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1.  Plasma lipidomics analysis finds long chain cholesteryl esters to be associated with Alzheimer's disease 
Translational Psychiatry  2015;5(1):e494-.
There is an urgent need for the identification of Alzheimer's disease (AD) biomarkers. Studies have now suggested the promising use of associations with blood metabolites as functional intermediate phenotypes in biomedical and pharmaceutical research. The aim of this study was to use lipidomics to identify a battery of plasma metabolite molecules that could predict AD patients from controls. We performed a comprehensive untargeted lipidomic analysis, using ultra-performance liquid chromatography/mass spectrometry on plasma samples from 35 AD patients, 40 elderly controls and 48 individuals with mild cognitive impairment (MCI) and used multivariate analysis methods to identify metabolites associated with AD status. A combination of 10 metabolites could discriminate AD patients from controls with 79.2% accuracy (81.8% sensitivity, 76.9% specificity and an area under curve of 0.792) in a novel test set. Six of the metabolites were identified as long chain cholesteryl esters (ChEs) and were reduced in AD (ChE 32:0, odds ratio (OR)=0.237, 95% confidence interval (CI)=0.10–0.48, P=4.19E−04; ChE 34:0, OR=0.152, 95% CI=0.05–0.37, P=2.90E−04; ChE 34:6, OR=0.126, 95% CI=0.03–0.35, P=5.40E−04; ChE 32:4, OR=0.056, 95% CI=0.01–0.24, P=6.56E−04 and ChE 33:6, OR=0.205, 95% CI=0.06–0.50, P=2.21E−03, per (log2) metabolite unit). The levels of these metabolites followed the trend control>MCI>AD. We, additionally, found no association between cholesterol, the precursor of ChE and AD. This study identified new ChE molecules, involved in cholesterol metabolism, implicated in AD, which may help identify new therapeutic targets; although, these findings need to be replicated in larger well-phenotyped cohorts.
doi:10.1038/tp.2014.127
PMCID: PMC4312824  PMID: 25585166
2.  A Dose Finding Study of Temsirolimus and Liposomal Doxorubicin for Patients with Recurrent and Refractory Bone and Soft Tissue Sarcoma 
There are few effective therapies for high-risk sarcomas. Initial chemosensitivity is often followed by relapse. In vitro, mTOR inhibition potentiates the efficacy of chemotherapy on resistant sarcoma cells. Although sarcoma trials using mTOR inhibitors have been disappointing, these drugs were used as maintenance. We conducted a Phase I/II clinical trial to test the ability of temsirolimus to potentiate the cytotoxic effect of liposomal doxorubicin and present here the dose-finding portion of this study. Adult and pediatrics patients with recurrent or refractory sarcomas were treated with increasing doses of liposomal doxorubicin and temsirolimus using a continual reassessment method for escalation, targeting a dose-limiting toxicity rate of 20%. Blood samples were drawn before and after the first dose of temsirolimus in Cycles 1 and 2 for pharmacokinetic analysis. The maximally tolerated dose combination was liposomal doxorubicin 30 mg/m2 monthly with temsirolimus 20 mg/m2 weekly. Hematologic toxicity was common but manageable. Dose-limiting toxicities were primarily renal. Concurrent administration of liposomal doxorubicin resulted in increased exposure to sirolimus, the active metabolite of temsirolimus. Thus, the combination of liposomal doxorubicin and temsirolimus is safe for heavily pretreated sarcoma patients. Coadministration with liposomal doxorubicin did not alter temsirolimus pharmacokinetics, but increased exposure to its active metabolite.
doi:10.1002/ijc.28083
PMCID: PMC3681832  PMID: 23382028
mTOR; cancer stem cell; chemoresistance; sarcoma; clinical trial
3.  Genomics and Successful Aging: Grounds for Renewed Optimism? 
Background.
Successful aging depends in part on delaying age-related disease onsets until later in life. Conditions including coronary artery disease, Alzheimer’s disease, prostate cancer, and type 2 diabetes are moderately heritable. Genome-wide association studies have identified many risk associated single-nucleotide polymorphisms for these conditions, but much heritability remains unaccounted for. Nevertheless, a great deal is being learned.
Methods.
Here, we review age-related disease associated single-nucleotide polymorphisms and identify key underlying pathways including lipid handling, specific immune processes, early tissue development, and cell cycle control.
Results.
