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1.  CYP1A1 and CYP1B1-mediated biotransformation of the antitrypanosomal methamidoxime prodrug DB844 forms novel metabolites through intramolecular rearrangement 
Journal of pharmaceutical sciences  2013;103(1):10.1002/jps.23765.
DB844 (CPD-594-12), N-methoxy-6-{5-[4-(N-methoxyamidino)phenyl]-furan-2-yl}-nicotinamidine, is an oral prodrug that has shown promising efficacy in both mouse and monkey models of second stage human African trypanosomiasis. However, gastrointestinal (GI) toxicity was observed with high doses in a vervet monkey safety study. In the current study, we compared the metabolism of DB844 by hepatic and extrahepatic cytochrome P450s to determine if differences in metabolite formation underlie the observed GI toxicity. DB844 undergoes sequential O-demethylation and N-dehydroxylation in the liver to form the active compound DB820 (CPD-593-12). However, extrahepatic CYP1A1 and CYP1B1 produced two new metabolites, MX and MY. Accurate mass and collision-induced dissociation mass spectrometry analyses of the metabolites supported proposed structures of MX and MY. In addition, MY was confirmed with a synthetic standard and detection of nitric oxide release when DB844 was incubated with CYP1A1. Taken altogether, we propose that MX is formed by insertion of an oxygen into the amidine C=N to form an oxaziridine, which is followed by intramolecular rearrangement of the adjacent O-methyl group and subsequent release of nitric oxide. The resulting imine ester, MX, is further hydrolyzed to form MY. These findings may contribute to furthering the understanding of toxicities associated with benzamidoxime- and benzmethamidoxime-containing molecules.
doi:10.1002/jps.23765
PMCID: PMC3865069  PMID: 24186380
CYP1A1; CYP1B1; cytochrome P450; intramolecular rearrangement; drug metabolism; prodrugs; human African trypanosomiasis; metabolite identification; nitric oxide
2.  Tianeptine interferes with microtubule organization and hormone secretion of pheochromocytoma cells 
Pheochromocytoma originates from chromaffin cells in the adrenal medulla and sympathetic paraganglia. 36–53% of pheochromocytoma becomes malignant and, thereafter, resistant to conventional treatments. Pheochromocytoma also causes hyper-secretion of catecholamines that cause severe hypertension. We found that an antidepressant, tianeptine, interfered with normal life cycle of pheochromocytoma cells at its clinical doses. Treatment with tianeptine caused microtubule bundling and specific degradation of cytoplasmic dynein, a retrograde microtubule motor that mediates various microtubule-dependent processes during interphase and mitosis, in the rat pheochromocytoma PC12 cells. Tianeptine also increased the levels of pro-apoptotic proteins, slowed cell cycle progression, and increased apoptosis in PC12 cells. Importantly, tianeptine treatment decreased high K+-stimulated secretion of norepinephrine and chromogranin A in PC12 cells and of epinephrine in the mouse pheochromocytoma MPC cells. Our study demonstrates, for the first time, that tianeptine interferes with normal life cycle of pheochromocytoma cells.
doi:10.1016/j.mce.2013.07.033
PMCID: PMC3796025  PMID: 23933152
pheochromocytoma; tianeptine; microtubule bundling; cytoplasmic dynein
3.  Oxidative stress, testosterone, and cognition among Caucasian and Mexican American men with and without Alzheimer’s disease 
Background
The use of testosterone among aging men has been increasing, but results from studies addressing the effectiveness of testosterone replacement therapy (TRT) have been equivocal.
Objective
Given our prior pre-clinical studies that reported a major influence of oxidative stress (OS) on testosterone’s neuroprotective effects, we investigated whether the negative effects of testosterone on brain function were predicted by oxidative load.
Methods
In order to test our hypothesis, we determined whether circulating total testosterone and luteinizing hormone (LH) correlated with cognition in a subset of the Texas Alzheimer’s Research & Care Consortium (TARCC) cohort, consisting of Caucasian (n=116) and Mexican-American (n=117) men. We also assessed whether OS (as indexed by homocysteine levels) modified this relationship between sex hormones and cognition, and whether the levels of two antioxidants, superoxide dismutase-1 (SOD), and glutathione S-transferase (GST) varied as a function of circulating testosterone.
Results
In a low OS environment, testosterone was positively associated with the level of the antioxidant, GST, while no deleterious effects on cognitive function were noted. In contrast, under conditions of high OS (homocysteine levels >12 μM), testosterone and LH were associated with cognitive impairment, but only among Caucasians. The ethnic difference was attributed to significantly higher GST levels among Mexican-Americans.
Conclusion
While testosterone may be beneficial under conditions of low OS, testosterone appears to have negative consequences under conditions of elevated OS, but only in Caucasians. Mexican-Americans, however, were protected from any deleterious effects of testosterone, potentially due to higher levels of endogenous antioxidant defenses such as GST.
doi:10.3233/JAD-131994
PMCID: PMC4219556  PMID: 24496073
androgens; homocysteine; Mexican American; antioxidants; luteinizing hormone
4.  Risk factors for mild cognitive impairment among Mexican Americans 
Background
While a great deal of literature has focused on risk factors for Mild Cognitive Impairment (MCI), little published work examines risk for MCI among Mexican Americans.
