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1.  Single-molecule analysis of diffusion and trapping of STIM1 and Orai1 at endoplasmic reticulum–plasma membrane junctions 
Molecular Biology of the Cell  2014;25(22):3672-3685.
STIM1 and Orai1 move to endoplasmic reticulum–plasma membrane (ER-PM) junctions to trigger store-operated Ca2+ entry. Single-molecule tracking reveals pure diffusion of STIM1 in the ER, subdiffusion of Orai1 in the PM, and trapping at junctions via STIM-Orai binding. STIM-Orai binding is loose, generating free proteins that can exchange freely with extrajunctional pools.
Following endoplasmic reticulum (ER) Ca2+ depletion, STIM1 and Orai1 complexes assemble autonomously at ER–plasma membrane (PM) junctions to trigger store-operated Ca2+ influx. One hypothesis to explain this process is a diffusion trap in which activated STIM1 diffusing in the ER becomes trapped at junctions through interactions with the PM, and STIM1 then traps Orai1 in the PM through binding of its calcium release-activated calcium activation domain. We tested this model by analyzing STIM1 and Orai1 diffusion using single-particle tracking, photoactivation of protein ensembles, and Monte Carlo simulations. In resting cells, STIM1 diffusion is Brownian, while Orai1 is slightly subdiffusive. After store depletion, both proteins slow to the same speeds, consistent with complex formation, and are confined to a corral similar in size to ER–PM junctions. While the escape probability at high STIM:Orai expression ratios is <1%, it is significantly increased by reducing the affinity of STIM1 for Orai1 or by expressing the two proteins at comparable levels. Our results provide direct evidence that STIM-Orai complexes are trapped by their physical connections across the junctional gap, but also reveal that the complexes are surprisingly dynamic, suggesting that readily reversible binding reactions generate free STIM1 and Orai1, which engage in constant diffusional exchange with extrajunctional pools.
doi:10.1091/mbc.E14-06-1107
PMCID: PMC4230625  PMID: 25057023
2.  Early Onset and Severe Clinical Course Associated with the m.5540G>A Mutation in MT-TW 
We report a patient harboring a de novo m.5540G>A mutation affecting the MT-TW gene coding for the mitochondrial tryptophan-transfer RNA. This patient presented with atonic-myoclonic epilepsy, bilateral sensorineural hearing loss, ataxia, motor regression, ptosis, and pigmentary retinopathy. Our proband had an earlier onset and more severe phenotype than the first reported patient harboring the same mutation. We discuss her clinical presentation and compare it with the only previously published case.
doi:10.1016/j.ymgmr.2013.12.001
PMCID: PMC4185924  PMID: 25302159
Mitochondrial DNA; MT-TW gene; sensorineural hearing loss; ataxia; pigmentary retinopathy
3.  Structure and function of a spectrin-like regulator of bacterial cytokinesis 
Nature Communications  2014;5:5421.
Bacterial cell division is facilitated by a molecular machine—the divisome—that assembles at mid-cell in dividing cells. The formation of the cytokinetic Z-ring by the tubulin homologue FtsZ is regulated by several factors, including the divisome component EzrA. Here we describe the structure of the 60-kDa cytoplasmic domain of EzrA, which comprises five linear repeats of an unusual triple helical bundle. The EzrA structure is bent into a semicircle, providing the protein with the potential to interact at both N- and C-termini with adjacent membrane-bound divisome components. We also identify at least two binding sites for FtsZ on EzrA and map regions of EzrA that are responsible for regulating FtsZ assembly. The individual repeats, and their linear organization, are homologous to the spectrin proteins that connect actin filaments to the membrane in eukaryotes, and we thus propose that EzrA is the founding member of the bacterial spectrin family.
EzrA regulates the polymerization of FtsZ, a tubulin-like protein and main component of the Z-ring, which drives cell division in bacteria. Here the authors describe the crystal structure of EzrA and demonstrate that it shares structural and functional properties with eukaryotic spectrins.
