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1.  Isolation and Chemical Characterization of Lipid A From the Outer Membrane of Gram-Negative Bacteria 
SHORT ABSTRACT
Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from Gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.
LONG ABSTRACT
Lipopolysaccharide (LPS) is the major cell surface molecule of Gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three components: the distal O-polysaccharide, a core oligosaccharide, and the lipid A hydrophobic anchor, where lipid A is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from Gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In additional to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of Gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response and have provided many new targets in the development of antibacterial compounds.
doi:10.3791/50623
PMCID: PMC3885993  PMID: 24084191
lipid A; Bligh-Dyer; thin layer chromatography (TLC); lipopolysaccharide; mass spectrometry; Collision Induced Dissociation (CID); Photodissociation (PD)
2.  Amyloid imaging for dementia in clinical practice 
BMC Medicine  2015;13:163.
In vivo imaging of brain amyloid using positron emission tomography (PET) scanning is widely used in research studies of dementia, with three amyloid PET ligands being licenced for clinical use. The main clinical use of PET is to help confirm or exclude the likely diagnosis of Alzheimer’s disease in challenging cases, where diagnostic uncertainty remains after current clinical and investigative work up. Whilst diagnostically valuable in such select cases, much wider clinical adoption, especially for very early disease, will be limited by both cost and the lack of a currently effective disease-modifying treatment that requires such early case identification. The use of amyloid imaging to appropriately stratify subjects for prognostic studies and therapeutic trials should increase the efficiency and potentially shorten the time of such studies, and its use combined with other biomarkers and genetics will likely lead to new ways of defining and classifying the dementias.
doi:10.1186/s12916-015-0404-6
PMCID: PMC4499896  PMID: 26170121
Amyloid; Dementia; Imaging; Positron emission tomography
3.  Characterization of a novel and potentially lethal designer drug, (±)-cis-para-methyl-4-methylaminorex (4,4'-DMAR, or “Serotoni”) 
Drug testing and analysis  2014;6(0):684-695.
During the second half of 2013, a total of 26 deaths involving para-methyl-4-methylaminorex (4,4’-DMAR) were reported to the European Monitoring Centre for Drugs and Drug Addiction. While aminorex and 4-methylaminorex (4-MAR) are known psychostimulants, nothing is known about the comparatively new para-methyl analogue. Analytical characterization of two independent samples obtained from online vendors confirmed the presence of the (±)-cis isomer that also appeared to be involved in at least 18 of the 26 deaths. Extensive characterizations included crystal structure analysis, single, tandem and high-resolution mass spectrometry, liquid and gas chromatography and nuclear magnetic resonance spectroscopy. For the work described here, both the (±)-cis and (±)-trans racemates were also synthesized, confirming that the differentiation between these two forms was straight-forward. Monoamine transporter activity was studied using rat brain synaptosomes. (±)-cis-4,4'-DMAR was a potent, efficacious substrate-type releaser at transporters for dopamine, norepinephrine and serotonin with EC50 values of 8.6 ± 1.1 nM (DAT), 26.9 ± 5.9 nM (NET) and 18.5 ± 2.8 nM (SERT), respectively. A comparison with d-amphetamine, aminorex and (±)-cis-4-MAR revealed that activity at SERT varied more than 100-fold across the four drugs, with (±)-cis-4,4’-DMAR exhibiting the highest potency for releasing 5-HT. The potent releasing activity of (±)-cis-4,4’-DMAR at all three monoamine transporters predicts a potential for serious side-effects such as psychotic symptoms, agitation, hyperthermia and cardiovascular stimulation, especially after high-dose exposure or following combination with other psychostimulants.
doi:10.1002/dta.1668
PMCID: PMC4128571  PMID: 24841869
Aminorex; 4-methylaminorex; para-methyl-4-methylaminorex; new psychoactive substances; psychostimulants; internet; monoamine transporters; synaptosomes
4.  Diagnostic criteria for vascular cognitive disorders: a VASCOG statement 
Background
Several sets of diagnostic criteria have been published for vascular dementia (VaD) since the 1960s. The continuing ambiguity in VaD definition warrants a critical re-examination.
Methods
Participants at a special symposium of the International Society for Vascular Behavioral and Cognitive Disorders (VASCOG) in 2009 critiqued the current criteria. They drafted a proposal for a new set of criteria, later reviewed through multiple drafts by the group, including additional experts and the members of the Neurocognitive Disorders Work Group of the DSM-5 Task Force.
Results
Cognitive disorders of vascular etiology are a heterogeneous group of disorders with diverse pathologies and clinical manifestations, discussed broadly under the rubric of vascular cognitive disorders (VCD). The continuum of vascular cognitive impairment is recognized by the categories of Mild Vascular Cognitive Disorder, and Vascular Dementia or Major Vascular Cognitive Disorder. Diagnostic thresholds are defined. Clinical and neuroimaging criteria are proposed for establishing vascular etiology. Subtypes of VCD are described, and the frequent co-occurrence of Alzheimer’s disease pathology emphasized.
