Search results for: liver markers

Authors: Lena Lapidot, Amir Amnon, Rita Nosenko, Veitsman Ella, Cohen-Ezra Oranit, Davidov Yana, Segev Shlomo, Koren Omry, Safran Michal, Ben-Ari Ziv

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Introduction: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer related mortality worldwide. Liver Cirrhosis is the main predisposing risk factor for the development of HCC. The factor(s) influencing disease progression from Cirrhosis to HCC remain unknown. Gut microbiota has recently emerged as a major player in different liver diseases, however its association with HCC is still a mystery. Moreover, there might be an important association between the gut microbiota, nutrition, life style and the progression of Cirrhosis and HCC. The aim of our study was to characterize the gut microbial signature in association with life style and nutrition of patients with Cirrhosis, HCC-Cirrhosis and healthy controls. Design: Stool samples were collected from 95 individuals (30 patients with HCC, 38 patients with Cirrhosis and 27 age, gender and BMI-matched healthy volunteers). All participants answered lifestyle and Food Frequency Questionnaires. 16S rRNA sequencing of fecal DNA was performed (MiSeq Illumina). Results: There was a significant decrease in alpha diversity in patients with Cirrhosis (qvalue=0.033) and in patients with HCC-Cirrhosis (qvalue=0.032) compared to healthy controls. The microbiota of patients with HCC-cirrhosis compared to patients with Cirrhosis, was characterized by a significant overrepresentation of Clostridium (pvalue=0.024) and CF231 (pvalue=0.010) and lower expression of Alphaproteobacteria (pvalue=0.039) and Verrucomicrobia (pvalue=0.036) in several taxonomic levels: Verrucomicrobiae, Verrucomicrobiales, Verrucomicrobiaceae and the genus Akkermansia (pvalue=0.039). Furthermore, we performed an analysis of predicted metabolic pathways (Kegg level 2) that resulted in a significant decrease in the diversity of metabolic pathways in patients with HCC-Cirrhosis (qvalue=0.015) compared to controls, one of which was amino acid metabolism. Furthermore, investigating the life style and nutrition habits of patients with HCC-Cirrhosis, we found significant correlations between intake of artificial sweeteners and Verrucomicrobia (qvalue=0.12), High sugar intake and Synergistetes (qvalue=0.021) and High BMI and the pathogen Campylobacter (qvalue=0.066). Furthermore, overweight in patients with HCC-Cirrhosis modified bacterial diversity (qvalue=0.023) and composition (qvalue=0.033). Conclusions: To the best of the our knowledge, we present the first report of the gut microbial composition in patients with HCC-Cirrhosis, compared with Cirrhotic patients and healthy controls. We have demonstrated in our study that there are significant differences in the gut microbiome of patients with HCC-cirrhosis compared to Cirrhotic patients and healthy controls. Our findings are even more pronounced because the significantly increased bacteria Clostridium and CF231 in HCC-Cirrhosis weren't influenced by diet and lifestyle, implying this change is due to the development of HCC. Further studies are needed to confirm these findings and assess causality.

Keywords: Cirrhosis, Hepatocellular carcinoma, life style, liver disease, microbiome, nutrition

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Authors: Marco Jan Rafael C. Sicam, Maria Daniella C. Yambao

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The Philippines is a tectonically active archipelagic country situated near the Circum-Pacific Belt. Hence, seismic hazard assessments are important in the nation-building. In 2014, the Philippines Institute of Volcanology and Seismology (PHIVOLCS) mapped a 12-km NW-trending unnamed active fault near Boso-Boso River, Rizal. Given the limited nature of their technical report, they would like to further consolidate relevant data about this fault. As such, this study aims to characterize the surface and subsurface expression of the fault along Boso-Boso River using rangefront morphology, structural criteria, and ground penetrating radar. This fault is subdivided into two segments: the first segment located in the city of Antipolo is mainly manifested in the upper Kinabuan Formation and terminating near Mt. Qutago, and the second segment in Baras, Pinugay, Rizal cuts through recent fluvial deposits and to the Guadalupe Formation. IfSAR-derived DTM data reveals the morphological expression of the fault defined by offset streams and ridges, linear sidehill valleys, and linear valleys. Fault gouges, fault breccia, transtentional flower structures, slickensides, and other shear sense markers observed in the units of the upper Cretaceous Kinabuan Formation indicate a sinistral sense of displacement. GPR radargram profiles revealed the presence of displacement in reflectors at 3-5 meters below the surface which may be suggestive of the fault within the area. Finally, the fault in Boso-Boso river may be a segment of the larger sinistral Montalban Fault in the north or largely affected by the movement from the Marikina Valley Fault System.

Keywords: NW unnamed fault, range-front morphology, shear sense markers, ground penetrating radar, boso-boso river, antipolo

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Authors: Alexander A. Stakheev, Larisa V. Samokhvalova, Sergey K. Zavriev

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Fusarium fungi are among the most devastating plant pathogens distributed all over the world. Significant reduction of grain yield and quality caused by Fusarium leads to multi-billion dollar annual losses to the world agricultural production. These organisms can also cause infections in immunocompromised persons and produce the wide range of mycotoxins, such as trichothecenes, fumonisins, and zearalenone, which are hazardous to human and animal health. Identification of Fusarium fungi based on the morphology of spores and spore-forming structures, colony color and appearance on specific culture media is often very complicated due to the high similarity of these features for closely related species. Modern Fusarium taxonomy increasingly uses data of crossing experiments (biological species concept) and genetic polymorphism analysis (phylogenetic species concept). A number of novel Fusarium sibling species has been established using DNA barcoding techniques. Species recognition is best made with the combined phylogeny of intron-rich protein coding genes and ribosomal DNA sequences. However, the internal transcribed spacer of (ITS), which is considered to be universal DNA barcode for Fungi, is not suitable for genus Fusarium, because of its insufficient variability between closely related species and the presence of non-orthologous copies in the genome. Nowadays, the translation elongation factor 1 alpha (TEF1α) gene is the “gold standard” of Fusarium taxonomy, but the search for novel informative markers is still needed. In this study, we used two novel DNA markers, frataxin (FXN) and heat shock protein 90 (HSP90) to discover phylogenetic relationships between Fusarium species. Multilocus phylogenetic analysis based on partial sequences of TEF1α, FXN, HSP90, as well as intergenic spacer of ribosomal DNA (IGS), beta-tubulin (β-TUB) and phosphate permease (PHO) genes has been conducted for 120 isolates of 19 Fusarium species from different climatic zones of Russia and neighboring countries using maximum likelihood (ML) and maximum parsimony (MP) algorithms. Our analyses revealed that FXN and HSP90 genes could be considered as informative phylogenetic markers, suitable for evolutionary and taxonomic studies of Fusarium genus. It has been shown that PHO gene possesses more variable (22 %) and parsimony informative (19 %) characters than other markers, including TEF1α (12 % and 9 %, correspondingly) when used for elucidating phylogenetic relationships between F. avenaceum and its closest relatives – F. tricinctum, F. acuminatum, F. torulosum. Application of novel DNA barcodes confirmed the fact that F. arthrosporioides do not represent a separate species but only a subspecies of F. avenaceum. Phylogeny based on partial PHO and FXN sequences revealed the presence of separate cluster of four F. avenaceum strains which were closer to F. torulosum than to major F. avenaceum clade. The strain F-846 from Moldova, morphologically identified as F. poae, formed a separate lineage in all the constructed dendrograms, and could potentially be considered as a separate species, but more information is needed to confirm this conclusion. Variable sites in PHO sequences were used for the first-time development of specific qPCR-based diagnostic assays for F. acuminatum and F. torulosum. This work was supported by Russian Foundation for Basic Research (grant № 15-29-02527).

