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1.  Host iron status and iron supplementation mediate susceptibility to erythrocytic stage Plasmodium falciparum 
Nature communications  2014;5:4446.
Iron deficiency and malaria have similar global distributions, and frequently co-exist in pregnant women and young children. Where both conditions are prevalent, iron supplementation is complicated by observations that iron deficiency anaemia protects against falciparum malaria, and that iron supplements increase susceptibility to clinically significant malaria, but the mechanisms remain obscure. Here, using an in vitro parasite culture system with erythrocytes from iron-deficient and replete human donors, we demonstrate that Plasmodium falciparum infects iron-deficient erythrocytes less efficiently. In addition, owing to merozoite preference for young erythrocytes, iron supplementation of iron-deficient individuals reverses the protective effects of iron deficiency. Our results provide experimental validation of field observations reporting protective effects of iron deficiency and harmful effects of iron administration on human malaria susceptibility. Because recovery from anaemia requires transient reticulocytosis, our findings imply that in malarious regions iron supplementation should be accompanied by effective measures to prevent falciparum malaria.
PMCID: PMC4249681  PMID: 25059846
2.  Robust Affinity Standards for Cu(I) Biochemistry 
Journal of the American Chemical Society  2013;135(49):18549-18559.
The measurement of reliable Cu(I) protein binding affinities requires competing reference ligands with similar binding strengths; however, the literature on such reference ligands is not only sparse but often conflicting. To address this deficiency, we have created and characterized a series of water-soluble monovalent copper ligands, MCL-1, MCL-2, and MCL-3, that form well-defined, air-stable, and colorless complexes with Cu(I) in aqueous solution. Concluding from X-ray structural data, electrochemical measurements, and an extensive network of equilibrium titrations, all three ligands form discrete Cu(I) complexes with 1:1 stoichiometry and are capable of buffering Cu(I) concentrations between 10−10 and 10−17 M. As most Cu(I) protein affinities have been obtained from competition experiments with bathocuproine disulfonate (BCS) or 2,2′-bicinchoninic acid (BCA), we further calibrated their Cu(I) stability constants against the MCL-series. To demonstrate the application of these reagents, we determined the Cu(I) binding affinity of CusF (logK = 14.3±0.1), a periplasmic metalloprotein required for the detoxification of elevated copper levels in E. coli. Altogether, this interconnected set of affinity standards establishes a reliable foundation that will facilitate the precise determination of Cu(I) binding affinities of proteins and small molecule ligands.
PMCID: PMC3983694  PMID: 24298878
copper; metallochaperone; stability constant; metal buffer
4.  An International, Prospective, Multicenter Evaluation of the Combination of AdvanDx Staphylococcus QuickFISH BC with mecA XpressFISH for Detection of Methicillin-Resistant Staphylococcus aureus Isolates from Positive Blood Cultures 
Journal of Clinical Microbiology  2014;52(11):3928-3932.
Sepsis caused by Staphylococcus aureus is a major health problem worldwide. Better outcomes are achieved when rapid diagnosis and determination of methicillin susceptibility enable early optimization of antimicrobial therapy. Eight large clinical laboratories, seven from the United States and one from Scotland, evaluated the combination of the Staphylococcus QuickFISH BC and the new mecA XpressFISH assay (both AdvanDx, Woburn, MA, USA) for the detection of methicillin-resistant S. aureus in positive blood cultures. Blood cultures flagged as positive by automated blood culture instruments and demonstrating only Gram-positive cocci in clusters on Gram stain were tested by QuickFISH, a 20-min assay. If only S. aureus was detected, mecA XpressFISH testing followed. The recovered S. aureus isolates were tested by cefoxitin disk diffusion as the reference method. The QuickFISH assay results were concordant with the routine phenotypic testing methods of the testing laboratories in 1,211/1,221 (99.1%) samples and detected 488/491 S. aureus organisms (sensitivity, 99.4%; specificity, 99.6%). Approximately 60% of the samples (730) contained coagulase-negative staphylococci or nonstaphylococci as assessed by the QuickFISH assay and were not tested further. The 458 compliant samples positive exclusively for S. aureus by the QuickFISH assay were tested by the mecA XpressFISH assay, which detected 209 of 211 methicillin-resistant S. aureus organisms (sensitivity, 99.1%; specificity, 99.6%). The mecA XpressFISH assay also showed high reproducibility, with 534/540 tests performed by 6 operators over 5 days achieving reproducible results (98.9% agreement). The combination of the Staphylococcus QuickFISH BC and mecA XpressFISH assays is sensitive, specific, and reproducible for the detection of methicillin-resistant S. aureus and yields complete results in 2 h after the blood culture turns positive.
