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1.  Towards a Quantitative Theory of Epidermal Calcium Profile Formation in Unwounded Skin 
PLoS ONE  2015;10(1):e0116751.
We propose and mathematically examine a theory of calcium profile formation in unwounded mammalian epidermis based on: changes in keratinocyte proliferation, fluid and calcium exchange with the extracellular fluid during these cells’ passage through the epidermal sublayers, and the barrier functions of both the stratum corneum and tight junctions localised in the stratum granulosum. Using this theory, we develop a mathematical model that predicts epidermal sublayer transit times, partitioning of the epidermal calcium gradient between intracellular and extracellular domains, and the permeability of the tight junction barrier to calcium ions. Comparison of our model’s predictions of epidermal transit times with experimental data indicates that keratinocytes lose at least 87% of their volume during their disintegration to become corneocytes. Intracellular calcium is suggested as the main contributor to the epidermal calcium gradient, with its distribution actively regulated by a phenotypic switch in calcium exchange between keratinocytes and extracellular fluid present at the boundary between the stratum spinosum and the stratum granulosum. Formation of the extracellular calcium distribution, which rises in concentration through the stratum granulosum towards the skin surface, is attributed to a tight junction barrier in this sublayer possessing permeability to calcium ions that is less than 15 nm s−1 in human epidermis and less than 37 nm s−1 in murine epidermis. Future experimental work may refine the presented theory and reduce the mathematical uncertainty present in the model predictions.
doi:10.1371/journal.pone.0116751
PMCID: PMC4308082  PMID: 25625723
3.  Protracted withdrawal from cocaine self-administration flips the switch on 5-HT1B receptor modulation of cocaine-abuse related behaviors 
Biological psychiatry  2012;72(5):396-404.
Background
The role of serotonin-1B receptors (5-HT1BRs) in modulating cocaine abuse-related behaviors has been controversial due to discrepancies between pharmacological and gene knockout approaches, and opposite influences on cocaine selfadministration versus cocaine-seeking behavior. We hypothesized that modulation of these behaviors via 5-HT1BRs in the mesolimbic pathway may vary depending on the stage of the addiction cycle.
Methods
To test this hypothesis, we examined the effects of increasing 5-HT1BR production by microinfusing a viral vector expressing either green fluorescent protein (GFP) and 5-HT1BR or GFP alone into the medial nucleus accumbens shell of rats either during maintenance of cocaine self-administration (i.e. active drug use) or during protracted withdrawal.
Results
5-HT1BR-gene transfer during maintenance shifted the dose–response curve for cocaine self-administration upward and to the left and increased break points and cocaine intake on a progressive ratio (PR) schedule, consistent with enhanced reinforcing effects of cocaine. In contrast, following 21 days of forced abstinence 5-HT1BR-gene transfer attenuated break points and cocaine intake on a PR schedule of reinforcement, as well as cue- and cocaine-primed reinstatement of cocaineseeking behavior.
Conclusions
This unique pattern of effects suggests that mesolimbic 5-HT1BRs differentially modulate cocaine abuse-related behaviors, with a facilitative influence during periods of active drug use in striking contrast to an inhibitory influence during protracted withdrawal. These findings suggest that targeting 5-HT1BRs may lead to a novel treatment for cocaine dependence and that the therapeutic efficacy of these treatments may vary depending on the stage of the addiction cycle.
doi:10.1016/j.biopsych.2012.03.024
PMCID: PMC4071622  PMID: 22541946
reward; addiction; reinforcement; relapse; craving; reinstatement
4.  External Quality Assurance of Malaria Nucleic Acid Testing for Clinical Trials and Eradication Surveillance 
PLoS ONE  2014;9(5):e97398.