Keywords: DNA barcode, fusarium, identification, phylogenetics, taxonomy

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Authors: Sharmeen Rahman, Daniel Schmidt, Jane Hughes

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Paratya australiensis Kemp (Decapoda: Atyidae) is a widely distributed indigenous freshwater shrimp, highly abundant in eastern Australia. This species has been considered as a model stream organism to study genetics, dispersal, biology, behaviour and evolution in Atyids. Paratya has a filter feeding and scavenging habit which plays a significant role in the formation of lotic community structure. It has been shown to reduce periphyton and sediment from hard substrates of coastal streams and hence acts as a strongly-interacting ecosystem macroconsumer. Besides, Paratya is one of the major food sources for stream dwelling fishes. Paratya australiensis is a cryptic species complex consisting of 9 highly divergent mitochondrial DNA lineages. Among them, one lineage has been observed to favour upstream sites at higher altitudes, with cooler water temperatures. This study aims to identify local adaptation in upstream and downstream populations of this lineage in three streams in the Conondale Range, North-eastern Brisbane, Queensland, Australia. Two populations (up and down stream) from each stream have been chosen to test for local adaptation, and a parallel pattern of adaptation is expected across all streams. Six populations each consisting of 24 individuals were sequenced using the Restriction Site Associated DNA-seq (RAD-seq) technique. Genetic markers (SNPs) were developed using double digest RAD sequencing (ddRAD-seq). These were used for de novo assembly of Paratya genome. De novo assembly was done using the STACKs program and produced 56, 344 loci for 47 individuals from one stream. Among these individuals, 39 individuals shared 5819 loci, and these markers are being used to test for local adaptation using Fst outlier tests (Arlequin) and Bayesian analysis (BayeScan) between up and downstream populations. Fst outlier test detected 27 loci likely to be under selection and the Bayesian analysis also detected 27 loci as under selection. Among these 27 loci, 3 loci showed evidence of selection at a significance level using BayeScan program. On the other hand, up and downstream populations are strongly diverged at neutral loci with a Fst =0.37. Similar analysis will be done with all six populations to determine if there is a parallel pattern of adaptation across all streams. Furthermore, multi-locus among population covariance analysis will be done to identify potential markers under selection as well as to compare single locus versus multi-locus approaches for detecting local adaptation. Adaptive genes identified in this study can be used for future studies to design primers and test for adaptation in related crustacean species.

Keywords: Paratya australiensis, rainforest streams, selection, single nucleotide polymorphism (SNPs)

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Authors: Lorenzo Buti

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This paper investigates the nature of democratic action through a critical reading of Claude Lefort’s notion of the democratic ‘regime’. Lefort provides one of the most innovative accounts of the essential features of a democratic regime. According to him, democracy is a political regime that acknowledges the indeterminacy of a society and stages it as a contestation between competing political actors. As such, democracy provides the symbolic markers of society’s openness towards the future. However, despite their democratic features, the recent decades in late capitalist societies attest to a sense of the future becoming fixed and predetermined. This suggests that Lefort’s conception of democracy harbours a misunderstanding of the character and experience of democratic action. This paper examines this underlying tension in Lefort’s work. It claims that Lefort underestimates how a democratic regime, next to its symbolic function, also takes a materially constituted form with its particular dynamics of power relations. Lefort’s systematic dismissal of this material dimension for democratic action can lead to the contemporary paradoxical situation where democracy’s symbolic markers are upheld (free elections, public debate, dynamic between government and opposition in parliament,…) but the room for political decision-making is constrained due to a myriad of material constraints (e.g., market pressures, institutional inertias). The paper draws out the implications for the notion of democratic action. Contra Lefort, it argues that democratic action necessarily targets the material conditions that impede the capacity for decision-making on the basis of equality and liberty. This analysis shapes our understanding of democratic action in two ways. First, democratic action takes an asymmetrical, insurgent form, as a contestation of material power relations from below. Second, it reveals an ambivalent position vis-à-vis the political regime: democratic action is symbolically made possible by the democratic dispositive, but it contests the constituted form that the democratic regime takes.

Keywords: Claude Lefort, democratic action, material constitution, political regime

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Authors: Dipali Dhawan

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Cancerous epithelial cells are confined to a primary site by the continued expression of adhesion molecules and the intact basal lamina. However, as the cancer progresses some cells are believed to undergo an epithelial-mesenchymal transition (EMT) event, leading to increased motility, invasion and, ultimately, metastasis of the cells from the primary tumour to secondary sites within the body. These disseminated cancer cells need the ability to self-renew, as stem cells do, in order to establish and maintain a heterogeneous metastatic tumour mass. Identification of the specific subpopulation of cancer stem cells amenable to the process of metastasis is highly desirable. In this study, we have isolated and characterized cancer stem cells from luminal and basal breast cancer cell lines (MDA-MB-231, MDA-MB-453, MDA-MB-468, MCF7 and T47D) on the basis of cell surface markers CD44 and CD24; as well as Side Populations (SP) using Hoechst 33342 dye efflux. The isolated populations were analysed for epithelial and mesenchymal markers like E-cadherin, N-cadherin, Sfrp1 and Vimentin by Western blotting and Immunocytochemistry. MDA-MB-231 cell lines contain a major population of CD44+CD24- cells whereas MCF7, T47D and MDA-MB-231 cell lines show a side population. We observed higher expression of N-cadherin in MCF-7 SP cells as compared to MCF-7NSP (Non-side population) cells suggesting that the SP cells are mesenchymal like cells and hence express increased N-cadherin with stem cell-like properties. There was an expression of Sfrp1 in the MCF7- NSP cells as compared to no expression in MCF7-SP cells, which suggests that the Wnt pathway is expressed in the MCF7-SP cells. The mesenchymal marker Vimentin was expressed only in MDA-MB-231 cells. Hence, understanding the breast cancer heterogeneity would enable a better understanding of the disease progression and therapeutic targeting.

Keywords: cancer stem cells, epithelial to mesenchymal transition, biomarkers, breast cancer

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Authors: Bahareh Yazdanparast Chaharmahali

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The study showed effect of low power laser therapy on knee range of motion (flexion and extension), resting angle of knee joint, knee circumference and rating of delayed onset muscle soreness induced pain, 24 and 48 hours after eccentric training of knee flexor muscle (hamstring muscle). We investigate the effects of pulsed ultrasound on swelling, relaxed, flexion and extension knee angle and pain. 20 volunteers among girl students of college voluntary participated in this research. After eccentric training, subjects were randomly divided into two groups, control and laser therapy. In day 1 and in order to induce delayed onset muscle soreness, subjects eccentrically trained their knee flexor muscles. In day 2, subjects were randomly divided into two groups: control and low power laser therapy. 24 and 48 hours after eccentric training. Variables (knee flexion and extension, srang of motion, resting knee joint angle and knee circumferences) were measured and analyzed. Data are reported as means ± standard error (SE) and repeated measured was used to assess differences within groups. Methods of treatment (low power laser therapy) have significant effects on delayed onset muscle soreness markers. 24 and 48 hours after training a significant difference was observed between mean pains of 2 groups. This difference was significant between low power laser therapy and C groups. The Bonferroni post hock is significant. Low power laser therapy trophy as used in this study did significantly diminish the effects of delayed – onset muscle soreness on swelling, relaxed – knee extension and flexion angle.

Keywords: creatine kinase, DOMS, eccentric training, low power laser

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Authors: Nandini Nalika, Suhel Parvez

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Nanotechnology has emerged to play a vital role in developing all through the industrial world with an immense production of nanomaterials including nanoparticles (NPs). Many toxicological studies have confirmed that due to unique small size and physico-chemical properties of NPs (1-100nm), they can be potentially hazardous. Metallic NPs of small size have been shown to induce higher levels of cellular oxidative stress and can easily pass through the Blood Brain Barrier (BBB) and significantly accumulate in brain. With the wide applications of titanium dioxide nanoparticles (TNPs) in day-to-day life in form of cosmetics, paints, sterilisation and so on, there is growing concern regarding the deleterious effects of TNPs on central nervous system and mitochondria appear to be important cellular organelles targeted to the pro-oxidative effects of NPs and an important source that contribute significantly for the production of reactive oxygen species after some toxicity or an injury. The aim of our study was to elucidate the effect of TNPs in anatase form with different concentrations (5-50 µg/ml) following with various oxidative stress markers in isolated brain mitochondria as an in vitro model. Oxidative stress was determined by measuring the different oxidative stress markers like lipid peroxidation as well as the protein carbonyl content which was found to be significantly increased. Reduced glutathione content and major glutathione metabolizing enzymes were also modulated signifying the role of glutathione redox cycle in the pathophysiology of TNPs. The study also includes the mitochondrial enzymes (Complex 1, Complex II, complex IV, Complex V ) and the enzymes showed toxicity in a relatively short time due to the effect of TNPs. The study provide a range of concentration that were toxic to the neuronal cells and data pointing to a general toxicity in brain mitochondria by TNPs, therefore, it is in need to consider the proper utilization of NPs in the environment.

