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1.  Phylogenetic placement of metagenomic reads using the minimum evolution principle 
BMC Genomics  2015;16(Suppl 1):S13.
A central problem of computational metagenomics is determining the correct placement into an existing phylogenetic tree of individual reads (nucleotide sequences of varying lengths, ranging from hundreds to thousands of bases) obtained using next-generation sequencing of DNA samples from a mixture of known and unknown species. Correct placement allows us to easily identify or classify the sequences in the sample as to taxonomic position or function.
Here we propose a novel method (PhyClass), based on the Minimum Evolution (ME) phylogenetic inference criterion, for determining the appropriate phylogenetic position of each read. Without using heuristics, the new approach efficiently finds the optimal placement of the unknown read in a reference phylogenetic tree given a sequence alignment for the taxa in the tree. In short, the total resulting branch length for the tree is computed for every possible placement of the unknown read and the placement that gives the smallest value for this total is the best (optimal) choice. By taking advantage of computational efficiencies and mathematical formulations, we are able to find the true optimal ME placement for each read in the phylogenetic tree. Using computer simulations, we assessed the accuracy of the new approach for different read lengths over a variety of data sets and phylogenetic trees. We found the accuracy of the new method to be good and comparable to existing Maximum Likelihood (ML) approaches.
In particular, we found that the consensus assignments based on ME and ML approaches are more correct than either method individually. This is true even when the statistical support for read assignments was low, which is inevitable given that individual reads are often short and come from only one gene.
PMCID: PMC4315155
2.  Accounting for eXentricities: Analysis of the X Chromosome in GWAS Reveals X-Linked Genes Implicated in Autoimmune Diseases 
PLoS ONE  2014;9(12):e113684.
Many complex human diseases are highly sexually dimorphic, suggesting a potential contribution of the X chromosome to disease risk. However, the X chromosome has been neglected or incorrectly analyzed in most genome-wide association studies (GWAS). We present tailored analytical methods and software that facilitate X-wide association studies (XWAS), which we further applied to reanalyze data from 16 GWAS of different autoimmune and related diseases (AID). We associated several X-linked genes with disease risk, among which (1) ARHGEF6 is associated with Crohn's disease and replicated in a study of ulcerative colitis, another inflammatory bowel disease (IBD). Indeed, ARHGEF6 interacts with a gastric bacterium that has been implicated in IBD. (2) CENPI is associated with three different AID, which is compelling in light of known associations with AID of autosomal genes encoding centromere proteins, as well as established autosomal evidence of pleiotropy between autoimmune diseases. (3) We replicated a previous association of FOXP3, a transcription factor that regulates T-cell development and function, with vitiligo; and (4) we discovered that C1GALT1C1 exhibits sex-specific effect on disease risk in both IBDs. These and other X-linked genes that we associated with AID tend to be highly expressed in tissues related to immune response, participate in major immune pathways, and display differential gene expression between males and females. Combined, the results demonstrate the importance of the X chromosome in autoimmunity, reveal the potential of extensive XWAS, even based on existing data, and provide the tools and incentive to properly include the X chromosome in future studies.
PMCID: PMC4257614  PMID: 25479423
3.  The human intestinal microbiome at extreme ages of life. Dietary intervention as a way to counteract alterations 
Frontiers in Genetics  2014;5:406.
The intestinal microbiome is defined as the assembly of genomes from microorganisms inhabiting the gut. This microbial ecosystem regulates important functions of the host and its correct composition and functionality is essential for a “healthy status.” Metagenomic studies have highlighted variations of the intestinal microbiota as a function of age and diet. Colonization of the infant gut starts at birth and is influenced by feeding habits (formula vs. breast-feeding), birth mode and antibiotic exposure. The intestinal microbiota of full-term vaginally delivered breast-fed infants is considered the gold-standard, representing the reference for studies of alterations in other pediatric populations. At 2–3 years of age, the intestinal microbiota reaches a composition similar to adults, remaining without noticeable variations until senescence, when microbial instability and changes reappear. Here we summarize the current knowledge on intestinal microbiota alterations at extreme stages of life and tools for designing differentiated nutritional strategies by the use of probiotics, prebiotics and specific nutrients in order to restore a balanced microbiota and to improve immune and nutritional status.
