ROP39 is an Irgb10-specific parasite effector that modulates acute Toxoplasma gondii virulence.

Toxoplasma gondii (T. gondii) is a zoonotic apicomplexan parasite that is an important cause of clinical disability in humans. On a global scale, one third of the human population is infected with T. gondii. Mice and other small rodents are believed to be responsible for transmission of T. gondii to the domestic cat, its definitive host. Interferon-inducible Immunity-Related GTPases (IRG proteins) are important for control of murine T. gondii infections. Virulence differences between T. gondii strains are linked to polymorphic rhoptry proteins (ROPs) that cooperate to inactivate individual IRG family members. In particular, the pseudokinase ROP5 isoform B is critically important in laboratory strains of mice. We identified T. gondii ROP39 in complex with ROP5B and demonstrate its contribution to acute T. gondii virulence. ROP39 directly targets Irgb10 and inhibits homodimer formation of the GTPase leading to an overall reduction of IRG protein loading onto the parasitophorous vacuolar membrane (PVM). Maintenance of PVM integrity rescues the parasite from IRG protein-mediated clearance in vitro and in vivo. This study identifies a novel T. gondii effector that is important for specific inactivation of the IRG resistance system. Our data reveal that yet unknown T. gondii effectors can emerge from identification of direct interaction partners of ROP5B.

Predicting transfer RNA gene activity from sequence and genome context.

Transfer RNA (tRNA) genes are among the most highly transcribed genes in the genome owing to their central role in protein synthesis. However, there is evidence for a broad range of gene expression across tRNA loci. This complexity, combined with difficulty in measuring transcript abundance and high sequence identity across transcripts, has severely limited our collective understanding of tRNA gene expression regulation and evolution. We establish sequence-based correlates to tRNA gene expression and develop a tRNA gene classification method that does not require, but benefits from, comparative genomic information and achieves accuracy comparable to molecular assays. We observe that guanine + cytosine (G + C) content and CpG density surrounding tRNA loci is exceptionally well correlated with tRNA gene activity, supporting a prominent regulatory role of the local genomic context in combination with internal sequence features. We use our tRNA gene activity predictions in conjunction with a comprehensive tRNA gene ortholog set spanning 29 placental mammals to estimate the evolutionary rate of functional changes among orthologs. Our method adds a new dimension to large-scale tRNA functional prediction and will help prioritize characterization of functional tRNA variants. Its simplicity and robustness should enable development of similar approaches for other clades, as well as exploration of functional diversification of members of large gene families.

Irgm2 and Gate-16 cooperatively dampen Gram-negative bacteria-induced caspase-11 response.

Inflammatory caspase-11 (rodent) and caspases-4/5 (humans) detect the Gram-negative bacterial component LPS within the host cell cytosol, promoting activation of the non-canonical inflammasome. Although non-canonical inflammasome-induced pyroptosis and IL-1-related cytokine release are crucial to mount an efficient immune response against various bacteria, their unrestrained activation drives sepsis. This suggests that cellular components tightly control the threshold level of the non-canonical inflammasome in order to ensure efficient but non-deleterious inflammatory responses. Here, we show that the IFN-inducible protein Irgm2 and the ATG8 family member Gate-16 cooperatively counteract Gram-negative bacteria-induced non-canonical inflammasome activation, both in cultured macrophages and in vivo. Specifically, the Irgm2/Gate-16 axis dampens caspase-11 targeting to intracellular bacteria, which lowers caspase-11-mediated pyroptosis and cytokine release. Deficiency in Irgm2 or Gate16 induces both guanylate binding protein (GBP)-dependent and GBP-independent routes for caspase-11 targeting to intracellular bacteria. Our findings identify molecular effectors that fine-tune bacteria-activated non-canonical inflammasome responses and shed light on the understanding of the immune pathways they control.

HNRNPA1 promotes recognition of splice site decoys by U2AF2 in vivo.