Keywords: mitochondria, nanoparticles, brain, in vitro

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Authors: M. Abuqebitah, H. Tanyildizi, N. Yeyin, I. Cavdar, M. Demir, L. Kabasakal

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Aim: Targeted radionuclide therapy (TRT) is an increasingly used treatment modality for wide range of cancers. Presently dosimetry is highly required either to plan treatment or to ascertain the absorbed dose delivered to critical organs during treatment. Methods and Materials: The study comprised 7 patients suffered from prostate cancer with progressive disease and candidate to undergo Lu-177-DOTA-617 therapy following to PSMA- PET/CT imaging for all patients. (5.2±0.3 mCi) was intravenously injected. To evaluate bone marrow absorbed dose 2 cc blood samples were withdrawn in short variable times (3, 15, 30, 60, 180 minutes) after injection. Furthermore, whole body scans were performed using scintillation gama camera in 4, 24, 48, and 120 hours after injection and in order to quantify the activity taken up in the body, kidneys , liver, right parotid, and left parotid the geometric mean of anterior and posterior counts were determined through ROI analysis, after that background subtraction and attenuation correction were applied using patients PSMA- PET/CT images taking in a consideration: organ thickness, body thickness, and Hounsfield unites from CT scan. OLINDA/EXM dosimetry program was used for curve fitting, residence time calculation, and absorbed dose calculations. Findings: Absorbed doses of bone marrow, left kidney, right kidney, liver, left parotid, right parotid, total body were 1.28±0.52, 32.36±16.36, 32.7±13.68, 10.35±3.45, 38.67±21.29, 37.55±19.77, 2.25±0.95 (mGy/mCi), respectively. Conclusion: Our first results clarify that Lu-177-DOTA-617 is safe and reliable therapy as there were no complications seen. In the other hand, the observable variation in the absorbed dose of the critical organs among the patients necessitate patient-specific dosimetry approach to save body organs and particularly highly exposed kidneys and parotid gland.

Keywords: Lu-177-PSMA, prostate cancer, radionuclide therapy

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Authors: Iliyasu Musa Omoyine, Musa Sunday Abraham, Oladele Sunday Blessing, Iliya Ibrahim Abdullahi, Ibegbu Augustine Oseloka, Nuhu Nana-Hawau, Animoku Abdulrazaq Amoto, Yusuf Abdullateef Onoruoiza, Sambo Sohnap James, Akpulu Steven Peter, Ajayi Abayomi

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The neuroprotective role of inflammation from detrimental intrinsic and extrinsic factors has been reported. However, the overactivation of astrocytes and microglia due to lead toxicity produce excessive pro-inflammatory cytokines, mediating neurodegenerative diseases. The present study investigated the mitigatory effects of quercetin on neuroinflammation, correlating with memory function in lead-exposed rats. In this study, Wistar rats were administered orally with Quercetin (Q: 60 mg/kg) and Succimer as a standard drug (S: 10 mg/kg) for 21 days after lead exposure (Pb: 125 mg/kg) of 21 days or in combination with Pb, once daily for 42 days. Working and reference memory was assessed using an Eight-arm radial water maze (8-ARWM). The changes in brain lead level, the neuronal nitric oxide synthase (nNOS) activity, and the level of neuroinflammatory markers such as tumour necrosis factor-alpha (TNF-α) and Interleukin 1 Beta (IL-1β) were determined. Immunohistochemically, astrocyte expression was evaluated. The results showed that the brain level of lead was increased significantly in lead-exposed rats. The expression of astrocytes increased in the CA3 and CA1 regions of the hippocampus, and the levels of brain TNF-α and IL-1β increased in lead-exposed rats. Lead impaired reference and working memory by increasing reference memory errors and working memory incorrect errors in lead-exposed rats. However, quercetin treatment effectively improved memory and inhibited neuroinflammation by reducing astrocytes’ expression and the levels of TNF-α and IL-1β. The expression of astrocytes and the levels of TNF-α and IL-1β correlated with memory function. The possible explanation for quercetin’s anti-neuroinflammatory effect is that it modulates the activity of cellular proteins involved in the inflammatory response; inhibits the transcription factor of nuclear factor-kappa B (NF-κB), which regulates the expression of proinflammatory molecules; inhibits kinases required for the synthesis of Glial fibrillary acidic protein (GFAP) and modifies the phosphorylation of some proteins, which affect the structure and function of intermediate filament proteins; and, lastly, induces Cyclic-AMP Response Element Binding (CREB) activation and neurogenesis as a compensatory mechanism for memory deficits and neuronal cell death. In conclusion, the levels of neuroinflammatory markers negatively correlated with memory function. Thus, quercetin may be a promising therapy in neuroinflammation and memory dysfunction in populations prone to lead exposure.

Keywords: lead, quercetin, neuroinflammation, memory

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Authors: Mohammad K. Parvez, Mohammed S. Al-Dosari

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Hepatitis E virus (HEV) is an emerging RNA virus that causes acute and chronic liver disease with a global mortality rate of about 2%. Despite milestone developments in understanding of HEV biology, there is still lack of a robust culture system or animal model. Therefore, in a novel approach, two recombinant-baculoviruses (vBac-ORF2 and vBac-ORF3) that could overexpress HEV ORF2 (structural/capsid) and ORF3 (nonstructural/regulatory) proteins, respectively were constructed. The established HEV-SAR55 (genotype 1) replicon that contained GFP gene, in place of ORF2/ORF3 sequences was in vitro transcribed, and GFP production in RNA transfected S10-3 cells was scored by FACS. Enhanced infectivity, if any, of nascent virions produced by exogenously-supplied ORF2 and viral RNA by co-expression of ORF3 was tested on naïve HepG2 cells. Co-transduction with vBac-ORF2/vBac-ORF3 (108 pfu/microL) produced high amounts of native ORF2/ORF3 in approximately 60% of S10-3 cells, determined by immunofluorescence microscopy and Western analysis. FACS analysis showed about 9% GFP positivity of S10-3 cells on day6 post-transfection (i.e, day5 post-transduction). Further, FACS scoring indicated that lysates from S10-3 cultures receiving the RNA plus vBac-ORF2 were capable of producing HEV particles with about 4% infectivity in HepG2 cells. However, lysates of cultures co-transduced with vBac-ORF3, were found to further enhance virion infectivity by approximately 17%. This supported a previously proposed role of ORF3 as a minor-structural protein in HEV virion assembly and infectivity. In conclusion, the present model for efficient genomic RNA packaging and production of infectious virions could be a valuable tool to study various aspects of HEV molecular biology, in vitro.

