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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.
PMCID: PMC3925465  PMID: 23955692
airway segmentation; rat lung; serial block-face imaging cryomicrotome
2.  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.
PMCID: PMC4277407  PMID: 25541994
3.  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.
PMCID: PMC3812345  PMID: 23820734
Intestinal stem cells; light and electron microscopy; viability; resistance to storage; enteroid culture
4.  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.
PMCID: PMC4153535  PMID: 19170536
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.
PMCID: PMC3970217  PMID: 23929624
apoptosis; BH3 domain; Mcl-1; foldamer; peptides; peptidomimetics; peptide design
6.  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.
PMCID: PMC3821998  PMID: 23819597
7.  Treatment of resistant metastatic melanoma using sequential epigenetic therapy (decitabine and panobinostat) combined with chemotherapy (temozolomide) 
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.
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 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.
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.
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.
PMCID: PMC4175037  PMID: 25062770
Melanoma; Epigenetics; Epigenetic priming; Resistance; Hypomethylation; Histone deacetylation
8.  Evaluation of an intervention aimed at improving voluntary incident reporting in hospitals 
Quality & Safety in Health Care  2007;16(3):169-175.
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.
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).
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.
PMCID: PMC2465009  PMID: 17545341
9.  Ketogenic Diets Enhance Oxidative Stress and Radio-Chemo-Therapy Responses in Lung Cancer Xenografts 
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.
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.
These results show that a KD enhances radio-chemo-therapy responses in lung cancer xenografts by a mechanism that may involve increased oxidative stress.
PMCID: PMC3954599  PMID: 23743570
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.
PMCID: PMC3375391  PMID: 22392697
11.  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.
PMCID: PMC3951424  PMID: 23619698
lymphoma; radioimmunotherapy; rituximab; ibritumomab tiuxetan; CpG 7909
12.  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.
PMCID: PMC4069224  PMID: 24604726
TPD52; Yeast two-hybrid; Pull down; PLP2; RAB5
13.  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.
PMCID: PMC4077696  PMID: 24983235
14.  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.
PMCID: PMC3901316  PMID: 24470786
air quality; AOD; autoregressive; Bayesian; spatial correlation; temporal correlation
15.  Geocoding accuracy and the recovery of relationships between environmental exposures and health 
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).
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.
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.
PMCID: PMC2359739  PMID: 18387189
16.  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.
PMCID: PMC3793637  PMID: 23302862
17.  Dynamics of Functionalized Surface Molecular Monolayers Studied with Ultrafast Infrared Vibrational Spectroscopy 
The structural dynamics of thin films consisting of tricarbonyl (1,10-phenanthroline)rhenium chloride (RePhen(CO)3Cl) linked to an alkyl silane monolayer through a triazole linker synthesized on silica-on-calcium-fluoride substrates are investigated using ultrafast infrared (IR) techniques. Ultrafast 2D IR vibrational echo experiments and polarization selective heterodyne detected transient grating (HDTG) measurements, as well as polarization dependent FT-IR and AFM experiments are employed to study the samples. The vibrational echo experiments measure spectral diffusion, while the HDTG experiments measure the vibrational excited state population relaxation and investigate the vibrational transition dipole orientational anisotropy decay. To investigate the anticipated impact of vibrational excitation transfer, which can be caused by the high concentration of RePhen(CO)3Cl in the monolayer, a concentration dependence of the spectral diffusion is measured. To generate a range of concentrations, mixed monolayers consisting of both hydrogen terminated and triazole/RePhen(CO)3Cl terminated alkyl silanes are synthesized. It is found that the measured rate of spectral diffusion is independent of concentration, with all samples showing spectral diffusion of 37 ± 6 ps. To definitively test for vibrational excitation transfer, polarization selective HDTG experiments are conducted. Excitation transfer will cause anisotropy decay. Polarization resolved heterodyne detected transient grating spectroscopy is sensitive to anisotropy decay (depolarization) caused by excitation transfer and molecular reorientation. The HDTG experiments show no evidence of anisotropy decay on the appropriate time scale, demonstrating the absence of excitation transfer the RePhen(CO)3Cl. Therefore the influence of excitation transfer on spectral diffusion is inconsequential in these samples, and the vibrational echo measurements of spectral diffusion report solely on structural dynamics. A small amount of very fast (~2 ps time scale) anisotropy decay is observed. The decay is concentration independent, and is assigned to wobbling-in-a-cone orientational motions of the RePhen(CO)3Cl. Theoretical calculations reported previously for experiments on a single concentration of the same type of sample suggested the presence of some vibrational excitation transfer and excitation transfer induced spectral diffusion. Possible reasons for the experimentally observed lack of excitation transfer in these high concentration samples are discussed.