Conclusions
The proposed criteria for VCD provide a coherent approach to the diagnosis of this diverse group of disorders, with a view to stimulating clinical and pathological validation studies. These criteria can be harmonized with the DSM-5 criteria such that an international consensus on the criteria for VCD may be achieved.
doi:10.1097/WAD.0000000000000034
PMCID: PMC4139434  PMID: 24632990
Vascular dementia; vascular cognitive disorder; vascular cognitive impairment; diagnostic criteria; cerebrovascular disease; multi-infarct dementia; post-stroke dementia; subcortical dementia
5.  Cortical Thickness in Dementia with Lewy Bodies and Alzheimer's Disease: A Comparison of Prodromal and Dementia Stages 
PLoS ONE  2015;10(6):e0127396.
Objectives
To assess and compare cortical thickness (CTh) of patients with prodromal Dementia with Lewy bodies (pro-DLB), prodromal Alzheimer's disease (pro-AD), DLB dementia (DLB-d), AD dementia (AD-d) and normal ageing.
Methods
Study participants(28 pro-DLB, 27 pro-AD, 31 DLB-d, 54 AD-d and 33 elderly controls) underwent 3Tesla T1 3D MRI and detailed clinical and cognitive assessments. We used FreeSurfer analysis package to measure CTh and investigate patterns of cortical thinning across groups.
Results
Comparison of CTh between pro-DLB and pro-AD (p<0.05, FDR corrected) showed more right anterior insula thinning in pro-DLB, and more bilateral parietal lobe and left parahippocampal gyri thinning in pro-AD. Comparison of prodromal patients to healthy elderly controls showed the involvement of the same regions. In DLB-d (p<0.05, FDR corrected) cortical thinning was found predominantly in the right temporo-parietal junction, and insula, cingulate, orbitofrontal and lateral occipital cortices. In AD-d(p<0.05, FDR corrected),the most significant areas affected included the entorhinal cortices, parahippocampal gyri and parietal lobes. The comparison of AD-d and DLB-d demonstrated more CTh in AD-d in the left entorhinal cortex (p<0.05, FDR corrected).
Conclusion
Cortical thickness is a sensitive measure for characterising patterns of grey matter atrophy in early stages of DLB distinct from AD. Right anterior insula involvement may be a key region at the prodromal stage of DLB and needs further investigation.
doi:10.1371/journal.pone.0127396
PMCID: PMC4489516  PMID: 26061655
6.  Neuroimaging characteristics of dementia with Lewy bodies 
This review summarises the findings and applications from neuroimaging studies in dementia with Lewy bodies (DLB), highlighting key differences between DLB and other subtypes of dementia. We also discuss the increasingly important role of imaging biomarkers in differential diagnosis and outline promising areas for future research in DLB. DLB shares common clinical, neuropsychological and pathological features with Parkinson’s disease dementia and other dementia subtypes, such as Alzheimer’s disease. Despite the development of consensus diagnostic criteria, the sensitivity for differential diagnosis of DLB in clinical practice remains low and many DLB patients will be misdiagnosed. The importance of developing accurate imaging markers in dementia is highlighted by the potential for treatments targeting specific molecular abnormalities as well as the responsiveness to cholinesterase inhibitors and marked neuroleptic sensitivity of DLB. We review various brain imaging techniques that have been applied to investigate DLB, including the characteristic nigrostriatal degeneration in DLB using positron emission tomography (PET) and single-photon emission computed tomography (SPECT) tracers. Dopamine transporter loss has proven to reliably differentiate DLB from other dementias and has been incorporated into the revised clinical diagnostic criteria for DLB. To date, this remains the 'gold standard' for diagnostic imaging of DLB. Regional cerebral blood flow, 18 F-fluorodeoxygluclose-PET and SPECT have also identified marked deficits in the occipital regions with relative sparing of the medial temporal lobe when compared to Alzheimer’s disease. In addition, structural, diffusion, and functional magnetic resonance imaging techniques have shown alterations in structure, white matter integrity, and functional activity in DLB. We argue that the multimodal identification of DLB-specific biomarkers has the potential to improve ante-mortem diagnosis and contribute to our understanding of the pathological background of DLB and its progression.
doi:10.1186/alzrt248
PMCID: PMC4055038  PMID: 25031634
7.  Identification of a broad family of lipid A late acyltransferases with non-canonical substrate specificity 
Molecular microbiology  2014;91(5):887-899.
Summary
Most gram-negative organisms produce lipopolysaccharide (LPS), a complex macromolecule anchored to the bacterial membrane by the lipid A moiety. Lipid A is synthesized via the Raetz pathway, a conserved nine-step enzymatic process first characterized in Escherichia coli. The Epsilonproteobacterium Helicobacter pylori uses the Raetz pathway to synthesize lipid A; however, only eight of nine enzymes in the pathway have been identified in this organism. Here, we identify the missing acyltransferase, Jhp0255, which transfers a secondary acyl chain to the 3′-linked primary acyl chain of lipid A, an activity similar to that of E. coli LpxM. This enzyme, reannotated as LpxJ due to limited sequence similarity with LpxM, catalyzes addition of a C12:0 or C14:0 acyl chain to the 3′-linked primary acyl chain of lipid A, complementing an E. coli LpxM mutant. Enzymatic assays demonstrate that LpxJ and homologs in Campylobacter jejuni and Wolinella succinogenes can act before the 2′ secondary acyltransferase, LpxL, as well as the 3-deoxy-D-manno-octulosonic acid (Kdo) transferase, KdtA. Ultimately, LpxJ is one member of a large class of acyltransferases found in a diverse range of organisms that lack an E. coli LpxM homolog, suggesting that LpxJ participates in lipid A biosynthesis in place of an LpxM homolog.
doi:10.1111/mmi.12501
PMCID: PMC3947442  PMID: 24372821
lipid A; acyltransferase; Helicobacter pylori; LpxJ; LpxM; DUF374; lipopolysaccharide; outer membrane
8.  Longitudinal assessment of global and regional atrophy rates in Alzheimer's disease and dementia with Lewy bodies 
NeuroImage : Clinical  2015;7:456-462.