Keywords: chronic liver disease, hepatitis E virus, ORF2, ORF3, replicon

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Authors: Constantinos S. Mammas, Andreas Lazaris, Adamantia S. Mamma-Graham, Georgia Kostopanagiotou, Chryssa Lemonidou, John Mantas, Eustratios Patsouris

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The process to build a better safety culture, methods of error analysis, and preventive measures, starts with an understanding of the effects when human factors engineering refer to remote microscopic diagnosis in surgery and specially in organ transplantation for the evaluation of the grafts. Α high percentage of solid organs arrive at the recipient hospitals and are considered as injured or improper for transplantation in the UK. Digital microscopy adds information on a microscopic level about the grafts (G) in Organ Transplant (OT), and may lead to a change in their management. Such a method will reduce the possibility that a diseased G will arrive at the recipient hospital for implantation. Aim: The aim of this study is to analyze the ergonomics of digital microscopy (DM) based on virtual slides, on telemedicine systems (TS) for tele-pathological evaluation (TPE) of the grafts (G) in organ transplantation (OT). Material and Methods: By experimental simulation, the ergonomics of DM for microscopic TPE of renal graft (RG), liver graft (LG) and pancreatic graft (PG) tissues is analyzed. In fact, this corresponded to the ergonomics of digital microscopy for TPE in OT by applying virtual slide (VS) system for graft tissue image capture, for remote diagnoses of possible microscopic inflammatory and/or neoplastic lesions. Experimentation included the development of an OTE-TS similar experimental telemedicine system (Exp.-TS) for simulating the integrated VS based microscopic TPE of RG, LG and PG Simulation of DM on TS based TPE performed by 2 specialists on a total of 238 human renal graft (RG), 172 liver graft (LG) and 108 pancreatic graft (PG) tissues digital microscopic images for inflammatory and neoplastic lesions on four electronic spaces of the four used TS. Results: Statistical analysis of specialist‘s answers about the ability to accurately diagnose the diseased RG, LG and PG tissues on the electronic space among four TS (A,B,C,D) showed that DM on TS for TPE in OT is elaborated perfectly on the ES of a desktop, followed by the ES of the applied Exp.-TS. Tablet and mobile-phone ES seem significantly risky for the application of DM in OT (p<.001). Conclusion: To make the largest reduction in errors and adverse events referring to the quality of the grafts, it will take application of human factors engineering to procurement, design, audit, and awareness-raising activities. Consequently, it will take an investment in new training, people, and other changes to management activities for DM in OT. The simulating VS based TPE with DM of RG, LG and PG tissues after retrieval, seem feasible and reliable and dependable on the size of the electronic space of the applied TS, for remote prevention of diseased grafts from being retrieved and/or sent to the recipient hospital and for post-grafting and pre-transplant planning.

Keywords: digital microscopy, organ transplantation, tele-pathology, virtual slides

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Authors: M. Bagheri Varzaneh, H. R. Rahmani, R. Jahanian

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The present study aimed to investigate the effects of copper-methionine on ascites incidence and hematological, morphological and enzymatic responses in broiler chickens. A total of 480 one-day-old Ross 308 broiler chicks were used in a completely randomized design in a 2×3 factorial arrangement of treatments including two ambient temperatures (thermoneutral and cold stress) and three copper levels (0, 100, and 200 mg/kg as copper-methionine) with 4 replicates (20 birds in each replicate). Broilers were kept in an environmentally-controlled room from 1 to 28 days; then, half of them exposed to cold temperature from 28 to 45 days of age. The birds were sacrificed at days 38 and 45 of age. Heparinized blood samples were collected to measure hematocrit, hemoglobin concentration, red blood cell (RBC) count, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Heart, lungs, liver, and spleen were collected and weighed separately on a sensitive digital scale. At d 38 of age, none of hematological variables, enzymatic parameters, and relative weights of organs were affected by treatments. Ascitic broilers were observed in group subjected to cold temperature and control diet (without supplemental copper) at d 45 of age. Relative weight of lungs and relative weight of heart in broilers fed on copper-methionine supplemented diets in cold temperature were lower compared with other groups. Relative liver weight, ALT, AST activities, and hematological parameters such as hematocrit, hemoglobin concentration, red blood cell count in ascitic broilers were significantly increased. In contrast, a significant decrease of the relative weight of spleen was shown in these chickens. The results showed that dietary supplementation with copper–methionine can decrease probability of ascites incidence in broilers chicks, especially under cold condition.

Keywords: ascites, cold temperature, copper-methionine, cold-stressed broiler

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Authors: Garima Anand, Rupam Kapoor

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Alternaria leaf spot caused by Alternaria carthami is one of the most devastating diseases of safflower. It has resulted in huge losses in crop production and cultivation leading to a fall out of India’s rank as the leading producer of safflower in the world. Understanding the diversity of any pathogen is essential for its management and for the development of disease control strategies. The diversity of A. carthami was therefore analysed on the basis of biochemical, pathogenicity and genetic lines using ISSR markers. Collections and isolations of 95 isolates of A. carthami were made from major safflower producing states of India. Virulence was analysed to evaluate the pathogenic potential of these isolates. The isolates from Bijapur, Dharwad districts (Karnataka), and Parbhani and Solapur districts (Maharashtra) were found to be highly virulent. The virulence assays showed low virulence levels (42%) for the largest part of the population. Biochemical characterization to assess aggressiveness of these isolates was done by estimating the activity of cell wall degrading enzymes where isolates from districts Dharwad, Bijapur of Karnataka and districts Parbhani and Latur of Maharashtra were found to be most aggressive. Genetic diversity among isolates of A. carthami was determined using eighteen ISSR markers. Distance analysis using neighbour joining method and PCoA analysis of the ISSR profiles divided the isolates into three sub-populations. The most virulent isolates clustered in one group in the dendrogram. The study provided no evidence for geographical clustering indicating that isolates are randomly spread across the states, signifying the high potential of the fungus to adapt to diverse regions. The study can, therefore, aid in the breeding and deployment of A. carthami resistant safflower varieties and in the management of Alternaria leaf spot disease.

Keywords: alternaria leaf spot, genetic diversity, pathogenic potential, virulence

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Authors: Vinay Mishra, Meena Malkani

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Objectives: To study the effect of physical maternal nutritional markers (viz. weight, height, gestational weight gain, BMI) and third-trimester haemoglobin concentration on anthropometry and cord blood haemoglobin of their newborn. Methods: Study area: Post-natal ward of a tertiary care hospital in an urban area. Study population: All post-partum women and their newborns. Sample size: 100. Maternal and neonatal data and anthropometric measurements were recorded in semi-structured case proforma. Data analysis: The data obtained was analysed using SPSS 20 software.The comparison between the groups was done using One-Way Analysis of Variance for two groups. For more than two groups comparisons further posthoc analysis was done using Tukey test. Pearson correlation coefficient was used for correlating the variables. A P value less than 0.05 was considered significant. Results: 1. Out of the 100 studied mothers, 52% were anaemic. 2. Cord blood haemoglobin values decreased significantly with the order of birth. 3. Cord blood haemoglobin of normal-weight newborns is significantly higher as compared to that of LBW newborns. 4. Cord blood haemoglobin has strong statistical significance with maternal anaemia. 5. Pre-pregnancy weight and gestational weight gain significantly influence the neonates anthropometry. Conclusions: 1. Birth order has a prominent inverse effect on the cord blood haemoglobin. 2. Majority of the expectant mothers are anaemic and give birth to LBW babies with reduced anthropometric markers. 3. Pre-pregnancy weight, height and gestational weight gain has a very significant impact on the neonatal anthropometry. 4. Thus, maternal nutrition during gestation and during the crucial periods of growth in the pre-child bearing age group has a very significant impact on foetal development.

Keywords: maternal nutrition, anthropometry, cord blood hemoglobin, newborn

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Authors: Paula Buldres, Jorge Toledo

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Objectives: This study aimed to characterize potential probiotic strains isolated from canine breast milk for use in dogs with enteropathies. Methodology: Six canine breast milk strains, one canine colostrum strain, and one control porcine breast milk strain were characterized. According to its functional properties of resistance to acids, different concentrations of bile salts, and pancreatin, its presumptive properties of safety and inhibitory effect on pathogens, non-cytotoxic characteristics, and adhesion to the intestine. The immunomodulatory effect of formulations with better probiotic characterization in vitro and in vivo was also analyzed. Results: Two strains characterized as potential probiotics were obtained, which corresponded to the canine strains (TUCO-16 and TUCO-17), presenting resistance to acidic pH, bile salts, and pancreatin, as well as an inhibitory effect on pathogenic Escherichia coli, Salmonella sp., and Clostridium perfringens. Strains TUCO-16 and TUCO-17 induced a significant increase in the expression of TNF-α and IL-8 in canine macrophages, respectively. Expression analyses of pattern recognition receptors in DH82 cells suggest that TUCO-16 and TUCO-17 might increase the TLR2 expression marker, and porcine strain (TUCO-4) increases the NOD2 expression marker. Based on the count obtained and the encapsulation yield, the best formulations correspond to FOS-Inulin for the TUCO-17 and TUCO-4 strains; Maltodextrin-Inulin for TUCO-16. All the strains are non-cytotoxic. The strain that showed the highest adhesion to intestinal epithelial cells was TUCO-17 with the FOS-Inulin formulation. On the other hand, the probiotics decreased the expression of pro-inflammatory markers in vivo, both in the intestine and in the spleen of mice. Conclusion: The combination of these three strains under study (TUCO-16, TUCO-17, and TUCO-4) would cover the probiotic properties in formulation and immunomodulation of all the markers under study.