Alternative pre-mRNA splicing plays a major role in expanding the transcript output of human genes. This process is regulated, in part, by the interplay of trans-acting RNA binding proteins (RBPs) with myriad cis-regulatory elements scattered throughout pre-mRNAs. These molecular recognition events are critical for defining the protein-coding sequences (exons) within pre-mRNAs and directing spliceosome assembly on noncoding regions (introns). One of the earliest events in this process is recognition of the 3' splice site (3'ss) by U2 small nuclear RNA auxiliary factor 2 (U2AF2). Splicing regulators, such as the heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), influence spliceosome assembly both in vitro and in vivo, but their mechanisms of action remain poorly described on a global scale. HNRNPA1 also promotes proofreading of 3'ss sequences though a direct interaction with the U2AF heterodimer. To determine how HNRNPA1 regulates U2AF-RNA interactions in vivo, we analyzed U2AF2 RNA binding specificity using individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) in control and HNRNPA1 overexpression cells. We observed changes in the distribution of U2AF2 crosslinking sites relative to the 3'ss of alternative cassette exons but not constitutive exons upon HNRNPA1 overexpression. A subset of these events shows a concomitant increase of U2AF2 crosslinking at distal intronic regions, suggesting a shift of U2AF2 to "decoy" binding sites. Of the many noncanonical U2AF2 binding sites, Alu-derived RNA sequences represented one of the most abundant classes of HNRNPA1-dependent decoys. We propose that one way HNRNPA1 regulates exon definition is to modulate the interaction of U2AF2 with decoy or bona fide 3'ss.

Improved genome assembly of American alligator genome reveals conserved architecture of estrogen signaling.

The American alligator, Alligator mississippiensis, like all crocodilians, has temperature-dependent sex determination, in which the sex of an embryo is determined by the incubation temperature of the egg during a critical period of development. The lack of genetic differences between male and female alligators leaves open the question of how the genes responsible for sex determination and differentiation are regulated. Insight into this question comes from the fact that exposing an embryo incubated at male-producing temperature to estrogen causes it to develop ovaries. Because estrogen response elements are known to regulate genes over long distances, a contiguous genome assembly is crucial for predicting and understanding their impact. We present an improved assembly of the American alligator genome, scaffolded with in vitro proximity ligation (Chicago) data. We use this assembly to scaffold two other crocodilian genomes based on synteny. We perform RNA sequencing of tissues from American alligator embryos to find genes that are differentially expressed between embryos incubated at male- versus female-producing temperature. Finally, we use the improved contiguity of our assembly along with the current model of CTCF-mediated chromatin looping to predict regions of the genome likely to contain estrogen-responsive genes. We find that these regions are significantly enriched for genes with female-biased expression in developing gonads after the critical period during which sex is determined by incubation temperature. We thus conclude that estrogen signaling is a major driver of female-biased gene expression in the post-temperature sensitive period gonads.

Chronic Dengue Virus Panencephalitis in a Patient with Progressive Dementia with Extrapyramidal Features.

OBJECTIVE: To determine the underlying etiology in a patient with progressive dementia with extrapyramidal signs and chronic inflammation referred to the National Institutes of Health Undiagnosed Diseases Program. METHODS: Extensive investigations included metabolic profile, autoantibody panel, infectious etiologies, genetic screening, whole exome sequencing, and the phage-display assay, VirScan, for viral immune responses. An etiological diagnosis was established postmortem. RESULTS: Using VirScan, enrichment of dengue viral antibodies was detected in cerebrospinal fluid as compared to serum. No virus was detected in serum or cerebrospinal fluid, but postmortem analysis confirmed dengue virus in the brain by immunohistochemistry, in situ hybridization, quantitative polymerase chain reaction, and sequencing. Dengue virus was also detectable by polymerase chain reaction and sequencing from brain biopsy tissue collected 33 months antemortem, confirming a chronic infection despite a robust immune response directed against the virus. Immunoprofiling and whole exome sequencing of the patient did not reveal any immunodeficiency, and sequencing of the virus demonstrated wild-type dengue virus in the central nervous system. INTERPRETATION: Dengue virus is the most common arbovirus worldwide and represents a significant public health concern. Infections with dengue virus are usually self-limiting, and chronic dengue infections have not been previously reported. Our findings suggest that dengue virus infections may persist in the central nervous system causing a panencephalitis and should be considered in patients with progressive dementia with extrapyramidal features in endemic regions or with relevant travel history. Furthermore, this work highlights the utility of comprehensive antibody profiling assays to aid in the diagnosis of encephalitis of unknown etiology. ANN NEUROL 2019;86:695-703.

