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1.  Airway Tree Segmentation in Serial Block-Face Cryomicrotome Images of Rat Lungs 
A highly-automated method for the segmentation of airways in serial block-face cryomicrotome images of rat lungs is presented. First, a point inside of the trachea is manually specified. Then, a set of candidate airway centerline points is automatically identified. By utilizing a novel path extraction method, a centerline path between the root of the airway tree and each point in the set of candidate centerline points is obtained. Local disturbances are robustly handled by a novel path extraction approach, which avoids the shortcut problem of standard minimum cost path algorithms. The union of all centerline paths is utilized to generate an initial airway tree structure, and a pruning algorithm is applied to automatically remove erroneous subtrees or branches. Finally, a surface segmentation method is used to obtain the airway lumen.
The method was validated on five image volumes of Sprague-Dawley rats. Based on an expert-generated independent standard, an assessment of airway identification and lumen segmentation performance was conducted. The average of airway detection sensitivity was 87.4% with a 95% confidence interval (CI) of (84.9, 88.6)%. A plot of sensitivity as a function of airway radius is provided. The combined estimate of airway detection specificity was 100% with a 95% CI of (99.4, 100)%. The average number and diameter of terminal airway branches was 1179 and 159 μm, respectively. Segmentation results include airways up to 31 generations. The regression intercept and slope of airway radius measurements derived from final segmentations were estimated to be 7.22 μm and 1.005, respectively. The developed approach enables quantitative studies of physiology and lung diseases in rats, requiring detailed geometric airway models.
doi:10.1109/TBME.2013.2277936
PMCID: PMC3925465  PMID: 23955692
airway segmentation; rat lung; serial block-face imaging cryomicrotome
2.  Pancreatectomy Predicts Improved Survival for Pancreatic Adenocarcinoma: Results of an Instrumental Variable Analysis 
Annals of surgery  2015;261(4):740-745.
Background and Objective
Pancreatic resection is the standard therapy for patients with stage I/II pancreatic ductal adenocarcinoma (PDA), yet many studies demonstrate low rates of resection. The objective of this study is to evaluate whether increasing resection rates would result in an increase in average survival in patients with stage I/II PDA.
Methods
SEER data were analyzed for patients with stage I/II pancreatic head cancers treated from 2004–2009. Pancreatectomy rates were examined within Health Service Areas (HSA) across 18 SEER regions. An instrumental variables (IV) analysis was performed, using HSA rates as an instrument, to determine the impact of increasing resection rates on survival.
Results
Pancreatectomy was performed in 4,322 of the 8,323 patients evaluated with stage I/II PDA (overall resection rate=51.9%). The resection rate across HSAs ranged from an average of 38.6% in the lowest quintile to 67.3% in the highest quintile. Median survival was improved in HSAs with higher resection rates. IV analysis revealed that, for patients whose treatment choices were influenced by the rates of resection in their geographic region, pancreatectomy was associated with a statistically significant increase in overall survival.
Conclusions
When controlling for confounders using IV analysis, pancreatectomy is associated with a statistically significant increase in survival for patients with resectable PDA. Based on these results, if resection rates were to increase in select patients, then average survival would also be expected to increase. It is important that this information be provided to physicians and patients so they can properly weigh the risks and advantages of pancreatectomy as treatment for PDA.
doi:10.1097/SLA.0000000000000796
PMCID: PMC4277740  PMID: 24979599
pancreatic cancer; pancreatectomy; SEER; instrumental variable analysis; epidemiology; Health Services Area
3.  Structure-Guided Rescaffolding of Selective Antagonists of BCL-XL 
ACS Medicinal Chemistry Letters  2014;5(6):662-667.
Because of the promise of BCL-2 antagonists in combating chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL), interest in additional selective antagonists of antiapoptotic proteins has grown. Beginning with a series of selective, potent BCL-XL antagonists containing an undesirable hydrazone functionality, in silico design and X-ray crystallography were utilized to develop alternative scaffolds that retained the selectivity and potency of the starting compounds.
doi:10.1021/ml500030p
PMCID: PMC4060937  PMID: 24944740
BCL-XL; BCL-2; apoptosis; cancer
4.  Digital imaging analysis to assess scar phenotype 
In order to understand the link between the genetic background of patients and wound clinical outcomes, it is critical to have a reliable method to assess the phenotypic characteristics of healed wounds. In this study, we present a novel imaging method that provides reproducible, sensitive and unbiased assessments of post-surgical scarring. We used this approach to investigate the possibility that genetic variants in orofacial clefting genes are associated with suboptimal healing. Red-green-blue (RGB) digital images of post-surgical scars of 68 patients, following unilateral cleft lip repair, were captured using the 3dMD image system. Morphometric and colorimetric data of repaired regions of the philtrum and upper lip were acquired using ImageJ software and the unaffected contralateral regions were used as patient-specific controls. Repeatability of the method was high with interclass correlation coefficient score > 0.8. This method detected a very significant difference in all three colors, and for all patients, between the scarred and the contralateral unaffected philtrum (P ranging from 1.20−05 to 1.95−14). Physicians’ clinical outcome ratings from the same images showed high inter-observer variability (overall Pearson coefficient = 0.49) as well as low correlation with digital image analysis results. Finally, we identified genetic variants in TGFB3 and ARHGAP29 associated with suboptimal healing outcome.