doi:10.1038/ncomms6421
PMCID: PMC4243239  PMID: 25403286
4.  Central Integration of Canal and Otolith Signals is Abnormal in Vestibular Migraine 
Vestibular migraine (VM), a common cause of vestibular symptoms within the general population, is a disabling and poorly understood form of dizziness. We sought to examine the underlying pathophysiology of VM with three studies, which involved the central synthesis of canal and otolith cues, and present preliminary results from each of these studies: (1) VM patients appear to have reduced motion perception thresholds when canal and otolith signals are modulated in a co-planar manner during roll tilt; (2) percepts of roll tilt appear to develop more slowly in VM patients than in control groups during a centrifugation paradigm that presents conflicting, orthogonal canal and otolith cues; and (3) eye movement responses appear to be different in VM patients when studied with a post-rotational tilt paradigm, which also presents a canal–otolith conflict, as the shift of the eye’s rotational axis was larger in VM and the relationship between the axis shift and tilt suppression of the vestibulo-ocular reflex differed in VM patients relative to control groups. Based on these preliminary perceptual and eye movement results obtained with three different motion paradigms, we present a hypothesis that the integration of canal and otolith signals by the brain is abnormal in VM and that this abnormality could be cerebellar in origin. We provide potential mechanisms that could underlie these observations, and speculate that one of more of these mechanisms contributes to the vestibular symptoms and motion intolerance that are characteristic of the VM syndrome.
doi:10.3389/fneur.2014.00233
PMCID: PMC4226145  PMID: 25426098
vestibular; migraine; cerebellum; perception; oculomotor
5.  Racial differences in cortical bone and their relationship to biochemical variables in black and white children in the early stages of puberty 
Introduction
Racial differences in bone structure likely have roots in childhood as bone size develops predominantly during growth. This study aimed to compare cortical bone health within the tibial diaphysis of black and white children in the early stages of puberty, and explore the contributions of biochemical variables in explaining racial variation in cortical bone properties.
Methods
A cross-sectional study was performed comparing peripheral quantitative computed tomography-derived cortical bone measures of the tibial diaphysis and biochemical variables in 314 participants (n=155 males; n=164 blacks) in the early stages of puberty.
Results
Blacks had greater cortical volumetric bone mineral density, mass and size compared to whites (all p<0.01), contributing to blacks having 17.0% greater tibial strength (polar strength-strain index [SSIP]) (p<0.001). Turnover markers indicated blacks had higher bone formation (osteocalcin [OC] and bone specific alkaline phosphatase) and lower bone resorption (N-terminal telopeptide) than whites (all p<0.01). Blacks also had lower 25-hydroxyvitamin D [25(OH)D], and higher 1,25-dihydroxyvitamin D [1,25(OH)2D] and parathyroid hormone (PTH) (all p<0.05). There were no correlations between tibial bone properties, and 25(OH)D and PTH in whites (all p≥0.10); however, SSIP was negatively and positively correlated with 25(OH)D and PTH in blacks, respectively (all p≤0.02). Variation in bone cross-sectional area and SSIP attributable to race was partially explained by tibial length, 25(OH)D/PTH and OC.
Conclusions
Divergence in tibial cortical bone properties between blacks and whites is established by the early stages of puberty with the enhanced cortical bone properties in black children possibly being explained by higher PTH and OC.
doi:10.1007/s00198-012-2174-8
PMCID: PMC4163020  PMID: 23093348
peripheral quantitative computed tomography; 25 hydroxy vitamin D; 1,25 dihydroxy vitamin D; PTH; bone turnover; race
6.  Missense Mutations in FBN1 Exons 41 and 42 Cause Weill-Marchesani Syndrome with Thoracic Aortic Disease and Marfan Syndrome 
American journal of medical genetics. Part A  2013;161(9):10.1002/ajmg.a.36044.
Mutations in FBN1 cause a range of overlapping but distinct conditions including Marfan syndrome (MFS), Weill-Marchesani syndrome (WMS), familial thoracic aortic aneurysms/dissections (FTAAD), acromicric dysplasia (AD), and geleophysic dysplasia (GD). Two forms of acromelic dysplasia, AD and GD, characterized by short stature, brachydactyly, reduced joint mobility, and characteristic facies, result from heterozygous missense mutations occurring in exons 41 and 42 of FBN1; missense mutations in these exons have not been reported to cause MFS or other syndromes. Here we report on probands with MFS and WMS who have heterozygous FBN1 missense mutations in exons 41 and 42, respectively. The proband with WMS has ectopia lentis, short stature, thickened pinnae, tight skin, striae atrophicae, reduced extension of the elbows, contractures of the fingers and toes, and brachydactyly and has a missense mutation in exon 42 of FBN1 (c.5242T>C ;p.C1748R). He also experienced a previously unreported complication of WMS, an acute thoracic aortic dissection. The second proband displays classic characteristics of MFS, including ectopia lentis, skeletal features and aortic root dilatation, and has a missense mutation in exon 41 of FBN1 (c.5084G>A; p.C1695Y). These phenotypes provide evidence that missense mutations in exons 41 and 42 of FBN1 lead to MFS and WMS in addition to AD and GD and also suggest that all individuals with pathogenic FBN1 mutations in these exons should be assessed for thoracic aortic disease and ectopia lentis. Further studies are necessary to elucidate the factors responsible for the different phenotypes associated with missense mutations in these exons of FBN1.
