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1.  Fasting induces ketoacidosis and hypothermia in PDHK2/PDHK4-double-knockout mice 
The Biochemical journal  2012;443(3):829-839.
The importance of PDHK (pyruvate dehydrogenase kinase) 2 and 4 in regulation of the PDH complex (pyruvate dehydrogenase complex) was assessed in single- and double-knockout mice. PDHK2 deficiency caused higher PDH complex activity and lower blood glucose levels in the fed, but not the fasted, state. PDHK4 deficiency caused similar effects, but only after fasting. Double deficiency intensified these effects in both the fed and fasted states. PDHK2 deficiency had no effect on glucose tolerance, PDHK4 deficiency produced only a modest effect, but double deficiency caused a marked improvement and also induced lower insulin levels and increased insulin sensitivity. In spite of these beneficial effects, the double-knockout mice were more sensitive than wild-type and single-knockout mice to long-term fasting, succumbing to hypoglycaemia, ketoacidosis and hypothermia. Stable isotope flux analysis indicated that hypoglycaemia was due to a reduced rate of gluconeogenesis and that slightly more glucose was converted into ketone bodies in the double-knockout mice. The findings establish that PDHK2 is more important in the fed state, PDHK4 is more important in the fasted state, and survival during long-term fasting depends upon regulation of the PDH complex by both PDHK2 and PDHK4.
doi:10.1042/BJ20112197
PMCID: PMC4323161  PMID: 22360721
glucose; heart; hypothermia; ketoacidosis; liver; pyruvate dehydrogenase complex (PDH complex); pyruvate dehydrogenase kinase (PDHK); skeletal muscle
2.  Solvation Free Energies of Alanine Peptides: The Effect of Flexibility 
The journal of physical chemistry. B  2013;117(51):16428-16435.
The electrostatic (ΔGel), van der Waals cavity-formation (ΔGvdw), and total (ΔG) solvation free energies for 10 alanine peptides ranging in length (n) from 1 to 10 monomers were calculated. The free energies were computed both with fixed, extended conformations of the peptides and again for some of the peptides without constraints. The solvation free energies, ΔGel, and components ΔGvdw, and ΔG, were found to be linear in n, with the slopes of the best-fit lines being γel, γvdw, and γ, respectively. Both γel and γ were negative for fixed and flexible peptides, and γvdw was negative for fixed peptides. That γvdw was negative was surprising, as experimental data on alkanes, theoretical models, and MD computations on small molecules and model systems generally suggest that γvdw should be positive. A negative γvdw seemingly contradicts the notion that ΔGvdw drives the initial collapse of the protein when it folds by favoring conformations with small surface areas. When we computed ΔGvdw for the flexible peptides, thereby allowing the peptides to assume natural ensembles of more compact conformations, γvdw was positive. Because most proteins do not assume extended conformations, a ΔGvdw that increases with increasing surface area may be typical for globular proteins. An alternative hypothesis is that the collapse is driven by intramolecular interactions. We find few intramolecular h-bonds but show that the intramolecular van der Waal’s interaction energy is more favorable for the flexible than for the extended peptides, seemingly favoring this hypothesis. The large fluctuations in the vdw energy may make attributing the collapse of the peptide to this intramolecular energy difficult.
doi:10.1021/jp409693p
PMCID: PMC3913540  PMID: 24328358
Folding free energy; Solvation effects
3.  PyTMs: a useful PyMOL plugin for modeling common post-translational modifications 
BMC Bioinformatics  2014;15(1):370.
Background
Post-translational modifications (PTMs) constitute a major aspect of protein biology, particularly signaling events. Conversely, several different pathophysiological PTMs are hallmarks of oxidative imbalance or inflammatory states and are strongly associated with pathogenesis of autoimmune diseases or cancers. Accordingly, it is of interest to assess both the biological and structural effects of modification. For the latter, computer-based modeling offers an attractive option. We thus identified the need for easily applicable modeling options for PTMs.
Results
We developed PyTMs, a plugin implemented with the commonly used visualization software PyMOL. PyTMs enables users to introduce a set of common PTMs into protein/peptide models and can be used to address research questions related to PTMs. Ten types of modification are currently supported, including acetylation, carbamylation, citrullination, cysteine oxidation, malondialdehyde adducts, methionine oxidation, methylation, nitration, proline hydroxylation and phosphorylation. Furthermore, advanced settings integrate the pre-selection of surface-exposed atoms, define stereochemical alternatives and allow for basic structure optimization of the newly modified residues.
Conclusion
PyTMs is a useful, user-friendly modelling plugin for PyMOL. Advantages of PyTMs include standardized generation of PTMs, rapid time-to-result and facilitated user control. Although modeling cannot substitute for conventional structure determination it constitutes a convenient tool that allows uncomplicated exploration of potential implications prior to experimental investments and basic explanation of experimental data. PyTMs is freely available as part of the PyMOL script repository project on GitHub and will further evolve.
Graphical AbstractPyTMs is a useful PyMOL plugin for modeling common post-translational modifications.
