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1.  Initial In-Vivo Analysis of 3D Heterogeneous Brain Computations for Model-Updated Image-Guided Neurosurgery 
Registration error resulting from intraoperative brain shift due to applied surgical loads has long been recognized as one of the most challenging problems in the field of frameless stereotactic neurosurgery. To address this problem, we have developed a 3-dimensional finite element model of the brain and have begun to quantify its predictive capability in an in vivo porcine model. Previous studies have shown that we can predict the average total displacement within 15% and 6.6% error using intraparenchymal and temporal deformation sources, respectively, under relatively simple model assumptions. In this paper, we present preliminary results using a heterogeneous model with an expanding temporally located mass and show that we are capable of predicting an average total displacement to 5.7% under similar model initial and boundary conditions. We also demonstrate that our approach can be viewed as having the capability of recapturing approximately 75% of the registration inaccuracy that may be generated by preoperative-based image-guided neurosurgery.
PMCID: PMC4548975  PMID: 26317118
2.  Model-Updated Image-Guided Neurosurgery Using the Finite Element Method: Incorporation of the Falx Cerebri 
Surgeons using neuronavigation have realized the value of image guidance for feature recognition as well as for the precise application of surgical instruments. Recently, there has been a growing concern about the extent of intraoperative misregistration due to tissue deformation. Intraoperative imaging is currently under evaluation but limitations related to cost effectiveness and image clarity have made its wide spread adoption uncertain. As a result, computational model-guided techniques have generated considerable appeal as an alternative approach. In this paper, we report our initial experience with enhancing our brain deformation model by explicitly adding the falx cerebri. The simulations reported show significant differences in subsurface deformation with the falx serving to damp the communication of displacement between hemispheres by as much as 4 mm. Additionally, these calculations, based on a human clinical case, demonstrate that while cortical shift predictions correlate well with various forms of the model (70–80% of surface motion recaptured), substantial differences in subsurface deformation occurs suggesting that subsurface validation of model-guided techniques will be important for advancing this concept.
PMCID: PMC4548980  PMID: 26317119
3.  Model-Updated Image-Guided Neurosurgery: Preliminary Analysis Using Intraoperative MR 
In this paper, initial clinical data from an intraoperative MR system are compared to calculations made by a three-dimensional finite element model of brain deformation. The preoperative and intraoperative MR data was collected on a patient undergoing a resection of an astrocytoma, grade 3 with non-enhancing and enhancing regions. The image volumes were co-registered and cortical displacements as well as subsurface structure movements were measured retrospectively. These data were then compared to model predictions undergoing intraoperative conditions of gravity and simulated tumor decompression. Computed results demonstrate that gravity and decompression effects account for approximately 40% and 30%, respectively, totaling a 70% recovery of shifting structures with the model. The results also suggest that a non-uniform decompressive stress distribution may be present during tumor resection. Based on this preliminary experience, model predictions constrained by intraoperative surface data appear to be a promising avenue for correcting brain shift during surgery. However, additional clinical cases where volumetric intraoperative MR data is available are needed to improve the understanding of tissue mechanics during resection.
PMCID: PMC4548986  PMID: 26317120
finite element modeling and simulation; image guided therapy; intraoperative image registration techniques
4.  Confocal Microscopy for the Histological Fluorescence Pattern of a Recurrent Atypical Meningioma: Case Report 
Neurosurgery  2011;68(6):E1768-E1773.
Background and Importance
Fluorescence-guided resection with 5-aminolevulinic acid (5-ALA), which has shown promising results in the resection of malignant gliomas, has been used for meningioma resection in an attempt to more clearly delineate the tumor margin. However, no article has investigated the fluorescence pattern of meningiomas on a histological level. Understanding the microscopic pattern of fluorescence could help assess the precision and utility of using 5-ALA for these tumors. We present the case of a recurrent atypical meningioma operated on with 5-ALA fluorescence-guided resection for delineation of tumor tissue from surrounding uninvolved dura.
Clinical Presentation
A 53-year-old woman presented with recurrent atypical meningioma of the falx. Prior treatment included surgical resection 6 years earlier with subsequent fractionated radiation therapy and radiosurgery for tumor progression. The patient was given 5-ALA 20 mg/kg body weight dissolved in 100 mL water 3 hours before induction of anesthesia. Intraoperative fluorescence was coregistered with preoperative imaging. Neuropathological analysis of the resected falx with confocal microscopy enabled correlation of fluorescence with the extent of tumor on a histological level.