doi:10.1111/wrr.12141
PMCID: PMC4411947  PMID: 24635173
wound healing; cleft palate; genes; genotype-phenotype; Tgfb3; Arhgap29; scar; imaging; digital
5.  Environmental DNA (eDNA) Sampling Improves Occurrence and Detection Estimates of Invasive Burmese Pythons 
PLoS ONE  2015;10(4):e0121655.
Environmental DNA (eDNA) methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR) for the Burmese python (Python molurus bivittatus), Northern African python (P. sebae), boa constrictor (Boa constrictor), and the green (Eunectes murinus) and yellow anaconda (E. notaeus). Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive constrictors. Generic sampling design and terminology are proposed to standardize and clarify interpretations of eDNA-based occupancy models.
doi:10.1371/journal.pone.0121655
PMCID: PMC4398459  PMID: 25874630
6.  A Comprehensive Assessment of Transfusion in Elective Pancreatectomy: Risk Factors and Complications 
Background
Specific data are needed regarding the impact of transfusion on operative complications in pancreatectomy. The objectives of this study were to determine risk factors for transfusion and to evaluate the potential association between transfusion and operative complications in elective pancreatectomy procedures.
Study Design
We reviewed our institution’s pancreatectomy and ACS-NSQIP databases. Multivariate analysis was used to determine clinicopathologic risk factors predictive of transfusion, and then a transfusion propensity score was developed to evaluate the impact of transfusion on post-pancreatectomy complications.
Results
Of the 173 patients who were treated from September 2007 to September 2011, 78 patients (45 %) were transfused≥1 unit of blood (median, 3.0 units; range, 1–55). Risk factors for transfusion included increasing Body Mass Index (BMI), smoking, increasing mortality risk score, preoperative anemia, intraoperative blood loss, and benign pathology. After controlling for these risk factors using a transfusion propensity score, transfusion was an independent predictor of increased complications, infectious complications, and hospital costs.
Conclusions
Multiple factors are predictive of transfusion in pancreatectomy, including increasing BMI and smoking. When controlling for transfusion propensity based on these risk factors, RBC transfusion is associated with worse operative outcomes including infectious complications. Development of protocols and strategies to minimize unnecessary transfusion in pancreatectomy are justified.
doi:10.1007/s11605-013-2169-6
PMCID: PMC4388036  PMID: 23423430
Transfusion; Pancreatectomy; Complications; Pancreatic cancer; Pancreatitis
8.  Homing of invasive Burmese pythons in South Florida: evidence for map and compass senses in snakes 
Biology Letters  2014;10(3):20140040.
Navigational ability is a critical component of an animal's spatial ecology and may influence the invasive potential of species. Burmese pythons (Python molurus bivittatus) are apex predators invasive to South Florida. We tracked the movements of 12 adult Burmese pythons in Everglades National Park, six of which were translocated 21–36 km from their capture locations. Translocated snakes oriented movement homeward relative to the capture location, and five of six snakes returned to within 5 km of the original capture location. Translocated snakes moved straighter and faster than control snakes and displayed movement path structure indicative of oriented movement. This study provides evidence that Burmese pythons have navigational map and compass senses and has implications for predictions of spatial spread and impacts as well as our understanding of reptile cognitive abilities.
doi:10.1098/rsbl.2014.0040
PMCID: PMC3982438  PMID: 24647727
invasive species; movement; navigation; snake
9.  SIRT6 Minor Allele Genotype Is Associated with >5-Year Decrease in Lifespan in an Aged Cohort 
PLoS ONE  2014;9(12):e115616.
Aging is a natural process involving complex interplay between environment, metabolism, and genes. Sirtuin genes and their downstream targets have been associated with lifespan in numerous organisms from nematodes to humans. Several target proteins of the sirtuin genes are key sensors and/or effectors of oxidative stress pathways including FOXO3, SOD3, and AKT1. To examine the relationship between single nucleotide polymorphisms (SNP) at candidate genes in these pathways and human lifespan, we performed a molecular epidemiologic study of an elderly cohort (≥65 years old.). Using age at death as a continuous outcome variable and assuming a co-dominant genetic model within the framework of multi-variable linear regression analysis, the genotype-specific adjusted mean age at death was estimated for individual SNP genotypes while controlling for age-related risk factors including smoking, body mass index, alcohol consumption and co-morbidity. Significant associations were detected between human lifespan and SNPs in genes SIRT3, SIRT5, SIRT6, FOXO3 and SOD3. Individuals with either the CC or CT genotype at rs107251 within SIRT6 displayed >5-year mean survival advantages compared to the TT genotype (5.5 and 5.9 years, respectively; q-value  = 0.012). Other SNPs revealed genotype-specific mean survival advantages ranging from 0.5 to 1.6 years. Gender also modified the effect of SNPs in SIRT3, SIRT5 and AKT1 on lifespan. Our novel findings highlight the impact of sirtuins and sirtuin-related genotypes on lifespan, the importance of evaluating gender and the advantage of using age as a continuous variable in analyses to report mean age at death.
doi:10.1371/journal.pone.0115616
PMCID: PMC4277407  PMID: 25541994
10.  Intestinal stem cells remain viable after prolonged tissue storage 
Cell and tissue research  2013;354(2):10.1007/s00441-013-1674-y.