Electronic supplementary material
The online version of this article (doi:10.1186/s12859-014-0370-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s12859-014-0370-6
PMCID: PMC4256751  PMID: 25431162
Post-translational modifications; PyMOL plugin; Structural bioinformatics; Modeling; Acetylation; Carbamylation; Citrullination; Oxidations; Malondialdehyde adducts; Nitration
4.  Adoptive transfer of cytokine-induced immunomodulatory adult microglia attenuates experimental autoimmune encephalomyelitis in DBA/1 mice 
Glia  2014;62(5):804-817.
Microglia are resident antigen-presenting cells in the central nervous system (CNS) that either suppress or promote disease depending on their activation phenotype and the microenvironment. Multiple sclerosis (MS) is a chronic inflammatory disease causing demyelination and nerve loss in the CNS, and experimental autoimmune encephalomyelitis (EAE) is an animal model of MS that is widely used to investigate pathogenic mechanisms and therapeutic effects. We isolated and cultured microglia from adult mouse brains and exposed them to specific combinations of stimulatory molecules and cytokines, the combination of IL-4, IL-10, and TGF-β yielding the optimal regime for induction of an immunosuppressive phenotype (M2). M2 microglia were characterized by decreased expression or production of CD86, PD-L1, nitric oxide, and IL-6, increased expression of PD-L2, and having a potent capacity to retain their phenotype on secondary proinflammatory stimulation. M2 microglia induced regulatory T cells, suppressed T-cell proliferation, and downmodulated M1-associated receptor expression in M1 macrophages. Myelin oligodendrocyte glycoprotein (MOG)-induced EAE was induced in DBA/1 mice and at different time points (0, 5, 12, or 15 days postimmunization) 3 × 105 M2 microglia were transferred intranasally. A single transfer of M2 microglia attenuated the severity of established EAE, which was particularly obvious when the cells were injected at 15 days postimmunization. M2 microglia-treated mice had reduced inflammatory responses and less demyelination in the CNS. Our findings demonstrate that adult M2 microglia therapy represents a novel intervention that alleviated established EAE and that this therapeutic principle may have relevance for treatment of MS patients.
doi:10.1002/glia.22643
PMCID: PMC4237117  PMID: 24677019
EAE; microglia; macrophages; phenotype; cell therapy
5.  Hypervascular tumor volume estimated by comparison to a large-scale cerebral blood volume radiographic atlas predicts survival in recurrent glioblastoma treated with bevacizumab 
Cancer Imaging  2014;14(1):31.
Background
Dynamic susceptibility contrast (DSC)-MRI is a well-established perfusion MR imaging technique for estimating relative cerebral blood volume (CBV) in primary brain tumors; however, tumors localized to regions with naturally elevated perfusion, including cortical tissue and common vascular territories, make evaluation of tumor vascularity difficult to assess. In the current study, we have constructed a large-scale radiographic atlas of CBV to assess treatment response to bevacizumab in individual patients with recurrent glioblastoma.
Methods
Z-score normalized CBV maps were registered to stereotactic atlas space in 450 patients with brain tumors. A CBV atlas was created by calculating the voxel-wise mean and variability in CBV. MRI and CBV maps from 32 recurrent glioblastoma patients were then obtained prior to and following treatment with bevacizumab, registered to and compared with the CBV atlas. The volume of tumor tissue with elevated CBV, percentage of enhancing tumor with elevated CBV, and the mean and maximum change in normalized CBV intensity relative to the atlas were computed.
Results
Voxel-wise comparison of individual patient CBV maps to the atlas allowed delineation of elevated tumor perfusion from artery and normal cortical tissue. An atlas-defined hypervascular tumor blood volume greater than 2.35 cc prior to treatment, 0.14 cc after treatment, and a decrease in atlas-defined hypervascular tumor volume less than 80% following treatment were characteristic of a shorter PFS and OS. Traditional measures of CBV were not predictive of PFS or OS.
Conclusions
This study highlights the advantages of large-scale population maps to identify abnormal biological tissues.
doi:10.1186/s40644-014-0031-z
PMCID: PMC4331836  PMID: 25608485
CBV; Recurrent GBM; Population map; Radiographic atlas; Bevacizumab
6.  Metabolic activation of CaMKII by Coenzyme A 
Molecular cell  2013;52(3):325-339.
SUMMARY
Active metabolism regulates oocyte cell death via calcium/calmodulin-dependent protein kinase II (CaMKII) mediated phosphorylation of caspase-2, but the link between metabolic activity and CaMKII is poorly understood. Here we identify coenzyme A (CoA) as the key metabolic signal that inhibits Xenopus laevis oocyte apoptosis, in a novel mechanism of CaMKII activation. We found that CoA directly binds to the CaMKII regulatory domain in the absence of Ca2+ to activate CaMKII in a calmodulin-dependent manner. Furthermore, we show that CoA inhibits apoptosis not only in X. laevis oocytes, but also in Murine oocytes. These findings uncover a novel mechanism of CaMKII regulation by metabolism and further highlight the importance of metabolism in preserving oocyte viability.
doi:10.1016/j.molcel.2013.08.043
PMCID: PMC3967247  PMID: 24095281
7.  A novel radiotracer to image glycogen metabolism in tumors by positron emission tomography 
Cancer research  2014;74(5):1319-1328.