Fluorescence guidance allowed clear intraoperative delineation of tumor tissue from adjacent, uninvolved dura. On a microscopic level, there was a very close correlation of fluorescence with tumor, but some tumor cells did not fluoresce.
PMCID: PMC4512294  PMID: 21389893
5-ALA; Brain tumor; Contrast enhancement; Fluorescence-guided resection; Meningioma; PpIX
5.  CSF hydrothorax without intrathoracic catheter migration in children with ventriculoperitoneal shunt 
Surgical Neurology International  2015;6(Suppl 11):S330-S333.
Thoracic complications of ventriculoperitoneal (VP) shunts have been extensively reported in the literature. Cerebrospinal fluid (CSF) hydrothorax without catheter migration, however, has been rarely described and poorly understood.
Case Description:
We describe development of pleural effusion and respiratory distress in a 3-year-old boy with no evidence of VP shunt catheter displacement on plain radiograph and stable ventricle size on rapid sequence magnetic resonance imaging (MRI) brain. Chest X-ray revealed complete opacity of right hemithorax. Pleural effusion was consistent with transudate. Beta-2 transferrin returned positive. The patient underwent externalization of VP shunt, and upon resolution of effusion, re-internalization with new distal shunt catheter. A literature review of CSF hydrothorax in children without intrathoracic shunt migration was performed. Eleven cases were identified in the English literature. Age at VP shunt placement ranged from birth to 8 years of age. Interval from VP shunt placement to CSF hydrothorax ranged from 1.5 months to 5 years. History of shunt revision was reported in two cases. Presenting symptoms also included ascites and inguinal hernia or hydrocele. Reported diagnostic studies consist of CSF culture, radionuclide shuntogram, beta-2 transferrin, and beta-trace protein. Laterality of the VP shunt and development of pleural effusion were predominantly right sided. Definitive surgical treatment included VA shunt, repositioning of the peritoneal catheter, and endoscopic choroid plexus coagulation.
CSF hydrothorax is a rare thoracic complication of VP shunt placement with no radiographic evidence of shunt migration or malfunction. Postulated mechanisms include limited peritoneal capacity to resorb CSF in children and microscopic communications present in congenital diaphragmatic hiatuses.
PMCID: PMC4521309
Cerebrospinal fluid hydrothorax; shunt malfunction; ventriculoperitoneal shunt
6.  The molecular dimension of microbial species: 1. Ecological distinctions among, and homogeneity within, putative ecotypes of Synechococcus inhabiting the cyanobacterial mat of Mushroom Spring, Yellowstone National Park 
Based on the Stable Ecotype Model, evolution leads to the divergence of ecologically distinct populations (e.g., with different niches and/or behaviors) of ecologically interchangeable membership. In this study, pyrosequencing was used to provide deep sequence coverage of Synechococcus psaA genes and transcripts over a large number of habitat types in the Mushroom Spring microbial mat. Putative ecological species [putative ecotypes (PEs)], which were predicted by an evolutionary simulation based on the Stable Ecotype Model (Ecotype Simulation), exhibited distinct distributions relative to temperature-defined positions in the effluent channel and vertical position in the upper 1 mm-thick mat layer. Importantly, in most cases variants predicted to belong to the same PE formed unique clusters relative to temperature and depth in the mat in canonical correspondence analysis, supporting the hypothesis that while the PEs are ecologically distinct, the members of each ecotype are ecologically homogeneous. PEs responded differently to experimental perturbations of temperature and light, but the genetic variation within each PE was maintained as the relative abundances of PEs changed, further indicating that each population responded as a set of ecologically interchangeable individuals. Compared to PEs that predominate deeper within the mat photic zone, the timing of transcript abundances for selected genes differed for PEs that predominate in microenvironments closer to upper surface of the mat with spatiotemporal differences in light and O2 concentration. All of these findings are consistent with the hypotheses that Synechococcus species in hot spring mats are sets of ecologically interchangeable individuals that are differently adapted, that these adaptations control their distributions, and that the resulting distributions constrain the activities of the species in space and time.
PMCID: PMC4475828  PMID: 26157420
Mushroom Spring; microbial species; microbial ecology; population genetics; thermophilic Synechococcus
7.  UNCLES: method for the identification of genes differentially consistently co-expressed in a specific subset of datasets 
BMC Bioinformatics  2015;16(1):184.