Intestinal stem cells (ISCs) are responsible for renewal of the epithelium both during normal homeostasis and following injury. As such they have significant therapeutic potential. However, it is unknown whether ISCs can survive tissue storage. We hypothesized that, although the majority of epithelial cells may die, ISCs would remain viable for at least 24 h at 4°C. To explore this hypothesis, jejuni of C57Bl6/J or Lgr5-LacZ mice were removed and either processed immediately or placed in phosphate buffered saline (PBS) at 4°C. Delayed isolations of epithelia were performed after 24, 30, or 48 h storage. At the light microscope level, despite extensive apoptosis of villus epithelial cells, small intestinal crypts remained morphologically intact through 30 h and ISCs were identifiable via Lgr5-LacZ positivity. Electron microscopy showed that ISCs retain high integrity through 24 h. When assessed by flow cytometry, ISCs were more resistant to degeneration than the rest of the epithelium, including neighboring Paneth cells, with higher viability across all time points. Culture of isolated crypts showed no loss of capacity to form complex enteroids after 24 h tissue storage, with efficiencies after 7 days of culture remaining above 80%. By 30 h storage, efficiencies declined but budding capability was retained. We conclude that, with delay in isolation, ISCs remain viable and retain their proliferative capacity. In contrast, the remainder of the epithelium, including the Paneth cells, exhibits degeneration and programmed cell death. If these findings are recapitulated with human tissue, storage at 4°C may offer a valuable temporal window for harvest of crypts or ISCs for therapeutic application.
doi:10.1007/s00441-013-1674-y
PMCID: PMC3812345  PMID: 23820734
Intestinal stem cells; light and electron microscopy; viability; resistance to storage; enteroid culture
11.  Crystal structure of PfRh5, an essential P. falciparum ligand for invasion of human erythrocytes 
eLife  null;3:e04187.
Plasmodium falciparum causes the most severe form of malaria in humans and is responsible for over 700,000 deaths annually. It is an obligate intracellular parasite and invades erythrocytes where it grows in a relatively protected niche. Invasion of erythrocytes is essential for parasite survival and this involves interplay of multiple protein–protein interactions. One of the most important interactions is binding of parasite invasion ligand families EBLs and PfRhs to host receptors on the surface of erythrocytes. PfRh5 is the only essential invasion ligand within the PfRh family and is an important vaccine candidate. PfRh5 binds the host receptor basigin. In this study, we have determined the crystal structure of PfRh5 using diffraction data to 2.18 Å resolution. PfRh5 exhibits a novel fold, comprising nine mostly anti-parallel α-helices encasing an N-terminal β-hairpin, with the overall shape being an elliptical disk. This is the first three-dimensional structure determined for the PfRh family of proteins.
DOI: http://dx.doi.org/10.7554/eLife.04187.001
eLife digest
Malaria is a disease caused by a single-celled parasite called Plasmodium, which is transmitted between humans by mosquitoes. It is estimated that 3.4 billion people worldwide live in regions where they are at risk of malaria, and malaria infections cause hundreds of thousands of deaths each year.
When a mosquito carrying Plasmodium parasites in its salivary glands bites a human, the parasite is injected into the person's bloodstream with the mosquito's saliva. The parasite then travels through the bloodstream to the liver, where it infects liver cells and multiplies without causing any symptoms for up to 4 weeks. After this period, the parasites break out of each infected liver cell, re-enter the bloodstream, and begin infecting red blood cells. When another mosquito bites the infected individual to feed on their blood, the parasite moves into the mosquito with the red blood cells and the cycle of infection continues.
While prevention and control measures have dramatically reduced the incidence of malaria in some countries, many people in African countries—and especially young children—die from malaria each year. Finding ways to reduce the spread of Plasmodium parasites, and in particular Plasmodium falciparum (which is responsible for the deadliest type of malaria), is critical for the global effort to control and eliminate this disease. As such, many researchers are trying to gain a better understanding of how the parasite both invades host cells and evades the immune system.
In this study, Chen et al. reveal the high-resolution structure of PfRh5, the protein from Plasmodium falciparum that forms a complex with other proteins to allow the parasite to bind to, and invade, red blood cells. This is one of the first three-dimensional structures that have been uncovered for this family of proteins—and reveals that the PfRh5 protein is shaped like an elliptical disk. Solving the structure of PfRh5 is the first step in understanding the role of this protein, and the other protein components, involved in invading red blood cells. These proteins are molecules that could potentially be used to vaccinate people against malaria, and understanding these proteins' functions will help efforts to design vaccines to prevent malarial disease.
