Gastrointestinal nematode parasites infect over 1 billion humans, with little evidence for generation of sterilising immunity. These helminths are highly adapted to their mammalian host, following a developmental program through successive niches, while effectively down-modulating host immune responsiveness. Larvae of Heligmosomoides polygyrus, for example, encyst in the intestinal submucosa, before emerging as adult worms into the duodenal lumen. Adults release immunomodulatory excretory-secretory (ES) products, but mice immunised with adult H. polygyrus ES become fully immune to challenge infection. ES products of the intestinal wall 4th stage (L4) larvae are similarly important in host-parasite interactions, as they readily generate sterile immunity against infection, while released material from the egg stage is ineffective. Proteomic analyses of L4 ES identifies protective antigen targets as well as potential tissue-phase immunomodulatory molecules, using as comparators the adult ES proteome and a profile of H. polygyrus egg-released material. While 135 proteins are shared between L4 and adult ES, 72 are L4 ES-specific; L4-specific proteins correspond to those whose transcription is restricted to larval stages, while shared proteins are generally transcribed by all life cycle forms. Two protein families are more heavily represented in the L4 secretome, the Sushi domain, associated with complement regulation, and the ShK/SXC domain related to a toxin interfering with T cell signalling. Both adult and L4 ES contain extensive but distinct arrays of Venom allergen/Ancylostoma secreted protein-Like (VAL) members, with acetylcholinesterases (ACEs) and apyrase APY-3 particularly abundant in L4 ES. Serum antibodies from mice vaccinated with L4 and adult ES react strongly to the VAL-1 protein and to ACE-1, indicating that these two antigens represent major vaccine targets for this intestinal nematode. We have thus defined an extensive and novel repertoire of H. polygyrus proteins closely implicated in immune modulation and protective immunity.
Intestinal helminth parasites are highly prevalent in humans and animals, and survive for long periods by deviating the host immune system. No vaccines are currently available to control these infections. Many helminths invade through barrier surfaces (such as the skin or the digestive tract) and develop through tissue larval stages before reaching their final niche such as the intestinal lumen. We studied the tissue larval stage of a mouse parasite, Heligmosomoides polygyrus, to test whether proteins released by this stage could elicit protective immunity, and found that they indeed constitute very effective vaccine antigens. Proteomic analysis to identify the individual proteins released by the larvae demonstrated that while many products are shared between tissue-dwelling larvae and adults occupying the intestinal lumen, larvae express higher levels of two gene families linked to immunomodulation, namely the Sushi protein family and the ShK toxin family. Antibody analysis of serum from vaccinated mice identified two major antigens recognised by the protective immune response as VAL-1 and ACE-1, which are respectively members of the venom allergen and acetylcholinesterase families. This work establishes that tissue larvae are the source of protective antigens for future vaccines, and highlights their production of two potentially immunomodulatory gene families.
Bacterial DNA is maintained in a supercoiled state controlled by the action of topoisomerases. Alterations in supercoiling affect fundamental cellular processes, including transcription. Here, we show that substitution at position 87 of GyrA of Salmonella influences sensitivity to antibiotics, including nonquinolone drugs, alters global supercoiling, and results in an altered transcriptome with increased expression of stress response pathways. Decreased susceptibility to multiple antibiotics seen with a GyrA Asp87Gly mutant was not a result of increased efflux activity or reduced reactive-oxygen production. These data show that a frequently observed and clinically relevant substitution within GyrA results in altered expression of numerous genes, including those important in bacterial survival of stress, suggesting that GyrA mutants may have a selective advantage under specific conditions. Our findings help contextualize the high rate of quinolone resistance in pathogenic strains of bacteria and may partly explain why such mutant strains are evolutionarily successful.
Fluoroquinolones are a powerful group of antibiotics that target bacterial enzymes involved in helping bacteria maintain the conformation of their chromosome. Mutations in the target enzymes allow bacteria to become resistant to these antibiotics, and fluoroquinolone resistance is common. We show here that these mutations also provide protection against a broad range of other antimicrobials by triggering a defensive stress response in the cell. This work suggests that fluoroquinolone resistance mutations may be beneficial under a range of conditions.