doi:10.1002/ajmg.a.36044
PMCID: PMC3829633  PMID: 23897642
geleophysic dysplasia; acromicric dysplasia; aortic aneurysm; aortic dissection; ectopia lentis
7.  An animal model of oxaliplatin-induced cold allodynia reveals a crucial role for Nav1.6 in peripheral pain pathways 
Pain  2013;154(9):1749-1757.
Cold allodynia, pain in response to cooling, occurs during or within hours of oxaliplatin infusion and is thought to arise from a direct effect of oxaliplatin on peripheral sensory neurons. To characterize the pathophysiological mechanisms underlying acute oxaliplatin-induced cold allodynia, we established a new intraplantar oxaliplatin mouse model that rapidly developed long-lasting cold allodynia mediated entirely through tetrodotoxin-sensitive Nav pathways. Using selective inhibitors and knockout animals, we found that Nav1.6 was the key isoform involved, while thermosensitive transient receptor potential channels were not involved. Consistent with a crucial role for delayed-rectifier potassium channels in excitability in response to cold, intraplantar administration of the K+-channel blocker 4-aminopyridine mimicked oxaliplatin-induced cold allodynia and was also inhibited by Navl.6 blockers. Intraplantar injection of the Nav1.6-activator Cn2 elicited spontaneous pain, mechanical allodynia and enhanced 4-aminopyridine-induced cold allodynia. These findings provide behavioural evidence for a crucial role of Nav1.6 in multiple peripheral pain pathways including cold allodynia.
doi:10.1016/j.pain.2013.05.032
PMCID: PMC3748219  PMID: 23711479
8.  High-Dosage Ascorbic Acid Treatment in Charcot-Marie-Tooth Disease Type 1A: Results of a Randomized, Double-Masked, Controlled Trial 
JAMA neurology  2013;70(8):981-987.
Importance
No current medications improve neuropathy in subjects with Charcot-Marie-Tooth disease type 1A (CMT1A). Ascorbic acid (AA) treatment improved the neuropathy of a transgenic mouse model of CMT1A and is a potential therapy. A lower dosage (1.5 g/d) did not cause improvement in humans. It is unknown whether a higher dosage would prove more effective.
Objective
To determine whether 4-g/d AA improves the neuropathy of subjects with CMT1A.
Design
A futility design to determine whether AA was unable to reduce worsening on the CMT Neuropathy Score (CMTNS) by at least 50% over a 2-year period relative to a natural history control group.
Setting
Three referral centers with peripheral nerve clinics (Wayne State University, Johns Hopkins University, and University of Rochester).
Participants
One hundred seventy-four subjects with CMT1A were assessed for eligibility; 48 did not meet eligibility criteria and 16 declined to participate. The remaining 110 subjects, aged 13 to 70 years, were randomly assigned in a double-masked fashion with 4:1 allocation to oral AA (87 subjects) or matching placebo (23 subjects). Sixty-nine subjects from the treatment group and 16 from the placebo group completed the study. Two subjects from the treatment group and 1 from the placebo group withdrew because of adverse effects.
Interventions
Oral AA (4 g/d) or matching placebo.
Main Outcomes and Measures
Change from baseline to year 2 in the CMTNS, a validated composite impairment score for CMT.
Results
The mean 2-year change in the CMTNS was −0.21 for the AA group and −0.92 for the placebo group, both better than natural history (+1.33). This was well below 50% reduction of CMTNS worsening from natural history, so futility could not be declared (P > .99).
Conclusions and Relevance
Both treated patients and those receiving placebo performed better than natural history. It seems unlikely that our results support undertaking a larger trial of 4-g/d AA treatment in subjects with CMT1A.
Trial Registration
clinicaltrials.gov Identifier: NCT00484510
doi:10.1001/jamaneurol.2013.3178
PMCID: PMC3752369  PMID: 23797954
9.  Differential contribution of chemotaxis and substrate restriction to segregation of immature and mature thymocytes 
Immunity  2009;31(6):986-998.