Collective analysis of the increasingly emerging gene expression datasets are required. The recently proposed binarisation of consensus partition matrices (Bi-CoPaM) method can combine clustering results from multiple datasets to identify the subsets of genes which are consistently co-expressed in all of the provided datasets in a tuneable manner. However, results validation and parameter setting are issues that complicate the design of such methods. Moreover, although it is a common practice to test methods by application to synthetic datasets, the mathematical models used to synthesise such datasets are usually based on approximations which may not always be sufficiently representative of real datasets.
Here, we propose an unsupervised method for the unification of clustering results from multiple datasets using external specifications (UNCLES). This method has the ability to identify the subsets of genes consistently co-expressed in a subset of datasets while being poorly co-expressed in another subset of datasets, and to identify the subsets of genes consistently co-expressed in all given datasets. We also propose the M-N scatter plots validation technique and adopt it to set the parameters of UNCLES, such as the number of clusters, automatically. Additionally, we propose an approach for the synthesis of gene expression datasets using real data profiles in a way which combines the ground-truth-knowledge of synthetic data and the realistic expression values of real data, and therefore overcomes the problem of faithfulness of synthetic expression data modelling. By application to those datasets, we validate UNCLES while comparing it with other conventional clustering methods, and of particular relevance, biclustering methods. We further validate UNCLES by application to a set of 14 real genome-wide yeast datasets as it produces focused clusters that conform well to known biological facts. Furthermore, in-silico-based hypotheses regarding the function of a few previously unknown genes in those focused clusters are drawn.
The UNCLES method, the M-N scatter plots technique, and the expression data synthesis approach will have wide application for the comprehensive analysis of genomic and other sources of multiple complex biological datasets. Moreover, the derived in-silico-based biological hypotheses represent subjects for future functional studies.
Electronic supplementary material
The online version of this article (doi:10.1186/s12859-015-0614-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4453228  PMID: 26040489
Genome-wide analysis; Consistent co-expression; Bi-CoPaM; UNCLES; Multiple datasets analysis
8.  Hbr1 Activates and Represses Hyphal Growth in Candida albicans and Regulates Fungal Morphogenesis under Embedded Conditions 
PLoS ONE  2015;10(6):e0126919.
Transitions between yeast and hyphae are essential for Candida albicans pathogenesis. The genetic programs that regulate its hyphal development can be distinguished by embedded versus aerobic surface agar invasion. Hbr1, a regulator of white-opaque switching, is also a positive and negative regulator of hyphal invasion. During embedded growth at 24°C, an HBR1/hbr1 strain formed constitutively filamentous colonies throughout the matrix, resembling EFG1 null colonies, and a subset of long unbranched hyphal aggregates enclosed in a spindle-shaped capsule. Inhibition of adenylate cyclase with farnesol perturbed the filamentation of HBR1/hbr1 cells producing cytokinesis-defective hyphae whereas farnesol treated EFG1 null cells produced abundant opaque-like cells. Point mutations in the Hbr1 ATP-binding domain caused distinct filamentation phenotypes including uniform radial hyphae, hyphal sprouts, and massive yeast cell production. Conversely, aerobic surface colonies of the HBR1 heterozygote on Spider and GlcNAc media lacked filamentation that could be rescued by growth under low (5%) O2. Consistent with these morphogenesis defects, the HBR1 heterozygote exhibited attenuated virulence in a mouse candidemia model. These data define Hbr1 as an ATP-dependent positive and negative regulator of hyphal development that is sensitive to hypoxia.
PMCID: PMC4454550  PMID: 26039220
9.  Endoscopic Transnasal Surgery as a Replacement for Maxillotomy Techniques to Approach the Central Skull Base: Fewer Complications and More Acceptable to Patients? 
Objectives To compare the complication rates of endoscopic transnasal and open maxillotomy approaches for the central skull base.
Design Retrospective review.
Setting Single-center study, London, United Kingdom.
Participants From 1992 to 2012, 81 patients underwent surgery for skull base lesions, 59 by maxillotomy and 22 by endoscopy.
Main Outcome Measures Total time of surgical anesthesia, blood loss, complications, duration of tracheal intubation, duration of hospital stay, myelopathy score, and mortality rate.
Results The surgical time, blood loss, and duration of the postoperative intubation period were significantly less with endoscopy (p < 0.001). Requirements for intensive care, ward stay, and total hospital stay were also significantly less in the endoscopic group (p = 0.01, p < 0.001, and p < 0.001, respectively). The complication rate was lower with transnasal endoscopic surgery.
Conclusion In patients for whom open maxillotomy or endoscopic surgery are both feasible, the preference should be to perform endoscopic surgery, with better visualization and fewer complications.