The gene expression of Trypanosoma brucei has been examined extensively in the blood of mammalian hosts and in forms found in the midgut of its arthropod vector, the tsetse fly. However, trypanosomes also undergo development within the mammalian bloodstream as they progress from morphologically ‘slender forms’ to transmissible ‘stumpy forms’ through morphological intermediates. This transition is temporally progressive within the first wave of parasitaemia such that gene expression can be monitored in relatively pure slender and stumpy populations as well as during the progression between these extremes. The development also represents the progression of cells from translationally active forms adapted for proliferation in the host to translationally quiescent forms, adapted for transmission. We have used metabolic labelling to quantitate translational activity in slender forms, stumpy forms and in forms undergoing early differentiation to procyclic forms in vitro. Thereafter we have examined the cohort of total mRNAs that are enriched throughout development in the mammalian bloodstream (slender, intermediate and stumpy forms), irrespective of strain, revealing those that exhibit consistent developmental regulation rather than sample specific changes. Transcripts that cosediment with polysomes in stumpy forms and slender forms have also been enriched to identify transcripts that escape translational repression prior to transmission. Combined, the expression and polysomal association of transcripts as trypanosomes undergo development in the mammalian bloodstream have been defined, providing a resource for trypanosome researchers. This facilitates the identification of those that undergo developmental regulation in the bloodstream and therefore those likely to have a role in the survival and capacity for transmission of stumpy forms.
The type of bacterial culture medium is an important consideration during design of any experimental protocol. The aim of this study was to understand the impact of medium choice on bacterial gene expression and physiology by comparing the transcriptome of Salmonella enterica SL1344 after growth in the widely used LB broth or the rationally designed MOPS minimal medium. Transcriptomics showed that after growth in MOPS minimal media, compared to LB, there was increased expression of 42 genes involved in amino acid synthesis and 23 genes coding for ABC transporters. Seven flagellar genes had decreased expression after growth in MOPS minimal medium and this correlated with a decreased motility. In both MOPS minimal medium and MEM expression of genes from SPI-2 was increased and the adhesion of S. Typhimurium to intestinal epithelial cells was higher compared to the levels after growth in LB. However, SL1344 invasion was not significantly altered by growth in either MOPs minimal media or MEM. Expression of SPI-2 was also measured using chromosomal GFP reporter fusions followed by flow cytometry which showed, for the first time, that the reduction in SPI-2 transcript after growth in different media related to a reduction in the proportion of the bacterial population expressing SPI-2. These data highlight the profound differences in the global transcriptome after in vitro growth in different media and show that choice of medium should be considered carefully during experimental design, particularly when virulence related phenotypes are being measured.
The genetics of sex determination remains mysterious in many organisms including some that are otherwise well-studied. Here we report the discovery and analysis of the mating-type locus of the model organism Dictyostelium discoideum. Three forms of a single genetic locus specifies this species’ three mating-types: two versions of the locus are entirely different in sequence, and the third resembles a composite of the other two. Single, unrelated genes are sufficient to determine two of the mating-types, while homologues of both these genes are required in the composite type. The key genes encode polypeptides that possess no recognisable similarity to established protein families. Sex determination in the social amoebae thus appears to use regulators unrelated to any currently known.
Cerebral hyperperfusion syndrome (CHS) may occur as a severe complication following surgical treatment of carotid stenosis. However, the mechanism inducing neurological symptoms in CHS remains unknown. We describe a patient with CHS presenting with seizures 24 h following carotid endarterectomy. Imaging demonstrated early ipsilateral blood–brain barrier (BBB) breakdown with electroencephalographic evidence of cortical dysfunction preceding brain edema. Using in vitro experiments on rat cortical tissue, we show that direct exposure of isolated brain slices to a serum-like medium induces spontaneous epileptiform activity, and that neuronal dysfunction is triggered by albumin. We propose BBB breakdown and subsequent albumin extravasation as a novel pathogenic mechanism underlying CHS and a potential target for therapy.