PMCID: PMC4313227  PMID: 25165083
5.  The Poggendorff illusion: a bias in the estimation of the orientation of virtual lines by second-stage filters 
Vision research  1999;39(14):2361-2380.
The veridical perception of collinearity between two separated lines is distorted by two parallel lines in the space between them (the Poggendorff illusion). This paper tests the conjecture that the perception of collinearity of separated lines is based on a two-stage mechanism. The first stage encodes the orientation of the virtual line between the proximal terminators of the target lines. The second stage compares this virtual orientation with the orientation of the target lines themselves. Errors can and do arise from either process. Two parallel lines, abutting against the target lines, cause the classical Poggendorff misalignment bias. The magnitude of the bias is increased by Gaussian blur, as is a version of the Poggendorff figure containing only acute angles. In the obtuse-angle figure, on the other hand, blur decreases the misalignment bias. We argue that the acute- and obtuse-angle biases depend upon different mechanisms, and that the obtuse-angle effect is more related to the obtuse-angle version of the Muller–Lyer illusion, which is also decreased by blur. If observers attempt to match the orientation of the virtual line between the two line intersections in the Poggendorff figure they make an error in the same direction as the Poggendorff bias. The orientation of the target lines in the figure, however, is veridically matched to a Gabor-patch probe, unless the target lines are very short, in which case the error is in the same direction as the Poggendorff bias. A small bend in the target lines where they abut the parallels increases the Poggendorff bias if it makes the line more orthogonal to the parallel, but has little effect in the opposite direction. The Poggendorff bias is unlikely to depend upon biases in first-stage linear filters because (a) it still exists in figures composed of short, luminance-balanced lines which are defined by contrast only; and (b) it also exists if the parallels are replaced by grating patches with the same mean luminance as the background. The orientation of the grating in the latter case affects the magnitude of the bias, but even an orientation which should reverse the Poggendorff bias by the mechanism of cross-orientation inhibition fails to do so. The Poggendorff bias is a complex effect arising from several sources. Blurring in second-stage filters with large receptive fields can explain many aspects of the phenomenon.
PMCID: PMC4213454  PMID: 10367057
Poggendorff bias; Muller–Lyer illusion; Gaussian blur
6.  A texture-processing model of the ‘visual sense of number’ 
Proceedings. Biological sciences / The Royal Society  2014;281(1790):10.1098/rspb.2014.1137 20141137.
It has been suggested that numerosity is an elementary quality of perception, similar to colour. If so (and despite considerable investigation), its mechanism remains unknown. Here, we show that observers require on average a massive difference of approximately 40% to detect a change in the number of objects that vary irrelevantly in blur, contrast and spatial separation, and that some naive observers require even more than this. We suggest that relative numerosity is a type of texture discrimination and that a simple model computing the contrast energy at fine spatial scales in the image can perform at least as well as human observers. Like some human observers, this mechanism finds it harder to discriminate relative numerosity in two patterns with different degrees of blur, but it still outpaces the human. We propose energy discrimination as a benchmark model against which more complex models and new data can be tested.
PMCID: PMC4123707  PMID: 25030988
psychophysics; numerosity; texture perception
7.  A texture-processing model of the ‘visual sense of number’ 
It has been suggested that numerosity is an elementary quality of perception, similar to colour. If so (and despite considerable investigation), its mechanism remains unknown. Here, we show that observers require on average a massive difference of approximately 40% to detect a change in the number of objects that vary irrelevantly in blur, contrast and spatial separation, and that some naive observers require even more than this. We suggest that relative numerosity is a type of texture discrimination and that a simple model computing the contrast energy at fine spatial scales in the image can perform at least as well as human observers. Like some human observers, this mechanism finds it harder to discriminate relative numerosity in two patterns with different degrees of blur, but it still outpaces the human. We propose energy discrimination as a benchmark model against which more complex models and new data can be tested.
PMCID: PMC4123707  PMID: 25030988
psychophysics; numerosity; texture perception
Experimental parasitology  2013;135(1):10.1016/j.exppara.2013.06.012.
Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine produced by many mammalian tissues including skin. It is also found in many invertebrate parasites of mammals including ticks and may function to aid the parasite to evade the innate and adaptive immune responses in the host. In this study, the cDNA for a MIF gene was sequenced from Sarcoptes scabiei, the scabies mite, using RT-PCR and RACE molecular techniques. The resulting nucleotide sequence had a length of 405 base pairs and the putative amino acid sequences for the mite and tick (Dermacentor variabilis) proteins were identical. The initial steps for the project resulted in the production of expressed scabies mite cDNAs. A real time (qPCR) assay was performed with MIF from scabies mites and various tick species. Results show that mRNA encoding MIF homologues was three times more abundant in the mite samples when compared to RNA prepared from D. variabilis salivary glands and 1.3 times more abundant when compared with RNA prepared from D. variabilis midgut.
PMCID: PMC3869457  PMID: 23831036
Sarcoptes scabiei; Macrophage migration inhibitory factor; scabies; tick; Dermacentor variabilis
9.  Linking HIV+ adolescents into care: The effects of relationships between local health departments and adolescent medicine clinics 
Journal of HIV/AIDS & social services  2013;12(3-4):10.1080/15381501.2013.817280.
The fragmentation of HIV-related diagnostic and treatment services, especially for youth, is a significant barrier for transitioning to care. The study identified key elements that affected care linkage efforts.
We conducted 64 interviews across 15 clinical sites. The constant comparative method was used.
Primary linkage to care processes are illustrated through three geographically diverse case studies. Factors included: inter-agency relationships, data sharing protocols, and service duplication concerns. Program improvement strategies were discussed.
A strong, citywide network is helpful in coordinating care linkage services. These partnerships will be critical in effectively realizing the goals of the National HIV/AIDS.
PMCID: PMC3835468  PMID: 24273461
Adolescents; HIV; Linkage to care; health departments
10.  “Youth friendly” clinics: Considerations for linking and engaging HIV-infected adolescents into care 
AIDS care  2013;26(2):199-205.
Linkage and engagement in care are critical corollaries to the health of HIV-infected adolescents. The adolescent HIV epidemic and adolescents’ unique barriers to care necessitates innovation in the provision of care, including the consideration of the clinical experience. Little research has addressed how “youth friendly” clinics may influence care retention for HIV-infected youth. We conducted 124 interviews with providers, outreach workers, and case managers, at 15 Adolescent Medicine Trials Network clinics. Photographs of each clinic documented the characteristics of the physical space. Constant comparison and content and visual narrative methods were utilized for data analysis. Three elements of youth friendliness were identified for clinics serving HIV-infected youth, including: (1) role of target population (e.g., pediatric, adolescent, HIV); (2) clinics’ physical environment; and (3) clinics’ social environment. Working to create ‘youth friendly’ clinics through changes in physical (e.g., space, entertainment, and educational materials) and social (e.g., staff training related to development, gender, sexual orientation) environments may help reduce HIV-infected adolescents’ unique barriers to care engagement. The integration of clinic design and staff training within the organization of a clinical program is helpful in meeting the specialized needs of HIV-infected youth.
PMCID: PMC4106414  PMID: 23782040
adolescents; HIV care; youth friendly; clinics; qualitative research
11.  RBC Barcoding Allows for the Study of Erythrocyte Population Dynamics and P. falciparum Merozoite Invasion 
PLoS ONE  2014;9(7):e101041.
Plasmodium falciparum invasion of host erythrocytes is essential for the propagation of the blood stage of malaria infection. Additionally, the brief extracellular merozoite stage of P. falciparum represents one of the rare windows during which the parasite is directly exposed to the host immune response. Therefore, efficient invasion of the host erythrocyte is necessary not only for productive host erythrocyte infection, but also for evasion of the immune response. Host traits, such as hemoglobinopathies and differential expression of erythrocyte invasion ligands, can protect individuals from malaria by impeding parasite erythrocyte invasion. Here we combine RBC barcoding with flow cytometry to study P. falciparum invasion. This novel high-throughput method allows for the (i) direct comparison of P. falciparum invasion into different erythrocyte populations and (ii) assessment of the impact of changing erythrocyte population dynamics on P. falciparum invasion.
PMCID: PMC4077748  PMID: 24984000
12.  Independent Lineages of Highly Sulfadoxine-Resistant Plasmodium falciparum Haplotypes, Eastern Africa 
Emerging Infectious Diseases  2014;20(7):1140-1148.
Parasites with increased resistance to sulfadoxine might undermine malaria control measures.