Nucleic acid testing (NAT) for malaria parasites is an increasingly recommended diagnostic endpoint in clinical trials of vaccine and drug candidates and is also important in surveillance of malaria control and elimination efforts. A variety of reported NAT assays have been described, yet no formal external quality assurance (EQA) program provides validation for the assays in use. Here, we report results of an EQA exercise for malaria NAT assays. Among five centers conducting controlled human malaria infection trials, all centers achieved 100% specificity and demonstrated limits of detection consistent with each laboratory's pre-stated expectations. Quantitative bias of reported results compared to expected results was generally <0.5 log10 parasites/mL except for one laboratory where the EQA effort identified likely reasons for a general quantitative shift. The within-laboratory variation for all assays was low at <10% coefficient of variation across a range of parasite densities. Based on this study, we propose to create a Molecular Malaria Quality Assessment program that fulfills the need for EQA of malaria NAT assays worldwide.
doi:10.1371/journal.pone.0097398
PMCID: PMC4023973  PMID: 24838112
5.  High-Content, High-Throughput Screening for the Identification of Cytotoxic Compounds Based on Cell Morphology and Cell Proliferation Markers 
PLoS ONE  2014;9(2):e88338.
Toxicity is a major cause of failure in drug discovery and development, and whilst robust toxicological testing occurs, efficiency could be improved if compounds with cytotoxic characteristics were identified during primary compound screening. The use of high-content imaging in primary screening is becoming more widespread, and by utilising phenotypic approaches it should be possible to incorporate cytotoxicity counter-screens into primary screens. Here we present a novel phenotypic assay that can be used as a counter-screen to identify compounds with adverse cellular effects. This assay has been developed using U2OS cells, the PerkinElmer Operetta high-content/high-throughput imaging system and Columbus image analysis software. In Columbus, algorithms were devised to identify changes in nuclear morphology, cell shape and proliferation using DAPI, TOTO-3 and phosphohistone H3 staining, respectively. The algorithms were developed and tested on cells treated with doxorubicin, taxol and nocodazole. The assay was then used to screen a novel, chemical library, rich in natural product-like molecules of over 300 compounds, 13.6% of which were identified as having adverse cellular effects. This assay provides a relatively cheap and rapid approach for identifying compounds with adverse cellular effects during screening assays, potentially reducing compound rejection due to toxicity in subsequent in vitro and in vivo assays.
doi:10.1371/journal.pone.0088338
PMCID: PMC3914966  PMID: 24505478
6.  Apolipoprotein(a) acts as a chemorepellent to human vascular smooth muscle cells via integrin αVβ3 and RhoA/ROCK-mediated mechanisms☆ 
Graphical abstract
Highlights
•Acute application of apo(a) to smooth muscle cells induced chemorepulsion.•Chronic application (>24 h) induced stress fibre formation and cell spreading.•Effects of apo(a) were mediated by integrin αVβ3, tyrosine kinases and RhoA/ROCK.•Apo(a) impaired SMC motility which potentially contributes to vascular dysfunction.
Lipoprotein(a) (Lp(a)) is an independent risk factor for the development of cardiovascular disease. Vascular smooth muscle cell (SMC) motility and plasticity, functions that are influenced by environmental cues, are vital to adaptation and remodelling in vascular physiology and pathophysiology. Lp(a) is reportedly damaging to SMC function via unknown molecular mechanisms. Apolipoprotein(a) (apo(a)), a unique glycoprotein moiety of Lp(a), has been demonstrated as its active component. The aims of this study were to determine functional effects of recombinant apo(a) on human vascular SMC motility and explore the underlying mechanism(s). Exposure of SMC to apo(a) in migration assays induced a potent, concentration-dependent chemorepulsion that was RhoA and integrin αVβ3-dependent, but transforming growth factor β-independent. SMC manipulation through RhoA gene silencing, Rho kinase inhibition, statin pre-treatment, αVβ3 neutralising antibody and tyrosine kinase inhibition all markedly inhibited apo(a)-mediated SMC migration. Our data reveal unique and potent activities of apo(a) that may negatively influence SMC remodelling in cardiovascular disease. Circulating levels of Lp(a) are resistant to lipid-lowering strategies and hence a greater understanding of the mechanisms underlying its functional effects on SMC may provide alternative therapeutic targets.
