bluuebunny/crossref_metadata_2025_split huggingface dataset:
Not a Simple Tether: Binding of Toxoplasma gondii AMA1 to RON2 during Invasion Protects AMA1 from Rhomboid-Mediated Cleavage and Leads to Dephosphorylation of Its Cytosolic Tail
Authors: Krishnamurthy Shruthi , Deng Bin , del Rio Roxana , Buchholz Kerry R. , Treeck Moritz , Urban Siniša , Boothroyd John , Lam Ying-Wai , Ward Gary E. 
Published: September 2016

ABSTRACT

Apical membrane antigen 1 (AMA1) is a receptor protein on the surface of
Toxoplasma gondii
that plays a critical role in host cell invasion. The ligand to which
T. gondii
AMA1 (TgAMA1) binds, TgRON2, is secreted into the host cell membrane by the parasite during the early stages of invasion. The TgAMA1-TgRON2 complex forms the core of the “moving junction,” a ring-shaped zone of tight contact between the parasite and host cell membranes, through which the parasite pushes itself during invasion. Paradoxically, the parasite also expresses rhomboid proteases that constitutively cleave the TgAMA1 transmembrane domain. How can TgAMA1 function effectively in host cell binding if its extracellular domain is constantly shed from the parasite surface? We show here that when TgAMA1 binds the domain 3 (D3) peptide of TgRON2, its susceptibility to cleavage by rhomboid protease(s) is greatly reduced. This likely serves to maintain parasite-host cell binding at the moving junction, a hypothesis supported by data showing that parasites expressing a hypercleavable version of TgAMA1 invade less efficiently than wild-type parasites do. Treatment of parasites with the D3 peptide was also found to reduce phosphorylation of S527 on the cytoplasmic tail of TgAMA1, and parasites expressing a phosphomimetic S527D allele of TgAMA1 showed an invasion defect. Taken together, these data suggest that TgAMA1-TgRON2 interaction at the moving junction protects TgAMA1 molecules that are actively engaged in host cell penetration from rhomboid-mediated cleavage and generates an outside-in signal that leads to dephosphorylation of the TgAMA1 cytosolic tail. Both of these effects are required for maximally efficient host cell invasion.

IMPORTANCE
Nearly one-third of the world’s population is infected with the protozoan parasite
Toxoplasma gondii
, which causes life-threatening disease in neonates and immunocompromised individuals.
T. gondii
is a member of the phylum Apicomplexa, which includes many other parasites of veterinary and medical importance, such as those that cause coccidiosis, babesiosis, and malaria. Apicomplexan parasites grow within their hosts through repeated cycles of host cell invasion, parasite replication, and host cell lysis. Parasites that cannot invade host cells cannot survive or cause disease. AMA1 is a highly conserved protein on the surface of apicomplexan parasites that is known to be important for invasion, and the work presented here reveals new and unexpected insights into AMA1 function. A more complete understanding of the role of AMA1 in invasion may ultimately contribute to the development of new chemotherapeutics designed to disrupt AMA1 function and invasion-related signaling in this important group of human pathogens.
Thrombin Cleavage of Plasmodium falciparum Erythrocyte Membrane Protein 1 Inhibits Cytoadherence
Authors: Gillrie Mark R. , Renaux Bernard , Russell-Goldman Eleanor , Avril Marion , Brazier Andrew J. , Mihara Koichiro , Di Cera Enrico , Milner Danny A. , Hollenberg Morley D. , Smith Joseph D. , Ho May 
Published: September 2016

