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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.

This study aimed at extraction of protein from the wastes of the threadfin bream, Nemipterus japonicus using pH shift solubilization and precipitation processes and evaluation of the influencing factors on protein extraction. Towards this objective, the wastes of head, skin and internal organs were collected, and their nutritional composition evaluated. Furthermore, the ratio of waste: water for protein extraction was screened and the effect of pH, centrifugation speed and time on protein solubility rate, the amino acid composition and the molecular weight of proteins via SDS-PAGE were determined. Results demonstrated that moisture was the highest percentage (~70-78%) of all wastes. Highest protein content (19.67±1.10%), fat content (1.81±0.09%) and carbohydrate content (4.43±0.23%) were observed in skin, head and internal organs, respectively. The optimum ratio of waste: water for protein extraction is 1:9 for head, 1:8 for internal organs and 1:6 for skin. The protein extraction efficiency for all three samples were high at pH 3 and pH 12. Protein solubility increased with increased centrifugation speed up to 10,000 ×g, and there was no significant difference (P>0.05) between the protein solubility at 10,000 ×g and 20’000 ×g. Furthermore, there was significant difference (P

This paper reports the prebiotic efficacy of soluble crude polysaccharides (SCP) from palm kernel cake on the growth rates and acidifying property of probiotic lactic acid bacteria (LAB), Lactobacillus plantarum ATCC 8014 and Lactobacillus rhamnosus ATCC 53103, to investigate into the potential SCP of an agriculture by-product as a novel prebiotic. The highest extraction yield of SCP (2.90%) was obtained using NaOH. The extracted polysaccharides were highly soluble (>97%). The highest percentage of protein (0.63 ± 0.09%) and the total carbohydrate content (55.36 ± 0.33%) were observed in NaOH-extracted SCP (SCPN) and citric acid-extracted SCP (SCPCA). Water-extracted SCP (SCPW) contained the highest percentage of glucose, whereas SCPCA demonstrated the presence of two major monosaccharides, galactose and mannose. Structural analysis of SCP showed peaks in the range of 1000–1077 cm−1, an indication of the presence of polysaccharides and alpha (α)- or beta (β)-glycosidic linkages. The degree of hydrolysis increased with the increase of pH from 5 to 8 following 4 h incubation. SCPW and SCPCA were more resistant (95%) to α-amylase as compared to fructooligosaccharides (FOS). As in the case of FOS (∼98%), SCPW (∼99%) and SCPCA (∼98%) were resistant to artificial human gastric juice and their degree of hydrolysis had negative correlation with the pH. The product was found to markedly stimulate the proliferation of the tested probiotics and acid production. All the above findings are supportive of the fact that polysaccharides extracted from CKC have a vast potential to be exploited as novel prebiotic.

Doxorubicin (DOX) is an effective and commonly used anthracycline anticancer drug for the treatment of osteosarcoma (OS). However, its antitumor effect is hampered by the nonspecific distribution and significant adverse effects. Nanoparticles based drug delivery systems are promising approaches to maximize the anticancer efficacy while decrease the side effects. In this study, biogenic aragonite nanoparticles (ANPs) were developed from cockle shells and loaded with DOX. An orthotopic rat OS model was induced by UMR-106 cells tibia cavity injection. The anticancer efficacy study included five groups: normal control group, OS model group, free DOX group (2 mg/kg), DOX-ANPs 1 group (2 mg of equivalent DOX/kg) and DOX-ANPs 2 group (1.5 mg of equivalent DOX/kg). This study demonstrates that the DOX-ANPs treatment groups can significantly reduce the tumor volume and increase the surviving ratio as compared to the OS model group. In addition, these two DOX-ANPs groups showed less toxicity to the normal organs compared to the free DOX group. Furthermore, DOX-ANPs 2 group showed the similar anticancer efficacy as DOX-ANPs 1 group, which suggested that DOX loaded onto the ANPs may allow the reduction of chemotherapy doses. These results highlight the promising application of ANPs derived from cockle shells as an effective drug delivery system for a successful chemotherapy against OS. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1898-1907, 2019.

