A novel composition for the culture of human adipose stem cells which includes complement C3

Adipose tissue is an easily accessible and abundant source of stem cells. Adipose stem cells (ASCs) are currently being researched as treatment options for repair and regeneration of damaged tissues. The standard culture conditions used for expansion of ASCs contain fetal bovine serum (FBS) which is undefined, could transmit known and unknown adventitious agents, and may cause adverse immune reactions. We have described a novel culture condition which excludes the use of FBS and characterised the resulting culture. Human ASCs were cultured in the novel culture medium, which included complement protein C3. These cultures, called C-ASCs, were compared with ASCs cultured in medium supplemented with FBS. Analysis of ASCs for surface marker profile, proliferation characteristics and differentiation potential indicated that the C-ASCs were similar to ASCs cultured in medium containing FBS. Using a specific inhibitor, we show that C3 is required for the survival of C-ASCs. This novel composition lends itself to being developed into a defined condition for the routine culture of ASCs for basic and clinical applications.     

Bayesian geostatistical analysis and prediction of Rhodesian human African trypanosomiasis

Background

The persistent spread of Rhodesian human African trypanosomiasis (HAT) in Uganda in recent years has increased concerns of a potential overlap with the Gambian form of the disease. Recent research has aimed to increase the evidence base for targeting control measures by focusing on the environmental and climatic factors that control the spatial distribution of the disease.

Objectives

One recent study used simple logistic regression methods to explore the relationship between prevalence of Rhodesian HAT and several social, environmental and climatic variables in two of the most recently affected districts of Uganda, and suggested the disease had spread into the study area due to the movement of infected, untreated livestock. Here we extend this study to account for spatial autocorrelation, incorporate uncertainty in input data and model parameters and undertake predictive mapping for risk of high HAT prevalence in future.

Materials and Methods

Using a spatial analysis in which a generalised linear geostatistical model is used in a Bayesian framework to account explicitly for spatial autocorrelation and incorporate uncertainty in input data and model parameters we are able to demonstrate a more rigorous analytical approach, potentially resulting in more accurate parameter and significance estimates and increased predictive accuracy, thereby allowing an assessment of the validity of the livestock movement hypothesis given more robust parameter estimation and appropriate assessment of covariate effects.

Results

Analysis strongly supports the theory that Rhodesian HAT was imported to the study area via the movement of untreated, infected livestock from endemic areas. The confounding effect of health care accessibility on the spatial distribution of Rhodesian HAT and the linkages between the disease''s distribution and minimum land surface temperature have also been confirmed via the application of these methods.

Conclusions

Predictive mapping indicates an increased risk of high HAT prevalence in the future in areas surrounding livestock markets, demonstrating the importance of livestock trading for continuing disease spread. Adherence to government policy to treat livestock at the point of sale is essential to prevent the spread of sleeping sickness in Uganda.     

Mdm2 is required for survival of hematopoietic stem cells/progenitors via dampening of ROS-induced p53 activity

Mdm2 is an E3 ubiquitin ligase that targets p53 for degradation. p53(515C) (encoding p53R172P) is a hypomorphic allele of p53 that rescues the embryonic lethality of Mdm2(-/-) mice. Mdm2(-/-) p53(515C/515C) mice, however, die by postnatal day 13 resulting from hematopoietic failure. Hematopoietic stem cells and progenitors of Mdm2(-/-) p53(515C/515C) mice were normal in fetal livers but were depleted in postnatal bone marrows. After birth, these mice had elevated reactive oxygen species (ROS) thus activating p53R172P. In the absence of Mdm2, stable p53R172P induced ROS and cell cycle arrest, senescence, and cell death in the hematopoietic compartment. This phenotype was partially rescued with antioxidant treatment and upon culturing of hematopoietic cells in methycellulose at 3% oxygen. p16 was also stabilized because of ROS, and its loss increased cell cycling and partially rescued hematopoiesis and survival. Thus, Mdm2 is required to control ROS-induced p53 levels for sustainable hematopoiesis.     

Orexin-1 Receptor Co-Localizes with Pancreatic Hormones in Islet Cells and Modulates the Outcome of Streptozotocin-Induced Diabetes Mellitus

