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1.  ST6Gal-I expression in ovarian cancer cells promotes an invasive phenotype by altering integrin glycosylation and function 
Ovarian adenocarcinoma is not generally discovered in patients until there has been widespread intraperitoneal dissemination, which is why ovarian cancer is the deadliest gynecologic malignancy. Though incompletely understood, the mechanism of peritoneal metastasis relies on primary tumor cells being able to detach themselves from the tumor, escape normal apoptotic pathways while free floating, and adhere to, and eventually invade through, the peritoneal surface. Our laboratory has previously shown that the Golgi glycosyltransferase, ST6Gal-I, mediates the hypersialylation of β1 integrins in colon adenocarcinoma, which leads to a more metastatic tumor cell phenotype. Interestingly, ST6Gal-I mRNA is known to be upregulated in metastatic ovarian cancer, therefore the goal of the present study was to determine whether ST6Gal-I confers a similarly aggressive phenotype to ovarian tumor cells.
Three ovarian carcinoma cell lines were screened for ST6Gal-I expression, and two of these, PA-1 and SKOV3, were found to produce ST6Gal-I protein. The third cell line, OV4, lacked endogenous ST6Gal-I. In order to understand the effects of ST6Gal-I on cell behavior, OV4 cells were stably-transduced with ST6Gal-I using a lentiviral vector, and integrin-mediated responses were compared in parental and ST6Gal-I-expressing cells.
Forced expression of ST6Gal-I in OV4 cells, resulting in sialylation of β1 integrins, induced greater cell adhesion to, and migration toward, collagen I. Similarly, ST6Gal-I expressing cells were more invasive through Matrigel.
ST6Gal-I mediated sialylation of β1 integrins in ovarian cancer cells may contribute to peritoneal metastasis by altering tumor cell adhesion and migration through extracellular matrix.
PMCID: PMC2584051  PMID: 19014651
2.  Dissociable causal roles for left and right parietal cortex in controlling attentional biases from the contents of working memory 
NeuroImage  2014;100:200-205.
The contents of working memory (WM) steer visual attention, but the extent of this guidance can be strategically enhanced or inhibited when WM content is reliably helpful or harmful to a visual task. Current understanding of the neural substrates mediating the cognitive control over WM biases is limited, however, by the correlational nature of functional MRI approaches. A recent fMRI study provided suggestive evidence for a functional lateralization of these control processes in posterior parietal cortex (PPC): activity in left PPC correlated with the presentation of WM cues that ought to be strategically enhanced to optimize performance, while activity in the right PPC correlated with the presentation of cues that ought to be inhibited to prevent detrimental attentional biases in a visual search. Here, we aimed to directly assess whether the left and right PPC are causally involved in the cognitive control of WM biases, and to clarify their precise functional contributions. We therefore applied 1Hz repetitive transcranial magnetic stimulation (rTMS) to left and right PPC (and a vertex control site) prior to administering a behavioral task assessing WM biasing control functions. We observed that the perturbation of left PPC eliminated the strategic benefit of predictably helpful WM cueing, while the perturbation of right PPC amplified the cost of unpredictable detrimental WM cueing. Left and right PPC thus play distinct causal roles in WM-attention interactions: left PPC to maximize benefits, and right PPC to minimize costs, of internally maintained content on visual attention.
PMCID: PMC4209476  PMID: 24945665
working memory; cognitive control; visual attention; parietal cortex
3.  The Role of Endocrine Disruptors in the Epigenetics of Reproductive Disease and Dysfunction: Potential Relevance to Humans 
In a murine model, we have linked early life toxicant exposure to reduced uterine sensitivity to progesterone, a phenotype we had previously associated with inflammation in endometriosis patients. Subsequent studies revealed that developmental toxicant exposure not only reduces fertility in male and female mice but also negatively impacts pregnancy leading to spontaneous preterm birth (PTB). An epigenetic alteration of the progesterone receptor gene correlated with reduced fertility and adverse pregnancy outcomes and persisted in multiple generations of mice in the absence of an additional toxicant exposure. Gene-environment interactions in women may explain why some patients “at risk” for PTB deliver at term while others without known risks deliver early. Our model provides a unique system to unravel the interactive influences of inflammation and reduced progesterone responsiveness on PTB and suggests that therapy needs to begin prior to pregnancy (and involve both partners) rather than once the inflammatory cascade has been initiated.
PMCID: PMC4056573  PMID: 24932427
4.  Dietary fish oil supplementation inhibits formation of endometriosis-associated adhesions in a chimeric mouse model 
Fertility and sterility  2012;99(2):543-550.e1.
To examine whether dietary fish oil supplementation reduces development of spontaneous endometriosis-associated adhesions using an established model.
Laboratory-based study.
Medical center research laboratory.
Disease-free women of reproductive age and nude mice.
Women were not provided any intervention. Mice were randomized to receive fish oil supplementation or control diet.