The bulk availability of banana waste in the world has made a move towards the development of alternative novel renewable sugars in present study as the pressed juice from banana frond and pseudostem was found to contain high level of renewable sugars such as glucose, sucrose and fructose. By using a simple sugarcane press, the glucose content in the banana frond juice was 16.6 g/L, which accounts for 55% of the total fermentable sugars. From the results, the lipase production reached ∼200 U/mL in medium comprising banana frond juice and banana pseudostem juice, comparable to basal medium with glucose (206.3 U/mL) as carbon source in basal media. As banana pseudostem possesses high content of starch, microwave heating pretreatment showed 36% enhancement of glucose content (10.4 g/L) in juice as compared to direct pressed juice (7.78 g/L). This result indicates that banana waste juice can be used as an alternative fermentable carbon source for lipase production and has potential as a fermentable carbon source.

This paper deliberates the modelling and validation of bacteriocin-like inhibitory substance (BLIS) secretion by Pediococcus acidilactici Kp10 at different agitation speeds in a stirred tank bioreactor. A range of models namely the re-parameterised logistic, Luedeking–Piret and maintenance energy were assessed to predict the culture performance of the said bacterium. Growth of P. acidilactici Kp10 was enhanced with increased agitation speed up to 600 rpm while BLIS secretion was maximum at 400 rpm but decreased at higher agitation speed. Growth of P. acidilactici aptly subscribed to the re-parameterised logistic model while BLIS secretion and lactose consumption fitted well with the Luedeking–Piret model. The models revealed a relationship between growth of the bacterium and BLIS secretion. Bacterial growth and BLIS secretion were largely affected by the agitation speed of the stirred tank bioreactor which regulated the oxygen transfer to the culture. BLIS secretion by P. acidilactici Kp10 was however enhanced in oxygen-limited culture. The study also assessed BLIS from the perspective of its stability when subjected to factors such as temperature, pH and detergents. Results showed that BLIS produced by this strain was not affected by heat (at 25–100 °C for 20 min and at 121 °C for 15 min), surfactant (Tween 40, 60 and 80 and urea), detergents (up to 1% SDS), organic solvents (50% each of acetone, methanol and ethanol) and stable in a wide range of pH (2–10). The above information are pertinent with reference to commercial applications of this bacterial product in food manufacturing which invariably involve various sterilization processes and subjected to a wide pH range.

Lactic acid bacteria (LAB) are of major interest in the food industry primarily by virtue of their biopreservative properties. LAB have ability to produce various types of antimicrobial compounds, the most important being bacteriocins. Bacteriocins and bacteriocin-producing cultures have the potential to increase the shelf-life of foods and contribute towards decreasing the incidence of food-borne diseases. In this respect, food preservation through in situ production of bacteriocins by LAB introduced into the food system would be the most logical approach. However, there is a need to understand the relationship between bacterial growth and bacteriocin production in various types of food system. Bacteriocin production by LAB is dependent on a number of factors such as the types of carbon and nitrogen sources and their concentrations in the media formulation. Other factors which need to be considered are the culture conditions which include pH, temperature and aeration which greatly influence the cultivation performance of bacteriocins producing LAB. Economic aspects pertaining to the optimization of fermentation process for the enhancement of bacteriocin production should also be given due considerations. Failure to acknowledge or recognize this hidden economic element would be a substantial financial loss to the industry especially from the point of view that the product is costly and highly sought after. Thus, the fermentation factors which influence the production of bacteriocins by LAB and the approaches to improve the production not only in term of yield and productivity but also in term of economic and regulation are reviewed in this paper.

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; TiO2) 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 (ΔE*) 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.

