Search

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 (DVL1, DVL2, and DVL3) are cytoplasmic mediators involved in canonical and non-canonical Wnt signaling that are important in 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 (qRT-PCR). 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.

Aim To determine the predictive value of phosphorylat - ed human epidermal growth factor receptor 2 (pHER2Y1248) status in breast cancer (BC) patients undergoing trastu - zumab-based adjuvant therapy. Methods Immunohistochemical status of pHER2Y1248, EGFR/HER1, HER3, and HER4 was determined in 124 consec - utive HER2-positive BC patients (median age [range] =57 years [49.0-64.0]) treated at the University Hospital for Tu - mors, Zagreb, between 2008 and 2011. The median followup 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, respective - ly. 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 2.0 cm ( P

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/β-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

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 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. European Union through the Europe Regional Development Fund, Operational Programme Competitiveness and Cohesion, under grant agreement No. KK.01.1.1.01.0008 Qatar University: QUSD-CMED-2018-2

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.

Purpose The present study aimed to compare the outcomes of obese patients who underwent HeartMate 3 implantation through less invasive surgery (LIS) with those who received full sternotomy (FS) implantation. We hypothesized that LIS is a safe and effective technique for LVAD implantation in obese patients. Methods We performed a retrospective review of all patients implanted with HeartMate 3 LVAD in our institution from September 2015 through Jun 2020. Obese patients (BMI≥ 30 kg/m2) were included in the study and dichotomized based on surgical approach into the full sternotomy (FS) cohort or LIS. Primary outcomes were index hospitalization and 30-day survival. Secondary outcomes were incidence of index hospitalization major complications. Results Of 231 patients implanted patients, 107 (46.3%) were obese and included in the study. FS was used in 26 (24.3%) patients and LIS approach in 81 (75.7%) patients. Preoperative patient characteristics were similar between the cohorts (Table 1). In the immediate postoperative course, fewer patients in the LIS cohort received units of packed red blood cells (34.6% vs 57.7%, p=0.042) and fresh frozen plasma (19.8 vs 46.2%, p=0.011). Duration of postoperative inotropic support was shorter in LIS cohort (p=0.049). The incidence of major complications was similar between the groups. Decreased incidence of severe RV failure (30.8% vs 11.1% LIS, p=0.028) and need for RVAD implantation (p=0.037) were observed in LIS cohort. Survival to discharge for the obese population was 87.5% overall and did not differ based on an approach (76.9% FS vs 91.4% LIS, p=0.079). Conclusion These data demonstrate multiple potential benefits of the less invasive surgical technique, including fewer postoperative complications and a trend towards better survival to discharge. This study suggests that less invasive HeartMate 3 implantation is a safe and effective approach for obese patients. Future prospective randomized trials are required to confirm our data.

Objective: We can observe an increase in incidence and prevalence of patients with aortic valve stenosis in the general population. The gold standard in aortic valve therapy is aortic valve replacement. Preoperative planning is essential for good outcomes, as the severity of stenosis and calcifications can sometimes be extremely progressive and even involve the aortic root and ascending aorta. There is not enough research on comparation of CT scan analysis of aortic valve stenosis and echocardiography which is the golden standard of disease diagnosis.1-3 Patients and Methods: We have analyzed 88 patients [age: 70.01±9.066 (mean±SD) ; female: n=45, 51.1%]. Among the patients, 12 had bicuspid aortic valve leaflet structure while the rest of the patients (n=76, 86.4%) exhibited TAV stenosis. Degree of aortic stenosis was assessed according to mean pressure gradient (MPG), peak pressure gradient (PPG), aortic valve area (AVA) indexed aortic valve area (AVAi) and maximum speed through aortic valve (Vmax). These were compared with calcium score (AVCS) calculated from CT scan. All of these patients were observed in the operating room during surgery and valves analyzed after explantation. All of the patients underwent aortic valve replacement. Results: Average AVCS values (median + IQ range) were 3306.3 (1995.4 – 4820.6) [female: 2215 (1463.35 – 3372.85) ; male: 4093.5 (3133.3 – 5274.4). Average AVCS values for BAV patients were 3063.5 (3323.125 – 4868.9) and 3106.55 (1965.375) – 4780.125) for TAV patients. There were significant correlations between AVCS and AVAi (Spearman’s ρ=−0.24, P=0.025), PPG (ρ= 0.38, P< 0.001), MPG (ρ= 0.36, P= 0.001) V max (ρ= 0.37, P < 0.001) and gender (ρ= 0.485, P < 0.001) while AVA values showed no significant correlation with AVCS (ρ= -0.066, P = 0.540). Overall survival was similar not depending of severity of calcifications and stenosis, however clamp time and surgery time were longer for patients with severely calcified valves which means calcium scoring as a parameter should also be taken in consideration during preoperative planning. Conclusion: Careful preoperative planning is essential for good outcome of surgery, here we have proven the connection between echocardiography parameters of aortic stenosis and calcium score calculated by CT scan.