Albumin; Blood–brain barrier; Cerebral hyperperfusion syndrome; Magnetic resonance imaging; Seizure; Astrocytes; TGF-βR
Upper extremity (UE) hemiparesis persists after stroke, limiting hand function. Neuromuscular electrical stimulation (NMES) is an effective intervention to improve UE recovery, although the underlying mechanisms are not fully understood. Our objective was to establish a reliable protocol to measure UE agonist–antagonist forearm monosynaptic reflexes in a pilot study to determine if NMES improves wrist function after stroke. We established the between-day reliability of the H-reflex in the extensor carpi radialis longus (ECRL) and flexor carpi radialis (FCR) musculature for individuals with prior stroke (n = 18). The same-day generation of ECRL/FCR H-reflex recruitment curves was well tolerated, regardless of age or UE spasticity. The between-day reliability of the ECRL H-reflex was enhanced above FCR, similar to healthy subjects , with the Hmax the most reliable parameter quantified in both muscles. H-reflex and functional measures following NMES show the potential for NMES-induced increases in ECRL Hmax, but confirmation requires a larger clinical study. Our initial results support the safe, easy, and efficacious use of in-home NMES, and establish a potential method to measure UE monosynaptic reflexes after stroke.
Stroke; H-reflex; FCR; ECRL; Upper extremity spasticity; Neuromuscular electrical stimulation
Decreased reciprocal inhibition (RI) of motor neurons may contribute to spasticity after stroke. However, decreased RI is not a uniform observation among stroke survivors, suggesting that this spinal circuit may be influenced by other stroke-related characteristics. The purpose of this study was to measure RI post-stroke and to examine the relationship between RI and other features of stroke.
RI was examined in 15 stroke survivors (PAR) and 10 control subjects by quantifying the effect of peroneal nerve stimulation on soleus H-reflex amplitude. The relationship between RI and age, time post-stroke, lesion side, walking velocity, Fugl-Meyer, Ashworth, and Achilles reflex scores was examined.
RI was absent and replaced by reciprocal facilitation in 10 of 15 PAR individuals. Reciprocal facilitation was associated with low Fugl-Meyer scores and slow walking velocities but not with hyperactive Achilles tendon reflexes. There was no relationship between RI or reciprocal facilitation and time post-stroke, lesion side, or Ashworth score.
Decreased RI is not a uniform finding post-stroke and is more closely related to walking ability and movement impairment than to spasticity.
Phenomena other than decreased RI may contribute to post-stroke spasticity.
Spasticity; Rehabilitation; Hemiparesis; CVA
Mutualistic interactions are wide-spread but the mechanisms underlying their evolutionary stability and ecological dynamics remain poorly understood. Cultivation mutualisms in which hosts consume symbionts occur in phylogenetically diverse groups, but often have symbiont monocultures for each host. This is consistent with the prediction that symbionts should avoid coexistence with other strains so that host services continue to benefit relatives, but it is less clear whether hosts should always favor monocultures and what mechanisms they might have to manipulate symbiont diversity. Few mutualisms have been studied in sufficient genetic detail to address these issues, so we decided to characterize symbiont diversity in the complex mutualism between multiple root aphid species and Lasius flavus ants. After showing elsewhere that three of these aphid species have low dispersal and mostly if not exclusively asexual reproduction, we here investigate aphid diversity within and between ant nest mounds.
The three focal species (Geoica utricularia, Forda marginata and Tetraneura ulmi) had considerable clonal diversity at the population level. Yet more than half of the ant mounds contained just a single aphid species, a significantly higher percentage than expected from a random distribution. Over 60% of these single-species mounds had a single aphid clone, and clones tended to persist across subsequent years. Whenever multiple species/clones co-occurred in the same mound, they were spatially separated with more than 95% of the aphid chambers containing individuals of a single clone.
L. flavus “husbandry” is characterized by low aphid “livestock” diversity per colony, especially at the nest-chamber level, but it lacks the exclusive monocultures known from other cultivation mutualisms. The ants appear to eat most of the early instar aphids, so that adult aphids are unlikely to face limited phloem resources and scramble competition with other aphids. We suggest that such culling of carbohydrate-providing symbionts for protein ingestion may maintain maximal host yield per aphid while also benefitting the domesticated aphids as long as their clone-mates reproduce successfully. The cost-benefit logic of this type of polyculture husbandry has striking analogies with human farming practices based on slaughtering young animals for meat to maximize milk-production by a carefully regulated adult livestock population.