The present study was aimed at enhancing the production of bacteriocin-like inhibitory substance (BLIS) produced by Pediococcus acidilactici Kp10 through optimizing the fermentation parameters. M17 was chosen in preliminary study as a culture medium because BLIS production was nine times higher (1427.7 AU mL−1) compared to that produced by MRS (160 AU mL−1). The fermentation parameters such as temperature, inoculum size, buffer strength, concentration of tween 80 and agitation speed were screened using two level half-factorial design. BLIS production is influenced by three most significant factors identified as temperature, inoculum size and agitation speed, which were further optimized using an artificial neural network (ANN). ANN predicted that a maximum activity of 5262.64 AU mL−1 would be obtained at optimum conditions of 120 rpm, 3% and 28.5 °C. The observed BLIS activity at the predicted optimum levels of the tested variables in ANN was 5118.5 AU mL−1, which was close to the predicted BLIS activity. Increased BLIS activity in the final solution, which resulted from the optimized process, would reduce downstream steps such as concentrating the product during purification.

This paper deliberates the modelling and validation of bacteriocin-like inhibitory substance (BLIS) secretion by Pediococcus acidilactici Kp10 at different agitation speeds in a stirred tank bioreactor. A range of models namely the re-parameterised logistic, Luedeking–Piret and maintenance energy were assessed to predict the culture performance of the said bacterium. Growth of P. acidilactici Kp10 was enhanced with increased agitation speed up to 600 rpm while BLIS secretion was maximum at 400 rpm but decreased at higher agitation speed. Growth of P. acidilactici aptly subscribed to the re-parameterised logistic model while BLIS secretion and lactose consumption fitted well with the Luedeking–Piret model. The models revealed a relationship between growth of the bacterium and BLIS secretion. Bacterial growth and BLIS secretion were largely affected by the agitation speed of the stirred tank bioreactor which regulated the oxygen transfer to the culture. BLIS secretion by P. acidilactici Kp10 was however enhanced in oxygen-limited culture. The study also assessed BLIS from the perspective of its stability when subjected to factors such as temperature, pH and detergents. Results showed that BLIS produced by this strain was not affected by heat (at 25–100 °C for 20 min and at 121 °C for 15 min), surfactant (Tween 40, 60 and 80 and urea), detergents (up to 1% SDS), organic solvents (50% each of acetone, methanol and ethanol) and stable in a wide range of pH (2–10). The above information are pertinent with reference to commercial applications of this bacterial product in food manufacturing which invariably involve various sterilization processes and subjected to a wide pH range.

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.

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.

Background Type 1 diabetes mellitus is a devastating disease for which there is currently no cure, but only lifetime management. Islet xenotransplantation is a promising technique for the restoration of blood glucose control in patients with diabetes mellitus. The purpose of this study was to explore the potential use of caprine (goat) islet cells as xenogeneic grafts in the treatment for diabetes in a mouse model. Methods Caprine pancreases were harvested and transported to the laboratory under conditions optimized to prevent ischemia. Islets were isolated, purified, and tested for functionality. Caprine islets (2000 islet equivalent) were transplanted beneath the kidney capsules of diabetic BALB/c mice under thalidomide-induced immunosuppression. Blood glucose and insulin levels of grafted mice were evaluated by glucometer and enzyme-linked immunosorbent assay kit, respectively. The functionality and quality of caprine pancreatic islet grafts were assessed by intraperitoneal glucose tolerance tests. Results The viability of purified islet cells exceeded 90%. Recipient mice exhibited normoglycemia (

