Exosomal microRNAs as a potential therapeutic strategy in hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is the fifth most com-mon cancer and the second cause of cancer-related death worldwide. The incidence of HCC is constantly increasing in correlation with the rise in diabetes and obesity, arguing for an urgent need for new developments in the treatment of this lethal cancer. Exosomes are small double-membrane vesicles loaded with distinct cargos, particularly small non-coding RNAs called microRNAs, representative of each donor cell and secreted to affect the features of neighboring cells or recipient cells located further away, like in the case of metastasis. A better understanding of the role of exosomes with a microRNA signature in cancer pathogenesis gave rise to the concept of their use as a noninvasive diagnostic biomarker and in the treatment of cancer, including HCC. In this communication, we review recent works that demonstrate that hepatic stellate cells establish an epigenetic communication with liver cancer cells, which affects their pro-malignant features. If naturally secreted patient-derived exosomes show major limitations concerning their clinical use, bio-engineered exosome mimetics that incorporate controlled components and exhibit no protumoral properties could be promising carriers for the treatment of liver cancers, which is the organ preferentially targeted by systemic injection of exosomes.

MicroRNA-feedback loop as a key modulator of liver tumorigenesis and inflammation

A recent work of Iliopoulos et al published in Cell highlighted a circuit orchestrated by microRNAs (miRNAs) that results in liver tumorigenesis and inflammation. This feedback loop, governed by miR-24 and miR-629, promotes a hepatocyte nuclear factor-4α transient inhibition resulting in miR-124 induction and signal transducer and activator of transcription 3 activation. These promising data support the use of miRNA mimics or inhibitors as potent therapeutic approaches in liver cancer.

Anti-hepatitis C virus potency of a new autophagy inhibitor using human liver slices model

AIM: To evaluate the antiviral potency of a new antihepatitis C virus(HCV) antiviral agent targeting the cellular autophagy machinery. METHODS: Non-infected liver slices, obtained from human liver resection and cut in 350 μm-thick slices(2.7 × 106 cells per slice) were infected with cell culture-grown HCV Con1b/C3 supernatant(multiplicity of infection = 0.1) cultivated for up to ten days. HCV infected slices were treated at day 4 post-infection with GNS-396 for 6 d at different concentrations. HCV replication was evaluated by strand-specific real-time quantitative reverse transcription- polymerase chain reaction. The infectivity titers of supernatants were evaluated by foci formation upon inoculation into naive Huh-7.5.1 cells. The cytotoxic effect of the drugs was evaluated by lactate dehydrogenase leakage assays. RESULTS: The antiviral efficacy of a new antiviral drug, GNS-396, an autophagy inhibitor, on HCV infection of adult human liver slices was evidenced in a dosedependent manner. At day 6 post-treatment, GNS-396 EC50 was 158 nmol/L without cytotoxic effect(compared to hydroxychloroquine EC50 = 1.17 μmol/L).CONCLUSION: Our results demonstrated that our ex vivo model is efficient for evaluation the potency of autophagy inhibitors, in particular a new quinoline derivative GNS-396 as antiviral could inhibit HCV infection in a dosedependent manner without cytotoxic effect.

Transient micro-elastography:A novel non-invasive approach to measure liver stiffness in mice

AIM:To develop and validate a transient micro-elastography device to measure liver stiffness(LS) in mice.METHODS:A novel transient micro-elastography(TME) device,dedicated to LS measurements in mice with a range of measurement from 1-170 kPa,was developed using an optimized vibration frequency of 300 Hz and a 2 mm piston.The novel probe was validated in a classical fibrosis model(CCl4) and in a transgenic murine model of systemic amyloidosis.RESULTS:TME could be successfully performed in control mice below the xiphoid cartilage,with a mean LS of 4.4 ± 1.3 kPa,a mean success rate of 88%,and an excellent intra-observer agreement(0.98).Treatment with CCl4 over seven weeks drastically increased LS as compared to controls(18.2 ± 3.7 kPa vs 3.6 ± 1.2 kPa).Moreover,fibrosis stage was highly correlated with LS(Spearman coefficient = 0.88,P < 0.01).In the amyloidosis model,much higher LS values were obtained,reaching maximum values of > 150 kPa.LS significantly correlated with the amyloidosis index(0.93,P < 0.0001) and the plasma concentration of mutant hapoA-□(0.62,P < 0.005).CONCLUSION:Here,we have established the first non-invasive approach to measure LS in mice,and have successfully validated it in two murine models of high LS.

