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2.  Protein and small non-coding RNA-enriched extracellular vesicles are released by the pathogenic blood fluke Schistosoma mansoni 
Journal of Extracellular Vesicles  2015;4:10.3402/jev.v4.28665.
Background
Penetration of skin, migration through tissues and establishment of long-lived intravascular partners require Schistosoma parasites to successfully manipulate definitive host defences. While previous studies of larval schistosomula have postulated a function for excreted/secreted (E/S) products in initiating these host-modulatory events, the role of extracellular vesicles (EVs) has yet to be considered. Here, using preparatory ultracentrifugation as well as methodologies to globally analyse both proteins and small non-coding RNAs (sncRNAs), we conducted the first characterization of Schistosoma mansoni schistosomula EVs and their potential host-regulatory cargos.
Results
Transmission electron microscopy analysis of EVs isolated from schistosomula in vitro cultures revealed the presence of numerous, 30–100 nm sized exosome-like vesicles. Proteomic analysis of these vesicles revealed a core set of 109 proteins, including homologs to those previously found enriched in other eukaryotic EVs, as well as hypothetical proteins of high abundance and currently unknown function. Characterization of E/S sncRNAs found within and outside of schistosomula EVs additionally identified the presence of potential gene-regulatory miRNAs (35 known and 170 potentially novel miRNAs) and tRNA-derived small RNAs (tsRNAs; nineteen 5′ tsRNAs and fourteen 3′ tsRNAs).
Conclusions
The identification of S. mansoni EVs and the combinatorial protein/sncRNA characterization of their cargo signifies that an important new participant in the complex biology underpinning schistosome/host interactions has now been discovered. Further work defining the role of these schistosomula EVs and the function/stability of intra- and extra-vesicular sncRNA components presents tremendous opportunities for developing novel schistosomiasis diagnostics or interventions.
doi:10.3402/jev.v4.28665
PMCID: PMC4595467  PMID: 26443722
extracellular vesicles; small non-coding RNAs; miRNAs; tsRNAs; proteome; Schistosoma mansoni; platyhelminth
3.  Association Between Nighttime Discharge from the Intensive Care Unit and Hospital Mortality: A Multi-Center Retrospective Cohort Study 
Background
We aimed to determine the impact of nighttime discharge from the intensive care unit (ICU) to the ward on hospital mortality and readmission rates in consecutive critically ill patients admitted to five Canadian ICUs. We hypothesized that hospital mortality and readmission rates would be higher for patients discharged after hours compared with discharge during the day.
Methods
A multi-center retrospective cohort study was carried out at five hospitals in Edmonton, Canada, between July 2002 and December 2009. Nighttime discharge was defined as discharge from the ICU occurring between 07:00 pm and 07:59 am. Logistic regression analysis was used to explore the associations between nighttime discharge and outcomes.
Results
Of 19,622 patients discharged alive from the ICU, 3,505 (17.9 %) discharges occurred during nighttime. Nighttime discharge occurred more commonly among medical than surgical patients (19.9 % vs. 13.8 %, P < 0.001) and among those with more comorbid conditions, compared with daytime discharged patients. Crude hospital mortality (11.8 % versus 8.8 %, P < 0.001) was greater for nighttime discharged as compared to daytime discharged patients. In a multivariable analysis, after adjustment for comorbidities, diagnosis and source of admission, nighttime discharge remains associated with higher mortality (odds ratio [OR] 1.29; 95 % CI, 1.14 to 1.46, P < 0.001). This finding was robust in two sensitivity analyses examining discharges occurring between 00:00 am and 04:59 am (OR 1.28; 1.12–1.47; P < 0.001) and for those who died within 48 h of ICU discharge without readmission (OR 1.24; 1.07–1.42, P = 0.002). There was no difference in ICU readmission for nighttime compared with daytime discharges (7.4 % vs. 6.9 %, p = 0.26). However, rates were higher for nighttime discharges in community compared with tertiary hospitals (7.7 % vs. 5.7 %, P = 0.023).
Conclusions
In a large integrated health region, 1 in 5 ICU patients are discharged at nighttime, a factor with increasing occurrence during our study and shown to be independently associated with higher hospital mortality.
Electronic supplementary material
The online version of this article (doi:10.1186/s12913-015-1044-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12913-015-1044-4
PMCID: PMC4570509  PMID: 26369933
4.  Quantitative Analysis of MicroRNAs in Vaccinia virus Infection Reveals Diversity in Their Susceptibility to Modification and Suppression 
PLoS ONE  2015;10(7):e0131787.
Vaccinia virus (VACV) is a large cytoplasmic DNA virus that causes dramatic alterations to many cellular pathways including microRNA biogenesis. The virus encodes a poly(A) polymerase which was previously shown to add poly(A) tails to the 3’ end of cellular miRNAs, resulting in their degradation by 24 hours post infection (hpi). Here we used small RNA sequencing to quantify the impact of VACV infection on cellular miRNAs in human cells at both early (6 h) and late (24 h) times post infection. A detailed quantitative analysis of individual miRNAs revealed marked diversity in the extent of their modification and relative change in abundance during infection. Some miRNAs became highly modified (e.g. miR-29a-3p, miR-27b-3p) whereas others appeared resistant (e.g. miR-16-5p). Furthermore, miRNAs that were highly tailed at 6 hpi were not necessarily among the most reduced at 24 hpi. These results suggest that intrinsic features of human cellular miRNAs cause them to be differentially polyadenylated and altered in abundance during VACV infection. We also demonstrate that intermediate and late VACV gene expression are required for optimal repression of some miRNAs including miR-27-3p. Overall this work reveals complex and varied consequences of VACV infection on host miRNAs and identifies miRNAs which are largely resistant to VACV-induced polyadenylation and are therefore present at functional levels during the initial stages of infection and replication.
doi:10.1371/journal.pone.0131787
PMCID: PMC4498801  PMID: 26161560
5.  Differences in the Faecal Microbiome in Schistosoma haematobium Infected Children vs. Uninfected Children 
PLoS Neglected Tropical Diseases  2015;9(6):e0003861.
