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1.  Ultrafast endocytosis at mouse hippocampal synapses 
Nature  2013;504(7479):242-247.
To sustain neurotransmission, synaptic vesicles and their associated proteins must be recycled locally at synapses. Synaptic vesicles are thought to be regenerated ~20 s after fusion by the assembly of clathrin scaffolds or in ~1 s by the reversal of fusion pores via ‘kiss-and-run’ endocytosis. Here we use optogenetics to stimulate cultured hippocampal neurons with a single stimulus, rapidly freeze them after fixed intervals and examine the ultrastructure using electron microscopy – ‘flash-and-freeze’ electron microscopy. Docked vesicles fuse and collapse into the membrane within 30 ms of the stimulus. Compensatory endocytosis occurs with 50-100 ms at sites flanking the active zone. Invagination is blocked by inhibition of actin polymerization, and scission is blocked by inhibiting dynamin. Because intact synaptic vesicles are not recovered, this form of recycling is not compatible with kiss-and-run endocytosis; moreover it is 200-fold faster than clathrin-mediated endocytosis. It is likely that ‘ultrafast endocytosis’ is specialized to rapidly restore the surface area of the membrane.
PMCID: PMC3957339  PMID: 24305055
2.  Anxiogenic effects of CGRP within the BNST may be mediated by CRF acting at BNST CRFR1 receptors 
Behavioural brain research  2013;243:286-293.
Calcitonin gene-related peptide (CGRP) acting within the bed nucleus of the stria terminalis (BNST) increases anxiety as well as neural activation in anxiety-related structures, and mediates behavioral stress responses. Similar effects have been described following intra-ventricular as well as intra-BNST infusions of the stress-responsive neuropeptide, corticotropin releasing factor (CRF). Interestingly, CGRP-positive terminals within the lateral division of the BNST form perisomatic baskets around neurons that express CRF, suggesting that BNST CGRP could exert its anxiogenic effects by increasing release of CRF from these neurons. With this in mind, the present set of experiments was designed to examine the role of CRFR1 signaling in the anxiogenic effects of CGRP within the BNST and to determine whether CRF from BNST neurons contributes to these effects. Consistent with previous studies, we found that 400 ng CGRP infused bilaterally into the BNST increased the acoustic startle response and induced anxiety-like behavior in the elevated plus maze compared to vehicle. Both of these effects were attenuated by 10 mg/kg PO of the CRFR1 antagonist, GSK876008. GSK876008 alone did not affect startle. An intra-BNST infusion of the CRFR1 antagonist CP376395 (2 μg) also blocked increases in acoustic startle induced by intra-BNST infusion of CGRP, as did virally-mediated siRNA knockdown of CRF expression locally within the BNST. Together, these results suggest that the anxiogenic effects of intra-BNST CGRP may be mediated by CRF from BNST neurons acting at local CRFR1 receptors.
PMCID: PMC3593796  PMID: 23376701
bed nucleus of the stria terminalis; calcitonin gene-related peptide; corticotropin releasing factor; anxiety; fear; startle
3.  Modularity in the motion system: Independent oculomotor and perceptual processing of brief moving stimuli 
Journal of Vision  2014;14(3):28.
In addition to motion perception per se, we utilize motion information for a wide range of brain functions. These varied functions place different demands on the visual system, and therefore a stimulus that provides useful information for one function may be inadequate for another. For example, the direction of motion of large high-contrast stimuli is difficult to discriminate perceptually, but other studies have shown that such stimuli are highly effective at eliciting directional oculomotor responses such as the ocular following response (OFR). Here, we investigated the degree of independence between perceptual and oculomotor processing by determining whether perceptually suppressed moving stimuli can nonetheless evoke reliable eye movements. We measured reflexively evoked tracking eye movements while observers discriminated the motion direction of large high-contrast stimuli. To quantify the discrimination ability of the oculomotor system, we used signal detection theory to generate associated oculometric functions. The results showed that oculomotor sensitivity to motion direction is not predicted by perceptual sensitivity to the same stimuli. In fact, in several cases oculomotor responses were more reliable than perceptual responses. Moreover, a trial-by-trial analysis indicated that, for stimuli tested in this study, oculomotor processing was statistically independent from perceptual processing. Evidently, perceptual and oculomotor responses reflect the activity of independent dissociable mechanisms despite operating on the same input. While results of this kind have traditionally been interpreted in the framework of perception versus action, we propose that these differences reflect a more general principle of modularity.