Most age-related disease associated single-nucleotide polymorphisms do not affect coding regions of genes or protein makeup but instead influence regulation of gene expression. Recent evidence indicates that evolution of gene regulatory sites is fundamental to interspecies differences. Animal models relevant to human aging may therefore need to focus more on gene regulation rather than testing major disruptions to fundamental pathway genes. Recent larger scale human studies of in vivo genome-wide expression (notably from the InCHIANTI aging study) have identified changes in splicing, the “fine tuning” of protein sequences, as a potentially important factor in decline of cellular function with age. Studies of expression with muscle strength and cognition have shown striking concordance with certain mice models of muscle repair and beta-amyloid phagocytosis respectively.
Conclusions.
The emerging clearer picture of the genetic architecture of age-related diseases in humans is providing new insights into the underlying pathophysiological pathways involved. Translation of genomics into new approaches to prevention, tests and treatments to extend successful aging is therefore likely in the coming decades.
doi:10.1093/gerona/gls091
PMCID: PMC3326244  PMID: 22454374
Longevity; Genetics; Public health
4.  Preclinical development of CAT-354, an IL-13 neutralizing antibody, for the treatment of severe uncontrolled asthma 
British Journal of Pharmacology  2012;166(1):177-193.
BACKGROUND AND PURPOSE
IL-13 is a pleiotropic Th2 cytokine considered likely to play a pivotal role in asthma. Here we describe the preclinical in vitro and in vivo characterization of CAT-354, an IL-13-neutralizing IgG4 monoclonal antibody (mAb), currently in clinical development.
EXPERIMENTAL APPROACH
In vitro the potency, specificity and species selectivity of CAT-354 was assayed in TF-1 cells, human umbilical vein endothelial cells and HDLM-2 cells. The ability of CAT-354 to modulate disease-relevant mechanisms was tested in human cells measuring bronchial smooth muscle calcium flux induced by histamine, eotaxin generation by normal lung fibroblasts, CD23 upregulation in peripheral blood mononuclear cells and IgE production by B cells. In vivo CAT-354 was tested on human IL-13-induced air pouch inflammation in mice, ovalbumin-sensitization and challenge in IL-13 humanized mice and antigen challenge in cynomolgus monkeys.
KEY RESULTS
CAT-354 has a 165 pM affinity for human IL-13 and functionally neutralized human, human variant associated with asthma and atopy (R130Q) and cynomolgus monkey, but not mouse, IL-13. CAT-354 did not neutralize human IL-4. In vitro CAT-354 functionally inhibited IL-13-induced eotaxin production, an analogue of smooth muscle airways hyperresponsiveness, CD23 upregulation and IgE production. In vivo in humanized mouse and cynomolgus monkey antigen challenge models CAT-354 inhibited airways hyperresponsiveness and bronchoalveolar lavage eosinophilia.
CONCLUSIONS AND IMPLICATIONS
CAT-354 is a potent and selective IL-13-neutralizing IgG4 mAb. The preclinical data presented here support the trialling of this mAb in patients with moderate to severe uncontrolled asthma.
doi:10.1111/j.1476-5381.2011.01659.x
PMCID: PMC3415647  PMID: 21895629
CAT-354; IL-13; IgG4; preclinical development for asthma; uncontrolled asthma; therapeutic antibody; asthma treatment
5.  Adenosine Receptors and Cancer 
The A1, A2A, A2B and A3 G-protein-coupled cell surface adenosine receptors (ARs) are found to be upregulated in various tumor cells. Activation of the receptors by specific ligands, agonists or antagonists, modulates tumor growth via a range of signaling pathways. The A1AR was found to play a role in preventing the development of glioblastomas. This antitumor effect of the A1AR is mediated via tumor-associated microglial cells. Activation of the A2AAR results in inhibition of the immune response to tumors via suppression of T regulatory cell function and inhibition of natural killer cell cytotoxicity and tumor-specific CD4+/CD8+ activity. Therefore, it is suggested that pharmacological inhibition by specific antagonists may enhance immunotherapeutics in cancer therapy. Activation of the A2BAR plays a role in the development of tumors via upregulation of the expression levels of angiogenic factors in microvascular endothelial cells. In contrast, it was evident that activation of A2BAR results in inhibition of ERK1/2 phosphorylation and MAP kinase activity, which are involved in tumor cell growth signals. Finally, A3AR was found to be highly expressed in tumor cells and tissues while low expression levels were noted in normal cells or adjacent tissue. Receptor expression in the tumor tissues was directly correlated to disease severity. The high receptor expression in the tumors was attributed to overexpression of NF-κB, known to act as an A3AR transcription factor. Interestingly, high A3AR expression levels were found in peripheral blood mononuclear cells (PBMCs) derived from tumor-bearing animals and cancer patients, reflecting receptor status in the tumors. A3AR agonists were found to induce tumor growth inhibition, both in vitro and in vivo, via modulation of the Wnt and the NF-κB signaling pathways. Taken together, A3ARs that are abundantly expressed in tumor cells may be targeted by specific A3AR agonists, leading to tumor growth inhibition. The unique characteristics of these A3AR agonists make them attractive as drug candidates.