PMCID: PMC4078146  PMID: 25072010
maxillotomy; endoscopy; skull base; surgery; complications
10.  CD47 Promotes Protective Innate and Adaptive Immunity in a Mouse Model of Disseminated Candidiasis 
PLoS ONE  2015;10(5):e0128220.
CD47 is a widely expressed receptor that regulates immunity by engaging its counter-receptor SIRPα on phagocytes and its secreted ligand thrombospondin-1. Mice lacking CD47 can exhibit enhanced or impaired host responses to bacterial pathogens, but its role in fungal immunity has not been examined. cd47-/- mice on a C57BL/6 background showed significantly increased morbidity and mortality following Candida albicans infection when compared with wild-type mice. Despite normal fungal colonization at earlier times, cd47-/- mice at four days post-infection had increased colonization of brain and kidneys accompanied by stronger inflammatory reactions. Neutrophil and macrophage numbers were significantly elevated in kidneys and neutrophils in the brains of infected cd47-/- mice. However, no defect in phagocytic activity towards C. albicans was observed in cd47-/- bone-marrow-derived macrophages, and neutrophil and macrophage killing of C. albicans was not impaired. CD47-deficiency did not alter the early humoral immune response to C. albicans. Th1, Th2, and Th17 population of CD4+ T cells were expanded in the spleen, and gene expression profiles of spleen and kidney showed stronger pro-inflammatory signaling in infected cd47-/- mice. The chemoattractant chemokines MIP-2α and MIP-2β were highly expressed in infected spleens of cd47-/- mice. G-CSF, GM-CSF, and the inflammasome component NLRP3 were more highly expressed in infected cd47-/- kidneys than in infected wild-type controls. Circulating pro- (TNF-α, IL-6) and anti-inflammatory cytokines (IL-10) were significantly elevated, but IL-17 was decreased. These data indicate that CD47 plays protective roles against disseminated candidiasis and alters pro-inflammatory and immunosuppressive pathways known to regulate innate and T cell immunity.
PMCID: PMC4444371  PMID: 26010544
11.  Observable Social Cognition: A Rating Scale (OSCARS): An Interview-Based Assessment for Schizophrenia 
Cognitive neuropsychiatry  2015;20(3):198-221.
Individuals with schizophrenia consistently show impairments in social cognition (SC). SC has become a potential treatment target due to its association with functional outcomes. An alternative method of assessment is to administer an observer-based scale incorporating an informant’s “first hand” impressions in ratings.
The present study used the Observable Social Cognition: A Rating Scale (OSCARS) in 62 outpatients and 50 non-psychiatric controls (NPCs) to assess performance in domains of SC (e.g. emotion perception, theory of mind).
The OSCARS demonstrated sufficient internal consistency and test-retest reliability. Construct validity was assessed through an exploratory factor analysis. Patient OSCARS indices were not significantly correlated with measures of SC with the exception of aggressive attributional style. Individuals with less impairment in SC reacted more aggressively to ambiguous situations. NPC OSCARS were significantly correlated with measures of theory of mind and attributional style. In a combined sample of patients and controls, six of eight items were significantly correlated with the SC task assessing the same domain, providing modest evidence of convergent validity. In patients, the OSCARS was significantly correlated with measures of functional outcome and neurocognition. Lastly, the OSCARS was found to be significantly associated with functional outcome after the influence of objective measures of SC was statistically removed.
The present study provides preliminary evidence that the OSCARS may be useful for clinicians in collecting data about patients’ potential real-world SC deficits, in turn increasing the degree to which these impairments may be targeted in treatment.
PMCID: PMC4372484  PMID: 25675960
social cognition; schizophrenia; schizophrenia spectrum illness; measurement; functional outcome
12.  Advancing Molecular-Guided Surgery through probe development and testing in a moderate cost evaluation pipeline 
Molecular guided oncology surgery has the potential to transform the way decisions about resection are done, and can be critically important in areas such as neurosurgery where the margins of tumor relative to critical normal tissues are not readily apparent from visual or palpable guidance. Yet there are major financial barriers to advancing agents into clinical trials with commercial backing. We observe that development of these agents in the standard biological therapeutic paradigm is not viable, due to the high up front financial investment needed and the limitations in the revenue models of contrast agents for imaging. The hypothesized solution to this problem is to develop small molecular biologicals tagged with an established fluorescent reporter, through the chemical agent approval pathway, targeting a phase 0 trials initially, such that the initial startup phase can be completely funded by a single NIH grant. In this way, fast trials can be completed to de-risk the development pipeline, and advance the idea of fluorescence-guided surgery (FGS) reporters into human testing. As with biological therapies the potential successes of each agent are still moderate, but this process will allow the field to advance in a more stable and productive manner, rather than relying upon isolated molecules developed at high cost and risk. The pathway proposed and tested here uses peptide synthesis of an epidermal growth factor receptor (EGFR)-binding Affibody molecules, uniquely conjugated to IRDye 800CW, developed and tested in academic and industrial laboratories with well-established records for GMP production, fill & finish, toxicity testing, and early phase clinical trials with image guidance.