PMCID: PMC4240173  PMID: 25484891
intestinal microbiome; elderly; newborns; dietary intervention; probiotics
4.  Gait Analysis in a Mecp2 Knockout Mouse Model of Rett Syndrome Reveals Early-Onset and Progressive Motor Deficits 
PLoS ONE  2014;9(11):e112889.
Rett syndrome (RTT) is a genetic disorder characterized by a range of features including cognitive impairment, gait abnormalities and a reduction in purposeful hand skills. Mice harbouring knockout mutations in the Mecp2 gene display many RTT-like characteristics and are central to efforts to find novel therapies for the disorder. As hand stereotypies and gait abnormalities constitute major diagnostic criteria in RTT, it is clear that motor and gait-related phenotypes will be of importance in assessing preclinical therapeutic outcomes. We therefore aimed to assess gait properties over the prodromal phase in a functional knockout mouse model of RTT. In male Mecp2 knockout mice, we observed alterations in stride, coordination and balance parameters at 4 weeks of age, before the onset of other overt phenotypic changes as revealed by observational scoring. These data suggest that gait measures may be used as a robust and early marker of MeCP2-dysfunction in future preclinical therapeutic studies.
PMCID: PMC4231076  PMID: 25392929
5.  Atypical Listeria innocua strains possess an intact LIPI-3 
BMC Microbiology  2014;14:58.
Listeria monocytogenes is a food-borne pathogen which is the causative agent of listeriosis and can be divided into three evolutionary lineages I, II and III. While all strains possess the well established virulence factors associated with the Listeria pathogenicity island I (LIPI-1), lineage I strains also possess an additional pathogenicity island designated LIPI-3 which encodes listeriolysin S (LLS), a post-translationally modified cytolytic peptide. Up until now, this pathogenicity island has been identified exclusively in a subset of lineage I isolates of the pathogen Listeria monocytogenes.
In total 64 L. innocua strains were screened for the presence of LIPI-3. Here we report the identification of an intact LIPI-3 in 11 isolates of L. innocua and the remnants of the cluster in several others. Significantly, we can reveal that placing the L. innocua lls genes under the control of a constitutive promoter results in a haemolytic phenotype, confirming that the cluster is capable of encoding a functional haemolysin.
Although the presence of the LIPI-3 gene cluster is confined to lineage I isolates of L. monocytogenes, a corresponding gene cluster or its remnants have been identified in many L. innocua strains.
PMCID: PMC3974016  PMID: 24606727
6.  High conjugated linoleic acid enriched ghee (clarified butter) increases the antioxidant and antiatherogenic potency in female Wistar rats 
Hypercholesterolemia and oxidative stress are the main stimulating factors responsible for coronary artery disease and progression of atherosclerosis. Dairy food products are rich in conjugated linoleic acid (CLA) which is considered as an important component due to its potential health benefits such as anticarcinogenic, antiatherogenic, antidiabetic and antiadipogenic properties. In the present study, the effect of CLA enriched ghee on the antioxidant enzyme system and antiatherogenic properties in Wistar rats has been studied.
Female Wistar rats of 21 days were taken for the study and fed with soybean diet (Control diet), low CLA diet and high CLA ghee diet (treatments) for thirty five days for studying antioxidative enzymes and sixteen weeks in case of antiatherogenic studies.
Feeding of high CLA enhanced ghee during pubescent period in rats lead to an increase in catalase (CAT) and superoxide dismutase (SOD) enzyme activities in blood and increased CAT, SOD and glutathione transferase (GST) enzymes activities in liver by 27, 130 and 168 percent, respectively. Plasma nitrate concentration and Haemoglobin levels remained the same in all the treatments. Feeding of high CLA ghee resulted in lower (P < 0.01) plasma cholesterol & triglyceride level (52.17 and 30.27%), and higher high density lipoproteins (33.26%) than feeding of soybean oil (control group) and thus manifested in decreased (P < 0.05) atherogenic index (from 0.472 to 0.244). Lesser cholesterol and triglyceride levels were observed in the liver and aorta of high CLA fed rats than in those of the other groups. Histopathological studies of liver showed normal hepatic cords with portal triad in the high CLA ghee fed rats whereas fatty degeneration of hepatocytes containing fat vacuoles was observed in the liver of the other groups.