Recent studies have shown that orexins play a critical role in the regulation of sleep/wake states, feeding behaviour, and reward processes. The exocrine and endocrine pancreas are involved in the regulation of food metabolism and energy balance. This function is deranged in diabetes mellitus. This study examined the pattern of distribution of orexin-1 receptor (OX₁R) in the endocrine cells of the pancreas of normal and diabetic Wistar (a model of type 1 diabetes), Goto-Kakizaki (GK, a model of type 2 diabetes) rats and in orexin-deficient (OX^−/−) and wild type mice. Diabetes mellitus (DM) was induced in Wistar rats and mice by streptozotocin (STZ). At different time points (12 h, 24 h, 4 weeks, 8 months and 15 months) after the induction of DM, pancreatic fragments of normal and diabetic rats were processed for immunohistochemistry and Western blotting. OX₁R-immunoreactive nerves were observed in the pancreas of normal and diabetic Wistar rats. OX₁R was also discernible in the pancreatic islets of normal and diabetic Wistar and GK rats, and wild type mice. OX₁R co-localized with insulin (INS) and glucagon (GLU) in the pancreas of Wistar and GK rats. The number of OX₁R-positive cells in the islets increased markedly (p<0.0001) after the onset of DM. The increase in the number of OX₁R-positive cells is associated with a high degree of co-localization with GLU. The number of GLU- positive cells expressing OX₁R was significantly (p<0.0001) higher after the onset of DM. The tissue level of OX₁R protein increased with the duration of DM especially in type 1 diabetes where it co-localized with cleaved caspase 3 in islet cells. In comparison to STZ-treated wild type mice, STZ-treated OX^−/− animals exhibited reduced hyperglycemia and handled glucose more efficiently in glucose tolerance test. The findings suggest an important role for the OX-OX₁R pathway in STZ-induced experimental diabetes.     

Overexpression of pyruvate decarboxylase in the yeast Hansenula polymorpha results in increased ethanol yield in high-temperature fermentation of xylose

Improvement of xylose fermentation is of great importance to the fuel ethanol industry. The nonconventional thermotolerant yeast Hansenula polymorpha naturally ferments xylose to ethanol at high temperatures (48-50 degrees C). Introduction of a mutation that impairs ethanol reutilization in H. polymorpha led to an increase in ethanol yield from xylose. The native and heterologous (Kluyveromyces lactis) PDC1 genes coding for pyruvate decarboxylase were expressed at high levels in H. polymorpha under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). This resulted in increased pyruvate decarboxylase activity and improved ethanol production from xylose. The introduction of multiple copies of the H. polymorpha PDC1 gene driven by the strong constitutive promoter led to a 20-fold increase in pyruvate decarboxylase activity and up to a threefold elevation of ethanol production.     

Removal of chlorophenols from synthetic solutions using Phanerochaete chrysosporium

The potential use of the fungus Phanerochaete chrysosporium to remove chlorophenols (phenol, o-chlorophenol, p-chlorophenol and 2,4,6-trichlorophenol) from aqueous solutions was evaluated. The kinetics of both adsorption and desorption of phenolic compounds was rapid for all adsorbates. The maximum adsorptions of phenol and chlorophenols onto the Phanerochaete chrysosporium were 1.23 mmol/g for phenol, 1.49 mmol/g for o-chlorophenol, 1.78 mmol/g for p-chlorophenol and 2.14 mmol/g for 2,4,6-trichlorophenol. The affinity order was as follows: 2,4,6-trichlorophenol > p-chlorophenol > o-chlorophenol > phenol. Phenol and chlorophenols binding with Phanerochaete chrysosporium were clearly pH dependent. The adsorption of phenol and chlorophenols increased as pH increased. Desorption of phenol or chlorophenols was achieved using methanol solution (30% (v/v)). Phanerochaete chrysosporium is suitable for reuse for more than ten cycles without noticeable loss of adsorption capacity.     

Epigenomic profiling reveals DNA-methylation changes associated with major psychosis

Epigenetic misregulation is consistent with various non-Mendelian features of schizophrenia and bipolar disorder. To date, however, few studies have investigated the role of DNA methylation in major psychosis, and none have taken a genome-wide epigenomic approach. In this study we used CpG-island microarrays to identify DNA-methylation changes in the frontal cortex and germline associated with schizophrenia and bipolar disorder. In the frontal cortex we find evidence for psychosis-associated DNA-methylation differences in numerous loci, including several involved in glutamatergic and GABAergic neurotransmission, brain development, and other processes functionally linked to disease etiology. DNA-methylation changes in a significant proportion of these loci correspond to reported changes of steady-state mRNA level associated with psychosis. Gene-ontology analysis highlighted epigenetic disruption to loci involved in mitochondrial function, brain development, and stress response. Methylome network analysis uncovered decreased epigenetic modularity in both the brain and the germline of affected individuals, suggesting that systemic epigenetic dysfunction may be associated with major psychosis. We also report evidence for a strong correlation between DNA methylation in the MEK1 gene promoter region and lifetime antipsychotic use in schizophrenia patients. Finally, we observe that frontal-cortex DNA methylation in the BDNF gene is correlated with genotype at a nearby nonsynonymous SNP that has been previously associated with major psychosis. Our data are consistent with the epigenetic theory of major psychosis and suggest that DNA-methylation changes are important to the etiology of schizophrenia and bipolar disorder.