DOI: http://dx.doi.org/10.7554/eLife.04187.002
doi:10.7554/eLife.04187
PMCID: PMC4356141  PMID: 25296023
malaria; plasmodium; erythrocyte; invasion; human
12.  Evaluation of an intervention aimed at improving voluntary incident reporting in hospitals 
Quality & Safety in Health Care  2007;16(3):169-175.
Objectives
To assess the effectiveness of an intervention package comprising intense education, a range of reporting options, changes in report management and enhanced feedback, in order to improve incident‐reporting rates and change the types of incidents reported.
Design, setting and participants
Non‐equivalent group controlled clinical trial involving medical and nursing staff working in 10 intervention and 10 control units in four major cities and two regional hospitals in South Australia.
Main outcome measures
Comparison of reporting rates by type of unit, profession, location of hospital, type of incident reported and reporting mechanism between baseline and study periods in control and intervention units.
Results
The intervention resulted in significant improvement in reporting in inpatient areas (additional 60.3 reports/10 000 occupied bed days (OBDs); 95% CI 23.8 to 96.8, p<0.001) and in emergency departments (EDs) (additional 39.5 reports/10 000 ED attendances; 95% CI 17.0 to 62.0, p<0.001). More reports were generated (a) by doctors in EDs (additional 9.5 reports/10 000 ED attendances; 95% CI 2.2 to 16.8, p = 0.001); (b) by nurses in inpatient areas (additional 59.0 reports/10 000 OBDs; 95% CI 23.9 to 94.1, p<0.001) and (c) anonymously (additional 20.2 reports/10 000 OBDs and ED attendances combined; 95% CI 12.6 to 27.8, p<0.001). Compared with control units, the study resulted in more documentation, clinical management and aggression‐related incidents in intervention units. In intervention units, more reports were submitted on one‐page forms than via the call centre (1005 vs 264 reports, respectively).
Conclusions
A greater variety and number of incidents were reported by the intervention units during the study, with improved reporting by doctors from a low baseline. However, there was considerable heterogeneity between reporting rates in different types of units.
doi:10.1136/qshc.2006.019349
PMCID: PMC2465009  PMID: 17545341
13.  Structure of the analgesic μ-conotoxin KIIIA and effects on structure and function of disulfide deletion†,‡ 
Biochemistry  2009;48(6):1210-1219.
The μ-conotoxin μ-KIIIA, from Conus kinoshitai, blocks mammalian neuronal voltage-gated sodium channels (VGSCs) and is a potent analgesic following systemic administration in mice. We have determined its solution structure using NMR spectroscopy. Key residues identified previously as being important for activity against VGSCs (Lys7, Trp8, Arg10, Asp11, His12 and Arg14) all reside on an α-helix with the exception of Arg14. To further probe structure-activity relationships of this toxin against VGSC subtypes, we have characterised the analogue μ-KIIIA[C1A,C9A], in which the Cys residues involved in one of the three disulfides in μ-KIIIA were replaced with Ala. Its structure is quite similar to that of μ-KIIIA, indicating that the Cys1-Cys9 disulfide bond could be removed without any significant distortion of the α-helix bearing the key residues. Consistent with this, μ-KIIIA[C1A,C9A] retained activity against VGSCs, with its rank order of potency being essentially the same as that of μ-KIIIA, namely, NaV1.2 > NaV1.4 > NaV1.7 ≥ NaV1.1 > NaV1.3 > NaV1.5. Kinetics of block were obtained for NaV1.2, NaV1.4 and NaV1.7, and in each case both kon and koff values of μ-KIIIA[C1A,C9A] were larger than those of μ-KIIIA. Our results show that the key residues for VGSC binding lie mostly on an α-helix and that the first disulfide bond can be removed without significantly affecting the structure of this helix, although the modification accelerates the on- and off-rates of the peptide against all tested VGSC subtypes. These findings lay the groundwork for the design of minimized peptides and helical mimetics as novel analgesics.
doi:10.1021/bi801998a
PMCID: PMC4153535  PMID: 19170536
14.  STRUCTURE-GUIDED RATIONAL DESIGN OF α/β-PEPTIDE FOLDAMERS WITH HIGH AFFINITY FOR BCL-2 FAMILY PRO-SURVIVAL PROTEINS 
We have used computational methods to improve the affinity of a foldamer ligand for its target protein. The effort began with a previously reported α/β-peptide based on the BH3 domain of the pro-apoptotic protein Puma; this foldamer binds tightly to Bcl-xL but weakly to Mcl-1. The crystal structure of the Puma-derived α/β-peptide complexed to Bcl-xL was used as the basis for computational design of variants intended to display improved binding to Mcl-1. Molecular modelling suggested modification of three α residues within the original α/β backbone. Individually, each substitution caused only a modest (4- to 15-fold) gain in affinity; however, together the three substitutions led to a 250-fold increase in binding to Mcl-1. These modifications had very little effect on affinity for Bcl-xL. Crystal structures of a number of the new α/β-peptides bound to either Mcl-1 or Bcl-xL validated the selection of each substitution. Overall, our findings demonstrate that structure-guided rational design can be used to improve affinity and alter partner selectivity of peptidic ligands with unnatural backbones that bind to specific protein partners.