doi:10.1016/j.biocel.2013.05.021
PMCID: PMC3731554  PMID: 23726972
Ao, aortic; α-SMA, alpha smooth muscle actin; apo(a), apolipoprotein(a); DMEM, Dulbecco's modified eagle medium; ERK, extracellular signal-regulated kinase; FCS, foetal calf serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HP, high power; Lp(a), lipoprotein(a); MAPK, mitogen activated protein kinase; NFκB, nuclear factor kappa B; PDGF, platelet-derived growth factor-BB; ROCK, Rho kinase; SV, saphenous vein; SMC, smooth muscle cell; TGFβ, transforming growth factor beta; Lipoprotein(a); Vascular smooth muscle cells; Migration; RhoA; Remodelling
7.  Successful Human Infection with P. falciparum Using Three Aseptic Anopheles stephensi Mosquitoes: A New Model for Controlled Human Malaria Infection 
PLoS ONE  2013;8(7):e68969.
Controlled human malaria infection (CHMI) is a powerful method for assessing the efficacy of anti-malaria vaccines and drugs targeting pre-erythrocytic and erythrocytic stages of the parasite. CHMI has heretofore required the bites of 5 Plasmodium falciparum (Pf) sporozoite (SPZ)-infected mosquitoes to reliably induce Pf malaria. We reported that CHMI using the bites of 3 PfSPZ-infected mosquitoes reared aseptically in compliance with current good manufacturing practices (cGMP) was successful in 6 participants. Here, we report results from a subsequent CHMI study using 3 PfSPZ-infected mosquitoes reared aseptically to validate the initial clinical trial. We also compare results of safety, tolerability, and transmission dynamics in participants undergoing CHMI using 3 PfSPZ-infected mosquitoes reared aseptically to published studies of CHMI using 5 mosquitoes. Nineteen adults aged 18–40 years were bitten by 3 Anopheles stephensi mosquitoes infected with the chloroquine-sensitive NF54 strain of Pf. All 19 participants developed malaria (100%); 12 of 19 (63%) on Day 11. The mean pre-patent period was 258.3 hours (range 210.5–333.8). The geometric mean parasitemia at first diagnosis by microscopy was 9.5 parasites/µL (range 2–44). Quantitative polymerase chain reaction (qPCR) detected parasites an average of 79.8 hours (range 43.8–116.7) before microscopy. The mosquitoes had a geometric mean of 37,894 PfSPZ/mosquito (range 3,500–152,200). Exposure to the bites of 3 aseptically-raised, PfSPZ-infected mosquitoes is a safe, effective procedure for CHMI in malaria-naïve adults. The aseptic model should be considered as a new standard for CHMI trials in non-endemic areas. Microscopy is the gold standard used for the diagnosis of Pf malaria after CHMI, but qPCR identifies parasites earlier. If qPCR continues to be shown to be highly specific, and can be made to be practical, rapid, and standardized, it should be considered as an alternative for diagnosis.
Trial Registration
ClinicalTrials.gov NCT00744133 NCT00744133
doi:10.1371/journal.pone.0068969
PMCID: PMC3712927  PMID: 23874828
8.  A meckelin–filamin A interaction mediates ciliogenesis 
Human Molecular Genetics  2011;21(6):1272-1286.
MKS3, encoding the transmembrane receptor meckelin, is mutated in Meckel–Gruber syndrome (MKS), an autosomal-recessive ciliopathy. Meckelin localizes to the primary cilium, basal body and elsewhere within the cell. Here, we found that the cytoplasmic domain of meckelin directly interacts with the actin-binding protein filamin A, potentially at the apical cell surface associated with the basal body. Mutations in FLNA, the gene for filamin A, cause periventricular heterotopias. We identified a single consanguineous patient with an MKS-like ciliopathy that presented with both MKS and cerebellar heterotopia, caused by an unusual in-frame deletion mutation in the meckelin C-terminus at the region of interaction with filamin A. We modelled this mutation and found it to abrogate the meckelin–filamin A interaction. Furthermore, we found that loss of filamin A by siRNA knockdown, in patient cells, and in tissues from FlnaDilp2 null mouse embryos results in cellular phenotypes identical to those caused by meckelin loss, namely basal body positioning and ciliogenesis defects. In addition, morpholino knockdown of flna in zebrafish embryos significantly increases the frequency of dysmorphology and severity of ciliopathy developmental defects caused by mks3 knockdown. Our results suggest that meckelin forms a functional complex with filamin A that is disrupted in MKS and causes defects in neuronal migration and Wnt signalling. Furthermore, filamin A has a crucial role in the normal processes of ciliogenesis and basal body positioning. Concurrent with these processes, the meckelin–filamin A signalling axis may be a key regulator in maintaining correct, normal levels of Wnt signalling.
