Stressing Mitosis to Death

The final stage of cell division (mitosis), involves the compaction of the duplicated genome into chromatid pairs. Each pair is captured by microtubules emanating from opposite spindle poles, aligned at the metaphase plate, and then faithfully segregated to form two identical daughter cells. Chromatids that are not correctly attached to the spindle are detected by the constitutively active spindle assembly checkpoint (SAC). Any stress that prevents correct bipolar spindle attachment, blocks the satisfaction of the SAC, and induces a prolonged mitotic arrest, providing the cell time to obtain attachment and complete segregation correctly. Unfortunately, during mitosis repairing damage is not generally possible due to the compaction of DNA into chromosomes, and subsequent suppression of gene transcription and translation. Therefore, in the presence of significant damage cell death is instigated to ensure that genomic stability is maintained. While most stresses lead to an arrest in mitosis, some promote premature mitotic exit, allowing cells to bypass mitotic cell death. This mini-review will focus on the effects and outcomes that common stresses have on mitosis, and how this impacts on the efficacy of mitotic chemotherapies

Insulin secreted from genetically engineered intestinal cells reduces blood glucose levels in diabetic mice

Poorly controlled diabetes mellitus can result in serious complications. Gene therapy is increasingly being considered as an alternative approach to treat diabetes, because of its ability to induce physiological insulin secretion and it allows patients to escape insulin injections. The properties of gut K and L-cells, including glucose sensitivity, the ability to process insulin and a regulated secretion pathway support their use as surrogate β-cells. Previous in vitro studies have provided sufficient evidence supporting the use of these cells for gene therapy studies. Therefore, we examined the ability of K and L-cells to produce insulin in diabetic mice. Chitosan nanoparticles were used to transfer the insulin gene into intestinal cells via oral administration. The efficiency of chitosan as a gene vehicle was investigated through the use of reporter gene. Insulin mRNA and protein expression levels were measured by RT-PCR and ELISA, respectively. Blood glucose testing revealed that this treatment reduced glucose levels in diabetic mice. The decrease in blood glucose level in the first week of treatment was greater in mice with K-cell specific insulin expression compared with mice with L-cellspecific insulin expression. These results indicate that inducing insulin secretion in K-cells conferred a quicker response to gene therapy

Engineering an L-cell line that expresses insulin under the control of the glucagon-like peptide-1 promoter for diabetes treatment

Background: Diabetes mellitus is a complicated disease with a pathophysiology that includes hyperinsulinemia, hyperglycemia and other metabolic impairments leading to many clinical complications. It is necessary to develop appropriate treatments to manage the disease and reduce possible acute and chronic side effects. The advent of gene therapy has generated excitement in the medical world for the possible application of gene therapy in the treatment of diabetes. The glucagon-like peptide-1 (GLP-1) promoter, which is recognised by gut L-cells, is an appealing candidate for gene therapy purposes. The specific properties of L-cells suggest that L-cells and the GLP-1 promoter would be useful for diabetes therapy approaches. Results: In this study, L-cells were isolated from a primary intestinal cell line to create suitable target cells for insulin expression studies. The isolated cells displayed L-cell properties and were therefore used as an L-cell surrogate. Next, the isolated L-cells were transfected with the recombinant plasmid consisting of an insulin gene located downstream of the GLP-1 promoter. The secretion tests revealed that an increase in glucose concentration from 5 mM to 25 mM induced insulin gene expression in the L-cells by 2.7-fold. Furthermore, L-cells quickly responded to the glucose stimulation; the amount of insulin protein increased 2-fold in the first 30 minutes and then reached a plateau after 90 minutes. Conclusion: Our data showed that L-cells efficiently produced the mature insulin protein. In addition, the insulin protein secretion was positively regulated with glucose induction. In conclusion, GLP-1 promoter and L-cell could be potential candidates for diabetes gene therapy agents

SR9009 administered for one day after myocardial ischemia-reperfusion prevents heart failure in mice by targeting the cardiac inflammasome

Reperfusion of patients after myocardial infarction (heart attack) triggers cardiac inflammation that leads to infarct expansion and heart failure (HF). We previously showed that the circadian mechanism is a critical regulator of reperfusion injury. However, whether pharmacological targeting using circadian medicine limits reperfusion injury and protects against HF is unknown. Here, we show that short-term targeting of the circadian driver REV-ERB with SR9009 benefits long-term cardiac repair post-myocardial ischemia reperfusion in mice. Gain and loss of function studies demonstrate specificity of targeting REV-ERB in mice. Treatment for just one day abates the cardiac NLRP3 inflammasome, decreasing immunocyte recruitment, and thereby allowing the vulnerable infarct to heal. Therapy is given in vivo, after reperfusion, and promotes efficient repair. This study presents downregulation of the cardiac inflammasome in fibroblasts as a cellular target of SR9009, inviting more targeted therapeutic investigations in the future