This paper is the first report of the antioxidant and antiatherogenic properties of the high CLA enriched ghee suggesting that high CLA ghee can be used as a potential food for decreasing the risk of cardiovascular diseases, particularly in India, where, ghee is widely used for culinary and medicinal purposes.
PMCID: PMC3766171  PMID: 23923985
High conjugated linoleic acid enriched ghee; Antiatherogenic; Antioxidant; Catalase; Superoxide dismutase; Cholesterol
7.  Improved Survival and Reduced Phenotypic Severity Following AAV9/MECP2 Gene Transfer to Neonatal and Juvenile Male Mecp2 Knockout Mice 
Typical Rett syndrome (RTT) is a pediatric disorder caused by loss-of-function mutations in the MECP2 gene. The demonstrated reversibility of RTT-like phenotypes in mice suggests that MECP2 gene replacement is a potential therapeutic option in patients. We report improvements in survival and phenotypic severity in Mecp2-null male mice after neonatal intracranial delivery of a single-stranded (ss) AAV9/CBA-MECP2 vector. Median survival was 16.6 weeks for MECP2-treated versus 9.3 weeks for GFP-treated mice. ssAAV9/CBA-MECP2-treated mice also showed significant improvement in the phenotype severity score, in locomotor function and in exploratory activity, as well as a normalization of neuronal nuclear volume in transduced cells. Wild-type mice receiving neonatal injections of the same ssAAV9/CBA-MECP2 vector did not show any significant deficits, suggesting a tolerance for modest MeCP2 overexpression. To test a MECP2 gene replacement approach in a manner more relevant for human translation, a self-complementary AAV vector designed to drive MeCP2 expression from a fragment of the Mecp2 promoter was injected intravenously into juvenile (4-5 week-old) Mecp2-null mice. While the brain transduction efficiency in juvenile mice was low (~2-4% of neurons), modest improvements in survival were still observed. These results support the concept of MECP2 gene therapy for RTT.
PMCID: PMC3536818  PMID: 23011033
AAV; Gene Therapy; MeCP2; Rett Syndrome
8.  The gut microbiota and its relationship to diet and obesity 
Gut Microbes  2012;3(3):186-202.
Obesity develops from a prolonged imbalance of energy intake and energy expenditure. However, the relatively recent discovery that the composition and function of the gut microbiota impacts on obesity has lead to an explosion of interest in what is now a distinct research field. Here, research relating to the links between the gut microbiota, diet and obesity will be reviewed under five major headings: (1) the gut microbiota of lean and obese animals, (2) the composition of the gut microbiota of lean and obese humans, (3) the impact of diet on the gut microbiota, (4) manipulating the gut microbiota and (5) the mechanisms by which the gut microbiota can impact on weight gain.
PMCID: PMC3427212  PMID: 22572830
gut microbiota; intervention; prebiotic; probiotic; diet and obesity
9.  Fast and Slow Implementations of Relaxed-Clock Methods Show Similar Patterns of Accuracy in Estimating Divergence Times 
Molecular Biology and Evolution  2011;28(9):2439-2442.
Phylogenetic analyses are using increasingly larger data sets for estimating divergence times. With this increase in data sizes, the computation time required is becoming a bottleneck in evolutionary investigations. Our recent study of two relaxed-clock programs (BEAST and MultiDivTime [MDT]) showed their usefulness in time estimation; however, they place a significant computational time burden on biologists even for moderately small data sets. Here, we report speed and accuracy of another relaxed-clock program (MCMCTree, MC2T). We find it to be much faster than both MDT and BEAST while producing comparable time estimates. These results will encourage the analysis of larger data sets as well as the evaluation of the robustness of estimated times to changes in the model of evolutionary rates and clock calibrations.
PMCID: PMC3203548  PMID: 21498604
10.  Colorectal cancer treatment 
Clinical Evidence  2010;2010:0401.
Colorectal cancer is the third most common malignancy in the developed countries, and about a quarter of people present with intestinal obstruction or perforation. Risk factors for colorectal cancer are mainly dietary and genetic. Overall 5-year survival is about 50%, with half of people having surgery experiencing recurrence of the disease.
