time PCR. DVL1 protein expression was significantly higher in LGSC compared with normal ovarian tissue, while DVL3 was overexpressed in both LGSC and HGSC. DVL2 and DVL3 protein expression was higher in HGSC cell lines when compared with normal control cell line FNE1, while DVL1, DVL2, and DVL3 mRNA expression was significantly increased only in OVSAHO cell line. Survival analysis revealed no significant impact of DVL proteins on patients' outcome. Our data show an active involvement of Dishevelled family proteins in serous ovarian carcinomas. Further studies should confirm the clinical relevance of these observations.

Neuropeptide Y is expressed by osteocytes and can inhibit osteoblastic activity.

Osteocytes are the most abundant osteoblast lineage cells within the bone matrix. They respond to mechanical stimulation and can participate in the release of regulatory proteins that can modulate the activity of other bone cells. We hypothesize that neuropeptide Y (NPY), a neurotransmitter with regulatory functions in bone formation, is produced by osteocytes and can affect osteoblast activity. To study the expression of NPY by the osteoblast lineage cells, we utilized transgenic mouse models in which we can identify and isolate populations of osteoblasts and osteocytes. The Col2.3GFP transgene is active in osteoblasts and osteocytes, while the DMP1 promoter drives green fluorescent protein (GFP) expression in osteocytes. Real-time PCR analysis of RNA from the isolated populations of cells derived from neonatal calvaria showed higher NPY mRNA in the preosteocytes/osteocytes fraction compared to osteoblasts. NPY immunostaining confirmed the strong expression of NPY in osteocytes (DMP1GFP(+)), and lower levels in osteoblasts. In addition, the presence of NPY receptor Y1 mRNA was detected in cavaria and long bone, as well as in primary calvarial osteoblast cultures, whereas Y2 mRNA was restricted to the brain. Furthermore, NPY expression was reduced by 30-40% in primary calvarial cultures when subjected to fluid shear stress. In addition, treatment of mouse calvarial osteoblasts with exogenous NPY showed a reduction in the levels of intracellular cAMP and markers of osteoblast differentiation (osteocalcin, BSP, and DMP1). These results highlight the potential regulation of osteoblast lineage differentiation by local NPY signaling.

Epigenome alterations in aortic valve stenosis and its related left ventricular hypertrophy.

Aortic valve stenosis is the most common cardiac valve disease, and with current trends in the population demographics, its prevalence is likely to rise, thus posing a major health and economic burden facing the worldwide societies. Over the past decade, it has become more than clear that our traditional genetic views do not sufficiently explain the well-known link between AS, proatherogenic risk factors, flow-induced mechanical forces, and disease-prone environmental influences. Recent breakthroughs in the field of epigenetics offer us a new perspective on gene regulation, which has broadened our perspective on etiology of aortic stenosis and other aortic valve diseases. Since all known epigenetic marks are potentially reversible this perspective is especially exciting given the potential for development of successful and non-invasive therapeutic intervention and reprogramming of cells at the epigenetic level even in the early stages of disease progression. This review will examine the known relationships between four major epigenetic mechanisms: DNA methylation, posttranslational histone modification, ATP-dependent chromatin remodeling, and non-coding regulatory RNAs, and initiation and progression of AS. Numerous profiling and functional studies indicate that they could contribute to endothelial dysfunctions, disease-prone activation of monocyte-macrophage and circulatory osteoprogenitor cells and activation and osteogenic transdifferentiation of aortic valve interstitial cells, thus leading to valvular inflammation, fibrosis, and calcification, and to pressure overload-induced maladaptive myocardial remodeling and left ventricular hypertrophy. This is especcialy the case for small non-coding microRNAs but was also, although in a smaller scale, convincingly demonstrated for other members of cellular epigenome landscape. Equally important, and clinically most relevant, the reported data indicate that epigenetic marks, particularly certain microRNA signatures, could represent useful non-invasive biomarkers that reflect the disease progression and patients prognosis for recovery after the valve replacement surgery.

Cardiac myxoma the great imitators: comprehensive histopathological and molecular approach.

Cardiac myxomas are rare benign and slowly proliferating neoplasms of uncertain histogenesis with heterogeneous histomorphology and variable and sometimes clinically quite malignant pathological manifestations. Majority of cardiac myxoma occur sporadically while a relatively small proportion of diagnosed cases develop as a part of Carney complex syndrome with established familial pattern of inheritance. Although histologically indistinguishable these two forms of cardiac myxoma exhibit distinct cytogenetic make-up and apparent pathological differences important for their clinical presentation and prognosis. Additional problem is presented with secondary lesions with more aggressive histology and significantly faster cell proliferation suggesting their successive malignant alteration. Surgical resection of cardiac myxoma is currently the only treatment of choice. However, to avoid potentially hazardous operating procedures and possible postoperative complications and to prevent recurrence of the neoplastic lesions it is necessary to develop alternative approaches and identify a possible drug targets for their successful pharmacological treatment. Due to the rarity of the disease, a small number of cases in one institution and lack of comprehensive experimental data particularly concerning the cases of metastatic dissemination and secondary lesions with malignant nature, a comprehensive multi-institutional approach is required for better understanding of their molecular pathology and illumination of key molecular, genetic as well as epigenetic markers and regulatory pathways responsible for their development. In this article we provide comprehensive pathohistological, molecular and cytogenetic overview of sporadic cardiac myxoma cases restating the major hypothesis concerning their histogenesis and emphasizing potential approaches for their further reexamination.