Context: Zerumbone (ZER) is known to exhibit anticancer properties on various cancer cells both in vitro and in vivo. However, the anti-angiogenesis effect of ZER on liver cancers in vivo is not yet addressed clearly. Aims: This study aimed to investigate the in vivo antiproliferative and antiangiogenesis effects of ZER using rats with diethylnitrosamine-induced hepatocellular carcinoma (HCC). Materials and Methods: The antiproliferative and anti-angiogenesis effects of ZER were determined using the hepatosomatic index, vascular endothelial growth factor (VEGF) immunoassay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, histopathology, and immunohistochemistry analysis. Results: Nontreated rats with HCC had higher median liver weight than those treated with ZER or suramin. The expression of VEGF, matrix metalloprotease, and Ki-67 that were high in nontreated HCC rats was down-regulated with ZER or suramin treatments. Statistical Analysis Used: Statistical analyses were performed using the Statistical Package for Social Science version 21.0 (SPSS Inc, IBM, Maryland, USA). The data were expressed as the mean ± standard deviation and analyzed using a one-way analysis of variance. P Abbreviations used: ZER: Zerumbone; HCC: Hepatocellular carcinoma; MMP-9: Matrix metalloproteinase-9; VEGF: Vascular endothelial growth factor; DEN: Diethylnitrosamine; PBS: Phosphate-buffered saline; ELISA: Enzyme-Linked Immunosorbent Assay; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling; FFPE: Formalin-fixed and paraffin-embedded; H and E: Hematoxylin and Eosin; DAB: Dako Envision®+Dual Link System; HRP: Horseradish peroxidase; H2O2: Hydrogen peroxide; PBST: PBS in Tween 20; SPSS: Statistical Package for Social Science; RUGS: Research University Grant Scheme. Correspondence

Drug delivery systems are designed to achieve drug therapeutic index and enhance the efficacy of controlled drug release targeting with specificity and selectivity by successful delivery of therapeutic agents at the desired sites without affecting the non-diseased neighbouring cells or tissues. In this research, we developed and demonstrated a bio-based calcium carbonate nanocrystals carrier that can be loaded with anticancer drug and selectively deliver it to cancer cells with high specificity by achieving the effective osteosarcoma cancer cell death without inducing specific toxicity. The results showed pH sensitivity of the controlled release characteristics of the drug at normal physiological pH 7.4 with approximately 80% released within 1,200 min but when exposed pH 4.8 the corresponding 80% was released in 50 min. This study showed that the DOX-loaded CaCO3 nanocrystals have promising applications in delivery of anticancer drugs.

HIGHLIGHTS: EDTA induces platelet microparticles (PMPs) vesiculation. Sodium citrated tubes are better than EDTA tubes in PMPs analysis. Sodium citrate has a minor impact on platelet structure. ABSTRACT: Platelet microparticles (PMPs) are small membrane fragments released from activated platelets in response to various stimuli. PMPs serve as biomarkers for several diseases and conditions and are useful tools for prognostic, diagnostic, and therapeutic purposes. The objective of our study was to compare the direct effects of ethylenediaminetetraacetic acid (EDTA) and sodium citrate anticoagulants on platelet structure and PMP vesiculation using transmission electron microscopy to visualize the morphologic changes in platelets. Micrographs revealed that platelets in the EDTA-anticoagulated tube manifested with significant morphologic changes and induced PMP vesiculation. On the other hand, the sodium citrate-anticoagulated tube showed a normal platelet structure and minor modifications in some cases, with poor indication of PMP vesiculation. In conclusion, EDTA induced platelet activation and PMP vesiculation and represents a major source of artifacts during the pre-analysis steps of PMP vesiculation.

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.

The study was conducted to investigate the effect of bone tissue engineering comprised of combination of bone marrow-derived osteoblasts and corticocancellous bone graft in repairing 15 mm compact bone critical sized defect. Implant of corticocancellous bone graft alone served as group I. For the second group, bone marrow-derived osteoblasts seeded into scaffold of corticocancellous bone graft and the third group with no treatment served as the control group. Rabbits were euthanized on the fifth (only in group II) and the eighth week (all groups) postoperative. Radiographs were taken on the weekly interval. After euthanasia at the fifth and eighth weeks, the implants were harvested for, gross, histology and scanning electron microscope observations. In group I, new bone formation was only observed at the margin of defect and in the defect centre, hypertrophied chondrocytes was detected and major bone formation pattern was creeping substitution. New bone formation at margin of defect and osteogenesis at th...

