Introduction to the Special Issue on Recent Developments on Computational Biology-I

In modern time,experts started to use interdisciplinary properties with the development of technology and science.Thus,these disciplines provide more sophisticated properties of real-world problems.In this sense,some models need to be investigated by using revised and modified traditional methods.The first discipline is the applied sciences such as physics,engineering,mechanics,electricity,biology,economy and mathematical applications[1-5].In this stage,many methods[5-10]are developed and modified.To uncover the deep properties of problems is to use the main properties of such interdisciplinary properties.Furthermore,works conducted on such mathematical models including non-local operators,partial,ordinary and integer order have introduced a deeper investigation of problems for experts.By using technological tools,expertsmay observe more realistic and exact results of models.

Advanced hepatocellular carcinoma and sorafenib: Diagnosis, indications, clinical and radiological follow-up

Advanced stage hepatocellular carcinoma(HCC) is a category of disease defined by radiological, clinical and hepatic function parameters, comprehending a wide range of patients with different general conditions. The main therapeutic option is represented by sorafenibtreatment, a multi-kinase inhibitor with anti-proliferative and anti-angiogenic effect. Trans-arterial Radio Embolization also represents a promising new approach to intermediate/advanced HCC. Post-marketing clinical studies showed that only a portion of patients actually benefits from sorafenib treatment, and an even smaller percentage of patients treated shows partial/complete response on follow-up examinations, up against relevant costs and an incidence of drug related adverse effects. Although the treatment with sorafenib has shown a significant increase in mean overall survival in different studies, only a part of patients actually shows real benefits, while the incidence of drug related significant adverse effects and the economic costs are relatively high. Moreover, only a small percentage of patients also shows a response in terms of lesion dimensions reduction. Being able to properly differentiate patients who are responding to the therapy from non-responders as early as possible is then still difficult and could be a pivotal challenge for the future; in fact it could spare several patients a therapy often difficult to bear, directing them to other second line treatments(many of which are at the moment still under investigation). For this reason, some supplemental criteria to be added to the standard modified Response Evaluation Criteria in Solid Tumors evaluation are being searched for. In particular, finding some parameters(cellular density, perfusion grade and enhancement rate) able to predict the sensitivity of the lesions to anti-angiogenic agents could help in stratifying patients in terms of treatment responsiveness before the beginning of the therapy itself, or in the first weeks of sorafenib treatment. This would bring a strongly desirable help in clinical managements of these patients.

Relationship between Crohn’s disease, infection with Mycobacterium avium subspecies paratuberculosis and SLC11A1 gene polymorphisms in Sardinian patients

AIM: To study the association between Crohn’s disease (CD), Mycobacterium avium subspecies paratubercu- losis (MAP), and genetic factors by examining the role of natural resistance-associated macrophage protein 1 (NRAMP1) gene polymorphisms (now SLC11A1) in Sar dinian patients with CD and controls. METHODS: Thirty-seven CD patients and 34 controls with no inflammatory bowel disease (IBD) were recruited at the University of Sassari after giving written con sent. Six SCL11A1 polymorphisms previously reported to be the most significantly associated with IBD were searched. M. paratuberculosis was identified by IS900 PCR and sequencing. Logistic regression was used to cal culate odds ratios (OR) for the associations among CD presence of MAP, and 6 loci described above. RESULTS: For the first time, a strong association was observed between polymorphisms at NRAMP1 locus 823C/T and CD. While CD was strongly associated withboth NRAMP1 and MAP, NRAMP1 polymorphisms and MAP themselves were not correlated. CONCLUSION: Combined with previous work on the NOD2/CARD15 gene, it is clear that the interplay of ge- netic, infectious, and immunologic factors in the etiology of CD is complex.