Cultivation mutualism; Host-symbiont conflict; Symbiont competition; Monoculture; Clonal mixing
Recent data have suggested that patient admission during intensive care unit (ICU) morning bedside rounds is associated with less favorable outcome. We undertook the present study to explore the association between morning round-time ICU admissions and hospital mortality in a large Canadian health region.
A multi-center retrospective cohort study was performed at five hospitals in Edmonton, Canada, between July 2002 and December 2009. Round-time ICU admission was defined as occurring between 8 and 11:59 a.m. Multivariable logistic regression analysis was used to explore the association between round-time admission and outcome.
Of 18,857 unique ICU admissions, 2,055 (10.9%) occurred during round time. Round-time admissions were more frequent in community hospitals compared with tertiary hospitals (12.0% vs. 10.5%; odds ratio [OR] 1.16; 95% CI, 1.05-1.29, P < 0.004) and from the ward compared with the emergency department (ED) or operating theater (17.5% vs. 9.2%; OR 2.1; 95% CI, 1.9-2.3, P < 0.0001). Round-time admissions were more often medical than surgical (12.6% vs. 6.6%; OR 2.06; 95% CI, 1.83-2.31, P < 0.0001), had more comorbid illness (11.9% vs. 10.5%; OR 1.15; 95% CI, 1.04-1.27, P < 0.008) and higher APACHE II score (22.2 vs. 21.3, P < 0.001), and were more likely to have a primary diagnosis of respiratory failure (37.0% vs. 31.3%, P < 0.001) or sepsis (11.1% vs. 9.0%, P = 0.002). Crude ICU mortality (15.3% vs. 11.6%; OR 1.38; 95% CI, 1.21-1.57, P < 0.0001) and hospital mortality (23.9% vs. 20.6%; OR 1.21; 95% CI, 1.09-1.35, P < 0.001) were higher for round-time compared with non-round-time admissions. In multi-variable analysis, round-time admission was associated with increased ICU mortality (OR 1.19, 95% CI, 1.03-1.38, P = 0.017) but was not significantly associated with hospital mortality (OR 1.02; 95% CI, 0.90-1.16, P = 0.700). In the subgroup admitted from the ED, round-time admission showed significantly higher ICU mortality (OR 1.54; 95% CI, 1.21-1.95; P < 0.001) and a trend for higher hospital mortality (OR 1.22; 95% CI, 0.99-1.51, P = 0.057).
Approximately 1 in 10 patients is admitted during morning rounds. These patients are more commonly admitted from the ward and are burdened by comorbidities, are non-operative, and have higher illness severity. These patients admitted during morning rounds have higher observed ICU mortality but no difference in hospital mortality.
To determine whether spasticity in persons with spinal cord injury (SCI) is associated with elevated monosynaptic reflex excitability.
Convenience sample of 9 subjects (8 men, 1 woman) with chronic and complete SCI and 20 persons (14 men, 6 women) with no neurologic impairment. Subjects with SCI exhibited lower-extremity spasticity as indicated by velocity-dependent increased resistance to passive muscle stretch, abnormally brisk deep tendon reflexes, involuntary lower-extremity flexion and/or extension spasms, and clonus.
Soleus H-reflex recruitment curves were elicited in all subjects.
Main Outcome Measures
Soleus H-reflex threshold (HTH), gain (HGN), and amplitude (HPP).
There was no difference between subjects with and without SCI in HTH, HGN, or HPP.
Spasticity in people with chronic and complete SCI was not associated with increased excitability of the connections between Ia afferent projections and motoneurons. Factors extrinsic to these connections may have a role in spasticity caused by SCI.