Keywords: probiotics, gastrointestinal infec, dog, probiotic formulation, immunomodulatory probiotics

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Authors: N. Umasuthan, S. D. N. K. Bathige, W. S. Thulasitha, I. Whang, J. Lee

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The MyD88 is an evolutionarily conserved host-expressed adaptor protein that is essential for proper TLR/ IL1R immune-response signaling. A previously identified complete cDNA (1626 bp) of OfMyD88 comprised an ORF of 867 bp encoding a protein of 288 amino acids (32.9 kDa). The gDNA (3761 bp) of OfMyD88 revealed a quinquepartite genome organization composed of 5 exons (with the sizes of 310, 132, 178, 92 and 155 bp) separated by 4 introns. All the introns displayed splice signals consistent with the consensus GT/AG rule. A bipartite domain structure with two domains namely death domain (24-103) coded by 1st exon, and TIR domain (151-288) coded by last 3 exons were identified through in silico analysis. Moreover, homology modeling of these two domains revealed a similar quaternary folding nature between human and rock bream homologs. A comprehensive comparison of vertebrate MyD88 genes showed that they possess a 5-exonic structure. In this structure, the last three exons were strongly conserved, and this suggests that a rigid structure has been maintained during vertebrate evolution. A cluster of TATA box-like sequences were found 0.25 kb upstream of cDNA starting position. In addition, putative 5'-flanking region of OfMyD88 was predicted to have TFBS implicated with TLR signaling, including copies of NFB1, APRF/ STAT3, Sp1, IRF1 and 2 and Stat1/2. Using qPCR technique, a ubiquitous mRNA expression was detected in liver and blood. Furthermore, a significantly up-regulated transcriptional expression of OfMyD88 was detected in head kidney (12-24 h; >2-fold), spleen (6 h; 1.5-fold), liver (3 h; 1.9-fold) and intestine (24 h; ~2-fold) post-Fla challenge. These data suggest a crucial role for MyD88 in antibacterial immunity of teleosts.

Keywords: MyD88, innate immunity, flagellin, genomic analysis

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Authors: Eva Karaskova, Jana Volejnikova, Dusan Holub, Maria Velganova-Veghova, Michaela Spenerova, Dagmar Pospisilova

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Background: Hepcidin is the central regulator of iron metabolism. Its production is mainly affected by an iron deficiency and the presence of inflammatory activity in the body. The aim of this study was to compare serum hepcidin levels in paediatric patients with newly diagnosed inflammatory bowel disease and hepcidin levels during maintenance therapy, correlate changes of serum hepcidin levels with selected markers of iron metabolism and inflammation and type of provided treatment. Methods: Children with newly diagnosed Crohn's disease (CD) and ulcerative colitis (UC) were included in this prospective study. Blood and stool samples were collected before treatment (baseline). Serum hepcidin, hemoglobin levels, platelet counts, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), interleukin-6 (IL 6), ferritin, iron, soluble transferrin receptors, and fecal calprotectin were assessed. The same parameters were measured and compared with the baseline levels in the follow-up period, during maintenance therapy (average of 39 months after diagnosis). Results: Patients with CD (n=30) had higher serum hepcidin levels (expressed as a median and interquartile range) at diagnosis than subjects with UC (n=13). These levels significantly decreased during the follow-up (from 36.5 (11.5-79.6) ng/ml to 2.1 (0.9-6.7) ng/ml). Contrarily, no significant serum hepcidin level changes were observed in UC (from 5.4 (3.4-16.6) ng/ml to 4.8 (0.9-8.1) ng/ml). While in children with CD hepcidin level dynamics correlated with disease activity and inflammatory markers (ESR, CRP), an only correlation with serum iron levels was observed in patients with UC. Conclusion: Children with CD had higher serum hepcidin levels at diagnosis compared to subjects with UC. Decrease of serum hepcidin in the CD group during anti-inflammatory therapy has been observed, whereas low hepcidin levels in children with UC have remained unchanged. Acknowledgment: This study was supported by grant MH CZ–DRO (FNOl, 00098892).

Keywords: children, Crohn's disease, ulcerative colitis, anaemia, hepcidin

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Authors: Maria Francesca Scannone, Martina Bochicchio

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One of the major environmental problems today is hydrocarbon contamination. The promising sustainable technologies for the treatment of these contaminated sites involves the use of biological organisms. In Agri Valley (Basilicata Region) there is a living laboratory (natural oil seeps) where the selective pressure has enriched the environmental matrices with microorganisms, fungi and plant species able to use the hydrocarbons as a source of metabolic energy, to degrade or tolerate hydrocarbons. Observers visiting this area are fascinated by its unspoiled nature, and the condition of the ecosystem does not appear to has been damaged. The amazing resiliency observed in Tramutola site is of key importance to try to bring green remediation technologies, but no research has been done to identify high-performing native species. The aim of this research was to study how natural processes affect the fate of released oil or how individual species or communities of plants and animals are capable of dealing with the burden of otherwise toxic chemicals. The survey of vegetation was carried out, more than 60 species have been identified and divided into tree, shrub and herb layer. Plant data sheets have been completed only for the species that showed the most appropriate properties for phytoremediation. In general, members of the Salicales, Cyperales, Poales, Fagales, Cornales, Equisetales orders were the most commonly identified orders. They are pioneer plants with high adaptive capacity and vegetative propagation. The literature review has highlighted the existence of rhizosphere effect and a green liver model on selected plants. The study provides significant information on the environmental stress adaptation processes of many indigenous plants that are living and growing on a natural leak of crude oil and gas that migrates up through subsurface.

Keywords: green liver, hydrocarbon degradation, oil seeps, phytoremediation

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Authors: Nayira A. Abd Elbaky, Amal J. Fatani, Hazar Yaqub, Nouf M. Al-Rasheed, Naglaa El-Orabi, Mai Osman

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The present work is aimed to evaluate the protective effect of N-acetyl cystiene (NAC), coenzyme Q10 (CoQ10), and their combination against carbon tetrachloride (CCl4)-induced cardiotoxicity in rats. CCl4 treatment significantly elevated the levels of cardiac oxidative stress bio markers including nitric oxide (NO) and malondialdehyde (MDA). A concomitant decrease in the level of reduced glutathione and the activity of membrane bound enzyme, calcium-adenosine triphosphatase were observed in the hearts of rats exposed to CCl4 compared to respective values in normal group. Quantitative analysis of myocardial energy metabolism revealed a significant decrease in the glucose content coupled with depletion in the activities of myocardial glycolytic enzymes as hexokinase (HK), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) after CCl4 treatment. In addition, a significant elevation in myocardial hydroxyproline level was observed in CCl4 intoxicated rats indicating interstitial collagen accumulation. Pretreatment with either NAC, CoQ10 or their combination successively alleviated the alterations in myocardial oxidative stress and antioxidant markers, as well as effectively up-regulated the decrease in cardiac energetic biomarkers in CCl4 intoxicated rats. Moreover, these antioxidants markedly reduced myocardial hydroxyproline level versus that of CCl4-treated animals. In conclusion, the present results illustrated that the prophylactic use of the current antioxidant resulted in a remarkable cardioprotective effect against CCl4 induced myocardial damage, which suggest that they may candidates as prophylactic agents against different cardio-toxins.