Sulfadoxine-resistant Plasmodium falciparum undermines malaria prevention with sulfadoxine/pyrimethamine. Parasites with a highly resistant mutant dihydropteroate synthase (dhps) haplotype have recently emerged in eastern Africa; they negated preventive benefits of sulfadoxine/pyrimethamine, and might exacerbate placental malaria. We explored emerging lineages of dhps mutant haplotypes in Malawi, the Democratic Republic of the Congo, and Tanzania by using analyses of genetic microsatellites flanking the dhps locus. In Malawi, a triple-mutant dhps SGEG (mutant amino acids are underlined) haplotype emerged in 2010 that was closely related to pre-existing double-mutant SGEA haplotypes, suggesting local origination in Malawi. When we compared mutant strains with parasites from the Democratic Republic of the Congo and Tanzania by multiple independent analyses, we found that SGEG parasites were partitioned into separate lineages by country. These findings support a model of local origination of SGEG dhps haplotypes, rather than geographic diffusion, and have implications for investigations of emergence and effects of parasite drug resistance.
PMCID: PMC4073871  PMID: 24960247
malaria; Plasmodium falciparum; parasites; sulfadoxine; drug resistance; lineages; genetics; haplotypes; population; eastern Africa
13.  Influence of host iron status on Plasmodium falciparum infection 
Iron deficiency affects one quarter of the world's population and causes significant morbidity, including detrimental effects on immune function and cognitive development. Accordingly, the World Health Organization (WHO) recommends routine iron supplementation in children and adults in areas with a high prevalence of iron deficiency. However, a large body of clinical and epidemiological evidence has accumulated which clearly demonstrates that host iron deficiency is protective against falciparum malaria and that host iron supplementation may increase the risk of malaria. Although many effective antimalarial treatments and preventive measures are available, malaria remains a significant public health problem, in part because the mechanisms of malaria pathogenesis remain obscured by the complexity of the relationships that exist between parasite virulence factors, host susceptibility traits, and the immune responses that modulate disease. Here we review (i) the clinical and epidemiological data that describes the relationship between host iron status and malaria infection and (ii) the current understanding of the biological basis for these clinical and epidemiological observations.
PMCID: PMC4018558  PMID: 24834053
malaria; iron; iron deficiency anemia; Plasmodium falciparum; iron supplementation
14.  Chronic dietary magnesium-L-threonate speeds extinction and reduces spontaneous recovery of a conditioned taste aversion 
Elevation of brain magnesium enhances synaptic plasticity and extinction of conditioned fear memories. This experiment examined the generalizability of this phenomenon by studying the effects of a novel magnesium compound, magnesium-L-threonate (MgT), on conditioned taste aversion (CTA) extinction and spontaneous recovery (SR). Adult male Sprague-Dawley rats were maintained on a 23-hour water deprivation cycle and acquired a CTA following the taste of a CS [0.3% saccharin + 16mg/ml MgT (SAC+MgT)] paired with a US [81 mg/kg (i.p.) Lithium Chloride (LiCl)]. Following CTA acquisition, rats drank a water + MgT solution for up to 1 hour/day over the next 31 days. For 14 additional days, some animals continued water + MgT treatment, but others drank water only to allow MgT to be eliminated from the body. We then employed 2 different extinction paradigms: (1) CS-Only (CSO), in which SAC was presented, every-other day, or (2) Explicitly Unpaired (EU), in which both SAC and LiCl were presented, but on alternate days. EU extinction procedures have been shown to speed CTA extinction and reduce spontaneous recovery of the aversion. Throughout extinction, half of the rats in each group continued to drink MgT (now in SAC or supplemental water+MgT solution), whereas the other half drank SAC only/water only until SAC drinking reached ≥ 90% of baseline (asymptotic extinction). Rats receiving MgT just before/during extinction drank less SAC on the first day of extinction suggesting that they had retained a stronger CTA. MgT enhanced the rate of extinction. Furthermore, the MgT-treated rats showed a relatively modest SR of the CTA 30 days later – indicating that the extinction procedure was more effective for these animals. Our data suggest that long-term dietary MgT may enhance the consolidation/retention of a CTA, speed extinction, and inhibit SR of this learned aversion.
PMCID: PMC3668337  PMID: 23474371
magnesium-L-threonate; conditioned taste aversion; extinction; spontaneous recovery; CTA; fear; magnesium
15.  Review article: the design of clinical trials in hepatic encephalopathy - an International Society for Hepatic Encephalopathy and Nitrogen Metabolism (ISHEN) consensus statement 
The clinical classification of hepatic encephalopathy is largely subjective, which has led to difficulties in designing trials in this field.