Background
Several infectious diseases and therapeutic interventions cause gut microbe dysbiosis and associated pathology. We characterised the gut microbiome of children exposed to the helminth Schistosoma haematobium pre- and post-treatment with the drug praziquantel (PZQ), with the aim to compare the gut microbiome structure (abundance and diversity) in schistosome infected vs. uninfected children.
Methods
Stool DNA from 139 children aged six months to 13 years old; with S. haematobium infection prevalence of 27.34% was extracted at baseline. 12 weeks following antihelminthic treatment with praziqunatel, stool DNA was collected from 62 of the 139 children. The 16S rRNA genes were sequenced from the baseline and post-treatment samples and the sequence data, clustered into operational taxonomic units (OTUs). The OTU data were analysed using multivariate analyses and paired T- test.
Results
Pre-treatment, the most abundant phyla were Bacteroidetes, followed by Firmicutes and Proteobacteria respectively. The relative abundance of taxa among bacterial classes showed limited variation by age group or sex and the bacterial communities had similar overall compositions. Although there were no overall differences in the microbiome structure across the whole age range, the abundance of 21 OTUs varied significantly with age (FDR<0.05). Some OTUs including Veillonella, Streptococcus, Bacteroides and Helicobacter were more abundant in children ≤ 1 year old compared to older children. Furthermore, the gut microbiome differed in schistosome infected vs. uninfected children with 27 OTU occurring in infected but not uninfected children, for 5 of these all Prevotella, the difference was statistically significant (p <0.05) with FDR <0.05. PZQ treatment did not alter the microbiome structure in infected or uninfected children from that observed at baseline.
Conclusions
There are significant differences in the gut microbiome structure of infected vs. uninfected children and the differences were refractory to PZQ treatment.
Author Summary
The role of the gut microbiome in host health is becoming clearer with better characterisation of the nutritional, biochemical and immunological function of the microbes. However, to date, there is a paucity of studies describing/ investigating the role of the gut microbiome in infection, pathology and acquired immunity in children exposed to helminths, in sub-Saharan Africa. We determined how the diversity and abundance of different gut bacteria taxonomic groups differ with age, sex and infection with the helminths parasites causing bilharzia/schistosomiasis in children aged 6 months -13 years. The parasites are controlled by treatment with the antihelminthic praziquantel (PZQ), which is associated with diarrhoea within the first 24 hours of treatment in some children. We therefore also investigated the effects of PZQ treatment on the gut microbiome of the children 12 weeks post-PZQ treatment. The composition of the gut microbiome changed with age within the 0–3 year age range and also differed significantly in abundance of the different taxonomic groups between infected and uninfected children prior to treatment with PZQ. However, the pre-treatment microbiome structure was not altered by antihelminthic treatment of infected or uninfected children. Further studies are required to investigate causality and mechanisms of interaction.
doi:10.1371/journal.pntd.0003861
PMCID: PMC4482744  PMID: 26114287
6.  A dominant role for the methyl-CpG-binding protein Mbd2 in controlling Th2 induction by dendritic cells 
Nature communications  2015;6:6920.
Dendritic cells (DCs) direct CD4+ T cell differentiation into diverse helper (Th) subsets that are required for protection against varied infections. However, the mechanisms used by DCs to promote Th2 responses, which are important both for immunity to helminth infection and in allergic disease, are currently poorly understood. We demonstrate a key role for the protein methyl-CpG-binding domain-2 (Mbd2), which links DNA methylation to repressive chromatin structure, in regulating expression of a range of genes that are associated with optimal DC activation and function. In the absence of Mbd2, DCs display reduced phenotypic activation and a dramatically impaired capacity to initiate Th2 immunity against helminths or allergens. These data identify an epigenetic mechanism that is central to activation of CD4+ T cell responses by DCs, particularly in Th2 settings, and reveal methyl-CpG-binding proteins and the genes under their control as possible therapeutic targets for type-2 inflammation.
doi:10.1038/ncomms7920
PMCID: PMC4413429  PMID: 25908537
7.  A dominant role for the methyl-CpG-binding protein Mbd2 in controlling Th2 induction by dendritic cells 
Nature Communications  2015;6:6920.
Dendritic cells (DCs) direct CD4+ T-cell differentiation into diverse helper (Th) subsets that are required for protection against varied infections. However, the mechanisms used by DCs to promote Th2 responses, which are important both for immunity to helminth infection and in allergic disease, are currently poorly understood. We demonstrate a key role for the protein methyl-CpG-binding domain-2 (Mbd2), which links DNA methylation to repressive chromatin structure, in regulating expression of a range of genes that are associated with optimal DC activation and function. In the absence of Mbd2, DCs display reduced phenotypic activation and a markedly impaired capacity to initiate Th2 immunity against helminths or allergens. These data identify an epigenetic mechanism that is central to the activation of CD4+ T-cell responses by DCs, particularly in Th2 settings, and reveal methyl-CpG-binding proteins and the genes under their control as possible therapeutic targets for type-2 inflammation.