Background & objective
Percent whole brain volume change (PBVC) measured from serial MRI scans is widely accepted as a sensitive marker of disease progression in Alzheimer's disease (AD). However, the utility of PBVC in the differential diagnosis of dementia remains to be established. We compared PBVC in AD and dementia with Lewy bodies (DLB), and investigated associations with clinical measures.
Methods
72 participants (14 DLBs, 25 ADs, and 33 healthy controls (HCs)) underwent clinical assessment and 3 Tesla T1-weighted MRI at baseline and repeated at 12 months. We used FSL-SIENA to estimate PBVC for each subject. Voxelwise analyses and ANCOVA compared PBVC between DLB and AD, while correlational tests examined associations of PBVC with clinical measures.
Results
AD had significantly greater atrophy over 1 year (1.8%) compared to DLB (1.0%; p = 0.01) and HC (0.9%; p < 0.01) in widespread regions of the brain including periventricular areas. PBVC was not significantly different between DLB and HC (p = 0.95). There were no differences in cognitive decline between DLB and AD. In the combined dementia group (AD and DLB), younger age was associated with higher atrophy rates (r = 0.49, p < 0.01).
Conclusions
AD showed a faster rate of global brain atrophy compared to DLB, which had similar rates of atrophy to HC. Among dementia subjects, younger age was associated with accelerated atrophy, reflecting more aggressive disease in younger people. PBVC could aid in differentiating between DLB and AD, however its utility as an outcome marker in DLB is limited.
Highlights
•AD showed faster global and regional brain atrophy in comparison to DLB.•Similar rates of global and regional atrophy were found in DLB and HC.•Longitudinal imaging could improve clinical differentiation of DLB from AD.•Atrophy rates might not be a useful marker to track disease progression in DLB.
doi:10.1016/j.nicl.2015.01.017
PMCID: PMC4325088  PMID: 25685712
Dementia; Alzheimer's disease; Lewy bodies; Neuroimaging; Atrophy
9.  193 nm Ultraviolet Photodissociation Mass Spectrometry for the Structural Elucidation of Lipid A Compounds in Complex Mixtures 
Analytical Chemistry  2014;86(4):2138-2145.
Here we implement ultraviolet photodissociation (UVPD) in an online liquid chromatographic tandem mass spectrometry (MS/MS) strategy to support analysis of complex mixtures of lipid A combinatorially modified during development of vaccine adjuvants. UVPD mass spectrometry at 193 nm was utilized to characterize the structures and fragment ion types of lipid A from Escherichia coli, Vibrio cholerae, and Pseudomonas aeruginosa using an Orbitrap mass spectrometer. The fragment ions generated by UVPD were compared to those from collision induced dissociation (CID) and higher energy collision dissociation (HCD) with respect to the precursor charge state. UVPD afforded the widest array of fragment ion types including acyl chain C–O, C–N, and C–C bond cleavages and glycosidic C–O and cross ring cleavages, thus providing the most comprehensive structural analysis of the lipid A. UVPD exhibited virtually no dependence on precursor ion charge state and was best at determining lipid A structure including acyl chain length and composition, giving it an advantage over collision based methods. UVPD was incorporated into an LC–MS/MS methodology for the analysis of a number of structural variants in a complex mixture of combinatorially engineered Escherichia coli lipid A.
doi:10.1021/ac403796n
PMCID: PMC3958132  PMID: 24446701
10.  Body mass index is associated with the maternal lines but height is heritable across family lines in the Lifeways Cross-Generation Cohort Study 
BMJ Open  2014;4(12):e005732.
Objectives
Overweight and obesity is a problem in children in particular and determining pathways of transmission is important in prevention. We aimed to examine associations for body mass index (BMI) across three generations of the same families.
Participants
Members of 556 families in the Lifeways Cross-Generation Cohort Study 2001–2014.
Setting
Community-based study with linkage to health records in the Republic of Ireland.
Methods
Employing a novel mixed-method approach which adjusts for age and familial group, BMI correlations were estimated at three ages of the index child, that is, at birth and at ages 5 and 9. Height was also examined for comparative purposes.
Results
Correlation of offspring's BMI with that of the mother increased with age (correlation coefficient 0.15 increasing to 0.28, p value <0.001 in all cases) while no consistent pattern was seen with offspring and fathers. There was an association also with each parent and their own mother. Offspring's BMI was correlated to a lesser extent with that of the maternal grandmother while for girls only there was an association with that of the paternal grandmother at ages 0 and 5 (correlation coefficients 0.25, 0.28, p values 0.02, 0.01, respectively). In contrast, height of the child was strongly associated with those of all family members at age 5, but at birth and at age 9 only there was an association with those of the parents and the paternal grandfather. Correlation of offspring's height with those of the mother and father increased with age.