ABSTRACT

Plasmodium falciparum
malaria remains one of the most deadly infections worldwide. The pathogenesis of the infection results from the sequestration of infected erythrocytes (IRBC) in vital organs, including the brain, with resulting impairment of blood flow, hypoxia, and lactic acidosis. Sequestration occurs through the adhesion of IRBC to host receptors on microvascular endothelium by
Plasmodium falciparum
erythrocyte membrane protein 1 (PfEMP1), a large family of variant surface antigens, each with up to seven extracellular domains that can bind to multiple host receptors. Consequently, antiadhesive therapies directed at single endothelial adhesion molecules may not be effective. In this study, we demonstrated that the serine protease thrombin, which is pivotal in the activation of the coagulation cascade, cleaved the major parasite adhesin on the surface of IRBC. As a result, adhesion under flow was dramatically reduced, and already adherent IRBC were detached. Thrombin cleavage sites were mapped to the Duffy binding-like δ1 (DBLδ1) domain and interdomains 1 and 2 in the PfEMP1 of the parasite line IT4var19. Furthermore, we observed an inverse correlation between the presence of thrombin and IRBC in cerebral malaria autopsies of children. We investigated a modified (R67A) thrombin and thrombin inhibitor, hirugen, both of which inhibit the binding of substrates to exosite I, thereby reducing its proinflammatory properties. Both approaches reduced the barrier dysfunction induced by thrombin without affecting its proteolytic activity on PfEMP1, raising the possibility that thrombin cleavage of variant PfEMP1 may be exploited as a broadly inhibitory antiadhesive therapy.

IMPORTANCE
Plasmodium falciparum
malaria is the third leading cause of mortality due to a pathogen, with 214 million people infected and 438,000 deaths annually. The adhesion of
Plasmodium falciparum
-infected erythrocytes (IRBC) to microvascular endothelium is a major pathological process in severe malaria. While the recent implementation of artemisinin-based antimalarial therapy for severe malaria improves patient survival by targeting all parasite stages, antiparasite drugs alone may not immediately reverse pathophysiological processes in occluded vessels. Here we show that thrombin, an enzyme intimately involved in the clotting process, cleaves the main parasite adhesin expressed on the surface of IRBC, thereby preventing and reversing the binding of IRBC to endothelial cells. This beneficial effect of thrombin can be achieved by modified thrombins that cause significantly less clotting and vessel leakage while preserving the ability to cleave the parasite protein. Our results provide the basis for using modified thrombins as adjunctive therapy in severe malaria.
Activation of the DNA Damage Response Is a Conserved Function of HIV-1 and HIV-2 Vpr That Is Independent of SLX4 Recruitment
Authors: Fregoso Oliver I. , Emerman Michael 
Published: September 2016

ABSTRACT
There has been extraordinary progress in understanding the roles of lentiviral accessory proteins in antagonizing host antiviral defense proteins. However, the precise primary function of the accessory gene Vpr remains elusive. Here we suggest that engagement with the DNA damage response is an important function of primate lentiviral Vpr proteins because of its conserved function among diverse lentiviral lineages. In contrast, we show that, for HIV-1, HIV-2, and related Vpr isolates and orthologs, there is a lack of correlation between DNA damage response activation and interaction with the host SLX4 protein complex of structure specific endonucleases; some Vpr proteins are able to interact with SLX4, but the majority are not. Using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 method to knock out SLX4, we formally showed that HIV-1 and HIV-2 Vpr orthologs can still activate the DNA damage response and cell cycle arrest in the absence of SLX4. Together, our data suggest that activation of the DNA damage response, but not SLX4 interaction, is conserved and therefore indicative of an important function of Vpr. Our data also indicate that Vpr activates the DNA damage response through an SLX4-independent mechanism that remains uncharacterized.

IMPORTANCE
HIV-1 and HIV-2 belong to a family of viruses called lentiviruses that infect at least 40 primate species, including humans. Lentiviruses have been circulating in primates for at least 5 million years. In order to better fight HIV, we must understand the viral and host factors necessary for infection, adaptation, and transmission of these viruses. Using the natural variation of HIV-1, HIV-2, and related lentiviruses, we have investigated the role of the DNA damage response in the viral life cycle. We have found that the ability of lentiviruses to activate the DNA damage response is largely conserved. However, we also found that the SLX4 host factor is not required for this activation, as was previously proposed. This indicates that the DNA damage response is an important player in the viral life cycle, and yet the mechanism(s) by which HIV-1, HIV-2, and other primate lentiviruses engage the DNA damage response is still unknown.
Global Identification of Biofilm-Specific Proteolysis in Candida albicans
Authors: Winter Michael B. , Salcedo Eugenia C. , Lohse Matthew B. , Hartooni Nairi , Gulati Megha , Sanchez Hiram , Takagi Julie , Hube Bernhard , Andes David R. , Johnson Alexander D. , Craik Charles S. , Nobile Clarissa J. 
Published: September 2016