Methods and outcomes
We conducted a systematic review and aimed to answer the following clinical question: What are the effects of treatments for colorectal cancer? We searched: Medline, Embase, The Cochrane Library, and other important databases up to August 2008 (Clinical Evidence reviews are updated periodically, please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA).
We found 57 systematic reviews, RCTs, or observational studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions.
In this systematic review we present information relating to the effectiveness and safety of the following interventions: adjuvant systemic chemotherapy, preoperative radiotherapy, and routine intensive follow-up.
Key Points
Colorectal cancer is the third most common malignancy in the developed world, and about a fifth of people present with intestinal obstruction or perforation. Risk factors for colorectal cancer are mainly dietary and genetic.Overall 5-year survival is about 50%, with half of people having surgery experiencing recurrence of the disease.
Adjuvant systemic chemotherapy reduces mortality compared with surgery alone in people who have Dukes' C colorectal cancer. We don't know whether adjuvant systemic chemotherapy improves mortality compared with surgery alone in people with Dukes' B colorectal cancer. It has been suggested that people with high-risk Dukes' B may derive some benefit with adjuvant systemic chemotherapy compared with surgery alone. However, we found no direct evidence on this group.We found some evidence that oral fluoropyrimidines (with or without leucovorin) may be as effective as intravenous fluorouracil (with or without leucovorin) regimens at reducing mortality. The addition of oxaliplatin to fluorouracil plus leucovorin improves disease-free survival at 3 and 4 years compared with fluorouracil plus leucovorin alone in people with Dukes' B or C colon cancer.Adding irinotecan to fluorouracil plus leucovorin does not reduce mortality any more than fluorouracil plus leucovorin alone in people with Dukes' C colorectal cancer and increases toxic effects.
Preoperative radiotherapy may modestly reduce local tumour recurrence and mortality compared with surgery alone in people with rectal cancer. Preoperative radiotherapy may reduce local recurrence compared with postoperative radiotherapy. There may be no difference in overall survival between preoperative and postoperative radiotherapy.
Routine intensive follow-up may reduce the time to detection of recurrence and may increase survival compared with less-intensive follow-up in people with colorectal cancer.
PMCID: PMC2907599  PMID: 21718569
11.  The Safety of EXPAREL ® (Bupivacaine Liposome Injectable Suspension) Administered by Peripheral Nerve Block in Rabbits and Dogs 
Journal of Drug Delivery  2012;2012:962101.
A sustained-release DepoFoam injection formulation of bupivacaine (EXPAREL, 15 mg/mL) is currently being investigated for postsurgical analgesia via peripheral nerve block (PNB). Single-dose toxicology studies of EXPAREL (9, 18, and 30 mg/kg), bupivacaine solution (Bsol, 9 mg/kg), and saline injected around the brachial plexus nerve bundle were performed in rabbits and dogs. The endpoints included clinical pathology, pharmacokinetics, and histopathology evaluation on Day 3 and Day 15 (2/sex/group/period). EXPAREL resulted in a nearly 4-fold lower Cmax versus Bsol at the same dose. EXPAREL was well tolerated at doses up to 30 mg/kg. The only EXPAREL-related effect seen was minimal to mild granulomatous inflammation of adipose tissue around nerve roots (8 of 24 rabbits and 7 of 24 dogs) in the brachial plexus sites. The results indicate that EXPAREL was well tolerated in these models and did not produce nerve damage after PNB in rabbits and dogs.
PMCID: PMC3270427  PMID: 22363842
12.  Safety Evaluation of EXPAREL (DepoFoam Bupivacaine) Administered by Repeated Subcutaneous Injection in Rabbits and Dogs: Species Comparison 
Journal of Drug Delivery  2011;2011:467429.