Conclusions
The results suggest that BMI is predominantly associated with the maternal line, possibly either with intrauterine development, or inherited through the X chromosome, or both, while height is a more complex trait with genetic influences of the parents and that of the paternal grandfather predominating.
doi:10.1136/bmjopen-2014-005732
PMCID: PMC4275682  PMID: 25518873
Body Mass Index; height; heritability; Intergenerational Relations; Population genetics
11.  Vascular risk factors and Alzheimer’s disease 
BMC Medicine  2014;12:218.
Vascular factors are now established risk factors for cognitive decline, both for dementia and its two main subtypes: Alzheimer’s disease (AD) and vascular dementia. Their impact likely goes beyond causing an increase in concurrent vascular pathology, since they have been associated with increasing the risk of degenerative Alzheimer (plaque and tangle) pathology, either by increasing its rate of formation or reducing elimination from the brain, or a mixture of the two. A comprehensive series of reviews published in BMC Medicine, investigates the relationship between AD and cardiovascular diseases and risk factors from a clinical, pathological and therapeutic perspective. Whilst links between vascular factors and AD have clearly been demonstrated at both the clinical and pathological level, the nature of the relationship remains to be fully established and there is a lack of high quality treatment studies examining the extent to which vascular risk modification alters AD disease course. Further longitudinal mechanistic and therapeutic studies are required, especially to determine whether treatment of vascular risk can prevent or delay the onset of AD and/or reduce its rate of clinical progression.
doi:10.1186/s12916-014-0218-y
PMCID: PMC4226870  PMID: 25385509
Alzheimer’s disease; Vascular risk; Stroke; Dementia; Risk factors; Prevention; Treatment; Hypertension
12.  Structural Characterization of Gangliosides and Glycolipids via Ultraviolet Photodissociation Mass Spectrometry 
Analytical chemistry  2013;85(21):10.1021/ac402379y.
Ultraviolet photodissociation (UVPD) mass spectrometry was used to characterize the structures of amphiphilic glycosphingolipids and gangliosides in comparison to collision induced dissociation (CID) and higher energy collision dissociation (HCD) in a high performance Orbitrap mass spectrometer. UVPD produced the widest array of fragment ions diagnostic for both the ceramide base and oligosaccharide moieties. CID and HCD generated mainly glycosidic B/Y and C/Z cleavages of the oligosaccharides moieties and very few informative fragments related to the hydrophobic ceramide base. Several unique cleavages at the sphingoid base and the fatty acid chain occurred upon UVPD, as well as a wider variety of cross ring cleavages (A/X ions), thus affording differentiation of isobaric gangliosides. An LC-UVPD-MS strategy allowed the elucidation of 27 gangliosides among five different classes.
doi:10.1021/ac402379y
PMCID: PMC3855282  PMID: 24083420
13.  Regulation of photoreceptor gap junction phosphorylation by adenosine in zebrafish retina 
Visual neuroscience  2014;31(3):237-243.
Electrical coupling of photoreceptors through gap junctions suppresses voltage noise, routes rod signals into cone pathways, expands the dynamic range of rod photoreceptors in high scotopic and mesopic illumination, and improves detection of contrast and small stimuli. In essentially all vertebrates, connexin 35/36 (gene homologues Cx36 in mammals, Cx35 in other vertebrates) is the major gap junction protein observed in photoreceptors, mediating rod-cone, cone-cone, and possibly rod-rod communication. Photoreceptor coupling is dynamically controlled by the day/night cycle and light/dark adaptation, and is directly correlated with phosphorylation of Cx35/36 at two sites, serine110 and serine 276/293 (homologous sites in teleost fish and mammals respectively). Activity of protein kinase A (PKA) plays a key role during this process. Previous studies have shown that activation of dopamine D4 receptors on photoreceptors inhibits adenylyl cyclase, down-regulates cAMP and PKA activity, and leads to photoreceptor uncoupling, imposing the daytime/light condition. In this study we explored the role of adenosine, a nighttime signal with a high extracellular concentration at night and a low concentration in the day, in regulating photoreceptor coupling by examining photoreceptor Cx35 phosphorylation in zebrafish retina. Adenosine enhanced photoreceptor Cx35 phosphorylation in daytime, but with a complex dose-response curve. Selective pharmacological manipulations revealed that adenosine A2a receptors provide a potent positive drive to phosphorylate photoreceptor Cx35 under the influence of endogenous adenosine at night. A2a receptors can be activated in the daytime as well by micromolar exogenous adenosine. However, the higher affinity adenosine A1 receptors are also present and have an antagonistic though less potent effect. Thus the nighttime/darkness signal adenosine provides a net positive drive on Cx35 phosphorylation at night, working in opposition to dopamine to regulate photoreceptor coupling via a push-pull mechanism. However, the lower concentration of adenosine present in the daytime actually reinforces the dopamine signal through action on the A1 receptor.
doi:10.1017/S095252381300062X
PMCID: PMC4109651  PMID: 24844306
adenosine; A2a receptor; A1 receptor; Cx36; photoreceptor
14.  Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun 
Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various heterologous genes could thus be investigated easily while maintaining many aspects of in vivo biology. There has been increasing interest to transfect terminally differentiated neurons for which conventional transfection methods have been fraught with difficulties such as low yields and significant losses in viability. Biolistic transfection can circumvent many of these difficulties yet only recently has this technique been modified so that it is amenable for use in mammalian tissues.