Main Outcome Measure(s)
Experimental endometriosis was established in mice via injection of human endometrial tissue within 16 hours of ovariectomy. Mice were provided standard or menhaden fish oil–supplemented diets for ≥2 weeks before initiation of experimental endometriosis and until killing them 1 week later. At necropsy, mice were examined for the presence and extent of adhesions and endometriotic-like lesions. Tissues were excised and morphologically characterized.
Adhesions/lesions were reduced in mice provided with dietary fish oil compared with control animals. Leukocytes were more numerous within the adhesions/lesions of the mice maintained on the standard diet compared with animals provided with fish oil. As indicated by staining intensity, collagen deposition was greater at adhesion sites within control mice compared with fish oil–supplemented animals.
Wound-healing associated with surgery created an inflammatory peritoneal microenvironment that promoted the development of both experimental endometriosis and adhesions in a murine model. Targeting excessive inflammation with fish oil may be an effective adjuvant therapy to reduce the development of postsurgical adhesions related to endometriosis.
PMCID: PMC3582352  PMID: 23103017
Omega-3 fatty acids; adhesions; endometriosis; mice; inflammation
5.  A multi-center, randomized, double blind placebo-controlled trial of estrogens to prevent Alzheimer’s disease and loss of memory in women: design and baseline characteristics 
Clinical trials (London, England)  2008;5(5):10.1177/1740774508096313.
Observational studies and small clinical trials suggested that hormone replacement therapy (HRT) decreases risk of cognitive loss and Alzheimer’s disease (AD) in postmenopausal women and may have value in primary prevention.
A clinical trial was designed to determine if HRT delays AD or memory loss. This report describes the rationale and original design of the trial and details extensive modifications that were required to respond to unanticipated findings that emerged from other studies during the course of the trial.
The trial was designed as a multi-center, placebo-controlled primary prevention trial for women 65 years of age or older with a family history of dementia. Recruitment from local sites was supplemented by centralized efforts to use names of Medicare beneficiaries. Inclusion criteria included good general health and intact memory functioning. Participants were randomized to HRT or placebo in a 1:1 ratio. Assignment was stratified by hysterectomy status and site. The primary outcomes were incident AD and memory decline on neuropsychological testing.
Enrollment began in March 1998. In response to the Women’s Health Initiative (WHI) May 2002 report of increased incidence of heart disease, stroke, pulmonary embolism, and breast cancer among women randomized to HRT, participants were re-consented with a revised consent form. Procedural modifications, including discontinuation of study medication and a modification of the planned primary outcome based on a final enrollment below the target enrollment (N = 477), were enacted in response to the subsequent WHI Memory Study report of increased risk of dementia and poorer cognitive function with HRT. The mean length of treatment exposure prior to discontinuation was 2.14 years. Participants’ mean age at baseline was 72.8; mean education was 14.2 years. Minority participation was 19% and 34% had a hysterectomy. The study continues to follow these participants for a total of 5 years blind to the original medication assignment.
Results reported from the WHI during the course of this study mandated extensive procedural modifications, including discontinuing recruitment before completion and halting study medication. Alternative strategies for study redesign that were considered are discussed.
PMCID: PMC3884686  PMID: 18827045
6.  Prothioconazole and Prothioconazole-Desthio Activities against Candida albicans Sterol 14-α-Demethylase 
Prothioconazole is a new triazolinthione fungicide used in agriculture. We have used Candida albicans CYP51 (CaCYP51) to investigate the in vitro activity of prothioconazole and to consider the use of such compounds in the medical arena. Treatment of C. albicans cells with prothioconazole, prothioconazole-desthio, and voriconazole resulted in CYP51 inhibition, as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol. We then compared the inhibitor binding properties of prothioconazole, prothioconazole-desthio, and voriconazole with CaCYP51. We observed that prothioconazole-desthio and voriconazole bind noncompetitively to CaCYP51 in the expected manner of azole antifungals (with type II inhibitors binding to heme as the sixth ligand), while prothioconazole binds competitively and does not exhibit classic inhibitor binding spectra. Inhibition of CaCYP51 activity in a cell-free assay demonstrated that prothioconazole-desthio is active, whereas prothioconazole does not inhibit CYP51 activity. Extracts from C. albicans grown in the presence of prothioconazole were found to contain prothioconazole-desthio. We conclude that the antifungal action of prothioconazole can be attributed to prothioconazole-desthio.
PMCID: PMC3591943  PMID: 23275516
7.  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 
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.
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.
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.
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.
PMCID: PMC3403860  PMID: 22396476
Alzheimer's disease; Dementia diagnosis; Ethnicity; Language; Mini-Mental State Examination
8.  Preconception Omega-3 Fatty Acid Supplementation of Adult Male Mice with a History of Developmental TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) Exposure Prevents Preterm Birth in Unexposed Female Partners 
Reproduction (Cambridge, England)  2011;142(2):235-241.
