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Fatemeh Abedini1, Maznah Ismail1,4, Hossein Hosseinkhani2, Tengku Azmi Tengku Ibrahim1,3, Abdul Rahman Omar1,3, Pei Pei Chong4, Mohd Hair Bejo3, Abraham J Domb51Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, Selangor Darul Ehsan, Malaysia; 2Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan; 3Faculty of Veterinary Medicine, 4Faculty of Medicine and Health Science, Universiti Putra Malaysia, Selangor Darul Ehsan, Malaysia; 5Department of Medicinal Chemistry and Natural Products, School of Pharmacy, Hebrew University-Hadassah Medical School, Jerusalem, IsraelAbstract: Liver metastasis is the main cause of mortality related to colorectal cancer. CXCR4 is necessary for the outgrowth of colon cancer micrometastases. In oncology, it has been demonstrated that several human tumors release lactate dehydrogenase (LDH) into the circulation. CXCR4 gene expression and serum LDH levels are often increased in patients with colorectal cancer. Despite technological advances in cancer therapy, five-year overall survival is still around 50%. Therefore, better treatment needs to be developed. RNA interference (RNAi) is a modern and powerful tool for inhibition of gene expression. However, the rate-limiting step in this technology is effective delivery of RNAi agents. We have investigated a novel strategy of CXCR4 siRNA therapy and its effect on serum LDH levels in a BALB/C mouse model of colorectal cancer metastasis to the liver. Hepatic metastasis was established by injecting a CT26.WT mouse colon carcinoma cell line via the tail vein. Our results demonstrated that CXCR4 siRNA/dextran-spermine nanoparticles achieved high silencing efficiency with low toxicity. Favorable localization of the nanoparticles was confirmed with CXCR4 gene expression in the liver, that was correlated with serum LDH levels. More research will be needed to determine the effect of CXCR4 silencing on serum LDH levels, which may be a useful marker for predicting liver metastasis in colorectal cancer.Keywords: colorectal cancer, CXCR4 siRNA, serum LDH, liver, dextran-spermine

Background: Severe leptospirosis is challenging as it could evolve rapidly and potentially fatal if appropriate management is not performed. An understanding of the progression and pathophysiology of Leptospira infection is important to determine the early changes that could be potentially used to predict the severe occurrence of leptospirosis. This study aimed to understand the kinetics pathogenesis of Leptospira interrogans strain HP358 in the hamster model and identify the early parameters that could be used as biomarkers to predict severe leptospirosis., Methodology/principal Findings: Male Syrian hamsters were infected with Leptospira interrogans strain HP358 and euthanized after 24 hours, 3, 4, 5, 6 and 7 days post-infection. Blood, lungs, liver and kidneys were collected for leptospiral detection, haematology, serum biochemistry and differential expression of pro- and anti-inflammatory markers. Macroscopic and microscopic organ damages were investigated. Leptospira interrogans strain HP358 was highly pathogenic and killed hamsters within 6-7 days post-infection. Pulmonary haemorrhage and blood vessel congestion in organs were noticed as the earliest pathological changes. The damages in organs and changes in biochemistry value were preceded by changes in haematology and immune gene expression., Conclusion/significance: This study deciphered haemorrhage as the earliest manifestation of severe leptospirosis and high levels of IL-1β, CXCL10/IP-10, CCL3/MIP-α, neutrophils and low levels of lymphocytes and platelets serve as a cumulative panel of biomarkers in severe leptospirosis., Competing Interests: The authors have declared that no competing interests exist.

Using only type B gelatin produces hard capsule shells which are too brittle. This study examines the blending of type B bovine gelatin with sodium alginate to produce hard capsule shells and through evaluation of their in vitro physicochemical properties provides a reflection on the role of gelatin and sodium alginate in the blend. The compositions and formulation of the capsule shells in this study comprised gelatin (10%, 20% and 30%), sodium alginate (1%, 2%, 3%, 4% and 5%), water, and opacifying agents (titanium dioxide; TiO 2 ) and polyethylene glycol (PEG) whose concentrations were kept constant. From the 15 films prepared, five were found to form hard capsule shells. Increased concentrations of sodium alginate increased the viscosity of the blends accompanied by capsule thickening. There was a good molecular compatibility between gelatin and sodium alginate. Increased gelatin and sodium alginate concentrations increased the water-holding capacity of the film, which decreased the redness ( a *), lightness ( L *), blueness ( b *), variation in the color parameters (Δ E *) and the whiteness index (WI). The weight of the capsule shells ranged between 0.080 g and 0.25 g and the moisture content was between 5% and 11%. Ash contents for all the formulations were below 5% and the sensitivity of capsules at pH 7 was higher than that at acidic pH. Highest rupture times were observed with simulated gastric fluid (SGF, pH 1) for all formulations. Increased gelatin concentration decreased the resistance of the capsule to force while increased sodium alginate concentration had no effect on resistance to force., Competing Interests: There is no competing interest as declared by authors., (This journal is © The Royal Society of Chemistry.)