Methods
Data from 1628 participants (non-Hispanic n= 1002; Mexican American n=626) were analyzed from two ongoing studies of cognitive aging and Alzheimer’s disease, Project FRONTIER and TARCC.
Results
When looking at the full cohorts (non-Hispanic and Mexican American), age, education, APOE ε4 status and gender were consistently related to MCI diagnosis across the two cohorts. However, when split by ethnicity advancing age was the only significant risk factor for MCI among Mexican Americans across both cohorts.
Conclusions
The current data suggests that many of the previously established risk factors for MCI among non-Hispanic cohorts may not be predictive of MCI among Mexican Americans and point to the need for additional work aimed at understanding factors related to cognitive aging among this underserved segment of the population.
doi:10.1016/j.jalz.2012.12.007
PMCID: PMC3737282  PMID: 23643456
Mexican American; Mild Cognitive Impairment; cognition; Alzheimer’s disease; ethnicity; cross-cultural; risk factors
5.  Normative Performance on the CLOX Task in a Multi-Ethnic Bilingual Cohort: A Project FRONTIER Study 
The CLOX test is a neuropsychological measure intended to aid in the assessment and detection of dementia in elderly populations. Few studies have provided normative data for this measure, with even less research available regarding the impact of socio-demographic factors on test scores. This study presents normative data for the CLOX in a sample of English- and Spanish-speaking Hispanic and non-Hispanic Whites. The total sample included 445 cognitively healthy older adults seen as part of an ongoing study of rural cognitive aging, Project FRONTIER. Unlike previous studies, criteria for “normality” (i.e., unimpaired) for CLOX1 and CLOX2 were based not merely on global impairment, but also on domain-specific impairment of executive functioning on the EXIT25 and/or Trail Making Test B (Trails B), or visuospatial/constructional impairment on the Line Orientation and Figure Copy subtests of the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), respectively. Hierarchical regression analyses revealed that CLOX1 scores require adjustment by Age across ethnicities, while Education and Gender are necessary stratification markers for CLOX1 performance only in non-Hispanic Whites. None of the demographic variables were valid predictors of CLOX2 performance, negating the need for such adjustments. In addition to being the first study to provide separate normative data for CLOX performance in Hispanic and non-Hispanic White samples, the current study offers a novel approach to defining “normal” by cognitive domain. We also highlight the need to directly examine the impact of socio-demographic factors before applying normative corrections based on factors that have negligible impact on test scores.
doi:10.1002/gps.2810
PMCID: PMC4142441  PMID: 22052628
Executive functioning; visuospatial skills; norms; geriatrics; cognition
6.  Shaker-Related Potassium Channels in the Central Medial Nucleus of the Thalamus Are Important Molecular Targets for Arousal Suppression by Volatile General Anesthetics 
The Journal of Neuroscience  2013;33(41):16310-16322.
The molecular targets and neural circuits that underlie general anesthesia are not fully elucidated. Here, we directly demonstrate that Kv1-family (Shaker-related) delayed rectifier K+ channels in the central medial thalamic nucleus (CMT) are important targets for volatile anesthetics. The modulation of Kv1 channels by volatiles is network specific as microinfusion of ShK, a potent inhibitor of Kv1.1, Kv1.3, and Kv1.6 channels, into the CMT awakened sevoflurane-anesthetized rodents. In heterologous expression systems, sevoflurane, isoflurane, and desflurane at subsurgical concentrations potentiated delayed rectifier Kv1 channels at low depolarizing potentials. In mouse thalamic brain slices, sevoflurane inhibited firing frequency and delayed the onset of action potentials in CMT neurons, and ShK-186, a Kv1.3-selective inhibitor, prevented these effects. Our findings demonstrate the exquisite sensitivity of delayed rectifier Kv1 channels to modulation by volatile anesthetics and highlight an arousal suppressing role of Kv1 channels in CMT neurons during the process of anesthesia.
doi:10.1523/JNEUROSCI.0344-13.2013
PMCID: PMC3792466  PMID: 24107962
7.  Overexpression of the Synthetic Chimeric Native-T-phylloplanin-GFP Genes Optimized for Monocot and Dicot Plants Renders Enhanced Resistance to Blue Mold Disease in Tobacco (N. tabacum L.) 
The Scientific World Journal  2014;2014:601314.
To enhance the natural plant resistance and to evaluate the antimicrobial properties of phylloplanin against blue mold, we have expressed a synthetic chimeric native-phylloplanin-GFP protein fusion in transgenic Nicotiana tabacum cv. KY14, a cultivar that is highly susceptible to infection by Peronospora tabacina. The coding sequence of the tobacco phylloplanin gene along with its native signal peptide was fused with GFP at the carboxy terminus. The synthetic chimeric gene (native-phylloplanin-GFP) was placed between the modified Mirabilis mosaic virus full-length transcript promoter with duplicated enhancer domains and the terminator sequence from the rbcSE9 gene. The chimeric gene, expressed in transgenic tobacco, was stably inherited in successive plant generations as shown by molecular characterization, GFP quantification, and confocal fluorescent microscopy. Transgenic plants were morphologically similar to wild-type plants and showed no deleterious effects due to transgene expression. Blue mold-sensitivity assays of tobacco lines were performed by applying P. tabacina sporangia to the upper leaf surface. Transgenic lines expressing the fused synthetic native-phyllopanin-GFP gene in the leaf apoplast showed resistance to infection. Our results demonstrate that in vivo expression of a synthetic fused native-phylloplanin-GFP gene in plants can potentially achieve natural protection against microbial plant pathogens, including P. tabacina in tobacco.