How anti-helminth and allergic immune responses are initiated is poorly understood. Here the authors show that to trigger these responses, dendritic cells specifically require methyl-CpG-binding domain-2, a protein promoting repressed chromatin state.
doi:10.1038/ncomms7920
PMCID: PMC4413429  PMID: 25908537
8.  Modulation of dendritic cell alternative activation and function by the vitamin A metabolite retinoic acid 
International Immunology  2015;27(11):589-596.
Retinoic acid modulates the functions of IL-4 in alternatively activated DCs
The archetypal Th2 cytokine IL-4 has previously been shown to alternatively activate murine macrophages and, more recently, dendritic cells (DCs) both in vitro and in vivo. IL-4 has also been shown to induce Aldh1a2 (aldehyde dehydrogenase 1a2) expression in murine macrophages recruited to the peritoneal cavity. However, the influence of IL-4 on DC Aldh1a2 induction in vivo has not yet been addressed. In this work, we found that DCs show enhanced aldehyde dehydrogenase enzyme activity in vivo, which led us to investigate the impact of the vitamin A metabolite all-trans retinoic acid (RA) on DC alternative activation and function. Antagonism of RA receptors reduced production of resistin-like molecule alpha by DCs responding to IL-4, while addition of exogenous RA enhanced production of this marker of alternative activation. Functionally, RA increased DC induction of CD4+ T-cell IL-10, while reducing CD4+ T-cell IL-4 and IL-13, revealing a previously unidentified role for RA in regulating the ability of alternatively activated DCs to influence Th2 polarization.
doi:10.1093/intimm/dxv020
PMCID: PMC4625886  PMID: 25899567
APC; IL-4; Th2
9.  NMD3 regulates both mRNA and rRNA nuclear export in African trypanosomes via an XPOI-linked pathway 
Nucleic Acids Research  2015;43(9):4491-4504.
Trypanosomes mostly regulate gene expression through post-transcriptional mechanisms, particularly mRNA stability. However, much mRNA degradation is cytoplasmic such that mRNA nuclear export must represent an important level of regulation. Ribosomal RNAs must also be exported from the nucleus and the trypanosome orthologue of NMD3 has been confirmed to be involved in rRNA processing and export, matching its function in other organisms. Surprisingly, we found that TbNMD3 depletion also generates mRNA accumulation of procyclin-associated genes (PAGs), these being co-transcribed by RNA polymerase I with the procyclin surface antigen genes expressed on trypanosome insect forms. By whole transcriptome RNA-seq analysis of TbNMD3-depleted cells we confirm the regulation of the PAG transcripts by TbNMD3 and using reporter constructs reveal that PAG1 regulation is mediated by its 5′UTR. Dissection of the mechanism of regulation demonstrates that it is not dependent upon translational inhibition mediated by TbNMD3 depletion nor enhanced transcription. However, depletion of the nuclear export factors XPO1 or MEX67 recapitulates the effects of TbNMD3 depletion on PAG mRNAs and mRNAs accumulated in the nucleus of TbNMD3-depleted cells. These results invoke a novel RNA regulatory mechanism involving the NMD3-dependent nuclear export of mRNA cargos, suggesting a shared platform for mRNA and rRNA export.
doi:10.1093/nar/gkv330
PMCID: PMC4482084  PMID: 25873624
10.  Clinical Outcomes and Microbiological Characteristics of Severe Pneumonia in Cancer Patients: A Prospective Cohort Study 
PLoS ONE  2015;10(3):e0120544.
Introduction
Pneumonia is the most frequent type of infection in cancer patients and a frequent cause of ICU admission. The primary aims of this study were to describe the clinical and microbiological characteristics and outcomes in critically ill cancer patients with severe pneumonia.
Methods
Prospective cohort study in 325 adult cancer patients admitted to three ICUs with severe pneumonia not acquired in the hospital setting. Demographic, clinical and microbiological data were collected.
Results
There were 229 (71%) patients with solid tumors and 96 (29%) patients with hematological malignancies. 75% of all patients were in septic shock and 81% needed invasive mechanical ventilation. ICU and hospital mortality rates were 45.8% and 64.9%. Microbiological confirmation was present in 169 (52%) with a predominance of Gram negative bacteria [99 (58.6%)]. The most frequent pathogens were methicillin-sensitive S. aureus [42 (24.9%)], P. aeruginosa [41(24.3%)] and S. pneumonia [21 (12.4%)]. A relatively low incidence of MR [23 (13.6%)] was observed. Adequate antibiotics were prescribed for most patients [136 (80.5%)]. In multivariate analysis, septic shock at ICU admission [OR 5.52 (1.92–15.84)], the use of invasive MV [OR 12.74 (3.60–45.07)] and poor Performance Status [OR 3.00 (1.07–8.42)] were associated with increased hospital mortality.