The high rate of glucose uptake to fuel the bioenergetic and anabolic demands of proliferating cancer cells is well recognized, and exploited with 18F-2-fluoro-2-deoxyglucose positron emission tomography (18F-FDG-PET) to image tumors clinically. In contrast, enhanced glucose storage as glycogen (glycogenesis) in cancer is less well understood and the availability of a non-invasive method to image glycogen in vivo could provide important biologic insights. Here, we demonstrate that 18F-N-(methyl-(2-fluoroethyl)-1H-[1,2,3]triazole-4-yl)glucosamine (18F-NFTG) annotates glycogenesis in cancer cells and tumors in vivo, measured by PET. Specificity of glycogen labeling was demonstrated by isolating 18F-NFTG-associated glycogen and with stable knockdown of glycogen synthase 1, which inhibited 18F-NFTG uptake, while oncogene (Rab25) activation-associated glycogen synthesis led to increased uptake. We further show that the rate of glycogenesis is cell cycle-regulated, enhanced during the non-proliferative state of cancer cells. We demonstrate that glycogen levels, 18F-NFTG, but not 18F-FDG uptake, increase proportionally with cell density and G1/G0 arrest with potential application in the assessment of activation of oncogenic pathways related to glycogenesis and the detection of post treatment tumor quiescence.
doi:10.1158/0008-5472.CAN-13-2768
PMCID: PMC3966281  PMID: 24590807
Glycogen; PET; 18F-FDG; 18F-NFTG; Tumor
8.  A novel radiotracer to image glycogen metabolism in tumors by positron emission tomography 
Cancer research  2014;74(5):1319-1328.
The high rate of glucose uptake to fuel the bioenergetic and anabolic demands of proliferating cancer cells is well recognized, and exploited with 18F-2-fluoro-2-deoxyglucose positron emission tomography (18F-FDG-PET) to image tumors clinically. In contrast, enhanced glucose storage as glycogen (glycogenesis) in cancer is less well understood and the availability of a non-invasive method to image glycogen in vivo could provide important biologic insights. Here, we demonstrate that 18F-N-(methyl-(2-fluoroethyl)-1H-[1,2,3]triazole-4-yl)glucosamine (18F-NFTG) annotates glycogenesis in cancer cells and tumors in vivo, measured by PET. Specificity of glycogen labeling was demonstrated by isolating 18F-NFTG-associated glycogen and with stable knockdown of glycogen synthase 1, which inhibited 18F-NFTG uptake, while oncogene (Rab25) activation-associated glycogen synthesis led to increased uptake. We further show that the rate of glycogenesis is cell cycle-regulated, enhanced during the non-proliferative state of cancer cells. We demonstrate that glycogen levels, 18F-NFTG, but not 18F-FDG uptake, increase proportionally with cell density and G1/G0 arrest with potential application in the assessment of activation of oncogenic pathways related to glycogenesis and the detection of post treatment tumor quiescence.
doi:10.1158/0008-5472.CAN-13-2768
PMCID: PMC3966281  PMID: 24590807
Glycogen; PET; 18F-FDG; 18F-NFTG; Tumor
9.  Diffusion tensor imaging (DTI) detects microstructural reorganization in the brain associated with chronic irritable bowel syndrome (IBS) 
Pain  2013;154(9):1528-1541.
Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by recurring abdominal pain associated with alterations in bowel habits. We hypothesized patients with chronic visceral pain associated with IBS may have microstructural differences in the brain compared with healthy control subjects (HCs), indicative of long-term neural reorganization of chronic pain pathways and regions associated with sensory integration. In the current study we performed population-based voxel-wise DTI comparisons and probabilistic tractography in a large sample of phenotyped patients with IBS (n=33) and HCs (n=93). Patients had lower fractional anisotropy (FA) in thalamic regions, the basal ganglia and sensory/motor association/integration regions as well as higher FA in frontal lobe regions and the corpus callosum. In addition, patients had reduced mean diffusivity (MD), within the globus pallidus, and higher MD in the thalamus, internal capsule, and coronal radiata projecting to sensory/motor regions, suggestive of differential changes in axon/dendritic density in these regions. Sex differences in FA and MD were also observed in the patients but not in HCs. Probabilistic tractography confirmed a higher degree of connectivity in patients between the thalamus and pre-frontal cortex, as well as the medial dorsal thalamic nuclei and anterior cingulate cortex, and a lower degree of connectivity between the globus pallidus and thalamus. Together, these results support the hypothesis that patients with chronically recurring visceral pain from IBS have long-term microstructural changes within the brain, particular in regions associated with integration of sensory information and cortico thalamic modulation.
