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1.  Epilepsy due to PNPO mutations: genotype, environment and treatment affect presentation and outcome 
Brain  2014;137(5):1350-1360.
Mutations in PNPO are a known cause of neonatal onset seizures that are resistant to pyridoxine but responsive to pyridoxal phosphate (PLP). Mills et al. show that PNPO mutations can also cause neonatal onset seizures that respond to pyridoxine but worsen with PLP, as well as PLP-responsive infantile spasms.
The first described patients with pyridox(am)ine 5’-phosphate oxidase deficiency all had neonatal onset seizures that did not respond to treatment with pyridoxine but responded to treatment with pyridoxal 5’-phosphate. Our data suggest, however, that the clinical spectrum of pyridox(am)ine 5’-phosphate oxidase deficiency is much broader than has been reported in the literature. Sequencing of the PNPO gene was undertaken for a cohort of 82 individuals who had shown a reduction in frequency and severity of seizures in response to pyridoxine or pyridoxal 5’-phosphate. Novel sequence changes were studied using a new cell-free expression system and a mass spectrometry-based assay for pyridoxamine phosphate oxidase. Three groups of patients with PNPO mutations that had reduced enzyme activity were identified: (i) patients with neonatal onset seizures responding to pyridoxal 5’-phosphate (n = 6); (ii) a patient with infantile spasms (onset 5 months) responsive to pyridoxal 5’-phosphate (n = 1); and (iii) patients with seizures starting under 3 months of age responding to pyridoxine (n = 8). Data suggest that certain genotypes (R225H/C and D33V) are more likely to result in seizures that to respond to treatment with pyridoxine. Other mutations seem to be associated with infertility, miscarriage and prematurity. However, the situation is clearly complex with the same combination of mutations being seen in patients who responded and did not respond to pyridoxine. It is possible that pyridoxine responsiveness in PNPO deficiency is affected by prematurity and age at the time of the therapeutic trial. Other additional factors that are likely to influence treatment response and outcome include riboflavin status and how well the foetus has been supplied with vitamin B6 by the mother. For some patients there was a worsening of symptoms on changing from pyridoxine to pyridoxal 5’-phosphate. Many of the mutations in PNPO affected residues involved in binding flavin mononucleotide or pyridoxal 5’-phosphate and many of them showed residual enzyme activity. One sequence change (R116Q), predicted to affect flavin mononucleotide binding and binding of the two PNPO dimers, and with high residual activity was found in Groups (ii) and (iii). This sequence change has been reported in the 1000 Genomes project suggesting it could be a polymorphism but alternatively it could be a common mutation, perhaps responsible for the susceptibility locus for genetic generalized epilepsy on 17q21.32 (close to rs72823592). We believe the reduction in PNPO activity and B6-responsive epilepsy in the patients reported here indicates that it contributes to the pathogenesis of epilepsy.
doi:10.1093/brain/awu051
PMCID: PMC3999720  PMID: 24645144
pyridoxal 5’-phosphate (PLP); pyridoxine; pyridox(am)ine 5’-phosphate oxidase (PNPO); seizures; epilepsy
2.  Trophic Relationships and Habitat Preferences of Delphinids from the Southeastern Brazilian Coast Determined by Carbon and Nitrogen Stable Isotope Composition 
PLoS ONE  2013;8(12):e82205.
To investigate the foraging habitats of delphinids in southeastern Brazil, we analyzed stable carbon (δ13C) and nitrogen (δ15N) isotopes in muscle samples of the following 10 delphinid species: Sotalia guianensis, Stenella frontalis, Tursiops truncatus, Steno bredanensis, Pseudorca crassidens, Delphinus sp., Lagenodelphis hosei, Stenella attenuata, Stenella longirostris and Grampus griseus. We also compared the δ13C and δ15N values among four populations of S. guianensis. Variation in carbon isotope results from coast to ocean indicated that there was a significant decrease in δ13C values from estuarine dolphins to oceanic species. S. guianensis from Guanabara Bay had the highest mean δ13C value, while oceanic species showed significantly lower δ13C values. The highest δ15N values were observed for P. crassidens and T. truncatus, suggesting that these species occupy the highest trophic position among the delphinids studied here. The oceanic species S. attenuata, G. griseus and L. hosei had the lowest δ15N values. Stable isotope analysis showed that the three populations of S. guianensis in coastal bays had different δ13C values, but similar δ15N results. Guiana dolphins from Sepetiba and Ilha Grande bays had different foraging habitat, with specimens from Ilha Grande showing more negative δ13C values. This study provides further information on the feeding ecology of delphinids occurring in southeastern Brazil, with evidence of distinctive foraging habitats and the occupation of different ecological niches by these species in the study area.
doi:10.1371/journal.pone.0082205
PMCID: PMC3864921  PMID: 24358155
3.  Comparison of central corneal thickness of primary open angle glaucoma patients with normal controls in South India 
Oman Journal of Ophthalmology  2013;6(1):33-36.