Locoregional therapies and their effects on the tumoral microenvironment of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma(PDAC) is expected to become the second leading cause of death from cancer by 2030. Despite intensive research in the field of therapeutics, the 5-year overall survival is approximately 8%, with only 20% of patients eligible for surgery at the time of diagnosis. The tumoral microenvironment(TME) of the PDAC is one of the main causes for resistance to antitumoral treatments due to the presence of tumor vasculature, stroma, and a modified immune response. The TME of PDAC is characterized by high stiffness due to fibrosis, with hypo microvascular perfusion, along with an immunosuppressive environment that constitutes a barrier to effective antitumoral treatment. While systemic therapies often produce severe side effects that can alter patients’ quality of life, locoregional therapies have gained attention since their action is localized to the pancreas and can thus alleviate some of the barriers to effective antitumoral treatment due to their physical effects. Local hyperthermia using radiofrequency ablation and radiation therapy-most commonly using a local high single dose-are the two main modalities holding promise for clinical efficacy. Recently, irreversible electroporation and focused ultrasound-derived cavitation have gained increasing attention. To date, most of the data are limited to preclinical studies, but ongoing clinical trials may help better define the role of these locoregional therapies in the management of PDAC patients.

Genome-wide differences in hepatitis C-vs alcoholism-associated hepatocellular carcinoma

AIM:To look at a comprehensive picture of etiology- dependent gene abnormalities in hepatocellular carcinoma in Western Europe. METHODS:With a liver-oriented microarray,transcript levels were compared in nodules and cirrhosis from a training set of patients with hepatocellular carcinoma (alcoholism,12;hepatitis C,10)and 5 controls.Loose or tight selection of informative transcripts with an abnormal abundance was statistically valid and the tightly selected transcripts were next quantified by qRTPCR in the nodules from our training set(12+10) and a test set(6+7). RESULTS:A selection of 475 transcripts pointed to significant gene over-representation on chromosome 8 (alcoholism)or-2(hepatitis C)and ontology indicated a predominant inflammatory response(alcoholism)or changes in cell cycle regulation,transcription factors and interferon responsiveness(hepatitis C).A stringent selection of 23 transcripts whose differences betweenetiologies were significant in nodules but not in cirrhotic tissue indicated that the above dysregulations take place in tumor but not in the surrounding cirrhosis.These 23 transcripts separated our test set according to etiologies. The inflammation-associated transcripts pointed to limited alterations of free iron metabolism in alcoholic vs hepatitis C tumors. CONCLUSION:Etiology-specific abnormalities(chromo- some preference;differences in transcriptomes and related functions)have been identified in hepatocellular carcinoma driven by alcoholism or hepatitis C.This may open novel avenues for differential therapies in this disease.