doi:10.1007/978-3-540-89615-9_14
PMCID: PMC3598010  PMID: 19639290
A1 adenosine receptor; A2A adenosine receptor; A2B adenosine receptor; A3 adenosine receptor; Expression; Tumor growth; Agonists; Antagonists
7.  Long-Term Exposure to Antibiotics Has Caused Accumulation of Resistance Determinants in the Gut Microbiota of Honeybees 
mBio  2012;3(6):e00377-12.
ABSTRACT
Antibiotic treatment can impact nontarget microbes, enriching the pool of resistance genes available to pathogens and altering community profiles of microbes beneficial to hosts. The gut microbiota of adult honeybees, a distinctive community dominated by eight bacterial species, provides an opportunity to examine evolutionary responses to long-term treatment with a single antibiotic. For decades, American beekeepers have routinely treated colonies with oxytetracycline for control of larval pathogens. Using a functional metagenomic screen of bacteria from Maryland bees, we detected a high incidence of tetracycline/oxytetracycline resistance. This resistance is attributable to known resistance loci for which nucleotide sequences and flanking mobility genes were nearly identical to those from human pathogens and from bacteria associated with farm animals. Surveys using diagnostic PCR and sequencing revealed that gut bacteria of honeybees from diverse localities in the United States harbor eight tetracycline resistance loci, including efflux pump genes (tetB, tetC, tetD, tetH, tetL, and tetY) and ribosome protection genes (tetM and tetW), often at high frequencies. Isolates of gut bacteria from Connecticut bees display high levels of tetracycline resistance. Resistance genes were ubiquitous in American samples, though rare in colonies unexposed for 25 years. In contrast, only three resistance loci, at low frequencies, occurred in samples from countries not using antibiotics in beekeeping and samples from wild bumblebees. Thus, long-term antibiotic treatment has caused the bee gut microbiota to accumulate resistance genes, drawn from a widespread pool of highly mobile loci characterized from pathogens and agricultural sites.
IMPORTANCE
We found that 50 years of using antibiotics in beekeeping in the United States has resulted in extensive tetracycline resistance in the gut microbiota. These bacteria, which form a distinctive community present in healthy honeybees worldwide, may function in protecting bees from disease and in providing nutrition. In countries that do not use antibiotics in beekeeping, bee gut bacteria contained far fewer resistance genes. The tetracycline resistance that we observed in American samples reflects the capture of mobile resistance genes closely related to those known from human pathogens and agricultural sites. Thus, long-term treatment to control a specific pathogen resulted in the accumulation of a stockpile of resistance capabilities in the microbiota of a healthy gut. This stockpile can, in turn, provide a source of resistance genes for pathogens themselves. The use of novel antibiotics in beekeeping may disrupt bee health, adding to the threats faced by these pollinators.
doi:10.1128/mBio.00377-12
PMCID: PMC3487773  PMID: 23111871
8.  Distinctive Gut Microbiota of Honey Bees Assessed Using Deep Sampling from Individual Worker Bees 
PLoS ONE  2012;7(4):e36393.
Surveys of 16S rDNA sequences from the honey bee, Apis mellifera, have revealed the presence of eight distinctive bacterial phylotypes in intestinal tracts of adult worker bees. Because previous studies have been limited to relatively few sequences from samples pooled from multiple hosts, the extent of variation in this microbiota among individuals within and between colonies and locations has been unclear. We surveyed the gut microbiota of 40 individual workers from two sites, Arizona and Maryland USA, sampling four colonies per site. Universal primers were used to amplify regions of 16S ribosomal RNA genes, and amplicons were sequenced using 454 pyrotag methods, enabling analysis of about 330,000 bacterial reads. Over 99% of these sequences belonged to clusters for which the first blastn hits in GenBank were members of the known bee phylotypes. Four phylotypes, one within Gammaproteobacteria (corresponding to “Candidatus Gilliamella apicola”) one within Betaproteobacteria (“Candidatus Snodgrassella alvi”), and two within Lactobacillus, were present in every bee, though their frequencies varied. The same typical bacterial phylotypes were present in all colonies and at both sites. Community profiles differed significantly among colonies and between sites, mostly due to the presence in some Arizona colonies of two species of Enterobacteriaceae not retrieved previously from bees. Analysis of Sanger sequences of rRNA of the Snodgrassella and Gilliamella phylotypes revealed that single bees contain numerous distinct strains of each phylotype. Strains showed some differentiation between localities, especially for the Snodgrassella phylotype.