PMCID: PMC3969025  PMID: 24665091
motion perception; eye movements; spatial suppression; ocular following response
4.  Social feeding in Caenorhabditis elegans is induced by neurons that detect aversive stimuli 
Nature  2002;419(6910):899-903.
Natural Caenorhabditis elegans isolates exhibit either social or solitary feeding on bacteria. We show here that social feeding is induced by nociceptive neurons that detect adverse or stressful conditions. Ablation of the nociceptive neurons ASH and ADL transforms social animals into solitary feeders. Social feeding is probably due to the sensation of noxious chemicals by ASH and ADL neurons; it requires the genes ocr-2 and osm-9, which encode TRP-related transduction channels, and odr-4 and odr-8, which are required to localize sensory chemoreceptors to cilia. Other sensory neurons may suppress social feeding, as social feeding in ocr-2 and odr-4 mutants is restored by mutations in osm-3, a gene required for the development of 26 ciliated sensory neurons. Our data suggest a model for regulation of social feeding by opposing sensory inputs: aversive inputs to nociceptive neurons promote social feeding, whereas antagonistic inputs from neurons that express osm-3 inhibit aggregation.
PMCID: PMC3955269  PMID: 12410303
5.  Lack of correlation between predicted and actual off-target effects of short-interfering RNAs targeting the human papillomavirus type 16 E7 oncogene 
British Journal of Cancer  2013;108(2):450-460.
When designing therapeutic short-interfering RNAs (siRNAs), off-target effects (OTEs) are usually predicted by computational quantification of messenger RNAs (mRNAs) that contain matches to the siRNA seed sequence in their 3′ UTRs. It is assumed that the higher the number of predicted transcriptional OTEs, the greater the size of the actual OTE signature and the more detrimental the phenotypic consequences in target-negative cells.
We tested this general assumption by investigating the OTEs of potential therapeutic siRNAs targeting the human papillomavirus (HPV) type-16 E7 oncogene. We studied HPV-negative squamous epithelial cells, from normal cervix (NCx) and skin (HaCaT), which would be vulnerable to ‘bystander' OTEs following transfection in vivo.
We observed no correlation between the number of computationally predicted OTEs and the actual number of seed-dependent OTEs (P=0.76). On average only 20.5% of actual transcriptional OTEs were seed-dependent (i.e., predicted). The unpredicted OTEs included stimulation of innate immune pathways, as well as indirect (downstream) effects of other OTEs, which affected important cancer-associated pathways. Although most significant OTEs observed were seen in both NCx and HaCaT cells, only 0–5.9% of differentially expressed genes overlapped between the two cell types.
These data do not support the assumption that actual OTEs correlate well with predicted OTEs.
PMCID: PMC3566827  PMID: 23299538
E7; HPV16; off-target effects; siRNA; Sylamer
6.  Visual context processing in schizophrenia 
Abnormal perceptual experiences are central to schizophrenia but the nature of these anomalies remains undetermined. We investigated contextual processing abnormalities across a comprehensive set of visual tasks. For perception of luminance, size, contrast, orientation and motion, we quantified the degree to which the surrounding visual context altered a center stimulus’ appearance. Across tasks, healthy participants showed robust contextual effects, as evidenced by pronounced misperceptions of center stimuli. Schizophrenia patients exhibited intact contextual modulations of luminance and size, but showed weakened contextual modulations of contrast, performing more accurately than controls. Strong motion and orientation context effects correlated with worse symptoms and social functioning. Importantly, the overall strength of contextual modulation across tasks did not differ between controls and schizophrenia patients. Additionally, performance measures across contextual tasks were uncorrelated, implying discrete underlying processes. These findings reveal that abnormal contextual modulation in schizophrenia is selective, arguing against the proposed unitary contextual processing dysfunction.
PMCID: PMC3756604  PMID: 23997995
Schizophrenia; center-surround; contextual effects; perception deficit; visual processing
7.  Prion-Seeding Activity in Cerebrospinal Fluid of Deer with Chronic Wasting Disease 
PLoS ONE  2013;8(11):e81488.