Conclusions
Severe pneumonia is associated with high mortality rates in cancer patients. A relatively low rate of MR pathogens is observed and severity of illness and organ dysfunction seems to be the best predictors of outcome in this population.
doi:10.1371/journal.pone.0120544
PMCID: PMC4372450  PMID: 25803690
11.  A Novel Gene Amplification Causes Upregulation of the PatAB ABC Transporter and Fluoroquinolone Resistance in Streptococcus pneumoniae 
Overexpression of the ABC transporter genes patA and patB confers efflux-mediated fluoroquinolone resistance in Streptococcus pneumoniae and is also linked to pneumococcal stress responses. Although upregulation of patAB has been observed in many laboratory mutants and clinical isolates, the regulatory mechanisms controlling expression of these genes are unknown. In this study, we aimed to identify the cause of high-level constitutive overexpression of patAB in M184, a multidrug-resistant mutant of S. pneumoniae R6. Using a whole-genome transformation and sequencing approach, we identified a novel duplication of a 9.2-kb region of the M184 genome which included the patAB genes. This duplication did not affect growth and was semistable with a low segregation rate. The expression levels of patAB in M184 were much higher than those that could be fully explained by doubling of the gene dosage alone, and inactivation of the first copy of patA had no effect on multidrug resistance. Using a green fluorescent protein reporter system, increased patAB expression was ascribed to transcriptional read-through from a tRNA gene upstream of the second copy of patAB. This is the first report of a large genomic duplication causing antibiotic resistance in S. pneumoniae and also of a genomic duplication causing antibiotic resistance by a promoter switching mechanism.
doi:10.1128/AAC.04858-14
PMCID: PMC4432121  PMID: 25779578
12.  Extracellular Onchocerca-derived small RNAs in host nodules and blood 
Parasites & Vectors  2015;8:58.
Background
microRNAs (miRNAs), a class of short, non-coding RNA can be found in a highly stable, cell-free form in mammalian body fluids. Specific miRNAs are secreted by parasitic nematodes in exosomes and have been detected in the serum of murine and dog hosts infected with the filarial nematodes Litomosoides sigmodontis and Dirofilaria immitis, respectively. Here we identify extracellular, parasite-derived small RNAs associated with Onchocerca species infecting cattle and humans.
Methods
Small RNA libraries were prepared from total RNA extracted from the nodule fluid of cattle infected with Onchocerca ochengi as well as serum and plasma from humans infected with Onchocerca volvulus in Cameroon and Ghana. Parasite-derived miRNAs were identified based on the criteria that sequences unambiguously map to hairpin structures in Onchocerca genomes, do not align to the human genome and are not present in European control serum.
Results
A total of 62 mature miRNAs from 52 distinct pre-miRNA candidates were identified in nodule fluid from cattle infected with O. ochengi of which 59 are identical in the genome of the human parasite O. volvulus. Six of the extracellular miRNAs were also identified in sequencing analyses of serum and plasma from humans infected with O. volvulus. Based on sequencing analysis the abundance levels of the parasite miRNAs in serum or plasma range from 5 to 127 reads/per million total host miRNA reads identified, comparable to our previous analyses of Schistosoma mansoni and L. sigmodontis miRNAs in serum. All six of the O. volvulus miRNAs identified have orthologs in other filarial nematodes and four were identified in the serum of mice infected with L. sigmodontis.
Conclusions
We have identified parasite-derived miRNAs associated with onchocerciasis in cattle and humans. Our results confirm the conserved nature of RNA secretion by diverse nematodes. Additional species-specific small RNAs from O. volvulus may be present in serum based on the novel miRNA sequences identified in the nodule fluid. In our analyses comparison to European control serum illuminates the scope for false-positives, warranting caution in criteria that should be applied to identification of biomarkers of infection.
Electronic supplementary material
The online version of this article (doi:10.1186/s13071-015-0656-1) contains supplementary material, which is available to authorized users.
doi:10.1186/s13071-015-0656-1
PMCID: PMC4316651  PMID: 25623184
microRNAs; Extracellular RNA; Filarial nematode; Onchocerciasis; Host-pathogen
13.  Genome and Phylogenetic Analyses of Trypanosoma evansi Reveal Extensive Similarity to T. brucei and Multiple Independent Origins for Dyskinetoplasty 
Two key biological features distinguish Trypanosoma evansi from the T. brucei group: independence from the tsetse fly as obligatory vector, and independence from the need for functional mitochondrial DNA (kinetoplast or kDNA). In an effort to better understand the molecular causes and consequences of these differences, we sequenced the genome of an akinetoplastic T. evansi strain from China and compared it to the T. b. brucei reference strain. The annotated T. evansi genome shows extensive similarity to the reference, with 94.9% of the predicted T. b. brucei coding sequences (CDS) having an ortholog in T. evansi, and 94.6% of the non-repetitive orthologs having a nucleotide identity of 95% or greater. Interestingly, several procyclin-associated genes (PAGs) were disrupted or not found in this T. evansi strain, suggesting a selective loss of function in the absence of the insect life-cycle stage. Surprisingly, orthologous sequences were found in T. evansi for all 978 nuclear CDS predicted to represent the mitochondrial proteome in T. brucei, although a small number of these may have lost functionality. Consistent with previous results, the F1FO-ATP synthase γ subunit was found to have an A281 deletion, which is involved in generation of a mitochondrial membrane potential in the absence of kDNA. Candidates for CDS that are absent from the reference genome were identified in supplementary de novo assemblies of T. evansi reads. Phylogenetic analyses show that the sequenced strain belongs to a dominant group of clonal T. evansi strains with worldwide distribution that also includes isolates classified as T. equiperdum. At least three other types of T. evansi or T. equiperdum have emerged independently. Overall, the elucidation of the T. evansi genome sequence reveals extensive similarity of T. brucei and supports the contention that T. evansi should be classified as a subspecies of T. brucei.