doi:10.1371/journal.pone.0036393
PMCID: PMC3338667  PMID: 22558460
9.  Interaction of catechol O-methyltransferase and serotonin transporter genes modulates effective connectivity in a facial emotion-processing circuitry 
Translational Psychiatry  2012;2(1):e70-.
Imaging genetic studies showed exaggerated blood oxygenation level-dependent response in limbic structures in carriers of low activity alleles of serotonin transporter-linked promoter region (5-HTTLPR) as well as catechol O-methyltransferase (COMT) genes. This was suggested to underlie the vulnerability to mood disorders. To better understand the mechanisms of vulnerability, it is important to investigate the genetic modulation of frontal-limbic connectivity that underlies emotional regulation and control. In this study, we have examined the interaction of 5-HTTLPR and COMT genetic markers on effective connectivity within neural circuitry for emotional facial expressions. A total of 91 healthy Caucasian adults underwent functional magnetic resonance imaging experiments with a task presenting dynamic emotional facial expressions of fear, sadness, happiness and anger. The effective connectivity within the facial processing circuitry was assessed with Granger causality method. We have demonstrated that in fear processing condition, an interaction between 5-HTTLPR (S) and COMT (met) low activity alleles was associated with reduced reciprocal connectivity within the circuitry including bilateral fusiform/inferior occipital regions, right superior temporal gyrus/superior temporal sulcus, bilateral inferior/middle prefrontal cortex and right amygdala. We suggest that the epistatic effect of reduced effective connectivity may underlie an inefficient emotion regulation that places these individuals at greater risk for depressive disorders.
doi:10.1038/tp.2011.69
PMCID: PMC3309546  PMID: 22832732
COMT; effective connectivity; facial emotion processing; 5-HTTLPR
10.  The orally active melanocortin-4 receptor antagonist BL-6020/979: a promising candidate for the treatment of cancer cachexia 
Background
Under physiological conditions, the melanocortin system is a crucial part of the complex network regulating food intake and energy expenditure. In pathological states, like cachexia, these two parameters are deregulated, i.e., food intake is decreased and energy expenditure is increased—a vicious combination leading to catabolism. Agouti-related protein (AgRP), the endogenous antagonist at the melanocortin-4 receptor (MC-4R), was found to increase food intake and to reduce energy expenditure. This qualifies MC-4R blockade as an attractive mode of action for the treatment of cachexia. Based on this rationale, a novel series of small-molecule MC-4R antagonists was designed, from which the orally active compound BL-6020/979 (formerly known as SNT207979) emerged as the first promising development candidate showing encouraging pre-clinical efficacy and safety properties which are presented here.
Methods and results
BL-6020/979 is an orally available, selective and potent MC-4R antagonist with a drug-like profile. It increased food intake and decreased energy expenditure in healthy wild-type but not in MC-4R deficient mice. More importantly, it ameliorated cachexia-like symptoms in the murine C26 adenocarcinoma model; with an effect on body mass and body composition and on the expression of catabolic genes. Moreover, BL-6020/979 showed antidepressant-like properties in the chronic mild stress model in rats and exhibits a favorable safety profile.
Conclusion
The properties of BL-6020/979 demonstrated in animal models and presented here make it a promising candidate suitable for further development towards a first-in-class treatment option for cachexia that potentially opens up the opportunity to treat two hallmarks of the disease, i.e., decreased food intake and increased energy expenditure, with one drug.
doi:10.1007/s13539-011-0039-1
PMCID: PMC3177041  PMID: 21966642
Cancer cachexia; C26 adenocarcinoma; Melanocortin-4 receptor antagonist; Mouse; Food intake; Metabolic rate
11.  The orally active melanocortin-4 receptor antagonist BL-6020/979: a promising candidate for the treatment of cancer cachexia 
Background
Under physiological conditions, the melanocortin system is a crucial part of the complex network regulating food intake and energy expenditure. In pathological states, like cachexia, these two parameters are deregulated, i.e., food intake is decreased and energy expenditure is increased—a vicious combination leading to catabolism. Agouti-related protein (AgRP), the endogenous antagonist at the melanocortin-4 receptor (MC-4R), was found to increase food intake and to reduce energy expenditure. This qualifies MC-4R blockade as an attractive mode of action for the treatment of cachexia. Based on this rationale, a novel series of small-molecule MC-4R antagonists was designed, from which the orally active compound BL-6020/979 (formerly known as SNT207979) emerged as the first promising development candidate showing encouraging pre-clinical efficacy and safety properties which are presented here.