Comparison of Muscle Activation Between Back Squats and Belt Squats.

Evans, TW, McLester, CN, Howard, JS, McLester, JR, and Calloway, JP. Comparison of muscle activation between back squats and belt squats. J Strength Cond Res 33(7S): S52-S59, 2019-A machine belt squat is a piece of equipment designed to allow the performance of squats while loading weight on the lifter's hips using a belt. The purpose of this investigation was to determine whether belt squats differ from back squats in activation of the primary movers and to determine the predictive capabilities of back squat load, training status, and anthropometric data on belt squat load. Thirty-one participants (16 men and 15 women) completed anthropometric measurements, a demographic questionnaire, a familiarization visit, and 2 testing visits, completing a 5 repetition maximum test for back squat and belt squat. Surface electromyography was used to measure muscle activation for the left and right vastus medialis, vastus lateralis, rectus femoris, and gluteus maximus (GM). Comparison of muscle activation between the 2 exercises showed significant differences in the left GM (back squat: 0.84 ± 0.45, belt squat: 0.69 ± 0.22, p = 0.015) and right GM (back squat: 0.86 ± 0.45, belt squat: 0.71 ± 0.29, p = 0.004). Regression analysis computed significant prediction equations for belt squat load for general population, men, women, and advanced lifters. Overall, results indicate that belt squats may significantly differ in GM activation from back squats. Back squat load, as well as other variables, may be effective in accurately estimating appropriate belt squat load. These findings may help to more appropriately program for training with machine belt squats as a back squat alternative.

Concurrent LETM and nerve root enhancement in spinal neurosarcoid: A case series.

Spinal neurosarcoidosis is a rare form of neurosarcoid which can be challenging to diagnose given its clinical or radiographic findings are often indistinguishable from other causes of spinal demyelinating disease. We present a series of three patients with spinal neurosarcoid, all of whom demonstrated concurrent longitudinally enhancing transverse myelitis as well as spinal nerve root enhancement. These findings may be suggestive of spinal neurosarcoid and may help clinicians make the diagnosis as well as reduce the need for invasive biopsy.

RabGDIα is a negative regulator of interferon-γ-inducible GTPase-dependent cell-autonomous immunity to Toxoplasma gondii.

IFN-γ orchestrates cell-autonomous host defense against various intracellular vacuolar pathogens. IFN-γ-inducible GTPases, such as p47 immunity-related GTPases (IRGs) and p65 guanylate-binding proteins (GBPs), are recruited to pathogen-containing vacuoles, which is important for disruption of the vacuoles, culminating in the cell-autonomous clearance. Although the positive regulation for the proper recruitment of IRGs and GBPs to the vacuoles has been elucidated, the suppressive mechanism is unclear. Here, we show that Rab GDP dissociation inhibitor α (RabGDIα), originally identified as a Rab small GTPase inhibitor, is a negative regulator of IFN-γ-inducible GTPases in cell-autonomous immunity to the intracellular pathogen Toxoplasma gondii. Overexpression of RabGDIα, but not of RabGDIß, impaired IFN-γ-dependent reduction of T. gondii numbers. Conversely, RabGDIα deletion in macrophages and fibroblasts enhanced the IFN-γ-induced clearance of T. gondii. Furthermore, upon a high dose of infection by T. gondii, RabGDIα-deficient mice exhibited a decreased parasite burden in the brain and increased resistance in the chronic phase than did control mice. Among members of IRGs and GBPs important for the parasite clearance, Irga6 and Gbp2 alone were more frequently recruited to T. gondii-forming parasitophorous vacuoles in RabGDIα-deficient cells. Notably, Gbp2 positively controlled Irga6 recruitment that was inhibited by direct and specific interactions of RabGDIα with Gbp2 through the lipid-binding pocket. Taken together, our results suggest that RabGDIα inhibits host defense against T. gondii by negatively regulating the Gbp2-Irga6 axis of IFN-γ-dependent cell-autonomous immunity.