The Impact of Sex, Circadian Disruption, and the Clock\u3csup\u3e∆19/∆19\u3c/sup\u3e Genotype on Alcohol Drinking in Mice

Shift work is associated with increased alcohol drinking, more so in males than females, and is thought to be a coping mechanism for disrupted sleep cycles. However, little is presently known about the causal influence of circadian rhythm disruptions on sex differences in alcohol consumption. In this study, we disrupted circadian rhythms in female and male mice using both environmental (i.e., shifting diurnal cycles) and genetic (i.e., Clock∆19/∆19 mutation) manipulations, and measured changes in alcohol consumption and preference using a two-bottle choice paradigm. Alcohol consumption and preference, as well as food and water consumption, total caloric intake, and weight were assessed in adult female and male Clock∆19/∆19 mutant mice or wild-type (WT) litter-mates, housed under a 12-hour:12-hour light:dark (L:D) cycle or a shortened 10-hour:10-hour L:D cycle. Female WT mice (under both light cycles) increased their alcohol consumption and preference over time, a pattern not observed in male WT mice. Compared to WT mice, Clock∆19/∆19 mice displayed increased alcohol consumption and preference. Sex differences were not apparent in Clock∆19/∆19 mice, with or without shifting diurnal cycles. In conclusion, sex differences in alcohol consumption patterns are evident and increase with prolonged access to alcohol. Disrupting circadian rhythms by mutating the Clock gene greatly increases alcohol consumption and abolishes sex differences present in WT animals

Development of Synbiotic Milk Chocolate Enriched with Lactobacillus paracasei, D-tagatose and Galactooligosaccharide

Background and Objective: Prebiotics are food ingredients that induce the growth or activity of beneficial bacteria (Bifidobacteria and Lactobacilli). Galactooligosaccharide and tagatose are two main prebiotic compounds which are used in the food industry. Chocolate is widely consumed all over the world and could be used as an excellent vehicle for delivery of prebiotics. Furthermore, the incorporation of probiotics into chocolate, allows broadening the health claims of chocolate. The aim of the current study was to investigate the effect of tagatose and galactooligosaccharide on the physicochemical and sensory properties of milk chocolate and the survivability of Lactobacillus paracasei in the optimized formulation. Material and Methods: Probiotic milk chocolate containing Lactobacillus paracasei were formulated by replacing a portion of the sucrose with the galactooligosaccharide powder and tagatose. For this purpose various concentrations of galactooligosaccharide and tagatose (2.5, 5 and 7.5% w w-1) along with stevia were used in chocolate formulation. Nine formulations were examined to determine some physicochemical, mechanical and sensory properties in order to find the optimum concentrations of these components. The lyophilized Lactobacillus paracasei were incorporated in the optimal formulation of prebiotic milk chocolate. The viability of probiotic bacteria in milk chocolate was carried out during storage at 22°C for up to 6 months.Results and Conclusion: In general, chocolate formulations with high levels of galactooligosaccharide, achieved the highest plastic viscosity and yield stress. The lowest viscosity and yield stress were observed for the samples containing high concentrations of tagatose and in control. In addition, galactooligosaccharide at higher ratios induced the least desirable sensorial effects, whereas tagatose improved the overall acceptability. It can be concluded that the overall acceptability of milk chocolate samples were with (7.5), tagatose: galactooligosaccharide ratios of 2.5%-2.5%, presenting the optimal applicable range as prebiotic compounds. Numbers of live Lactobacillus paracasei cells remained above 8.0 log CFU g-1 until 6 months under ambient conditions. Milk chocolate was shown to be an excellent vehicle for the delivery of Lactobacillus paracasei, and the prebiotic ingredients galactooligosaccharide and tagatose did not interfere in its viability. Conflict of interest: The authors declare no conflict of interest

Rev-erbα Knockout Reduces Ethanol Consumption and Preference in Male and Female Mice

Alcohol use is a contributor in the premature deaths of approximately 3 million people annually. Among the risk factors for alcohol misuse is circadian rhythm disruption; however, this connection remains poorly understood. Inhibition of the circadian nuclear receptor REV-ERBα is known to disrupt molecular feedback loops integral to daily oscillations, and impact diurnal fluctuations in the expression of proteins required for reward-related neurotransmission. However, the role of REV-ERBα in alcohol and substance use-related phenotypes is unknown. Herein, we used a Rev-erbα knockout mouse line and ethanol two-bottle choice preference testing to show that disruption of Rev-erbα reduces ethanol preference in male and female mice. Rev-erbα null mice showed the lowest ethanol preference in a two-bottle choice test across all genotypes, whereas there were no ethanol preference differences between heterozygotes and wildtypes. In a separate experiment, alcohol-consuming wildtype C57Bl/6N mice were administered the REV-ERBα/β inhibitor SR8278 (25 mg/kg or 50 mg/kg) for 7 days and alcohol preference was evaluated daily. No differences in alcohol preference were observed between the treatment and vehicle groups. Our data provides evidence that genetic variation in REV-ERBα may contribute to differences in alcohol drinking