doi:10.1016/j.pain.2013.04.010
PMCID: PMC3758125  PMID: 23721972
10.  Influence of Grid Spacing in Poisson-Boltzmann Equation Binding Energy Estimation 
Grid-based solvers of the Poisson-Boltzmann, PB, equation are routinely used to estimate electrostatic binding, ΔΔGel, and solvation, ΔGel, free energies. The accuracies of such estimates are subject to grid discretization errors from the finite difference approximation to the PB equation. Here, we show that the grid discretization errors in ΔΔGel are more significant than those in ΔGel, and can be divided into two parts: (i) errors associated with the relative positioning of the grid and (ii) systematic errors associated with grid spacing. The systematic error in particular is significant for methods, such as the molecular mechanics PB surface area, MM-PBSA, approach that predict electrostatic binding free energies by averaging over an ensemble of molecular conformations. Although averaging over multiple conformations can control for the error associated with grid placement, it will not eliminate the systematic error, which can only be controlled by reducing grid spacing. The present study indicates that the widely-used grid spacing of 0.5 Å produces unacceptable errors in ΔΔGel, even though its predictions of ΔGel are adequate for the cases considered here. Although both grid discretization errors generally increase with grid spacing, the relative sizes of these errors differ according to the solute-solvent dielectric boundary definition. The grid discretization errors are generally smaller on the Gaussian surface used in the present study than on either the solvent-excluded or van der Waals surfaces, which both contain more surface discontinuities (e.g., sharp edges and cusps). Additionally, all three molecular surfaces converge to very different estimates of ΔΔGel.
doi:10.1021/ct300765w
PMCID: PMC3756143  PMID: 23997692
11.  HECTOR: a parallel multistage homopolymer spectrum based error corrector for 454 sequencing data 
BMC Bioinformatics  2014;15:131.
Background
Current-generation sequencing technologies are able to produce low-cost, high-throughput reads. However, the produced reads are imperfect and may contain various sequencing errors. Although many error correction methods have been developed in recent years, none explicitly targets homopolymer-length errors in the 454 sequencing reads.
Results
We present HECTOR, a parallel multistage homopolymer spectrum based error corrector for 454 sequencing data. In this algorithm, for the first time we have investigated a novel homopolymer spectrum based approach to handle homopolymer insertions or deletions, which are the dominant sequencing errors in 454 pyrosequencing reads. We have evaluated the performance of HECTOR, in terms of correction quality, runtime and parallel scalability, using both simulated and real pyrosequencing datasets. This performance has been further compared to that of Coral, a state-of-the-art error corrector which is based on multiple sequence alignment and Acacia, a recently published error corrector for amplicon pyrosequences. Our evaluations reveal that HECTOR demonstrates comparable correction quality to Coral, but runs 3.7× faster on average. In addition, HECTOR performs well even when the coverage of the dataset is low.
Conclusion
Our homopolymer spectrum based approach is theoretically capable of processing arbitrary-length homopolymer-length errors, with a linear time complexity. HECTOR employs a multi-threaded design based on a master-slave computing model. Our experimental results show that HECTOR is a practical 454 pyrosequencing read error corrector which is competitive in terms of both correction quality and speed. The source code and all simulated data are available at: http://hector454.sourceforge.net.
doi:10.1186/1471-2105-15-131
PMCID: PMC4023493  PMID: 24885381
NGS error correction; Homopolymer-length error; 454 sequencing; Parallelization
12.  Identifying the mesenchymal molecular subtype of glioblastoma using quantitative volumetric analysis of anatomic magnetic resonance images 
Neuro-Oncology  2013;15(5):626-634.
Background
Subtypes of glioblastoma multiforme (GBM) based on genetic and molecular alterations are thought to cause alterations in anatomic MRI owing to downstream biological changes, such as edema production, blood–brain barrier breakdown, and necrosis. The purpose of the current study was to identify a potential relationship between imaging features and the mesenchymal (MES) GBM subtype, which has the worst patient prognosis.
Methods
MRIs from 46 patients with histologically confirmed GBM were retrospectively analyzed. The volume of contrast enhancement, regions of central necrosis, and hyperintensity of T2/fluid attenuated inversion recovery (FLAIR) were measured. Additionally, the ratio of T2/FLAIR hyperintense volume to the volume of contrast enhancement and necrosis was calculated.
Results
The volume of contrast enhancement, volume of central necrosis, combined volume of contrast enhancement and central necrosis, and the ratio of T2/FLAIR to contrast enhancement and necrosis were significantly different in MES compared with non-MES GBM (Mann–Whitney, P < .05). Receiver-operator characteristics indicated that these 4 metrics were all significant predictors of the MES phenotype. The volume ratio of T2 hyperintensity to contrast enhancement and central necrosis was significantly lower in MES vs non-MES GBM (P < .0001), was a significant predictor of the MES phenotype (area under the curve = 0.93, P < .001), and could be used to stratify short- and long-term overall survival (log-rank, P = .0064 using cutoff of 3.0). These trends were also present when excluding isocitrate dehydrogenase 1 mutant tumors and incorporating covariates such as age and KPS score.