The Toxoplasma gondii rhoptry protein ROP18 is an Irga6-specific kinase and regulated by the dense granule protein GRA7.

In mice, avirulent strains (e.g. types II and III) of the protozoan parasite Toxoplasma gondii are restricted by the immunity-related GTPase (IRG) resistance system. Loading of IRG proteins onto the parasitophorous vacuolar membrane (PVM) is required for vacuolar rupture resulting in parasite clearance. In virulent strain (e.g. type I) infections, polymorphic effector proteins ROP5 and ROP18 cooperate to phosphorylate and thereby inactivate mouse IRG proteins to preserve PVM integrity. In this study, we confirmed the dense granule protein GRA7 as an additional component of the ROP5/ROP18 kinase complex and identified GRA7 association with the PVM by direct binding to ROP5. The absence of GRA7 results in reduced phosphorylation of Irga6 correlated with increased vacuolar IRG protein amounts and attenuated virulence. Earlier work identified additional IRG proteins as targets of T. gondii ROP18 kinase. We show that the only specific target of ROP18 among IRG proteins is in fact Irga6. Similarly, we demonstrate that GRA7 is strictly an Irga6-specific virulence effector. This identifies T. gondii GRA7 as a regulator for ROP18-specific inactivation of Irga6. The structural diversity of the IRG proteins implies that certain family members constitute additional specific targets for other yet unknown T. gondii virulence effectors.

The immunity-related GTPase Irga6 dimerizes in a parallel head-to-head fashion.

BACKGROUND: The immunity-related GTPases (IRGs) constitute a powerful cell-autonomous resistance system against several intracellular pathogens. Irga6 is a dynamin-like protein that oligomerizes at the parasitophorous vacuolar membrane (PVM) of Toxoplasma gondii leading to its vesiculation. Based on a previous biochemical analysis, it has been proposed that the GTPase domains of Irga6 dimerize in an antiparallel fashion during oligomerization. RESULTS: We determined the crystal structure of an oligomerization-impaired Irga6 mutant bound to a non-hydrolyzable GTP analog. Contrary to the previous model, the structure shows that the GTPase domains dimerize in a parallel fashion. The nucleotides in the center of the interface participate in dimerization by forming symmetric contacts with each other and with the switch I region of the opposing Irga6 molecule. The latter contact appears to activate GTP hydrolysis by stabilizing the position of the catalytic glutamate 106 in switch I close to the active site. Further dimerization contacts involve switch II, the G4 helix and the trans stabilizing loop. CONCLUSIONS: The Irga6 structure features a parallel GTPase domain dimer, which appears to be a unifying feature of all dynamin and septin superfamily members. This study contributes important insights into the assembly and catalytic mechanisms of IRG proteins as prerequisite to understand their anti-microbial action.

Loss of the interferon-γ-inducible regulatory immunity-related GTPase (IRG), Irgm1, causes activation of effector IRG proteins on lysosomes, damaging lysosomal function and predicting the dramatic susceptibility of Irgm1-deficient mice to infection.

BACKGROUND: The interferon-γ (IFN-γ)-inducible immunity-related GTPase (IRG), Irgm1, plays an essential role in restraining activation of the IRG pathogen resistance system. However, the loss of Irgm1 in mice also causes a dramatic but unexplained susceptibility phenotype upon infection with a variety of pathogens, including many not normally controlled by the IRG system. This phenotype is associated with lymphopenia, hemopoietic collapse, and death of the mouse. RESULTS: We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family). In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes. Another GMS protein, Irgm3, is localized to endoplasmic reticulum (ER) membranes; in the Irgm3-deficient mouse, activated GKS proteins are found at the ER. The Irgm3-deficient mouse does not show the drastic phenotype of the Irgm1 mouse. In the Irgm1/Irgm3 double knock-out mouse, activated GKS proteins associate with lipid droplets, but not with lysosomes, and the Irgm1/Irgm3(-/-) does not have the generalized immunodeficiency phenotype expected from its Irgm1 deficiency. CONCLUSIONS: The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles. Our data suggest that the generalized lymphomyeloid collapse that occurs in Irgm1(-/-) mice upon infection with a variety of pathogens may be due to lysosomal damage caused by off-target activation of GKS proteins on lysosomal membranes and consequent failure of autophagosomal processing.