Considering that expression of miRNAs in maternal- placental-fetal unit may signal prenatal exposure to common environmental chemical pollutants, one of the aim of our research project on the assessment of daily exposure to main toxic metals and maternal individual susceptibility as factors of developmental origins of health and disease is to evaluate expression of selected miRNAs as epigenetic markers of prenatal exposure to tobacco smoke (TS) that is a mixture of harmful chemicals including highly reactive metal ions. Expression of miR-1537, miR-190b, miR-16, miR-21 and miR- 146a were measured by quantitative PCR using custom miScript miRNA PCR Array (Qiagen, DE) and cDNA synthesis (miScript II Reverse Transcription Kit) from isolated total miRNA (miRNeasy Plasma Kit) in the samples of blood plasma from healthy smoking (n = 13) and nonsmoking (n = 13) postpartum women who had vaginal term childbirth in the University Hospital in Zagreb, Croatia. Results were analyzed by ΔΔCT method of relative quantification for miRNA PCR arrays. MiR-1537 was not expressed, whereas fold changes for other four miRNAs indicated downregulation of miR-190b and miR-21 and upregulation of miR-146a in maternal plasma related to TS exposure. Our preliminary results on selected miRNAs add to a novel research approach in environmental epigenetics of metals. For final conclusions the data on biomarkers of TS and metal exposure and effects along with epigenetic markers should be taken into consideration.

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.

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 sexadjusted 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.

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.

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.

Background: Patients with preserved left ventricular ejection fraction (EF) and aortic stenosis have often increased global longitudinal strain as a sign of intrinsic left ventricular impairment. Heart failure due to left ventricular deformation is also shown to be predicted with simple blood count test like red cell distribution width (RDW). We evaluated the correlation of GLS and RDW patients with different severity stage of aortic stenosis and signs of left ventricular strain. Methods: We recorded relevant clinical, laboratory, and echocardiographic parameters, and measured global longitudinal strain (GLS) in 83 patients with EF > 45% and mild, moderate, and severe aortic stenosis (AS) in whom coronary artery disease was previously excluded. GLS was obtained using 2D speckle tracking echocardiography. RDW was easily obtained from full blood count that is routinely measured. Results: Mean velocity and aortic valve area was 2.73 m/s and 1.72 cm2, 3.37 m/s and 1, 33 cm2, and 4.62 m/s and 0.74 cm2 in patients with mild, moderate and severe AS, respectively. Mean GLS was -18.82%, -17.7% and -16.85% in patients with mild, moderate and severe AS, and did not differ significantly. Patients with severe aortic stenosis had significantly higher left ventricular mass index and NT-pro BNP levels compared to patients with mild and moderate AS (186.66 g/m2 vs 133.57 and 169.57 g/m2, and 2644.69 pg/ml vs 312.71 vs 403.2 pg/ml, respectively). Among patients with mild aortic stenosis regression analysis showed significant positive correlation of GLS with RDW (beta 0.590, R square 0.34, p=0.005). That correlation was not confirmed for GLS and NT-pro BNP. Conclusion: GLS did not differ significantly among patients with different severity of AS, although patients with severe AS had highest values of GLS. Among patients with moderate AS, RDW showed to be a good predictor of impaired left ventricular function measured by GLS, and therefore it could be further evaluated as a tool in early selection for treatment of patients with moderate AS, preserved EF and no coronary artery disease.