PMCID: PMC4405779  PMID: 25914500
surgery; guidance; fluorescence; emission; filter; luminescence; intervention; cancer; oncology; Affibody
14.  The incidence of deep vein thrombosis and pulmonary embolism with the elective use of external fixators 
Little evidence exists about the incidence of deep vein thrombosis (DVT) and pulmonary embolism (PE) with the use of external fixators. We investigated this in a cohort of 207 consecutive patients undergoing 258 elective frame applications by case note review. Case notes were obtained for 84 % of the sample population. The type of surgery, demographic data, thromboembolic risk factors and the incidence of DVT/PE were recorded. One patient experienced DVT (0.39 %) and one a PE (0.39 %). Both were of high risk and had received mechanical and chemical thromboprophylaxis during their inpatient stay. These complications were identified at least 3 months post-operatively. These findings help to more accurately counsel patients undergoing elective frame surgery on the risks of DVT/PE and also contribute to the discussion between surgeons about whether or not extended course chemical thromboprophylaxis would be of overall benefit.
PMCID: PMC4570881  PMID: 25899134
Thrombosis; Prophylaxis; Frame; Elective; Incidence
15.  Bayesian Models for Screening and TB Mobile for Target Inference with Mycobacterium tuberculosis 
The search for compounds active against Mycobacterium tuberculosis is reliant upon high throughput screening (HTS) in whole cells. We have used Bayesian machine learning models which can predict anti-tubercular activity to filter an internal library of over 150,000 compounds prior to in vitro testing. We used this to select and test 48 compounds in vitro; 11 were active with MIC values ranging from 0.4 µM to 10.2 µM, giving a high hit rate of 22.9%. Among the hits, we identified several compounds belonging to the same series including five quinolones (including ciprofloxacin), three molecules with long aliphatic linkers and three singletons. This approach represents a rapid method to prioritize compounds for testing that can be used alongside medicinal chemistry insight and other filters to identify active molecules. Such models can significantly increase the hit rate of HTS, above the usual 1% or lower rates seen. In addition, the potential targets for the 11 molecules were predicted using TB Mobile and clustering alongside a set of over 740 molecules with known M. tuberculosis target annotations. These predictions may serve as a mechanism for prioritizing compounds for further optimization.
PMCID: PMC4394018  PMID: 24440548
Bayesian models; Collaborative Drug Discovery Tuberculosis database; function class fingerprints; Virtual Screening; Mycobacterium tuberculosis
16.  Biochar from commercially cultivated seaweed for soil amelioration 
Scientific Reports  2015;5:9665.
Seaweed cultivation is a high growth industry that is primarily targeted at human food and hydrocolloid markets. However, seaweed biomass also offers a feedstock for the production of nutrient-rich biochar for soil amelioration. We provide the first data of biochar yield and characteristics from intensively cultivated seaweeds (Saccharina, Undaria and Sargassum – brown seaweeds, and Gracilaria, Kappaphycus and Eucheuma – red seaweeds). While there is some variability in biochar properties as a function of the origin of seaweed, there are several defining and consistent characteristics of seaweed biochar, in particular a relatively low C content and surface area but high yield, essential trace elements (N, P and K) and exchangeable cations (particularly K). The pH of seaweed biochar ranges from neutral (7) to alkaline (11), allowing for broad-spectrum applications in diverse soil types. We find that seaweed biochar is a unique material for soil amelioration that is consistently different to biochar derived from ligno-cellulosic feedstock. Blending of seaweed and ligno-cellulosic biochar could provide a soil ameliorant that combines a high fixed C content with a mineral-rich substrate to enhance crop productivity.
PMCID: PMC4391317  PMID: 25856799
17.  Extremely Low Microsatellite Diversity but Distinct Population Structure in a Long-Lived Threatened Species, the Australian Lungfish Neoceratodus forsteri (Dipnoi) 
PLoS ONE  2015;10(4):e0121858.