H-reflex; Muscle spasticity; Paralysis; Rehabilitation; Soleus muscle; Spinal cord injuries
We have examined the transcriptional response of Caenorhabditis elegans following exposure to the anthelmintic drug ivermectin (IVM) using whole genome microarrays and real-time QPCR. Our original aim was to identify candidate molecules involved in IVM metabolism and/or excretion. For this reason the IVM tolerant strain, DA1316, was used to minimise transcriptomic changes related to the phenotype of drug exposure. However, unlike equivalent work with benzimidazole drugs, very few of the induced genes were members of xenobiotic metabolising enzyme families. Instead, the transcriptional response was dominated by genes associated with fat mobilization and fatty acid metabolism including catalase, esterase, and fatty acid CoA synthetase genes. This is consistent with the reduction in pharyngeal pumping, and consequential reduction in food intake, upon exposure of DA1316 worms to IVM. Genes with the highest fold change in response to IVM exposure, cyp-37B1, mtl-1 and scl-2, were comparably up-regulated in response to short–term food withdrawal (4 hr) independent of IVM exposure, and GFP reporter constructs confirm their expression in tissues associated with fat storage (intestine and hypodermis). These experiments have serendipitously identified novel genes involved in an early response of C. elegans to reduced food intake and may provide insight into similar processes in higher organisms.
The infective schistosome cercaria develops within the intramolluscan daughter sporocyst from an undifferentiated germ ball, during which synthesis of proteins essential for infection occurs. When the aquatic cercaria locates the mammalian host it rapidly penetrates into the epidermis using glandular secretions. It then undergoes metamorphosis into the schistosomulum, including replacement of its tegument surface membranes, a process taking several days before it exits the skin. Patterns of gene expression underlying this transition have been characterised.
Methods and Principal Findings
All gene models from the S. mansoni genome (www.GeneDB.org) were incorporated into a high-density oligonucleotide array. Double-stranded cDNA from germ balls, cercariae, and day 3 schistosomula was hybridised to the array without amplification. Statistical analysis was performed using Bioconductor to reveal differentially transcribed loci. Genes were categorised on the basis of biological process, tissue association or molecular function to aid understanding of the complex processes occurring. Genes necessary for DNA replication were enriched only in the germ ball, while those involved in translation were up-regulated in the germ ball and/or day 3 schistosomulum. Different sets of developmental genes were up-regulated at each stage. A large number of genes encoding elastases and invadolysins, and some venom allergen-like proteins were up-regulated in the germ ball, those encoding cysteine and aspartic proteases in the cercaria and schistosomulum. Micro exon genes encoding variant secreted proteins were highly up-regulated in the schistosomulum along with tegument and gut-associated genes, coincident with remodelling of the parasite body. Genes encoding membrane proteins were prominently up-regulated in the cercaria and/or day 3 schistosomulum.
Our study highlights an expanded number of transcripts encoding proteins potentially involved in skin invasion. It illuminates the process of metamorphosis into the schistosomulum and highlights the very early activation of gut-associated genes whilst revealing little change in the parasite's energy metabolism or stress responses.
The schistosome cercaria develops from undifferentiated germ balls within the daughter sporocyst located in the hepatopancreas of its snail intermediate host. This is where the proteins it uses to infect humans are synthesised. After a brief free life in fresh water, if the cercaria locates a host, it infects by direct penetration through the skin. It then transforms into the schistosomulum stage, adapted for life in human tissues. We have designed a large scale array comprising probes representing all known schistosome genes and used it in hybridisation experiments to establish which genes are turned on or off in the parasite during these stages in its life cycle. Genes encoding proteins involved in cell division were prominent in the germ ball along with those for proteases and potential immunomodulators, deployed during skin penetration. The non-feeding cercaria was the least active at synthesising proteins. Conversion to the schistosomulum was accompanied by transcription of genes involved in body remodeling, including production of a new outer surface, and gut activation long before ingestion of red blood cells begins. Our data help us to understand better the proteins deployed to achieve infection, and subsequent adaptations necessary for establishment of the parasite in the human host.