ABSTRACT

Candida albicans
is a fungal species that is part of the normal human microbiota and also an opportunistic pathogen capable of causing mucosal and systemic infections.
C. albicans
cells proliferate in a planktonic (suspension) state, but they also form biofilms, organized and tightly packed communities of cells attached to a solid surface. Biofilms colonize many niches of the human body and persist on implanted medical devices, where they are a major source of new
C. albicans
infections. Here, we used an unbiased and global substrate-profiling approach to discover proteolytic activities produced specifically by
C. albicans
biofilms, compared to planktonic cells, with the goal of identifying potential biofilm-specific diagnostic markers and targets for therapeutic intervention. This activity-based profiling approach, coupled with proteomics, identified Sap5 (Candidapepsin-5) and Sap6 (Candidapepsin-6) as major biofilm-specific proteases secreted by
C. albicans
. Fluorogenic peptide substrates with selectivity for Sap5 or Sap6 confirmed that their activities are highly upregulated in
C. albicans
biofilms; we also show that these activities are upregulated in other
Candida
clade pathogens. Deletion of the
SAP5
and
SAP6
genes in
C. albicans
compromised biofilm development
in vitro
in standard biofilm assays and
in vivo
in a rat central venous catheter biofilm model. This work establishes secreted proteolysis as a promising enzymatic marker and potential therapeutic target for
Candida
biofilm formation.

IMPORTANCE
Biofilm formation by the opportunistic fungal pathogen
C. albicans
is a major cause of life-threatening infections. This work provides a global characterization of secreted proteolytic activity produced specifically by
C. albicans
biofilms. We identify activity from the proteases Sap5 and Sap6 as highly upregulated during
C. albicans
biofilm formation and develop Sap-cleavable fluorogenic substrates that enable the detection of biofilms from
C. albicans
and also from additional pathogenic
Candida
species. Furthermore,
SAP5
and
SAP6
deletions confirm that both proteases are required for proper biofilm development
in vitro
and
in vivo
. We propose that secreted proteolysis is a promising marker for the diagnosis and potential therapeutic targeting of
Candida
biofilm-associated infections.
Does integrated training in evidence-based medicine (EBM) in the general practice (GP) specialty training improve EBM behaviour in daily clinical practice? A cluster randomised controlled trial
Authors: Kortekaas M F , Bartelink M E L , Zuithoff N P A , van der Heijden G J M G , de Wit N J , Hoes A W 
Published: September 2016