Predictors of survival in patients with established cirrhosis and hepatocellular carcinoma treated with sorafenib

AIM:To investigate in greater detail the efficacy and safety of sorafenib for the treatment of hepatocellular carcinoma(HCC)in patients with established cirrhosis.METHODS:From October 2009 to July 2012 patients with an established diagnosis of cirrhosis and HCC treated with sorafenib were consecutively enrolled.According to the Barcelona Clinic Liver Cancer(BCLC)classification,patients were in the advanced stage(BCLC-C)or in the intermediate stage(BCLC-B)but unfit or unresponsive to other therapeutic strategies.Treatment was evaluated performing a 4-phase computed tomography or magnetic resonance imaging scan every 2-3 mo,and analyzed according to the modified Response Evaluation Criteria in Solid Tumors.Sorafenib was administered at 800 mg/d,until radiological progression or occurrence of unacceptable adverse events(AEs).Univariate and multivariate analyses identified predictors of 16-wk clinical benefit and overall survival.RESULTS:Forty-four patients were enrolled,15 had intermediate HCC and 14 a Child-Pugh score of B7.AEs caused treatment interruption in 19 patients(43%),and median treatment duration was shorter in this subset(5 wk vs 19 wk,P<0.001)and in the BCLC-C subgroup(13 wk vs 40 wk,P=0.015).No significant differences in the reason for treatment interruption or in treatment duration were found comparing patients in Child-Pugh class A vs B or in patients older or younger than 70 years.After 16 wk of treatment,18 patients(41%)had stable disease or partial response.Patients with viral infection or BCLC-C were at higher risk of disease progression.ECOG,extrahepatic spread,macrovascular invasion,alpha-fetoprotein or alkaline phosphatase levels at admission were independent predictors of overall survival.CONCLUSION:In patients with cirrhosis and HCC treated with sorafenib,AEs are a common cause of early treatment withdrawal.Vascular invasion and extrahepatic spread condition early response to treatment and survival.Baseline biochemical parameters may be helpful to identify patients at higher risk of shorter overall survival.

Relevant incidental findings at abdominal multi-detector contrast-enhanced computed tomography:A collateral screening?

AIM: To investigate the prevalence of relevant incidental findings(RIFs) detected during routine abdominal contrast-enhanced computed tomography(Ce CT).METHODS: We retrospectively evaluated the reports of a consecutive series of abdominal Ce CT studies performed between January and May 2013. For each report, patients' age and sex, admission as inpatient or outpatient, clinical suspicion as indicated by the requesting physician, availability of a previous abdominal examination, and name of the reporting radiologist were recorded. Based on the clinical suspicion, the presence and features of any RIFs(if needing additional workup) was noted.RESULTS: One thousand forty abdominal Ce CT were performed in 949 patients(528 males, mean age 66 ±14 years). No significant difference was found between inpatients and outpatients age and sex distribution(P > 0.472). RIFs were found in 195/1040(18.8%) Ce CT [inpatients = 108/470(23.0%); outpatients = 87/570(15.2%); P = 0.002]. RIFs were found in 30/440(6.8%) Ce CT with a previous exam and in 165/600(27.5%) without a previous exam(P < 0.001). Radiologists' distribution between inpatients or outpatients was significantly different(P < 0.001). RIFs prevalence increased with aging, except for a peak in 40-49 year group. Most involved organs were kidneys, gallbladder, and lungs.CONCLUSION: A RIF is detected in 1/5 patients undergoing abdominal Ce CT. Risk of overdiagnosis should be taken into account.

Endoscopic ultrasound-guided drainage of pancreatic fluid collections: The impact of evolving experience and new technologies in diagnosis and treatment over the last two decades

Background: Endoscopic ultrasound (EUS)-guided drainage is the preferred approach for drainage of pan- creatic uid collections (PFCs) due to the better experience and signi cant progress using newer stents and access devices during last decade. This study aimed to evaluate the role of the evolving experience and possible in uence of new technological devices on the outcome of patients evaluated for PFCs and submitted to EUS-guided drainage during two different periods: the early period at the beginning of experience when a standardized technique was used and the late period when the increased experience of the operator, combined with different stents quality were introduced in the management of PFCs. Methods: We retrospectively analyzed the clinical data of a cohort of 91 consecutive patients, who underwent EUS-guided drainage of symptomatic PFCs from October 2001 to September 2017. Demographic, therapeutic results, complications, and outcomes were compared between early years’ group (2001 2008) and late years’ group (2009 2017). Results: Endoscopic treatment was successfully achieved in 55.6% (20/36) of patients in the early years’ group, and in 96.4% (53/55) in the late years’ group. Eighteen patients (12 in early years’ and 6 in the late year’s group) required additional open surgery. Procedural complications were observed in 5 patients, 4 in early years’ and 1 in late years’ group. Mortality was registered in two patients (2.2%), one for each group. Conclusions: During our long-term survey using EUS-guided endoscopic drainage of PFCs, signi cantly better outcomes in term of improved success rate and decrease complications rate were observed during the late period.