EXPAREL (bupivacaine extended-release liposome injection), DepoFoam bupivacaine, is in development for prolonged postsurgical analgesia. Repeat-dose toxicity studies were conducted in rabbits and dogs to compare the potential local and systemic toxicities of EXPAREL and bupivacaine HCl (Bsol), and the reversibility of any effects. Dogs tolerated much larger doses than rabbits. EXPAREL-related minimal-to-moderate granulomatous inflammation was noted at the injection sites. In recovery animals, the granulomatous inflammation was observed less frequently and was characterized by an increased number of multinucleated giant cells. These effects were considered a normal response to liposomes and nonadverse. Rabbits are more sensitive than dogs. In rabbits, convulsions were noted with EXPAREL and more frequently with Bsol; a NOAEL was not identified. In dogs, EXPAREL was well tolerated (NOAEL > 30 mg/kg/dose). The cumulative exposure of EXPAREL in these studies is well in excess of the proposed maximum single-dose exposure that is intended in humans.
PMCID: PMC3189577  PMID: 22013534
13.  Carbohydrate catabolic flexibility in the mammalian intestinal commensal Lactobacillus ruminis revealed by fermentation studies aligned to genome annotations 
Microbial Cell Factories  2011;10(Suppl 1):S12.
Lactobacillus ruminis is a poorly characterized member of the Lactobacillus salivarius clade that is part of the intestinal microbiota of pigs, humans and other mammals. Its variable abundance in human and animals may be linked to historical changes over time and geographical differences in dietary intake of complex carbohydrates.
In this study, we investigated the ability of nine L. ruminis strains of human and bovine origin to utilize fifty carbohydrates including simple sugars, oligosaccharides, and prebiotic polysaccharides. The growth patterns were compared with metabolic pathways predicted by annotation of a high quality draft genome sequence of ATCC 25644 (human isolate) and the complete genome of ATCC 27782 (bovine isolate). All of the strains tested utilized prebiotics including fructooligosaccharides (FOS), soybean-oligosaccharides (SOS) and 1,3:1,4-β-D-gluco-oligosaccharides to varying degrees. Six strains isolated from humans utilized FOS-enriched inulin, as well as FOS. In contrast, three strains isolated from cows grew poorly in FOS-supplemented medium. In general, carbohydrate utilisation patterns were strain-dependent and also varied depending on the degree of polymerisation or complexity of structure. Six putative operons were identified in the genome of the human isolate ATCC 25644 for the transport and utilisation of the prebiotics FOS, galacto-oligosaccharides (GOS), SOS, and 1,3:1,4-β-D-Gluco-oligosaccharides. One of these comprised a novel FOS utilisation operon with predicted capacity to degrade chicory-derived FOS. However, only three of these operons were identified in the ATCC 27782 genome that might account for the utilisation of only SOS and 1,3:1,4-β-D-Gluco-oligosaccharides.
This study has provided definitive genome-based evidence to support the fermentation patterns of nine strains of Lactobacillus ruminis, and has linked it to gene distribution patterns in strains from different sources. Furthermore, the study has identified prebiotic carbohydrates with the potential to promote L. ruminis growth in vivo.
PMCID: PMC3231919  PMID: 21995520
14.  Recombinant bacteriophage lysins as antibacterials 
Bioengineered Bugs  2010;1(1):9-16.
With the increasing worldwide prevalence of antibiotic resistant bacteria, bacteriophage endolysins (lysins) represent a very promising novel alternative class of antibacterial in the fight against infectious disease. Lysins are phage-encoded peptidoglycan hydrolases which, when applied exogenously (as purified recombinant proteins) to Gram-positive bacteria, bring about rapid lysis and death of the bacterial cell. A number of studies have recently demonstrated the strong potential of these enzymes in human and veterinary medicine to control and treat pathogens on mucosal surfaces and in systemic infections. They also have potential in diagnostics and detection, bio-defence, elimination of food pathogens and control of phytopathogens. This review discusses the extensive research on recombinant bacteriophage lysins in the context of antibacterials, and looks forward to future development and potential.
PMCID: PMC3035150  PMID: 21327123
lysin; endolysin; bacteriophage; pathogen; antibacterial; infection; lytic; enzyme
15.  Late presentation of sorafenib-associated rash: a case report 
Sorafenib, an oral multitargeted tyrosine kinase inhibitor, is licensed for the treatment of hepatocellular carcinoma. Rash is one of the most common side effects of its use, generally appearing within days to a few weeks of commencing treatment. We report the first case of rash appearing nine months after starting treatment with sorafenib.