SUMMARY
T cell development requires sequential localization of thymocyte subsets to distinct thymic microenvironments. To address mechanisms governing this segregation, we used 2-photon microscopy to visualize the migration of purified thymocyte subsets in defined microenvironments within thymic slices. Double-negative (CD4−8−) and double-positive (CD4+8+; DP) thymocytes were strictly confined to cortex where they moved slowly without directional bias. DP cells accumulated and migrated more rapidly in a specialized inner-cortical microenvironment, but were excluded from the medulla by an inability to migrate on medullary substrates. In contrast, CD4 single-positive (SP) thymocytes migrated directionally towards the medulla, where they accumulated and moved very rapidly. Our results reveal a requisite two-step process governing CD4 SP medullary localization: the chemokine receptor CCR7 mediated chemotaxis of CD4 SP cells towards the medulla, whereas a distinct pertussis-toxin sensitive pathway was required for medullary entry. These findings suggest that developmentally regulated responses to both chemotactic signals and specific migratory substrates guide thymocytes to specific locations in the thymus as they mature.
doi:10.1016/j.immuni.2009.09.020
PMCID: PMC4106268  PMID: 19962328
10.  Mitochondrial Haplogroups Are Associated With Risk of Neuroretinal Disorder in HIV-Positive Patients 
Although highly active antiretroviral therapy has improved survivorship dramatically and decreased the incidence of cytomegalovirus retinitis among patients with AIDS, other ophthalmic complications continue to occur. One complication observed in ~12% of HIV-infected patients is a presumed neuroretinal disorder (NRD), manifested as decreased contrast sensitivity and associated with vague subjective complaints of hazy vision. Pathologically, patients with AIDS even without ocular opportunistic infections have loss of optic nerve axons, suggestive of mitochondrial dysfunction. We explored whether variation in mitochondrial DNA was associated with time to NRD in HIV-infected patients in the Longitudinal Study of Ocular Complications of AIDS cohort. Within the Western European, or “N”, mitochondrial DNA macrohaplogroup, haplogroup J, was associated with 80% decrease in the risk of progression to NRD during the study (hazard ratio = 0.20, P = 0.039) and suggested an independent association with protection against NRD in a cross-section of all patients taken at enrollment (1.5% vs. 8.9% in patients with vs. without haplogroup J, respectively, P = 0.05). These data suggest that mitochondrial genotype may influence propensity to develop HIV-associated NRD in patients with AIDS.
doi:10.1097/QAI.0b013e3181cb8319
PMCID: PMC4096800  PMID: 20098332
AIDS; mitochondrial DNA; neuroretinal disorder
11.  A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression 
Human Molecular Genetics  2014;23(23):6345-6355.
The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes.
doi:10.1093/hmg/ddu352
PMCID: PMC4222368  PMID: 25008111
12.  Structure of the LdcB LD-Carboxypeptidase Reveals the Molecular Basis of Peptidoglycan Recognition 
Structure(London, England:1993)  2014;22(7):949-960.
Summary
Peptidoglycan surrounds the bacterial cytoplasmic membrane to protect the cell against osmolysis. The biosynthesis of peptidoglycan, made of glycan strands crosslinked by short peptides, is the target of antibiotics like β-lactams and glycopeptides. Nascent peptidoglycan contains pentapeptides that are trimmed by carboxypeptidases to tetra- and tripeptides. The well-characterized DD-carboxypeptidases hydrolyze the terminal D-alanine from the stem pentapeptide to produce a tetrapeptide. However, few LD-carboxypeptidases that produce tripeptides have been identified, and nothing is known about substrate specificity in these enzymes. We report biochemical properties and crystal structures of the LD-carboxypeptidases LdcB from Streptococcus pneumoniae, Bacillus anthracis, and Bacillus subtilis. The enzymes are active against bacterial cell wall tetrapeptides and adopt a zinc-carboxypeptidase fold characteristic of the LAS superfamily. We have also solved the structure of S. pneumoniae LdcB with a product mimic, elucidating the residues essential for peptidoglycan recognition and the conformational changes that occur on ligand binding.
Highlights
•A peptidoglycan, peptide stem-trimming carboxypeptidase, LdcB, has been characterized•The crystal structure of LdcB has been solved with a peptidoglycan mimic bound•The LdcB structure undergoes significant conformational change on binding ligand•The exquisite substrate specificity of LdcB has also been demonstrated in vitro
Peptidoglyan is an essential layer surrounding the bacterial cytoplasmic membrane that is matured and trimmed by carboxypeptidases. Hoyland et al. describe the structure of one such carboxypeptidase in the presence of a product mimic, explaining the molecular specificity of the enzyme family.
doi:10.1016/j.str.2014.04.015
PMCID: PMC4087270  PMID: 24909784
13.  Elongated Structure of the Outer-Membrane Activator of Peptidoglycan Synthesis LpoA: Implications for PBP1A Stimulation 
Structure(London, England:1993)  2014;22(7):1047-1054.