Keywords: carbon tetrachloride, lipid peroxidation, antioxidant, energy metabolism, hydroxyproline

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Authors: Shahira El Shafie, Hanaa Eldash, Engy Ghabbour, Mohamed Eid

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Several immunologic defects can be found in patients with beta-thalassemia, among which the impairment of neutrophil phagocytic function is of utmost importance. Attention has been directed to the role of proinflammatory cytokines in neutrophil chemotaxis and phagocytosis. Interleukin-8 (IL-8) is an important chemotactic and activation peptide for neutrophils; changes in IL-8 level and potential correlation with neutrophil function can be relevant to immunomodulation pathophysiology in beta-thalassemia patients. This case-control study aimed to evaluate IL-8 level and to assess granulocyte recruitment, as markers of immunomodulation, in poly-transfused thalassemia patients attending Fayoum University Hospitals. The study was conducted on 50 patients with ß thalassemia and 32 age-matched controls. 21/50 patients were transfused more than ten times, and 29/50 were transfused in a lower frequency. Patients and controls were subjected to thorough history taking and clinical examination, measurement of IL-8 level using human IL-8 ELISA kit, and Rebuck skin window technique (RSWT) to assess granulocyte recruitment. Our data showed statistically significant higher levels of IL-8 in ß thalassemia patients compared to control with a much higher difference in patients transfused more than ten times. Neutrophil recruitment was significantly lower in ß thalassemia patients compared to control at 4 hours and 24 hours test time. Although IL-8, the main chemotactic pro-inflammatory cytokine showed a higher level in thalassemia patients, neutrophils recruitment was significantly lower, especially in those receiving more than ten transfusion times. Our findings suggest a possible role of other neutrophil chemotactic factors, defective neutrophil response, or increased IL-8 as compensation of abnormal function. We recommend the use of IL-8 and Rebuck skin window technique as useful markers of immunomodulation in thalassemia and further study for these biomarkers to assess their clinical implications and impact on the management of thalassemia patients.

Keywords: beta-thalassemia, Interleukin-8, Rebuck skin window technique, immunomodulation

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Authors: Mengying Wang, Dongjing Liu, Holger Schwender, Ping Wang, Hongping Zhu, Tao Wu, Terri H Beaty

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Background: Maternal smoking is a recognized risk factor for nonsyndromic cleft lip with or without cleft palate (NSCL/P). It has been reported that the effect of maternal smoking on oral clefts is mediated through genes that influence nicotine dependence. The polymorphisms of cholinergic receptor nicotinic alpha (CHRNA) and beta (CHRNB) subunits genes have previously shown strong associations with nicotine dependence. Here, we attempted to investigate whether the above genes are associated with clefting risk through testing for potential gene-gene (G×G) and gene-environment (G×E) interaction. Methods: We selected 120 markers in 14 genes associated with nicotine dependence to conduct transmission disequilibrium tests among 806 Chinese NSCL/P case-parent trios ascertained in an international consortium which conducted a genome-wide association study (GWAS) of oral clefts. We applied Cordell’s method using “TRIO” package in R to explore G×G as well as G×E interaction involving environmental tobacco smoke (ETS) based on conditional logistic regression model. Results: while no SNP showed significant association with NSCL/P after Bonferroni correction, we found signals for G×G interaction between 10 pairs of SNPs in CHRNA3, CHRNA5, and CHRNB4 (p<10-8), among which the most significant interaction was found between RS3743077 (CHRNA3) and RS11636753 (CHRNB4, p<8.2×10-12). Linkage disequilibrium (LD) analysis revealed only low level of LD between these markers. However, there were no significant results for G×ETS interaction. Conclusion: This study fails to detect association between nicotine dependence genes and NSCL/P, but illustrates the importance of taking into account potential G×G interaction for genetic association analysis in NSCL/P. This study also suggests nicotine dependence genes should be considered as important candidate genes for NSCL/P in future studies.

Keywords: Gene-Gene Interaction, Maternal Smoking, Nicotine Dependence, Non-Syndromic Cleft Lip with or without Cleft Palate

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Authors: Marcela Parra-Vargas, Ana Sandoval-Rodriguez, Roberto Rodriguez-Echevarria, Jose Dominguez-Rosales, Juan Armendariz-Borunda

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Non-alcoholic fatty liver disease (NAFLD) is characterized by an excess of hepatic lipids, and it is to author’s best knowledge, the most prevalent chronic liver disorder. Anthocyanin-rich food consumption is linked to health benefits in metabolic disorders associated with obesity and NAFLD, although the precise functional role of anthocyanidin delphinidin (Dp) has yet to be established. The aim of this study was to investigate the effect of the Dp in NAFLD metabolic alterations by evaluating prevention or amelioration of hepatic lipid accumulation, as well as molecular mechanisms in two experimental obesity-related models of NALFD. In vitro: HepG2 cells were incubated with sodium palmitate (PA, 1 mM) to induce lipotoxic damage, and concomitantly treated with Dp (180 uM) for 24 h. Subsequently, total lipid accumulation was measured by colorimetric staining with Oil Red O, and total intrahepatic triglycerides were determined by an enzymatic assay. To assess molecular mechanisms, cells were pre-treated with PA for 24 h and then exposed to Dp for 1 h. In vivo: four-week-old male C57BL/6Nhsd mice were allocated in two main groups. Mice were fed with standard diet (control) or high-fat and high-carbohydrate diet (45% fat, HFD) for 16 wk to induce NAFLD. Then HFD was divided into subgroups: one treated orally with Dp (15 mg/kg bw, HFD-Dp) every day for 4 wk, while HFD group treated with vehicle (DMSO). Weight and fasting glucose were recorded weekly, while dietary ingestion was measured daily. Insulin tolerance test was performed at the end of treatment. Liver histology was evaluated with H&E and Masson’s trichrome stain. RT-PCR was used to evaluate gene expression and Western Blot to determine levels of protein in both experimental models. Parametric data were analyzed with one-way ANOVA and Tukey’s post-hoc test. Kruskal-Wallis and Mann-Whitney U test for non-parametric data, and P < 0.5 were considered significant. Dp prevented hepatic lipid accumulation by PA in HepG2 hepatocytes. Furthermore, Dp down-regulated gene expression of SREBP1c, FAS, and CPT1a without modifying AMPK phosphorylation levels. In vivo, Dp oral administration did not ameliorate lipid metabolic alterations raised by HFD. Adiposity, dietary ingestion, fasting glucose, and insulin sensitivity after Dp treatment remained similar to HFD group. Histological analysis showed hepatic damage in HFD groups and no differences between HFD and HFD-Dp groups were found. Hepatic gene expression of ACC and FAS were not altered by HFD. SREBP1c was similar in both HFD and HFD-Dp groups. No significant changes were observed in SREBP1c, ACC, and FAS adipose tissue gene expression by HFD or Dp treatment. Additionally, immunoblotting analysis revealed no changes in pathway SIRT1-LKB-AMPK and PPAR alpha by both HFD groups compared to control. In conclusion, the antioxidant Dp may provoke beneficial effects in the prevention of hepatic lipid accumulation. Nevertheless, the oral dose administrated in mice that simulated the total intake of anthocyanins consumed daily by humans has no effect as a treatment on hepatic lipid metabolic alterations and histological abnormalities associated with exposure to chronic HFD. A healthy lifestyle with regular intake of antioxidants such as anthocyanins may prevent metabolic alterations in NAFLD.

Keywords: anthocyanins, antioxidants, delphinidin, non-alcoholic fatty liver disease, obesity

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Authors: Hongping Lu, Kelbinur Tursun, Yaru Li, Yu Zhang, Shunai Liu, Ming Han