Dictyostelium is the only non-metazoan with functionally analyzed SH2 domains and studying them can give insights into their evolution and wider potential. LrrB has a novel domain configuration with leucine-rich repeat, 14-3-3 and SH2 protein–protein interaction modules. It is required for the correct expression of several specific genes in early development and here we characterize its role in later, multicellular development. During development in the light, slug formation in LrrB null (lrrB-) mutants is delayed relative to the parental strain, and the slugs are highly defective in phototaxis and thermotaxis. In the dark the mutant arrests development as an elongated mound, in a hitherto unreported process we term dark stalling. The developmental and phototaxis defects are cell autonomous and marker analysis shows that the pstO prestalk sub-region of the slug is aberrant in the lrrB- mutant. Expression profiling, by parallel micro-array and deep RNA sequence analyses, reveals many other alterations in prestalk-specific gene expression in lrrB- slugs, including reduced expression of the ecmB gene and elevated expression of ampA. During culmination ampA is ectopically expressed in the stalk, there is no expression of ampA and ecmB in the lower cup and the mutant fruiting bodies lack a basal disc. The basal disc cup derives from the pstB cells and this population is greatly reduced in the lrrB- mutant. This anatomical feature is a hallmark of mutants aberrant in signaling by DIF-1, the polyketide that induces prestalk and stalk cell differentiation. In a DIF-1 induction assay the lrrB- mutant is profoundly defective in ecmB activation but only marginally defective in ecmA induction. Thus the mutation partially uncouples these two inductive events. In early development LrrB interacts physically and functionally with CldA, another SH2 domain containing protein. However, the CldA null mutant does not phenocopy the lrrB- in its aberrant multicellular development or phototaxis defect, implying that the early and late functions of LrrB are affected in different ways. These observations, coupled with its domain structure, suggest that LrrB is an SH2 adaptor protein active in diverse developmental signaling pathways.
► LrrB null (lrrB-) slugs are highly defective in phototaxis and thermotaxis, perhaps because the anterior, pstO prestalk sub-region of the slug is aberrant. ► There are many changes in gene expression in the null mutant, especially in prestalk-specific transcripts. ► The posterior, pstB sub-population of the slug is absent in the lrrB- mutant and the derivative structures are aberrant or missing. ► The lrrB- mutant is highly defective in ecmB induction by the polyketide DIF-1 but only marginally defective in ecmA induction, so the mutation partially uncouples these two inductive events. ► SH2 domain signaling in Dictyostelium mirrors that of animal cells in utilizing single adaptor proteins in multiple signaling pathways.
Dictyostelium; SH2 domain; Adaptor protein; DIF-1; Prestalk; Basal disc
Clinical and experimental data suggest that stress contributes to the pathology of epilepsy. We review mechanisms by which stress, primarily via stress hormones, may exacerbate epilepsy, focusing on the intersection between stress-induced pathways and the progression of pathological events that occur before, during, and after the onset of epileptogenesis. In addition to this temporal nuance, we discuss other complexities in stress-epilepsy interactions, including the role of blood-brain barrier dysfunction, neuron-glia interactions, and inflammatory/cytokine pathways that may be protective or damaging depending on context. We advocate the use of global analytical tools, such as microarray, in support of a shift away from a narrow focus on seizures and towards profiling the complex, early process of epileptogenesis, in which multiple pathways may interact to dictate the ultimate onset of chronic, recurring seizures.
Cdk8 is a component of the mediator complex which facilitates transcription by RNA polymerase II and has been shown to play an important role in development of Dictyostelium discoideum. This eukaryote feeds as single cells but starvation triggers the formation of a multicellular organism in response to extracellular pulses of cAMP and the eventual generation of spores. Strains in which the gene encoding Cdk8 have been disrupted fail to form multicellular aggregates unless supplied with exogenous pulses of cAMP and later in development, cdk8- cells show a defect in spore production.
Microarray analysis revealed that the cdk8- strain previously described (cdk8-HL) contained genome duplications. Regeneration of the strain in a background lacking detectable gene duplication generated strains (cdk8-2) with identical defects in growth and early development, but a milder defect in spore generation, suggesting that the severity of this defect depends on the genetic background. The failure of cdk8- cells to aggregate unless rescued by exogenous pulses of cAMP is consistent with a failure to express the catalytic subunit of protein kinase A. However, overexpression of the gene encoding this protein was not sufficient to rescue the defect, suggesting that this is not the only important target for Cdk8 at this stage of development. Proteomic analysis revealed two potential targets for Cdk8 regulation, one regulated post-transcriptionally (4-hydroxyphenylpyruvate dioxygenase (HPD)) and one transcriptionally (short chain dehydrogenase/reductase (SDR1)).