Author Summary
The single-cell parasite Trypanosoma evansi is the disease-causing trypanosome with the widest geographical distribution. The disease, called surra, has significant economic impact primarily due to infections of cattle, horses, and camels. Morphologically the parasite is indistinguishable from bloodstream stage T. brucei, a parasite causing sleeping sickness in humans and the disease nagana in animals. T. brucei, however, is strictly bound to sub-Saharan Africa where its obligate vector, the tsetse fly, resides. The lack of a complete mitochondrial genome in T. evansi further distinguishes this parasite from T. brucei. Important questions regarding the biology of T. evansi include how it escaped from Africa, whether this has happened more than once, and how exactly it is related to T. brucei. To help answer these questions we have sequenced the T. evansi nuclear genome. Our phylogenetic analysis demonstrates that T. evansi, and the closely related horse parasite T. equiperdum, evolved more than once from T. brucei. We also demonstrate extensive similarity to T. brucei, including the maintenance of numerous genes that T. evansi no longer requires. Therefore, despite the significant functional and pathological differences between T. evansi and T. brucei, our analysis supports the notion that T. evansi is not an independent species.
doi:10.1371/journal.pntd.0003404
PMCID: PMC4288722  PMID: 25568942
14.  A novel fMRI paradigm suggests that pedaling-related brain activation is altered after stroke 
The purpose of this study was to examine the feasibility of using functional magnetic resonance imaging (fMRI) to measure pedaling-related brain activation in individuals with stroke and age-matched controls. We also sought to identify stroke-related changes in brain activation associated with pedaling. Fourteen stroke and 12 control subjects were asked to pedal a custom, MRI-compatible device during fMRI. Subjects also performed lower limb tapping to localize brain regions involved in lower limb movement. All stroke and control subjects were able to pedal while positioned for fMRI. Two control subjects were withdrawn due to claustrophobia, and one control data set was excluded from analysis due to an incidental finding. In the stroke group, one subject was unable to enter the gantry due to excess adiposity, and one stroke data set was excluded from analysis due to excessive head motion. Consequently, 81% of subjects (12/14 stroke, 9/12 control) completed all procedures and provided valid pedaling-related fMRI data. In these subjects, head motion was ≤3 mm. In both groups, brain activation localized to the medial aspect of M1, S1, and Brodmann’s area 6 (BA6) and to the cerebellum (vermis, lobules IV, V, VIII). The location of brain activation was consistent with leg areas. Pedaling-related brain activation was apparent on both sides of the brain, with values for laterality index (LI) of –0.06 (0.20) in the stroke cortex, 0.05 (±0.06) in the control cortex, 0.29 (0.33) in the stroke cerebellum, and 0.04 (0.15) in the control cerebellum. In the stroke group, activation in the cerebellum – but not cortex – was significantly lateralized toward the damaged side of the brain (p = 0.01). The volume of pedaling-related brain activation was smaller in stroke as compared to control subjects. Differences reached statistical significance when all active regions were examined together [p = 0.03; 27,694 (9,608) μL stroke; 37,819 (9,169) μL control]. When individual regions were examined separately, reduced brain activation volume reached statistical significance in BA6 [p = 0.04; 4,350 (2,347) μL stroke; 6,938 (3,134) μL control] and cerebellum [p = 0.001; 4,591 (1,757) μL stroke; 8,381 (2,835) μL control]. Regardless of whether activated regions were examined together or separately, there were no significant between-group differences in brain activation intensity [p = 0.17; 1.30 (0.25)% stroke; 1.16 (0.20)% control]. Reduced volume in the stroke group was not observed during lower limb tapping and could not be fully attributed to differences in head motion or movement rate. There was a tendency for pedaling-related brain activation volume to increase with increasing work performed by the paretic limb during pedaling (p = 0.08, r = 0.525). Hence, the results of this study provide two original and important contributions. First, we demonstrated that pedaling can be used with fMRI to examine brain activation associated with lower limb movement in people with stroke. Unlike previous lower limb movements examined with fMRI, pedaling involves continuous, reciprocal, multijoint movement of both limbs. In this respect, pedaling has many characteristics of functional lower limb movements, such as walking. Thus, the importance of our contribution lies in the establishment of a novel paradigm that can be used to understand how the brain adapts to stroke to produce functional lower limb movements. Second, preliminary observations suggest that brain activation volume is reduced during pedaling post-stroke. Reduced brain activation volume may be due to anatomic, physiology, and/or behavioral differences between groups, but methodological issues cannot be excluded. Importantly, brain action volume post-stroke was both task-dependent and mutable, which suggests that it could be modified through rehabilitation. Future work will explore these possibilities.
doi:10.3389/fnhum.2015.00324
PMCID: PMC4454878  PMID: 26089789
rehabilitation; hemiparesis; hemiplegia; locomotion; lower extremity; fMRI; imaging; plasticity
15.  Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity 
Nature Communications  2014;5:5488.
In mammalian systems RNA can move between cells via vesicles. Here we demonstrate that the gastrointestinal nematode Heligmosomoides polygyrus, which infects mice, secretes vesicles containing microRNAs (miRNAs) and Y RNAs as well as a nematode Argonaute protein. These vesicles are of intestinal origin and are enriched for homologues of mammalian exosome proteins. Administration of the nematode exosomes to mice suppresses Type 2 innate responses and eosinophilia induced by the allergen Alternaria. Microarray analysis of mouse cells incubated with nematode exosomes in vitro identifies Il33r and Dusp1 as suppressed genes, and Dusp1 can be repressed by nematode miRNAs based on a reporter assay. We further identify miRNAs from the filarial nematode Litomosoides sigmodontis in the serum of infected mice, suggesting that miRNA secretion into host tissues is conserved among parasitic nematodes. These results reveal exosomes as another mechanism by which helminths manipulate their hosts and provide a mechanistic framework for RNA transfer between animal species.