We recently reported that adult male C57BL/6 mice exposed in utero to the environmental toxicant TCDD confer an increased risk of preterm birth (PTB) to unexposed females. Risk of PTB was coincident with decreased placental progesterone receptor (PR) mRNA expression and increased toll-like receptor-4 (TLR-4) mRNA expression, suggesting toxicant exposure induced a heightened inflammatory response at the maternal-fetal interface. Since omega-3 fatty acids exhibit anti-inflammatory activity, herein, we provided TCDD-exposed males a fish oil-enriched diet prior to mating. Although PTB was common in control females mated to TCDD-exposed males on the standard diet, fish oil supplementation of TCDD-exposed males eliminated PTB in unexposed partners. We also determined the influence of preconception, paternal fish oil supplementation on the placental inflammatory response in late pregnancy (E18.5) by examining expression of PR and TLR-4 mRNA as well as expression of 15-hydroxy prostaglandin dehydrogenase (PGDH). PGDH catabolizes the inflammatory PGE2 to an inactive form; thus, reduced expression of this enzyme would promote tissue inflammation. Compared to control pregnancies, examination of E18.5 placentas arising from TCDD-exposed males on the standard diet revealed a significant increase in TLR-4 mRNA expression corresponding to a reduction in PR mRNA and PGDH protein expression. In contrast, fish oil supplementation of toxicant-exposed males led to normalization of placental expression of both PR and TLR-4 mRNA and a marked increase in PGDH expression. These studies suggest that a paternal preconception diet which includes omega-3 fatty acids prevents the toxicant-associated increase in the placental inflammatory response at late gestation, preventing PTB.
PMCID: PMC3730265  PMID: 21653731
9.  Reliable Change on the Boston Naming Test 
Serial assessments are commonplace in neuropsychological practice and used to document cognitive trajectory for many clinical conditions. However, true change scores may be distorted by measurement error, repeated exposure to the assessment instrument, or person variables. The present study provides reliable change indices (RCI) for the Boston Naming Test, derived from a sample of 844 cognitively normal adults aged 56 years and older. All participants were retested between 9 and 24 months after their baseline exam. Results showed that a 4-point decline during a 9–15 month retest period or a 6-point decline during a 16–24 month retest period represents reliable change. These cutoff values were further characterized as a function of a person’s age and family history of dementia. These findings may help clinicians and researchers to characterize with greater precision the temporal changes in confrontation naming ability.
PMCID: PMC3617478  PMID: 22264406
BNT; RCI; Aging; Dementia; Serial; Assessment
10.  Diagnostic Validity of Age- And Education-Corrections for the Mini-Mental State Examination (MMSE) in African American Elders 
To investigate whether demographic (age and education) adjustments for the Mini-Mental State Examination (MMSE) attenuate mean score discrepancies between African American and Caucasian adults, and to determine whether demographically-adjusted MMSE scores improve the diagnostic classification accuracy of dementia in African American adults when compared to unadjusted MMSE scores.
Cross-sectional study.
Community-dwelling adults participating in the Mayo Clinic Alzheimer’s Disease Patient Registry (ADPR) and Alzheimer’s Disease Research Center (ADRC).
Three thousand two hundred fifty-four adults (2819 Caucasian, 435 African American) aged 60 and older.
MMSE at study entry.
African American adults obtained significantly lower unadjusted MMSE scores (23.0 ± 7.4) compared to Caucasian adults (25.3 ± 5.4). This discrepancy persisted despite adjustment of MMSE scores for age and years of education using established regression weights or newly-derived weights. However, controlling for dementia severity at baseline and adjusting MMSE scores for age and quality of education attenuated this discrepancy. Among African American adults, an age- and education-adjusted MMSE cut score of 23/24 provided optimal dementia classification accuracy, but this represented only a modest improvement over an unadjusted MMSE cut score of 22/23. The posterior probability of dementia in African American adults is presented for various unadjusted MMSE cut scores and prevalence rates of dementia.
Age, dementia severity at study entry, and quality of educational experience are important explanatory factors to understand the existing discrepancies in MMSE performance between Caucasian and African American adults. Our findings support the use of unadjusted MMSE scores when screening African American elders for dementia, with an unadjusted MMSE cut score of 22/23 yielding optimal classification accuracy.
PMCID: PMC3288600  PMID: 22150301
MMSE; African American; ethnicity; dementia; cognition
11.  Difficulty and Discrimination Parameters of Boston Naming Test Items in a Consecutive Clinical Series 
The Boston Naming Test is one of the most widely used neuropsychological instruments; yet, there has been limited use of modern psychometric methods to investigate its properties at the item level. The current study used Item response theory to examine each item's difficulty and discrimination properties, as well as the test's measurement precision across the range of naming ability. Participants included 300 consecutive referrals to the outpatient neuropsychology service at Mayo Clinic in Florida. Results showed that successive items do not necessarily reflect a monotonic increase in psychometric difficulty, some items are inadequate to distinguish individuals at various levels of naming ability, multiple items provide redundant psychometric information, and measurement precision is greatest for persons within a low-average range of ability. These findings may be used to develop short forms, improve reliability in future test versions by replacing psychometrically poor items, and analyze profiles of intra-individual variability.