This analysis has confirmed the importance of Cdk8 at multiple stages of Dictyostelium development, although the severity of the defect in spore production depends on the genetic background. Potential targets of Cdk8-mediated gene regulation have been identified in Dictyostelium which will allow the mechanism of Cdk8 action and its role in development to be determined.
To investigate how much movement practice occurred during stroke rehabilitation, and what factors might influence doses of practice provided.
Observational survey of stroke therapy sessions.
7 inpatient and outpatient rehabilitation sites.
We observed a convenience sample of 312 physical and occupational therapy sessions for people with stroke.
Main Outcome Measures
We recorded numbers of repetitions in specific movement categories and data on potential modifying factors (patient age, side affected, time since stroke, Functional Independence Measure item scores, and years of therapist experience). Descriptive statistics were used to characterize amounts of practice. Correlation and regression analyses were used to determine if potential factors were related to the amount of practice in the two important categories of upper extremity functional movements and gait steps.
Practice of task-specific, functional upper extremity movements occurred in 51% of the sessions that addressed upper limb rehabilitation and the average number of repetitions/session was 32 (95% CI = 20–44). Practice of gait occurred in 84% of sessions that addressed lower limb rehabilitation and the average number of gait steps/session was 357 (95% CI = 296–418). None of the potential factors listed above accounted for significant variance in the amount of practice in either of these two categories.
The amount of practice provided during post-stroke rehabilitation is small compared to animal models. It is possible that current doses of task-specific practice during rehabilitation are not adequate to drive the neural reorganization needed to optimally promote function post stroke.
Intensity; Rehabilitation; Stroke; Motor; Physical Therapy; Occupational Therapy
TMC435 is a small-molecule inhibitor of the NS3/4A serine protease of hepatitis C virus (HCV) currently in phase 2 development. The in vitro resistance profile of TMC435 was characterized by selection experiments with HCV genotype 1 replicon cells and the genotype 2a JFH-1 system. In 80% (86/109) of the sequences from genotype 1 replicon cells analyzed, a mutation at NS3 residue D168 was observed, with changes to V or A being the most frequent. Mutations at NS3 positions 43, 80, 155, and 156, alone or in combination, were also identified. A transient replicon assay confirmed the relevance of these positions for TMC435 inhibitory activity. The change in the 50% effective concentrations (EC50s) observed for replicons with mutations at position 168 ranged from <10-fold for those with the D168G or D168N mutation to ∼2,000-fold for those with the D168V or D168I mutation, compared to the EC50 for the wild type. Of the positions identified, mutations at residue Q80 had the least impact on the activity of TMC435 (<10-fold change in EC50s), while greater effects were observed for some replicons with mutations at positions 43, 155, and 156. TMC435 remained active against replicons with the specific mutations observed after in vitro or in vivo exposure to telaprevir or boceprevir, including most replicons with changes at positions 36, 54, and 170 (<3-fold change in EC50s). Replicons carrying mutations affecting the activity of TMC435 remained fully susceptible to alpha interferon and NS5A and NS5B inhibitors. Finally, combinations of TMC435 with alpha interferon and NS5B polymerase inhibitors prevented the formation of drug-resistant replicon colonies.