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Objective: To investigate whether NS5ATP9 is involved in IL-6 mediated autophagy and the relationship between IL-6 and NS5ATP9 in liver cancer cells. Methods: 1. Detect the mRNA and protein levels of Beclin 1 after HepG2 cells were treated with or without recombinant human IL-6 protein. 2. Measure and compare of the changes of autophagy-related genes with their respective control, after IL-6 was silenced or neutralized with monoclonal antibody against human IL-6. 3. HepG2 cells were incubated with 50 ng/ml of IL-6 in the presence or absence of PDTC. The expression of NS5ATP9 was analyzed by Western blot after 48 h. 4. After NS5ATP9-silenced HepG2 cells had been treated with 50 ng/ml recombinant IL-6 protein, we detected the Beclin 1 and LC3B (LC3Ⅱ/Ⅰ) expression. 5. HepG2 cells were transfected with pNS5ATP9, si-NS5ATP9, and their respective control. Total RNA was isolated from cells and analyzed for IL-6. 6. Silence or neutralization of IL-6 in HepG2 cells which has been transfected with NS5ATP9. Beclin 1 and LC3 protein levels were analyzed by Western blot. Result: 1. After HepG2 were treated with recombinant human IL-6 protein, the expression of endogenous Beclin 1 was up-regulated at mRNA and protein level, and the conversion of endogenous LC3-I to LC3-II was also increased. These results indicated that IL-6 could induce autophagy. 2. When HepG2 cells were treated with IL-6 siRNA or monoclonal antibody against human IL-6, the expression of autophagy-related genes were decreased. 3. Exogenous human IL-6 recombinant protein up-regulated NS5ATP9 via NF-κB activation. 4. The expression of Beclin 1 and LC3B was down-regulated after IL-6 treated NS5ATP9-silenced HepG2 cells. 5. NS5ATP9 could reverse regulates IL-6 expression in HepG2 cells. 6. Silence or neutralization of IL-6 attenuates NS5ATP9-induced autophagy slightly. Conclusion: Our results implied that in HCC patients, maybe the higher level of IL-6 in the serum promoted the expression of NS5ATP9 and induced autophagy in cancer cells. And the over-expression of NS5ATP9 which induced by IL-6, in turn, increased IL-6 expression, further, promotes the IL-6/NS5ATP9-mediated autophagy and affects the progression of tumor. Therefore, NS5ATP9 silence might be a potential target for HCC therapy.

Keywords: autophagy, Hepatocellular carcinoma, IL-6, microenvironment, NS5ATP9

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Authors: Abdolamir Allameh, Shahnaz Esmaeili, Mina Allameh, Safoura Khajeniazi

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Stem cells with therapeutic applications can be isolated from human placenta/umblical cord blood (UCB) as well as the cord tissue (UC). Stem cells in culture are vulnerable to oxidative stress, particularly when subjected to differentiation process. The aim of this study was to examine the chnages in the rate of oxidation that occurs to cellular macromolecules during hepatic differentiation of mononuclear cells (MSCs). In addition, the impact of the hepatic differentiation process of MSC on cellular and biological activity of the cells will be undertaken. For this purpose, first mononuclear cells (MNCs) were isolated from human UCB which was obtained from a healthy full-term infant. The cells were cultured at a density of 3×10⁵ cells/cm² in DMEM- low-glucose culture media supplemented with 20% FBS, 2 mM L-glutamine, 100 μg/ml streptomycin and 100 U/ml penicillin. Cell cultures were then incubated at 37°C in a humidified 5% CO₂ incubator. After removing non-adherent cells by replacing culture medium, fibroblast-like adherent cells were resuspended in 0.25% trypsin-EDTA and plated in 25 cm² flasks (1×10⁴/ml). Characterization of the MSCs was routinely done by observing their morphology and growth curve. MSCs were subjected to a 2-step hepatocyte differentiation protocol in presence of hepatocyte growth factor (HGF), dexamethazone (DEX) and oncostatin M (OSM). The hepatocyte-like cells derived from MSCs were checked every week for 3 weeks for changes in lipid peroxidation, protein carbonyl formation and DNA oxidation i.e., 8-hydroxy-2'-deoxyguanosine (8-OH-dG) assay. During the 3-week differentiation process of MSCs to hepatocyte-like cells we found that expression liver-specific markers such as albumin, was associated with increased levels of lipid peroxidation and protein carbonyl formation. Whereas, undifferentiated MSCs has relatively low levels of lipid peroxidation products. There was a significant increase ( p < 0.05) in lipid peroxidation products in hepatocytes on days 7, 14, and 21 of differentiation. Likewise, the level of protein carbonyls in the cells was elevated during the differentiation. The level of protein carbonyls measured in hepatocyte-like cells obtained 3 weeks after differentiation induction was estimated to be ~6 fold higher compared to cells recovered on day 7 of differentiation. On the contrary, there was a small but significant decrease in DNA damage marker (8-OH-dG) in hepatocytes recovered 3 weeks after differentiation onset. The level of 8-OHdG which was in consistent with formation of reactive oxygen species (ROS). In conclusion, this data suggest that despite the elevation in oxidation of lipid and protein molecules during hepatocyte development, the cells were normal in terms of DNA integrity, morphology, and biologically activity.

Keywords: adult stem cells, DNA integrity, free radicals, hepatic differentiation

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Authors: Mahesh M. Patil, K. A. Anu-Appaiah

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Overweight and obesity is a serious medical problem, increasing in prevalence, and affecting millions worldwide. Investigators have been trying from decades to articulate the burden of obesity and related risk factors. To answer this problem, we suggest a new therapeutic anti-obesity compounds from Garcinia xanthochymus fruit. However, there is little published scientific information on non-hydroxycitric acid Garcinia species. Our findings include biochemical characterization of the fruit; in vivo toxicity and bio-efficacy study of G. xanthochymus in high fat diet wistar rat model. We observed that Garcinia pericarp is a rich source of organic acids, polyphenols, mono- (40.63%) and poly-unsaturated fatty acids (16.45%; omega-3: 10.02%). Toxicological studies have showed that Garcinia is safe and had no observed adverse effect level up to 400 mg/kg/day. Body weight and food intake was significantly (P<0.05) reduced in oral gavage treated rats (sonicated Garcinia powder) in 13 weeks. Subcutaneous fat was significantly (P<0.05) reduced in Garcinia treated rats. Hepatocytes significantly (p<0.05) overexpressed sterol regulatory element binding protein 2, liver X receptor- α, liver X receptor- β, lipoprotein lipase and monoacylglycerol lipase. Fatty acid binding protein 1 and peroxisome proliferator activated receptor- α were down regulated as assessed by real time qPCR. Currently our research is focused on the adipocyte obesity related gene expressions, effect of Garcinia on 3T3-adipocyte cell lines and high fat diet induced mice model. This in vivo pre-clinical data suggests that G. xanthochymus may have clinical utility for the treatment of obesity. However, further studies are required to establish its potency.

Keywords: Garcinia xanthochymus, anti-obesity, high fat diet, real time qPCR

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Authors: ZhenlinJu, Christopher P. Vellano, RehanAkbani, Yiling Lu, Gordon B. Mills

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The epithelial–mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal characteristics, such as profound disruption of cell-cell junctions, loss of apical-basolateral polarity, and extensive reorganization of the actin cytoskeleton to induce cell motility and invasion. A hallmark of EMT is its capacity to promote metastasis, which is due in part to activation of several transcription factors and subsequent downregulation of E-cadherin. Unfortunately, current approaches have yet to uncover robust protein marker sets that can classify tumors as possessing strong EMT signatures. In this study, we utilize reverse phase protein array (RPPA) data and consensus clustering methods to successfully classify a subset of cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) tumors into an EMT protein signaling group (EMT group). The overall survival (OS) of patients in the EMT group is significantly worse than those in the other Hormone and PI3K/AKT signaling groups. In addition to a shrinkage and selection method for linear regression (LASSO), we applied training/test set and Monte Carlo resampling approaches to identify a set of protein markers that predicts the EMT status of CESC tumors. We fit a logistic model to these protein markers and developed a classifier, which was fixed in the training set and validated in the testing set. The classifier robustly predicted the EMT status of the testing set with an area under the curve (AUC) of 0.975 by Receiver Operating Characteristic (ROC) analysis. This method not only identifies a core set of proteins underlying an EMT signature in cervical cancer patients, but also provides a tool to examine protein predictors that drive molecular subtypes in other diseases.