Changes on lysosomal compartment during PMA-induced differentiation of THP-1 monocytic cells: Influence of type I and type IV collagens

In this work, the influence of different substrate adhesion during phorbol-12-myristate-13-acetate (PMA)-induced differentiation of THP-1 monocytic cell line was studied. In particular, by morphocytochemical and cytometric approaches, the influence of type I and type IV collagens in an experimental model representative of three phases (initial, intermediate and terminal) of monocyte-macrophage transition was analyzed. The cells in these three phases of differentiation were obtained by using 6, 30 e 60 nM PMA. In this experimental model, referring to adhesion to glass as control, by using the azo-dye coupling method, we have considered the analysis of Acid Phosphatase (AcP) activity as a marker of differentiated status expression, in relation to the acquisition of macrophagic phenotype. Endosomal/lysosomal system was further characterized by taking into account the uptake of fluorescent probe LysoTracker Red. Fluorochromization in the various experimental conditions was analyzed morphologically (fluorescence microscopy) and quantitatively (static cytometry). Data related to lysosome compartment were integrated, from a cytokinetic point of view, by flow cytometry measurements of DNA/protein content. Our results have indicated that type I and type IV collagens were able to influence, with respect to glass adhesion, various differentiation phases. Type I collagen showed the higher effects in the condition of high differentiation (60 nM PMA), causing an increase in AcP activity and lysosomal system. Type IV collagen, besides determining effects on lysosomal compartment of intermediate and terminally differentiated cells, influenced mainly proliferative activity of cells with initial differentiation level (6 nM PMA).

Pioglitazone does not modify ANP levels of type 2 diabetic patients

Background: The atrial natriuretic peptide (ANP) regulates fluid volume redistribution between heart and the pulmonary vessels. In diabetic patients the physiological action of ANP appears to be seriously altered. Methods: 12 subjects (gender 6M/6F, age 47 ± 2 years, BMI 29.1 ± 0.1 kg/m2), with type 2 diabetes and under stable conditions, were studied after one month of pioglitazione treatment (30 mg/die) by means of isotonic blood volume expansion. Results: After one month of pioglitazone treatment the meta- bolic profile of the subjects improved (decrease dia- stolic blood pressure: p = 0.05, total cholesterol: p = 0.01, triglycerides: p = 0.03 and blood glucose: p = 0.01) as the expansion of their plasma volume was found associated with the decrease of hematocrit (p < 0.05). The statistical comparison before versus after pioglitazone showed a significant decrease in the ba- sal aldosterone levels post-treatment (p < 0.04). Nonetheless ANP plasma levels were similar before and after therapy. Conclusions: The inappropriately high concentrations of ANP induced by hyperglyce-mia and the abnormal responses to a physiological sti- mulus like an isotonic blood volume expansion are not reverted by one month of pioglitazone. This is in contrast with the brisk improvement of the metabolic profile seen for the same period of treatment. ANP secretion is modified by fluid load in diabetic patients. This anomaly is not reverted by pioglitazone.

Measurement of Glutathionylated Haemoglobin by MAL-DI-ToF Mass Spectrometry as a Biomarker of Oxidative Stress in Heavy Smokers and in Occupational Obese Subjects

Glutathionyl-haemoglobin (Hb-SSG) is a minor form of haemoglobin characterized by the presence of a disulfide bond between the β-93 cysteine residue and the thiol group of glutathione. Hb-SSG is naturally present in the erythrocytes at levels comparable to those of glycated haemoglobin and can be measured by MALDI mass spectrometry on very small samples of erythrocytes from peripheral blood. Since Hb-SSG has been recognized as a sensitive biomarker of oxidative stress in several degenerative diseases (diabetes, hyperlipidemia, kidney disease) and in healthy workers exposed to glutathione-depleting toxic agents such as butadiene, we have measured for the first time the levels of Hb-SSG in two groups: healthy heavy cigarette smokers and overweight-obese. For both classes of subjects, the measured levels (6.4%±1.7%, n=30 for smokers;3.0%±0.8%, n=20 for overweight-obese) are in the upper 97thpercentile of those measured in the Italian general population. Levels in smokers show a small, yet statistically significant dependence on the level of smoking addiction (>20 cig./day vs. ￡20 cig./day: 7.0% ± 1.4% vs. 5.7% ± 1.1%;p < 0.05). This biomarker thus adds to those presently available to rationally assess the extent of biological damage caused by tobacco smoking.