PMCID: PMC3142950  PMID: 21593059
Boston Naming Test; Item response theory; Item difficulty; Item discriminability
12.  Immune interactions in endometriosis 
Endometriosis is a common, complex gynecologic disorder characterized by the presence of endometrial glands and stroma at extrauterine (ectopic) sites. In women who develop this disease, alterations in specific biological processes involving both the endocrine and immune systems have been observed, which may explain the survival and growth of displaced endometrial tissue in affected women. In the past decade, a considerable amount of research has implicated a role for alterations in progesterone action at both eutopic and ectopic sites of endometrial growth which may contribute to the excessive inflammation associated with progression of endometriosis; however, it remains unclear whether these anomalies induce the condition or are simply a consequence of the disease process. In this article, we summarize current knowledge of alterations within the immune system of endometriosis patients and discuss how endometrial cells from women with this disease not only have the capacity to escape immunosurveillance, but also use inflammatory mechanisms to promote their growth within the peritoneal cavity. Finally, we discuss evidence that exposure to an environmental endocrine disruptor, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, can mediate the development of an endometrial phenotype that exhibits both reduced progesterone responsiveness and hypersensitivity to proinflammatory stimuli mimicking the endometriosis phenotype. Future studies in women with endometriosis should consider whether a heightened inflammatory response within the peritoneal microenvironment contributes to the development and persistence of this disease.
PMCID: PMC3204940  PMID: 21895474
chemokines; cytokines; dioxin; endocrine-disrupting chemicals; endometriosis; estrogen; immune–endocrine interactions; inflammation; peritoneum; progesterone; TCDD
13.  Validation of the Mayo Sleep Questionnaire to Screen for REM Sleep Behavior Disorder in an Aging and Dementia Cohort 
Sleep medicine  2011;12(5):445-453.
To validate a questionnaire focused on REM sleep behavior disorder (RBD) among participants in an aging and dementia cohort.
RBD is a parasomnia that can develop in otherwise neurologically-normal adults as well as in those with a neurodegenerative disease. Confirmation of RBD requires polysomnography (PSG). A simple screening measure for RBD would be desirable for clinical and research purposes.
We had previously developed the Mayo Sleep Questionnaire (MSQ), a 16 item measure, to screen for the presence of RBD and other sleep disorders. We assessed the validity of the MSQ by comparing the responses of patients’ bed partners with the findings on PSG. All subjects recruited in the Mayo Alzheimer’s Disease Research Center at Mayo Clinic Rochester and Mayo Clinic Jacksonville from 1/00 to 7/08 who had also undergone a PSG were the focus of this analysis.
The study sample was comprised of 176 subjects [150 male; median age 71 years (range 39–90)], with the following clinical diagnoses: normal (n=8), mild cognitive impairment (n=44), Alzheimer’s disease (n=23), dementia with Lewy bodies (n=74), as well as other dementia and/or parkinsonian syndromes (n=27). The core question on recurrent dream enactment behavior yielded a sensitivity (SN) of 98% and specificity (SP) of 74% for the diagnosis of RBD. The profile of responses on four additional subquestions on RBD and one on obstructive sleep apnea improved specificity.
These data suggest that among aged subjects with cognitive impairment and/or parkinsonism, the MSQ has adequate SN and SP for the diagnosis of RBD. The utility of this scale in other patient populations will require further study.
PMCID: PMC3083495  PMID: 21349763
sleep disorders; parasomnias; dementia; Alzheimer’s disease; dementia with Lewy bodies; parkinsonism
14.  Association of common KIBRA variants with episodic memory and AD risk 
Neurobiology of aging  2010;32(3):557.e1-557.e9.
KIBRA SNP rs17070145 was identified in a GWAS of memory performance, with some but not all follow-up studies confirming association of its T allele with enhanced memory. This allele was associated with reduced Alzheimer's disease (AD) risk in one study, which also found overexpression of KIBRA in memory-related brain regions of ADs. We genotyped rs17070145 and 14 additional SNPs in 2571 LOADs vs. 2842 controls, including African-Americans. We found significantly reduced risk for rs17070145 T allele in the older African-American subjects (p=0.007) and a suggestive effect in the older Caucasian series. Meta-analysis of this allele in >8000 subjects from our and published series showed a suggestive protective effect (p=0.07). Analysis of episodic memory in control subjects did not identify associations with rs17070145, though other SNPs showed significant associations in one series. KIBRA showed evidence of overexpression in the AD temporal cortex (p=0.06) but not cerebellum. These results suggest a modest role for KIBRA as a cognition and AD risk gene, and also highlight the multifactorial complexity of its genetic associations.