Transmissible spongiform encephalopathies (TSEs), or prion diseases, are a uniformly fatal family of neurodegenerative diseases in mammals that includes chronic wasting disease (CWD) of cervids. The early and ante-mortem identification of TSE-infected individuals using conventional western blotting or immunohistochemistry (IHC) has proven difficult, as the levels of infectious prions in readily obtainable samples, including blood and bodily fluids, are typically beyond the limits of detection. The development of amplification-based seeding assays has been instrumental in the detection of low levels of infectious prions in clinical samples. In the present study, we evaluated the cerebrospinal fluid (CSF) of CWD-exposed (n=44) and naïve (n=4) deer (n=48 total) for CWD prions (PrPd) using two amplification assays: serial protein misfolding cyclic amplification with polytetrafluoroethylene beads (sPMCAb) and real-time quaking induced conversion (RT-QuIC) employing a truncated Syrian hamster recombinant protein substrate. Samples were evaluated blindly in parallel with appropriate positive and negative controls. Results from amplification assays were compared to one another and to obex immunohistochemistry, and were correlated to available clinical histories including CWD inoculum source (e.g. saliva, blood), genotype, survival period, and duration of clinical signs. We found that both sPMCAb and RT-QuIC were capable of amplifying CWD prions from cervid CSF, and results correlated well with one another. Prion seeding activity in either assay was observed in approximately 50% of deer with PrPd detected by IHC in the obex region of the brain. Important predictors of amplification included duration of clinical signs and time of first tonsil biopsy positive results, and ultimately the levels of PrPd identified in the obex by IHC. Based on our findings, we expect that both sPMCAb and RT-QuIC may prove to be useful detection assays for the detection of prions in CSF.
PMCID: PMC3839929  PMID: 24282599
8.  A Mutation in the C. elegans EXP-2 Potassium Channel That Alters Feeding Behavior 
Science (New York, N.Y.)  1999;286(5449):2501-2504.
The nematode pharynx has a potassium channel with unusual properties, which allows the muscles to repolarize quickly and with the proper delay. Here, the Caenorhabditis elegans exp-2 gene is shown to encode this channel. EXP-2 is a Kv-type (voltage-activated) potassium channel that has inward-rectifying properties resembling those of the structurally dissimilar human ether-à-go-go–related gene (HERG) channel. Null and gain-of-function mutations affect pharyngeal muscle excitability in ways that are consistent with the electrophysiological behavior of the channel, and thereby demonstrate a direct link between the kinetics of this unusual channel and behavior.
PMCID: PMC3791429  PMID: 10617464
9.  Ultrafast endocytosis at Caenorhabditis elegans neuromuscular junctions 
eLife  2013;2:e00723.
Synaptic vesicles can be released at extremely high rates, which places an extraordinary demand on the recycling machinery. Previous ultrastructural studies of vesicle recycling were conducted in dissected preparations using an intense stimulation to maximize the probability of release. Here, a single light stimulus was applied to motor neurons in intact Caenorhabditis elegans nematodes expressing channelrhodopsin, and the animals rapidly frozen. We found that docked vesicles fuse along a broad active zone in response to a single stimulus, and are replenished with a time constant of about 2 s. Endocytosis occurs within 50 ms adjacent to the dense projection and after 1 s adjacent to adherens junctions. These studies suggest that synaptic vesicle endocytosis may occur on a millisecond time scale following a single physiological stimulus in the intact nervous system and is unlikely to conform to current models of endocytosis.
eLife digest
Neurons communicate with one another at junctions called synapses. When an electrical signal travels along a neuron and arrives at a synapse, vesicles filled with small neurotransmitter molecules fuse with the cell membrane and release the neurotransmitter. These chemicals rapidly bind to receptors on the downstream neuron that induce an electrical response in that cell.
Vesicles can be consumed at prodigious rates, up to 500 a second, so the cell must recover the membrane rapidly and regenerate more vesicles filled with neurotransmitter. Experiments in the 1970s and 1980s suggested that when vesicles empty their contents into the synapse, they fuse completely with the membrane and are lost. To recover the membrane, the cell forms ‘pits’, by means of a coat protein called clathrin, which then bud off into the cell as new vesicles. It takes roughly 15–20 s for vesicles to be recycled in this way. By contrast, synapses with very high firing rates are thought to recycle vesicles through a faster process known as ‘kiss and run’, in which vesicles are not fully integrated into the membrane, but instead fuse transiently with it to form a reversible pore within about a second.
However, these studies triggered vesicle release using conditions that are unlikely to occur naturally inside cells. Now, Watanabe et al. have used optogenetics to study vesicle recycling in response to single stimuli at the synapse between neurons and muscles in an intact living animal, the nematode C. elegans. The worms had been genetically modified to express a light-sensitive ion channel called channelrhodopsin in their motor neurons. Watanabe et al. used a single pulse of light to stimulate vesicle release, and then rapidly froze the worms before studying their synapses with electron microscopy.