Summary
The bacterial cell envelope contains the stress-bearing peptidoglycan layer, which is enlarged during cell growth and division by membrane-anchored synthases guided by cytoskeletal elements. In Escherichia coli, the major peptidoglycan synthase PBP1A requires stimulation by the outer-membrane-anchored lipoprotein LpoA. Whereas the C-terminal domain of LpoA interacts with PBP1A to stimulate its peptide crosslinking activity, little is known about the role of the N-terminal domain. Herein we report its NMR structure, which adopts an all-α-helical fold comprising a series of helix-turn-helix tetratricopeptide-repeat (TPR)-like motifs. NMR spectroscopy of full-length LpoA revealed two extended flexible regions in the C-terminal domain and limited, if any, flexibility between the N- and C-terminal domains. Analytical ultracentrifugation and small-angle X-ray scattering results are consistent with LpoA adopting an elongated shape, with dimensions sufficient to span from the outer membrane through the periplasm to interact with the peptidoglycan synthase PBP1A.
Graphical Abstract
Highlights
•LpoA’s N-terminal domain features an all-α-helical fold similar to TPR domains•The C-terminal domain of E. coli LpoA contains two extensive flexible regions•Full-length LpoA adopts an elongated structure with low interdomain flexibility•LpoA can span the periplasm to stimulate the peptidoglycan synthase PBP1A
Jean et al. determine the NMR structure of LpoA N-terminal domain and identify two unique flexible regions in the C-terminal domain. SAXS data suggest little flexibility between the two domains leading to an elongated shape for the full-length protein, required in the activation of peptidoglycan synthase PBP1A.
doi:10.1016/j.str.2014.04.017
PMCID: PMC4111904  PMID: 24954617
14.  The YmdB Phosphodiesterase Is a Global Regulator of Late Adaptive Responses in Bacillus subtilis 
Journal of Bacteriology  2014;196(2):265-275.
Bacillus subtilis mutants lacking ymdB are unable to form biofilms, exhibit a strong overexpression of the flagellin gene hag, and are deficient in SlrR, a SinR antagonist. Here, we report the functional and structural characterization of YmdB, and we find that YmdB is a phosphodiesterase with activity against 2′,3′- and 3′,5′-cyclic nucleotide monophosphates. The structure of YmdB reveals that the enzyme adopts a conserved phosphodiesterase fold with a binuclear metal center. Mutagenesis of a catalytically crucial residue demonstrates that the enzymatic activity of YmdB is essential for biofilm formation. The deletion of ymdB affects the expression of more than 800 genes; the levels of the σD-dependent motility regulon and several sporulation genes are increased, and the levels of the SinR-repressed biofilm genes are decreased, confirming the role of YmdB in regulating late adaptive responses of B. subtilis.
doi:10.1128/JB.00826-13
PMCID: PMC3911264  PMID: 24163345
15.  Responses evoked by a vestibular implant providing chronic stimulation 
Patients with bilateral vestibular loss experience dehabilitating visual, perceptual, and postural difficulties, and an implantable vestibular prosthesis that could improve these symptoms would be of great benefit to these patients. In previous work, we have shown that a one-dimensional, unilateral canal prosthesis can improve the vestibulooccular reflex (VOR) in canal-plugged squirrel monkeys. In addition to the VOR, the potential effects of a vestibular prosthesis on more complex, highly integrative behaviors, such as the perception of head orientation and posture have remained unclear. We tested a one-dimensional, unilateral prosthesis in a rhesus monkey with bilateral vestibular loss and found that chronic electrical stimulation partially restored the compensatory VOR and also that percepts of head orientation relative to gravity were improved. However, the one-dimensional prosthetic stimulation had no clear effect on postural stability during quiet stance, but sway evoked by head-turns was modestly reduced. These results suggest that not only can the implementation of a vestibular prosthesis provide partial restitution of VOR but may also improve perception and posture in the presence of bilateral vestibular hypofunction (BVH). In this review, we provide an overview of our previous and current work directed towards the eventual clinical implementation of an implantable vestibular prosthesis.