doi:10.1002/cbic.201300351
PMCID: PMC3970217  PMID: 23929624
apoptosis; BH3 domain; Mcl-1; foldamer; peptides; peptidomimetics; peptide design
15.  Low-Dose Radiation-Induced Enhancement of Thymic Lymphomagenesis in Lck-Bax Mice is Dependent on LET and Gender 
Radiation research  2013;180(2):10.1667/RR3293.1.
The hypothesis that mitochondrial dysfunction and increased superoxide levels in thymocytes over expressing Bax (Lck-Bax1 and Lck-Bax38&1) contributes to lymphomagenesis after low-dose radiation was tested. Lck-Bax1 single-transgenic and Lck-Bax38&1 double-transgenic mice were exposed to single whole-body doses of 10 or 100 cGy of 137Cs or iron ions (1,000 MeV/n, 150 keV/μm) or silicon ions (300 MeV/n, 67 keV/μm). A 10 cGy dose of 137Cs significantly increased the incidence and onset of thymic lymphomas in female Lck-Bax1 mice. In Lck-Bax38&1 mice, a 100 cGy dose of high-LET iron ions caused a significant dose dependent acceleration of lymphomagenesis in both males and females that was not seen with silicon ions. To determine the contribution of mitochondrial oxidative metabolism, Lck-Bax38&1 over expressing mice were crossed with knockouts of the mitochondrial protein deacetylase, Sirtuin 3 (Sirt3), which regulates superoxide metabolism. Sirt3−/−/Lck-Bax38&1 mice demonstrated significant increases in thymocyte superoxide levels and acceleration of lymphomagenesis (P < 0.001). These results show that lymphomagenesis in Bax over expressing animals is enhanced by radiation exposure in both an LET and gender dependent fashion. These findings support the hypothesis that mitochondrial dysfunction leads to increased superoxide levels and accelerates lymphomagenesis in Lck-Bax transgenic mice.
doi:10.1667/RR3293.1
PMCID: PMC3821998  PMID: 23819597
16.  Treatment of resistant metastatic melanoma using sequential epigenetic therapy (decitabine and panobinostat) combined with chemotherapy (temozolomide) 
Purpose
To explore the safety and tolerability of combining two epigenetic drugs: decitabine (a DNA methyltransferase inhibitor) and panobinostat (a histone deacetylase inhibitor), with chemotherapy with temozolomide (an alkylating agent). The purpose of such combination is to evaluate the use of epigenetic priming to overcome resistance of melanoma to chemotherapy.
Methods
A Phase I clinical trial enrolling patients aged 18 years or older, with recurrent or unresectable stage III or IV melanoma of any site. This trial was conducted with full Institutional Review Board approval and was registered with the National Institutes of Health under the clinicaltrials.gov identifier NCT00925132. Patients were treated with subcutaneous decitabine 0.1 or 0.2 mg/kg three times weekly for 2 weeks (starting on day 1), in combination with oral panobinostat 10, 20, or 30 mg every 96 h (starting on day 8), and oral temozolomide 150 mg/m2/day on days 9 through 13. In cycle 2, temozolomide was increased to 200 mg/m2/day if neutropenia or thrombocytopenia had not occurred. Each cycle lasted 6 weeks, and patients could receive up to six cycles. Patients who did not demonstrate disease progression were eligible to enter a maintenance protocol with combination of weekly panobinostat and thrice-weekly decitabine until tumor progression, unacceptable toxicity, or withdrawal of consent.
Results
Twenty patients were initially enrolled, with 17 receiving treatment. The median age was 56 years. Eleven (65 %) were male, and 6 (35 %) were female. Eleven (64.7 %) had cutaneous melanoma, 4 (23.5 %) had ocular melanoma, and 2 (11.8 %) had mucosal melanoma. All patients received at least one treatment cycle and were evaluable for toxicity. Patients received a median of two 6-week treatment cycles (range 1–6). None of the patients experienced DLT. MTD was not reached. Adverse events attributed to treatment included grade 3 lymphopenia (24 %), anemia (12 %), neutropenia (12 %), and fatigue (12 %), as well as grade 2 leukopenia (30 %), neutropenia (23 %), nausea (23 %), and lymphopenia (18 %). The most common reason for study discontinuation was disease progression.
