Efficient Generation of Functional Dopaminergic Neurons from Human Induced Pluripotent Stem Cells Under Defined Conditions

Human induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells represent a promising unlimited cell source for generating patient-specific cells for biomedical research and personalized medicine. As a first step, critical to clinical applications, we attempted to develop defined culture conditions to expand and differentiate human iPSCs into functional progeny such as dopaminergic neurons for treating or modeling Parkinson's disease (PD). We used a completely defined (xeno-free) system that we previously developed for efficient generation of authentic dopaminergic neurons from human embryonic stem cells (hESCs), and applied it to iPSCs. First, we adapted two human iPSC lines derived from different somatic cell types for the defined expansion medium and showed that the iPSCs grew similarly as hESCs in the same medium regarding pluripotency and genomic stability. Second, by using these two independent adapted iPSC lines, we showed that the process of differentiation into committed neural stem cells (NSCs) and subsequently into dopaminergic neurons was also similar to hESCs. Importantly, iPSC-derived dopaminergic neurons were functional as they survived and improved behavioral deficits in 6-hydroxydopamine-leasioned rats after transplantation. In addition, iPSC-derived NSCs and neurons could be efficiently transduced by a baculoviral vector delivering episomal DNA for future gene function study and disease modeling using iPSCs. We also performed genome-wide microarray comparisons between iPSCs and hESCs, and we derived NSC and dopaminergic neurons. Our data revealed overall similarity and visible differences at a molecular level. Efficient generation of functional dopaminergic neurons under defined conditions will facilitate research and applications using PD patient-specific iPSCs. Stem Cells 2010;28:1893–190

S6K1 regulates hematopoietic stem cell self-renewal and leukemia maintenance.

Hyperactivation of the mTOR pathway impairs hematopoietic stem cell (HSC) functions and promotes leukemogenesis. mTORC1 and mTORC2 differentially control normal and leukemic stem cell functions. mTORC1 regulates p70 ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E–binding (eIF4E-binding) protein 1 (4E-BP1), and mTORC2 modulates AKT activation. Given the extensive crosstalk that occurs between mTORC1 and mTORC2 signaling pathways, we assessed the role of the mTORC1 substrate S6K1 in the regulation of both normal HSC functions and in leukemogenesis driven by the mixed lineage leukemia (MLL) fusion oncogene MLL-AF9. We demonstrated that S6K1 deficiency impairs self-renewal of murine HSCs by reducing p21 expression. Loss of S6K1 also improved survival in mice transplanted with MLL-AF9–positive leukemic stem cells by modulating AKT an

Role of p85α in neutrophil extra- and intracellular reactive oxygen species generation

Drug resistance is a growing problem that necessitates new strategies to combat pathogens. Neutrophil phagocytosis and production of intracellular ROS, in particular, has been shown to cooperate with antibiotics in the killing of microbes. This study tested the hypothesis that p85α, the regulatory subunit of PI3K, regulates production of intracellular ROS. Genetic knockout of p85α in mice caused decreased expression of catalytic subunits p110α, p110β, and p110δ, but did not change expression levels of the NADPH oxidase complex subunits p67phox, p47phox, and p40phox. When p85α, p55α, and p50α (all encoded by Pik3r1) were deleted, there was an increase in intracellular ROS with no change in phagocytosis in response to both Fcγ receptor and complement receptor stimulation. Furthermore, the increased intracellular ROS correlated with significantly improved neutrophil killing of both methicillin-susceptible and methicillin-resistant S. aureus. Our findings suggest inhibition of p85α as novel approach to improving the clearance of resistant pathogens

Association Between Pre-operative BUN and Post-operative 30-Day Mortality in Patients Undergoing Craniotomy for Tumors: Data From the ACS NSQIP Database

ObjectiveThere is limited evidence to clarify the specific relationship between pre-operative blood urea nitrogen (BUN) and post-operative 30-day mortality in patients undergoing craniotomy for tumors. Therefore, we aimed to investigate this relationship in detail.MethodsElectronic medical records of 18,642 patients undergoing craniotomy for tumors in the ACS NSQIP from 2012 to 2015 were subjected to secondary retrospective analysis. The principal exposure was pre-operative BUN. Outcome measures were post-operative 30-day mortality. We used binary logistic regression modeling to evaluate the association between them and conducted a generalized additive model and smooth curve fitting (penalized spline method) to explore the potential relationship and its explicit curve shape. We also conducted sensitivity analyses to ensure the robustness of the results and performed subgroup analyses.ResultsA total of 16,876 patients were included in this analysis. Of these, 47.48% of patients were men. The post-operative 30-day mortality of the included cases was 2.49% (420/16,876), and the mean BUN was 16.874 ± 6.648 mg/dl. After adjusting covariates, the results showed that pre-operative BUN was positively associated with post-operative 30-day mortality (OR = 1.020, 95% CI: 1.004, 1.036). There was also a non-linear relationship between BUN and post-operative 30-day mortality, and the inflection point of the BUN was 9.804. For patients with BUN < 9.804 mg/dl, a 1 unit decrease in BUN was related to a 16.8% increase in the risk of post-operative 30-day mortality (OR = 0.832, 95% CI: 0.737, 0.941); for patients with BUN > 9.804 mg/dl, a 1 unit increase in BUN was related to a 2.8% increase in the risk of post-operative 30-day mortality (OR = 1.028, 95% CI: 1.011, 1.045). The sensitivity analysis proved that the results were robust. The subgroup analysis revealed that all listed subgroups did not affect the relationship between pre-operative BUN and post-operative 30-day mortality (P > 0.05).ConclusionOur study demonstrated that pre-operative BUN (mg/dl) has specific linear and non-linear relationships with post-operative 30-day mortality in patients over 18 years of age who underwent craniotomy for tumors. Proper pre-operative management of BUN and maintenance of BUN near the inflection point (9.804 mg/dl) could reduce the risk of post-operative 30-day mortality in these cases

Soil microbiome structure and function in ecopiles used to remediate petroleum-contaminated soil

The soil microbiome consists of a vast variety of microorganisms which contribute to essential ecosystem services including nutrient recycling, protecting soil structure, and pathogen suppression. Recalcitrant organic compounds present in soils contaminated with fuel oil can lead to a decrease in functional redundancy within soil microbiomes. Ecopiling is a passive bioremediation technique involving biostimulation of indigenous hydrocarbon degraders, bioaugmentation through inoculation with known petroleum-degrading consortia, and phytoremediation. The current study investigates the assemblage of soil microbial communities and pollutant-degrading potential in soil undergoing the Ecopiling process, through the amplicon marker gene and metagenomics analysis of the contaminated soil. The analysis of key community members including bacteria, fungi, and nematodes revealed a surprisingly diverse microbial community composition within the contaminated soil. The soil bacterial community was found to be dominated by Alphaproteobacteria (60–70%) with the most abundant genera such as Lysobacter, Dietzia, Pseudomonas, and Extensimonas. The fungal community consisted mainly of Ascomycota (50–70% relative abundance). Soil sequencing data allowed the identification of key enzymes involved in the biodegradation of hydrocarbons, providing a novel window into the function of individual bacterial groups in the Ecopile. Although the genus Lysobacter was identified as the most abundant bacterial genus (11–46%) in all of the contaminated soil samples, the metagenomic data were unable to confirm a role for this group in petrochemical degradation. Conversely, genera with relatively low abundance such as Dietzia (0.4–9.0%), Pusillimonas (0.7–2.3%), and Bradyrhizobium (0.8–1.8%) did possess genes involved in aliphatic or aromatic compound degradation