Conclusions
Results suggest that volume ratio may be a simple, cost-effective, and noninvasive biomarker for quickly identifying MES GBM.
doi:10.1093/neuonc/not008
PMCID: PMC3635524  PMID: 23444259
GBM; glioblastoma; IDH1; mesenchymal; molecular subtypes; MRI; radiogenomics
13.  Cerebral Activations Related to Audition-Driven Performance Imagery in Professional Musicians 
PLoS ONE  2014;9(4):e93681.
Functional Magnetic Resonance Imaging (fMRI) was used to study the activation of cerebral motor networks during auditory perception of music in professional keyboard musicians (n = 12). The activation paradigm implied that subjects listened to two-part polyphonic music, while either critically appraising the performance or imagining they were performing themselves. Two-part polyphonic audition and bimanual motor imagery circumvented a hemisphere bias associated with the convention of playing the melody with the right hand. Both tasks activated ventral premotor and auditory cortices, bilaterally, and the right anterior parietal cortex, when contrasted to 12 musically unskilled controls. Although left ventral premotor activation was increased during imagery (compared to judgment), bilateral dorsal premotor and right posterior-superior parietal activations were quite unique to motor imagery. The latter suggests that musicians not only recruited their manual motor repertoire but also performed a spatial transformation from the vertically perceived pitch axis (high and low sound) to the horizontal axis of the keyboard. Imagery-specific activations in controls were seen in left dorsal parietal-premotor and supplementary motor cortices. Although these activations were less strong compared to musicians, this overlapping distribution indicated the recruitment of a general ‘mirror-neuron’ circuitry. These two levels of sensori-motor transformations point towards common principles by which the brain organizes audition-driven music performance and visually guided task performance.
doi:10.1371/journal.pone.0093681
PMCID: PMC3979724  PMID: 24714661
14.  Image Quality in Real-Time Teleultrasound of Infant Hip Exam Over Low-Bandwidth Internet Links: a Transatlantic Feasibility Study 
Journal of Digital Imaging  2012;26(2):209-216.
Evolution of communication systems, especially internet-based technologies, has probably affected Radiology more than any other medical specialty. Tremendous increase in internet bandwidth has enabled a true revolution in image transmission and easy remote viewing of the static images and real-time video stream. Previous reports of real-time telesonography, such as the ones developed for emergency situations and humanitarian work, rely on high compressions of images utilized by remote sonologist to guide and supervise the unexperienced examiner. We believe that remote sonology could be also utilized in teleultrasound exam of infant hip. We tested feasibility of a low-cost teleultrasound system for infant hip and performed data analysis on the transmitted and original images. Transmission of data was accomplished with Remote Ultrasound (RU), a software package specifically designed for teleultrasound transmission through limited internet bandwidth. While image analysis of image pairs revealed statistically significant loss of information, panel evaluation failed to recognize any clinical difference between the original saved and transmitted still images.
doi:10.1007/s10278-012-9512-4
PMCID: PMC3597954  PMID: 22847913
Telemedicine; Image quality analysis; Ultrasound; Teleradiology
15.  Pre- and post-contrast three-dimensional double inversion-recovery MRI in human glioblastoma 
Journal of neuro-oncology  2013;112(2):257-266.
Fluid attenuated inversion recovery (FLAIR) MRI sequences have become an indispensible tool for defining the malignant boundary in patients with brain tumors by nulling the signal contribution from cerebro-spinal fluid allowing both regions of edema and regions of non-enhancing, infiltrating tumor to become hyperintense on resulting images. In the current study we examined the utility of a three-dimensional double inversion recovery (DIR) sequence that additionally nulls the MR signal associated with white matter, implemented either pre-contrast or post-contrast, in order to determine whether this sequence allows for better differentiation between tumor and normal brain tissue. T1- and T2-weighted, FLAIR, dynamic susceptibility contrast (DSC)-MRI estimates of cerebral blood volume (rCBV), contrast-enhanced T1-weighted images (T1+C), and DIR data (pre- or post-contrast) were acquired in 22 patients with glioblastoma. Contrast-to-noise (CNR) and tumor volumes were compared between DIR and FLAIR sequences. Line profiles across regions of tumor were generated to evaluate similarities between image contrasts. Additionally, voxel-wise associations between DIR and other sequences were examined. Results suggested post-contrast DIR images were hyperintense (bright) in regions spatially similar those having FLAIR hyperintensity and hypointense (dark) in regions with contrast-enhancement or elevated rCBV due to the high sensitivity of 3D turbo spin echo sequences to susceptibility differences between different tissues. DIR tumor volumes were statistically smaller than tumor volumes as defined by FLAIR (Paired t test, P = 0.0084), averaging a difference of approximately 14 mL or 24 %. DIR images had approximately 1.5× higher lesion CNR compared with FLAIR images (Paired t test, P = 0.0048). Line profiles across tumor regions and scatter plots of voxel-wise coherence between different contrasts confirmed a positive correlation between DIR and FLAIR signal intensity and a negative correlation between DIR and both post-contrast T1-weighted image signal intensity and rCBV. Additional discrepancies between FLAIR and DIR abnormal regions were also observed, together suggesting DIR may provide additional information beyond that of FLAIR.
doi:10.1007/s11060-013-1057-y
PMCID: PMC3616610  PMID: 23344788
Double inversion-recovery; DIR; MRI; Glioblastoma; Multiparametric MRI
16.  Complete Khoisan and Bantu genomes from southern Africa 
Nature  2010;463(7283):943-947.