PMCID: PMC3065956  PMID: 21185624
Alzheimer's disease; Association studies in genetics; Case control studies
15.  Characterization of frontotemporal dementia and/or amyotrophic lateral sclerosis associated with the GGGGCC repeat expansion in C9ORF72 
Brain  2012;135(3):765-783.
Numerous kindreds with familial frontotemporal dementia and/or amyotrophic lateral sclerosis have been linked to chromosome 9, and an expansion of the GGGGCC hexanucleotide repeat in the non-coding region of chromosome 9 open reading frame 72 has recently been identified as the pathogenic mechanism. We describe the key characteristics in the probands and their affected relatives who have been evaluated at Mayo Clinic Rochester or Mayo Clinic Florida in whom the hexanucleotide repeat expansion were found. Forty-three probands and 10 of their affected relatives with DNA available (total 53 subjects) were shown to carry the hexanucleotide repeat expansion. Thirty-six (84%) of the 43 probands had a familial disorder, whereas seven (16%) appeared to be sporadic. Among examined subjects from the 43 families (n = 63), the age of onset ranged from 33 to 72 years (median 52 years) and survival ranged from 1 to 17 years, with the age of onset <40 years in six (10%) and >60 in 19 (30%). Clinical diagnoses among examined subjects included behavioural variant frontotemporal dementia with or without parkinsonism (n = 30), amyotrophic lateral sclerosis (n = 18), frontotemporal dementia/amyotrophic lateral sclerosis with or without parkinsonism (n = 12), and other various syndromes (n = 3). Parkinsonism was present in 35% of examined subjects, all of whom had behavioural variant frontotemporal dementia or frontotemporal dementia/amyotrophic lateral sclerosis as the dominant clinical phenotype. No subject with a diagnosis of primary progressive aphasia was identified with this mutation. Incomplete penetrance was suggested in two kindreds, and the youngest generation had significantly earlier age of onset (>10 years) compared with the next oldest generation in 11 kindreds. Neuropsychological testing showed a profile of slowed processing speed, complex attention/executive dysfunction, and impairment in rapid word retrieval. Neuroimaging studies showed bilateral frontal abnormalities most consistently, with more variable degrees of parietal with or without temporal changes; no case had strikingly focal or asymmetric findings. Neuropathological examination of 14 patients revealed a range of transactive response DNA binding protein molecular weight 43 pathology (10 type A and four type B), as well as ubiquitin-positive cerebellar granular neuron inclusions in all but one case. Motor neuron degeneration was detected in nine patients, including five patients without ante-mortem signs of motor neuron disease. While variability exists, most cases with this mutation have a characteristic spectrum of demographic, clinical, neuropsychological, neuroimaging and especially neuropathological findings.
PMCID: PMC3286335  PMID: 22366793
frontotemporal dementia; amyotrophic lateral sclerosis; motor neuron disease; TDP-43; neurogenetics; chromosome 9
16.  Impact of Recently Emerged Sterol 14α-Demethylase (CYP51) Variants of Mycosphaerella graminicola on Azole Fungicide Sensitivity▿ 
Applied and Environmental Microbiology  2011;77(11):3830-3837.
The progressive decline in the effectiveness of some azole fungicides in controlling Mycosphaerella graminicola, causal agent of the damaging Septoria leaf blotch disease of wheat, has been correlated with the selection and spread in the pathogen population of specific mutations in the M. graminicola CYP51 (MgCYP51) gene encoding the azole target sterol 14α-demethylase. Recent studies have suggested that the emergence of novel MgCYP51 variants, often harboring substitution S524T, has contributed to a decrease in the efficacy of prothioconazole and epoxiconazole, the two currently most effective azole fungicides against M. graminicola. In this study, we establish which amino acid alterations in novel MgCYP51 variants have the greatest impact on azole sensitivity and protein function. We introduced individual and combinations of identified alterations by site-directed mutagenesis and functionally determined their impact on azole sensitivity by expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a regulatable promoter controlling native CYP51 expression. We demonstrate that substitution S524T confers decreased sensitivity to all azoles when introduced alone or in combination with Y461S. In addition, S524T restores the function in S. cerevisiae of MgCYP51 variants carrying the otherwise lethal alterations Y137F and V136A. Sensitivity tests of S. cerevisiae transformants expressing recently emerged MgCYP51 variants carrying combinations of alterations D134G, V136A, Y461S, and S524T reveal a substantial impact on sensitivity to the currently most widely used azoles, including epoxiconazole and prothioconazole. Finally, we exploit a recently developed model of the MgCYP51 protein to predict that the substantial structural changes caused by these novel combinations reduce azole interactions with critical residues in the binding cavity, thereby causing resistance.
PMCID: PMC3127603  PMID: 21478305
17.  Continuous Three-Dimensional Control of a Virtual Helicopter Using a Motor Imagery Based Brain-Computer Interface 
PLoS ONE  2011;6(10):e26322.