doi:10.1093/hmg/ddr557
PMCID: PMC3284117  PMID: 22121117
9.  CONSENSUS TREATMENT PLANS FOR INDUCTION THERAPY OF NEWLY-DIAGNOSED PROLIFERATIVE LUPUS NEPHRITIS IN JUVENILE SYSTEMIC LUPUS ERYTHEMATOSUS 
Arthritis care & research  2012;64(3):375-383.
Objective
To formulate consensus treatment plans (CTPs) for induction therapy of newly-diagnosed proliferative lupus nephritis (LN) in juvenile systemic lupus erythematosus (jSLE).
Methods
A structured consensus formation process was employed by the members of the Childhood Arthritis and Rheumatology Research Alliance (CARRA) after considering the existing medical evidence and current treatment approaches.
Results
After an initial Delphi survey (response rate 70%), a 2-day consensus conference, and two follow-up Delphi surveys (response rates 63–79%), consensus was achieved for a limited set of CTPs addressing the induction therapy of proliferative LN. These CTPs were developed for prototypic patients defined by eligibility characteristics, and included immunosuppressive therapy with either mycophenolic acid orally twice per day, or intravenous cyclophosphamide once per month at standardized doses for six months. Additionally, the CTPs describe three options for standardized use of glucocorticoids; including a primarily oral, a mixed oral/intravenous, and a primarily intravenous regimen. There was consensus on measures of effectiveness and safety of the CTPs. The CTPs were well accepted by the pediatric rheumatology providers treating children with LN, and up to 300 children per year in North America are expected to be candidates for the treatment with the CTPs.
Conclusion
CTPs for induction therapy of proliferative LN in jSLE based on the available scientific evidence and pediatric rheumatology group experience have been developed. Consistent use of the CTPs may improve the prognosis of proliferative LN, and support the conduct of comparative effectiveness studies aimed at optimizing therapeutic strategies for proliferative LN in jSLE.
doi:10.1002/acr.21558
PMCID: PMC3457803  PMID: 22162255
children; SLE; lupus nephritis; induction therapy; consensus
10.  Mutations in TMEM216 perturb ciliogenesis and cause Joubert, Meckel and related syndromes 
Nature genetics  2010;42(7):619-625.
Joubert syndrome (JBTS), related disorders (JSRD) and Meckel syndrome (MKS) are ciliopathies. We now report that MKS2 and JBTS2 loci are allelic and due to mutations in TMEM216, encoding an uncharacterized tetraspan transmembrane protein. JBTS2 patients displayed frequent nephronophthisis and polydactytly, and two cases conformed to the Oro-Facio-Digital type VI phenotype, whereas skeletal dysplasia was common in MKS fetuses. A single p.R73L mutation was identified in all patients of Ashkenazi Jewish descent (n=10). TMEM216 localized to the base of primary cilia, and loss of TMEM216 in patient fibroblasts or following siRNA knockdown caused defective ciliogenesis and centrosomal docking, with concomitant hyperactivation of RhoA and Dishevelled. TMEM216 complexed with Meckelin, encoded by a gene also mutated in JSRD and MKS. Abrogation of tmem216 expression in zebrafish led to gastrulation defects that overlap with other ciliary morphants. The data implicate a new family of proteins in the ciliopathies, and further support allelism between ciliopathy disorders.
doi:10.1038/ng.594
PMCID: PMC2894012  PMID: 20512146
11.  Plasmodium falciparum Malaria Challenge by the Bite of Aseptic Anopheles stephensi Mosquitoes: Results of a Randomized Infectivity Trial 
PLoS ONE  2010;5(10):e13490.