Frac-seq reveals isoform-specific recruitment to polyribosomes.

Pre-mRNA splicing is required for the accurate expression of virtually all human protein coding genes. However, splicing also plays important roles in coordinating subsequent steps of pre-mRNA processing such as polyadenylation and mRNA export. Here, we test the hypothesis that nuclear pre-mRNA processing influences the polyribosome association of alternative mRNA isoforms. By comparing isoform ratios in cytoplasmic and polyribosomal extracts, we determined that the alternative products of ∼30% (597/1954) of mRNA processing events are differentially partitioned between these subcellular fractions. Many of the events exhibiting isoform-specific polyribosome association are highly conserved across mammalian genomes, underscoring their possible biological importance. We find that differences in polyribosome association may be explained, at least in part by the observation that alternative splicing alters the cis-regulatory landscape of mRNAs isoforms. For example, inclusion or exclusion of upstream open reading frames (uORFs) in the 5'UTR as well as Alu-elements and microRNA target sites in the 3'UTR have a strong influence on polyribosome association of alternative mRNA isoforms. Taken together, our data demonstrate for the first time the potential link between alternative splicing and translational control of the resultant mRNA isoforms.

Identification of the microsporidian Encephalitozoon cuniculi as a new target of the IFNγ-inducible IRG resistance system.

The IRG system of IFNγ-inducible GTPases constitutes a powerful resistance mechanism in mice against Toxoplasma gondii and two Chlamydia strains but not against many other bacteria and protozoa. Why only T. gondii and Chlamydia? We hypothesized that unusual features of the entry mechanisms and intracellular replicative niches of these two organisms, neither of which resembles a phagosome, might hint at a common principle. We examined another unicellular parasitic organism of mammals, member of an early-diverging group of Fungi, that bypasses the phagocytic mechanism when it enters the host cell: the microsporidian Encephalitozoon cuniculi. Consistent with the known susceptibility of IFNγ-deficient mice to E. cuniculi infection, we found that IFNγ treatment suppresses meront development and spore formation in mouse fibroblasts in vitro, and that this effect is mediated by IRG proteins. The process resembles that previously described in T. gondii and Chlamydia resistance. Effector (GKS subfamily) IRG proteins accumulate at the parasitophorous vacuole of E. cuniculi and the meronts are eliminated. The suppression of E. cuniculi growth by IFNγ is completely reversed in cells lacking regulatory (GMS subfamily) IRG proteins, cells that effectively lack all IRG function. In addition IFNγ-induced cells infected with E. cuniculi die by necrosis as previously shown for IFNγ-induced cells resisting T. gondii infection. Thus the IRG resistance system provides cell-autonomous immunity to specific parasites from three kingdoms of life: protozoa, bacteria and fungi. The phylogenetic divergence of the three organisms whose vacuoles are now known to be involved in IRG-mediated immunity and the non-phagosomal character of the vacuoles themselves strongly suggests that the IRG system is triggered not by the presence of specific parasite components but rather by absence of specific host components on the vacuolar membrane.

Clinical utility of the list sign as a predictor of non-demyelinating disorders in a multiple sclerosis (MS) practice.

OBJECTIVES: Not all patients referred for evaluation of multiple sclerosis (MS) meet criteria required for MS or related entities. Identification of markers to exclude demyelinating disease may help detect patients whose presenting symptoms are inconsistent with MS. In this study, we evaluate whether patients who present a self-prepared list of symptoms during an initial visit are less likely to have demyelinating disease and whether this action, which we term the "list sign," may help exclude demyelinating disease. METHODS: Using chart review, 300 consecutive new patients who presented for evaluation to a neurologist at a tertiary MS referral center were identified retrospectively. Patients were defined as having demyelinating disease if diagnosed with MS or a related demyelinating condition. RESULTS: Of the 233 enrolled subjects, 157 were diagnosed with demyelinating disease and 74 did not meet criteria for demyelinating disease. Fifteen (8.4%) subjects had a positive list sign, of which 1 patient had demyelinating disease. The 15 subjects described a mean of 12.07 symptoms, and 8 of these patients met Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) criteria for somatic symptom disorder. The specificity and positive predictive value of the list sign for non-demyelinating disease were 0.99 (95% confidence interval (CI) 0.96-0.99) and 0.93 (95% CI 0.66-0.99), respectively. CONCLUSION: A positive list sign may be useful to exclude demyelinating disease and to guide diagnostic evaluations for other conditions. Patients with a positive list sign also have a high incidence of somatic symptom disorder.