doi:10.3233/VES-2012-0442
PMCID: PMC4041130  PMID: 22699148
vestibular; vestibular prosthesis; implant; vestibulooccular reflex; psychophysics; balance; posture
16.  Adaptation of Vestibular Tone Studied with Electrical Stimulation of Semicircular Canal Afferents 
Damage to one vestibular labyrinth or nerve causes a central tone imbalance, reflected by prominent spontaneous nystagmus. Central adaptive mechanisms eliminate the nystagmus over several days, and the mechanisms underlying this process have received extensive study. The characteristics of vestibular compensation when the tone imbalance is presented gradually or repeatedly have never been studied. We used high-frequency electrical stimulation of semicircular canal afferents to generate a vestibular tone imbalance and recorded the nystagmus produced when the stimulation was started abruptly or gradually and when it was repeatedly cycled on and off. In the acute-onset protocol, brisk nystagmus occurred when stimulation started, gradually resolved within 1 day, and reversed direction when the stimulation was stopped after 1 week. Repeated stimulation cycles resulted in progressively smaller nystagmus responses. In the slow-onset protocol, minimal nystagmus occurred while the stimulation ramped-up to its maximum rate over 12 h, but a reversal still occurred when the stimulation was stopped after 1 week, and repeated stimulation cycles did not affect this pattern. The absence of nystagmus during the 12 h ramp of stimulation demonstrates that central vestibular tone can rebalance relatively quickly, and the reduction in the stimulation-off nystagmus with repeated cycles of the acute-onset but not the slow-onset stimulation suggests that dual-state adaptation may have occurred with the former paradigm but not the latter.
doi:10.1007/s10162-013-0376-1
PMCID: PMC3642268  PMID: 23423561
vestibular; nystagmus; compensation; adaptation; electrical stimulation
17.  In Search of Decay in Verbal Short-Term Memory 
Is forgetting in the short term due to decay with the mere passage of time, interference from other memoranda, or both? Past research on short-term memory has revealed some evidence for decay and a plethora of evidence showing that short-term memory is worsened by interference. However, none of these studies has directly contrasted decay and interference in short-term memory in a task that rules out the use of rehearsal processes. In this article the authors present a series of studies using a novel paradigm to address this problem directly, by interrogating the operation of decay and interference in short-term memory without rehearsal confounds. The results of these studies indicate that short-term memories are subject to very small decay effects with the mere passage of time but that interference plays a much larger role in their degradation. The authors discuss the implications of these results for existing models of memory decay and interference.
doi:10.1037/a0014873
PMCID: PMC3980403  PMID: 19271849
short-term memory; decay; interference; recognition; verbal working memory
18.  Anterior Tibialis CMAP Amplitude Correlations with Impairment in CMT1A 
Muscle & nerve  2013;47(4):493-496.
Introduction
CMT1A is a slowly progressive neuropathy in which impairment is length dependent. Fibular nerve conduction studies to the anterior tibialis muscle (AT) may serve as a physiological marker of disease progression in patients with CMT1A.
Objective
Determine whether the AT compound muscle action potential (CMAP) amplitude correlates with impairment in patients with CMT1A.
Methods
We correlated AT CMAP amplitudes and impairment measured by the CMT Neuropathy Score (CMTNS) in a cross-section of 121 patients with CMT1A and a subset of 27 patients with longitudinal data.
Results
AT CMAP amplitudes correlated with impairment as measured by the CMTNS in cross sectional analysis. Longitudinal changes in the AT CMAP showed a strong inverse correlation with leg strength but not other components of the CMTNS.
Discussion
AT CMAP amplitude may serve as a useful outcome measure for physiological changes in natural history studies and clinical trials for patients with CMT1A.
doi:10.1002/mus.23614
PMCID: PMC3608739  PMID: 23456782
Neuropathy; Charcot-Marie-Tooth Disease (CMT); Outcome measure; Charcot-Marie-Tooth Neuropathy Score (CMTNS); Nerve Conduction Studies (NCS)
19.  The Mind and Brain of Short-Term Memory 
Annual review of psychology  2008;59:193-224.