Context: Due to increase in the number of patients with impaired immunity, the incidence of liver cancer has increased considerably., Aims: The aim of this study is the investigation the in vitro anticancer effect of zerumbone (ZER) on hepatocellular carcinoma (HCC)., Materials and Methods: The anticancer mechanism of ZER was determined by the rat aortic ring, human umbilical vein endothelial cells (HUVECs) proliferation, chorioallantoic membrane, cell migration, and proliferation inhibition assays., Results: Our results showed that ZER reduced tube formation by HUVECs effectively inhibits new blood vessel and tissue matrix formation. Western blot analysis revealed that ZER significantly ( P < 0.05) decreased expression of molecular effectors of angiogenesis, the matrix metalloproteinase-9, vascular endothelial growth factor (VEGF), and VEGF receptor proteins. We found that ZER inhibited the proliferation and suppressed migration of HepG2 cell in dose-dependent manner., Statistical Analysis Used: Statistical analyses were performed according to the Statistical Package for Social Science (SPSS) version 17.0. The data were expressed as the mean ± standard deviation and analyzed using a one-way analysis of variance. A P < 0.05 was considered statistically significant., Conclusion: The study for the first time showed that ZER is an inhibitor angiogenesis, tumor growth, and spread, which is suggested to be the mechanisms for its anti-HCC effect., Summary: Tumor angiogenesis has currently become an important research area for the control of cancer growth and metastasis. The current study determined the effect of zerumbone on factors associated with angiogenesis that occurs in tumor formation. Abbreviations used: ZER: Zerumbone, MMP-9: Matrix metalloproteinase-9, VEGF: Vascular endothelial growth factor, VEGFR: Vascular endothelial growth factor receptor, HUVECs: Human umbilical vein endothelial cells, HCC: Hepatocellular carcinoma, HIFCS: Heat inactivated fetal calf serum, DMSO: Dimethyl sulfoxide, EDTA: Ethyldiaminetetraacetic acid, Ig: Immunoglobulin, CAM: Chorioallantoic membrane, HRP: Horseradish peroxidase, NIH: National Institutes of Health, MTT: Microtetrazolium, SPSS: Statistical Package for Social Science., Competing Interests: There are no conflicts of interest.

The present study investigates the detection of lard in cocoa butter through changes in fatty acids composition, triacylglycerols profile, and thermal characteristics. Cocoa butter was mixed with 1% to 30% (v/v ) of lard and analyzed using a gas chromatography flame ionization detector, high performance liquid chromatography, and differential scanning calorimetry. The results revealed that the mixing of lard in cocoa butter showed an increased amount of oleic acid in the cocoa butter while there was a decrease in the amount of palmitic acid and stearic acids. The amount of POS, SOS, and POP also decreased with the addition of lard. A heating thermogram from the DSC analysis showed that as the concentration of lard increased from 3% to 30%, two minor peaks at -26 °C and 34.5 °C started to appear and a minor peak at 34.5 °C gradually overlapped with the neighbouring major peak. A cooling thermogram of the above adulterated cocoa butter showed a minor peak shift to a lower temperature of -36 °C to -41.5 °C. Values from this study could be used as a basis for the identification of lard from other fats in the food authentication process., Competing Interests: The authors declare no conflict of interest.