ObjectivesEvidence-based medicine (EBM) is an important element in the general practice (GP) specialty training. Studies show that integrating EBM training into clinical practice brings larger benefits than stand-alone modules. However, these studies have neither been performed in GP nor assessed EBM behaviour of former trainees in daily clinical practice.SettingGP specialty training in the Netherlands.ParticipantsAll 82 third year GP trainees who started their final third year in 2011 were approached for inclusion, of whom 79 (96%) participated: 39 in the intervention group and 40 in the control group.InterventionIntegrated EBM training, in which EBM is embedded closely within the clinical context by joint assignments for the trainee and supervisor in daily practice, and teaching sessions based on dilemmas from actual patient consultations.ComparisonStand-alone EBM training at the institute only.Primary and secondary outcomesOur primary outcome was EBM behaviour, assessed by measuring guideline adherence (incorporating rational, motivated deviation) and information-seeking behaviour. Our secondary outcomes were EBM attitude and EBM knowledge. Data were acquired using logbooks and questionnaires, respectively. Analyses were performed using mixed models.ResultsLogbook data were available from 76 (96%) of the participating trainees at baseline (7614 consultations), 60 (76%) at the end of the third year (T1, 4973 consultations) and 53 (67%) 1 year after graduation (T2, 3307 consultations). We found no significant differences in outcomes between the 2 groups, with relative risks for guideline adherence varying between 0.96 and 0.99 (95% CI 0.86 to 1.11) at T1, and 0.99 and 1.10 (95% CI 0.92 to 1.25) at T2, and for information-seeking behaviour between 0.97 and 1.16 (95% CI 0.70 to 1.91) and 0.90 and 1.10 (95% CI 0.70 to 1.32), respectively.ConclusionsIntegrated EBM training compared with stand-alone EBM training does not improve EBM behaviour, attitude or knowledge of (future) GPs.
Sequential simulation (SqS) of clinical pathways: a tool for public and patient engagement in point-of-care diagnostics
Authors: Huddy Jeremy R , Weldon Sharon-Marie , Ralhan Shvaita , Painter Tim , Hanna George B , Kneebone Roger , Bello Fernando 
Published: September 2016

ObjectivesPublic and patient engagement (PPE) is fundamental to healthcare research. To facilitate effective engagement in novel point-of-care tests (POCTs), the test and downstream consequences of the result need to be considered. Sequential simulation (SqS) is a tool to represent patient journeys and the effects of intervention at each and subsequent stages. This case study presents a process evaluation of SqS as a tool for PPE in the development of a volatile organic compound-based breath test POCT for the diagnosis of oesophagogastric (OG) cancer.SettingThree 3-hour workshops in central London.Participants38 members of public attended a workshop, 26 (68%) had no prior experience of the OG cancer diagnostic pathway.InterventionsClinical pathway SqS was developed from a storyboard of a patient, played by an actor, noticing symptoms of oesophageal cancer and following a typical diagnostic pathway. The proposed breath testing strategy was then introduced and incorporated into a second SqS to demonstrate pathway impact. Facilitated group discussions followed each SqS.Primary and secondary outcome measuresEvaluation was conducted through pre-event and postevent questionnaires, field notes and analysis of audiovisual recordings.Results38 participants attended a workshop. All participants agreed they were able to contribute to discussions and like the idea of an OG cancer breath test. Five themes emerged related to the proposed new breath test including awareness of OG cancer, barriers to testing and diagnosis, design of new test device, new clinical pathway and placement of test device. 3 themes emerged related to the use of SqS: participatory engagement, simulation and empathetic engagement, and why participants attended.ConclusionsSqS facilitated a shared immersive experience for participants and researchers that led to the coconstruction of knowledge that will guide future research activities and be of value to stakeholders concerned with the invention and adoption of POCT.
Modelling study to estimate the health burden of foodborne diseases: cases, general practice consultations and hospitalisations in the UK, 2009
Authors: O'Brien Sarah J , Larose Tricia L , Adak Goutam K , Evans Meirion R , Tam Clarence C 
Published: September 2016