Methods and results
BL-6020/979 is an orally available, selective and potent MC-4R antagonist with a drug-like profile. It increased food intake and decreased energy expenditure in healthy wild-type but not in MC-4R deficient mice. More importantly, it ameliorated cachexia-like symptoms in the murine C26 adenocarcinoma model; with an effect on body mass and body composition and on the expression of catabolic genes. Moreover, BL-6020/979 showed antidepressant-like properties in the chronic mild stress model in rats and exhibits a favorable safety profile.
Conclusion
The properties of BL-6020/979 demonstrated in animal models and presented here make it a promising candidate suitable for further development towards a first-in-class treatment option for cachexia that potentially opens up the opportunity to treat two hallmarks of the disease, i.e., decreased food intake and increased energy expenditure, with one drug.
doi:10.1007/s13539-011-0039-1
PMCID: PMC3177041  PMID: 21966642
Cancer cachexia; C26 adenocarcinoma; Melanocortin-4 receptor antagonist; Mouse; Food intake; Metabolic rate
12.  Immunologic recovery following autologous stem-cell transplantation with pre- and posttransplantation rituximab for low-grade or mantle cell lymphoma 
Annals of Oncology  2009;21(6):1203-1210.
Background: Rituximab may improve transplant outcomes but may delay immunologic recovery.
Patients and methods: Seventy-seven patients with low-grade or mantle cell lymphoma received autologous stem-cell transplantation (ASCT) on a phase II study. Rituximab 375 mg/m2 was administered 3 days before mobilization-dose cyclophosphamide, then weekly for four doses after count recovery from ASCT. Immune reconstitution was assessed.
Results: Sixty percent of transplants occurred in first remission. Actuarial event-free survival (EFS) and overall survival (OS) were 60% and 73%, respectively, at 5 years, with 7.2-year median follow-up for OS in surviving patients. Median EFS was 8.3 years. Older age and transformed lymphomas were independently associated with inferior EFS, whereas day 60 lymphocyte counts did not predict EFS or late infections. Early and late transplant-related mortality was 1% and 8%, with secondary leukemia in two patients. B-cell counts recovered by 1–2 years; however, the median IgG level remained low at 2 years. Late-onset idiopathic neutropenia, generally inconsequential, was noted in 43%.
Conclusion: ASCT with rituximab can produce durable remissions on follow-up out to 10 years. Major infections do not appear to be significantly increased or to be predicted by immune monitoring.
doi:10.1093/annonc/mdp484
PMCID: PMC2875548  PMID: 19880437
immune reconstitution; lymphoma; rituximab; transplantation
13.  The receptor SIGIRR suppresses Th17 cell proliferation via inhibition of the interleukin-1 receptor pathway and mTOR kinase activation 
Immunity  2010;32(1):54-66.
Interleukin-1 (IL-1)-mediated signaling in T cells is essential for T helper 17 (Th17) cell differentiation. We showed here that SIGIRR, a negative regulator of IL-1 receptor and Toll-like receptor signaling, was induced during Th17 cell lineage commitment and governed Th17 cell differentiation and expansion through its inhibitory effects on IL-1 signaling. The absence of SIGIRR in T cells resulted in increased Th17 cell polarization in vivo upon myelin oligodendrocyte glycoprotein (MOG35–55) peptide immunization. Recombinant IL-1 promoted a marked increase in the proliferation of SIGIRR-deficient T cells under an in vitro Th17 cell-polarization condition. Importantly, we detected increased IL-1-induced phosphorylation of JNK and mTOR kinase in SIGIRR-deficient Th17 cells compared to wild-type Th17 cells. IL-1-induced proliferation was abolished in mTOR-deficient Th17 cells, indicating the essential role of mTOR activation. Our results demonstrate an important mechanism by which SIGIRR controls Th17 cell expansion and effector function through the IL-1-induced mTOR signaling pathway.
doi:10.1016/j.immuni.2009.12.003
PMCID: PMC3015141  PMID: 20060329
14.  Osteonectin/SPARC polymorphisms in Caucasian men with idiopathic osteoporosis 
Summary
Animal models suggest a role for osteonectin/SPARC in determination of bone mass. We found haplotypes consisting of three single nucleotide polymorphisms (SNPs) in the 3′ untranslated region (UTR) of the osteonectin gene are associated with bone density in Caucasian men with idiopathic osteoporosis.