New modifications to the accelerator chamber have enhanced the gene gun's firing accuracy and increased its depths of penetration while also allowing the use of lower gas pressure (50 psi) without loss of transfection efficiency as well as permitting a focused regioselective spread of the particles to within 3 mm. In addition, this technique is straight forward and faster to perform than tedious microinjections. Both transient and stable expression are possible with nanoparticle bombardment where episomal expression can be detected within 24 hr and the cell survival was shown to be better than, or at least equal to, conventional methods. This technique has however one crucial advantage: it permits the transfection to be localized within a single restrained radius thus enabling the user to anatomically isolate the heterologous gene's effects. Here we present an in-depth protocol to prepare viable adult organotypic slices and submit them to regioselective transfection using an improved gene gun.
doi:10.3791/52148
PMCID: PMC4249736  PMID: 25407047
Neuroscience; Issue 92; Biolistics; gene gun; organotypic brain slices; Diolistic; gene delivery; staining
15.  Patterns of cerebellar volume loss in dementia with Lewy bodies and Alzheimer׳s disease: A VBM-DARTEL study 
Psychiatry Research  2014;223(3):187-191.
Evidence suggests that the cerebellum contributes to cognition as well as motor function. We investigated cerebellar grey matter (GM) and white matter (WM) changes from magnetic resonance images in dementia with Lewy bodies (DLB), Alzheimer׳s disease (AD) and healthy older subjects using voxel-based morphometry (VBM). Subjects (39 controls, 41 DLB, and 48 AD) underwent magnetic resonance imaging as well as clinical and cognitive assessments. VBM used SPM8 with a cerebellar brain mask to define the subspace for voxel analysis. Statistical analyses were conducted using the general linear model. Relative to findings in controls, VBM analysis revealed cerebellar GM loss in lobule VI bilaterally in AD and in left Crus I and right Crus II regions in DLB. WM deficits were confined to AD in the bilateral middle cerebellar peduncles. DLB demonstrates a different pattern of cerebellar GM loss which, although not significantly different from that in AD, could be an important feature in understanding the neurobiology of DLB and warrants further investigation.
Highlights
•We studied cerebellar grey and white matter changes from MR images in dementia.•Cerebellar volumes loss occurred in both DLB and AD compared to controls.•Cerebellar pathology under both conditions warrants further consideration.
doi:10.1016/j.pscychresns.2014.06.006
PMCID: PMC4333903  PMID: 25037902
DARTEL-VBM; Dementia with Lewy bodies; Alzheimer’s disease; MRI; Cerebellum
16.  Molecular and functional asymmetry at a vertebrate electrical synapse 
Neuron  2013;79(5):957-969.
SUMMARY
Electrical synapses are abundant in the vertebrate brain, but their functional and molecular complexity are still poorly understood. We report here that electrical synapses between auditory afferents and goldfish Mauthner cells are constructed by apposition of hemichannels formed by two homologs of mammalian connexin36 (Cx36), and that while Cx35 is restricted to presynaptic hemiplaques, Cx34.7 is restricted to postsynaptic hemiplaques, forming heterotypic junctions. This molecular asymmetry is associated with rectification of electrical transmission that may act to promote cooperativity between auditory afferents. Our data suggest that in similarity to pre- and postsynaptic sites at chemical synapses, one side in electrical synapses should not necessarily be considered the mirror image of the other. While asymmetry based on the presence of two Cx36 homologs is restricted to teleost fish, it might also be based on differences in posttranslational modifications of individual connexins or in the complement of gap junction-associated proteins.
doi:10.1016/j.neuron.2013.06.037
PMCID: PMC4020187  PMID: 24012008
electrical transmission; connexin36; auditory; rectification; gap junction
17.  Genetic impact on cognition and brain function in newly diagnosed Parkinson’s disease: ICICLE-PD study 
Brain  2014;137(10):2743-2758.
See Dujardin (doi:10.1093/brain/awu218) for a scientific commentary on this article. Nombela et al. present data from the ICICLE-PD study of cognition in newly diagnosed Parkinson’s disease. Consistent with the ‘Dual Syndrome’ hypothesis, impairments in executive function reflect a frontal dopaminergic syndrome modulated by COMT genotype, while visuospatial and memory deficits reflect disruption of temporo-parietal systems modulated by MAPT and APOE.