Background: Dead islets replaced with viable islets are a promising offer to restore normal insulin production to a person with diabetes. The main reason for establishing a new islet source for transplantation is the insufficiency of human donor pancreas while using xenogeneic islets perhaps assists this problem. The expression of PDX1 is essential for the pancreas expansion. In mature β-cells, PDX1 has several critical roles such as glucose sensing, insulin synthesis, and insulin secretion. In this study, we aimed to evaluate the expression of pancreatic duodenal homeobox-1 (PDX1) in treated caprine islets in culture and to assess the protective effects of antioxidant factors on the PDX1 gene in cultured caprine islets., Materials and Methods: Purified islets were treated with serum-free, serum, IBMX, tocopherol, or IBMX and tocopherol media. Quantitative polymerase chain reaction and Western blotting were carried out to compare the expression levels of PDX1 in treated purified islets cultured with different media., Results: Islets treated with IBMX/tocopherol exhibited the highest fold change in the relative expression of PDX1 on day 5 post-treatment (relative expression: 6.80±2.08), whereas serum-treated islets showed the lowest fold changes in PDX1 expression on day 5 post-treatment (0.67±0.36), as compared with the expression on day 1 post-treatment. Insulin production and viability tests of purified islets showed superiority of islet at supplemented serum-free media with IBMX/tocopherol compared to other cultures (53.875%±1.59%)., Conclusions: Our results indicated that supplemented serum-free medium with tocopherol and IBMX enhances viability and PDX1 gene expression compared to serum-added and serum-free media., (© 2017 The Authors. Xenotransplantation published by John Wiley & Sons Ltd.)

The edible red seaweed Kappaphycus alvarezii or Eucheuma cottonii is commercially cultivated in the pristine tropical seas for carrageenan production. The systemic, cellular, and molecular effects of E. cottonii 50% alcohol extract [seaweed E. cottonii ethanol extract (SECE)] on breast cancer were investigated in a rat model. Mammary tumor was induced by subcutaneously injecting LA7 cells in female rat mammary pads. After 2 weeks of cancer growth, the rats received oral administration of either SECE [150 mg/kg body weight (BW) and 300 mg/kg BW] or tamoxifen. Electron microscopy imaging results confirmed macrophage activity and hematoxylin and eosin staining indicated that tumor histopathological alterations were restored toward normal structures by the seaweed extract. The extract suppressed tumor development and modulated the immune responses. This was evidenced by the microscopic observations, the increased spleen weight, size, spleen CD19 B cells, and blood immunoglobulin G (IgG) levels. The extract also increased the circulating total white blood cells, lymphocytes, segmented neutrophils count, T cells (CD3), T-helper cells (CD4), cytotoxic T cell (CD8), and nuclear factor-kappa beta expressions. The extract enhanced cancer cell death, by upregulating the Birc5, Chk1, and p53 levels and downregulating the tumor growth cellular Mdm2 (transformed mouse 3T3 cell double minute 2) messenger RNA (mRNA) expression. The extract showed no toxicity at 150 mg/kg BW in rats. The lectin-rich SECE showed tumor suppression by enhancing immune responses and upregulating the cancer cell apoptosis mRNA expressions.

Background: Pancreatic islets are composed of different hormone-secreting cell types. A finely balanced combination of endocrine cells in the islets regulates intraportal vein secretions and plasma nutrient levels. Every islet cell type is distinguished by its specific secretory granule pattern and hormone content, endocrine and cell signaling mechanisms, and neuronal interactions. The scarcity of pancreatic islet donors for patients with diabetes has caused a considerable interest in the field of islet xenotransplantation. Previous studies have shown that cell arrangement in the pancreatic islets of ruminants differs from that of other mammals; however, caprine islet cytoarchitecture has not yet been comprehensively described. This investigation aimed to characterize caprine islets in regard to better understanding of caprine islet structure and compare with previously reported species, by conducting a detailed analysis of islet architecture and composition using confocal microscopy and immunofluorescence staining for pancreatic islet hormones., Methodology: After collection and purification of caprine islets with Euro-Ficoll density gradients, islets were considered for viability and functionality procedures with DTZ (dithizone) staining and GSIST (glucose-stimulated insulin secretion test) subsequently. Batches of islet were selected for immunostaining and study through confocal microscopy and flow cytometry., Results: Histological sections of caprine pancreatic islets showed that α-cells were segregated at the periphery of β-cells. In caprine islets, α- and δ-cells remarkably were intermingled with β-cells in the mantle. Such cytoarchitecture was observed in all examined caprine pancreatic islets and was also reported for the islets of other ruminants. In both small and large caprine islets (< 150 and > 150 μm in diameter, respectively), the majority of β-cells were positioned at the core and α-cells were arranged at the mantle, while some single α-cells were also observed in the islet center. We evaluated the content of β-, α-, and δ-cells by confocal microscopy (n = 35, mean ± SD; 38.01 ± 9.50%, 30.33 ± 10.11%, 2.25 ± 1.10%, respectively) and flow cytometry (n = 9, mean ± SD; 37.52 ± 9.74%, 31.72 ± 4.92%, 2.70 ± 2.81%, respectively). Our findings indicate that the caprine islets are heterogeneous in cell composition. The difference could be attributed to species-specific interaction between endocrine cells and blood., Conclusions: Comparative studies of islet architecture may lead to better understanding of islet structure and cell type population arrangement. These results suggest the use of caprine islets as an addition to the supply of islets for diabetes research., (© 2016 The Authors. Xenotransplantation. Published by John Wiley & Sons Ltd.)