To review the current classification of hepatic encephalopathy and to develop consensus guidelines on the design and conduct of future clinical trials.
A round table was convened at the 14th International Society for Hepatic Encephalopathy and Nitrogen Metabolism (ISHEN) meeting. Key discussion points were the nomenclature of hepatic encephalopathy and the selection of patients, standards of care and end-points for assessing the treatment and secondary prevention of hepatic encephalopathy.
It was generally agreed that severity assessment of hepatic encephalopathy in patients with cirrhosis, whether made clinically or more objectively, should be continuous rather than categorical, and a system for assessing the SONIC (Spectrum of Neuro-cognitive Impairment in Cirrhosis) was proposed. Within this system, patients currently classified as having minimal hepatic encephalopathy and Grade I hepatic encephalopathy would be classified as having Covert hepatic encephalopathy, whereas those with apparent clinical abnormalities would continue to be classified as overt hepatic encephalopathy. Some aspects of the terminology require further debate. Consensus was also reached on the patient populations, standards of care and endpoints to assess clinical trial outcomes. However, some compromises had to be made as there is considerable inter- and intravariability in the availability of some of the more objective surrogate performance markers.
The objectives of the round table were met. Robust, defendable guidelines for the conduct of future studies into hepatic encephalopathy have been provided. Outstanding issues are few and will continue to be discussed.
PMCID: PMC3971432  PMID: 21306407
16.  Prediction of Staphylococcus aureus Antimicrobial Resistance by Whole-Genome Sequencing 
Journal of Clinical Microbiology  2014;52(4):1182-1191.
Whole-genome sequencing (WGS) could potentially provide a single platform for extracting all the information required to predict an organism's phenotype. However, its ability to provide accurate predictions has not yet been demonstrated in large independent studies of specific organisms. In this study, we aimed to develop a genotypic prediction method for antimicrobial susceptibilities. The whole genomes of 501 unrelated Staphylococcus aureus isolates were sequenced, and the assembled genomes were interrogated using BLASTn for a panel of known resistance determinants (chromosomal mutations and genes carried on plasmids). Results were compared with phenotypic susceptibility testing for 12 commonly used antimicrobial agents (penicillin, methicillin, erythromycin, clindamycin, tetracycline, ciprofloxacin, vancomycin, trimethoprim, gentamicin, fusidic acid, rifampin, and mupirocin) performed by the routine clinical laboratory. We investigated discrepancies by repeat susceptibility testing and manual inspection of the sequences and used this information to optimize the resistance determinant panel and BLASTn algorithm. We then tested performance of the optimized tool in an independent validation set of 491 unrelated isolates, with phenotypic results obtained in duplicate by automated broth dilution (BD Phoenix) and disc diffusion. In the validation set, the overall sensitivity and specificity of the genomic prediction method were 0.97 (95% confidence interval [95% CI], 0.95 to 0.98) and 0.99 (95% CI, 0.99 to 1), respectively, compared to standard susceptibility testing methods. The very major error rate was 0.5%, and the major error rate was 0.7%. WGS was as sensitive and specific as routine antimicrobial susceptibility testing methods. WGS is a promising alternative to culture methods for resistance prediction in S. aureus and ultimately other major bacterial pathogens.
PMCID: PMC3993491  PMID: 24501024
17.  High-contrast fluorescence sensing of aqueous Cu(I) with triaryl-pyrazoline probes: Dissecting the roles of ligand donor strength and excited state proton transfer 
Cu(I)-responsive fluorescent probes based on a photoinduced electron transfer (PET) mechanism generally show incomplete fluorescence recovery relative to the intrinsic quantum yield of the fluorescence reporter. Previous studies on probes with an N-aryl thiazacrown Cu(I)-receptor revealed that the recovery is compromised by incomplete Cu(I)-N coordination and resultant ternary complex formation with solvent molecules. Building upon a strategy that successfully increased the fluorescence contrast and quantum yield of Cu(I) probes in methanol, we integrated the arylamine PET donor into the backbone of a hydrophilic thiazacrown ligand with a sulfonated triarylpyrazoline as a water-soluble fluorescence reporter. This approach was not only expected to disfavor ternary complex formation in aqueous solution but also to maximize PET switching through a synergistic Cu(I)-induced conformational change. The resulting water-soluble probe 1 gave a strong 57-fold fluorescence enhancement upon saturation with Cu(I) with high selectivity over other cations, including Cu(II), Hg(II), and Cd(II); however, the recovery quantum yield did not improve over probes with the original N-aryl thiazacrown design. Concluding from detailed photophysical data, including responses to acidification, solvent isotope effects, quantum yields, and time-resolved fluorescence decay profiles, the fluorescence contrast of 1 is compromised by inadequate coordination of Cu(I) to the weakly basic arylamine nitrogen of the PET donor and by fluorescence quenching via two distinct excited state proton transfer pathways operating under neutral and acidic conditions.
