DMBT1 expression is down-regulated in breast cancer.

BACKGROUND: We studied the expression of DMBT1 (deleted in malignant brain tumor 1), a putative tumor suppressor gene, in normal, proliferative, and malignant breast epithelium and its possible relation to cell cycle. METHODS: Sections from 17 benign lesions and 55 carcinomas were immunostained with anti DMBT1 antibody (DMBTh12) and sections from 36 samples, were double-stained also with anti MCM5, one of the 6 pre-replicative complex proteins with cell proliferation-licensing functions. DMBT1 gene expression at mRNA level was assessed by RT-PCR in frozen tissues samples from 39 patients. RESULTS: Normal glands and hyperplastic epithelium in benign lesions displayed a luminal polarized DMBTh12 immunoreactivity. Normal and hyperplastic epithelium adjacent to carcinomas showed a loss of polarization, with immunostaining present in basal and perinuclear cytoplasmic compartments. DMBT1 protein expression was down-regulated in the cancerous lesions compared to the normal and/or hyperplastic epithelium adjacent to carcinomas (3/55 positive carcinomas versus 33/42 positive normal/hyperplastic epithelia; p = 0.0001). In 72% of cases RT-PCR confirmed immunohistochemical results. Most of normal and hyperplastic mammary cells positive with DMBTh12 were also MCM5-positive. CONCLUSIONS: The redistribution and up-regulation of DMBT1 in normal and hyperplastic tissues flanking malignant tumours and its down-regulation in carcinomas suggests a potential role in breast cancer. Moreover, the concomitant expression of DMTB1 and MCM5 suggests its possible association with the cell-cycle regulation

Therapeutic Effect of Iron Citrate in Blocking Calcium Deposition in High Pi-Calcified VSMC: Role of Autophagy and Apoptosis

In chronic kidney disease (CKD), the first cause of mortality is cardiovascular disease induced mainly by vascular calcification (VC). Recently, iron-based phosphate binders have been proposed in advanced CKD to treat hyperphosphatemia. We studied the effect of iron citrate (iron) on the progression of calcification in high-phosphate (Pi) calcified VSMC. Iron arrested further calcification when added on days 7\u201315 in the presence of high Pi (1.30 \ub1 0.03 vs 0.61 \ub1 0.02; OD/mg protein; day 15; Pi vs Pi + Fe, p < 0.01). We next investigated apoptosis and autophagy. Adding iron to high-Pi-treated VSMC, on days 7\u201311, decreased apoptotic cell number (17.3 \ub1 2.6 vs 11.6 \ub1 1.6; Annexin V; % positive cells; day 11; Pi vs Pi + Fe; p < 0.05). The result was confirmed thorough analysis of apoptotic nuclei both in VSMCs and aortic rings treated on days 7\u201315 (3.8 \ub1 0.2 vs 2.3 \ub1 0.3 and 4.0 \ub1 0.3 vs 2.2 \ub1 0.2; apoptotic nuclei; arbitrary score; day 15; Pi vs Pi + Fe; VSMCs and aortic rings; p < 0.05). Studying the prosurvival axis GAS6/AXL, we found that iron treatment on days 9\u201314 counteracted protein high-Pi-stimulated down-regulation and induced its de novo synthesis. Moreover, iron added on days 9\u201315 potentiated autophagy, as detected by an increased number of autophagosomes with damaged mitochondria and an increase in autophagic flux. Highlighting the effect of iron on apoptosis, we demonstrated its action in blocking the H2O2-induced increase in calcification added both before high Pi treatment and when the calcification was already exacerbated. In conclusion, we demonstrate that iron arrests further high Pi-induced calcium deposition through an anti-apoptotic action and the induction of autophagy on established calcified VSMC

Dynamic acetylation profile during mammalian neurulation

Neural tube defects (NTDs) result from failure of neural tube closure during embryogenesis. These severe birth defects of the central nervous system include anencephaly and spina bifida, and affect 0.5-2 per 1,000 pregnancies worldwide in humans. It has been demonstrated that acetylation plays a pivotal role during neural tube closure, as animal models for defective histone acetyltransferase proteins display NTDs. Acetylation represents an important component of the complex network of posttranslational regulatory interactions, suggesting a possible fundamental role during primary neurulation events. This study aimed to assess protein acetylation contribution to early patterning of the central nervous system both in human and murine specimens

Integrin ανβ5 in vitro inhibition limits pro-fibrotic response in cardiac fibroblasts of spontaneously hypertensive rats