Introduction
Osteonectin is a matricellular protein regulating matrix assembly, osteoblast differentiation, and survival. Animal studies indicate that osteonectin is essential for normal bone mass. The 3′ UTR is a regulatory region controlling mRNA stability, trafficking, and translation, and we determined whether osteonectin 3′ UTR haplotypes could be associated with bone mass and/or idiopathic osteoporosis.
Methods
Single strand conformation polymorphism and allele-specific PCR analysis were used to assess alleles at osteonectin cDNA bases 1046, 1599, and 1970, using genomic DNA from middle-aged Caucasian men with idiopathic, low turnover osteoporosis (n=56) and matched controls (n=59). Bone density was measured by DXA at spine, hip and radius. Allele and haplotype frequencies were analyzed by Chi square analysis and Fisher's exact test.
Results
Five common osteonectin 3′ UTR haplotypes were identified. The frequency of one haplotype (1046C-1599C-1970T) was higher in controls compared with patients, and this haplotype was also associated with higher bone densities at multiple sites in patients. In contrast, a second haplotype (1046C-1599G-1970T) was associated with lower bone densities in patients at multiple sites.
Conclusions
Osteonectin regulates skeletal remodeling and bone mass in animals, and haplotypes in the 3′ UTR of this gene are associated with bone density in Caucasian men with idiopathic osteoporosis.
doi:10.1007/s00198-007-0523-9
PMCID: PMC2888145  PMID: 18084690
3′ Untranslated region; Bone mineral density; Idiopathic osteoporosis; Non-collagen matrix protein; Osteonectin/SPARC; SNP/polymorphism
15.  Improved spiral tube assembly for high-speed counter-current chromatography 
Journal of chromatography. A  2008;1216(19):4193-4200.
The original spiral tube support (STS) assembly is improved by changing the shape of the tubing, with 1-cm presses perpendicularly along the length. This modification interrupts the laminar flow of the mobile phase. The tubing in the 4 return grooves to the center of the rotor is flattened by a specially made pressing tool to decrease the dead volume and thus increase the column efficiency. The performance of this spiral tube assembly was tested in separations of dipeptides and proteins with suitable polar two-phase solvent systems. The results revealed that the present system yields high partition efficiency with a satisfactory level of stationary phase retention in a short elution time. The present high-speed counter-current chromatographic system will be efficiently applied to a broad spectrum of two-phase solvent systems including aqueous-aqueous polymer phase systems which are used for separation of biopolymers such as proteins and nucleic acids. .
doi:10.1016/j.chroma.2008.10.126
PMCID: PMC2680281  PMID: 19062024
spiral tube support assembly; high-speed counter-current chromatography; Separation of proteins and peptides; polymer phase system (TPAS)
16.  Spiral Tube Assembly for High-Speed Countercurrent Chromatography 
Optimal elution modes were determined for four typical two-phase solvent systems each with different physical parameters to achieve the best peak resolution and retention of the stationary phase by spiral tube high-speed countercurrent chromatography using a suitable set of test samples. Both retention of the stationary phase and partition efficiency are governed by an interplay between two forces, i.e., Archimedean Screw force and radial centrifugal force gradient of the spiral channel. In the polar solvent system represented by 1-butanol./acetic acid/water (4:1:5, v/v/v) with settling time of over 30 s, the effect by the radial centrifugal gradient force dominates giving the best separation of dipeptides either by pumping the lower phase from the inner terminal or the upper phase from the outer terminal of the spiral channel. In the moderately hydrophobic two-phase solvent system represented by hexane/ethyl acetate/methanol/0.1 M HCl (1:1:1:1) with settling time of 19 s, and two hydrophobic solvent systems of hexane/ethanol/water (5:4:1, v/v/v) and non-aqueous binary system of hexane/acetonitrile both having settling time of 9, the effect of the Archimedean screw force play a major role in hydrodynamic equilibrium, giving the best separations by pumping the lower phase from the head or the upper phase from the tail of the spiral channel.
doi:10.1080/10826070802019913
PMCID: PMC2664165  PMID: 19343107
Spiral tube assembly; high-speed countercurrent chromatography; two-phase solvent systems; elution modes
19.  Delayed detection of cleft palate: an audit of newborn examination 
Archives of Disease in Childhood  2005;91(3):238-240.