Parkinson’s disease is associated with multiple cognitive impairments and increased risk of dementia, but the extent of these deficits varies widely among patients. The ICICLE-PD study was established to define the characteristics and prevalence of cognitive change soon after diagnosis, in a representative cohort of patients, using a multimodal approach. Specifically, we tested the ‘Dual Syndrome’ hypothesis for cognitive impairment in Parkinson’s disease, which distinguishes an executive syndrome (affecting the frontostriatal regions due to dopaminergic deficits) from a posterior cortical syndrome (affecting visuospatial, mnemonic and semantic functions related to Lewy body pathology and secondary cholinergic loss). An incident Parkinson’s disease cohort (n = 168, median 8 months from diagnosis to participation) and matched control group (n = 85) were recruited to a neuroimaging study at two sites in the UK. All participants underwent clinical, neuropsychological and functional magnetic resonance imaging assessments. The three neuroimaging tasks (Tower of London, Spatial Rotations and Memory Encoding Tasks) were designed to probe executive, visuospatial and memory encoding domains, respectively. Patients were also genotyped for three polymorphisms associated with cognitive change in Parkinson’s disease and related disorders: (i) rs4680 for COMT Val158Met polymorphism; (ii) rs9468 for MAPT H1 versus H2 haplotype; and (iii) rs429358 for APOE-ε2, 3, 4. We identified performance deficits in all three cognitive domains, which were associated with regionally specific changes in cortical activation. Task-specific regional activations in Parkinson’s disease were linked with genetic variation: the rs4680 polymorphism modulated the effect of levodopa therapy on planning-related activations in the frontoparietal network; the MAPT haplotype modulated parietal activations associated with spatial rotations; and APOE allelic variation influenced the magnitude of activation associated with memory encoding. This study demonstrates that neurocognitive deficits are common even in recently diagnosed patients with Parkinson’s disease, and that the associated regional brain activations are influenced by genotype. These data further support the dual syndrome hypothesis of cognitive change in Parkinson’s disease. Longitudinal data will confirm the extent to which these early neurocognitive changes, and their genetic factors, influence the long-term risk of dementia in Parkinson’s disease. The combination of genetics and functional neuroimaging provides a potentially useful method for stratification and identification of candidate markers, in future clinical trials against cognitive decline in Parkinson’s disease.
doi:10.1093/brain/awu201
PMCID: PMC4163033  PMID: 25080285
Parkinson’s disease; cognition; functional MRI; genetics
18.  Is ioflupane I123 injection diagnostically effective in patients with movement disorders and dementia? Pooled analysis of four clinical trials 
BMJ Open  2014;4(7):e005122.
Objectives
To pool clinical trials of similar design to assess overall sensitivity and specificity of ioflupane I123 injection (DaTSCAN or ioflupane (123I)) to detect or exclude a striatal dopaminergic deficit disorder (SDDD), such as parkinsonian syndrome and dementia with Lewy bodies.
Design
Pooled analysis of three phase 3 and one phase 4 clinical trials. These four trials were selected because they were the four studies used for the US new drug application to the Food and Drug Administration (FDA).
Setting
Multicentre, open-label, non-randomised.
Participants
Patients with either a movement disorder or dementia, and healthy volunteers.
Interventions
Ioflupane (123I) was administered.
Outcome measures
Images were assessed by panels of 3–5 blinded experts and/or on-site nuclear medicine physicians, classified as normal or abnormal and compared with clinical diagnosis (reference standard) to determine sensitivity and specificity.
Results
Pooling the four studies, 928 participants were enrolled, 849 were dosed and 764 completed their study. Across all studies, when images were assessed by on-site readers, ioflupane (123I) diagnostic effectiveness had an overall (95% CI) sensitivity of 91.9% (88.7% to 94.5%) and specificity of 83.6% (78.7% to 87.9%). When reads were conducted blindly by a panel of independent experts, the overall sensitivity was 88.7% (86.8% to 90.4%) and specificity was 91.2% (89.0% to 93.0%).
Conclusions
In this pooled analysis, the visual assessment of ioflupane (123I) images provided high levels of sensitivity and specificity in detecting the presence/absence of an SDDD. Ioflupane (123I) imaging has the potential to improve diagnostic accuracy in patients with signs and symptoms of a movement disorder and/or dementia.
Trial registration number
NCT00209456.
doi:10.1136/bmjopen-2014-005122
PMCID: PMC4091455  PMID: 24993764
19.  The STRIDE (Strategies to Increase confidence, InDependence and Energy) study: cognitive behavioural therapy-based intervention to reduce fear of falling in older fallers living in the community - study protocol for a randomised controlled trial 
Trials  2014;15:210.
Background
Around 30% to 62% of older individuals fall each year, with adverse consequences of falls being by no means limited to physical injury and escalating levels of dependence. Many older individuals suffer from a variety of adverse psychosocial difficulties related to falling including fear, anxiety, loss of confidence and subsequent increasing activity avoidance, social isolation and frailty. Such ‘fear of falling’ is common and disabling, but definitive studies examining the effective management of the syndrome are lacking. Cognitive behavioural therapy has been trialed with some success in a group setting, but there is no adequately powered randomised controlled study of an individually based cognitive behavioural therapy intervention, and none using non-mental health professionals to deliver the intervention.
Methods/Design
We are conducting a two-phase study examining the role of individual cognitive behavioural therapy delivered by healthcare assistants in improving fear of falling in older adults. In Phase I, the intervention was developed and taught to healthcare assistants, while Phase II is the pragmatic randomised controlled study examining the efficacy of the intervention in improving fear of falling in community-dwelling elders attending falls services. A qualitative process evaluation study informed by Normalization Process Theory is being conducted throughout to examine the potential promoters and inhibitors of introducing such an intervention into routine clinical practice, while a health economic sub-study running alongside the trial is examining the costs and benefits of such an approach to the wider health economy.