A Toxoplasma gondii pseudokinase inhibits host IRG resistance proteins.

The ability of mice to resist infection with the protozoan parasite, Toxoplasma gondii, depends in large part on the function of members of a complex family of atypical large GTPases, the interferon-gamma-inducible immunity-related GTPases (IRG proteins). Nevertheless, some strains of T. gondii are highly virulent for mice because, as recently shown, they secrete a polymorphic protein kinase, ROP18, from the rhoptries into the host cell cytosol at the moment of cell invasion. Depending on the allele, ROP18 can act as a virulence factor for T. gondii by phosphorylating and thereby inactivating mouse IRG proteins. In this article we show that IRG proteins interact not only with ROP18, but also strongly with the products of another polymorphic locus, ROP5, already implicated as a major virulence factor from genetic crosses, but whose function has previously been a complete mystery. ROP5 proteins are members of the same protein family as ROP18 kinases but are pseudokinases by sequence, structure, and function. We show by a combination of genetic and biochemical approaches that ROP5 proteins act as essential co-factors for ROP18 and present evidence that they work by enforcing an inactive GDP-dependent conformation on the IRG target protein. By doing so they prevent GTP-dependent activation and simultaneously expose the target threonines on the switch I loop for phosphorylation by ROP18, resulting in permanent inactivation of the protein. This represents a novel mechanism in which a pseudokinase facilitates the phosphorylation of a target by a partner kinase by preparing the substrate for phosphorylation, rather than by upregulation of the activity of the kinase itself.

Irgm1 (LRG-47), a regulator of cell-autonomous immunity, does not localize to mycobacterial or listerial phagosomes in IFN-γ-induced mouse cells.

The IFN-inducible protein Irgm1 (LRG-47) belongs to the family of immunity-related GTPases that function in cell-autonomous resistance against intracellular pathogens in mice. Irgm1 deficiency is associated with a severe immunodeficiency syndrome. The protein has been variously interpreted as a direct effector molecule on bacterial phagosomes or on other organelles or as an inducer of autophagy. In this study, we re-examined one of these claims, namely that Irgm1 targets mycobacterial and listerial phagosomes. We found no colocalization of endogenous Irgm1, using two immunofluorescent staining techniques, either in fibroblasts or in macrophages. We demonstrated the predicted existence of two protein isoforms of Irgm1 derived from differential splicing and described immunological reagents for their detection. Both Irgm1 isoforms localize to the Golgi apparatus and weakly to mitochondria; however, only the long Irgm1 isoforms can be detected on endolysosomal membranes. Together with the previous observation that the general immunodeficiency phenotype of Irgm1(-/-) mice is reversed in Irgm1/Irgm3 double-deficient mice, our results argue against a direct effector function of Irgm1 at the bacterial phagosome. We discuss these findings in the context of evidence that Irgm1 functions as a negative regulator of other members of the immunity-related GTPase protein family.

Loss of exon identity is a common mechanism of human inherited disease.

It is widely accepted that at least 10% of all mutations causing human inherited disease disrupt splice-site consensus sequences. In contrast to splice-site mutations, the role of auxiliary cis-acting elements such as exonic splicing enhancers (ESE) and exonic splicing silencers (ESS) in human inherited disease is still poorly understood. Here we use a top-down approach to determine rates of loss or gain of known human exonic splicing regulatory (ESR) sequences associated with either disease-causing mutations or putatively neutral single nucleotide polymorphisms (SNPs). We observe significant enrichment toward loss of ESEs and gain of ESSs among inherited disease-causing variants relative to neutral polymorphisms, indicating that exon skipping may play a prominent role in aberrant gene regulation. Both computational and biochemical approaches underscore the relevance of exonic splicing enhancer loss and silencer gain in inherited disease. Additionally, we provide direct evidence that both SRp20 (SRSF3) and possibly PTB (PTBP1) are involved in the function of a splicing silencer that is created de novo by a total of 83 different inherited disease mutations in 67 different disease genes. Taken together, we find that ~25% (7154/27,681) of known mis-sense and nonsense disease-causing mutations alter functional splicing signals within exons, suggesting a much more widespread role for aberrant mRNA processing in causing human inherited disease than has hitherto been appreciated.