Electrophysiology of Sodium Receptors in Taste Cells

Sodium intake is important to maintain proper osmolarity and volume of extracellular fluid in vertebrates. The ability to find sources of sodium ions for managing electrolyte homeostasis relies on the activity of the taste system to sense salt. Several studies have been performed to understand the mechanisms underlying Na+ reception in taste cells, the peripheral detectors for food chemicals. It is now generally accepted that Na+ interacts with specific ion channels in taste cell membrane, called sodium receptors. As ion channels, these proteins mediate transmembrane ion fluxes (that is, electrical currents) during their operation. Thus, a lot of information on the functional properties of sodium receptors has been obtained by using electrophysiological techniques. Here, I review our current knowledge on the biophysical and physiological features of these receptors obtained by applying the patch-clamp recording techniques to single taste cells.

challenges of advanced hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is an aggressive malignancy,resulting as the third cause of death by cancer each year. The management of patients with HCC is complex,as both the tumour stage and any underlying liver disease must be considered conjointly. Although surveillance by imaging,clinical and biochemical parameters is routinely performed,a lot of patients suffering from cirrhosis have an advanced stage HCC at the first diagnosis. Advanced stage HCC includes heterogeneous groups of patients with different clinical condition and radiological features and sorafenib is the only approved treatment according to Barcelona Clinic Liver Cancer. Since the introduction of sorafenib in clinical practice,several phase Ⅲ clinical trials have failed to demonstrate any superiority over sorafenib in the frontline setting. Locoregional therapies have also been tested as first line treatment,but their role in advanced HCC is still matter of debate. No single agent or combination therapies have been shown to impact outcomes after sorafenib failure. Therefore this review will focus on the range of experimental therapeutics for patients with advanced HCC and highlights the successes and failures of these treatments as well as areas for future development. Specifics such as dose limiting toxicity and safety profile in patients with liver dysfunction related to the underlying chronic liver disease should be considered when developing therapies in HCC. Finally,robust validated and reproducible surrogate end-points as well as predictive biomarkers should be defined in future randomized trials.

Therapeutic and clinical aspects of portal vein thrombosis in patients with cirrhosis

Portal vein thrombosis(PVT) is a frequent complication in cirrhosis, particularly in advanced stages of the disease. As for general venous thromboembolism, risk factors for PVT are slow blood flow, vessel wall damage and hypercoagulability, all features of advanced cirrhosis. Actually, the old dogma of a hemorrhagic tendency in cirrhosis has been challenged by new laboratory tools and the clinical evidence that venous thrombosis also occurs in cirrhosis. The impaired hepatic synthesis of both pro- and anticoagulants leads to a rebalanced hemostasis, more liable to be tipped towards thrombosis or even bleeding. Conventional anticoagulant drugs(low molecular weight heparin or vitamin K antagonists) may be used in cirrhosis patients with PVT, particularly in those eligible for liver transplantation, to prevent thrombosis progression thus permitting/facilitating liver transplant. However, several doubts exist on the level of anticoagulation achieved as estimated by coagulation tests, on the efficacy of treatment monitoring and on the correct timing for discontinuation in non-transplant candidates, while in transplant candidates there is expert consensus on continuing anticoagulation until transplantation. The recent introduction of direct acting oral anticoagulant drugs(DOACs) in other clinical settings generates much interest on their possible application in patients with cirrhosis and PVT. However, DOACs were not evaluated yet in patients with liver disease and cannot be recommended for the present time.