The Australian lungfish is a unique living representative of an ancient dipnoan lineage, listed as ‘vulnerable’ to extinction under Australia’s Environment Protection and Biodiversity Conservation Act 1999. Historical accounts indicate this species occurred naturally in two adjacent river systems in Australia, the Burnett and Mary. Current day populations in other rivers are thought to have arisen by translocation from these source populations. Early genetic work detected very little variation and so had limited power to answer questions relevant for management including how genetic variation is partitioned within and among sub-populations. In this study, we use newly developed microsatellite markers to examine samples from the Burnett and Mary Rivers, as well as from two populations thought to be of translocated origin, Brisbane and North Pine. We test whether there is significant genetic structure among and within river drainages; assign putatively translocated populations to potential source populations; and estimate effective population sizes. Eleven polymorphic microsatellite loci genotyped in 218 individuals gave an average within-population heterozygosity of 0.39 which is low relative to other threatened taxa and for freshwater fishes in general. Based on FST values (average over loci = 0.11) and STRUCTURE analyses, we identify three distinct populations in the natural range, one in the Burnett and two distinct populations in the Mary. These analyses also support the hypothesis that the Mary River is the likely source of translocated populations in the Brisbane and North Pine rivers, which agrees with historical published records of a translocation event giving rise to these populations. We were unable to obtain bounded estimates of effective population size, as we have too few genotype combinations, although point estimates were low, ranging from 29 - 129. We recommend that, in order to preserve any local adaptation in the three distinct populations that they be managed separately.
PMCID: PMC4390199  PMID: 25853492
18.  Microsatellite primers for vulnerable and thriving Acacia (Fabaceae) species from Australia’s arid zone1 
Applications in Plant Sciences  2015;3(4):apps.1400121.
Premise of the study:
Microsatellite markers were developed for the common arid Australian shrub Acacia ligulata (Fabaceae) and the threatened overstory trees A. melvillei and A. pendula.
Methods and Results:
DNA sequence data generated by 454 sequencing were used to identify microsatellite nucleotide repeat motifs. Including previously developed primer sets, we report on the development of 10 polymorphic microsatellite loci for each species. Six of these were novel for A. melvillei and A. ligulata, and five were novel for A. pendula, while five more each were transferred from primers developed for related species (A. carneorum and A. loderi). We found three to 17 alleles per locus for each species, with high multilocus genotypic diversity within each of two A. ligulata and A. pendula stands, and one A. melvillei population. A second A. melvillei stand appeared to be monoclonal.
These markers will allow assessment of population genetics, mating systems, and connectedness of populations of these and possibly other arid-zone acacias.
PMCID: PMC4406836  PMID: 25909043
Acacia; Fabaceae; genetic diversity; perennial plant; recruitment failure; sexual and asexual reproduction
19.  Human induced pluripotent stem cell derived erythroblasts can undergo definitive erythropoiesis and co-express gamma and beta globins 
British Journal of Haematology  2014;166(3):435-448.
Human induced pluripotent stem cells (hiPSCs), like embryonic stem cells, are under intense investigation for novel approaches to model disease and for regenerative therapies. Here, we describe the derivation and characterization of hiPSCs from a variety of sources and show that, irrespective of origin or method of reprogramming, hiPSCs can be differentiated on OP9 stroma towards a multi-lineage haemo-endothelial progenitor that can contribute to CD144+ endothelium, CD235a+ erythrocytes (myeloid lineage) and CD19+ B lymphocytes (lymphoid lineage). Within the erythroblast lineage, we were able to demonstrate by single cell analysis (flow cytometry), that hiPSC-derived erythroblasts express alpha globin as previously described, and that a sub-population of these erythroblasts also express haemoglobin F (HbF), indicative of fetal definitive erythropoiesis. More notably however, we were able to demonstrate that a small sub-fraction of HbF positive erythroblasts co-expressed HbA in a highly heterogeneous manner, but analogous to cord blood-derived erythroblasts when cultured using similar methods. Moreover, the HbA expressing erythroblast population could be greatly enhanced (44·0 ± 6·04%) when a defined serum-free approach was employed to isolate a CD31+ CD45+ erythro-myeloid progenitor. These findings demonstrate that hiPSCs may represent a useful alternative to standard sources of erythrocytes (RBCs) for future applications in transfusion medicine.
PMCID: PMC4375519  PMID: 24837254
pluripotent stem cell; erythropoiesis; globin expression
20.  Distinct Mechanisms of Inadequate Erythropoiesis Induced by Tumor Necrosis Factor Alpha or Malarial Pigment 
PLoS ONE  2015;10(3):e0119836.