The effect of medium composition (type and concentration of carbon and nitrogen sources) and culture conditions (pH, aeration and temperature) on the production of bacteriocin-like inhibitory substances (BLIS) by Lactobacillus paracasei LA07 was studied in shake flask culture. The highest BLIS production was obtained from de Man, Rogosa and Sharpe (MRS) broth, which was 4%, 22%, 51% and 63% higher than those obtained in M17 Broth, Tryptic Soy Broth (TSB), Brain Heart Infusion Broth (BHIB) and Nutrient Broth (NB) respectively. The addition of meat extract (5 g/L) and yeast extract (5 g/L) to MRS broth increased further BLIS production by 5% and 8%, respectively. The production of BLIS was further improved by 11% and 13% through the addition of tryptone (3 g/L) and glucose (10 g/L), respectively. The growth of Lb. paracasei LA07 and BLIS production were not significantly affected by agitation, either in anaerobic or in aerobic conditions. The optimum initial pH and temperature for BLIS production by...

The main aim of the present study was to investigate the effect of combination of bone marrow as the primary origin of osteoblast and at the same time as the seed cell and corticocancellous bone graft as the natural scaffold in repair of compact bone full thickness segmental critical sized defect in rabbits. Twelve rabbits had been divided into two groups; In Group one, fresh autogenous bone marrow aspirate has been seeded into the scaffold of autogenous corticocancellous bone graft which was utilized to repair critical size compact bone defect in mid shaft of radius. Corticocancellous bone graft alone was used as the Group 2 or control group. Up to 8 weeks, radiographs were taken to evaluate the level of osteogenicity in both groups. Rabbits were euthanized on week eight postoperative and the implants were harvested for gross, histological and scanning electron microscope observations. New bone formation and osteogenesis was observed at the margins and centre of the Group 1. Combination of mature and immature trabecullae covered the defect and bone formation pattern included osteogenesis, osteoinduction and osteocunduction. In the implant of corticocancellous bone graft alone or group 2, the major new bone formation was at the margins of the defect and osteogenesis was not observed at the centre of the defect and the major bone formation pattern was creeping substitution. As the conclusion, combination of bone marrow and corticocancellous bone graft had better results than corticocancellous bone graft alone in osteogenesis potential. Bone formation capability and critical sized defect repair was faster and more efficient and successful in Group 2 defect.

Exogenesis (cross-species) germ cell transplantation provides an opportunity to investigate fundamental aspects of spermatogenesis. In this study, testis biopsies of patients with maturation arrest of spermatogenesis during a one year ago were first minced mechanically into small pieces and then Spermatogonial Stem Cells (SSCs) and Sertoli cells isolated by the two- step enzymatic digestion, were plated and grown on DSA-Lectin coated dishes in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal calf serum. Transplantation of human spermatogonial cells into mouse recipient testis was performed on day 7 (before colony formation) and 2 weeks after culturing (colony formation). The effects of different concentrations of spermatogonial cell on quantity of transplantation and percent of colonized seminiferous tubules were assayed during 8 weeks after transplantation. The result showed that SSCs can be observed on the basement membrane of the seminiferous tubules in place of spermatogonial stem cells and proliferation occurs about 4 weeks after transplantation. The difference in donor cells concentration had more effect on colonization of mouse recipient testis (p

Hani H, TengkuAzmi TI, Abas MO, Mohd-Azmi ML, Zeenathul NA. Isolation, density purification, and in vitro culture maintenance of functional caprine islets of Langerhans as an alternative islet source for diabetes study. Xenotransplantation 2010; 17: 469–480. © 2010 John Wiley & Sons A/S. Abstract: Background: Insufficient availability of human donors makes the search for alternative source of islet cells mandatory for future developments in pancreatic transplantation. The present study investigates the potential of caprine as an alternative source of pancreatic islets. The objectives of the study were to optimize techniques for caprine islet isolation and purification for culture establishment, and to subsequently assess their viable and functional potential. Methods: Caprine pancreatic tissues were collected from a local slaughterhouse and prior transported to the laboratory by maintaining the cold chain. Islets were obtained by a collagenase-based digestion and optimized isolation technique. Islet cell purity and viability were determined by dithizone and trypan blue staining, respectively. Islet clusters of different sizes were positively identified by staining methods and demonstrated 90% viability in the culture system. Following static incubation, an in vitro insulin secretion assay was carried out and analyzed by ELISA. Results: The islets remained satisfactorily viable for 5 days in the culture system following regular media changes. The current study has successfully optimized the isolation, purification and culture maintenance of caprine islets. Conclusion: The successful yield, viability and functionality of islets isolated from the optimized protocol provide promising potential as an alternative source of islets for diabetes and transplantation researches.