Keywords: consensus clustering, TCGA CESC, Silhouette, Monte Carlo LASSO

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Authors: Shahira M. Ezzat, Marwa I. Ezzat, Mona M. Okba, Esther T. Menze, Ashraf B. Abdel-Naim, Shahnas O. Mohamed

Abstract:

Background: In order to decrease the burden of the high cost of synthetic drugs, it is important to focus on phytopharmaceuticals. The aim of our study was to search for the mechanism of ginger (Zingiber officinale Roscoe) anti-inflammatory potential and to correlate it to its biophytochemicals. Methods: Various extracts viz. water, 50%, 70%, 80%, and 90% ethanol were prepared from ginger rhizomes. Fractionation of the aqueous extract (AE) was accomplished using Diaion HP-20. In vitro anti-inflammatory activity of the different extracts and isolated compounds was evaluated by protein denaturation inhibition, membrane stabilization, protease inhibition, and anti-lipoxygenase assays. In vivo anti-inflammatory activity of AE was estimated by assessment of rat paw oedema after carrageenan injection. Prostaglandin E2 (PGE2), certain inflammation markers (TNF-α, IL-6, IL-1α, IL-1β, INFr, MCP-1MIP, RANTES, and Nox) levels and MPO activity in the paw edema exudates were measured. Total antioxidant capacity (TAC) was also determined. Histopathological alterations of paw tissues were scored. Results: All the tested extracts showed significant (p < 0.1) anti-inflammatory activities. The highest percentage of heat induced albumin denaturation (66%) was exhibited by the 50% ethanol (250 μg/ml). The 70 and 90% ethanol extracts (500 μg/ml) were more potent as membrane stabilizers (34.5 and 37%, respectively) than diclofenac (33%). The 80 and 90% ethanol extracts (500 μg/ml) showed maximum protease inhibition (56%). The strongest anti-lipoxygenase activity was observed for the AE. It showed more significant lipoxygenase inhibition activity than that of diclofenac (58% and 52%, respectively) at the same concentration (125 μg/ml). Fractionation of AE yielded four main fractions (Fr I-IV) which showed significant in vitro anti-inflammatory. Purification of Fr-III and IV led to the isolation of 6-poradol (G1), 6-shogaol (G2); methyl 6- gingerol (G3), 5-gingerol (G4), 6-gingerol (G5), 8-gingerol (G6), 10-gingerol (G7), and 1-dehydro-6-gingerol (G8). G2 (62.5 ug/ml), G1 (250 ug/ml), and G8 (250 ug/ml) exhibited potent anti-inflammatory activity in all studied assays, while G4 and G5 exhibited moderate activity. In vivo administration of AE ameliorated rat paw oedema in a dose-dependent manner. AE (at 200 mg/kg) showed significant reduction (60%) of PGE2 production. The AE at different doses (at 25-200 mg/kg) showed significant reduction in inflammatory markers except for IL-1α. AE (at 25 mg/kg) is superior to indomethacin in reduction of IL-1β. Treatment of animals with the AE (100, 200 mg/kg) or indomethacin (10 mg/kg) showed significant reduction in TNF-α, IL-6, MCP-1, and RANTES levels, and MPO activity by about (31, 57 and 32% ) (65, 60 and 57%) (27, 41 and 28%) (23, 32 and 23%) (66, 67 and 67%) respectively. AE at 100 and 200 mg/kg was equipotent to indomethacin in reduction of NOₓ level and in increasing the TAC. Histopathological examination revealed very few inflammatory cells infiltration and oedema after administration of AE (200 mg/kg) prior to carrageenan. Conclusion: Ginger anti-inflammatory activity is mediated by inhibiting macrophage and neutrophils activation as well as negatively affecting monocyte and leukocyte migration. Moreover, it produced dose-dependent decrease in pro-inflammatory cytokines and chemokines and replenished the total antioxidant capacity. We strongly recommend future investigations of ginger in the potential signal transduction pathways.

Keywords: anti-lipoxygenase activity, inflammatory markers, 1-dehydro-6-gingerol, 6-shogaol

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Authors: Suang K. Lau, Ismail Goolam, Rafid Al-Asady

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Background: The majority of patients with hepatocellular carcinoma (HCC) are not suitable for curative treatment, in the form of surgical resection or transplantation, due to tumour extent and underlying liver dysfunction. In these non-resectable cases, a variety of non-surgical therapies are available, including microwave ablation (MWA), which has shown increasing popularity due to its low morbidity, low reported complication rate, and the ability to perform multiple ablations simultaneously. Objective: The aim of this study was to evaluate the validity of MWA as a viable treatment option in the management of HCC and hepatic metastatic disease, by assessing its efficacy and complication rate at a tertiary hospital situated in Westmead (Australia). Methods: A retrospective observational study was performed evaluating patients that underwent MWA between 1/1/2017–31/12/2018 at Westmead Hospital, NSW, Australia. Outcome measures, including residual disease, recurrence rates, as well as major and minor complication rates, were retrospectively analysed over a 12-months period following MWA treatment. Excluded patients included those whose lesions were treated on the basis of residual or recurrent disease from previous treatment, which occurred prior to the study window (11 patients) and those who were lost to follow up (2 patients). Results: Following treatment of 106 new hepatic lesions, the complete response rate (CR) was 86% (91/106) at 12 months follow up. 10 patients had the residual disease at post-treatment follow up imaging, corresponding to an incomplete response (ICR) rate of 9.4% (10/106). The local recurrence rate (LRR) was 4.6% (5/106) with follow-up period up to 12 months. The minor complication rate was 9.4% (10/106) including asymptomatic pneumothorax (n=2), asymptomatic pleural effusions (n=2), right lower lobe pneumonia (n=3), pain requiring admission (n=1), hypotension (n=1), cellulitis (n=1) and intraparenchymal hematoma (n=1). There was 1 major complication reported, with pleuro-peritoneal fistula causing recurrent large pleural effusion necessitating repeated thoracocentesis (n=1). There was no statistically significant association between tumour size, location or ablation factors, and risk of recurrence or residual disease. A subset analysis identified 6 segment VIII lesions, which were treated via a trans-pleural approach. This cohort demonstrated an overall complication rate of 33% (2/6), including 1 minor complication of asymptomatic pneumothorax and 1 major complication of pleuro-peritoneal fistula. Conclusions: Microwave ablation therapy is an effective and safe treatment option in cases of non-resectable hepatocellular carcinoma and liver metastases, with good local tumour control and low complication rates. A trans-pleural approach for high segment VIII lesions is associated with a higher complication rate and warrants greater caution.

Keywords: hepatocellular carcinoma, liver metastases, microwave ablation, trans-pleural approach

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Authors: Yu-Tzu Hsu, Alice Wu, Hsiang-Kuang Tseng

Abstract:

Introduction: Hepatitis A virus causes acute hepatitis and is usually transmitted by a fecal-oral route of food contamination, which is more prevalent in areas with poor hygienic practices. However, we described a hepatitis A outbreak associated with a fecal-oral route of sexual behavior in men who has sex with men (MSM) in Northern Taiwan. Methods: We retrospectively collected patients with acute HAV infection in MacKay Memorial Hospital, Taipei, Taiwan between July 2015 and November 2016. Demographic data (age, gender, onset time and infection risk), laboratory data (GOT, GPT, bilirubin, HIV status, HBsAg, HCV antibody and syphilis), clinical symptoms and travel history with a foreign tour were analyzed. We compared variables between HIV and non-HIV group. Unless otherwise stated, continuous variables were expressed as mean ± SD, and categorical variables were expressed as number (percentage) for each item. The t test for continuous variables was applied for the comparison between two groups and chi-square for categorical variables were applied for measures of association. Results: We collected 80 cases during the study period. Among them, 54 (67.5%) cases were MSM and 43 (53.8%) cases were HIV positive. The average age was 32.6±7.59 years-old. The average value of initial liver function was 1324 IU/L for AST (GOT), 2100 IU/L for ALT (GPT), and 5.82 mg/dL for bilirubin. We found seven (8.6%) cases were in the status of HBV carrier, five (6.3%) cases were positive for HCV antibody, and 15 (18.6%) cases were co-infected with syphilis. With regards to associated symptoms, 32 (40%) had fever, 46 (57.5%) had nausea, 34 (42.5%) had abdominal discomfort and 46 (57.5%) had general malaise. To compare the non-HIV patients with HIV patients, HIV patients were more likely to be male (p=0.008), MSM (p=0.000), co-infected syphilis (p=0.000) and slowly improving liver function of transaminases (p=0.033, 0.027). Conclusion: The HAV outbreak in Northern Taiwan was mainly occurred in MSM population. Hereafter, our cohort data support a policy in Taiwan to provide one dose of free HAV vaccine shot in this population. Hopefully, the outbreak could be stop by the free vaccine policy and public education.

Keywords: acute hepatitis A, men who has sex with men, human immunodeficiency virus, vaccine

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