Trial registration
Current Controlled Trials ISRCTN78396615
doi:10.1186/1745-6215-15-210
PMCID: PMC4059076  PMID: 24906406
Fear of falling; Falls; Elders; Community; Randomised controlled trial; Cognitive behavioural therapy; Complex intervention; Normalization process theory
20.  Dynamin1 concentration in the prefrontal cortex is associated with cognitive impairment in Lewy body dementia 
F1000Research  2014;3:108.
Dementia with Lewy Bodies (DLB) and Parkinson’s Disease Dementia (PDD) together, represent the second most common cause of dementia, after Alzheimer’s disease (AD). The synaptic dysfunctions underlying the cognitive decline and psychiatric symptoms observed throughout the development of PDD and DLB are still under investigation. In this study we examined the expression level of Dynamin1 and phospho-CaMKII, key proteins of endocytosis and synaptic plasticity respectively, as potential markers of molecular processes specifically deregulated with DLB and/or PDD. In order to measure the levels of these proteins, we isolated grey matter from post-mortem prefrontal cortex area (BA9), anterior cingulated gyrus (BA24) and parietal cortex (BA40) from DLB and PDD patients in comparison to age-matched controls and a group of AD cases. Clinical and pathological data available included the MMSE score, neuropsychiatric history, and semi-quantitative scores for AD pathology (plaques - tangles) and for α-synuclein (Lewy bodies).
Changes in the expression of the synaptic markers, and correlates with neuropathological features and cognitive decline were predominantly found in the prefrontal cortex. On one hand, levels of Dynamin1 were significantly reduced, and correlated with a higher rate of cognitive decline observed in cases from three dementia groups. On the other hand, the fraction of phospho-CaMKII was decreased, and correlated with a high score of plaques and tangles in BA9. Interestingly, the correlation between the rate of cognitive decline and the level of Dynamin1 remained when the analysis was restricted to the PDD and DLB cases, highlighting an association of Dynamin1 with cognitive decline in people with Lewy Body dementia.
doi:10.12688/f1000research.3786.1
PMCID: PMC4309165  PMID: 25671083
Alzheimer’s disease; Dementia with Lewy bodies; Parkinson’s disease with dementia; synaptic dysfunction; vesicle recycling; synaptic plasticity; beta amyloid; tau; cognitive impairment
21.  A Genome Wide Association Study of Plasmodium falciparum Susceptibility to 22 Antimalarial Drugs in Kenya 
PLoS ONE  2014;9(5):e96486.
Background
Drug resistance remains a chief concern for malaria control. In order to determine the genetic markers of drug resistant parasites, we tested the genome-wide associations (GWA) of sequence-based genotypes from 35 Kenyan P. falciparum parasites with the activities of 22 antimalarial drugs.
Methods and Principal Findings
Parasites isolated from children with acute febrile malaria were adapted to culture, and sensitivity was determined by in vitro growth in the presence of anti-malarial drugs. Parasites were genotyped using whole genome sequencing techniques. Associations between 6250 single nucleotide polymorphisms (SNPs) and resistance to individual anti-malarial agents were determined, with false discovery rate adjustment for multiple hypothesis testing. We identified expected associations in the pfcrt region with chloroquine (CQ) activity, and other novel loci associated with amodiaquine, quinazoline, and quinine activities. Signals for CQ and primaquine (PQ) overlap in and around pfcrt, and interestingly the phenotypes are inversely related for these two drugs. We catalog the variation in dhfr, dhps, mdr1, nhe, and crt, including novel SNPs, and confirm the presence of a dhfr-164L quadruple mutant in coastal Kenya. Mutations implicated in sulfadoxine-pyrimethamine resistance are at or near fixation in this sample set.
Conclusions/Significance
Sequence-based GWA studies are powerful tools for phenotypic association tests. Using this approach on falciparum parasites from coastal Kenya we identified known and previously unreported genes associated with phenotypic resistance to anti-malarial drugs, and observe in high-resolution haplotype visualizations a possible signature of an inverse selective relationship between CQ and PQ.
doi:10.1371/journal.pone.0096486
PMCID: PMC4014544  PMID: 24809681
22.  A Bayesian Approach to Inferring the Phylogenetic Structure of Communities from Metagenomic Data 
Genetics  2014;197(3):925-937.