Zerumbone (ZER), a monosesquiterpene found in the subtropical ginger (Zingiber zerumbet Smith), possesses antiproliferative properties to several cancer cells lines, including the cervical, skin and colon cancers. In this study, the antitumourigenic effects of ZER were assessed in rats induced to develop liver cancer with a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg) and dietary 2-acetylaminofluorene (AAF) (0.02%). The rats also received intraperitoneal ZER injections at 15, 30 or 60 mg/kg body wt. twice a week for 11 weeks, beginning week four post-DEN injection. The hepatocytes of positive control (DEN/AAF) rats were smaller with larger hyperchromatic nuclei than normal, showing cytoplasmic granulation and intracytoplasmic violaceous material, which were characteristics of hepatocarcinogenesis. Histopathological evaluations showed that ZER protects the rat liver from the carcinogenic effects of DEN and AAF. Serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (AP) and alpha-fetoprotein (AFP) were significantly lower (P < 0.05) in ZER-treated than untreated rats with liver cancer. The liver malondialdehyde (MDA) concentrations significantly (P < 0.05) increased in the untreated DEN/AAF rats indicating hepatic lipid peroxidation. There was also significant (P < 0.05) reduction in the hepatic tissue glutathione (GSH) concentrations. The liver sections of untreated DEN/AAF rats also showed abundant proliferating cell nuclear antigen (PCNA), while in ZER-treated rats the expression of this antigen was significantly (P < 0.05) lowered. By the TUNEL assay, there were significantly (P < 0.05) higher numbers of apoptotic cells in DEN/AAF rats treated with ZER than those untreated. Zerumbone treatment had also increased Bax and decreased Bcl-2 protein expression in the livers of DEN/AAF rats, which suggested increased apoptosis. Even after 11 weeks of ZER treatment, there was no evidence of abnormality in the liver of normal rats. This study suggests that ZER reduces oxidative stress, inhibits proliferation, induces mitochondria-regulated apoptosis, thus minimising DEN/AAF-induced carcinogenesis in rat liver. Therefore, ZER has great potential in the treatment of liver cancers.

Restoring skeletal integrity and bone tissue regeneration is still a significant challenging issue. In this regard, bone grafting have been used to augment orthopedic repairs in human and veterinary surgery for several decades and still being under many investigation to hunt for new approaches to improve bone healing following incidences of bone complications. Bone graft is bone transplant and is categorized into autogenous and allogenic grafts as well as synthetic bone graft which are bone graft substitutes. Each of these classified grafts have some advantageous as well as a range of drawbacks, which researchers are still looking to remove those disadvantageous. Finding new instruments and new sites for graft harvests are the major concerns of researchers to diminish the morbidities of donor site in autografts. Looking for agents boosting inductivity of the allografts is the main worries of these kinds of grafting materials and finally new fabrication techniques by new pore sizes are the significant bothering for synthetic bone graft substitutes. This review would consider all grafting methods and materials that would open new windows to the bone grafting techniques.

Osteoporosis is a metabolic bone disorder, which is characterized by a decreased in the overall amount of bone mass. This is major public problem with 30% mortality rate within the first year following osteoporotic hip fracture. The altered microarchitectural structure of bone would lead to increased susceptibility to fractures. By improving the public health and aging of the population, the incidence of this disorder is also increasing. Different treatments costs of patients is showing an increasing trend, which implies that there should be a public education regarding this threatening abnormality. Prevention rather than treatment has to be publicized by correct instructing the population and especially in the women society. This review has a brief simple but concise outlook at osteoporosis its manifestation, treatment and prevention.