The role of infection in erythropoietic dysfunction is poorly understood. In children with P. falciparum malaria, the by-product of hemoglobin digestion in infected red cells (hemozoin) is associated with the severity of anemia which is independent of circulating levels of the inflammatory cytokine tumor necrosis alpha (TNF-α). To gain insight into the common and specific effects of TNF-α and hemozoin on erythropoiesis, we studied the gene expression profile of purified primary erythroid cultures exposed to either TNF-α (10ng/ml) or to hemozoin (12.5μg/ml heme units) for 24 hours. Perturbed gene function was assessed using co-annotation of associated gene ontologies and expression of selected genes representative of the profile observed was confirmed by real time PCR (rtPCR). The changes in gene expression induced by each agent were largely distinct; many of the genes significantly modulated by TNF-α were not affected by hemozoin. The genes modulated by TNF-α were significantly enriched for those encoding proteins involved in the control of type 1 interferon signalling and the immune response to viral infection. In contrast, genes induced by hemozoin were significantly enriched for functional roles in regulation of transcription and apoptosis. Further analyses by rtPCR revealed that hemozoin increases expression of transcription factors that form part of the integrated stress response which is accompanied by reduced expression of genes involved in DNA repair. This study confirms that hemozoin induces cellular stress on erythroblasts that is additional to and distinct from responses to inflammatory cytokines and identifies new genes that may be involved in the pathogenesis of severe malarial anemia. More generally the respective transcription profiles highlight the varied mechanisms through which erythropoiesis may be disrupted during infectious disease.
PMCID: PMC4363658  PMID: 25781011
21.  Methylphenidate and Cocaine Self-Administration Produce Distinct Dopamine Terminal Alterations 
Addiction biology  2012;19(2):145-155.
Methylphenidate (MPH) is a commonly abused psychostimulant prescribed for the treatment of attention deficit hyperactivity disorder. MPH has a mechanism of action similar to cocaine (COC) and is commonly characterized as a dopamine transporter (DAT) blocker. While there has been extensive work aimed at understanding dopamine (DA) nerve terminal changes following COC self-administration, very little is known about the effects of MPH self-administration on the DA system. We used fast scan cyclic voltammetry in nucleus accumbens core slices from animals with a five-day self-administration history of 40 injections/day of either MPH (0.56 mg/kg) or COC (1.5 mg/kg) to explore alterations in baseline DA release and uptake kinetics as well as alterations in the interaction of each compound with the DAT. Although MPH and COC have similar behavioral effects, the consequences of self-administration on DA system parameters were found to be divergent. We show that COC self-administration reduced DAT levels and maximal rates of DA uptake, as well as reducing electrically stimulated release, suggesting decreased DA terminal function. In contrast, MPH self-administration increased DAT levels, DA uptake rates, and DA release, suggesting enhanced terminal function, which was supported by findings of increased metabolite/DA tissue content ratios. Tyrosine hydroxylase mRNA, protein and phosphorylation levels were also assessed in both groups. Additionally, COC self-administration reduced COC-induced DAT inhibition, while MPH self-administration increased MPH-induced DAT inhibition, suggesting opposite pharmacodynamic effects of these two drugs. These findings suggest that the factors governing DA system adaptations are more complicated than simple DA uptake blockade.
PMCID: PMC3390453  PMID: 22458761
Cocaine; Dopamine; Dopamine Transporter; Methylphenidate; Nucleus Accumbens; Self-administration
22.  Reduction of the reinforcing effectiveness of cocaine by continuous d-amphetamine treatment in rats: importance of active self-administration during treatment period 
Psychopharmacology  2013;231(5):949-954.
Continuous administration of d-amphetamine has shown promise as a treatment for psychostimulant addiction. In rodent studies, constant infusion of d-amphetamine (5 mg/kg/day) has been shown to reduce cocaine-reinforced responding in the dose range of 0.19 - 0.75 mg/kg/inf.
The present study tested whether these effects were a reflection of pharmacological interactions between d-amphetamine and cocaine or if they resulted from associative learning mechanisms.
After stable progressive ratio (PR) baselines were established, rats were implanted with subcutaneous osmotic mini-pumps filled with either d-amphetamine (5 mg/kg/day - groups 1 and 2) or saline (group 3). During the treatment period, groups 1 and 3 self-administered cocaine at a dose that was previously shown to produce the most robust effects in combination with d-amphetamine treatment (0.19 mg/kg/inf), while group 2 received passive cocaine infusions.