PMCID: PMC3755598  PMID: 23169532
18.  Exploring Stakeholder Perceptions of Facilitators and Barriers to Using Needle Exchange Programs in Yunnan Province, China 
PLoS ONE  2014;9(2):e86873.
Injection drug use is an ongoing urban health crisis in China and one of the largest drivers of the transmission of HIV/AIDS. Sentinel surveillance sites in Yunnan province show upwards of 20% of injection drug users (IDUs) are HIV positive. Though the Ministry of Health has scaled-up needle exchange programs (NEPs), they have not received official government recognition nor have they been extensively evaluated to explore factors influencing their acceptability and feasibility. Using in-depth qualitative interviews conducted from February to July 2008 with 35 participants consisting of IDUs and other key stakeholders, we explored facilitators and barriers to accessing needle exchange programs in Kunming, the capital of Yunnan province. Content analysis was conducted to identify themes including attitudes toward NEPs and harm reduction, barriers to access, and suggestions for improvement. Themes that emerged included fears of breached confidentiality and police interference at the exchange sites and tensions between the public health and law enforcement perspective. Low levels of NEP-related knowledge and awareness were uniformly reported among interviewees. Suggestions to facilitate an increase in NEP acceptance included raising awareness of harm reduction and HIV more generally, offering services such as psychological counseling, job training and behavioral therapy at NEPs, and increasing communication between police, government, and public health officials. High rates of HIV infection among injection drug users in China have prompted rapid scale up of NEPs. Additional adaptations are necessary, however, to increase needle exchange use among injection drug users. This study finds that an urgent need to raise awareness of NEPs among policy makers and IDUs and act upon identified steps for developing social-structural interventions to create enabling environments that facilitate increased access to NEPs among injection drug users in Kunming.
PMCID: PMC3911934  PMID: 24498286
19.  Stimulation of the dorsal periaqueductal gray enhances spontaneous recovery of a conditioned taste aversion 
Brain research  2012;1493:27-39.
Due to its relevance to clinical practice, extinction of learned fears has been a major focus of recent research. However, less is known about the means by which conditioned fears re-emerge (i.e., spontaneously recover) as time passes or contexts change following extinction. The periaqueductal gray represents the final common pathway mediating defensive reactions to fear and we have reported previously that the dorsolateral PAG (dlPAG) exhibits a small but reliable increase in neural activity (as measured by c-fos protein immunoreactivity) when spontaneous recovery (SR) of a conditioned taste aversion (CTA) is reduced. Here we extend these correlational studies to determine if inducing dlPAG c-fos expression through electrical brain stimulation could cause a reduction in SR of a CTA. Male Sprague-Dawley rats acquired a strong aversion to saccharin (conditioned stimulus; CS) and then underwent CTA extinction through multiple non-reinforced exposures to the CS. Following a 30-day latency period after asymptotic extinction was achieved; rats either received stimulation of the dorsal PAG (dPAG) or stimulation of closely adjacent structures. Sixty minutes following the stimulation, rats were again presented with the saccharin solution as we tested for SR of the CTA. The brain stimulation evoked c-fos expression around the tip of the electrodes. However, stimulation of the dPAG failed to reduce SR of the previously extinguished CTA. In fact, dPAG stimulation caused rats to significantly suppress their saccharin drinking (relative to controls) – indicating an enhanced SR. These data refute a cause-and-effect relationship between enhanced dPAG c-fos expression and a reduction in SR. However, they highlight a role for the dPAG in modulating SR of extinguished CTAs.
PMCID: PMC3544555  PMID: 23183042
Periaqueductal gray; PAG; Central gray; Conditioned taste aversion; CTA; Extinction; Spontaneous recovery; c-fos
20.  A bias-free measure of retinotopic tilt adaptation 
Journal of Vision  2014;14(1):7.