Conclusions
This triple agent of dual epigenetic therapy in combination with traditional chemotherapy was generally well tolerated by the cohort and appeared safe to be continued in a Phase II trial. No DLTs were observed, and MTD was not reached.
doi:10.1007/s00280-014-2501-1
PMCID: PMC4175037  PMID: 25062770
Melanoma; Epigenetics; Epigenetic priming; Resistance; Hypomethylation; Histone deacetylation
17.  Local mapping of detector response for reliable quantum state estimation 
Nature Communications  2014;5:4332.
Improved measurement techniques are central to technological development and foundational scientific exploration. Quantum physics relies on detectors sensitive to non-classical features of systems, enabling precise tests of physical laws and quantum-enhanced technologies including precision measurement and secure communications. Accurate detector response calibration for quantum-scale inputs is key to future research and development in these cognate areas. To address this requirement, quantum detector tomography has been recently introduced. However, this technique becomes increasingly challenging as the complexity of the detector response and input space grow in a number of measurement outcomes and required probe states, leading to further demands on experiments and data analysis. Here we present an experimental implementation of a versatile, alternative characterization technique to address many-outcome quantum detectors that limits the input calibration region and does not involve numerical post processing. To demonstrate the applicability of this approach, the calibrated detector is subsequently used to estimate non-classical photon number states.
The successful realization of quantum information protocols relies on characterization of quantum states and measurements. Here, Cooper et al. experimentally demonstrate a technique enabling calibration of a detector with a sizeable number of outcomes using a limited amount of resources.
doi:10.1038/ncomms5332
PMCID: PMC4104434  PMID: 25019300
18.  Ketogenic Diets Enhance Oxidative Stress and Radio-Chemo-Therapy Responses in Lung Cancer Xenografts 
Purpose
Ketogenic diets (KDs) are high in fat and low in carbohydrates as well as protein which forces cells to rely on lipid oxidation and mitochondrial respiration rather than glycolysis for energy metabolism. Cancer cells (relative to normal cells) are believed to exist in a state of chronic oxidative stress mediated by mitochondrial metabolism. The current study tests the hypothesis that KDs enhance radio-chemo-therapy responses in lung cancer xenografts by enhancing oxidative stress.
Experimental Design
Mice bearing NCI-H292 and A549 lung cancer xenografts were fed a KD (KetoCal® 4:1 fats: proteins+carbohydrates) and treated with either conventionally fractionated (1.8-2 Gy) or hypofractionated (6 Gy) radiation as well as conventionally fractionated radiation combined with carboplatin. Mice weights and tumor size were monitored. Tumors were assessed for immuno-reactive 4-hydroxy-2-nonenal-(4HNE) modified proteins as a marker of oxidative stress as well as PCNA and γH2AX as indices of proliferation and DNA damage, respectively.
Results
The KD combined with radiation resulted in slower tumor growth in both NCI-H292 and A549 xenografts (p<0.05), relative to radiation alone. The KD also slowed tumor growth when combined with carboplatin and radiation, relative to control. Tumors from animals fed a KD in combination with radiation demonstrated increases in oxidative damage mediated by lipid peroxidation as determined by 4HNE-modified proteins as well as decreased proliferation as assessed by decreased immunoreactive PCNA.
Conclusions
These results show that a KD enhances radio-chemo-therapy responses in lung cancer xenografts by a mechanism that may involve increased oxidative stress.
doi:10.1158/1078-0432.CCR-12-0287
PMCID: PMC3954599  PMID: 23743570
19.  REGULATION OF PANCREATIC CANCER GROWTH BY SUPEROXIDE 
Molecular carcinogenesis  2012;52(7):555-567.
K-ras mutations have been identified in up to 95% of pancreatic cancers, implying their critical role in the molecular pathogenesis. Expression of K-ras oncogene in an immortalized human pancreatic ductal epithelial cell line, originally derived from normal pancreas (H6c7), induced the formation of carcinoma in mice. We hypothesized that K-ras oncogene correlates with increased non-mitochondrial-generated superoxide (O2·−), which could be involved in regulating cell growth contributing to tumor progression. In the H6c7 cell line and its derivatives, H6c7er-Kras+ (H6c7 cells expressing K-ras oncogene), and H6c7eR-KrasT (tumorigenic H6c7 cells expressing K-ras oncogene), there was an increase in hydroethidine fluorescence in cell lines that express K-ras. Western blots and activity assays for the antioxidant enzymes that detoxify O2·− were similar in these cell lines suggesting that the increase in hydroethidine fluorescence was not due to decreased antioxidant capacity. To determine a possible non-mitochondrial source of the increased levels of O2·−, Western analysis demonstrated the absence of NADPH oxidase-2 (NOX2) in H6c7 cells but present in the H6c7 cell lines expressing K-ras and other pancreatic cancer cell lines. Inhibition of NOX2 decreased hydroethidine fluorescence and clonogenic survival. Furthermore, in the cell lines with the K-ras oncogene, overexpression of superoxide dismutases, that detoxify non-mitochondrial sources of O2·−, and treatment with the small molecule O2·− scavenger Tempol, also decreased hydroethidine fluorescence, inhibited clonogenic survival and inhibited growth of tumor xenografts. Thus, O2·− produced by NOX2 in pancreatic cancer cells with K-ras, may regulate pancreatic cancer cell growth.
doi:10.1002/mc.21891
PMCID: PMC3375391  PMID: 22392697
20.  A Phase I Trial of Immunostimulatory CpG 7909 Oligodeoxynucleotide and 90Yttrium Ibritumomab Tiuxetan Radioimmunotherapy for Relapsed B-cell Non-Hodgkin Lymphoma 
American journal of hematology  2013;88(7):589-593.