Use of non-selective beta blockers in cirrhosis:The evidence we need before closing(or not) the window

Non selective beta blockers(NSBBs)are used in primary and secondary prophylaxis of portal hypertensionrelated bleeding in patients with cirrhosis.The efficacy of NSBBs treatment is predicted by hemodynamic response in term of reduction of the hepatic venouspressure gradient(HVPG)below 12 mm Hg or at least20%of the basal value.Nevertheless a relevant number of patients who do not achieve this HVPG reduction during NSBBs therapy do not bleed during follow up;this evidence suggests an additional non-hemodynamic advantage of NSBBs treatment to modify the natural history of cirrhosis.Recent studies have questioned the efficacy and safety of NSBBs in patients with advanced stage of liver disease characterized by refractory ascites and/or spontaneous bacterial peritonitis.These studies have suggested the existence of a defined and limited period to modify the natural history of cirrhosis by NSBBs:the"window hypothesis".According with this hypothesis,patients with cirrhosis benefit from the use of NSBBs from the appearance of varices up to the development of an advanced stage of cirrhosis.Indeed,in patients with refractory ascites and/or spontaneous bacterial peritonitis the hemodynamic effects of NSBBs may expose to a high risk of further complications such as renal insufficiency and/or death.Methodological concerns and contrasting results counterbalance the evidence produced up to now on this issue and are the main topic of this editorial.

Reporting rotator cuff tears on magnetic resonance arthrography using the Snyder's arthroscopic classification

AIM To determine diagnostic performance of magnetic resonance arthrography(MRA) in evaluating rotator cuff tears(RCTs) using Snyder's classification for reporting.METHODS One hundred and twenty-six patients(64 males, 62 females; median age 55 years) underwent shoulder MRA and arthroscopy, which represented our reference standard. Surgical arthroscopic reports were reviewed and the reported Snyder's classification was recorded. MRA examinations were evaluated by two independent radiologists(14 and 5 years' experience) using Snyder's classification system, blinded to arthroscopy. Agreement between arthroscopy and MRA on partial-and fullthickness tears was calculated, first regardless of their extent. Then, analysis took into account also the extent of the tear. Interobserver agreement was also calculated the quadratically-weighted Cohen kappa statistics.RESULTS On arthroscopy, 71/126 patients(56%) had a fullthickness RCT. The remaining 55/126 patients(44%) had a partial-thickness RCT. Regardless of tear extent, out of 71 patients with arthroscopically-confirmed fullthickness RCTs, 66(93%) were correctly scored by both readers. All 55 patients with arthroscopic diagnosis of partial-thickness RCT were correctly assigned as having a partial-thickness RCT at MRA by both readers. Interobserver reproducibility analysis showed total agreement between the two readers in distinguishing partial-thickness from full-thickness RCTs, regardless of tear extent(k = 1.000). With regard to tear extent, in patients in whom a complete tear was correctly diagnosed, correct tear extent was detected in 61/66 cases(92%); in the remaining 5/66 cases(8%), tear extent was underestimated. Agreement was k = 0.955. Interobserver agreement was total(k = 1.000).CONCLUSION MRA shows high diagnostic accuracy and reproducibility in evaluating RCTs using the Snyder's classification for reporting. Snyder's classification may be adopted for routine reporting of MRA.

Nuclear imaging to characterize adrenal tumors: Comparison with MRI

AIM:To describe the role of nuclear imaging modalities using nor-cholesterol,metaiodobenzylguanidine(MIBG)and fluorine-deoxy-glucose(FDG)in adrenal tumors for lesion characterization in comparison with magnetic resonance(MR).METHODS:Population was classified in group 1 consisting of 30 patients with non-hypersecreting unilateral adrenal masses,in group 2 consisting of 34 patients with hypersecreting(n=19)or non-hypersecreting(n=15)adrenal adenomas and in group 3 consisting of 18 patients with chromaffin-tissue tumors(CTT),of which 14 were pheochromocytomas while 4 were paragangliomas(n=4).All patients underwent MR and nuclear studies(nor-cholesterol,MIBG and FDG).Pathology samples(n=63)or follow-up data in adenomas(n=19)were used as standard of reference forimaging studies interpretation.RESULTS:In group 1,MR findings were not highly accurate for lesion characterization,while the results of nuclear scans showed abnormal nor-cholesterol,MIBG and FDG concentration in all cases of adenomas,pheos and malignant tumors,respectively.In group 2,no differences in MR parameters were found between hyperfunctioning and non-hyperfunctioning adenomas,while nor-cholesterol uptake was significantly higher in hyperfunctioning compared to non-hyperfunctioning lesions.In group 3,no differences in MR parameters were found between benign and malignant CCT,while MIBG uptake was significantly higher in malignant compared to benign tumors.CONCLUSION:On the basis of our findings,nuclear imaging modalities using specific target agents are able to better characterize adrenal tumors,compared with MR.In particular,radionuclide techniques are able to identify the nature of adrenal incidentalomas and to differentiate between hypersecreting and non-hypersecreting adenomas as well as between benign and malignant CTT.