Background:
Studies mainly in the western population have compared central corneal thickness in primary open angle glaucoma and normal individuals have found variable results. We did this study to compare the central corneal thickness of primary open angle glaucoma patients with normal controls in a south Indian population.
Materials and Methods:
This was a masked, cross-sectional study undertaken in a tertiary care center in South India. A total of 50 controls and 50 primary open angle glaucoma patients were studied. Central corneal thickness between the two groups was compared using Wilcoxon two sample test and the signed rank test.
Results:
The mean central corneal thickness in the control group was 536 μm (462–608 μm) and in the primary open angle glaucoma group was 531 μm (476–609 μm).
Conclusion:
There was no significant difference in the central corneal thickness between primary open angle glaucoma patients and the normal controls.
doi:10.4103/0974-620X.111907
PMCID: PMC3678195  PMID: 23772123
Central corneal thickness; open angle glaucoma; ocular tonometry
4.  A Huge Epidermoid Cyst Endangering Life 
Epidermoid cyst of the neck or auricular area are relatively more common than that of the oral cavity. In most cases about 80% they remain asymptomatic. But in about 20% cases it becomes painful because of secondary infection seeking treatment. Here we present a case report of biopsy proved Epidermoid cyst with life threatening infection.
doi:10.1007/s12663-010-0106-8
PMCID: PMC3238561  PMID: 22942595
Epidermoid cyst; Oral cavity; Life threatening infection
5.  Cloning, Soluble Expression and Purification of High Yield Recombinant hGMCSF in Escherichia coli 
Expression of human granulocyte macrophage colony stimulating factor (hGMCSF), a cytokine of therapeutic importance, as a thioredoxin (TRX) fusion has been investigated in Escherichia coli BL21 (DE3) codon plus cells. The expression of this protein was low when cloned under the T7 promoter without any fusion tags. High yield of GMCSF was achieved (∼88 mg/L of fermentation broth) in the shake flask when the gene was fused to the E. coli TRX gene. The protein was purified using a single step Ni2+-NTA affinity chromatography and the column bound fusion tag was removed by on-column cleavage with enterokinase. The recombinant hGMCSF was expressed as a soluble and biologically active protein in E. coli, and upon purification, the final yield was ∼44 mg/L in shake flask with a specific activity of 2.3 × 108 U/mg. The results of Western blot and RP-HPLC analyses, along with biological activity using the TF-1 cell line, established the identity of the purified hGMCSF. In this paper, we report the highest yield of hGMCSF expressed in E. coli. The bioreactor study shows that the yield of hGMCSF could be easily scalable with a yield of ∼400 mg/L, opening up new opportunities for large scale production hGMCSF in E. coli.
doi:10.3390/ijms12032064
PMCID: PMC3111651  PMID: 21673940
human granulocyte macrophage colony stimulating factor; on-column cleavage; TRX fusion; IMAC; enterokinase
6.  Correction: Isotope Analysis Reveals Foraging Area Dichotomy for Atlantic Leatherback Turtles 
PLoS ONE  2009;4(8):10.1371/annotation/17755b0c-3597-4da2-be87-08a14caba677.
doi:10.1371/annotation/17755b0c-3597-4da2-be87-08a14caba677
PMCID: PMC2727541
7.  Correction: Isotope Analysis Reveals Foraging Area Dichotomy for Atlantic Leatherback Turtles 
PLoS ONE  2009;4(8):10.1371/annotation/72a9b040-40d8-495a-a4aa-a7ea8fd00de8.
doi:10.1371/annotation/72a9b040-40d8-495a-a4aa-a7ea8fd00de8
PMCID: PMC2727527
8.  Correction: Isotope Analysis Reveals Foraging Area Dichotomy for Atlantic Leatherback Turtles 
PLoS ONE  2009;4(7):10.1371/annotation/de853c89-d3eb-441f-8d87-0366bb1533b5.