doi:10.1155/2014/601314
PMCID: PMC3980785  PMID: 24778589
8.  Arsenic exposure, AS3MT polymorphism, and neuropsychological functioning among rural dwelling adults and elders: a cross-sectional study 
Environmental Health  2014;13:15.
Background
The aim was to examine the link between low-level arsenic exposure and cognitive functioning, and the potential role of a single nucleotide polymorphism (SNP A35991G, rs10748835) of the AS3MT gene in modifying this link.
Methods
Data were analyzed on 526 participants from Project FRONTIER. Hierarchical linear regressions were created with neuropsychological raw index scores as the outcome variable and arsenic exposure and AS3MT SNP as different predictor variables.
Results
Within the total sample, arsenic exposure was negatively associated with language (p < 0.001) and executive functioning (p < 0.001). Among those with the AA genotype of the AS3MT gene, arsenic levels were negatively associated with language (p < 0.001), attention (p = 0.01), and executive functioning (p = 0.04). Among those with the AG genotype, arsenic levels were positively associated with immediate (p = 0.04) and delayed memory (p < 0.001) and negatively associated with executive functioning (p = 0.03). Among those with the GG genotype, arsenic levels were negatively associated with visuospatial functioning (p = 0.02).
Conclusions
Low-level arsenic exposure is associated with cognitive functioning; however, this association is modified by an AS3MT gene.
doi:10.1186/1476-069X-13-15
PMCID: PMC4234288  PMID: 24621105
Arsenic; AS3MT; Neuropsychology; Cognition
9.  Allosteric mechanism of water channel gating by Ca2+–calmodulin 
Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, our understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudo-atomic structure of full-length mammalian aquaporin-0 (AQP0, Bos Taurus) in complex with CaM using electron microscopy to understand how this signaling protein modulates water channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 water permeability by allosterically closing the cytoplasmic gate of AQP0. Our mechanistic model provides new insight, only possible in the context of the fully assembled channel, into how CaM regulates multimeric channels by facilitating cooperativity between adjacent subunits.
doi:10.1038/nsmb.2630
PMCID: PMC3766450  PMID: 23893133
aquaporin (AQP); gating; calmodulin (CaM); electron microscopy (EM); molecular dynamics (MD); calcium regulation; water channel; membrane protein complex
10.  Serum osmolality and effects of water deprivation in captive Asian elephants (Elephas maximus) 
Serum from 21 healthy, captive Asian elephants (Elephas maximus) was evaluated by measured and calculated osmolality. Serum osmolality results for this population of Asian elephants had a median of 261 mOsm/kg and an interquartile interval of 258–269 mOsm/kg when measured by freezing point osmometry and a median of 264 mOsm/kg and an interquartile interval of 257–269 mOsm/kg when measured by vapor pressure osmometry. These values are significantly lower than values reported in other mammalian species and have important diagnostic and therapeutic implications. Calculated osmolality produced unreliable results and needs further study to determine an appropriate formula and its clinical application in this species. A 16-hr water deprivation test in 16 Asian elephants induced a small, subclinical, but statistically significant increase in measured serum osmolality. Serum osmolality, blood urea nitrogen, and total protein by refractometer were sensitive indicators of hydration status. Serum osmolality measurement by freezing point or vapor pressure osmometry is a useful adjunct to routine clinical tests in the diagnostic evaluation of elephants.
doi:10.1177/1040638712445770
PMCID: PMC3886624  PMID: 22643341
Elephants; Elephas maximus; osmolality; osmolarity; water deprivation
11.  Characterization of Mexican Americans with Mild Cognitive Impairment and Alzheimer’s Disease 
Objective
To provide characterization of Mexican Americans who meet criteria for Alzheimer’s Disease (AD) and Mild Cognitive Impairment (MCI).
Methods
1069 participants ages 40 and above who self-identified as either non-Hispanic white (n=633) or Mexican American (n=436); were recruited using a community-based participatory research (CBPR) approach. Global cognition was assessed via the Mini Mental State Exam (MMSE), dementia severity by the Clinical Dementia Rating Scale (CDR) and depression via the Geriatric Depression Scale 30-item version. Age, gender, education, ApoEε4 allele frequency and diabetic diagnoses were also analyzed.
Results
Mexican Americans (normal controls, MCI and AD) were younger, less highly educated, performed more poorly on the MMSE, endorsed more symptoms of depression, were more likely to be diagnosed with diabetes, and possessed the ApoEε4 allele less frequently. Age was the only significant risk factor for cognitive dysfunction (AD/MCI) among Mexican Americans (OR=1.06, 95% CI = 1.03–1.09). Age (B=0.07, std=0.02, p<0.001) and ApoEε4 presence (B=0.9, std=0.4, p=0.02) were significantly related to increased disease severity.