Correlation of phosphorylated HER2 with clinicopathological characteristics and efficacy of trastuzumab treatment for breast cancer.

AIM: To determine the correlation of phosphorylated human epidermal growth factor receptor-2 (pHER2) with clinicopathological characteristics of breast cancer (BC) and patients' response to trastuzumab-based therapy. PATIENTS AND METHODS: pHER2 was determinated immuno-histochemically in 88 cases of HER2-positive and 50 cases of HER2-negative BC. All patients with HER2-positive BC received trastuzumab-based therapy and 16 of them (18.2%) had disease progression during therapy treatment (i.e. trastuzumab-resistant). RESULTS: pHER2 was predominantly expressed in HER2-positive BC, with 55 cases (62.5%) of tumours expressing pHER2. Six cases of HER2-negative cancer (12.5%) displayed positive expression of pHER2. Expression of pHER2 correlated with younger age of patients and negative oestrogen receptor status. Acquisition of resistance to trastuzumab correlated with negativity for pHER2 (p=0.028). CONCLUSION: Positive expression of pHER2 may yield additional information regarding the poor prognosis of BC and could be used for pre-selection of patients with HER2-overexpressing BC displaying resistance to trastuzumab treatment.

Identification of differentially expressed genes between osteoblasts and osteocytes.

Osteocytes represent the most abundant cellular component of mammalian bones with important functions in bone mass maintenance and remodeling. To elucidate the differential gene expression between osteoblasts and osteocytes we completed a comprehensive analysis of their gene profiles. Selective identification of these two mature populations was achieved by utilization of visual markers of bone lineage cells. We have utilized dual GFP reporter mice in which osteocytes are expressing GFP (topaz) directed by the DMP1 promoter, while osteoblasts are identified by expression of GFP (cyan) driven by 2.3 kb of the Col1a1 promoter. Histological analysis of 7-day-old neonatal calvaria confirmed the expression pattern of DMP1GFP in osteocytes and Col2.3 in osteoblasts and osteocytes. To isolate distinct populations of cells we utilized fluorescent activated cell sorting (FACS). Cell suspensions were subjected to RNA extraction, in vitro transcription and labeling of cDNA and gene expression was analyzed using the Illumina WG-6v1 BeadChip. Following normalization of raw data from four biological replicates, 3444 genes were called present in all three sorted cell populations: GFP negative, Col2.3cyan(+) (osteoblasts), and DMP1topaz(+) (preosteocytes and osteocytes). We present the genes that showed in excess of a 2-fold change for gene expression between DMP1topaz(+) and Col2.3cyan(+) cells. The selected genes were classified and grouped according to their associated gene ontology terms. Genes clustered to osteogenesis and skeletal development such as Bmp4, Bmp8a, Dmp1, Enpp1, Phex and Ank were highly expressed in DMP1topaz(+)cells. Most of the genes encoding extracellular matrix components and secreted proteins had lower expression in DMP1topaz(+) cells, while most of the genes encoding plasma membrane proteins were increased. Interestingly a large number of genes associated with muscle development and function and with neuronal phenotype were increased in DMP1topaz(+) cells, indicating some new aspects of osteocyte biology. Although a large number of genes differentially expressed in DMP1topaz(+) and Col2.3cyan(+) cells in our study have already been assigned to bone development and physiology, for most of them we still lack any substantial data. Therefore, isolation of osteocyte and osteoblast cell populations and their subsequent microarray analysis allowed us to identify a number or genes and pathways with potential roles in regulation of bone mass.

Twelve-year experience in identification of skeletal remains from mass graves.

AIM: To present twelve-year (1993-2005) experience in identification of human remains found in mass graves in Croatia and Bosnia and Herzegovina (BH), as well as remains that presumably belonged to Croatian citizens given by Serbia and Montenegro. The unique experience of identification of more than a thousand of skeletal samples is valuable for better organization of post-mortem identifications. METHODS: Standard forensic methods and methods based on DNA analysis were used for identification of human remains from mass graves. DNA was isolated using standard phenol/chloroform/isoamyl alcohol extraction. In some cases, decalcification and repurification were used prior to the extraction to overcome inhibition of amplification process. Different DNA systems were used for DNA quantitation and amplification (AluQuant, short tandem repeats (STR) commercial systems, Y chromosome STRs, and mitochondrial DNA [mtDNA]). Typing of PCR products was performed on AmpliType PM and AmpliType DQA1 DNA probe strips, ABI PRISM(R) 310 Genetic Analyzer and immobilized sequence-specific oligonucleotide (SSO) probes. RESULTS: Up-to-date analysis of 1,155 skeletal samples resulted in 703 positively identified bodies: 577 using standard forensic methods, 109 by DNA typing, and 17 by combination of these two methods. The majority of identifications from 1993 to 1999 was, as usual, achieved by standard forensic methods. Later on, these methods were not sufficient and DNA analysis was requested. It was performed in 42% of all cases in 12 years. The crucial step in DNA analysis is extraction of genomic DNA. Standard phenol/chloroform/isoamyl alcohol extraction, complemented with other methods and modifications, proved as the most successful method for this step. In certain cases, the quality and/or quantity of nDNA was not satisfying and the analysis of the mtDNA was performed. CONCLUSION: Our experience demonstrated that the advent of forensic DNA analysis methods greatly increased our ability to positively identify previously unknown skeletal remains by a comparative genetic analysis with presumptive relatives.