Aims
To identify prevalence of delayed detection of cleft palate, and associated factors that could lead to improved identification at neonatal clinical examination.
Methods
Audit of hospital notes, parental questionnaire incorporating open ended questions, and telephone questionnaire of junior doctors in the referring hospitals incorporating fixed choice questions.
Results
Of 344 cleft palate patients without cleft lip or submucous cleft palate, the day the cleft was detected was recorded in 92%. Delayed detection, after the first day, was 28% overall, distributed as 37% with isolated cleft palate and 23% with syndromic cleft palate. Narrow V shaped clefts were more likely to be delayed in detection compared with broad U shaped clefts, as were soft palate clefts compared with hard palate clefts. Five with isolated cleft palates were not detected until after the first year. Babies born at home were unlikely to be detected on day 1. Symptoms were significantly increased in the delayed detection group for feeding problems and nasal regurgitation. A telephone questionnaire of trainee paediatricians in referring units revealed that digital examination was more commonly practised than visual inspection, and few recalled receiving specific instruction on examination of the palate.
Conclusion
Delayed detection of cleft palate was not uncommon, and the features of those more likely to be missed suggested digital examination was related. Trainee doctors and midwives should be instructed to inspect visually using a light and tongue depressor, then digitally if submucous cleft palate is suspected.
doi:10.1136/adc.2005.077958
PMCID: PMC2065953  PMID: 16352626
audit; cleft palate; examination; neonatal; palate
20.  Efficacious Recombinant Influenza Vaccines Produced by High Yield Bacterial Expression: A Solution to Global Pandemic and Seasonal Needs 
PLoS ONE  2008;3(5):e2257.
It is known that physical linkage of TLR ligands and vaccine antigens significantly enhances the immunopotency of the linked antigens. We have used this approach to generate novel influenza vaccines that fuse the globular head domain of the protective hemagglutinin (HA) antigen with the potent TLR5 ligand, flagellin. These fusion proteins are efficiently expressed in standard E. coli fermentation systems and the HA moiety can be faithfully refolded to take on the native conformation of the globular head. In mouse models of influenza infection, the vaccines elicit robust antibody responses that mitigate disease and protect mice from lethal challenge. These immunologically potent vaccines can be efficiently manufactured to support pandemic response, pre-pandemic and seasonal vaccines.
doi:10.1371/journal.pone.0002257
PMCID: PMC2373928  PMID: 18493310
21.  Small cell oesophageal carcinoma: an institutional experience and review of the literature 
British Journal of Cancer  2007;96(5):708-711.
Primary small cell oesophageal carcinoma (SCOC) is rare, prognosis is poor and there is no established optimum treatment strategy. It shares many clinicopathologic features with small cell carcinoma of the lung; therefore, a similar staging and treatment strategy was adopted. Sixteen cases referred to Velindre hospital between 1998 and 2005 were identified. Patients received platinum-based combination chemotherapy if appropriate. Those with limited disease (LD) received radical radiotherapy (RT) to all sites of disease on completion of chemotherapy. Median survival of all patients was 13.2 months. Median survival of patients with LD was significantly longer than those with extensive disease (24.4 vs 9.1 months, P=0.034). This is one of the largest single institution series in the world literature. Combined modality therapy using platinum-based combination chemotherapy and radical RT may allow a nonsurgical approach to management, avoiding the morbidity of oesophagectomy. Prophylactic cranial irradiation is controversial, and should be discussed on an individual basis.
doi:10.1038/sj.bjc.6603611
PMCID: PMC2360086  PMID: 17299393
small cell; oesophagus; chemotherapy; radiotherapy
22.  Chlamydia trachomatis prevalence in men in the mid-west of Ireland 
Sexually Transmitted Infections  2004;80(5):349-353.
Objectives: To estimate the prevalence of chlamydia infection in young men in the Mid-Western Health Board Region of Ireland, and to determine risk factors for its acquisition.
Methods: Consecutive men attending orthopaedic clinics (OPD), and a university sports arena (UL) were recruited to a chlamydia prevalence study. All men aged 17–35 who had been sexually active and had not passed urine in the last hour were eligible. Information about chlamydia was given, informed consent obtained, and a self administered questionnaire was completed. A first void urine (FVU) was collected and tested by ligase chain reaction (LCR).