Background
Experimental infection of malaria-naïve volunteers by the bite of Plasmodium falciparum-infected mosquitoes is a preferred means to test the protective effect of malaria vaccines and drugs. The standard model relies on the bite of five infected mosquitoes to induce malaria. We examined the efficacy of malaria transmission using mosquitoes raised aseptically in compliance with current Good Manufacturing Practices (cGMPs).
Methods and Findings
Eighteen adults aged 18–40 years were randomized to receive 1, 3 or 5 bites of Anopheles stephensi mosquitoes infected with the chloroquine-sensitive NF54 strain of P. falciparum.
Seventeen participants developed malaria; fourteen occurring on Day 11. The mean prepatent period was 10.9 days (9–12 days). The geometric mean parasitemia was 15.7 parasites/µL (range: 4–70) by microscopy. Polymerase chain reaction (PCR) detected parasites 3.1 (range: 0–4) days prior to microscopy. The geometric mean sporozoite load was 16,753 sporozoites per infected mosquito (range: 1,000–57,500). A 1-bite participant withdrew from the study on Day 13 post-challenge and was PCR and smear negative.
Conclusions
The use of aseptic, cGMP-compliant P. falciparum-infected mosquitoes is safe, is associated with a precise prepatent period compared to the standard model and appears more efficient than the standard approach, as it led to infection in 100% (6/6) of volunteers exposed to three mosquito bites and 83% (5/6) of volunteers exposed to one mosquito bite.
Trial Registration
ClinicalTrials.gov NCT00744133
doi:10.1371/journal.pone.0013490
PMCID: PMC2958836  PMID: 21042404
12.  Loss of the Metalloprotease ADAM9 Leads to Cone-Rod Dystrophy in Humans and Retinal Degeneration in Mice 
Cone-rod dystrophy (CRD) is an inherited progressive retinal dystrophy affecting the function of cone and rod photoreceptors. By autozygosity mapping, we identified null mutations in the ADAM metallopeptidase domain 9 (ADAM9) gene in four consanguineous families with recessively inherited early-onset CRD. We also found reduced photoreceptor responses in Adam9 knockout mice, previously reported to be asymptomatic. In 12-month-old knockout mice, photoreceptors appear normal, but the apical processes of the retinal pigment epithelium (RPE) cells are disorganized and contact between photoreceptor outer segments (POSs) and the RPE apical surface is compromised. In 20-month-old mice, there is clear evidence of progressive retinal degeneration with disorganized POS and thinning of the outer nuclear layer (ONL) in addition to the anomaly at the POS-RPE junction. RPE basal deposits and macrophages were also apparent in older mice. These findings therefore not only identify ADAM9 as a CRD gene but also identify a form of pathology wherein retinal disease first manifests at the POS-RPE junction.
doi:10.1016/j.ajhg.2009.04.005
PMCID: PMC2681008  PMID: 19409519
13.  Loss of the Metalloprotease ADAM9 Leads to Cone-Rod Dystrophy in Humans and Retinal Degeneration in Mice 
Cone-rod dystrophy (CRD) is an inherited progressive retinal dystrophy affecting the function of cone and rod photoreceptors. By autozygosity mapping, we identified null mutations in the ADAM metallopeptidase domain 9 (ADAM9) gene in four consanguineous families with recessively inherited early-onset CRD. We also found reduced photoreceptor responses in Adam9 knockout mice, previously reported to be asymptomatic. In 12-month-old knockout mice, photoreceptors appear normal, but the apical processes of the retinal pigment epithelium (RPE) cells are disorganized and contact between photoreceptor outer segments (POSs) and the RPE apical surface is compromised. In 20-month-old mice, there is clear evidence of progressive retinal degeneration with disorganized POS and thinning of the outer nuclear layer (ONL) in addition to the anomaly at the POS-RPE junction. RPE basal deposits and macrophages were also apparent in older mice. These findings therefore not only identify ADAM9 as a CRD gene but also identify a form of pathology wherein retinal disease first manifests at the POS-RPE junction.
doi:10.1016/j.ajhg.2009.04.005
PMCID: PMC2681008  PMID: 19409519
14.  Examination of actin and microtubule dependent APC localisations in living mammalian cells 
BMC Cell Biology  2006;7:3.