Integrative rodent models for assessing male reproductive toxicity of environmental endocrine active substances

In the present review, we first summarize the main benefits, limitations and pitfalls of conventional in vivo approaches to assessing male reproductive structures and functions in rodents in cases of endocrine active substance （EAS） exposure from the postulate that they may provide data that can be extrapolated to humans. Then, we briefly present some integrated approaches in rodents we have recently developed at the organism level. We particularly focus on the possible effects and modes of action （MOA） of these substances at low doses and in mixtures, real-life conditions and at the organ level, deciphering the precise effects and MOA on the fetal testis. It can be considered that the in vivo experimental EAS exposure of rodents remains the first choice for studies and is a necessary tool （together with the epidemiological approach） for understanding the reproductive effects and MOA of EASs, provided the pitfalls and limitations of the rodent models are known and considered. We also provide some evidence that classical rodent models may be refined for studying the multiple consequences of EAS exposure, not only on the reproductive axis but also on various hormonally regulated organs and tissues, among which several are implicated in the complex process of mammalian reproduction. Such models constitute an interesting way of approaching human exposure conditions. Finally, we show that organotypic culture models are powerful complementary tools, especially when focusing on the MOA. All these approaches have contributed in a combinatorial manner to a better understanding of the impact of EAS exposure on human reproduction.

Protein Phosphatase 2C of Toxoplasma Gondii Interacts with Human SSRP1 and Negatively Regulates Cell Apoptosis

Objective The protozoan Toxoplasma gondii expresses large amounts of a 37 kDa Type 2C serine-threonine phosphatase,the so-called TgPP2 C which has been suggested to contribute to parasite growth regulation.Ectopic expression in mammalian cells also indicated that the enzyme could regulate growth and survival.In this study,we aimed to investigate the interaction of TgPP2 C with human SSRP1（structure-specific recognition protein 1） and the effects of TgPP2 C on cell viability.Methods The yeast two hybrid system,His-tag pull-down and co-immunoprecipitation assays were used to confirm the interaction of TgPP2 C with SSRP1 and determine the binding domain on SSRP1.The evaluation of cell apoptosis was performed using cleaved caspase-3 antibody and Annexin-V/PI kit combined with flow cytometry.Results We identified human SSRP1 as an interacting partner of TgPP2 C.The C-terminal region of SSRP1 including the amino acids 471 to 538 was specifically mapped as the region responsible for interaction with TgPP2 C.The overexpression of TgPP2 C down-regulated cell apoptosis and negatively regulated apoptosis induced by DRB,casein kinase II（CKII） inhibitor,through enhanced interaction with SSRP1.Conclusion TgPP2 C may be a parasitic factor capable of promoting cell survival through interaction with the host protein SSRP1,thereby creating a favorable environment for parasite growth.

Visualization of Activated BAT in Mice,with FDG-PET and Its Relation to UCP1

The visualization of symmetric structure by [18F]-FluoroDeoxyGlucose-Positron Emission Tomography (FDG-PET), corresponding to adipose density in computed tomography (CT), has led to the idea that Brown Adipose Tissue (BAT) could be present in adult human. This article studies the FDG uptake in a mice model deficient on Uncoupling Protein 1 (UCP1), in a simple thermal activation protocol. Methods: FDG were injected in mice, control and knock out (K.O.) for the UCP1. Before imaging mice were placed either in cold or warm environment. BAT uptake was evaluated by ratio named RISC. Results: In warm condition, mean value of the Ratio of Inter-Scapular uptake (RISC) was 1.34 +/﹣ 0.27. After cold exposure, RISC increased 2 fold for control mice, male K.O. did not increase their RISC, female K.O. increased their RISC up to 2.45. Conclusion: Our study brought a further confirmation that FDG-PET visualised activated Brown Adipose Tissue. It gives a direct proof of the role of UCP1 in this process. The FDG uptake by cold female K.O. mice was unexpected.