Mammalian cell-derived exosomes can carry RNA and proteins from cell to cell, but this mode of transport has not been shown in nematodes. Here the authors show that a gastrointestinal parasite secretes exosomes that transfer microRNAs to mammalian cells and regulate innate immunity.
doi:10.1038/ncomms6488
PMCID: PMC4263141  PMID: 25421927
16.  Strain Classification of Mycobacterium tuberculosis Isolates in Brazil Based on Genotypes Obtained by Spoligotyping, Mycobacterial Interspersed Repetitive Unit Typing and the Presence of Large Sequence and Single Nucleotide Polymorphism 
PLoS ONE  2014;9(10):e107747.
Rio de Janeiro is endemic for tuberculosis (TB) and presents the second largest prevalence of the disease in Brazil. Here, we present the bacterial population structure of 218 isolates of Mycobacterium tuberculosis, derived from 186 patients that were diagnosed between January 2008 and December 2009. Genotypes were generated by means of spoligotyping, 24 MIRU-VNTR typing and presence of fbpC103, RDRio and RD174. The results confirmed earlier data that predominant genotypes in Rio de Janeiro are those of the Euro American Lineages (99%). However, we observed differences between the classification by spoligotyping when comparing to that of 24 MIRU-VNTR typing, being respectively 43.6% vs. 62.4% of LAM, 34.9% vs. 9.6% of T and 18.3% vs. 21.5% of Haarlem. Among isolates classified as LAM by MIRU typing, 28.0% did not present the characteristic spoligotype profile with absence of spacers 21 to 24 and 32 to 36 and we designated these conveniently as “LAM-like”, 79.3% of these presenting the LAM-specific SNP fbpC103. The frequency of RDRio and RD174 in the LAM strains, as defined both by spoligotyping and 24 MIRU-VNTR loci, were respectively 11% and 15.4%, demonstrating that RD174 is not always a marker for LAM/RDRio strains. We conclude that, although spoligotyping alone is a tool for classification of strains of the Euro-American lineage, when combined with MIRU-VNTRs, SNPs and RD typing, it leads to a much better understanding of the bacterial population structure and phylogenetic relationships among strains of M. tuberculosis in regions with high incidence of TB.
doi:10.1371/journal.pone.0107747
PMCID: PMC4196770  PMID: 25314118
18.  Changes in hemodynamic responses in chronic stroke survivors do not affect fMRI signal detection in a block experimental design 
Magnetic resonance imaging  2013;31(7):10.1016/j.mri.2013.02.009.
The use of canonical functions to model BOLD-fMRI data in people post-stroke may lead to inaccurate descriptions of task-related brain activity. The purpose of this study was to determine whether the spatiotemporal profile of hemodynamic responses (HDRs) obtained from stroke survivors during an event-related experiment could be used to develop individualized HDR functions that would enhance BOLD-fMRI signal detection in block experiments. Our long term goal was to use this information to develop individualized HDR functions for stroke survivors that could be used to analyze brain activity associated with locomotor-like movements. We also aimed to examine the reproducibility of HDRs obtained across two scan sessions in order to determine whether data from a single event-related session could be used to analyze block data obtained in subsequent sessions. Results indicate that the spatiotemporal profile of HDRs measured with BOLD-fMRI in stroke survivors was not the same as that observed in individuals without stroke. We observed small between-group differences in the rates of rise and decline of HDRs that were more apparent in individuals with cortical as compared to subcortical stroke. There were no differences in the peak or time to peak of HDRs in people with and without stroke. Of interest, differences in HDRs were not as substantial as expected from previous reports and were not large enough to necessitate the use of individualized HDR functions to obtain valid measures of movement-related brain activity. We conclude that all strokes do not affect the spatiotemporal characteristics of HDRs in such a way as to produce inaccurate representations of brain activity as measured by BOLD-fMRI. However, care should be taken to identify individuals whose BOLD-fMRI data may not provide an accurate representation of underlying brain activation when canonical models are used. Examination of HDRs need not be done for each scan session, as our data suggest that the characteristics of HDRs in stroke survivors are reproducible across days.
doi:10.1016/j.mri.2013.02.009
PMCID: PMC3822766  PMID: 23642802
Stroke; CVA; fMRI; Hemodynamic response function; Locomotion; Methods
19.  Identification of a human neonatal immune-metabolic network associated with bacterial infection 
Nature Communications  2014;5:4649.
Understanding how human neonates respond to infection remains incomplete. Here, a system-level investigation of neonatal systemic responses to infection shows a surprisingly strong but unbalanced homeostatic immune response; developing an elevated set-point of myeloid regulatory signalling and sugar-lipid metabolism with concomitant inhibition of lymphoid responses. Innate immune-negative feedback opposes innate immune activation while suppression of T-cell co-stimulation is coincident with selective upregulation of CD85 co-inhibitory pathways. By deriving modules of co-expressed RNAs, we identify a limited set of networks associated with bacterial infection that exhibit high levels of inter-patient variability. Whereas, by integrating immune and metabolic pathways, we infer a patient-invariant 52-gene-classifier that predicts bacterial infection with high accuracy using a new independent patient population. This is further shown to have predictive value in identifying infection in suspected cases with blood culture-negative tests. Our results lay the foundation for future translation of host pathways in advancing diagnostic, prognostic and therapeutic strategies for neonatal sepsis.