Brain-computer interfaces (BCIs) allow a user to interact with a computer system using thought. However, only recently have devices capable of providing sophisticated multi-dimensional control been achieved non-invasively. A major goal for non-invasive BCI systems has been to provide continuous, intuitive, and accurate control, while retaining a high level of user autonomy. By employing electroencephalography (EEG) to record and decode sensorimotor rhythms (SMRs) induced from motor imaginations, a consistent, user-specific control signal may be characterized. Utilizing a novel method of interactive and continuous control, we trained three normal subjects to modulate their SMRs to achieve three-dimensional movement of a virtual helicopter that is fast, accurate, and continuous. In this system, the virtual helicopter's forward-backward translation and elevation controls were actuated through the modulation of sensorimotor rhythms that were converted to forces applied to the virtual helicopter at every simulation time step, and the helicopter's angle of left or right rotation was linearly mapped, with higher resolution, from sensorimotor rhythms associated with other motor imaginations. These different resolutions of control allow for interplay between general intent actuation and fine control as is seen in the gross and fine movements of the arm and hand. Subjects controlled the helicopter with the goal of flying through rings (targets) randomly positioned and oriented in a three-dimensional space. The subjects flew through rings continuously, acquiring as many as 11 consecutive rings within a five-minute period. In total, the study group successfully acquired over 85% of presented targets. These results affirm the effective, three-dimensional control of our motor imagery based BCI system, and suggest its potential applications in biological navigation, neuroprosthetics, and other applications.
PMCID: PMC3202533  PMID: 22046274
18.  Regulation of Invasive Behavior by Vascular Endothelial Growth Factor is HEF1-dependent 
Oncogene  2010;29(31):4449-4459.
We previously reported a VEGF autocrine loop in head and neck squamous cell carcinoma (HNSCC) cell lines, supporting a role for VEGF in HNSCC tumorigenesis. Using a phosphotyrosine proteomics approach we screened the HNSCC cell line, SCC-9 for effectors of VEGFR2 signaling. A cluster of proteins involved in cell migration and invasion, including the p130Cas paralog, human enhancer of filamentation1 (HEF1/Cas-L/Nedd9) was identified. HEF1 silencing and overexpression studies revealed a role for VEGF in regulating cell migration, invasion, and MMP expression in a HEF1-dependent manner. Moreover, cells plated on extracellular matrix coated coverslips exhibited enhanced invadopodia formation in response to VEGF that was HEF1-dependent. Immunolocalization revealed that HEF1 colocalized to invadopodia with MT1-MMP. Analysis of HNSCC tissue microarrays for HEF1 immunoreactivity revealed a 6.5-fold increase in the odds of having a metastasis with a high HEF1 score compared to a low HEF1 score. These findings suggest that HEF1 may be prognostic for advanced stage HNSCC. They also demonstrate for the first time, that HEF1 is required for VEGF-mediated HNSCC cell migration and invasion, consistent with HEF1’s recent identification as a metastatic regulator. These results support a strategy targeting VEGF:VEGFR2 in HNSCC therapeutics.
PMCID: PMC2921319  PMID: 20498643
VEGF; Invadopodia; Cell migration; invasion; HEF1; NEDD9; CAS-L; tyrosine phosphorylation
19.  Robust and Expanded Norms for the Dementia Rating Scale 
The Dementia Rating Scale (DRS) is a widely used measure of global cognition, with age- and education-corrected norms derived from a cross-sectional sample of adults participating in Mayo's Older Americans Normative Studies (MOANS). In recent years, however, studies have indicated that cross-sectional normative samples of older adults represent an admixture of individuals who are indeed cognitively normal (i.e., disease-free) and individuals with incipient neurodegenerative disease. Theoretically, the “contamination” of cross-sectional normative samples with cases of preclinical dementia can lead to underestimation of the test mean and overestimation of the variance, thus reducing the clinical utility of the norms. Robust norming, in which dementia cases are removed from the normative cohort through longitudinal follow-up, is an alternative approach to norm development. The current study presents a reappraisal of the original MOANS DRS norms, provides robust and expanded norms based on a sample of 894 adults age 55 and over, and critically evaluates the benefits of robust norming.