Conclusions
Given the rapidly growing and aging Mexican American population, there is a substantial need for research into cognitive aging, MCI and AD among this ethnic group. The current findings hold important implications for both clinic and research settings and point to additional research needs.
doi:10.3233/JAD-2012-121420
PMCID: PMC3524411  PMID: 22976076
Mild Cognitive Impairment; Alzheimer’s disease; Mexican American; Hispanic; cognition; depression; diabetes
12.  The Link between C-Reactive Protein and Alzheimer’s Disease Among Mexican Americans 
Background
The aim of this study is to evaluate the link between CRP and Alzheimer’s disease (AD) and Mild Cognitive Impairment (MCI) among Mexican Americans.
Methods
Non-fasting serum CRP levels, MMSE scores and CDR scores were analyzed from 1,066 participants (Mexican American n=471, non-Hispanic n=595) of the Texas Alzheimer’s Research & Care Consortium (TARCC).
Results
Among the total cohort, CRP levels among AD cases were significantly decreased as compared to normal controls (p<0.001) and MCI cases (p=0.002). CRP levels among MCI cases were decreased relative to controls (p=0.03). Among Mexican American and non-Hispanic AD cases, CRP levels were significantly decreased among AD cases as compared to controls. CRP levels were only associated with disease severity (CDR scores) among non-Hispanics (p=0.03) AD cases.
Conclusions
These results show that, while CRP levels are decreased among Mexican American AD cases, CRP appears to not be related to clinical variables as it is among non-Hispanic whites.
doi:10.3233/JAD-122071
PMCID: PMC3608400  PMID: 23254637
C-Reactive Protein; Alzheimer’s disease; Mexican American; Neuropsychology
13.  Biomarkers of Vascular Risk, Systemic Inflammation and Microvascular Pathology and Neuropsychiatric Symptoms in Alzheimer’s Disease 
Numerous serum and plasma based biomarkers of systemic inflammation have been linked to both neuropsychiatric disorders and Alzheimer’s disease. The present study investigated the relationship of clinical biomarkers of cardiovascular risk (cholesterol, triglycerides and homocysteine) and a panel of markers of systemic inflammation (CRP, TNF-α, IL1-ra, IL-7, IL-10, IL-15, IL18) and microvascular pathology (ICAM-1, VCAM-1) to neuropsychiatric symptoms in a sample with mild Alzheimer’s disease. Biomarker data was analyzed on a sample of 194 diagnosed with mild to moderate probable Alzheimer’s disease. The sample was composed of 127 females and 67 males. The presence of neuropsychiatric symptoms was gathered from interview with caretakers/family members using the Neuropsychiatric Inventory. For total sample IL15, VCAM (Vascular Adhesion Molecule) and triglycerides were significantly and negatively related to number of neuropsychiatric symptoms and total cholesterol and homocysteine were positively related and as a group accounted for 16.1% of the variance. When stratified by gender different patterns of significant biomarkers were found with relationships more robust for males for both total symptoms and symptom clusters. A combination of biomarkers of systemic inflammation, microvascular pathology and clinical biomarkers of cardiovascular risk can account for a significant portion of the variance in the occurrence of neuropsychiatric symptoms in Alzheimer’s disease supporting a vascular and inflammatory component of psychiatric disorders found in Alzheimer’s disease. Gender differences suggest distinct impact of specific risks with total cholesterol a measure of cardiovascular risk being the strongest marker for males and IL-15 a marker of inflammation being the strongest for females.
doi:10.3233/JAD-122359
PMCID: PMC3631280  PMID: 23403534
Alzheimer’s Disease; neuropsychiatric symptoms; biomarkers; gender
14.  In Vivo Analysis of Aquaporin 0 Function in Zebrafish: Permeability Regulation Is Required for Lens Transparency 
Purpose.
The zebrafish lens is well suited for studies of physiology and development due to its rapid formation in the embryo and genetic accessibility. Aquaporin 0 (AQP0), a lens-specific membrane protein, is required for lens clarity. Zebrafish have two copies of AQP0 (Aqp0a and b), whereas mammals have a single, multifunctional protein. Here we demonstrate a reliable knockdown/rescue system in zebrafish and use it to provide evidence for subfunctionalization of Aqp0a and b, as well as to show that calcium-mediated regulation of Aqp0a in zebrafish lenses is necessary for transparency.
Methods.
Coinjection of antisense oligonucleotides and DNA rescue constructs into zebrafish embryos, followed by evaluation of the developing fish for cataracts, was used to analyze the functions of Aqp0a and b. The water permeability and regulation characteristics of each rescue protein were tested in a Xenopus oocyte swelling assay.
Results.
Both copies of AQP0 are necessary for lens clarity in the zebrafish, and neither is sufficient. Water permeability is necessary but also insufficient. Phosphorylation and regulation of Aqp0a are required for its function.
Conclusions.
In the zebrafish lens, the two closely related AQP0s have acquired distinct functions that are both necessary for lens development and clarity. Regulation of AQP0 water permeability, a well-studied phenomenon in vitro, may be physiologically relevant in the living lens.