The genetic structure of the indigenous hunter-gatherer peoples of southern Africa, the oldest known lineage of modern human, is important for understanding human diversity. Studies based on mitochondrial1 and small sets of nuclear markers2 have shown that these hunter-gatherers, known as Khoisan, San, or Bushmen, are genetically divergent from other humans1,3. However, until now, fully sequenced human genomes have been limited to recently diverged populations4–8. Here we present the complete genome sequences of an indigenous hunter-gatherer from the Kalahari Desert and a Bantu from southern Africa, as well as protein-coding regions from an additional three hunter-gatherers from disparate regions of the Kalahari. We characterize the extent of whole-genome and exome diversity among the five men, reporting 1.3 million novel DNA differences genome-wide, including 13,146 novel amino acid variants. In terms of nucleotide substitutions, the Bushmen seem to be, on average, more different from each other than, for example, a European and an Asian. Observed genomic differences between the hunter-gatherers and others may help to pinpoint genetic adaptations to an agricultural lifestyle. Adding the described variants to current databases will facilitate inclusion of southern Africans in medical research efforts, particularly when family and medical histories can be correlated with genome-wide data.
doi:10.1038/nature08795
PMCID: PMC3890430  PMID: 20164927
17.  Spatial, Temporal, and Functional Aspects of Macrophages during “The Good, the Bad, and the Ugly” Phases of Inflammation 
doi:10.3389/fimmu.2014.00612
PMCID: PMC4253962  PMID: 25520719
macrophage activation; M1/M2 macrophages; microenvironment; phenotype; immunotherapy; adoptive
18.  TLTF in Cerebrospinal Fluid for Detection and Staging of T. b. gambiense Infection 
PLoS ONE  2013;8(11):e79281.
Background
Trypanosome-derived lymphocyte triggering factor (TLTF) is a molecule released by African trypanosomes that interacts with the host immune system, resulting in increased levels of IFN-γ production.
Methodology/Principal findings
TLTF and anti-TLTF antibodies were assessed in sera and cerebrospinal fluid (CSF) from patients infected with Trypanosoma brucei gambiense (T. b. gambiense) in an attempt to identify alternative markers for diagnosis and stage determination of human African trypanosomiasis or sleeping sickness. Seventy-four serum and sixty-one CSF samples from patients with parasitologically confirmed infection and known disease stage along with 13 sera and CSF from uninfected controls were tested. In serum the levels of anti-TLTF antibodies were unrelated to the disease stage. In contrast, levels of anti-TLTF antibodies in CSF were higher in intermediate/late stages than in early stage disease patients. Specificity of the detected antibodies was assessed by inhibition of TLTF bioactivity as represented by its ability to induce IFN-γ production. Additionally, TLTF was detected in CSF from late stage patients by Western blotting with the anti-TLTF specific monoclonal antibody MO3.
Conclusions/Significance
These findings suggest a new possibility for disease diagnosis with focus on involvement of the CNS through detection of TLTF and anti-TLTF antibodies in the CSF.
doi:10.1371/journal.pone.0079281
PMCID: PMC3834137  PMID: 24260185
19.  Scoparone Exerts Anti-Tumor Activity against DU145 Prostate Cancer Cells via Inhibition of STAT3 Activity 
PLoS ONE  2013;8(11):e80391.
Scoparone, a natural compound isolated from Artemisia capillaris, has been used in Chinese herbal medicine to treat neonatal jaundice. Signal transducer and activator of transcription 3 (STAT3) contributes to the growth and survival of many human tumors. This study was undertaken to investigate the anti-tumor activity of scoparone against DU145 prostate cancer cells and to determine whether its effects are mediated by inhibition of STAT3 activity. Scoparone inhibited proliferation of DU145 cells via cell cycle arrest in G1 phase. Transient transfection assays showed that scoparone repressed both constitutive and IL-6-induced transcriptional activity of STAT3. Western blot and quantitative real-time PCR analyses demonstrated that scoparone suppressed the transcription of STAT3 target genes such as cyclin D1, c-Myc, survivin, Bcl-2, and Socs3. Consistent with this, scoparone decreased phosphorylation and nuclear accumulation of STAT3, but did not reduce phosphorylation of janus kinase 2 (JAK2) or Src, the major upstream kinases responsible for STAT3 activation. Moreover, transcriptional activity of a constitutively active mutant of STAT3 (STAT3C) was inhibited by scoparone, but not by AG490, a JAK2 inhibitor. Furthermore, scoparone treatment suppressed anchorage-independent growth in soft agar and tumor growth of DU145 xenografts in nude mice, concomitant with a reduction in STAT3 phosphorylation. Computational modeling suggested that scoparone might bind the SH2 domain of STAT3. Our findings suggest that scoparone elicits an anti-tumor effect against DU145 prostate cancer cells in part through inhibition of STAT3 activity.
doi:10.1371/journal.pone.0080391
PMCID: PMC3829856  PMID: 24260381
20.  Adoptive Transfer of Immunomodulatory M2 Macrophages Prevents Type 1 Diabetes in NOD Mice 
Diabetes  2012;61(11):2881-2892.