PMCID: PMC2904669  PMID: 20427376
Dementia Rating Scale; DRS; Alzheimer's disease; Robust; Norms
20.  Mechanism of Binding of Prothioconazole to Mycosphaerella graminicola CYP51 Differs from That of Other Azole Antifungals ▿  
Prothioconazole is one of the most important commercially available demethylase inhibitors (DMIs) used to treat Mycosphaerella graminicola infection of wheat, but specific information regarding its mode of action is not available in the scientific literature. Treatment of wild-type M. graminicola (strain IPO323) with 5 μg of epoxiconazole, tebuconazole, triadimenol, or prothioconazole ml−1 resulted in inhibition of M. graminicola CYP51 (MgCYP51), as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol in azole-treated cells. Successful expression of MgCYP51 in Escherichia coli enabled us to conduct spectrophotometric assays using purified 62-kDa MgCYP51 protein. Antifungal-binding studies revealed that epoxiconazole, tebuconazole, and triadimenol all bound tightly to MgCYP51, producing strong type II difference spectra (peak at 423 to 429 nm and trough at 406 to 409 nm) indicative of the formation of classical low-spin sixth-ligand complexes. Interaction of prothioconazole with MgCYP51 exhibited a novel spectrum with a peak and trough observed at 410 nm and 428 nm, respectively, indicating a different mechanism of inhibition. Prothioconazole bound to MgCYP51 with 840-fold less affinity than epoxiconazole and, unlike epoxiconazole, tebuconazole, and triadimenol, which are noncompetitive inhibitors, prothioconazole was found to be a competitive inhibitor of substrate binding. This represents the first study to validate the effect of prothioconazole on the sterol composition of M. graminicola and the first on the successful heterologous expression of active MgCYP51 protein. The binding affinity studies documented here provide novel insights into the interaction of MgCYP51 with DMIs, especially for the new triazolinethione derivative prothioconazole.
PMCID: PMC3067226  PMID: 21169436
21.  Molecular Modelling of the Emergence of Azole Resistance in Mycosphaerella graminicola 
PLoS ONE  2011;6(6):e20973.
A structural rationale for recent emergence of azole (imidazole and triazole) resistance associated with CYP51 mutations in the wheat pathogen Mycosphaerella graminicola is presented, attained by homology modelling of the wild type protein and 13 variant proteins. The novel molecular models of M. graminicola CYP51 are based on multiple homologues, individually identified for each variant, rather than using a single structural scaffold, providing a robust structure-function rationale for the binding of azoles, including important fungal specific regions for which no structural information is available. The wild type binding pocket reveals specific residues in close proximity to the bound azole molecules that are subject to alteration in the variants. This implicates azole ligands as important agents exerting selection on specific regions bordering the pocket, that become the focus of genetic mutation events, leading to reduced sensitivity to that group of related compounds. Collectively, the models account for several observed functional effects of specific alterations, including loss of triadimenol sensitivity in the Y137F variant, lower sensitivity to tebuconazole of I381V variants and increased resistance to prochloraz of V136A variants. Deletion of Y459 and G460, which brings about removal of that entire section of beta turn from the vicinity of the binding pocket, confers resistance to tebuconazole and epoxiconazole, but sensitivity to prochloraz in variants carrying a combination of A379G I381V ΔY459/G460. Measurements of binding pocket volume proved useful in assessment of scope for general resistance to azoles by virtue of their accommodation without bonding interaction, particularly when combined with analysis of change in positions of key amino acids. It is possible to predict the likely binding orientation of an azole molecule in any of the variant CYPs, providing potential for an in silico screening system and reliable predictive approach to assess the probability of particular variants exhibiting resistance to particular azole fungicides.
PMCID: PMC3124474  PMID: 21738598
22.  Mild cognitive impairment associated with limbic and neocortical lewy body disease: a clinicopathological study 
Brain  2009;133(2):540-556.
There are little data on the relationship between Lewy body disease and mild cognitive impairment syndromes. The Mayo Clinic aging and dementia databases in Rochester, Minnesota, and Jacksonville, Florida were queried for cases who were diagnosed with mild cognitive impairment between 1 January 1996 and 30 April 2008, were prospectively followed and were subsequently found to have autopsy-proven Lewy body disease. The presence of rapid eye movement sleep behaviour disorder was specifically assessed. Mild cognitive impairment subtypes were determined by clinical impression and neuropsychological profiles, based on prospective operational criteria. The diagnosis of clinically probable dementia with Lewy bodies was based on the 2005 McKeith criteria. Hippocampal volumes, rate of hippocampal atrophy, and proton magnetic resonance spectroscopy were assessed on available magnetic resonance imaging and spectroscopy scans. Eight subjects were identified; six were male. Seven developed dementia with Lewy bodies prior to death; one died characterized as mild cognitive impairment. The number of cases and median age of onset (range) for specific features were: seven with rapid eye movement sleep behaviour disorder—60 years (27–91 years), eight with cognitive symptoms—69 years (62–89 years), eight with mild cognitive impairment—70.5 years (66–91 years), eight with parkinsonism symptoms—71 years (66–92 years), six with visual hallucinations—72 years (64–90 years), seven with dementia—75 years (67–92 years), six with fluctuations in cognition and/or arousal—76 years (68–92 years) and eight dead—76 years (71–94 years). Rapid eye movement sleep behaviour disorder preceded cognitive symptom onset in six cases by a median of 10 years (2–47 years) and mild cognitive impairment diagnosis by a median of 12 years (3–48 years). The mild cognitive impairment subtypes represented include: two with single domain non-amnestic mild cognitive impairment, three with multi-domain non-amnestic mild cognitive impairment, and three with multi-domain amnestic mild cognitive impairment. The cognitive domains most frequently affected were attention and executive functioning, and visuospatial functioning. Hippocampal volumes and the rate of hippocampal atrophy were, on average, within the normal range in the three cases who underwent magnetic resonance imaging, and the choline/creatine ratio was elevated in the two cases who underwent proton magnetic resonance spectroscopy when they were diagnosed as mild cognitive impairment. On autopsy, six had neocortical-predominant Lewy body disease and two had limbic-predominant Lewy body disease; only one had coexisting high-likelihood Alzheimer's disease. These findings indicate that among Lewy body disease cases that pass through a mild cognitive impairment stage, any cognitive pattern or mild cognitive subtype is possible, with the attention/executive and visuospatial domains most frequently impaired. Hippocampal volume and proton magnetic resonance spectroscopy data were consistent with recent data in dementia with Lewy bodies. All cases with rapid eye movement sleep behaviour disorder and mild cognitive impairment were eventually shown to have autopsy-proven Lewy body disease, indicating that rapid eye movement sleep behaviour disorder plus mild cognitive impairment probably reflects brainstem and cerebral Lewy body disease.