A novel knockdown/rescue assay in zebrafish demonstrates that regulation of AQP0 water permeability is required for lens transparency and shows subfunctionalization of the two copies of zebrafish AQP0.
doi:10.1167/iovs.13-12337
PMCID: PMC3729242  PMID: 23800763
cataract; AQP0; zebrafish; subfunctionalization
15.  Regulation of AQP0 water permeability is enhanced by cooperativity 
The Journal of General Physiology  2013;141(3):287-295.
Aquaporin 0 (AQP0), essential for lens clarity, is a tetrameric protein composed of four identical monomers, each of which has its own water pore. The water permeability of AQP0 expressed in Xenopus laevis oocytes can be approximately doubled by changes in calcium concentration or pH. Although each monomer pore functions as a water channel, under certain conditions the pores act cooperatively. In other words, the tetramer is the functional unit. In this paper, we show that changes in external pH and calcium can induce an increase in water permeability that exhibits either a positive cooperativity switch-like increase in water permeability or an increase in water permeability in which each monomer acts independently and additively. Because the concentrations of calcium and hydrogen ions increase toward the center of the lens, a concentration signal could trigger a regulatory change in AQP0 water permeability. It thus seems plausible that the cooperative modes of water permeability regulation by AQP0 tetramers mediated by decreased pH and elevated calcium are the physiologically important ones in the living lens.
doi:10.1085/jgp.201210884
PMCID: PMC3581697  PMID: 23440275
16.  Diagnostic Accuracy of the MMSE in Detecting Probable and Possible Alzheimer's Disease in Ethnically Diverse Highly Educated Individuals: An Analysis of the NACC Database 
Background.
To validate and extend the findings of a raised cut score of O’Bryant and colleagues (O’Bryant SE, Humphreys JD, Smith GE, et al. Detecting dementia with the mini-mental state examination in highly educated individuals. Arch Neurol. 2008;65(7):963–967.) for the Mini-Mental State Examination in detecting cognitive dysfunction in a bilingual sample of highly educated ethnically diverse individuals.
Methods.
Archival data were reviewed from participants enrolled in the National Alzheimer's Coordinating Center minimum data set. Data on 7,093 individuals with 16 or more years of education were analyzed, including 2,337 cases with probable and possible Alzheimer's disease, 1,418 mild cognitive impairment patients, and 3,088 nondemented controls. Ethnic composition was characterized as follows: 6,296 Caucasians, 581 African Americans, 4 American Indians or Alaska natives, 2 native Hawaiians or Pacific Islanders, 149 Asians, 43 “Other,” and 18 of unknown origin.
Results.
Diagnostic accuracy estimates (sensitivity, specificity, and likelihood ratio) of Mini-Mental State Examination cut scores in detecting probable and possible Alzheimer's disease were examined. A standard Mini-Mental State Examination cut score of 24 (≤23) yielded a sensitivity of 0.58 and a specificity of 0.98 in detecting probable and possible Alzheimer's disease across ethnicities. A cut score of 27 (≤26) resulted in an improved balance of sensitivity and specificity (0.79 and 0.90, respectively). In the cognitively impaired group (mild cognitive impairment and probable and possible Alzheimer's disease), the standard cut score yielded a sensitivity of 0.38 and a specificity of 1.00 while raising the cut score to 27 resulted in an improved balance of 0.59 and 0.96 of sensitivity and specificity, respectively.
Conclusions.
These findings cross-validate our previous work and extend them to an ethnically diverse cohort. A higher cut score is needed to maximize diagnostic accuracy of the Mini-Mental State Examination in individuals with college degrees.
doi:10.1093/gerona/gls006
PMCID: PMC3403860  PMID: 22396476
Alzheimer's disease; Dementia diagnosis; Ethnicity; Language; Mini-Mental State Examination
17.  Mosaic VSGs and the Scale of Trypanosoma brucei Antigenic Variation 
PLoS Pathogens  2013;9(7):e1003502.
A main determinant of prolonged Trypanosoma brucei infection and transmission and success of the parasite is the interplay between host acquired immunity and antigenic variation of the parasite variant surface glycoprotein (VSG) coat. About 0.1% of trypanosome divisions produce a switch to a different VSG through differential expression of an archive of hundreds of silent VSG genes and pseudogenes, but the patterns and extent of the trypanosome diversity phenotype, particularly in chronic infection, are unclear. We applied longitudinal VSG cDNA sequencing to estimate variant richness and test whether pseudogenes contribute to antigenic variation. We show that individual growth peaks can contain at least 15 distinct variants, are estimated computationally to comprise many more, and that antigenically distinct ‘mosaic’ VSGs arise from segmental gene conversion between donor VSG genes or pseudogenes. The potential for trypanosome antigenic variation is probably much greater than VSG archive size; mosaic VSGs are core to antigenic variation and chronic infection.