Background
The trafficking of the adenomatous polyposis coli (APC) tumour suppressor protein in mammalian cells is a perennially controversial topic. Immunostaining evidence for an actin-associated APC localisation at intercellular junctions has been previously presented, though live imaging of mammalian junctional APC has not been documented.
Results
Using live imaging of transfected COS-7 cells we observed intercellular junction-associated pools of GFP-APC in addition to previously documented microtubule-associated GFP-APC and a variety of minor localisations. Although both microtubule and junction-associated populations could co-exist within individual cells, they differed in their subcellular location, dynamic behaviour and sensitivity to cytoskeletal poisons. GFP-APC deletion mutant analysis indicated that a protein truncated immediately after the APC armadillo repeat domain retained the ability to localise to adhesive membranes in transfected cells. Supporting this, we also observed junctional APC immunostaining in cultures of human colorectal cancer cell line that express truncated forms of APC.
Conclusion
Our data indicate that APC can be found in two spatially separate populations at the cell periphery and these populations can co-exist in the same cell. The first localisation is highly dynamic and associated with microtubules near free edges and in cell vertices, while the second is comparatively static and is closely associated with actin at sites of cell-cell contact. Our imaging confirms that human GFP-APC possesses many of the localisations and behaviours previously seen by live imaging of Xenopus GFP-APC. However, we report the novel finding that GFP-APC puncta can remain associated with the ends of shrinking microtubules. Deletion analysis indicated that the N-terminal region of the APC protein mediated its junctional localisation, consistent with our observation that truncated APC proteins in colon cancer cell lines are still capable of localising to the cell cortex. This may have implications for the development of colorectal cancer.
doi:10.1186/1471-2121-7-3
PMCID: PMC1386658  PMID: 16423286
15.  Trapping of normal EB1 ligands in aggresomes formed by an EB1 deletion mutant 
BMC Cell Biology  2005;6:17.
Background
EB1 is a microtubule tip-associated protein that interacts with the APC tumour suppressor protein and the p150glued subunit of dynactin. We previously reported that an EB1 deletion mutant that retains both of these interactions but does not directly associate with microtubules (EB1-ΔN2-GFP) spontaneously formed perinuclear aggregates when expressed in COS-7 cells.
Results
In the present study live imaging indicated that EB1-ΔN2-GFP aggregates underwent dynamic microtubule-dependent changes in morphology and appeared to be internally cohesive. EB1-ΔN2-GFP aggregates were phase-dense structures that displayed microtubule-dependent accumulation around the centrosome, were immunoreactive for both the 20s subunit of the proteasome and ubiquitin, and induced the collapse of the vimentin cytoskeleton. Fractionation studies revealed that a proportion of EB1-ΔN2-GFP was detergent-insoluble and ubiquitylated, indicating that EB1-ΔN2-GFP aggregates are aggresomes. Immunostaining also revealed that APC and p150glued were present in EB1-ΔN2-GFP aggregates, whereas EB3 was not. Furthermore, evidence for p150glued degradation was found in the insoluble fraction of EB1-ΔN2-GFP transfected cultures.
Conclusion
Our data indicate that aggresomes can be internally cohesive and may not represent a simple "aggregate of aggregates" assembled around the centrosome. Our observations also indicate that a partially misfolded protein may retain the ability to interact with its normal physiological ligands, leading to their co-assembly into aggresomes. This supports the idea that the trapping and degradation of co-aggregated proteins might contribute to human pathologies characterised by aggresome formation.
doi:10.1186/1471-2121-6-17
PMCID: PMC1090566  PMID: 15813965
16.  The Horamagraph 
British Medical Journal  1868;2(400):235.
PMCID: PMC2310966
17.  Suction-Curettes 
British Medical Journal  1866;1(273):319.
PMCID: PMC2309115

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