They found that vesicle recycling occurred at the edges of the synapse or at a specialized structure in the middle of the synapse. Vesicle recycling took less than 50 ms—much faster than anything previously observed. This ultrafast recycling is unlikely to occur via ‘kiss and run’ since recycling occurred at sites lateral to the sites of fusion and because the recycled vesicles were larger than the originals, implying that they had not simply re-formed after a brief fusion event.
By using physiologically relevant stimuli in an intact animal, Watanabe et al. reveal that vesicles can be recycled at synapses much more rapidly than previously thought, suggesting that our current models of this process may need to be reassessed.
PMCID: PMC3762212  PMID: 24015355
synaptic vesicle endocytosis; optogenetics; time-resolved electron microscopy; high-pressure freezing; synaptic vesicle exocytosis; active zone; C. elegans
10.  Visual context processing in bipolar disorder: a comparison with schizophrenia 
Anomalous perception has been investigated extensively in schizophrenia, but it is unclear whether these impairments are specific to schizophrenia or extend to other psychotic disorders. Recent studies of visual context processing in schizophrenia (Tibber et al., 2013; Yang et al., 2013) point to circumscribed, task-specific abnormalities. Here we examined visual contextual processing across a comprehensive set of visual tasks in individuals with bipolar disorder and compared their performance with that of our previously published results from schizophrenia and healthy participants tested on those same tasks. We quantified the degree to which the surrounding visual context alters a center stimulus' appearance for brightness, size, contrast, orientation and motion. Across these tasks, healthy participants showed robust contextual effects, as indicated by pronounced misperceptions of the center stimuli. Participants with bipolar disorder showed contextual effects similar in magnitude to those found in healthy participants on all tasks. This result differs from what we found in schizophrenia participants (Yang et al., 2013) who showed weakened contextual modulations of contrast but intact contextual modulations of perceived luminance and size. Yet in schizophrenia participants, the magnitude of the contrast illusion did not correlate with symptom measures. Performance on the contrast task by the bipolar disorder group also could not be distinguished from that of the schizophrenia group, and this may be attributed to the result that bipolar patients who presented with greater manic symptoms showed weaker contrast modulation. Thus, contrast gain control may be modulated by clinical state in bipolar disorder. Stronger motion and orientation context effects correlated with worse clinical symptoms across both patient groups and especially in schizophrenia participants. These results highlight the complexity of visual context processing in schizophrenia and bipolar disorder.
PMCID: PMC3757289  PMID: 24009596
bipolar disorder; contextual effects; perception deficit; visual processing; schizophrenia
11.  Frequency of mutations in the genes associated with hereditary sensory and autonomic neuropathy in a UK cohort 
Journal of neurology  2012;259(8):1673-1685.
The hereditary sensory and autonomic neuropathies (HSAN, also known as the hereditary sensory neuropathies) are a clinically and genetically heterogeneous group of disorders, characterised by a progressive sensory neuropathy often complicated by ulcers and amputations, with variable motor and autonomic involvement. To date, mutations in twelve genes have been identified as causing HSAN. To study the frequency of mutations in these genes and the associated phenotypes, we screened 140 index patients in our inherited neuropathy cohort with a clinical diagnosis of HSAN for mutations in the coding regions of SPTLC1, RAB7, WNK1/HSN2, FAM134B, NTRK1 (TRKA) and NGFB. We identified 25 index patients with mutations in six genes associated with HSAN (SPTLC1, RAB7, WNK1/HSN2, FAM134B, NTRK1 and NGFB); 20 of which appear to be pathogenic giving an overall mutation frequency of 14.3%. Mutations in the known genes for HSAN are rare suggesting that further HSAN genes are yet to be identified. The p.Cys133Trp mutation in SPTLC1 is the most common cause of HSAN in the UK population and should be screened first in all patients with sporadic or autosomal dominant HSAN.
PMCID: PMC3752368  PMID: 22302274
12.  Mother to Offspring Transmission of Chronic Wasting Disease in Reeves’ Muntjac Deer 
PLoS ONE  2013;8(8):e71844.