Author Summary
Trypanosoma brucei—a deadly parasite of humans and animals—owes its success to its ability to cope with host immunity, and the mechanism it uses to do so is a remarkable example of biological variation. Immune responses that develop against the parasite surface coat are only partially effective against the parasite population; some individual parasites will have already switched to a different variant of the coat antigen, and thus survive to prolong infection. Little is known about how the pattern of antigen variation unfolds, particularly after the early stage of infection. Here, we examined different antigen variants that appeared over the course of infection, to estimate their diversity and to see whether the parasites are able to generate new antigen variants by combination. We found antigen diversity was much greater than expected, and that ‘mosaic’ variants—produced by combining bits of more than one antigen gene—played a central role in the later stages of infection. These results provide important evidence for the robustness of this key survival strategy, provide clues about its evolution, and allow us to identify patterns in common with other antigenically variable pathogens.
doi:10.1371/journal.ppat.1003502
PMCID: PMC3708902  PMID: 23853603
18.  A Depressive Endophenotype of Mild Cognitive Impairment and Alzheimer’s Disease 
PLoS ONE  2013;8(7):e68848.
Background
Alzheimer’s disease (AD) is a devastating public health problem that affects over 5.4 million Americans. Depression increases the risk of Mild Cognitive Impairment (MCI) and AD. By understanding the influence of depression on cognition, the potential exists to identify subgroups of depressed elders at greater risk for cognitive decline and AD. The current study sought to: 1) clinically identify a sub group of geriatric patients who suffer from depression related cognitive impairment; 2) cross validate this depressive endophenotype of MCI/AD in an independent cohort.
Methods and Findings
Data was analyzed from 519 participants of Project FRONTIER. Depression was assessed with the GDS30 and cognition was assessed using the EXIT 25 and RBANS. Five GDS items were used to create the Depressive endophenotype of MCI and AD (DepE). DepE was significantly negatively related to RBANS index scores of Immediate Memory (B=-2.22, SE=.37, p<0.001), visuospatial skills (B=-1.11, SE=0.26, p<0.001), Language (B=-1.03, SE=0.21, p<0.001), Attention (B=-2.56, SE=0.49, p<0.001), and Delayed Memory (B=-1.54, SE = 037, p<0.001), and higher DepE scores were related to poorer executive functioning (EXIT25; B=0.65, SE=0.19, p=0.001). DepE scores significantly increased risk for MCI diagnosis (odds ratio [OR] = 2.04; 95% CI=1.54-2.69). Data from 235 participants in the TARCC (Texas Alzheimer’s Research & Care Consortium) were analyzed for cross-validation of findings in an independent cohort. The DepE was significantly related to poorer scores on all measures, and a significantly predicted of cognitive change over 12- and 24-months.
Conclusion
The current findings suggest that a depressive endophenotype of MCI and AD exists and can be clinically identified using the GDS-30. Higher scores increased risk for MCI and was cross-validated by predicting AD in the TARCC. A key purpose for the search for distinct subgroups of individuals at risk for AD and MCI is to identify novel treatment and preventative opportunities.
doi:10.1371/journal.pone.0068848
PMCID: PMC3708919  PMID: 23874786
19.  Safety, Pharmacokinetic, and Efficacy Studies of Oral DB868 in a First Stage Vervet Monkey Model of Human African Trypanosomiasis 
There are no oral drugs for human African trypanosomiasis (HAT, sleeping sickness). A successful oral drug would have the potential to reduce or eliminate the need for patient hospitalization, thus reducing healthcare costs of HAT. The development of oral medications is a key objective of the Consortium for Parasitic Drug Development (CPDD). In this study, we investigated the safety, pharmacokinetics, and efficacy of a new orally administered CPDD diamidine prodrug, 2,5-bis[5-(N-methoxyamidino)-2-pyridyl]furan (DB868; CPD-007-10), in the vervet monkey model of first stage HAT. DB868 was well tolerated at a dose up to 30 mg/kg/day for 10 days, a cumulative dose of 300 mg/kg. Mean plasma levels of biomarkers indicative of liver injury (alanine aminotransferase, aspartate aminotransferase) were not significantly altered by drug administration. In addition, no kidney-mediated alterations in creatinine and urea concentrations were detected. Pharmacokinetic analysis of plasma confirmed that DB868 was orally available and was converted to the active compound DB829 in both uninfected and infected monkeys. Treatment of infected monkeys with DB868 began 7 days post-infection. In the infected monkeys, DB829 attained a median Cmax (dosing regimen) that was 12-fold (3 mg/kg/day for 7 days), 15-fold (10 mg/kg/day for 7 days), and 31-fold (20 mg/kg/day for 5 days) greater than the IC50 (14 nmol/L) against T. b. rhodesiense STIB900. DB868 cured all infected monkeys, even at the lowest dose tested. In conclusion, oral DB868 cured monkeys with first stage HAT at a cumulative dose 14-fold lower than the maximum tolerated dose and should be considered a lead preclinical candidate in efforts to develop a safe, short course (5–7 days), oral regimen for first stage HAT.