Endoscopic ultrasound-guided fine-needle aspiration for suspected malignancies adjacent to the gastrointestinal tract

AIM:To investigate the impact of endoscopic ultrasound-guided fine-needle aspiration(EUS-FNA)in association with a multidisciplinary team evaluation for the detection of gastrointestinal malignancies.METHODS:A cohort of 1019 patients with suspected malignant lesions adjacent to the gastrointestinal tract received EUS-FNA after a standardized multidisciplinary team evaluation(MTE)and were divided into 4 groups according to their specific malignant risk score(MRS).Patients with a MRS of 0(without detectable risk of malignancy)received only EUS without FNA.For patients with a MRS score ranging from 1(low risk)-through 2(intermediate risk)-to 3(high risk),EUS-FNA cytology of the lesion was planned for a different time and was prioritized for those patients at higher risk for cancer.The accuracy,efficiency and quality assessment for the early detection of patients with potentially curable malignant lesions were evaluated for the whole cohort and in the different classes of MRSs.The time to definitive cytological diagnosis(TDCD),accuracy,sensitivity,specificity,positive and negative predictive values,and the rate of inconclusive tests were calculated for all patients and for each MRS group.RESULTS:A total of 1019 patients with suspected malignant lesions were evaluated by EUS-FNA.In 515patients of 616 with true malignant lesions the tumor was diagnosed by EUS-FNA;421 patients with resectable lesions received early surgical treatment,and 94patients received chemo-radiotherapy.The overall diagnostic accuracy for the 1019 lesions in which a final diagnosis was obtained by EUS-FNA was 0.95.When patients were stratified by MTE into 4 classes of MRSs,a higher rate of patients in the group with higher cancer risk(MRS-3)received early treatment and EUSFNA showed the highest level of accuracy(1.0).TDCD was also shorter in the MRS-3 group.The number of patients who received surgical treatment or chemo-radiotherapy was significantly higher in the MRS-3 patient group(36.3%in MRS-3,10.7%in MRS-2,and 3.5%in MRS-1).CONCLUSION:EUS-FNA can effectively detect a curable malignant lesions at an earlier time and at a higher rate in patients with a higher cancer risk that were evaluated using MTE.

Prednisolone inhibits SaOS2 osteosarcoma cell proliferation by activating inducible nitric oxide synthase

AIM:To investigate the effect of prednisolone,a synthetic glucocorticoid used in inflammatory diseases,on the growth of cultured osteosarcoma cells.METHODS:Two osteosarcoma cell lines with different degree of differentiation were used.SaOS2 show a rather mature phenotype,while U2 OS are negative for almost all osteoblastic markers.The cells were exposed to different concentrations of prednisolone(1-9 μmol/L) with or without antioxidants or the inhibitor of inducible nitric oxide synthase(i NOS) l-N6-(iminoethyl)-lysine-HCl(L-NIL).Cell growth was assessed by counting viable cells.The production of nitric oxide(NO) was measured in the conditioned media by the Griess method.The production of reactive oxygen species was quantified using 2'-7'-dichlorofluorescein diacetate.Western blot with specific antibodies against NOSs was performed on cell extracts.RESULTS:Prednisolone inhibited SaOS2 cell growth in a dose dependent manner.No significant effects were observed in U2OS.The inhibition of SaOS2 growth is not due to oxidative stress,because antioxidants do not rescue cell proliferation.Since high concentrations of NO inhibit bone formation,we also measured NO and found it induced in SaOS2,but not in U2 OS,exposed to prednisolone,because of the upregulation of i NOS as detected by western blot.Therefore,we treated SaOS2 with prednisolone in the presence or in the absence of L-NIL.L-NIL prevented NO release induced by prednisolone at all the concentrations apart from 9 μmol/L.At the same concentrations,we found that L-NIL rescued SaOS2 growth after exposure to prednisolone.In U2 OS cells,prednisolone did not induce NO production nor affected cell growth.All together,these data indicate that a link exists between increased amounts of NO and growth inhibition in response to prednisolone in SaOS2.CONCLUSION:Prednisolone inhibited SaOS2 proliferation by increasing the release of NO through the upregulation of i NOS,while no effect was exerted on U2OS.