doi:10.1371/annotation/de853c89-d3eb-441f-8d87-0366bb1533b5
PMCID: PMC2727544
9.  Use of Clinical Decision Support to Increase Influenza Vaccination: Multi-year Evolution of the System 
Despite recognition that clinical decision support (CDS) can improve patient care, there has been poor penetration of this technology into healthcare settings. We used CDS to increase inpatient influenza vaccination during implementation of an electronic medical record, in which pharmacy and nursing transactions increasingly became electronic. Over three influenza seasons we evaluated standing orders, provider reminders, and pre-selected physician orders. A pre-intervention cross-sectional survey showed that most patients (95%) met criteria for vaccination. During our intervention, physicians were increasingly likely to accept pre-selected vaccination orders, Year 1 (47%), Year 2 (77%), Year 3 (83%); however vaccine administration by nurses was suboptimal. As electronic medical record functionality improved, patient receipt of vaccine increased dramatically, Year 1 [0/36; 0%], Year 2 [8/66; 12%], Year 3 [286/805; 36%]. Successful use of clinical decision support to increase inpatient influenza vaccination only occurred after initiation of CPOE for all medications and integration of an electronic medication administration record. Also, since most patients met criteria for influenza vaccination, complicated logic to identify high-risk patients was unnecessary.
doi:10.1197/jamia.M2698
PMCID: PMC2585533  PMID: 18756001
10.  Mercury immune toxicity in harbour seals: links to in vitro toxicity 
Environmental Health  2008;7:52.
Background
Mercury is known to bioaccumulate and to magnify in marine mammals, which is a cause of great concern in terms of their general health. In particular, the immune system is known to be susceptible to long-term mercury exposure. The aims of the present study were (1) to determine the mercury level in the blood of free-ranging harbour seals from the North Sea and (2) to examine the link between methylmercury in vitro exposure and immune functions using seal and human mitogen-stimulated peripheral blood mononuclear cells (T-lymphocytes).
Methods
Total mercury was analysed in the blood of 22 harbour seals. Peripheral blood mononuclear cells were isolated from seals (n = 11) and from humans (n = 9). Stimulated lymphocytes of both species were exposed to functional tests (proliferation, metabolic activity, radioactive precursor incorporation) under increasing doses of methylmercury (0.1 to 10 μM). The expression of cytokines (IL-2, IL-4 and TGF-β) was investigated in seal lymphocytes by RT-PCR and by real time quantitative PCR (n = 5) at methylmercury concentrations of 0.2 and 1 μM. Finally, proteomics analysis was attempted on human lymphocytes (cytoplasmic fraction) in order to identify biochemical pathways of toxicity at concentration of 1 μM (n = 3).
Results
The results showed that the number of seal lymphocytes, viability, metabolic activity, DNA and RNA synthesis were reduced in vitro, suggesting deleterious effects of methylmercury concentrations naturally encountered in free-ranging seals. Similar results were found for human lymphocytes. Functional tests showed that a 1 μM concentration was the critical concentration above which lymphocyte activity, proliferation and survival were compromised. The expression of IL-2 and TGF-β mRNA was weaker in exposed seal lymphocytes compared to control cells (0.2 and 1 μM). Proteomics showed some variation in the protein expression profile (e.g. vimentin).
Conclusion
Our results suggest that seal and human PBMCs react in a comparable way to MeHg in vitro exposure with, however, larger inter-individual variations. MeHg could be an additional cofactor in the immunosuppressive pollutant cocktail generally described in the blood of seals and this therefore raises the possibility of additional additive effects in the marine mammal immune system.
doi:10.1186/1476-069X-7-52
PMCID: PMC2600635  PMID: 18959786
11.  Isotope Analysis Reveals Foraging Area Dichotomy for Atlantic Leatherback Turtles 
PLoS ONE  2008;3(3):e1845.
Background
The leatherback turtle (Dermochelys coriacea) has undergone a dramatic decline over the last 25 years, and this is believed to be primarily the result of mortality associated with fisheries bycatch followed by egg and nesting female harvest. Atlantic leatherback turtles undertake long migrations across ocean basins from subtropical and tropical nesting beaches to productive frontal areas. Migration between two nesting seasons can last 2 or 3 years, a time period termed the remigration interval (RI). Recent satellite transmitter data revealed that Atlantic leatherbacks follow two major dispersion patterns after nesting season, through the North Gulf Stream area or more eastward across the North Equatorial Current. However, information on the whole RI is lacking, precluding the accurate identification of feeding areas where conservation measures may need to be applied.