BackgroundTo date the TGF-1 activation mediated by integrin 5 during fibrosis is well-known. This process has been shown also in the heart, where cardiac fibroblasts (CF) differentiate into -smooth muscle actin (-SMA)-positive myofibroblasts (MyoFB). Here, we studied the effects on CF, isolated by spontaneously hypertensive rats (SHR), of integrin 5 inhibition in MyoFB differentiation.MethodsStaining and immunohistochemistry were performed on rat cardiac tissue. CF were isolated by enzymatic digestion from SHR (SHR-CF) and normotensive WKY (WKY-CF) rat hearts and then treated for in vitro evaluation.ResultsSHR heart tissues revealed a higher TGF-1 expression vs. WKY samples. SHR-CF showed an enhanced SMAD2/3 activation and an up-regulated expression of -SMA, a typical MyoFB marker, especially after TGF-1 treatment. Immunostaining on cardiac tissues revealed a higher expression of integrin 5 in SHR vs. WKY rat hearts. In vitro results confirmed the up-regulation of integrin 5 expression in SHR-CF at basal condition and after TGF-1 treatment, in comparison with WKY-CF. Inhibition of integrin 5 by cilengitide treatment led a decreased expression of 5, collagen I, and -SMA in SHR-CF vs. WKY-CF, resulting in a diminished differentiation of CF into MyoFB. Taking together, results suggested that SHR-CF are more susceptible to TGF-1, showing an up-regulated activation of SMAD2/3 signaling, and an increased 5, -SMA, and collagen I expression. Hypertension stimulus promoted an up-regulation of integrin 5 on SHR cardiac tissue and its in vitro inhibition reverted pro-fibrotic events of SHR-CF.ConclusionInhibition of integrin 5 exerted by cilengitide strongly diminished SHR-CF differentiation into detrimental MyoFB. So, integrin 5 might be considered a novel therapeutic target and cilengitide an effective pharmacological tool to limit the progression of hypertension-induced cardiac fibrosis

Advanced Differentiated Thyroid Cancer. A Complex Condition Needing a Tailored Approach

Differentiated thyroid cancers (DTCs) are slow-growing malignant tumours, including papillary and follicular carcinomas. Overall, prognosis is good, although it tends to worsen when local invasion occurs with bulky cervical nodes, or in the case of distant metastases. Surgery represents the main treatment for DTCs. However, radical excision is challenging and significant morbidity and functional loss can follow the treatment of the more advanced forms. Literature on advanced thyroid tumours, both differentiated and undifferentiated, does not provide clear and specific guidelines. This emerges the need for a tailored and multidisciplinary approach. In the present study, we report our single-centre experience of 111 advanced (local, regional, and distant) DTCs, investigating the rate of radical excision, peri-procedural and post-procedural complications, quality of life, persistence, recurrence rates, and survival rates. Results are critically appraised and compared to the existing published evidence review

Intraoperative ultrasound for prediction of hepatocellular carcinoma biological behavior : prospective comparison with pathology

Background & Aims Preoperative prediction of both microinvasive hepatocellular carcinoma and histological grade of hepatocellular carcinoma is pivotal to treatment planning and prognostication. The aim of this study was to evaluate whether some intraoperative ultrasound features correlate with both the presence of same histological patterns and differentiation grade of hepatocellular carcinoma on the histological features of the primary resected tumour. Methods All patients with single, small hepatocellular carcinoma that underwent hepatic resection were included in this prospective double-blind study: the intraoperative ultrasound patterns of nodule were registered and compared with similar histological features. Results A total of 179 patients were enclosed in this study: 97 (54%) patients (34% in HCC 642 cm) had a microinvasive hepatocellular carcinoma at ultrasound examination, while 82 (46%) patients (41% in HCC 642 cm) at histological evaluation. Statistical analysis showed that diameters 642 cm, presence of satellites and microinvasive hepatocellular carcinoma at ultrasound examination were the variables with the strongest association with the histological findings. In the multivariate analysis, the vascular microinfiltration and infiltrative hepatocellular carcinoma aspect were independent predictors for grading. Conclusions In patients with cirrhosis and hepatocellular carcinoma, the prevalence of microinvasive hepatocellular carcinoma is high, even in cases of HCC 642 cm. Intraoperative ultrasound findings strongly correlated with histopathological criteria in detecting microinvasive patterns and are useful to predict neoplastic differentiation. The knowledge of these features prior to treatment are highly desired (this can be obtained by an intraoperative ultrasound examination), as they could help in providing optimal management of patients with hepatocellular carcinoma

PKA regulatory subunit R2B is required for murine and human adipocyte differentiation

ADIPOGENESIS IS A COMPLEX PROCESS MODULATED BY SEVERAL FACTORS, INCLUDING CAMP SIGNALING. THE MAIN CAMP TARGET IS PROTEIN KINASE A (PKA), A TETRAMERIC ENZYME WITH FOUR REGULATORY SUBUNITS SHOWING TISSUE-SPECIFIC EXPRESSION AND FUNCTION: PRKAR2B is the main regulatory subunit in adipose tissue in mice and in adult humans. This study aimed to evaluate the expression of PKA regulatory subunits in human adipose tissue during fetal development and to investigate their role in the differentiation of 3T3-L1 and primary human preadipocytes. The expression of PKA regulatory subunits was evaluated in fetal adipose tissue (immunohistochemistry) and in cultured 3T3-L1 and primary human preadipocytes (western blot analysis). Cultured cells were transiently transfected with siRNA against PRKAR2B and induced to differentiate. Differentiation was evaluated by intracellular triglyceride staining (Oil Red O) and expression of molecular markers of adipocyte differentiation. In this study, we found that PRKAR2B is the main regulatory subunit in human adipose tissue during fetal development, from 12 weeks of gestation to the end of gestation, as well as in 3T3-L1 and primary human preadipocytes. The expression of PRKAR2B increases progressively during in vitro differentiation. The silencing of PRKAR2B abolishes the increase in the expression of peroxisome proliferator-activated receptor gamma (PPAR\u3b3 (PPARG)), fatty acid synthase, aP2 (FABP4), and lipoprotein lipase, as well as intracellular triglyceride accumulation, resulting in impaired adipocyte differentiation in both mouse and human cell systems. In conclusion, PRKAR2B is the key PKA regulatory subunit involved in mouse and human adipose tissue development. The physiological increase in the expression of PRKAR2B is an essential event in adipogenesis in both mice and humans, and it might represent a possible target for future strategies for obesity treatment