Macrophages are multifunctional immune cells that may either drive or modulate disease pathogenesis depending on their activation phenotype. Autoimmune type 1 diabetes (T1D) is a chronic proinflammatory condition characterized by unresolved destruction of pancreatic islets. Adoptive cell transfer of macrophages with immunosuppressive properties represents a novel immunotherapy for treatment of such chronic autoimmune diseases. We used a panel of cytokines and other stimuli to discern the most effective regimen for in vitro induction of immunosuppressive macrophages (M2r) and determined interleukin (IL)-4/IL-10/transforming growth factor-β (TGF-β) to be optimal. M2r cells expressed programmed cell death 1 ligand-2, fragment crystallizable region γ receptor IIb, IL-10, and TGF-β, had a potent deactivating effect on proinflammatory lipopolysaccharide/interferon-γ–stimulated macrophages, and significantly suppressed T-cell proliferation. Clinical therapeutic efficacy was assessed after adoptive transfer in NOD T1D mice, and after a single transfer of M2r macrophages, >80% of treated NOD mice were protected against T1D for at least 3 months, even when transfer was conducted just prior to clinical onset. Fluorescent imaging analyses revealed that adoptively transferred M2r macrophages specifically homed to the inflamed pancreas, promoting β-cell survival. We suggest that M2r macrophage therapy represents a novel intervention that stops ongoing autoimmune T1D and may have relevance in a clinical setting.
doi:10.2337/db11-1635
PMCID: PMC3478537  PMID: 22745325
21.  Metformin Inhibits Growth Hormone–Mediated Hepatic PDK4 Gene Expression Through Induction of Orphan Nuclear Receptor Small Heterodimer Partner 
Diabetes  2012;61(10):2484-2494.
Growth hormone (GH) is a counter-regulatory hormone that plays an important role in preventing hypoglycemia during fasting. Because inhibition of the pyruvate dehydrogenase complex (PDC) by pyruvate dehydrogenase kinase 4 (PDK4) conserves substrates for gluconeogenesis, we tested whether GH increases PDK4 expression in liver by a signaling pathway sensitive to inhibition by metformin. The effects of GH and metformin were determined in the liver of wild-type, small heterodimer partner (SHP)-, PDK4-, and signal transducer and activator of transcription 5 (STAT5)-null mice. Administration of GH in vivo increased PDK4 expression via a pathway dependent on STAT5 phosphorylation. Metformin inhibited the induction of PDK4 expression by GH via a pathway dependent on AMP-activated protein kinase (AMPK) and SHP induction. The increase in PDK4 expression and PDC phosphorylation by GH was reduced in STAT5-null mice. Metformin decreased GH-mediated induction of PDK4 expression and metabolites in wild-type but not in SHP-null mice. In primary hepatocytes, dominant-negative mutant-AMPK and SHP knockdown prevented the inhibitory effect of metformin on GH-stimulated PDK4 expression. SHP directly inhibited STAT5 association on the PDK4 gene promoter. Metformin inhibits GH-induced PDK4 expression and metabolites via an AMPK-SHP–dependent pathway. The metformin-AMPK-SHP network may provide a novel therapeutic approach for the treatment of hepatic metabolic disorders induced by the GH-mediated pathway.
doi:10.2337/db11-1665
PMCID: PMC3447904  PMID: 22698918
22.  Clinical and Immunologic Effects of Intranodal Autologous Tumor Lysate-Dendritic Cell Vaccine with Aldesleukin (Interleukin 2) and IFN-α2a Therapy in Metastatic Renal Cell Carcinoma Patients 
Purpose
To evaluate the clinical and immunologic outcomes of DC (dendritic cell) vaccine with interleukin (IL)-2 and IFN-α 2a in metastatic renal cell carcinoma patients.
Experimental Design
Eighteen consented and eligible patients were treated. Peripheral blood monocytes were cultured ex vivo into mature DCs and loaded with autologous tumor lysate. Treatment consisted of five cycles of intranodal vaccination of DCs (1 × 107 cells/1 mL Lactated Ringer’s solution), 5-day continuous i.v. infusion of IL-2 (18MiU/m2), and three s.c. injections of IFN-α 2a (6MiU) every other day. Response Evaluation Criteria in Solid Tumors criteria were used for disease assessment. Correlative immunologic end points included peripheral blood lymphocyte cell phenotype and function as well as peripheral blood anti–renal cell carcinoma antibody and cytokine levels.
Results
All patients received between two and five treatment cycles. Toxicities consisted of known and expected cytokine side effects. Overall objective clinical response rate was 50% with three complete responses. Median time to progression for all patients was 8 months, and median survival has not been reached (median follow up of 37+ months). Treatment-related changes in correlative immunologic end points were noted and the level of circulating CD4+ T regulatory cells had a strong association with outcome. Pre–IP-10 serum levels approached significance for predicting outcome.