Methodology/Principal Findings
Using stable isotopes as dietary tracers we determined the characteristics of feeding grounds of leatherback females nesting in French Guiana. During migration, 3-year RI females differed from 2-year RI females in their isotope values, implying differences in their choice of feeding habitats (offshore vs. more coastal) and foraging latitude (North Atlantic vs. West African coasts, respectively). Egg-yolk and blood isotope values are correlated in nesting females, indicating that egg analysis is a useful tool for assessing isotope values in these turtles, including adults when not available.
Conclusions/Significance
Our results complement previous data on turtle movements during the first year following the nesting season, integrating the diet consumed during the year before nesting. We suggest that the French Guiana leatherback population segregates into two distinct isotopic groupings, and highlight the urgent need to determine the feeding habitats of the turtle in the Atlantic in order to protect this species from incidental take by commercial fisheries. Our results also emphasize the use of eggs, a less-invasive sampling material than blood, to assess isotopic data and feeding habits for adult female leatherbacks.
doi:10.1371/journal.pone.0001845
PMCID: PMC2267998  PMID: 18365003
12.  Long-term feeding ecology and habitat use in harbour porpoises Phocoena phocoena from Scandinavian waters inferred from trace elements and stable isotopes 
BMC Ecology  2007;7:1.
Background
We investigated the feeding ecology and habitat use of 32 harbour porpoises by-caught in 4 localities along the Scandinavian coast from the North Sea to the Barents Sea using time-integrative markers: stable isotopes (δ13C, δ15N) and trace elements (Zn, Cu, Fe, Se, total Hg and Cd), in relation to habitat characteristics (bathymetry) and geographic position (latitude).
Results
Among the trace elements analysed, only Cd, with an oceanic specific food origin, was found to be useful as an ecological tracer. All other trace elements studied were not useful, most likely because of physiological regulation and/or few specific sources in the food web. The δ13C, δ15N signatures and Cd levels were highly correlated with each other, as well as with local bathymetry and geographic position (latitude). Variation in the isotopic ratios indicated a shift in harbour porpoise's feeding habits from pelagic prey species in deep northern waters to more coastal and/or demersal prey in the relatively shallow North Sea and Skagerrak waters. This result is consistent with stomach content analyses found in the literature. This shift was associated with a northward Cd-enrichment which provides further support to the Cd 'anomaly' previously reported in polar waters and suggests that porpoises in deep northern waters include Cd-contaminated prey in their diet, such as oceanic cephalopods.
Conclusion
As stable isotopes and Cd provide information in the medium and the long term respectively, the spatial variation found, shows that harbour porpoises experience different ecological regimes during the year along the Scandinavian coasts, adapting their feeding habits to local oceanographic conditions, without performing extensive migration.
doi:10.1186/1472-6785-7-1
PMCID: PMC1781931  PMID: 17229317
13.  Zn, Cu, Cd and Hg binding to metallothioneins in harbour porpoises Phocoena phocoena from the southern North Sea 
BMC Ecology  2006;6:2.
Background
Harbour porpoises Phocoena phocoena from the southern North Sea are known to display high levels of Zn and Hg in their tissues linked to their nutritional status (emaciation). The question arises regarding a potential role of metallothioneins (MTs) with regard to these high metal levels. In the present study, metallothionein detection and associated Zn, Cd, Cu and Hg concentrations were investigated in the liver and kidney of 14 harbour porpoises collected along the Belgian coast.
Results
Metallothioneins seemed to play a key role in essential metal homeostasis, as they were shown to bind 50% of the total hepatic Zn and 36% of the total hepatic Cu concentrations. Renal MTs also participated in Cd detoxification, as they were shown to bind 56% of the total renal Cd. Hg was mainly found in the insoluble fraction of both liver and kidney. Concomitant increases in total Zn concentration and Zn bound to MTs were observed in the liver, whereas Zn concentration bound to high molecular weight proteins remained constant. Cu, Zn and Cd were accumulated preferentially in the MT fraction and their content in this fraction increased with the amount in the hepatocytosol.
Conclusion
MTs have a key role in Zn and Cu homeostasis in harbour porpoises. We demonstrated that increasing hepatic Zn concentration led to an increase in Zn linked to MTs, suggesting that these small proteins take over the Zn overload linked to the poor body condition of debilitated harbour porpoises.
doi:10.1186/1472-6785-6-2
PMCID: PMC1434725  PMID: 16464247

Results 1-14 (14)