Background: Strain imaging is an established method for the accurate quantification of left ventricular function and left ventricular global longitudinal strain (GLS) has been shown to be a superior marker of contractility than ejection fraction (EF), and also a good marker for prognosis of patients with aortic stenosis (AS). We prospectively analyzed GLS in context to other relevant clinical parameters in patients with severe aortic stenosis and preserved EF. Patients and Methods: We preoperatively measured GLS in 42 patients with EF > 45% and severe AS. GLS was obtained using 2D speckle tracking echocardiography and patients were divided into four groups according to GLS quartiles. Relevant clinical and echocardiographic parameters were analyzed in relation to GLS, together with curve estimation regression analyses using GLS as independent variable. Results: In all, mean EF was 59.3% (SD 8.4%) and mean aortic valve area (AVA) was 0.74 cm2 (SD 0.17 cm2). Mean GLS was -16.85% (SD -3.58%) with four groups of patients according to quartiles. Group 1 consisted of 12 patients with mean GLS -20.46%, group 2 of 11 patients with mean GLS -18.85%, group 3 of 10 patients with mean GLS -15.43%, and group 4 represented 8 patients with mean GLS -10.67%. Among many clinical and echocardiographic parameters analyzed, groups differed significantly only by eGFR with group 3 having the lowest value (Kruskal Wallis, p=0.034). Curve estimation regression analysis using GLS as independent variable showed only statistically significant negative correlation of GLS with AVA (beta -0.347, R square 0.12, p=0.026). Left atrial volume index (LAVI) showed nearly significant positive correlation with GLS (beta 0.283, R square 0.08, p=0.06) whereas other parameters showed insignificant correlation. Conclusions: Almost one third of patients with preserved EF and severe AS have impaired left ventricular function measured with GLS. AVA and LAVI showed to be good predictors of impaired global ejection fraction. GLS should be used as an additional marker of left ventricular disfunction in all patients with severe aortic stenosis in order to select asymptomatic candidates for early treatment.

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.

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.

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.

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.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.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.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.

Heterochromatin characteristics have been studied in Leptinotarsa decemlineata belonging to the leaf beetles (Chrysomelidae) and compared to the thoroughly analysed heterochromatin of the mealworm beetle Tenebrio molitor, representing darkling beetles (Tenebrionidae). C-banding reveals heterochromatin in pericentromeric regions of all chromosomes of both species; however heterochromatic blocks of L. decemlineata are significantly smaller in size. Digestion of L. decemlineata genomic DNA with 12 restriction enzymes reveals presence of bands characteristic for repetitive DNA, but again in a significantly lower amount than those found in genomic DNA of T. molitor. While T. molitor exhibits complete correspondence of restriction enzyme effects on naked DNA and mitotic chromosomes, L. decemlineata mitotic chromosomes are resistant to the digestion with restriction enzymes. The results show that two species have similar organization of heterochromatin on chromosomes but differ significantly in its amount. In addition, difference in accessibility to in situ restriction enzyme digestion indicates specificity of heterochromatin structure in the two species., Proučavan je heterokromatin u krumpirove zlatice Leptinotarsa decemlineata kao predstavnice zlatnica (Chrysomelidae), te je uspoređen s detaljno opisanim heterokromatinom brašnara Tenebrio molitor, kao predstavnika crnokrilaca (Tenebrionidae). Metoda C-pruganja je pokazala prisutnost heterokromatina u pericentromernim područjima svih kromosoma obiju vrsta, iako je njegov udio znatno manji u krumpirove zlatice. Razgradnja genomske DNA krumpirove zlatice sa 12 restrikcijskih enzima otkrila je prisutnost ponovljene DNA, ali opet u znatno manjoj količini u odnosu na brašnara. Iako kod brašnara T. molitor postoji podudarnost u razgradnji DNA i mitotskih kromosoma restrikcijskim enzimima, mitotski kromosomi krumpirove zlatice su otporni na razgradnju restrikcijskim enzimima. Rezultati pokazuju da obje vrste imaju podjednaku organizaciju heterokromatina na kromosomima unatoč velikoj razlici u količini. Pored toga, razlika u razgradnji mitotskih kromosoma restrikcijskim enzimima ukazuje na specifičnost heterokromatinske strukture u ovih vrsta.

U ovom radu je lokaliziran i vizualiziran kostitutivni heterokromatin na kromosomima vrsta Tenebrio molitor, Tribolium madens i Palorus subdepressus, pri čemu su korištene metode C-pruganja, razgradnje restrikcijskim enzimima in situ i bojanja A-T pb bogatih regija kromosoma fluorokromima. Ujedno je određen smještaj satelitske DNA na kromosomima vrste Tenebrio molitor primjenom metode PRINS-a.