Conclusions
The clinical and immunologic responses observed in this trial suggest an interaction between DC vaccination and cytokine therapy. Our data support the hypothesis that modulation of inflammatory, regulatory, and angiogenic pathways are necessary to optimize therapeutic benefit in renal cell carcinoma patients. Further exploration of this approach is warranted.
doi:10.1158/1078-0432.CCR-08-3240
PMCID: PMC3775650  PMID: 19622576
23.  Genetic Inactivation of Pyruvate Dehydrogenase Kinases Improves Hepatic Insulin Resistance Induced Diabetes 
PLoS ONE  2013;8(8):e71997.
Pyruvate dehydrogenase kinases (PDK1-4) play a critical role in the inhibition of the mitochondrial pyruvate dehydrogenase complex especially when blood glucose levels are low and pyruvate can be conserved for gluconeogenesis. Under diabetic conditions, the Pdk genes, particularly Pdk4, are often induced, and the elevation of the Pdk4 gene expression has been implicated in the increased gluconeogenesis in the liver and the decreased glucose utilization in the peripheral tissues. However, there is no direct evidence yet to show to what extent that the dysregulation of hepatic Pdk genes attributes to hyperglycemia and insulin resistance in vivo. To address this question, we crossed Pdk2 or Pdk4 null mice with a diabetic model that is deficient in hepatic insulin receptor substrates 1 and 2 (Irs1/2). Metabolic analyses reveal that deletion of the Pdk4 gene had better improvement in hyperglycemia and glucose tolerance than knockout of the Pdk2 gene whereas the Pdk2 gene deletion showed better insulin tolerance as compared to the Pdk4 gene inactivation on the Irs1/2 knockout genetic background. To examine the specific hepatic effects of Pdks on diabetes, we also knocked down the Pdk2 or Pdk4 gene using specific shRNAs. The data also indicate that the Pdk4 gene knockdown led to better glucose tolerance than the Pdk2 gene knockdown. In conclusion, our data suggest that hepatic Pdk4 may be critically involved in the pathogenesis of diabetes.
doi:10.1371/journal.pone.0071997
PMCID: PMC3733847  PMID: 23940800
24.  18F-FDOPA and 18F-FLT positron emission tomography parametric response maps predict response in recurrent malignant gliomas treated with bevacizumab 
Neuro-Oncology  2012;14(8):1079-1089.
The current study examined the use of voxel-wise changes in 18F-FDOPA and 18F-FLT PET uptake, referred to as parametric response maps (PRMs), to determine whether they were predictive of response to bevacizumab in patients with recurrent malignant gliomas. Twenty-four patients with recurrent malignant gliomas who underwent bevacizumab treatment were analyzed. Patients had MR and PET images acquired before and at 2 time points after bevacizumab treatment. PRMs were created by examining the percentage change in tracer uptake between time points in each image voxel. Voxel-wise increase in PET uptake in areas of pretreatment contrast enhancement defined by MRI stratified 3-month progression-free survival (PFS) and 6-month overall survival (OS) according to receiver-operating characteristic curve analysis. A decrease in PET tracer uptake was associated with longer PFS and OS, whereas an increase in PET uptake was associated with short PFS and OS. The volume fraction of increased 18F-FDOPA PET uptake between the 2 posttreatment time points also stratified long- and short-term PFS and OS (log-rank, P < .05); however, 18F-FLT uptake did not stratify OS. This study suggests that an increase in FDOPA or FLT PET uptake on PRMs after bevacizumab treatment may be a useful biomarker for predicting PFS and that FDOPA PET PRMs are also predictive of OS in recurrent gliomas treated with bevacizumab.
doi:10.1093/neuonc/nos141
PMCID: PMC3408264  PMID: 22711609
Bevacizumab; 18F-FDOPA; 18F-FLT, glioblastoma; PRMs
25.  Mutations in BCKD-kinase Lead to a Potentially Treatable Form of Autism with Epilepsy 
Science (New York, N.Y.)  2012;338(6105):394-397.
Autism spectrum disorders are a genetically heterogeneous constellation of syndromes characterized by impairments in reciprocal social interaction. Available somatic treatments have limited efficacy. We have identified inactivating mutations in the gene BCKDK (Branched Chain Ketoacid Dehydrogenase Kinase) in consanguineous families with autism, epilepsy, and intellectual disability. The encoded protein is responsible for phosphorylation-mediated inactivation of the E1α subunit of branched-chain ketoacid dehydrogenase (BCKDH). Patients with homozygous BCKDK mutations display reductions in BCKDK messenger RNA and protein, E1α phosphorylation, and plasma branched-chain amino acids. Bckdk knockout mice show abnormal brain amino acid profiles and neurobehavioral deficits that respond to dietary supplementation. Thus, autism presenting with intellectual disability and epilepsy caused by BCKDK mutations represents a potentially treatable syndrome.
doi:10.1126/science.1224631
PMCID: PMC3704165  PMID: 22956686

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