PMCID: PMC3523711  PMID: 23259027
surface; dynamics; monolayer; infrared; spectroscopy; 2D IR; vibrational; energy transfer
18.  Identification of Candidate B-Lymphoma Genes by Cross-Species Gene Expression Profiling 
PLoS ONE  2013;8(10):e76889.
Comparative genome-wide expression profiling of malignant tumor counterparts across the human-mouse species barrier has a successful track record as a gene discovery tool in liver, breast, lung, prostate and other cancers, but has been largely neglected in studies on neoplasms of mature B-lymphocytes such as diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma (BL). We used global gene expression profiles of DLBCL-like tumors that arose spontaneously in Myc-transgenic C57BL/6 mice as a phylogenetically conserved filter for analyzing the human DLBCL transcriptome. The human and mouse lymphomas were found to have 60 concordantly deregulated genes in common, including 8 genes that Cox hazard regression analysis associated with overall survival in a published landmark dataset of DLBCL. Genetic network analysis of the 60 genes followed by biological validation studies indicate FOXM1 as a candidate DLBCL and BL gene, supporting a number of studies contending that FOXM1 is a therapeutic target in mature B cell tumors. Our findings demonstrate the value of the “mouse filter” for genomic studies of human B-lineage neoplasms for which a vast knowledge base already exists.
PMCID: PMC3793908  PMID: 24130802
19.  RABL6A Promotes Oxaliplatin Resistance in Tumor Cells and Is a New Marker of Survival for Resected Pancreatic Ductal Adenocarcinoma Patients 
Genes & Cancer  2013;4(7-8):273-284.
Pancreatic ductal adenocarcinoma (PDAC) is characterized by early recurrence following pancreatectomy, rapid progression, and chemoresistance. Novel prognostic and predictive biomarkers are urgently needed to both stratify patients for clinical trials and select patients for adjuvant therapy regimens. This study sought to determine the biological significance of RABL6A (RAB, member RAS oncogene family-like protein 6 isoform A), a novel pancreatic protein, in PDAC. Analyses of RABL6A protein expression in PDAC specimens from 73 patients who underwent pancreatic resection showed that RABL6A levels are altered in 74% of tumors relative to adjacent benign ductal epithelium. Undetectable RABL6A expression, found in 7% (5/73) of patients, correlated with improved overall survival (range 41 to 118 months with 3/5 patients still living), while patients with RABL6A expression had a worse outcome (range 3.3 to 100 months, median survival 20.3 months) (P = 0.0134). In agreement with those findings, RABL6A expression was increased in pancreatic cancer cell lines compared to normal pancreatic epithelial cells, and its knockdown inhibited pancreatic cancer cell proliferation and induced apoptosis. Moreover, RABL6A depletion selectively sensitized cells to oxaliplatin-induced arrest and death. This work reveals that RABL6A promotes the proliferation, survival, and oxaliplatin resistance of PDAC cells, whereas its loss is associated with extended survival in patients with resected PDAC. Such data suggest RABL6A is a novel biomarker of PDAC and potential target for anticancer therapy.