The horizontal transmission of prion diseases has been well characterized in bovine spongiform encephalopathy (BSE), chronic wasting disease (CWD) of deer and elk and scrapie of sheep, and has been regarded as the primary mode of transmission. Few studies have monitored the possibility of vertical transmission occurring within an infected mother during pregnancy. To study the potential for and pathway of vertical transmission of CWD in the native cervid species, we used a small cervid model–the polyestrous breeding, indoor maintainable, Reeves’ muntjac deer–and determined that the susceptibility and pathogenesis of CWD in these deer reproduce that in native mule and white-tailed deer. Moreover, we demonstrate here that CWD prions are transmitted from doe to fawn. Maternal CWD infection also appears to result in lower percentage of live birth offspring. In addition, evolving evidence from protein misfolding cyclic amplification (PMCA) assays on fetal tissues suggest that covert prion infection occurs in utero. Overall, our findings demonstrate that transmission of prions from mother to offspring can occur, and may be underestimated for all prion diseases.
PMCID: PMC3743758  PMID: 23977159
13.  Aerosol Transmission of Chronic Wasting Disease in White-Tailed Deer 
Journal of Virology  2013;87(3):1890-1892.
While the facile transmission of chronic wasting disease (CWD) remains incompletely elucidated, studies in rodents suggest that exposure of the respiratory mucosa may be an efficient pathway. The present study was designed to address this question in the native cervid host. Here, we demonstrate aerosol transmission of CWD to deer with a prion dose >20-fold lower than that used in previous oral inoculations. Inhalation of prions may facilitate transmission of CWD and, perhaps, other prion infections.
PMCID: PMC3554158  PMID: 23175370
14.  Susceptibility of Domestic Cats to Chronic Wasting Disease 
Journal of Virology  2013;87(4):1947-1956.
Domestic and nondomestic cats have been shown to be susceptible to feline spongiform encephalopathy (FSE), almost certainly caused by consumption of bovine spongiform encephalopathy (BSE)-contaminated meat. Because domestic and free-ranging nondomestic felids scavenge cervid carcasses, including those in areas affected by chronic wasting disease (CWD), we evaluated the susceptibility of the domestic cat (Felis catus) to CWD infection experimentally. Cohorts of 5 cats each were inoculated intracerebrally (i.c.) or orally (p.o.) with CWD-infected deer brain. At 40 and 42 months postinoculation, two i.c.-inoculated cats developed signs consistent with prion disease, including a stilted gait, weight loss, anorexia, polydipsia, patterned motor behaviors, head and tail tremors, and ataxia, and the cats progressed to terminal disease within 5 months. Brains from these two cats were pooled and inoculated into cohorts of cats by the i.c., p.o., and intraperitoneal and subcutaneous (i.p./s.c.) routes. Upon subpassage, feline CWD was transmitted to all i.c.-inoculated cats with a decreased incubation period of 23 to 27 months. Feline-adapted CWD (FelCWD) was demonstrated in the brains of all of the affected cats by Western blotting and immunohistochemical analysis. Magnetic resonance imaging revealed abnormalities in clinically ill cats, which included multifocal T2 fluid attenuated inversion recovery (FLAIR) signal hyperintensities, ventricular size increases, prominent sulci, and white matter tract cavitation. Currently, 3 of 4 i.p./s.c.- and 2 of 4 p.o. secondary passage-inoculated cats have developed abnormal behavior patterns consistent with the early stage of feline CWD. These results demonstrate that CWD can be transmitted and adapted to the domestic cat, thus raising the issue of potential cervid-to-feline transmission in nature.
PMCID: PMC3571486  PMID: 23236066
15.  Improved Mos1-mediated transgenesis in C. elegans 
Nature methods  2012;9(2):117-118.
PMCID: PMC3725292  PMID: 22290181
16.  Protons act as a transmitter for muscle contraction in C. elegans 
Cell  2008;132(1):149-160.
Muscle contraction is normally mediated by the release of neurotransmitters from motor neurons. Here we demonstrate that protons can act as a direct transmitter from intestinal cells to stimulate muscle contraction. During the C. elegans defecation motor program the posterior body muscles contract even in the absence of neuronal inputs or vesicular neurotransmission. In this study, we demonstrate that the space between the intestine and the muscle is acidified just prior to muscle contraction and that the release of caged protons is sufficient to induce muscle contraction. PBO-4 is a putative Na+/H+ ion exchanger expressed on the basolateral membrane of the intestine, juxtaposed to the posterior body muscles. In pbo-4 mutants the extracellular space is not acidified and the muscles fail to contract. The pbo-5 and pbo-6 genes encode subunits of a ‘cys-loop’ proton-gated cation channel required for muscles to respond to acidification. In heterologous expression assays the PBO receptor is half-maximally activated at a pH of 6.8. The identification of the mechanisms for release and reception of proton signals establishes a highly unusual mechanism for intercellular communication.