Bone marrow-derived mesenchymal stem cell and simvastatin treatment leads to improved functional recovery and modified c-Fos expression levels in the brain following ischemic stroke

Objective(s): The beneficial outcomes of bone marrow-derived mesenchymal stem cell (BMSC) treatment on functional recovery following stroke has been well established. Furthermore, 5-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors have also been shown to increase neuronal survival and promote the movement of BMSCs towards the sites of inflammation. However, the precise mechanisms mediating the improved neurological functional recovery in stoke models following a combination treatment of Simvastatin and BMSCs still remained poorly understood. Materials and Methods: Here, an embolic stroke model was used to experimentally induce a focal ischemic brain injury by inserting a preformed clot into the middle cerebral artery (MCA). Following stroke, animals were treated either with an intraperitoneal injection of Simvastatin, an intravenous injection of 3 ×106 BMSCs, or a combination of these two treatments.Results: Seven days after ischemia, the combination of Simvastatin and BMSCs led to a significant increase in BMSC relocation, endogenous neurogenesis, arteriogenesis and astrocyte activation while also reducing neuronal damage when compared to BMSC treatment alone (PConclusion: These results further demonstrate the synergistic benefits of a combination treatment and help to improve our understanding of the underlying mechanisms mediating this beneficial effect

Improvement of isolated caprine islet survival and functionality in vitro by enhancing of PDX1 gene expression

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

Comparison of GIP and GLP-1 promoter-mediated insulin expression in Gut K and L-cells for the potential treatment of diabetes mellitus

Diabetes mellitus is characterized by abnormally high blood glucose levels, which causes serious complications. It is hypothesized that gut K and L-cells could make potential candidates for diabetes gene therapy manipulation. The project was carried out to determine and compare the quality and quantity of the human insulin expressed in both mice K and L-cells in vitro and in vivo which have been transfected with human insulin gene construct with GIP and GLP-1 promoters. These cells do not naturally produce insulin but respond to glucose and other nutrients in the gut by secreting GIP (glucose-dependent insulinotropic polypeptide) and GLP-1 (glucagon-like peptide-1) which then stimulate beta cells of pancreas to produce insulin. For this purpose, two plasmids containing neomycin with GIP or GLP-1 were constructed to isolate pure surrogate models for in vitro studies. The next two plasmids containing insulin with GIP or GLP-1 were constructed in order to study efficiency and efficacy of insulin expression in vitro and in vivo and the last two plasmids containing GFP with GIP or GLP-1 were constructed to study delivery efficiency of gene vehicle in vivo. We also studied different nutrients stimulus effects and time expression of the insulin secretion in both the K and L cells. Finally, the insulin expression efficacy of the reconstructed plasmids with GIP and GLP-1 promoters in lowering the glucose levels was compared in diabetes animal model. To study these hypotheses, neomycin, insulin and GFP genes were inserted downstream of the promoters in 6 different recombinant constructs. QRT- PCR was used to authenticate the purity of the isolated K and L-cell lines. Isolated K and L-cell lines were then transfected by recombinant insulin constructs. RT-PCR, immunocytostaining and Western blotting were used to analyze qualification of mature insulin while QRT- PCR and ELISA were employed to quantify the secreted insulin from both cell-lines. Glucose and meat hydrolysate (MH) were used to study different stimulant effects on insulin expression in both cell lines. The secretion of insulin was investigated for 180 min in 6 different time points. For in vivo studies, chitosan was employed to transfer the recombinant insulin constructs into the target cells orally. The ability of chitosan to deliver the constructs was investigated using immunohistostaining on the intestine of the mice. The effect of secreted insulin on lowering the glucose levels in STZ-induced diabetic mice was investigated by blood glucose testing in two weeks duration. QRT-PCR results proved that the isolated cells were pure K and L-cell. The Western blotting and immunocytostaining results of insulin secretion studies revealed that both cells were able to produce mature and active insulin. Statistical analysis revealed that the difference between insulin expressions from K and L-cells in relation to the glucose and MH stimulations were not significant. The immunohistostaining results showed that chitosan was an efficient gene vehicle to transfect the intestinal cells. In addition, RT-PCT and ELISA confirmed that pure human insulin was expressed in vivo. Blood glucose testing results showed that the treatment with the recombinant insulin gene significantly reduced glucose levels in diabetic mice. As a conclusion, recombinant neomycin constructs successfully produced pure K and L-cells. Recombinant insulin constructs comparably efficiently produced mature insulin protein in K and L-cells in vitro and in vivo. The response of K and L-cells to induction of both stimulators was mostly comparable. However, meat hydrolysate is found to be a more potent insulin secretion agonist for both cells than glucose. In addition, our in vivo results provided successful preliminary data that proved the analogous ability of both GIP and GLP-1 insulin constructs in lowering blood glucose levels of the diabetic mice