Mitochondrial dysfunction activates the AMPK signaling and autophagy to promote cell survival

Autophagy is a cellular self-eating process essential for stress response and maintainingtissue homeostasis by lysosomal degradation of unwanted or damaged proteins and organelles.Here, we show that cells with defective mitochondria induce autophagy to promotecell survival through activating the AMPK pathway. Loss of mitochondrial complex III proteincytochrome b activates the AMPK signaling and induced autophagy. Inhibiting mitochondria energeticsby mitochondria-targeted agents activates the AMPK signaling and induced autophagy.Genetic inhibition of AMPK inhibits autophagy induction in cells with defective mitochondria,while genetic inhibition of autophagy has no effect on AMPK activation. Mitochondria dysfunctionhas no effect of DNA repair of UV-induced DNA damage. However, mitochondria dysfunctionsensitizes cells to apoptosis induced by UV radiation. Genetic inhibition of autophagy orAMPK sensitized cells to apoptosis in cells with defective mitochondria. Our results demonstratethat AMPK and autophagy senses mitochondria dysfunction and serves as a mechanismfor survival. Our findings may provide new insights into the interplay between mitochondriafunction and autophagy process in maintaining tissue homeostasis, and suggest that this interactionmay play important roles in diseases such as cancer and neurodegeneration.

Genetics of congenital hypothyroidism: Modern concepts

Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder and one of the most common preventable causes of intellectual disability in the world. CH may be due to developmental or functional thyroid defects (primary or peripheral CH) or be hypothalamic-pituitary in origin (central CH). In most cases, primary CH is caused by a developmental malformation of the gland (thyroid dysgenesis, TD) or by a defect in thyroid hormones synthesis (dyshormonogenesis, DH). TD represents about 65% of CH and a genetic cause is currently identified in fewer than 5% of patients. The remaining 35% are cases of DH and are explained with certainty at the molecular level in more than 50% of cases. The etiology of CH is mostly unknown and may include contributions from individual and environmental factors. In recent years, the detailed phenotypic description of patients, high-throughput sequencing technologies, and the use of animal models have made it possible to discover new genes involved in the development or function of the thyroid gland. This paper reviews all the genetic causes of CH. The modes by which CH is transmitted will also be discussed, including a new oligogenic model. CH is no longer simply a dominant disease for cases of CH due to TD and recessive for cases of CH due to DH, but a far more complex disorder.

Sulphated galactopyran derived fromGracilaria opuntia, a marine macroalgae restores the antioxidant metabolic enzymes during STZ induced diabetic rats

Objective:To screen the effect of sulphated galactopyran fraction isolated from Gracilaria opuntia (G. opuntia) (FM4) in streptozotocin (STZ) induced diabetic rats. Methods:In vitro antioxidant assays of FM4 were estimated byDPPH,ABTS, hydroxyl free radical and Nitric oxide free radical activities.FM4 was purified and characterized by1H-NMR spectra andFTIR as sulphated galactopyran. Diabetes was induced intraperitonially by single dose ofSTZ (55 mg/kg body weight).FM4 was administrated orally (80, 100, 125 mg/kgBW) to diabetic rats for 60 days. The enzymatic and non-enzymatic antioxidants such as superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione-S-transferase (GST), lipid peroxidase (LPx), glutathione reduced (GSH), vitamin-C (VIT-C) and vitamin-E (VIT-E) levels were estimated. Glibenclamide was used as standard drug. Results:Our results demonstrated that the aqueous extract ofG. opuntia possess free radical scavenging activity. During FM4 fraction treatment (100 mg/kgBW), theSOD,GPx,CAT,GST, GSH,VIT-C and VIT-E levels were significantly (P < 0.05) increased, and theLPx levels were decreased in different organs such as liver, kidney, brain and pancreas of diabetic rats. Conclusions: The sulphated galactopyran fraction of the marine macroalgae (G. opuntia) possesses the antioxidant activity which might help in the prevention of oxidative damage that occurs during diabetes.