PMCID: PMC2258244  PMID: 18191228
17.  Differential Effects of the CRF-R1 Antagonist GSK876008 on Fear-Potentiated, Light- and CRF-Enhanced Startle Suggest Preferential Involvement in Sustained versus Phasic Threat Responses 
The amplitude of the acoustic startle response is increased when elicited in the presence of brief cues that predict shock (fear-potentiated startle) and also when elicited during sustained exposure to bright light (light-enhanced startle). Although both effects are thought to reflect fear or anxiety, their neuroanatomical substrates differ. Whereas fear-potentiated startle is disrupted by reversible inactivation of the central nucleus of the amygdala (CeA) but not the closely related bed nucleus of the stria terminalis (BNST), light-enhanced startle is disrupted by BNST inactivation but not by CeA inactivation. Intra-ventricular infusions of corticotropin releasing factor (CRF) also increase startle (CRF-enhanced startle) and this effect is mediated by CRF receptors within the BNST, with no involvement of the CeA. Together, these observations suggest that CeA- and BNST-dependent fear and anxiety may be differentially sensitive to CRF receptor blockade. We tested this by orally administering the novel, potent, and selective CRF-R1 antagonist GSK876008 to rats prior to CRF-enhanced, light-enhanced, or fear-potentiated startle testing. GSK876008 disrupted CRF-enhanced startle with a linear dose-response curve, and light-enhanced startle with a U-shaped dose-response curve, but did not disrupt fear-potentiated startle to a visual stimulus at any dose tested, and even augmented the response in some animals. GSK876008 also disrupted shock-related ‘baseline’ startles increases, which may have reflected context conditioning (shown elsewhere to also be BNST-dependent). Overall, these results suggest that short-duration CeA-dependent threat responses can be pharmacologically dissociated from longer-duration BNST-dependent responses in terms of their sensitivity to CRF1 receptor antagonists.
PMCID: PMC3586210  PMID: 19078950
fear; anxiety; startle; amygdala; bed nucleus of the stria terminalis; corticotropin releasing factor
18.  Cardiovascular diseases in older patients with osteoporotic hip fracture: prevalence, disturbances in mineral and bone metabolism, and bidirectional links 
Considerable controversy exists regarding the contribution of mineral/bone metabolism abnormalities to the association between cardiovascular diseases (CVDs) and osteoporotic fractures.
Aims and methods
To determine the relationships between mineral/bone metabolism biomarkers and CVD in 746 older patients with hip fracture, clinical data were recorded and serum concentrations of parathyroid hormone (PTH), 25-hydroxyvitamin D, calcium, phosphate, magnesium, troponin I, parameters of bone turnover, and renal, liver, and thyroid functions were measured.
CVDs were diagnosed in 472 (63.3%) patients. Vitamin D deficiency was similarly prevalent in patients with (78.0%) and without (82.1%) CVD. The CVD group had significantly higher mean PTH concentrations (7.6 vs 6.0 pmol/L, P < 0.001), a higher prevalence of secondary hyperparathyroidism (SPTH) (PTH > 6.8 pmol/L, 43.0% vs 23.3%, P < 0.001), and excess bone resorption (urinary deoxypyridinoline corrected by creatinine [DPD/Cr] > 7.5 nmol/μmol, 87.9% vs 74.8%, P < 0.001). In multivariate regression analysis, SHPT (odds ratio [OR] 2.6, P = 0.007) and high DPD/Cr (OR 2.8, P = 0.016) were independent indictors of CVD. Compared to those with both PTH and DPD/Cr in the normal range, multivariate-adjusted ORs for the presence of CVD were 17.3 (P = 0.004) in subjects with SHPT and 9.7 (P < 0.001) in patients with high DPD/Cr. CVD was an independent predicator of SHPT (OR 2.8, P = 0.007) and excess DPD/Cr (OR 2.5, P = 0.031). CVD was predictive of postoperative myocardial injury, while SHPT was also an independent predictor of prolonged hospital stay and in-hospital death.
SHPT and excess bone resorption are independent pathophysiological mediators underlying the bidirectional associations between CVD and hip fracture, and therefore are important diagnostic and therapeutic targets.