The ultrastructure of the cell wall of extensively drug-resistant (XDR), multidrug-resistant (MDR) and susceptible tuberculosis (TB) bacilli was viewed under transmission electron microscopy (TEM). Under the TEM, marked differences were observed in the thickness of their cell wall: 20.2 ±1.5 and 17.1 ±1.03 nm for the XDR and MDR TB bacilli, respectively, and 15.6 ± 1.3 nm for the susceptible isolates (p < 0.05). In MDR bacilli, thickening of the cell wall was observed in the intermediate electron-transparent layer (ETL) and outer electron-opaque layer, whereas in XDR TB cells the basal peptidoglycan layer was denser and almost fused with the ETL. Five to seven percent of XDR TB bacilli had the appearance of the stationary phase with cell wall thickness ranging from 21 to 26 nm (p < 0.001). Information provided in this study is of significant importance in terms of drug selection for effective treatment of resistant strains.

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 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.

Characteristics of Human Amniotic Epithelial Cells and Bone Marrow Mesenchymal Stem Cells in a Parallel Study: Hanging Drop Colony Formation and Doubling Time Amniotic epithelial cells exhibit pluripotent capacity with some unique properties. They are considered as a good candidate in regenerative medicine. Human bone marrow mesenchymal stromal cells (hBM-MSC) are the gold standard in cell therapy procedures, but whether these cells could be replaced with hAEC remains a debate? We compared the stem cell characteristics of hAEC with hBM-MSC considering isolation, morphology, population doubling time, colony formation on substratum and in suspension, alkaline phosphatase activity and expression of some CD Markers. hAEC were round and cubical but hBM-MSC appeared spindle-shaped in culture. Both groups of cells expressed mesenchymal stem cells markers (CD44, CD90) and they did not express CD34. OCT4 was highly expressed in hAEC but not in hBM-MSC. Their proliferation rate was slower than hBM-MSC. Either of the cell groups could form colonies on substratum as well as suspension with a positive alkaline phosphatase activity. Our results showed that biological properties of human amniotic epithelial cells are to some extent similar to human bone marrow mesenchymal cells.

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.

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 [150mg/kg body weight (BW) and 300mg/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 150mg/kg BW in rats. The lectin-rich SECE showed tumor suppression by enhancing immune responses and upregulating the cancer cell apoptosis mRNA expressions.

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

A polymer–salt aqueous two-phase system (ATPS) consisting of polyethylene–glycol (PEG) with sodium citrate was developed for direct recovery of a bacteriocin-like inhibitory substance (BLIS) from a culture of Pediococcus acidilactici Kp10. The influences of phase composition, tie-line length (TLL), volume ratio (VR), crude sample loading, pH and sodium chloride (NaCl) on the partition behaviour of BLIS was investigated. Under optimum conditions of ATPS, the purification of BLIS was achieved at 26.5% PEG (8000)/11% sodium citrate with a TLL of 46.38% (w/w), VR of 1.8, and 1.8% crude load at pH 7 without the presence of NaCl. BLIS from P. acidilactici Kp10 was successfully purified by the ATPS up to 8.43-fold with a yield of 81.18%. Given that the operation of ATPS is simple, environmentally friendly and cost-effective, as it requires only salts and PEG, it may have potential for industrial applications in the recovery of BLIS from fermentation broth.

Tocotrienols are isomers of the vitamin E family, which have been reported to exert cytotoxic effects in various cancer cells. Although there have been some reports on the effects of tocotrienols in leukemic cells, ultrastructural evidence of tocotrienol-induced apoptotic cell death in leukemic cells is lacking. The present study investigated the effects of three isomers of tocotrienols (alpha, delta, and gamma) on a human T lymphoblastic leukemic cell line (CEM-SS). Cell viability assays showed that all three isomers had cytotoxic effects (p < 0.05) on CEM-SS cells with delta-tocotrienol being the most potent. Transmission electron microscopy showed that the cytotoxic effects by delta- and gamma-tocotrienols were through the induction of an apoptotic pathway as demonstrated by the classical ultrastructural apoptotic changes characterized by peripheral nuclear chromatin condensation and nuclear fragmentation. These findings were confirmed biochemically by the demonstration of phosphatidylserine externalization via flow cytometry analysis. This is the first study showing classical ultrastructural apoptotic changes induced by delta- and gamma-tocotrienols in human T lymphoblastic leukemic cells.