A histological procedure to study fungal infection in the wax moth Galleria mellonella

The invertebrate model Galleria mellonella is a widely used factitious host to study the microbial pathogenesis in vivo. However, a specific procedure for the recovery and the processing of the infected tissues, important for a better understanding of the host-pathogen interactions, has not been reported to our knowledge. In the present study we describe a new procedure of fixation and processing of larval tissue that allows studying the larval topographic anatomy and assessing the morphological changes due to the fungal infection. Lepidopteran larvae were infected with Candida albicans strains displaying various biofilm-forming abilities. The whole larvae were then examined for tissue changes by histological techniques. We show that comparing cutting planes, serial transversal sections of paraffin-embedded larva result in better accuracy and information recovering. Using this technique, it was possible to preserve the integrity of G. mellonella internal structures allowing the detailed analysis of morphological differences in different experimental groups (i.e., healthy vs infected larvae). We were also able to study strain-related differences in the pathogenesis of C. albicans by observing the immune response elicited and the invasiveness of two isolates within the larval tissues.\ua0In general, by processing the whole larva and optimizing routinely histochemical stainings, it is possible to visualize and analyse infected tissues. Various degrees of pathogenicity (strain- or inoculum-related), and the infection time course can be described in details. Moreover, the host immune response events can be followed throughout the infectious process leading to a comprehensive picture of the studied phenomenon

Численное моделирование конвективного теплообмена с различным расположением испарителя теплового насоса в промышленном водоёме.

В процессе исследования проводилось математическое моделирование естественной конвекции в водоеме с теплообменником испарителем теплового насоса В результате исследования проведен анализ влияния местоположения теплообменника-испарителя теплового насоса на тепловой режим водоема. Предложенная модель естественной конвекции жидкости в водоеме, используемого в качестве низкопотенциального источника энергии испарителя теплового насоса, может быть использована для усовершенствования методики расчета тепловых режимов водоемов – источников низкопотенциальной теплоты.In the process of research was conducted mathematical simulation of natural convection in a reservoir with heat exchanger, the evaporator of the heat pump The study analyzes the impact location of the heat exchanger-evaporator of the heat pump on the thermal regime of the reservoir. The proposed model of natural convection of fluid in a reservoir to be used as low-grade energy source of the heat pump evaporator can be used to improve the calculation of thermal regimes of reservoirs – the sources of low-grade heat

Prognostic significance of a comprehensive histologic evaluation of reticulin fibrosis, collagen deposition and osteosclerosis in primary myelofibrosis patients.

AimsTo evaluate whether a comprehensive histological evaluation of reticulin fibrosis, collagen deposition and osteosclerosis in bone marrow trephine biopsies (BMBs) of primary myelofibrosis (PMF) patients may have prognostic implications. Methods and resultsReticulin fibrosis, collagen deposition and osteosclerosis were graded from 0 to 3 in a series of 122 baseline BMBs. Then, we assigned to each case a comprehensive score [reticulin, collagen, osteosclerosis (RCO) score, ranging from 0 to 9] that allowed us to distinguish two groups of patients, with low-grade (RCO score 0-4) and high-grade (RCO score 5-9) stromal changes. Of 122 patients, 88 displayed a low-grade and 34 a high-grade RCO score. The latter was associated more frequently with anaemia, thrombocytopenia, peripheral blood blasts and increased lactate dehydrogenase levels. The RCO score was correlated strictly with overall mortality (P = 0.013) and International Prognostic Scoring System (IPSS) risk categories, and was able to discriminate the overall survival of both low- and high-grade patients (log-rank test: P < 0.001). Moreover, it proved to be more accurate than the European Consensus on Grading of Bone Marrow Fibrosis (ECGMF grade) in identifying high-risk patients with poor prognosis. Finally, a combined analysis of RCO scores and IPSS risk categories in an integrated clinical-pathological evaluation was able to increase the positive predictive value (PPV) for mortality in high-risk patients. ConclusionThe comprehensive RCO score, obtained by histological evaluation of reticulin fibrosis, collagen deposition and osteosclerosis was prognostically significant and more accurate than ECGMF grade in identifying high-risk patients and improved PPV when applied in addition to IPSS