PMCID: PMC2822633  PMID: 19889717
mild cognitive impairment; dementia; dementia with Lewy bodies; Lewy body disease; neuropathology
23.  Internal Consistency and Test-Retest Stability of the Geriatric Depression Scale-Short Form in African American Older Adults 
The Geriatric Depression Scale (GDS) is one of the most widely used self-rated mood questionnaires for older adults. It is highly correlated with clinical diagnoses of depression and has demonstrated validity across different patient populations. However, the reliability of the GDS among African American older adults remains to be firmly established. In a baseline sample of 401 African American adults age 51 and over, the GDS-15 item short form demonstrates good internal consistency (KR20=.71). Stability over a 15-month interval in a retest sample of 51 adults is deemed adequate (r=.68). These findings support the use of the GDS-15 item short form as a reliable mood questionnaire among African American older adults.
PMCID: PMC2794552  PMID: 20161488
Geriatric depression scale; GDS; Depression; African American; Reliability
24.  Verbal Learning and Memory in Patients with Dementia with Lewy Bodies or Parkinson's Disease with Dementia 
This study compared verbal learning and memory in patients with autopsy-confirmed dementia with Lewy Bodies (DLB) and patients with Parkinson's disease with dementia (PDD). Twenty-four DLB patients, 24 PDD patients, and 24 normal comparison participants were administered the California Verbal Learning Test. The three groups were matched on demographic variables and the two patient groups were matched on the Mattis Dementia Rating Scale. The results indicated that DLB patients recalled less information than PDD patients on all but one recall measure and displayed a more rapid rate of forgetting. In contrast, the PDD patients committed a greater percent of perseveration errors than the DLB patients. The two groups did not differ in the percentage of recall intrusion errors or any measures of recognition. A discriminant function analysis (DFA) using short delay cued recall, percent perseveration errors, and list b recall, differentiated the DLB and PDD groups with 81.3% accuracy. The application of the DFA algorithm to another sample of 42 PDD patients resulted in a 78.6% correct classification rate. The results suggest that, despite equivalent levels of general cognitive impairment, patients with DLB or PDD exhibit a different pattern of verbal learning and memory deficits.
PMCID: PMC2935683  PMID: 19221922
25.  Differential Item Functioning of the Boston Naming Test in Cognitively Normal African American and Caucasian Older Adults 
Scores on the Boston Naming Test (BNT) are frequently lower for African American when compared to Caucasian adults. Although demographically-based norms can mitigate the impact of this discrepancy on the likelihood of erroneous diagnostic impressions, a growing consensus suggests that group norms do not sufficiently address or advance our understanding of the underlying psychometric and sociocultural factors that lead to between-group score discrepancies. Using item response theory and methods to detect differential item functioning (DIF), the current investigation moves beyond comparisons of the summed total score to examine whether the conditional probability of responding correctly to individual BNT items differs between African American and Caucasian adults. Participants included 670 adults age 52 and older who took part in Mayo's Older Americans and Older African Americans Normative Studies. Under a 2-parameter logistic IRT framework and after correction for the false discovery rate, 12 items where shown to demonstrate DIF. Six of these 12 items (“dominoes,” “escalator,” “muzzle,” “latch,” “tripod,” and “palette”) were also identified in additional analyses using hierarchical logistic regression models and represent the strongest evidence for race/ethnicity-based DIF. These findings afford a finer characterization of the psychometric properties of the BNT and expand our understanding of between-group performance.
PMCID: PMC2835360  PMID: 19570311
Boston Naming Test; Item response theory; Differential item functioning; Ethnicity; Race; Bias

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