Infection remains a leading cause of morbidity and mortality in neonates worldwide. Here the authors report disproportionate immune stimulatory, co-inhibitory and metabolic pathway responses that specifically mark bacterial infection and can be used to predict sepsis in neonatal patients at the first clinical signs of infection.
doi:10.1038/ncomms5649
PMCID: PMC4143936  PMID: 25120092
20.  A draft genome for the African crocodilian trypanosome Trypanosoma grayi  
Scientific Data  2014;1:140024.
The availability of genome sequence data has greatly enhanced our understanding of the adaptations of trypanosomatid parasites to their respective host environments. However, these studies remain somewhat restricted by modest taxon sampling, generally due to focus on the most important pathogens of humans. To address this problem, at least in part, we are releasing a draft genome sequence for the African crocodilian trypanosome, Trypanosoma grayi ANR4. This dataset comprises genomic DNA sequences assembled de novo into contigs, encompassing over 10,000 annotated putative open reading frames and predicted protein products. Using phylogenomic approaches we demonstrate that T. grayi is more closely related to Trypanosoma cruzi than it is to the African trypanosomes T. brucei, T. congolense and T. vivax, despite the fact T. grayi and the African trypanosomes are each transmitted by tsetse flies. The data are deposited in publicly accessible repositories where we hope they will prove useful to the community in evolutionary studies of the trypanosomatids.
doi:10.1038/sdata.2014.24
PMCID: PMC4322581  PMID: 25977781
21.  Genome wide dissection of the quorum sensing signaling pathway in Trypanosoma brucei 
Nature  2013;505(7485):681-685.
The protozoan parasites Trypanosoma brucei spp. cause important human and livestock diseases in sub Saharan Africa. In the mammalian blood, two developmental forms of the parasite exist: proliferative ‘slender’ forms and arrested ‘stumpy’ forms that are responsible for transmission to tsetse flies. The slender to stumpy differentiation is a density-dependent response that resembles quorum sensing (QS) in microbial systems and is crucial for the parasite life cycle, ensuring both infection chronicity and disease transmission1. This response is triggered by an elusive ‘stumpy induction factor’ (SIF) whose intracellular signaling pathway is also uncharacterized. Laboratory-adapted (monomorphic) trypanosome strains respond inefficiently to SIF but can generate forms with stumpy characteristics when exposed to cell permeable cAMP and AMP analogues. Exploiting this, we have used a genome-wide RNAi library screen to identify the signaling components driving stumpy formation. In separate screens, monomorphic parasites were exposed to 8-(4-chlorophenylthio)-cAMP (pCPTcAMP) or 8-pCPT-2′-O-Me-5′-AMP to select cells that were unresponsive to these signals and hence remained proliferative. Genome-wide ion torrent-based RNA interference Target sequencing identified cohorts of genes implicated in each step of the signaling pathway, from purine metabolism, through signal transducers (kinases, phosphatases) to gene expression regulators. Genes at each step were independently validated in cells naturally capable of stumpy formation, confirming their role in density sensing in vivo, whilst the putative RNA-binding protein, RBP7, was required for normal QS and promoted cell-cycle arrest and transmission competence when overexpressed. This study reveals that QS signaling in trypanosomes shares similarities to fundamental quiescence pathways in eukaryotic cells, its components providing targets for QS-interference based therapeutics.
doi:10.1038/nature12864
PMCID: PMC3908871  PMID: 24336212
22.  Low frequency depression of H-reflexes in humans with acute and chronic spinal-cord injury 
Experimental brain research  2000;133(2):233-241.
We measured low-frequency depression of soleus H-reflexes in individuals with acute (n=5) and chronic (n=7) spinal-cord injury and in able-bodied controls (n=7). In one acute subject, we monitored longitudinal changes in low-frequency depression of H-reflexes over 44 weeks and examined the relationship between H-reflex depression and soleus-muscle fatigue properties. Soleus H-reflexes were elicited at 0.1, 0.2, 1, 5, and 10 Hz. The mean peak-to-peak amplitude of ten reflexes at each frequency was calculated, and values obtained at each frequency were normalized to 0.1 Hz. H-reflex amplitude decreased with increasing stimulation frequency in all three groups, but H-reflex suppression was significantly larger in the able-bodied and acute groups than in the chronic group. The acute subject who was monitored longitudinally displayed reduced low-frequency depression with increasing time post injury. At 44 weeks post injury, the acute subject’s H-reflex depression was similar to that of chronic subjects, and his soleus fatigue index (assessed with a modified Burke fatigue protocol) dropped substantially, consistent with transformation to faster muscle. There was a significant inverse correlation over the 44 weeks between the fatigue index and the mean normalized H-reflex amplitude at 1, 5, and 10 Hz. We conclude that: (1) the chronically paralyzed soleus muscle displays impaired low-frequency depression of H-reflexes, (2) attenuation of rate-sensitive depression in humans with spinal-cord injury occurs gradually, and (3) changes in H-reflex excitability are generally correlated with adaptation of the neuromuscular system. Possible mechanisms underlying changes in low-frequency depression and their association with neuromuscular adaptation are discussed.
PMCID: PMC4034370  PMID: 10968224
Spasticity; Paralysis; Plasticity; Muscle; Fatigue
23.  Combinatorial Communication in Bacteria: Implications for the Origins of Linguistic Generativity 
PLoS ONE  2014;9(4):e95929.