CD5 expression promotes IL-10 production through activation of the MAPK/Erk pathway and upregulation of TRPC1 channels in B lymphocytes

CD5 is constitutively expressed on T cells and a subset of mature normal and leukemic B cells in patients with chronic lymphocytic leukemia(CLL).Important functional properties are associated with CD5 expression in B cells,including signal transducer and activator of transcription 3 activation,IL-10 production and the promotion of B-lymphocyte survival and transformation.However,the pathway(s)by which CD5 influences the biology of B cells and its dependence on B-cell receptor(BCR)co-signaling remain unknown.In this study,we show that CD5 expression activates a number of important signaling pathways,including Erk1/2,leading to IL-10 production through a novel pathway independent of BCR engagement.This pathway is dependent on extracellular calcium(Ca2+)entry facilitated by upregulation of the transient receptor potential channel 1(TRPC1)protein.We also show that Erk1/2 activation in a subgroup of CLL patients is associated with TRPC1 overexpression.In this subgroup of CLL patients,small inhibitory RNA(siRNA)for CD5 reduces TRPC1 expression.Furthermore,siRNAs for CD5 or for TRPC1 inhibit IL-10 production.These findings provide new insights into the role of CD5 in B-cell biology in health and disease and could pave the way for new treatment strategies for patients with B-CLL.

The Ability of Different Imputation Methods to Preserve the Significant Genes and Pathways in Cancer

Deciphering important genes and pathways from incomplete gene expression data could facilitate a better understanding of cancer. Different imputation methods can be applied to estimate the missing values. In our study, we evaluated various imputation methods for their performance in preserving significant genes and pathways. In the first step, 5% genes are considered in random for two types of ignorable and non-ignorable missingness mechanisms with various missing rates. Next, 10 well-known imputation methods were applied to the complete datasets. The significance analysis of microarrays （SAM） method was applied to detect the significant genes in rectal and lung cancers to showcase the utility of imputation approaches in preserving significant genes. To determine the impact of different imputation methods on the identification of important genes, the chi-squared test was used to compare the proportions of overlaps between significant genes detected from original data and those detected from the imputed datasets. Additionally, the significant genes are tested for their enrichment in important pathways, using the ConsensusPathDB. Our results showed that almost all the significant genes and pathways of the original dataset can be detected in all imputed datasets, indicating that there is no significant difference in the performance of various imputation methods tested. The source code and selected datasets are available on http：//profiles.bs.ipm.ir/soft- wares/imputationmethods/.

Multiparametric characterization of red blood cell physiology after hypotonic dialysis based drug encapsulation process

Red blood cells(RBCs)can act as carriers for therapeutic agents and can substantially improve the safety,pharmacokinetics,and pharmacodynamics of many drugs.Maintaining RBCs integrity and lifespan is important for the efficacy of RBCs as drug carrier.We investigated the impact of drug encapsulation by hypotonic dialysis on RBCs physiology and integrity.Several parameters were compared between processed RBCs loaded with l-asparaginase(“eryaspase”),processed RBCs without drug and non-processed RBCs.Processed RBCs were less hydrated and displayed a reduction of intracellular content.We observed a change in the metabolomic but not in the proteomic profile of processed RBCs.Encapsulation process caused moderate morphological changes and was accompanied by an increase of RBCs-derived Extracellular Vesicles release.Despite a decrease in deformability,processed RBCs were not mechanically retained in a spleen-mimicking device and had increased surface-to-volume ratio and osmotic resistance.Processed RBCs half-life was not significantly affected in a mouse model and our previous phase 1 clinical study showed that encapsulation of asparaginase in RBCs prolonged its in vivo half-life compared to free forms.Our study demonstrated that encapsulation by hypotonic dialysis may affect certain characteristics of RBCs but does not significantly affect the in vivo longevity of RBCs or their drug carrier function.

Theranostics:纳米热疗法或可软化肿瘤,提高癌症靶向疗法的治疗效率

肿瘤的机械阻力和标准疗法的附带损伤往往会阻碍抵御癌症的效果，近日，来自法国国家健康与医学研究所等机构的研究人员通过对肿瘤加热成功软化了恶性肿瘤，这种方法称之为纳米热疗法，其能够让肿瘤对治疗制剂更加敏感，相关研究刊登于国际杂志Theranostics上。