Case presentation
A 75-year-old Caucasian man initially presented with asymptomatic transient jaundice. He was diagnosed with Barcelona Clinic Liver Cancer stage B hepatocellular carcinoma after extensive investigation. He tolerated sorafenib 400 mg twice a day before presenting nine months later with a rash, confirmed to be drug-induced.
Sorafenib is a drug of choice in Barcelona Clinic Liver Cancer stage B hepatocellular carcinoma. It can cause protracted rash quite late into treatment. Successful management of the rash could contribute to achieving stable disease in hepatocellular carcinoma over a significant period of time.
PMCID: PMC2974748  PMID: 20973944
16.  Heterologous expression of linoleic acid isomerase from Propionibacterium acnes and anti-proliferative activity of recombinant trans-10, cis-12 conjugated linoleic acid 
Microbiology  2007;153(Pt 8):2483-2490.
The linoleic acid isomerase enzyme from Propionibacterium acnes responsible for bioconversion of linoleic acid to trans-10, cis-12 conjugated linoleic acid (t10, c12 CLA) was cloned and overexpressed in Lactococcus lactis and Escherichia coli, resulting in between 30 and 50 % conversion rates of the substrate linoleic acid to t10, c12 CLA. The anti-proliferative activities of the fatty acids produced following isomerization of linoleic acid by L. lactis and E. coli were assessed using the human SW480 colon cancer cell line. Fatty acids generated from both L. lactis and E. coli contained a mixture of linoleic acid and t10, c12 CLA at a ratio of ∼1.35 : 1. Following 5 days of incubation of SW480 cells with 5–20 μg ml−1 (17.8–71.3 μM) of the t10, c12 CLA, there was a significant (P<0.001) reduction in growth of the SW480 cancer cells compared with the linoleic acid control. Cell viability after treatment with the highest concentration (20 μg ml−1) of the t10, c12 CLA was reduced to 7.9 % (L. lactis CLA) and 19.6 % (E. coli CLA), compared with 95.4 % (control linoleic acid) and 31.7 % (pure t10, c12 CLA). In conclusion, this is believed to represent the first report in which recombinant strains are capable of producing CLA with an anti-proliferative potential.
PMCID: PMC2885616  PMID: 17660413
17.  Dynamics of ATP-induced Calcium Signaling in Single Mouse Thymocytes  
The Journal of Cell Biology  1997;138(5):987-998.
Extracellular ATP (ATPo) elicits a robust change in the concentration of intracellular Ca2+ ([Ca2+]i) in fura-2–loaded mouse thymocytes. Most thymocytes (60%) exposed to ATPo exhibited a biphasic rise in [Ca2+]i; [Ca2+]i rose slowly at first to a mean value of 260 nM after 163 s and then increased rapidly to a peak level of 735 nM. In many cells, a declining plateau, which lasted for more than 10 min, followed the crest in [Ca2+]i. Experiments performed in the absence of extracellular [Ca2+]o abolished the rise in thymocyte [Ca2+]i, indicating that Ca2+ influx, rather than the release of stored Ca2+, is stimulated by ATPo. ATPo- mediated Ca2+ influx was potentiated as the [Mg2+]o was reduced, confirming that ATP4− is the active agonist form. In the absence of Mg2+o, 3′-O-(4-benzoyl)benzoyl-ATP (BzATP) proved to be the most effective agonist of those tested. The rank order of potency for adenine nucleotides was BzATP4−>ATP4−>MgATP2−>ADP3−, suggesting purinoreceptors of the P2X7/P2Z class mediate the ATPo response. Phenotyping experiments illustrate that both immature (CD4−CD8−, CD4+CD8+) and mature (CD4+CD8−, CD4−CD8+) thymocyte populations respond to ATP. Further separation of the double-positive population by size revealed that the ATPo-mediated [Ca2+]i response was much more pronounced in large (actively dividing) than in small (terminally differentiated) CD4+CD8+ thymocytes. We conclude that thymocytes vary in sensitivity to ATPo depending upon the degree of maturation and suggest that ATPo may be involved in processes that control cellular differentiation within the thymus.
PMCID: PMC2136769  PMID: 9281578

Results 1-17 (17)