The past 10 years have brought near-revolutionary changes in psychological theories about short-term memory, with similarly great advances in the neurosciences. Here, we critically examine the major psychological theories (the “mind”) of short-term memory and how they relate to evidence about underlying brain mechanisms. We focus on three features that must be addressed by any satisfactory theory of short-term memory. First, we examine the evidence for the architecture of short-term memory, with special attention to questions of capacity and how—or whether—short-term memory can be separated from long-term memory. Second, we ask how the components of that architecture enact processes of encoding, maintenance, and retrieval. Third, we describe the debate over the reason about forgetting from short-term memory, whether interference or decay is the cause. We close with a conceptual model tracing the representation of a single item through a short-term memory task, describing the biological mechanisms that might support psychological processes on a moment-by-moment basis as an item is encoded, maintained over a delay with some forgetting, and ultimately retrieved.
doi:10.1146/annurev.psych.59.103006.093615
PMCID: PMC3971378  PMID: 17854286
working memory; attention; encoding; storage; retrieval
20.  Autoantibodies to Agrin in Myasthenia Gravis Patients 
PLoS ONE  2014;9(3):e91816.
To determine if patients with myasthenia gravis (MG) have antibodies to agrin, a proteoglycan released by motor neurons and is critical for neuromuscular junction (NMJ) formation, we collected serum samples from 93 patients with MG with known status of antibodies to acetylcholine receptor (AChR), muscle specific kinase (MuSK) and lipoprotein-related 4 (LRP4) and samples from control subjects (healthy individuals and individuals with other diseases). Sera were assayed for antibodies to agrin. We found antibodies to agrin in 7 serum samples of MG patients. None of the 25 healthy controls and none of the 55 control neurological patients had agrin antibodies. Two of the four triple negative MG patients (i.e., no detectable AChR, MuSK or LRP4 antibodies, AChR-/MuSK-/LRP4-) had antibodies against agrin. In addition, agrin antibodies were detected in 5 out of 83 AChR+/MuSK-/LRP4- patients but were not found in the 6 patients with MuSK antibodies (AChR-/MuSK+/LRP4-). Sera from MG patients with agrin antibodies were able to recognize recombinant agrin in conditioned media and in transfected HEK293 cells. These sera also inhibited the agrin-induced MuSK phosphorylation and AChR clustering in muscle cells. Together, these observations indicate that agrin is another autoantigen in patients with MG and agrin autoantibodies may be pathogenic through inhibition of agrin/LRP4/MuSK signaling at the NMJ.
doi:10.1371/journal.pone.0091816
PMCID: PMC3954737  PMID: 24632822
21.  Whole-Exome Sequencing Identifies ALMS1, IQCB1, CNGA3, and MYO7A Mutations in Patients with Leber Congenital Amaurosis 
Human mutation  2011;32(12):1450-1459.
It has been well documented that mutations in the same retinal disease gene can result in different clinical phenotypes due to difference in the mutant allele and/or genetic background. To evaluate this, a set of consanguineous patient families with Leber congenital amaurosis (LCA) that do not carry mutations in known LCA disease genes was characterized through homozygosity mapping followed by targeted exon/whole-exome sequencing to identify genetic variations. Among these families, a total of five putative disease-causing mutations, including four novel alleles, were found for six families. These five mutations are located in four genes, ALMS1, IQCB1, CNGA3, and MYO7A. Therefore, in our LCA collection from Saudi Arabia, three of the 37 unassigned families carry mutations in retinal disease genes ALMS1, CNGA3, and MYO7A, which have not been previously associated with LCA, and 3 of the 37 carry novel mutations in IQCB1, which has been recently associated with LCA. Together with other reports, our results emphasize that the molecular heterogeneity underlying LCA, and likely other retinal diseases, may be highly complex. Thus, to obtain accurate diagnosis and gain a complete picture of the disease, it is essential to sequence a larger set of retinal disease genes and combine the clinical phenotype with molecular diagnosis.
doi:10.1002/humu.21587
PMCID: PMC3943164  PMID: 21901789
Leber congenital amaurosis; LCA; whole-exome sequencing; SNP; padlock
22.  Miniaturized Bioaffinity Assessment Coupled to Mass Spectrometry for Guided Purification of Bioactives from Toad and Cone Snail 
Biology  2014;3(1):139-156.
A nano-flow high-resolution screening platform, featuring a parallel chip-based microfluidic bioassay and mass spectrometry coupled to nano-liquid chromatography, was applied to screen animal venoms for nicotinic acetylcholine receptor like (nAChR) affinity by using the acetylcholine binding protein, a mimic of the nAChR. The potential of this microfluidic platform is demonstrated by profiling the Conus textile venom proteome, consisting of over 1,000 peptides. Within one analysis (<90 min, 500 ng venom injected), ligands are detected and identified. To show applicability for non-peptides, small molecular ligands such as steroidal ligands were identified in skin secretions from two toad species (Bufo alvarius and Bufo marinus). Bioactives from the toad samples were subsequently isolated by MS-guided fractionation. The fractions analyzed by NMR and a radioligand binding assay with α7-nAChR confirmed the identity and bioactivity of several new ligands.
doi:10.3390/biology3010139
PMCID: PMC4009767  PMID: 24833338
on-line microfluidics; nano-liquid chromatography-mass spectrometry (nano-LC-MS); Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP); Conus textile; Bufo alvarius; Bufo marinus
23.  Abnormal Calcium Handling Properties Underlie Familial Hypertrophic Cardiomyopathy Pathology in Patient-Specific Induced Pluripotent Stem Cells 
Cell stem cell  2013;12(1):101-113.