Species recognition is an important aspect of an organism's biology. Here, we consider how parasitoid wasps vary their reproductive decisions when their offspring face intra- and interspecific competition for resources and mates. We use host acceptance and sex ratio behaviour to test whether female Nasonia vitripennis and Nasonia longicornis discriminate between conspecifics and heterospecifics when ovipositing. We tested pairs of conspecific or heterospecific females ovipositing either simultaneously or sequentially on a single host, using strains varying in their recent history of sympatry. Both N. vitripennis and N. longicornis rejected parasitized hosts more often than unparasitized hosts, although females were more likely to superparasitize their own species in the sequential treatment. However, sex ratio behaviour did not vary, suggesting similar responses towards conspecifics and heterospecifics. This contrasts with theory predicting that heterospecifics should not influence sex ratios as their offspring do not influence local mate competition, where conspecifics would. These non-adaptive sex ratios reinforce the lack of adaptive kin discrimination in N. vitripennis and suggest a behavioural constraint. Discrimination between closely related species is therefore context dependent in Nasonia. We suggest that isolating mechanisms associated with the speciation process have influenced behaviour to a greater extent than selection on sex ratios.
species recognition; speciation; adaptation; sex ratios; superparasitism; multiparasitism
The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 lacking a functional ramA or ramR or with plasmid-mediated high-level overexpression of ramA were compared to those of the wild-type parental strain. Inactivation of ramA led to increased expression of 14 SPI-1 genes and decreased expression of three SPI-2 genes, and it altered expression of ribosomal biosynthetic genes and several amino acid biosynthetic pathways. Furthermore, disruption of ramA led to decreased survival within RAW 264.7 mouse macrophages and attenuation within the BALB/c ByJ mouse model. Highly overexpressed ramA led to increased expression of genes encoding multidrug resistance (MDR) efflux pumps, including acrAB, acrEF, and tolC. Decreased expression of 34 Salmonella pathogenicity island (SPI) 1 and 2 genes, decreased SipC production, decreased adhesion to and survival within macrophages, and decreased colonization of Caenorhabditis elegans were also seen. Disruption of ramR led to the increased expression of ramA, acrAB, and tolC, but not to the same level as when ramA was overexpressed on a plasmid. Inactivation of ramR had a more limited effect on pathogenicity gene expression. In silico analysis of a suggested RamA-binding consensus sequence identified target genes, including ramR, acrA, tolC, sipABC, and ssrA. This study demonstrates that the regulation of a mechanism of MDR and expression of virulence genes show considerable overlap, and we postulate that such a mechanism is dependent on transcriptional activator concentration and promoter sensitivity. However, we have no evidence to support the hypothesis that increased MDR via RamA regulation of AcrAB-TolC gives rise to a hypervirulent strain.
Focal epilepsy often develops following traumatic, ischemic or infectious brain injury. While the electrical activity of the epileptic brain is well characterized, the mechanisms underlying epileptogenesis are poorly understood. We have recently shown that in the rat neocortex, long-lasting breakdown of the blood-brain barrier (BBB) or direct exposure of the neocortex to serum-derived albumin leads to rapid up-regulation of the astrocytic marker, glial fibrillary acidic protein (GFAP), followed by delayed (within 4–7 days) development of an epileptic focus. We investigated the role of astrocytes in epileptogenesis in the BBB-breakdown and albumin models of epileptogenesis. We found similar, robust changes in astrocytic gene expression in the neocortex within hours following treatment with deoxycholic acid (BBB breakdown) or albumin. These changes predict reduced clearance capacity for both extracellular glutamate and potassium. Electrophysiological recordings in-vitro confirmed the reduced clearance of activity-dependent accumulation of both potassium and glutamate 24 h following exposure to albumin. We used a NEURON model to simulate the consequences of reduced astrocytic uptake of potassium and glutamate on excitatory postsynaptic potentials (EPSPs). The model predicted that the accumulation of glutamate is associated with frequency-dependent (>100 Hz) decreased facilitation of EPSPs, while potassium accumulation leads to frequency-dependant (10–50 Hz) and N-methyl-D-aspartic acid (NMDA)-dependent synaptic facilitation. In-vitro electrophysiological recordings during epileptogenesis confirmed frequency-dependant synaptic facilitation leading to seizure-like activity. Our data indicate a transcription-mediated astrocytic transformation early during epileptogenesis. We suggest that the resulting reduction in the clearance of extracellular potassium underlies frequency-dependent neuronal hyper-excitability and network synchronization.
Albumin; Astrocytes; Blood-brain barrier; Glutamate; Post-synaptic potentials; Potassium