Results: 82% (207/252) of men from OPD, and 60% (186/310) from UL participated. 6.3% (13/207) from the OPD and 5.4% (10/186) from UL tested LCR positive, giving an overall prevalence of 5.9% (23/393). Proved risk factors for chlamydial positivity were: (1) more than one sexual partner in previous 6 months, (2) more than eight lifetime sexual partners, (3) current symptoms (dysuria or discharge). No statistical significance was found for age, condom use, smoking, days since last sexual intercourse and previous GUM clinic attendance. No statistically significant difference to cost effective prevalence of 6% was shown.
Conclusions: A 5.9% prevalence of Chlamydia trachomatis was found which is cost effective to screen and treat. Non-invasive screening of men in the community was possible. Numbers of sexual partners and current symptoms were significant risk factors. Since only 25% of men in this laboratory were diagnosed with chlamydia outside the GUM clinic, compared with 59% of women, it is important that community screening of men is promoted.
doi:10.1136/sti.2003.008615
PMCID: PMC1744887  PMID: 15459401
23.  Psychosocial outcomes at 18 months after good neurological recovery from aneurysmal subarachnoid haemorrhage 
Objectives: To evaluate functioning, 18 months after surgery, of 49 patients with good neurological recovery following aneurysmal subarachnoid haemorrhage (SAH), and to determine the extent of any improvements in disturbances of mood, cognitive functioning, and levels of activity and participation previously observed at 9 month follow up.
Methods: SAH patients, matched for age, gender, and occupation with healthy control participants, completely quantitative measures of mood (HADS, FIES, BDI) and activity/participation (BICRO-39 scales), and a brief cognitive assessment battery (verbal fluency, digit span, prose recall). Controls completed the HADS and the BICRO-39.
Results: Patients showed some recovery of cognitive functioning, though impairments of prose recall persisted. Anxiety and depression symptoms were higher in patients than in controls, but fewer than 20% scored in the clinical range on any questionnaires except for RIES-Intrusive thoughts (22%); only three showed signs of full blown post-traumatic stress disorder. Almost half showed elevated dependence on others for domestic activities and organisation and abnormally low levels of employment. Very little variance in outcome was predicted by demographic variables, neurological or cognitive impairment, prior life stress, or mood. However, levels of social activity and self-organisation were related to persisting fatigue.
Conclusions: The observed decline in intrusive thoughts and avoidance over time is consistent with that seen after life threatening illness or trauma. The persistent reductions in independence and levels of employment may in some cases reflect considered lifestyle adjustments rather than adverse and unwanted changes but in others indicate a need for focused rehabilitation.
doi:10.1136/jnnp.2002.000414
PMCID: PMC1739192  PMID: 15258212
24.  Impact of pharmacometrics on drug approval and labeling decisions: A survey of 42 new drug applications 
The AAPS Journal  2005;7(3):E503-E512.
The value of quantitative thinking in drug development and regulatory review is increasingly being appreciated. Modeling and simulation of data pertaining to pharmacokinetic, pharmacodynamic, and disease progression is often referred to as the pharmacometrics analyses. The objective of the current report is to assess the role of pharmacometrics at the US Food and Drug Administration (FDA) in making drug approval and labeling decisions. The New Drug Applications (NDAs) submitted between 2000 and 2004 to the Cardio-renal, Oncology, and Neuropharmacology drug products divisions were surveyed. For those NDA reviews that included a pharmacometrics consultation, the clinical pharmacology scientists ranked the impact on the regulatory decision(s). Of about a total of 244 NDAs, 42 included a pharmacometrics component. Review of NDAs involved independent, quantitative evaluation by FDA pharmacometricians, even when such analysis was not conducted by the sponsor. Pharmacometric analyses were pivotal in regulatory decision making in more than half of the 42 NDAs. Of the 14 reviews that were pivotal to approval related decisions, 5 identified the need for additional trials, whereas 6 reduced the burden of conducting additional trials. Collaboration among the FDA clinical pharmacology, medical, and statistical reviewers and effective communication with the sponsors was critical for the impact to occur. The survey and the case studies emphasize the need for early interaction between the FDA and sponsors to plan the development more efficiently by appreciating the regulatory expectations better.
doi:10.1208/aapsj070351
PMCID: PMC2751253  PMID: 16353928
regulatory decisions; modeling; simulation; FDA; dose-response

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