The traditional method of single stimuli for measuring perceptual illusions and context effects confounds perceptual effects with changes in the observer's decision criterion. By deciding consciously or unconsciously to select one of the two response alternatives more than the other when unsure of the correct response, the observer can shift his or her psychometric function in a manner indistinguishable from a genuine perceptual shift. Here, a spatial two-alternative forced-choice method is described to measure a perceptual aftereffect by its influence on the shape of the psychometric function rather than the mean. The method was tested by measuring the effect of motion adaptation on the apparent Vernier offset of stationary Gabor patterns. The shift due to adaptation was found to be comparable in size to the internal noise, estimated from the slope of the psychometric function. By moving the eyes between adaptation and test, it was determined that adaptation is retinotopic rather than spatiotopic.
PMCID: PMC3886440  PMID: 24403393
visual adaptation; signal detection theory; methods
21.  Linking HIV-positive adolescents to care in 15 different clinics across the United States: Creating solutions to address structural barriers for linkage to care 
AIDS care  2013;26(1):10.1080/09540121.2013.808730.
Linkage to care is a critical corollary to expanded HIV testing, but many adolescents are not successfully linked to care, in part due to fragmented care systems. Through a collaboration of the National Institutes of Health (NIH), Centers for Disease Control and Prevention (CDC) and the Adolescent Trials Network (ATN), a linkage to care outreach worker was provided to ATN clinics. Factors related to linkage were explored to better understand how to improve retention rates and health outcomes for HIV-positive adolescents. We conducted 124 interviews with staff at 15 Adolescent Trials Network clinics to better understand linkage to care processes, barriers, and facilitators. Content analysis was conducted focusing on structural barriers to care and potential solutions, specifically at the macro-, meso-, and micro-levels. Macro-level barriers included navigating health insurance policies, transportation to appointments, and ease of collecting and sharing client-level contact information between testing agencies, local health departments and clinics; meso-level barriers included lack of youth friendliness within clinic space and staff, and duplication of linkage services; micro-level barriers included adolescents’ readiness for care and adolescent developmental capacity. Staff initiated solutions included providing transportation for appointments and funding clinic visits and tests with a range of grants and clinic funds while waiting for insurance approval. However, such solutions were often ad hoc and partial, using micro-level solutions to address macro-level barriers. Comprehensive initiatives to improve linkage to care are needed to address barriers to HIV-care for adolescents, whose unique developmental needs make accessing care particularly challenging. Matching the level of structural solution to the level of structural barriers (i.e., macro-level with macro-level), such as creating policy to address needed youth healthcare entitlements versus covering uninsured patients with clinic funds is imperative to achieving the goal of increasing linkage to care rates for newly diagnosed adolescents.
PMCID: PMC3872213  PMID: 23777542
adolescents; HIV/AIDS; linkage to care; structural barriers; qualitative methods
22.  Developmentally dynamic colocalization patterns of DSCAM with adhesion and synaptic proteins in the mouse retina 
Molecular Vision  2014;20:1422-1433.
The Down syndrome cell adhesion molecule (Dscam) gene is required for normal dendrite arborization and lamination in the mouse retina. In this study, we characterized the developmental localization of the DSCAM protein to better understand the postnatal stages of retinal development during which laminar disorganization occur in the absence of the protein.
Immunohistochemistry and colocalization analysis software were used to assay the localization of the DSCAM protein during development of the retina.
We found that DSCAM was initially localized diffusely throughout mouse retinal neurites but then adopted a punctate distribution. DSCAM colocalized with catenins in the adult retina but was not detected at the active zone of chemical synapses, electrical synapses, and tight junctions. Further analysis identified a wave of colocalization between DSCAM and numerous synaptic and junction proteins coinciding with synaptogenesis between bipolar and retinal ganglion cells.
Research presented in this study expands our understanding of DSCAM function by characterizing its location during the development of the retina and identifies temporally regulated localization patterns as an important consideration in understanding the function of adhesion molecules in neural development.
PMCID: PMC4191645  PMID: 25352748
23.  Lenticular cytoprotection, part 2: Link between glycogen synthase kinase-3β, epithelial to mesenchymal transition, and mitochondrial depolarization 
Molecular Vision  2014;20:1758-1775.