PMCID: PMC3585505  PMID: 23460043
cardiovascular disease; hip fracture; PTH; 25(OH)D; secondary hyperparathyroidism; bone turnover; mineral metabolism
19.  Task modulation of brain responses in visual word recognition as studied using EEG/MEG and fMRI 
Do task demands change the way we extract information from a stimulus, or only how we use this information for decision making? In order to answer this question for visual word recognition, we used EEG/MEG as well as fMRI to determine the latency ranges and spatial areas in which brain activation to words is modulated by task demands. We presented letter strings in three tasks (lexical decision, semantic decision, silent reading), and measured combined EEG/MEG as well as fMRI responses in two separate experiments. EEG/MEG sensor statistics revealed the earliest reliable task effects at around 150 ms, which were localized, using minimum norm estimates (MNE), to left inferior temporal, right anterior temporal and left precentral gyri. Later task effects (250 and 480 ms) occurred in left middle and inferior temporal gyri. Our fMRI data showed task effects in left inferior frontal, posterior superior temporal and precentral cortices. Although there was some correspondence between fMRI and EEG/MEG localizations, discrepancies predominated. We suggest that fMRI may be less sensitive to the early short-lived processes revealed in our EEG/MEG data. Our results indicate that task-specific processes start to penetrate word recognition already at 150 ms, suggesting that early word processing is flexible and intertwined with decision making.
PMCID: PMC3719031  PMID: 23888133
top–down control; lexical decision; semantic decision; reading; source estimation
20.  Closure of skin lacerations under tension 
PMCID: PMC3954205  PMID: 22524947
21.  A new explanation for recessive myotonia congenita 
Neurology  2012;78(24):1953-1958.
To assess whether exon deletions or duplications in CLCN1 are associated with recessive myotonia congenita (MC).
We performed detailed clinical and electrophysiologic characterization in 60 patients with phenotypes consistent with MC. DNA sequencing of CLCN1 followed by multiplex ligation-dependent probe amplification to screen for exon copy number variation was undertaken in all patients.
Exon deletions or duplications in CLCN1 were identified in 6% of patients with MC. Half had heterozygous exonic rearrangements. The other 2 patients (50%), with severe disabling infantile onset myotonia, were identified with both a homozygous mutation, Pro744Thr, which functional electrophysiology studies suggested was nonpathogenic, and a triplication/homozygous duplication involving exons 8–14, suggesting an explanation for the severe phenotype.
These data indicate that copy number variation in CLCN1 may be an important cause of recessive MC. Our observations suggest that it is important to check for exon deletions and duplications as part of the genetic analysis of patients with recessive MC, especially in patients in whom sequencing identifies no mutations or only a single recessive mutation. These results also indicate that additional, as yet unidentified, genetic mechanisms account for cases not currently explained by either CLCN1 point mutations or exonic deletions or duplications.
PMCID: PMC3369509  PMID: 22649220
22.  Acetazolamide efficacy in hypokalemic periodic paralysis and the predictive role of genotype 
Neurology  2011;77(22):1960-1964.
Acetazolamide has been the most commonly used treatment for hypokalemic periodic paralysis since 1968. However, its mechanism of efficacy is not fully understood, and it is not known whether therapy response relates to genotype. We undertook a clinical and genetic study to evaluate the response rate of patients treated with acetazolamide and to investigate possible correlations between response and genotype.
We identified a total of 74 genotyped patients for this study. These included patients who were referred over a 15-year period to the only UK referral center or to a Chinese center and who underwent extensive clinical evaluation. For all genotyped patients, the response to acetazolamide therapy in terms of attack frequency and severity was documented. Direct DNA sequencing of CACNA1S and SCN4A was performed.
Only 46% of the total patient cohort (34 of 74) reported benefit from acetazolamide. There was a greater chance of benefit in patients with mutations in CACNA1S (31 responded of 55 total) than in those with mutations in SCN4A (3 responded of 19 total). Patients with mutations that resulted in amino acids being substituted by glycine in either gene were the least likely to report benefit.
This retrospective study indicates that only approximately 50% of genotyped patients with hypokalemic periodic paralysis respond to acetazolamide. We found evidence supporting a relationship between genotype and treatment response. Prospective randomized controlled trials are required to further evaluate this relationship. Development of alternative therapies is required.
PMCID: PMC3235354  PMID: 22094484
23.  Suitability of PSA-detected localised prostate cancers for focal therapy: experience from the ProtecT study 
British Journal of Cancer  2011;105(7):931-937.