HSP90、CDC37、CRM1作为P16^(INK4A)活性调节剂在大鼠肝癌发生与人类肝癌中的作用

Current evidence indicates that neoplastic nodules induced in liver of Brown Norway (BN) rats genetically resistant to hepatocarcinogenesis are not prone to evolve into hepatocellular carcinoma. We show that BN rats subjected to diethylnitrosamine/ 2- acetylaminofluorene/partial hepatectomy treatment with a “ resistant hepatocyte" protocol displayed higher number of glutathione-S-transferase 7- 7(+ ) hepatocytes when compared with susceptible Fisher 344 (F344) rats, both during and at the end of 2- acetylaminofluorene treatment. However, DNA synthesis declined in BN but not F344 rats after completion of reparative growth. Upregulation of p16INK4A Hsp90 and Cdc37 genes; an increase in Cdc37- Cdk4 complexes; and a decrease in p16INK4A-Cdk4 complexes occurred in preneoplastic liver, nodules, and hepatocellular carcinoma of F344 rats. These parameters did not change significantly in BN rats. E2f4 was equally expressed in the lesions of both strains, but Crm1 expression and levels of E2f4- Crml complex were higher in F344 rats. Marked upregulation of P16INK4A was associated with moderate over-expression of HSP90,CDC37, E2F4, and CRM1 in human hepatocellular carcinomas with a better prognosis. In contrast, strong induction of HSP90, CDC37, and E2F4 was paralleled by P16INK4A downregulation and high levels of HSP90- CDK4 and CDC37- CDK4 complexes in hepatocellular carcinomas with poorer prognosis. CDC37 downregulation by small interfering RNA inhibited in vitro growth of HepG2 cells. In conclusion, our findings underline the role of Hsp90/Cdc37 and E2f4/Crm1 systems in the acquisition of a susceptible or resistant carcinogenic phenotype. The results also suggest that protection by CDC37 and CRM1 against growth restraint by P16INK4A influences die prognosis of human hepatocellular carcinoma.

Short- and long-term effects of silver nanoparticles on human microvascular endothelial cells

AIM: To study the response to silver nanoparticles(Ag NP) of human microvascular endothelial cells, protagonists of angiogenesis. METHODS: We cultured human microvascular endothelial cells and endothelial colony-forming cells in their corresponding growth medium. Stock solutions of Ag NP were prepared in culture medium and sonicated before use. They were added at different concentrations and for different times to culture media. The toxicity of Ag NP was investigated by measuring the reduction of yellow tetrazolium salt to dark purple formazan(MTT assay) at 575 nm. After staining with trypan blue, cell proliferation was assessed by counting viable cells. The lactate dehydrogenase leakage assay was performed on culture media by following the oxidation of NADH to NAD+ and monitoring the reaction kinetically at 340 nm. Reactive oxygen species production was quantified using 2'-7'-dichlorofluorescein diacetate. The alkaline comet assay was performed after mixing the cells with low melting-point agarose. Electrophoresis was then conducted and the samples were stained with ethidium bromide and analyzed with a fluorescence microscope.RESULTS: Ag NP are cytotoxic in a dose and time dependent fashion for HMEC. At high concentrations, Ag NP determine loss of membrane integrity as demonstrated by the increased activity of lactate dehydrogenase in the culture medium. Ag NP rapidly stimulate the formation of free radicals. However, pre-incubation with Trolox, apocynin, or N-acetyl-L-cysteine, antioxidants which have different structure and act through different mechanisms, is not sufficient to prevent cytotoxicity. Ag NP also induce DNA damage dose-dependently, as shown by comet assay. When exposed to sublethal concentrations of Ag NP for long times, the cells remain viable but are growth retarded. Interestingly, removal of Ag NP partially rescues cell growth. Also genotoxicity is reversible upon removal of Ag NP from culture medium, suggesting that no permanent modifications occur. It is noteworthy that Ag NP are cytotoxic and genotoxic also for endothelial progenitors, in particular for endothelial colony-forming cells, which participate to angiogenesis.CONCLUSION: Silver nanoparticles are cytotoxic and genotoxic for human microvascular endothelial cells and might become a useful tool to control excessive angiogenesis.