Pancreatic islet transplantation is commonly used to treat diabetes. Cell isolation and purification methods can affect the structure and function of the isolated islet cells. Thus, the development of cell isolation techniques that preserve the structure and function of pancreatic islet cells is essential for enabling successful transplantation procedures. The impact of purification procedures on cell function can be assessed by performing ultrastructure and in vivo studies. Thus, the aim of this study was to evaluate the effect of caprine islets purification procedure on islet cell ultrastructure and functional integrity prior to and post-isolation/purification. The islets were isolated from caprine pancreas by using an optimized collagenase XI-S concentration, and the cells were subsequently purified using Euro-Ficoll density gradient. In vitro viability of islets was determined by fluorescein diacetate and propidium iodide staining. Static incubation was used to assess functionality and insulin production by islet cells in culture media when exposed to various levels of glucose. Pancreatic tissues were examined by using light microscopy, fluorescence microscopy, scanning, and transmission electron microscopy. In vivo viability and functionality of caprine islets were assessed by evaluating the transplanted islets in diabetic mice. Insulin assay of glucose-stimulated insulin secretion test showed that the insulin levels increased with increasing concentration of glucose. Thus, purified islets stimulated with high glucose concentration (25 mM) secreted higher levels of insulin (0.542 ± 0.346 μg/L) than the insulin levels (0.361 ± 0.219, 0.303 ± 0.234 μg/L) secreted by exposure to low glucose concentrations (1.67 mM). Furthermore, insulin levels of recipient mice were significantly higher (p < 0.001) than those prior to xenotransplantation. In addition, following islets transplantation, there was significant enhancement in blood glucose levels of diabetic recipient mice. Overall, although the purified caprine islets had minor deformations in the plasma membrane and changes in cell integrity of peripheral region, the alterations did not significantly alter the functionality and viability of the purified islets.

Influenza A virus is one of the most important health risks that lead to significant respiratory infections. Continuous antigenic changes and lack of promising vaccines are the reasons for the unsuccessful treatment of influenza. Statins are pleiotropic drugs that have recently served as anti-influenza agents due to their anti-inflammatory activity. In this study, the effect of simvastatin on influenza A-infected cells was investigated. Based on the MTT cytotoxicity test, hemagglutination (HA) assay and qPCR it was found that simvastatin maintained cell viability and decreased the viral load significantly as compared to virus-inoculated cells. The expression of important pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-6 and interferon-γ), which was quantified using ELISA showed that simvastatin decreased the expression of pro-inflammatory cytokines to an average of 2-fold. Furthermore, the modulation of actin filament polymerization was determined using rhodamine staining. Endocytosis and autophagy processes were examined by detecting Rab and RhoA GTPase protein prenylation and LC3 lipidation using western blotting. The results showed that inhibiting GTPase and LC3 membrane localization using simvastatin inhibits influenza replication. Findings of this study provide evidence that modulation of RhoA, Rabs and LC3 may be the underlying mechanisms for the inhibitory effects of simvastatin as an anti-influenza compound.