Author Summary
Development of orally administered medicines for human African trypanosomiasis (HAT) would potentially reduce the need for patient hospitalization, thus lowering healthcare costs. In this study, we investigated the potential of a novel diamidine prodrug, DB868 (CPD-007-10), as an oral treatment for first stage HAT. When administered to uninfected monkeys by oral gavage, DB868 was well tolerated up to a maximum dose of 30 mg/kg/day for 10 days (cumulative dose [CD] = 300 mg/kg). DB868 was absorbed into the systemic circulation and was converted to the active compound DB829 in concentrations that were potentially therapeutic for blood trypanosomes. Subsequently, DB868 was evaluated for efficacy in the first stage vervet monkey model of HAT in which treatment was initiated at 7 days post-infection with T. b. rhodesiense KETRI 2537. All infected monkeys were cured, even at the lowest of the three dose regimens tested: 3 mg/kg/day for 7 days (CD = 21 mg/kg), 10 mg/kg/day for 7 days (CD = 70 mg/kg) and 20 mg/kg/day for 5 days (CD = 100 mg/kg). DB868 conversion to DB829 was comparable between uninfected and infected monkeys. In view of its favourable safety and oral efficacy profile, we conclude that DB868 is a suitable candidate for development as a new treatment for first stage HAT.
doi:10.1371/journal.pntd.0002230
PMCID: PMC3674995  PMID: 23755309
20.  The impact of GPX1 on the association of groundwater selenium and depression: a project FRONTIER study 
BMC Psychiatry  2013;13:7.
Background
Prior animal model and human-based studies have linked selenium concentrations to decreased risk for depression; however, this work has not focused on household groundwater levels or specific depressive symptoms. The current study evaluated the link between groundwater selenium levels and depression. We also sought to determine if a functional polymorphism in the glutathione peroxidase 1 (GPX1) gene impacted this link.
Methods
We used a cross-sectional design to analyze data from 585 participants (183 men and 402 women) from Project FRONTIER, a study of rural health in West Texas. Residential selenium concentrations were estimated using Geospatial Information System (GIS) analyses. Linear regression models were created using Geriatric Depression Scale (GDS-30) total and subfactor scores as outcome variables and selenium concentrations as predictor variables. Analyses were re-run after stratification of the sample on GPX1 Pro198Leu genotype (rs1050454).
Results
Selenium levels were significantly and negatively related to all GDS and subfactor scores accounting for up to 17% of the variance beyond covariates. Selenium was most strongly protective against depression among homozygous carriers of the C allele at the Pro198Leu polymorphism of the GPX1 gene. Analyses also point towards a gene-environmental interaction between selenium exposure and GPX1 polymorphism.
Conclusion
Our results support the link between groundwater selenium levels and decreased depression symptoms. These findings also highlight the need to consider the genetics of the glutathione peroxidase system when examining this relationship, as variation in the GPX1 gene is related to depression risk and significantly influences the protective impact of selenium, which is indicative of a gene-environment interaction.
doi:10.1186/1471-244X-13-7
PMCID: PMC3566946  PMID: 23289525
Aging; Depression; Environmental factors; Selenium; GPX1
21.  The Relation between Inflammation and Neuropsychological Test Performance 
Background. Considerable research documents an association between pro- and anti-inflammatory markers and Alzheimer's disease (AD), yet the differential relation between these markers and neuropsychological functioning in AD and nondemented controls has received less attention. The current study sought to evaluate the relationship between peripheral markers of inflammation (both pro- and anti-inflammatory) and neuropsychological functioning through the Texas Alzheimer's Research and Care Consortium (TARCC) cohort. Methods. There were 320 participants (Probable AD n = 124, Controls n = 196) in the TARCC Longitudinal Research Cohort available for analysis. Regression analyses were utilized to examine the relation between proinflammatory and anti-inflammatory markers and neuropsychological functioning. Follow-up analyses were conducted separately by case versus control status. Results. Proinflammatory and anti-inflammatory markers were found to be associated with neuropsychological testing. Third tertile proinflammatory markers were negatively associated with measures of attention and language, and anti-inflammatory markers were positively associated with measures of immediate verbal memory and delayed verbal and visual memory. Conclusions. These findings support the link between peripheral inflammatory markers and neuropsychological functioning and suggest the utility of examining profiles of inflammatory markers in the future.
doi:10.1155/2012/703871
PMCID: PMC3449133  PMID: 23008797
22.  A Blood-Based Algorithm for the Detection of Alzheimer's Disease 
Background
We previously created a serum-based algorithm that yielded excellent diagnostic accuracy in Alzheimer's disease. The current project was designed to refine that algorithm by reducing the number of serum proteins and by including clinical labs. The link between the biomarker risk score and neuropsychological performance was also examined.
Methods
Serum-protein multiplex biomarker data from 197 patients diagnosed with Alzheimer's disease and 203 cognitively normal controls from the Texas Alzheimer's Research Consortium were analyzed. The 30 markers identified as the most important from our initial analyses and clinical labs were utilized to create the algorithm.
Results
The 30-protein risk score yielded a sensitivity, specificity, and AUC of 0.88, 0.82, and 0.91, respectively. When combined with demographic data and clinical labs, the algorithm yielded a sensitivity, specificity, and AUC of 0.89, 0.85, and 0.94, respectively. In linear regression models, the biomarker risk score was most strongly related to neuropsychological tests of language and memory.