Contemporary screening for prostate cancer frequently identifies small volume, low-grade lesions. Some clinicians have advocated focal prostatic ablation as an alternative to more aggressive interventions to manage these lesions. To identify which patients might benefit from focal ablative techniques, we analysed the surgical specimens of a large sample of population-detected men undergoing radical prostatectomy as part of a randomised clinical trial.
Surgical specimens from 525 men who underwent prostatectomy within the ProtecT study were analysed to determine tumour volume, location and grade. These findings were compared with information available in the biopsy specimen to examine whether focal therapy could be provided appropriately.
Solitary cancers were found in prostatectomy specimens from 19% (100 out of 525) of men. In addition, 73 out of 425 (17%) men had multiple cancers with a solitary significant tumour focus. Thus, 173 out of 525 (33%) men had tumours potentially suitable for focal therapy. The majority of these were small, well-differentiated lesions that appeared to be pathologically insignificant (38–66%). Criteria used to select patients for focal prostatic ablation underestimated the cancer's significance in 26% (34 out of 130) of men and resulted in overtreatment in more than half. Only 18% (24 out of 130) of men presumed eligible for focal therapy, actually had significant solitary lesions.
Focal therapy appears inappropriate for the majority of men presenting with prostate-specific antigen-detected localised prostate cancer. Unifocal prostate cancers suitable for focal ablation are difficult to identify pre-operatively using biopsy alone. Most lesions meeting criteria for focal ablation were either more aggressive than expected or posed little threat of progression.
PMCID: PMC3185935  PMID: 21863028
prostate cancer; unifocal; regional ablation; prostatectomy
24.  American Indian Methamphetamine and other Drug use in the Southwestern United States 
To investigate the extent of methamphetamine and other drug use among American Indians (AI) in the Four Corners region, we developed collaborations with Southwestern tribal entities and treatment programs in and around New Mexico.
(1) We held nine focus groups, mostly with Southwest AI participants (N=81) from three diverse New Mexico communities to understand community members, treatment providers, and clients/relatives views on methamphetamine (2) We conducted a telephone survey of staff (N=100) from agencies across New Mexico to assess perceptions of methamphetamine use among people working with AI populations. (3) We collected and analyzed self-reported drug use data from 300 AI clients/relatives who completed the Addiction Severity Index (ASI) in the context of treatment at three diverse addiction treatment programs.
Each focus group offered a unique perspective about the effect of drugs and alcohol on each respective community. Though data from the phone surveys and ASIs suggested concerning rates of methamphetamine use, with women more adversely affected by substance use in general, alcohol was identified as the biggest substance use problem for AI populations in the Southwest.
There appears to be agreement that methamphetamine use is a significant problem in these communities, but that alcohol is much more prevalent and problematic. There was less agreement about what should be done to prevent and treat methamphetamine use. Future research should attend to regional and tribal differences due to variability in drug use patterns, and should focus on identifying and improving dissemination of effective substance use interventions.
PMCID: PMC3415471  PMID: 21988577
American Indian; Tribal Based Participatory Research; Methamphetamine; Substance Use; Addiction Severity Index; Phone Survey; Qualitative; Focus Group; Protective Factors; Southwest
25.  UNC119 is required for G protein trafficking in sensory neurons 
Nature neuroscience  2011;14(7):874-880.
UNC119 is widely expressed among vertebrates and invertebrates. Here we report that UNC119 recognized the acylated N-terminus of the rod photoreceptor transducin α-subunit (Tα) as well as C. elegans G proteins Odr-3 and Gpa-13. The crystal structure of human UNC119 at 1.95 Å resolution revealed an immunoglobulin-like β-sandwich fold. Pulldowns and isothermal titration calorimetry revealed a tight interaction between UNC119 and acylated Gα peptides. Co-crystallization of UNC119 with an acylated Tα N-terminal peptide at 2.0 Å revealed that the lipid chain is buried deeply into UNC119's hydrophobic cavity. UNC119 bound TαGTP inhibiting its GTPase activity, thereby providing a stable UNC119-TαGTP complex that is capable of diffusing from the inner segment back to the outer segment following light-induced translocation. UNC119 deletion in both mouse and C. elegans lead to G protein mislocalization. These results establish UNC119 as a novel Gα-subunit cofactor that is essential for G-protein trafficking in sensory cilia.
PMCID: PMC3178889  PMID: 21642972

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