Radioimmunotherapy (RIT) for relapsed indolent non-Hodgkin lymphoma produces overall response rates (ORR) of 80% with mostly partial remissions. Synthetic CpG oligonucleotides change the phenotype of malignant B-cells, are immunostimulatory, and can produce responses when injected intratumorally and combined with conventional radiation. In this phase I trial we tested systemic administration of both CpG and RIT. Eligible patients had biopsy-proven previously treated CD20+ B-cell NHL and met criteria for RIT. Patients received rituximab 250 mg/m2 days 1,8, and 15; 111In-ibritumomab tiuxetan days 1, 8; CpG 7909 days 6, 13, 20, 27; and 0.4 mCi/kg of 90Y-ibritumomab tiuxetan day 15. The doses of CpG 7909 tested were 0.08, 0.16, 0.32 (six patients each) and 0.48 mg/kg (12 patients) IV over 2 hours without dose limiting toxicity. The ORR was 93% (28/30) with 63% (19/30) complete remission (CR); median progression free survival of 42.7 months (95% CI 18-NR); and median duration of response (DR) of 35 months (4.6-76+). Correlative studies demonstrated a decrease in IL10 and TNFα, and an increase in IL1β, in response to therapy. CpG 7909 at a dose of 0.48 mg/kg is safe with standard RIT and produces a high CR rate and long DR; these results warrant confirmation.
doi:10.1002/ajh.23460
PMCID: PMC3951424  PMID: 23619698
lymphoma; radioimmunotherapy; rituximab; ibritumomab tiuxetan; CpG 7909
21.  Identification of PLP2 and RAB5C as novel TPD52 binding partners through yeast two-hybrid screening 
Molecular biology reports  2014;41(7):4565-4572.
Tumor protein D52 (TPD52) is overexpressed in different cancers, but its molecular functions are poorly defined. A large, low-stringency yeast two-hybrid screen using full-length TPD52 bait identified known partners (TPD52, TPD52L1, TPD52L2, MAL2) and four other preys that reproducibly bound TPD52 and TPD52L1 baits (PLP2, RAB5C, GOLGA5, YIF1A). PLP2 and RAB5 interactions with TPD52 were confirmed in pull down assays, with interaction domain mapping experiments indicating that both proteins interact with a novel binding region of TPD52. This study provides insights into TPD52 functions, and ways to maximise the efficiency of lowstringency yeast two-hybrid screens.
doi:10.1007/s11033-014-3327-y
PMCID: PMC4069224  PMID: 24604726
TPD52; Yeast two-hybrid; Pull down; PLP2; RAB5
22.  Inhibition of Plasmepsin V Activity Demonstrates Its Essential Role in Protein Export, PfEMP1 Display, and Survival of Malaria Parasites 
PLoS Biology  2014;12(7):e1001897.
A small molecule inhibitor of the malarial protease Plasmepsin V impairs protein export and cellular remodeling, reducing parasite survival in human erythrocytes.
The malaria parasite Plasmodium falciparum exports several hundred proteins into the infected erythrocyte that are involved in cellular remodeling and severe virulence. The export mechanism involves the Plasmodium export element (PEXEL), which is a cleavage site for the parasite protease, Plasmepsin V (PMV). The PMV gene is refractory to deletion, suggesting it is essential, but definitive proof is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks the activity of PMV isolated from P. falciparum and Plasmodium vivax. Assessment of PMV activity in P. falciparum revealed PEXEL cleavage occurs cotranslationaly, similar to signal peptidase. Treatment of P. falciparum–infected erythrocytes with the inhibitor caused dose-dependent inhibition of PEXEL processing as well as protein export, including impaired display of the major virulence adhesin, PfEMP1, on the erythrocyte surface, and cytoadherence. The inhibitor killed parasites at the trophozoite stage and knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of PMV increased resistance. This provides the first direct evidence that PMV activity is essential for protein export in Plasmodium spp. and for parasite survival in human erythrocytes and validates PMV as an antimalarial drug target.