Metagenomics provides a powerful new tool set for investigating evolutionary interactions with the environment. However, an absence of model-based statistical methods means that researchers are often not able to make full use of this complex information. We present a Bayesian method for inferring the phylogenetic relationship among related organisms found within metagenomic samples. Our approach exploits variation in the frequency of taxa among samples to simultaneously infer each lineage haplotype, the phylogenetic tree connecting them, and their frequency within each sample. Applications of the algorithm to simulated data show that our method can recover a substantial fraction of the phylogenetic structure even in the presence of high rates of migration among sample sites. We provide examples of the method applied to data from green sulfur bacteria recovered from an Antarctic lake, plastids from mixed Plasmodium falciparum infections, and virulent Neisseria meningitidis samples.
doi:10.1534/genetics.114.161299
PMCID: PMC4096371  PMID: 24793089
metagenomics; Bayesian phylogenetics; microevolution
23.  Multivariate spatial covariance analysis of 99mTc-exametazime SPECT images in dementia with Lewy bodies and Alzheimer's disease: utility in differential diagnosis 
We examined 99mTc-exametazime brain blood flow single-photon emission computed tomography (SPECT) images using a spatial covariance analysis (SCA) approach to assess its diagnostic value in distinguishing dementia with Lewy bodies (DLB) from Alzheimer's disease (AD). Voxel SCA was simultaneously applied to a set of preprocessed images (AD, n=40; DLB, n=26), generating a series of eigenimages representing common intercorrelated voxels in AD and DLB. Linear regression derived a spatial covariance pattern (SCP) that discriminated DLB from AD. To investigate the diagnostic value of the model SCP, the SCP was validated by applying it to a second, independent, AD and DLB cohort (AD, n=34; DLB, n=29). Mean SCP expressions differed between AD and DLB (F1,64=36.2, P<0.001) with good diagnostic accuracy (receiver operating characteristic (ROC) curve area 0.87, sensitivity 81%, specificity 88%). Forward application of the model SCP to the independent cohort revealed similar differences between groups (F1,61=38.4, P<0.001), also with good diagnostic accuracy (ROC 0.86, sensitivity 80%, specificity 80%). Multivariate analysis of blood flow SPECT data appears to be robust and shows good diagnostic accuracy in two independent cohorts for distinguishing DLB from AD.
doi:10.1038/jcbfm.2013.2
PMCID: PMC3618400  PMID: 23361395
Alzheimer's disease; differential diagnosis; dementia with Lewy bodies; perfusion; spatial covariance; SPECT
24.  fMRI resting state networks and their association with cognitive fluctuations in dementia with Lewy bodies 
NeuroImage : Clinical  2014;4:558-565.
Cognitive fluctuations are a core symptom in dementia with Lewy bodies (DLB) and may relate to pathological alterations in distributed brain networks. To test this we analysed resting state fMRI changes in a cohort of fluctuating DLB patients (n = 16) compared with age matched controls (n = 17) with the aim of finding functional connectivity (FC) differences between these two groups and whether these associate with cognitive fluctuations in DLB. Resting state networks (RSNs) were estimated using independent component analysis and FC between the RSN maps and the entirety of the brain was assessed using dual regression. The default mode network (DMN) appeared unaffected in DLB compared to controls but significant cluster differences between DLB and controls were found for the left fronto-parietal, temporal, and sensory–motor networks. Desynchronization of a number of cortical and subcortical areas related to the left fronto-parietal network was associated with the severity and frequency of cognitive fluctuations. Our findings provide empirical evidence for the potential role of attention–executive networks in the aetiology of this core symptom in DLB.
Highlights
•We report resting state network (RSN) alterations in dementia with Lewy bodies (DLB).•The default mode network was intact in DLB compared to healthy controls (HC).•Fronto-parietal, temporal, and sensory–motor RSNs showed differences (DLB < HC).•The left fronto-parietal network (FPN) correlated with cognitive fluctuations in DLB.•The FPN therefore may be a potential marker for cognitive fluctuations in DLB.
doi:10.1016/j.nicl.2014.03.013
PMCID: PMC3984441  PMID: 24818081
Cognitive fluctuations; Visual hallucinations; Resting state network; Lewy bodies; Dementia
25.  Characterizing mild cognitive impairment in incident Parkinson disease 
Neurology  2014;82(4):308-316.
Objective:
To describe the frequency of mild cognitive impairment (MCI) in Parkinson disease (PD) in a cohort of newly diagnosed incident PD cases and the associations with a panel of biomarkers.
Methods:
Between June 2009 and December 2011, 219 subjects with PD and 99 age-matched controls participated in clinical and neuropsychological assessments as part of a longitudinal observational study. Consenting individuals underwent structural MRI, lumbar puncture, and genotyping for common variants of COMT, MAPT, SNCA, BuChE, EGF, and APOE. PD-MCI was defined with reference to the new Movement Disorder Society criteria.
Results:
The frequency of PD-MCI was 42.5% using level 2 criteria at 1.5 SDs below normative values. Memory impairment was the most common domain affected, with 15.1% impaired at 1.5 SDs. Depression scores were significantly higher in those with PD-MCI than the cognitively normal PD group. A significant correlation was found between visual Pattern Recognition Memory and cerebrospinal β-amyloid 1–42 levels (β standardized coefficient = 0.350; p = 0.008) after controlling for age and education in a linear regression model, with lower β-amyloid 1–42 and 1–40 levels observed in those with PD-MCI. Voxel-based morphometry did not reveal any areas of significant gray matter loss in participants with PD-MCI compared with controls, and no specific genotype was associated with PD-MCI at the 1.5-SD threshold.
Conclusions:
In a large cohort of newly diagnosed PD participants, PD-MCI is common and significantly correlates with lower cerebrospinal β-amyloid 1–42 and 1–40 levels. Future longitudinal studies should enable us to determine those measures predictive of cognitive decline.
doi:10.1212/WNL.0000000000000066
PMCID: PMC3929202  PMID: 24363137

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