PMCID: PMC3807645  PMID: 24167655
pancreatic ductal adenocarcinoma; ARF; RABL6A; oxaliplatin; chemoresistance
20.  Current and Emerging Pharmacotherapeutic Options for Smoking Cessation 
Tobacco smoking remains the single most preventable cause of morbidity and mortality in developed countries and poses a significant threat across developing countries where tobacco use prevalence is increasing. Nicotine dependence is a chronic disease often requiring multiple attempts to quit; repeated interventions with pharmacotherapeutic aids have become more popular as part of cessation therapies. First-line medications of known efficacy in the general population include varenicline tartrate, bupropion hydrochloride, nicotine replacement therapy products, or a combination thereof. However, less is known about the use of these products in marginalized groups such as the indigenous, those with mental illnesses, youth, and pregnant or breastfeeding women. Despite the efficacy and safety of these first line pharmacotherapies, many smokers continue to relapse and alternative pharmacotherapies and cessation options are required. Thus, the aim of this review is to summarize the existing and developing pharmacotherapeutic and other options for smoking cessation, to identify gaps in current clinical practice, and to provide recommendations for future evaluations and research.
PMCID: PMC3668891  PMID: 23772176
smoking; smoking cessation; pharmacotherapy; pharmacotherapeutic; nicotine; varenicline tartrate; Champix; nicotine patches; bupropion; zyban
21.  Increased Oxidative Stress Created by Adenoviral MnSOD or CuZnSOD plus BCNU Inhibits Breast Cancer Cell Growth 
Free radical biology & medicine  2007;44(5):856-867.
Superoxide dismutases (SODs) have been found to decrease tumor formation and angiogenesis. SOD gene therapy, as with many other gene transfer strategies, may not completely inhibit tumor growth on its own. Thus, concomitant therapies are necessary to completely control the spread of this disease. We hypothesized that intratumoral injection of AdSOD in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) chemotherapy would synergistically inhibit breast cancer growth.
Our data indicate that BCNU when combined with SOD overexpression increased oxidative stress as suggested by elevated glutathione disulfide (GSSG) production in one of three breast cancer cell lines tested, at least in part due to glutathione reductase (GR) inactivation. The increased oxidative stress caused by BCNU combined with adenovirally-expressed SODs, manganese or copper zinc SOD, decreased growth and survival in the three cell lines tested in vitro, but had the largest effect in the MDA-MB231 cell line, which showed the largest amount of oxidative stress.
Delivery of MnSOD and BCNU intratumorally completely inhibited MDA-MB231 xenograft growth and increased nude mouse survival in vivo. Intravenous (IV) BCNU, recapitulating clinical usage, and intratumoral AdMnSOD delivery, to provide tumor specificity, provided similar decreased growth and survival in our nude mouse model. This cancer therapy produced impressive results suggesting the potential use of oxidative stress induced growth inhibitory treatments for breast cancer patients.
PMCID: PMC3649000  PMID: 18155673
superoxide dismutase; adenovirus; xenografts; BCNU; breast cancer
22.  Population-Based Exploration of Academic Achievement Outcomes in Pediatric Acute Lymphoblastic Leukemia Survivors 
Journal of Pediatric Psychology  2012;37(4):458-466.
Objective Examine academic achievement among pediatric acute lymphoblastic leukemia survivors diagnosed during the years 1993–2008. Method A deterministic linkage of the Iowa Cancer Registry and Iowa Testing Programs databases was performed and yielded 147 survivors. Achievement data, in the form of Iowa Percentile Rank scores, were obtained and analyzed by grade and content domain. Results Children diagnosed before age 5 evidenced more underachievement than those diagnosed later (p = .05). Underachievement was noted in mathematics in grades 8 and 11 (p's < .05), in addition to a longitudinal decrease in scores from grades 4 through 11 (p = .01). No differences were found in academic achievement between males and females. Conclusions Utilization of a population-based approach with a nationally recognized, standardized instrument indicates that academic underachievement is subtle yet exists, most notably in mathematics.
PMCID: PMC3334536  PMID: 22271791
academic achievement; cancer survivorship; cognition; leukemia; population-based
23.  Antibody response to pneumococcal vaccination as a function of preimmunization titer 
The interpretation of an adequate response to the unconjugated 23-valent pneumococcal vaccine for serotypes having high preimmunization titers remains challenging.
We sought to determine whether high preimmunization titers preclude a 4-fold or greater response to vaccination. Moreover, we sought to determine the effect of the following covariates on this response: absolute preimmunization titer value, age, sex, serum IgG level, and serum IgG subclasses.