Conclusions
Our previously published diagnostic algorithm can be restricted to only 30 serum proteins and still retain excellent diagnostic accuracy. Additionally, the revised biomarker risk score is significantly related to neuropsychological test performance.
doi:10.1159/000330750
PMCID: PMC3169374  PMID: 21865746
Algorithm, blood-based; Alzheimer's disease; Diagnosis
23.  Serum Granulocyte Colony-Stimulating Factor and Alzheimer's Disease 
Background
Granulocyte colony-stimulating factor (G-CSF) promotes the survival and function of neutrophils. G-CSF is also a neurotrophic factor, increasing neuroplasticity and suppressing apoptosis.
Methods
We analyzed G-CSF levels in 197 patients with probable Alzheimer's disease (AD) and 203 cognitively normal controls (NCs) from a longitudinal study by the Texas Alzheimer's Research and Care Consortium (TARCC). Data were analyzed by regression with adjustment for age, education, gender and APOE4 status.
Results
Serum G-CSF was significantly lower in AD patients than in NCs (β = −0.073; p = 0.008). However, among AD patients, higher serum G-CSF was significantly associated with increased disease severity, as indicated by lower Mini-Mental State Examination scores (β = −0.178; p = 0.014) and higher scores on the global Clinical Dementia Rating (CDR) scale (β = 0.170; p = 0.018) and CDR Sum of Boxes (β = 0.157; p = 0.035).
Conclusions
G-CSF appears to have a complex relationship with AD pathogenesis and may reflect different pathophysiologic processes at different illness stages.
doi:10.1159/000341780
PMCID: PMC3457029  PMID: 23012618
Granulocyte colony-stimulating factor; Alzheimer's disease; Inflammation; Serum proteins; Mini-Mental State Examination; Clinical Dementia Rating-Sum of Boxes
24.  X-ray Crystal Structure of Arsenite-Inhibited Xanthine Oxidase: μ-Sulfido μ-Oxo Double-Bridge between Molybdenum and Arsenic in the Active Site 
Journal of the American Chemical Society  2011;133(32):12414-12417.
Xanthine oxidoreductase is a molybdenum-containing enzyme which catalyzes the hydroxylation reaction on sp2-hybridized carbon centers of a variety of substrates including purines, aldehydes and other heterocyclic compounds. The complex of arsenite-inhibited xanthine oxidase has been characterized previously by UV-visible, EPR and X-ray absorption spectroscopy, and the catalytically essential sulfido ligand of the square-pyrimidal molybdenum center suggested to be involved in arsenite binding through either μ-sulfido, μ-oxo double-bridge or a single μ-sulfido bridge. However, this is contrary to the crystallographically observed single μ-oxo bridge between molybdenum and arsenic in the desulfo-form of aldehyde oxidoreductase from Desulfovibrio gigas (an enzyme closely related to xanthine oxidase) whose molybdenum center has an oxo ligand replacing the catalytically essential sulfur as seen in the functional form of xanthine oxidase. Here, we use X-ray crystallography to characterize the molybdenum center of arsenite-inhibited xanthine oxidase and solve the structures of oxidized and reduced inhibition complexes at 1.82 and 2.11 Å resolution, respectively. We observe μ-sulfido, μ-oxo double-bridge between molybdenum and arsenic in the active sites of both complexes. Arsenic exhibits four-coordinate with a distorted trigonal-prismatic geometry in the oxidized complex and three-coordinate with a distorted trigonal-planar geometry in the reduced complex. The doubly-bridged binding mode is in agreement with previous XAS data that the catalytically essential sulfur is also essential for high affinity of reduced xanthine oxidoreductase for arsenite.
doi:10.1021/ja2050265
PMCID: PMC3163118  PMID: 21761899
25.  Staging Dementia Using Clinical Dementia Rating Scale Sum of Boxes Scores 
Archives of neurology  2008;65(8):1091-1095.
Background
The Clinical Dementia Rating Scale Sum of Boxes (CDR-SOB) score is commonly used, although the utility regarding this score in staging dementia severity is not well established.
Obiective
To investigate the effectiveness of CDRSOB scores in staging dementia severity compared with the global CDR score.
Design
Retrospective study.
Setting
Texas Alzheimer's Research Consortium minimum data set cohort.
Participants
A total of 1577 participants (110 controls, 202 patients with mild cognitive impairment, and 1265 patients with probable Alzheimer disease) were available for analysis.
Main Outcome Measures
Receiver operating characteristic curves were generated from a derivation sample to determine optimal cutoff scores and ranges, which were then applied to the validation sample.
Results
Optimal ranges of CDR-SOB scores corresponding to the global CDR scores were 0.5 to 4.0 for a global score of 0.5, 4.5 to 9.0 for a global score of 1.O, 9.5 to 15.5 for a global score of 2.0, and 16.0 to 18.0 for a global score of 3.0. When applied to the validation sample, κ scores ranged from 0.86 to 0.94 (P <.001 for all), with 93.0% of the participants falling within the new staging categories.
Conclusions
The CDR-SOB score compares well with the global CDR score for dementia staging. Owing to the increased range of values, the CDR-SOB score offers several advantages over the global score, including increased utility in tracking changes within and between stages of dementia severity. Interpretive guidelines for CDR-SOB scores are provided.
doi:10.1001/archneur.65.8.1091
PMCID: PMC3409562  PMID: 18695059

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