ObjectiveTo generate estimates of the burden of UK-acquired foodborne disease accounting for uncertainty.DesignA modelling study combining data from national public health surveillance systems for laboratory-confirmed infectious intestinal disease (IID) and outbreaks of foodborne disease and 2 prospective, population-based studies of IID in the community. The underlying data sets covered the time period 1993–2008. We used Monte Carlo simulation and a Bayesian approach, using a systematic review to generate Bayesian priors. We calculated point estimates with 95% credible intervals (CrI).SettingUK, 2009.Outcome measuresPathogen-specific estimates of the number of cases, general practice (GP) consultations and hospitalisations for foodborne disease in the UK in 2009.ResultsBayesian approaches gave slightly more conservative estimates of overall health burden (∼511 000 cases vs 566 000 cases).Campylobacteris the most common foodborne pathogen, causing 280 400 (95% CrI 182 503–435 693) food-related cases and 38 860 (95% CrI 27 160–55 610) GP consultations annually. Despite this, there are only around 562 (95% CrI 189–1330) food-related hospital admissions due toCampylobacter, reflecting relatively low disease severity.Salmonellacauses the largest number of hospitalisations, an estimated 2490 admissions (95% CrI 607–9631), closely followed byEscherichia coliO157 with 2233 admissions (95% CrI 170–32 159). Other common causes of foodborne disease includeClostridium perfringens, with an estimated 79 570 cases annually (95% CrI 30 700–211 298) and norovirus with 74 100 cases (95% CrI 61 150–89 660). Other viruses and protozoa ranked much lower as causes of foodborne disease.ConclusionsThe 3 models yielded similar estimates of the burden of foodborne illness in the UK and show that continued reductions inCampylobacter,Salmonella,E. coliO157,C. perfringensand norovirus are needed to mitigate the impact of foodborne disease.
Self-reported physical and mental health of Australian carers: a cross-sectional study
Authors: Hussain Rafat , Wark Stuart , Dillon Gina , Ryan Peta 
Published: September 2016

ObjectiveTo report on self-reported physical and mental health of informal carers in rural regions of New South Wales, Australia.MethodsA cross-sectional community-based sample (n=222) of carers completed a questionnaire incorporating self-reported measures of health from validated international instruments including Medical Outcomes Study Scale (SF-36), the Centre for Epidemiology-Depression (CES-D) and Kessler-10 (K-10) Psychological Distress Scales, along with information on participant demographics and other key caregiving characteristics such as health condition of care recipient.ResultsRural carers’ self-reported health was poor as evident on the SF-36 Physical and Mental Health component scores as well as each individual domain of the SF-36. Results from the CES-D and K-10 scores indicated very high rates of depressive symptoms and psychological distress. Over 70% of carers within the current study had CES-D scores indicative of depressive symptoms. Scores on the K-10 indicate almost half the carers were experiencing high levels of psychological distress, which is over 4 times the rate reported in the general Australian population.Conclusions and implicationsResults from this study were compared to Australian population normative data and were found to be significantly below Australian age-matched population norms for SF-36, CES-D and K-10. These findings illustrate the poor health profile of informal carers relative to the general Australian population, especially in terms of depressive symptoms and psychological distress. This highlights the need for additional support for rural carers in order to ease the accumulated mental and physical health burdens of this group.
Patients’ experiences of a multidisciplinary team-led community case management programme: a qualitative study
Authors: Gowing Alice , Dickinson Claire , Gorman Tom , Robinson Louise , Duncan Rachel 
Published: September 2016

ObjectivesTo explore the views and experiences of patients on the care they have received while enrolled on the Northumberland High Risk Patient Programme (NHRPP). This programme involved case finding of frail patients using a multidisciplinary team (MDT)-led community case management programme, and support of patients through care planning and regular reviews using primary, community, secondary and social care professionals.DesignA qualitative study using semistructured interviews, which were digitally recorded, transcribed and subject to thematic analysis.SettingCommunity patients receiving primary care in the county of Northumberland, England.Participants23 participants took part, of which 16 were patients enrolled on the NHRPP, and 7 carers. GP practices were selected purposively by size, deprivation and location, and patients identified and invited by General Practitioners to participate.Results4 main themes emerged from the data: awareness and understanding of the NHRPP, confidence in the primary healthcare team, limitations of home care and the active role of being a patient. Despite having a low level of awareness of the details of the NHRPP, participants did think that its broad aim made sense. Participants discussed their high level of satisfaction with their care and access to team members. However, some limitations of alternatives to hospital care were identified, including the need to consider psychological as well as medical needs, the importance of overnight care and the needs of those without informal carers. Finally, participants discussed the active nature of being a patient under the NHRPP if they were to contribute fully to planning and managing their own care.ConclusionsThis study has identified that a programme of MDT-led case management was generally very well received by patients and their families. However, a number of factors were identified that could improve the implementation of the programme and further research needs to be undertaken to address these.