Summary
Familial hypertrophic cardiomyopathy (HCM) is a prevalent hereditary cardiac disorder linked to arrhythmia and sudden cardiac death. While the causes of HCM have been identified as genetic mutations in the cardiac sarcomere, the pathways by which sarcomeric mutations engender myocyte hypertrophy and electrophysiological abnormalities are not understood. To elucidate the mechanisms underlying HCM development, we generated patient-specific induced pluripotent stem cell cardiomyocytes (iPSC-CMs) from a ten-member family cohort carrying a hereditary HCM missense mutation (Arg663His) in the MYH7 gene. Diseased iPSC-CMs recapitulated numerous aspects of the HCM phenotype including cellular enlargement and contractile arrhythmia at the single-cell level. Calcium (Ca2+) imaging indicated dysregulation of Ca2+ cycling and elevation in intracellular Ca2+ ([Ca2+]i) are central mechanisms for disease pathogenesis. Pharmacological restoration of Ca2+ homeostasis prevented development of hypertrophy and electrophysiological irregularities. We anticipate that these findings will help elucidate the mechanisms underlying HCM development and identify novel therapies for the disease.
doi:10.1016/j.stem.2012.10.010
PMCID: PMC3638033  PMID: 23290139
24.  Store-Operated Calcium Channels: New Perspectives on Mechanism and Function 
Store-operated calcium channels (SOCs) are a nearly ubiquitous Ca2+ entry pathway stimulated by numerous cell surface receptors via the reduction of Ca2+ concentration in the ER. The discovery of STIM proteins as ER Ca2+ sensors and Orai proteins as structural components of the Ca2+ release-activated Ca2+ (CRAC) channel, a prototypic SOC, opened the floodgates for exploring the molecular mechanism of this pathway and its functions. This review focuses on recent advances made possible by the use of STIM and Orai as molecular tools. I will describe our current understanding of the store-operated Ca2+ entry mechanism and its emerging roles in physiology and disease, areas of uncertainty in which further progress is needed, and recent findings that are opening new directions for research in this rapidly growing field.
Store-operated Ca2+ channels (SOCs) in the plasma membrane are activated by reduced ER luminal Ca2+ concentrations. This involves the rearrangement of STIM (Ca2+ sensor) and Orai proteins (calcium channel components) in the cell.
doi:10.1101/cshperspect.a003970
PMCID: PMC3225942  PMID: 21791698
25.  Electrical stimulation of semicircular canal afferents affects the perception of head orientation 
Patients with vestibular dysfunction have visual, perceptual, and postural deficits. While there is considerable evidence that a semicircular canal prosthesis that senses angular head velocity and stimulates canal ampullary nerves can improve vision by augmenting the vestibulo-ocular reflex, no information is available regarding the potential utility of a canal prosthesis to improve perceptual deficits. In this study we investigated the possibility that electrical stimulation of canal afferents could be used to modify percepts of head orientation. Two rhesus monkeys were trained to align a light bar parallel to gravity, and were tested in the presence and absence of electrical stimulation provided by an electrode implanted in the right posterior canal. While the monkeys aligned the light bar close to the true earth-vertical without stimulation, when the right posterior canal was stimulated their responses deviated towards their left ear, consistent with a misperception of head tilt towards the right. The deviation of the light bar from the earth-vertical exceeded the torsional deviation of the eyes, indicating that the perceptual changes were not simply visual in origin. Eye movements recorded during electrical stimulation in the dark were consistent with isolated activation of right posterior canal afferents, with no evidence of otolith stimulation. These results demonstrate that electrical stimulation of canal afferents affects the perception of head orientation, and therefore suggest that motion-modulated stimulation of canal afferents by a vestibular prosthesis could potentially improve vestibular percepts in patients lacking normal vestibular function.
doi:10.1523/JNEUROSCI.0112-13.2013
PMCID: PMC3718451  PMID: 23719819

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