Bacillus thuringiensis (Bt) parasporal proteins with selective anticancer activity have recently garnered interest. This study determines the efficacy and mode of cell death of Bt 18 parasporal proteins against 3 leukemic cell lines (CEM-SS, CCRF-SB and CCRF-HSB-2).Cell-based biochemical analysis aimed to determine cell viability and the percentage of apoptotic cell death in treated cell lines; ultrastructural analysis to study apoptotic changes and Western blot to identify the parasporal proteins' binding site were performed. Bt 18 parasporal proteins moderately decreased viability of leukemic cells but not that of normal human T lymphocytes. Further purification of the proteins showed changes in inhibition selectivity. Phosphatidylserine externalization, active caspase-3, cell cycle, and ultrastructural analysis confirmed apoptotic activity and S-phase cell-cycle arrest. Western blot analysis demonstrated glyceraldehyde 3-phosphate dehydrogenase as a binding protein. We suggest that Bt 18 parasporal proteins inhibit leukemic cell viability by cell-cycle arrest and apoptosis and that glyceraldehyde 3-phosphate dehydrogenase binding initiates apoptosis.

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

Background: Currently, there has been extensive research interest for inorganic nanocrystals such as calcium phosphate, iron oxide, silicone, carbon nanotube and layered double hydroxide as a drug delivery system especially in cancer therapy. However, toxicological screening of such particles is paramount importance before use as delivery carrier. In this study we examine the biocompatibility of CaCO3 nanocrystal on NIH 3T3 cell line. Material and Methods: Transmission and field emission scanning electron microscopy (TEM and FESEM) were used for the characterisation of CaCO3 nanocrystals. Cytotoxicity and genotoxic effect of calcium carbonate nanocrystals in cultured mouse embryonic fibroblast NIH 3T3 cell line using various bioassays including MTT, and Neutral red/Trypan blue double-staining assays. LDH, BrdU and reactive oxygen species were used for toxicity analysis. Cellular morphology was examined by scanning electron microscopy (SEM) and confocal fluorescence microscope. Results: The outcome of the analyses revealed a clear rod-shaped aragonite polymorph of calcium carbonate nanocrystal. The analysed cytotoxic and genotoxicity of CaCO3 nanocrystal on NIH 3T3 cells using different bioassays revealed no significance differences as compared to control. A slight decrease in cell viability was noticed when the cells were exposed to higher concentrations of 200 to 400 µg/ml, while increase in ROS generation and LDH released at 200 and 400 µg/ml was observed. Conclusions: The study has shown that CaCO3 nanocrystal is biocompatible and non toxic to NIH 3T3 fibroblast cells. The analysed results offer a promising potential of CaCO3 nanocrystal for the development of intracellular drugs, genes and other macromolecule delivery systems.

There has been increasing evidence that cancers are dependent on a number of altered molecular pathways, which can develop diverse mechanisms of resistance to therapy with single agents. Therefore, combination regimens may provide the best hope for effective therapies with durable effects. In this current investigation, the anti-cancer effect of zerumbone (ZER), a natural compound from Zingiber zerumbet smith of Zingiberaceae family and curcumin (a yellow pigment from Curcuma longa) were used in combination for probable treatment in liver cancer. Both compounds were investigated in MTT cell proliferative assay as well as flow cytometric analysis (annexinV-FITC, cell cycle and TUNEL assay) on HepG2 cells in 3 ratios of ZER: CUR (1:3, 3:1 and 1:1). The IC50 obtained were 13μg/mL, 4 μg/mL and 7μg/mL respectively and the combinations further showed no toxic effect on normal liver cell lines (WRL68). The current findings revealed that the best combination of ZER: CUR is 3:1. The outcome of this study demonstrates that this combination has the ability to increased efficacy with limited toxicity in liver cancer treatment.

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.