Combinatorial communication, in which two signals are used together to achieve an effect that is different to the sum of the effects of the component parts, is apparently rare in nature: it is ubiquitous in human language, appears to exist in a simple form in some non-human primates, but has not been demonstrated in other species. This observed distribution has led to the pair of related suggestions, that (i) these differences in the complexity of observed communication systems reflect cognitive differences between species; and (ii) that the combinations we see in non-human primates may be evolutionary pre-cursors of human language. Here we replicate the landmark experiments on combinatorial communication in non-human primates, but in an entirely different species, unrelated to humans, and with no higher cognition: the bacterium Pseudomonas aeruginosa. Using the same general methods as the primate studies, we find the same general pattern of results: the effect of the combined signal differs from the composite effect of the two individual signals. This suggests that advanced cognitive abilities and large brains do not necessarily explain why some species have combinatorial communication systems and others do not. We thus argue that it is premature to conclude that the systems observed in non-human primates are evolutionarily related to language. Our results illustrate the value of an extremely broad approach to comparative research.
doi:10.1371/journal.pone.0095929
PMCID: PMC3997515  PMID: 24759740
24.  Lower Extremity Passive Range of Motion in Community-Ambulating Stroke Survivors 
Background
Physical therapists may prescribe stretching exercises for individuals with stroke to improve joint integrity and to reduce the risk of secondary musculoskeletal impairment. While deficits in passive range of motion (PROM) exist in stroke survivors with severe hemiparesis and spasticity, the extent to which impaired lower extremity PROM occurs in community-ambulating stroke survivors remains unclear. This study compared lower extremity PROM in able-bodied individuals and independent community-ambulatory stroke survivors with residual stroke-related neuromuscular impairments. Our hypothesis was that the stroke group would show decreased lower extremity PROM in the paretic but not the nonparetic side and that decreased PROM would be associated with increased muscle stiffness and decreased muscle length.
Methods
Individuals with chronic poststroke hemiparesis who reported the ability to ambulate independently in the community (n = 17) and age-matched control subjects (n = 15) participated. PROM during slow (5 degrees/sec) hip extension, hip flexion, and ankle dorsiflexion was examined bilaterally using a dynamometer that measured joint position and torque. The maximum angular position of the joint (ANGmax), torque required to achieve ANGmax (Tmax), and mean joint stiffness (K) were measured. Comparisons were made between able-bodied and paretic and able-bodied and nonparetic limbs.
Results
Contrary to our expectations, between-group differences in ANGmax were observed only during hip extension in which ANGmax was greater bilaterally in people post-stroke compared to control subjects (P ≤ 0.05; stroke = 13 degrees, able-bodied = −1 degree). Tmax, but not K, was also significantly higher during passive hip extension in paretic and nonparetic limbs compared to control limbs (P ≤ 0.05; stroke = 40 Nm, able-bodied = 29 Nm). Compared to the control group, Tmax was increased during hip flexion in the paretic and nonparetic limbs of post-stroke subjects (P ≤ 0.05, stroke = 25 Nm, able-bodied = 18 Nm). K in the nonparetic leg was also increased during hip flexion (P ≤ 0.05, nonparetic = 0.52 Nm/degree, able-bodied = 0.37 Nm/degree.)
Conclusion
This study demonstrates that community-ambulating stroke survivors with residual neuromuscular impairments do not have decreased lower extremity PROM caused by increased muscle stiffness or decreased muscle length. In fact, the population of stroke survivors examined here appears to have more hip extension PROM than age-matched able-bodied individuals. The clinical implications of these data are important and suggest that lower extremity PROM may not interfere with mobility in community-ambulating stroke survivors. Hence, physical therapists may choose to recommend activities other than stretching exercises for stroke survivors who are or will become independent community ambulators.
doi:10.1097/NPT.0b013e31816594ea
PMCID: PMC3963266  PMID: 18463552
cerebral vascular accident (CVA); hemiparesis; muscle; range of motion (ROM); spasticity
25.  High-Throughput Chemical Screening for Antivirulence Developmental Phenotypes in Trypanosoma brucei 
Eukaryotic Cell  2014;13(3):412-426.
In the bloodstream of mammalian hosts, the sleeping sickness parasite, Trypanosoma brucei, exists as a proliferative slender form or a nonproliferative, transmissible, stumpy form. The transition between these developmental forms is controlled by a density-dependent mechanism that is important for the parasite's infection dynamics, immune evasion via ordered antigenic variation, and disease transmissibility. However, stumpy formation has been lost in most laboratory-adapted trypanosome lines, generating monomorphic parasites that proliferate uncontrolled as slender forms in vitro and in vivo. Nonetheless, these forms are readily amenable to cell culture and high-throughput screening for trypanocidal lead compounds. Here, we have developed and exploited a high-throughput screen for developmental phenotypes using a transgenic monomorphic cell line expressing a reporter under the regulation of gene control signals from the stumpy-specific molecule PAD1. Using a whole-cell fluorescence-based assay to screen over 6,000 small molecules from a kinase-focused compound library, small molecules able to activate stumpy-specific gene expression and proliferation arrest were assayed in a rapid assay format. Independent follow-up validation identified one hit able to induce modest, yet specific, changes in mRNA expression indicative of a partial differentiation to stumpy forms in monomorphs. Further, in pleomorphs this compound induced a stumpy-like phenotype, entailing growth arrest, morphological changes, PAD1 expression, and enhanced differentiation to procyclic forms. This not only provides a potential tool compound for the further understanding of stumpy formation but also demonstrates the use of high-throughput screening in the identification of compounds able to induce specific phenotypes, such as differentiation, in African trypanosomes.
doi:10.1128/EC.00335-13
PMCID: PMC3957582  PMID: 24442893

Results 1-25 (135)