In replication of previous studies, d-amphetamine treatment resulted in a significant (35%) decrease in breakpoints relative to saline controls. By contrast, no reductions in breakpoints were observed in animals that received passive cocaine infusions during the treatment period (group 2).
Active self-administration of cocaine during the treatment period appears to be an important factor in reducing cocaine-reinforced breakpoints. These findings suggest learning mechanisms are involved in the therapeutic effects of continuous d-amphetamine, and pharmacological interaction mechanisms such as cross-tolerance cannot completely account for the observed decreases in cocaine seeking.
PMCID: PMC4327829  PMID: 24146137
Addiction; agonist therapy; breakpoint; cocaine; d-amphetamine; dose; progressive ratio; rat; reinforcing efficacy; self-administration
23.  Therapeutic targeting of the thrombospondin-1 receptor CD47 to treat liver cancer 
CD47 is a signaling receptor for the matricellular protein thrombospondin-1 and a counter-receptor for signal regulatory protein-α (SIRPα) on macrophages. Following its initial discovery in 1992 as a cell surface protein that is over-expressed by ovarian carcinoma, elevated CD47 expression has emerged as a negative prognostic factor for a variety of cancers. CD47 is also a potential therapeutic target based on the ability of CD47 blockade to cause regression of tumors in mice, and a humanized CD47 antibody has recently entered phase I clinical trials. CD47 blockade may control tumor growth by inhibiting thrombospondin-1 signaling or by preventing inhibitory SIRPα signaling in tumor-associated macrophages. A recent publication by Lee et al. (Hepatology 60:179–191, 2014) provides evidence that blocking CD47 signaling specifically depletes tumor-initiating stem cells in hepatocellular carcinoma and implicates cathepsin-S/protease-activated receptor-2 signaling in mediating this therapeutic response.
PMCID: PMC4414848  PMID: 25779848
CD47; Tumor-initiating cells; Cathepsin-S; NFκB; Chemoresistance
24.  Efficient Stereo Image Geometrical Reconstruction at Arbitrary Camera Settings from a Single Calibration 
Camera calibration is central to obtaining a quantitative image-to-physical-space mapping from stereo images acquired in the operating room (OR). A practical challenge for cameras mounted to the operating microscope is maintenance of image calibration as the surgeon’s field-of-view is repeatedly changed (in terms of zoom and focal settings) throughout a procedure. Here, we present an efficient method for sustaining a quantitative image-to-physical space relationship for arbitrary image acquisition settings (S) without the need for camera re-calibration. Essentially, we warp images acquired at S into the equivalent data acquired at a reference setting, S0, using deformation fields obtained with optical flow by successively imaging a simple phantom. Closed-form expressions for the distortions were derived from which 3D surface reconstruction was performed based on the single calibration at S0. The accuracy of the reconstructed surface was 1.05 mm and 0.59 mm along and perpendicular to the optical axis of the operating microscope on average, respectively, for six phantom image pairs, and was 1.26 mm and 0.71 mm for images acquired with a total of 47 arbitrary settings during three clinical cases. The technique is presented in the context of stereovision; however, it may also be applicable to other types of video image acquisitions (e.g., endoscope) because it does not rely on any a priori knowledge about the camera system itself, suggesting the method is likely of considerable significance.
PMCID: PMC4339948  PMID: 25333148
25.  Hexokinase-II positively regulates glucose starvation induced autophagy through TORC1 inhibition 
Molecular cell  2014;53(4):521-533.
Hexokinase-II (HK-II) catalyzes the first step of glycolysis and also functions as a protective molecule, however, its role in protective autophagy has not been determined. Results showed that inhibition of HK-II diminished, while overexpression of HK-II potentiated, autophagy induced by glucose deprivation in cardiomyocyte and non-cardiomyocyte cells. Immunoprecipitation studies revealed that HK-II binds to and inhibits the autophagy suppressor, mTOR complex 1 (TORC1), and this binding was increased by glucose deprivation. The TOS motif, a scaffold sequence responsible for binding TORC1 substrates, is present in HK-II and mutating it blocked its ability to bind to TORC1 and regulate protective autophagy. The transition from glycolysis to autophagy appears to be regulated by a decrease in glucose-6 phosphate. We suggest that HK-II binds TORC1 as a decoy substrate and provides a previously unrecognized mechanism for switching cells from a metabolic economy based on plentiful energy, to one of conservation, under starvation.
PMCID: PMC3943874  PMID: 24462113

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