The inhibition of GSK-3β blocks mitochondrial membrane permeability transition (mMPT) for HLE-B3 cells in atmospheric oxygen. GSK-3β, as part of a multifactorial complex, also regulates nuclear levels of β-catenin, a known coordinator of cell survival and adhesion. The purpose of these studies was to demonstrate a novel, but likely disadvantageous, link between β-catenin’s influence on the expression of the pro-survival protein, vascular endothelial growth factor (VEGF), resulting in enhanced lens epithelial cell mitochondrial protection against depolarization and nuclear β-catenin as an inducer of epithelial to mesenchymal transition (EMT).
Virally transformed human lens epithelial cells (HLE-B3) were treated with SB216763, a specific inhibitor of GSK-3β catalytic activity and XAV939, a specific β-catenin inhibitor that bars the translocation of β-catenin from cytoplasm to the nucleus. Western blot analysis was employed to detect the levels of cytoplasmic and nuclear β-catenin and phospho-β-catenin, pBcl-2 and the EMT proteins, α-smooth muscle actin (α-SMA), and fibronectin. ELISA was used to measure the levels of VEGF in cell culture supernatants. JC-1 analysis was performed to analyze the influence of either SB216763 or XAV939 on mitochondrial depolarization.
Cultured lens epithelial cells maintained in hypoxia (1% oxygen) and subsequently reintroduced into atmospheric oxygen and treated with the GSK-3β inhibitor SB216763 illustrated a marked inhibition of phosphorylation of glycogen synthase (downstream substrate of GSK-3β) and significant increase in nuclear translocation of β-catenin. The augmented nuclear β-catenin levels positively correlated with increased expression of α-SMA and fibronectin, both marker proteins indicative of EMT. The enhanced nuclear β-catenin activity also elicited increased VEGF and pBcl-2 expression, resulting in increased resistance to mitochondrial depolarization. Treatment of the cells with the β-catenin inhibitor XAV939 resulted in decreased expression of nuclear β-catenin, VEGF levels, pBcl-2, and EMT proteins, as well as increased mitochondrial depolarization.
The data support a model whereby the onset of epithelial to mesenchymal transition may circuitously benefit from the enhanced synthesis of VEGF by setting up a potentially harmful situation whereby the resulting mesenchymal cell population may be more resistant to mitochondrial depolarization than the lens epithelial cell population from which it originated. These findings support the potential therapeutic relevance of developing strategies to undermine the progression of normal cells to mesenchymal transition without subverting cell viability.
PMCID: PMC4287703  PMID: 25593505
24.  Hepatitis B and Liver Cancer Among Three Asian American Sub-Groups: A Focus Group Inquiry 
Prevalence of hepatitis B among Asian Americans is higher than for any other ethnic group in the United States. Since more than 50% of liver cancer is hepatitis B related, the burden of morbidity and mortality is extremely high among Asian Americans, highlighting the need for culturally appropriate interventions. We conducted focus groups (n = 8) with a total of 58 Korean, Vietnamese, and Chinese immigrants in Maryland to explore knowledge, awareness and perceived barriers toward hepatitis B screening and vaccinations. Thematic analysis uncovered generally low levels of knowledge and awareness of hepatitis B risks, screening, and vaccination; inter-generational differences; and barriers to prevention. Some differences arose across ethnic groups, particularly toward perceived orientation to preventive activities and the role of religious groups. High rates of hepatitis B infection among Asian Americans highlight the need for tailored interventions. These findings may assist policy strategists in implementing interventions that will facilitate the integration and scale-up of hepatitis B education, screening, and vaccination campaigns.
PMCID: PMC3804298  PMID: 21901445
Hepatitis B risk; HBV screening and vaccinations; Asian Americans; Qualitative; Immigrant health
25.  Perceived pattern regularity computed as a summary statistic: implications for camouflage 
Why do the equally spaced dots in figure 1 appear regularly spaced? The answer ‘because they are’ is naive and ignores the existence of sensory noise, which is known to limit the accuracy of positional localization. Actually, all the dots in figure 1 have been physically perturbed, but in the case of the apparently regular patterns to an extent that is below threshold for reliable detection. Only when retinal pathology causes severe distortions do regular grids appear perturbed. Here, we present evidence that low-level sensory noise does indeed corrupt the encoding of relative spatial position, and limits the accuracy with which observers can detect real distortions. The noise is equivalent to a Gaussian random variable with a standard deviation of approximately 5 per cent of the inter-element spacing. The just-noticeable difference in positional distortion between two patterns is smallest when neither of them is perfectly regular. The computation of variance is statistically inefficient, typically using only five or six of the available dots.
PMCID: PMC3367773  PMID: 22438499
vision; texture; camouflage

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