Author Summary
To survive within human red blood cells, malaria parasites must export a catalog of proteins that remodel the host cell and its surface. This enables parasites to acquire nutrients from outside the cell and to modify the cell surface in order to evade host defenses. Protein export involves proteolytic cleavage of the Plasmodium Export Element (PEXEL) by the aspartyl protease Plasmepsin V. We report here the development of a small molecule inhibitor that closely mimics the natural PEXEL substrate and blocks the activity of Plasmepsin V from the malarial parasites Plasmodium falciparum and Plasmodium vivax. The inhibitor impairs export and cellular remodeling and kills P. falciparum at the ring-trophozoite transition, providing direct evidence that Plasmepsin V activity is essential for export of PEXEL proteins and parasite survival within the host. These findings validate Plasmepsin V as a highly conserved antimalarial drug target.
doi:10.1371/journal.pbio.1001897
PMCID: PMC4077696  PMID: 24983235
23.  Spatiotemporal modeling of irregularly spaced Aerosol Optical Depth data 
Many advancements have been introduced to tackle spatial and temporal structures in data. When the spatial and/or temporal domains are relatively large, assumptions must be made to account for the sheer size of the data. The large data size, coupled with realities that come with observational data, make it difficult for all of these assumptions to be met. In particular, air quality data are very sparse across geographic space and time, due to a limited air pollution monitoring network. These “missing” values make it diffcult to incorporate most dimension reduction techniques developed for high-dimensional spatiotemporal data. This article examines aerosol optical depth (AOD), an indirect measure of radiative forcing, and air quality. The spatiotemporal distribution of AOD can be influenced by both natural (e.g., meteorological conditions) and anthropogenic factors (e.g., emission from industries and transport). After accounting for natural factors influencing AOD, we examine the spatiotemporal relationship in the remaining human influenced portion of AOD. The presented data cover a portion of India surrounding New Delhi from 2000 – 2006. The proposed method is demonstrated showing how it can handle the large spatiotemporal structure containing so much missing data for both meteorologic conditions and AOD over time and space.
doi:10.1007/s10651-012-0221-4
PMCID: PMC3901316  PMID: 24470786
air quality; AOD; autoregressive; Bayesian; spatial correlation; temporal correlation
24.  Geocoding accuracy and the recovery of relationships between environmental exposures and health 
Background
This research develops methods for determining the effect of geocoding quality on relationships between environmental exposures and health. The likelihood of detecting an existing relationship – statistical power – between measures of environmental exposures and health depends not only on the strength of the relationship but also on the level of positional accuracy and completeness of the geocodes from which the measures of environmental exposure are made. This paper summarizes the results of simulation studies conducted to examine the impact of inaccuracies of geocoded addresses generated by three types of geocoding processes: a) addresses located on orthophoto maps, b) addresses matched to TIGER files (U.S Census or their derivative street files); and, c) addresses from E-911 geocodes (developed by local authorities for emergency dispatch purposes).
Results
The simulated odds of disease using exposures modelled from the highest quality geocodes could be sufficiently recovered using other, more commonly used, geocoding processes such as TIGER and E-911; however, the strength of the odds relationship between disease exposures modelled at geocodes generally declined with decreasing geocoding accuracy.
Conclusion
Although these specific results cannot be generalized to new situations, the methods used to determine the sensitivity of results can be used in new situations. Estimated measures of positional accuracy must be used in the interpretation of results of analyses that investigate relationships between health outcomes and exposures measured at residential locations. Analyses similar to those employed in this paper can be used to validate interpretation of results from empirical analyses that use geocoded locations with estimated measures of positional accuracy.
doi:10.1186/1476-072X-7-13
PMCID: PMC2359739  PMID: 18387189
25.  How insulin engages its primary binding site on the insulin receptor 
Nature  2013;493(7431):241-245.
Insulin receptor signalling has a central role in mammalian biology, regulating cellular metabolism, growth, division, differentiation and survival1,2. Insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus and the onset of Alzheimer’s disease3; aberrant signalling occurs in diverse cancers, exacerbated by crosstalk with the homologous type 1 insulin-like growth factor receptor (IGF1R)4. Despite more than three decades of investigation, the three-dimensional structure of the insulin–insulin receptor complex has proved elusive, confounded by the complexity of producing the receptor protein. Here we present the first view, to our knowledge, of the interaction of insulin with its primary binding site on the insulin receptor, on the basis of four crystal structures of insulin bound to truncated insulin receptor constructs. The direct interaction of insulin with the first leucine-rich-repeat domain (L1) of insulin receptor is seen to be sparse, the hormone instead engaging the insulin receptor carboxy-terminal α-chain (αCT) segment, which is itself remodelled on the face of L1 upon insulin binding. Contact between insulin and L1 is restricted to insulin B-chain residues. The αCT segment displaces the B-chain C-terminal β-strand away from the hormone core, revealing the mechanism of a long-proposed conformational switch in insulin upon receptor engagement. This mode of hormone–receptor recognition is novel within the broader family of receptor tyrosine kinases5. We support these findings by photo-crosslinking data that place the suggested interactions into the context of the holoreceptor and by isothermal titration calorimetry data that dissect the hormone–insulin receptor interface. Together, our findings provide an explanation for a wealth of biochemical data from the insulin receptor and IGF1R systems relevant to the design of therapeutic insulin analogues.
doi:10.1038/nature11781
PMCID: PMC3793637  PMID: 23302862

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