We conducted a retrospective analysis of patients who were seen in our immune disorders clinic between 2001 and 2007 who had received the unconjugated 23-valent pneumococcal vaccine. Logistic regression was used to estimate the effect of different covariates, including preimmunization titer values, age, sex, IgG levels, and IgG subclass values, on the odds of a 4-fold or greater antibody response.
Per serotype, 10% to 40% of subjects with a high preimmunization titer attained at least a 4-fold response to vaccination. However, the odds of a 4-fold or greater response were found to decrease as a function of the absolute preimmunization titer value with an absolute value for each serotype beyond which the odds ratio approached zero.
High pneumococcal preimmunization titers do not necessarily preclude a 4-fold or greater response to vaccination. However, there appear to be serotype-specific preimmunization titer values, ranging from 4.4 to 10.3 μg/mL, above which a 4-fold or greater response would not be expected. This response does not seem to be significantly affected by age, sex, IgG level, or IgG subclass value.
PMCID: PMC3613280  PMID: 18951616
Pneumococcal vaccine; antibody; high preimmunization titer; fold response; age; sex; IgG; IgG subclass
24.  Discrete complexes immobilized onto Click-SBA-15 silica: Controllable loadings and the impact of surface coverage on catalysis 
Azidopropyl functionalized mesoporous silica SBA-15 were prepared with variable azide loadings of 0.03 – 0.7 mmol g−1 (ca. 2 – 50% of maximal surface coverage) through a direct synthesis, co-condensation approach. These materials are functionalized selectively with ethynylated organic moieties through the copper-catalyzed azide alkyne cycloaddition (CuAAC) or “click” reaction. Specific loading within a material can be regulated by either the azide loading or limiting the alkyne reagent relative to the azide loading. The immobilization of ferrocene, pyrene, tris(pyridylmethyl)amine (TPA), and iron porphyrin (FeTPP) demonstrates the robust nature and reproducibility of this two step synthetic attachment strategy. Loading-sensitive pyrene fluorescence correlates with a theoretically random. surface distribution, rather than a uniform one; full site-isolation of tethered moieties ca. 15 Å in length. occurs at loadings less than 0.02 mmol g−1. The effect of surface loading on reactivity is observed in oxygenation of SBA-15-[CuI(TPA)]. SBA-15-[MnII(TPA)]-catalyzed epoxidation exhibits a systematic dependence on surface loading. A comparison of homogeneous, site-isolated and site-dense complexes provides insight into catalyst speciation and ligand activity.
PMCID: PMC3277213  PMID: 22277027
25.  Evaluation of Diverse α/β Backbone Patterns for Functional α-Helix Mimicry: Analogues of the Bim BH3 Domain 
Peptidic oligomers that contain both α- and β-amino acid residues, in regular patterns throughout the backbone, are emerging as structural mimics of α-helix-forming conventional peptides (composed exclusively of α-amino acid residues). Here we describe a comprehensive evaluation of diverse α/β-peptide homologues of the Bim BH3 domain in terms of their ability to bind to the BH3-recognition sites on two partner proteins, Bcl-xL and Mcl-1. These proteins are members of the anti-apoptotic Bcl-2 family, and both bind tightly to the Bim BH3 domain itself. All α/β-peptide homologues retain the side chain sequence of the Bim BH3 domain, but each homologue contains periodic α-residue → β3-residue substitutions. Previous work has shown that the ααβαααβ pattern, which aligns the β3-residues in a 'stripe' along one side of the helix, can support functional α-helix mimicry, and the results reported here support this conclusion. The present study provides the first evaluation of functional mimicry by ααβ and αααβ patterns, which cause the β3-residues to spiral around the helix periphery. We find that the αααβ pattern can support effective mimicry of the Bim BH3 domain, as manifested by the crystal structure of an α/β-peptide bound to Bcl-xL, affinity for a variety of Bcl-2 family proteins, and induction of apoptotic signaling in mouse embryonic fibroblast extracts. The best αααβ homologue shows substantial protection from proteolytic degradation relative to the Bim BH3 α-peptide.
PMCID: PMC3364022  PMID: 22040025

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