Comparative genomics of the apicomplexan parasites Toxoplasma gondii and Neospora caninum: Coccidia differing in host range and transmission strategy.

Toxoplasma gondii is a zoonotic protozoan parasite which infects nearly one third of the human population and is found in an extraordinary range of vertebrate hosts. Its epidemiology depends heavily on horizontal transmission, especially between rodents and its definitive host, the cat. Neospora caninum is a recently discovered close relative of Toxoplasma, whose definitive host is the dog. Both species are tissue-dwelling Coccidia and members of the phylum Apicomplexa; they share many common features, but Neospora neither infects humans nor shares the same wide host range as Toxoplasma, rather it shows a striking preference for highly efficient vertical transmission in cattle. These species therefore provide a remarkable opportunity to investigate mechanisms of host restriction, transmission strategies, virulence and zoonotic potential. We sequenced the genome of N. caninum and transcriptomes of the invasive stage of both species, undertaking an extensive comparative genomics and transcriptomics analysis. We estimate that these organisms diverged from their common ancestor around 28 million years ago and find that both genomes and gene expression are remarkably conserved. However, in N. caninum we identified an unexpected expansion of surface antigen gene families and the divergence of secreted virulence factors, including rhoptry kinases. Specifically we show that the rhoptry kinase ROP18 is pseudogenised in N. caninum and that, as a possible consequence, Neospora is unable to phosphorylate host immunity-related GTPases, as Toxoplasma does. This defense strategy is thought to be key to virulence in Toxoplasma. We conclude that the ecological niches occupied by these species are influenced by a relatively small number of gene products which operate at the host-parasite interface and that the dominance of vertical transmission in N. caninum may be associated with the evolution of reduced virulence in this species.

The extended impact of co-designed personalised aids for people living with chronic conditions: an exploratory study in a healthcare setting.

Assistive technology has great potential to help individuals living with chronic health conditions, however devices often fail to align with the unique requirements of users. These results in device abandonment and missed opportunities to benefit people. This exploratory study aims to evaluate the short and longer-term satisfaction, psychological benefit, use and resources involved in co-designed customised assistive devices within a current healthcare service. Individuals with chronic health conditions identified daily living challenges. Eleven individuals completed the trial and were involved throughout the design process. Outcome measures evaluated the impact of the devices provided, healthcare utilisation, help required, and resources used. Nineteen custom assistive devices were produced for twenty-four challenges in daily living identified. At 3-months, eighteen devices were still being used. Daily challenges had become easier for individuals to complete and required less help from informal carers. Individuals were satisfied with the devices and service provided. Improvements in competence, adaptability and self-esteem were sustained long-term. The average clinician's time required to produce a device was 5 h 55 min, with an average cost of £203.79. People with chronic conditions were able to benefit from the co-design process resulting in satisfaction and long-term utilisation of the device, and positive psycho-social benefits. The costs associated with embedding this approach in a healthcare service were calculated. Scaling up the co-design process reduced the associated costs per device compared to previous work. Further work is required to evaluate co-designing across larger samples and explore opportunities to further improve the cost-efficiency.

By involving users in the design process, healthcare professionals can create devices that better meet users' expectations, preferences and functional needs, thereby increasing overall usability, satisfaction and utilisation long-term of the devices.Incorporating the individual's perspective and needs into the design process enabled users to better understand the solutions that could be produced and thus encouraged users to identify other challenges in daily living they faced where an assistive device could assist them.Through being provided with devices to support them with specific challenges they faced, individuals were able to perform more tasks independently, reducing the need for help from family members and informal carers for the associated tasks.Re-evaluating the solutions generated with other previous research may help identify common design solutions and features to enable further scaling-up of this co-design approach.