Proliferation of spermatogonial stem cells (SSCs) in vitro system is very important. It can enhance SSCs numbers for success of transplantation and treatment of infertility in cancer patients. In this study, testicular cells that obtained from azoospermia patients (n=8) by enzymatic digestion were cryopreserved at the beginning and after 2 weeks of culture. Then, frozen-thawed SSCs were co-cultured on fresh Sertoli cells (experimental group 1), and frozen-thawed Sertoli cells (experimental group 2) for another 3 weeks. In control group, fresh SSCs were co-cultured on fresh Sertoli cells. Viability rate after enzymatic digestion was 93.4%±5.0. Frozen-thawed testicular cells after 2 weeks of culture had a significantly (P<0.05) higher percentage of living cells compared to frozen-thawed testicular cells at the beginning of culture (59.2±7.05 and 46.3±8.40 respectively). The number of colonies in the experimental group 1 was significantly higher than experimental group 2 (19.6±2.8 and 8.33±1.5, respectively, P<0.05). The diameter of the colonies in the experimental group 1 was significantly higher than control and experimental group 2 (P<0.05) after 3 weeks of culture (269.7±52.1, 204.34±24.1 and 112.52±23.5 μm, respectively). Cryopreservation technique will raise the possibility of banking SSCs for men who have a cancer-related illness and waiting for radiotherapy and/or chemotherapy., (© 2012 Blackwell Verlag GmbH.)

The synthesised biobased calcium carbonate nanocrystals had demonstrated to be an effective carrier for delivery of anticancer drug doxorubicin (DOX). The use of these nanocrystals displayed high levels of selectivity and specificity in achieving effective cancer cell death without nonspecific toxicity. These results confirmed that DOX was intercalated into calcium carbonate nanocrystals at high loading and encapsulation efficiency (4.8 and 96%, resp.). The CaCO₃/DOX nanocrystals are relatively stable at neutral pH (7.4), resulting in slow release, but the nanocrystals progressively dissociated in acidic pH (4.8) regimes, triggering faster release of DOX. The CaCO₃/DOX nanocrystals exhibited high uptake by MDA MB231 breast cancer cells and a promising potential delivery of DOX to target cells. In vitro chemosensitivity using MTT, modified neutral red/trypan blue assay, and LDH on MDA MB231 breast cancer cells revealed that CaCO₃/DOX nanocrystals are more sensitive and gave a greater reduction in cell growth than free DOX. Our findings suggest that CaCO₃ nanocrystals hold tremendous promise in the areas of controlled drug delivery and targeted cancer therapy.

In this study, isolated spermatogonial stem cells (SSCs) and Sertoli cells using enzymatic digestion from patients with maturation arrest of spermatogenesis were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum in three different groups: (1) SSCs cultured without Sertoli cells (2) SSCs co-cultured with Sertoli cells (as control group), (3) SSCs co-cultured with Sertoli cells and adding different concentrations of basic fibroblast growth factor (0.1, 1, 10 ng ml(-1)) and human leukaemia inhibitory factor (1000, 1200, 1500 unit ml(-1)) as experimental groups. The assessment of colonies every 10 days during 5-week cultures showed that in the first group, the average number and diameter of the colonies were significantly lower than in the other groups (P < 0.05). The largest number of colonies was observed in control condition (32.29 ± 9.15) in day 30. The largest diameter of colonies was formed in combination dosages of 1 ng ml(-1) basic fibroblast growth factor (bFGF) + 1500 unit ml(-1) leukaemia inhibitory factor (LIF) (302.93 ± 37.68) and 10 ng ml(-1) bFGF and 1200 unit ml(-1) LIF (262.87 ± 35.54) in day 30 respectively. Isolated SSCs were positive for spermatogonial cell markers such as Oct4, Stra8, Piwil2 and Vasa but negative for Nanog. Transplantation technique indicated that hSSCs have good efficiency for colonisation of mouse seminiferous tubules after proliferation in culture system., (© 2011 Blackwell Verlag GmbH.)

Feline coronavirus (FCoV) consists of two biotypes based on their growth in cell culture and their antigenicity. Infections with FCoV are highly prevalent in the cat population worldwide. In this study, Felis catus whole fetus (Fcwf-4)cell culture was infected with FCoV UPM11C/08. Virus multiplication in cell culture was monitored and examined under the transmission electron microscope. The virus particles revealed the characteristic morphology of feline FCoV represented by envelope viruses surrounded by peplomers. Virus attachment and entry into the cell occurred 15 h post-infection (pi), and the myriad of virus particles were observed both extracellularly and intracellularly after 48 h pi. Thereafter, intracellular virus particles were observed to be present in vacuoles or present freely in the cytoplasm.