A novel, stain-free, natural auto-fluorescent signal, Sig M, identified from cytometric and transcriptomic analysis of infectivity of Cryptosporidium hominis and Cryptosporidium parvum

Cryptosporidiosis is a worldwide diarrheal disease caused by the protozoan Cryptosporidium. The primary symptom is diarrhea, but patients may exhibit different symptoms based on the species of the Cryptosporidium parasite they are infected with. Furthermore, some genotypes within species are more transmissible and apparently virulent than others. The mechanisms underpinning these differences are not understood, and an effective in vitro system for Cryptosporidium culture would help advance our understanding of these differences. Using COLO-680N cells, we employed flow cytometry and microscopy along with the C. parvum-specific antibody Sporo-Glo™ to characterize infected cells 48 h following an infection with C. parvum or C. hominis. The Cryptosporidium parvum-infected cells showed higher levels of signal using Sporo-Glo™ than C. hominis-infected cells, which was likely because Sporo-Glo™ was generated against C. parvum. We found a subset of cells from infected cultures that expressed a novel, dose-dependent auto-fluorescent signal that was detectable across a range of wavelengths. The population of cells that expressed this signal increased proportionately to the multiplicity of infection. The spectral cytometry results confirmed that the signature of this subset of host cells closely matched that of oocysts present in the infectious ecosystem, pointing to a parasitic origin. Present in both C. parvum and C. hominis cultures, we named this Sig M, and due to its distinct profile in cells from both infections, it could be a better marker for assessing Cryptosporidium infection in COLO-680N cells than Sporo-Glo™. We also noted Sig M’s impact on Sporo-Glo™ detection as Sporo-Glo™ uses fluoroscein–isothiocynate, which is detected where Sig M also fluoresces. Lastly, we used NanoString nCounter® analysis to investigate the transcriptomic landscape for the two Cryptosporidium species, assessing the gene expression of 144 host and parasite genes. Despite the host gene expression being at high levels, the levels of putative intracellular Cryptosporidium gene expression were low, with no significant difference from controls, which could be, in part, explained by the abundance of uninfected cells present as determined by both Sporo-Glo™ and Sig M analyses. This study shows for the first time that a natural auto-fluorescent signal, Sig M, linked to Cryptosporidium infection can be detected in infected host cells without any fluorescent labeling strategies and that the COLO-680N cell line and spectral cytometry could be useful tools to advance the understanding of Cryptosporidium infectivity

High Throughput Genome-Wide Survey of Small RNAs from the Parasitic Protists Giardia intestinalis and Trichomonas vaginalis

RNA interference (RNAi) is a set of mechanisms which regulate gene expression in eukaryotes. Key elements of RNAi are small sense and antisense RNAs from 19 to 26 nt generated from double-stranded RNAs. MicroRNAs (miRNAs) are a major type of RNAi-associated small RNAs and are found in most eukaryotes studied to date. To investigate whether small RNAs associated with RNAi appear to be present in all eukaryotic lineages, and therefore present in the ancestral eukaryote, we studied two deep-branching protozoan parasites, Giardia intestinalis and Trichomonas vaginalis. Little is known about endogenous small RNAs involved in RNAi of these organisms. Using Illumina Solexa sequencing and genome-wide analysis of small RNAs from these distantly related deep-branching eukaryotes, we identified 10 strong miRNA candidates from Giardia and 11 from Trichomonas. We also found evidence of Giardia short-interfering RNAs potentially involved in the expression of variant-specific surface proteins. In addition, eight new small nucleolar RNAs from Trichomonas are identified. Our results indicate that miRNAs are likely to be general in ancestral eukaryotes and therefore are likely to be a universal feature of eukaryotes

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Clinical spectrum and features of activated phosphoinositide 3-kinase δ syndrome: A large patient cohort study.

BACKGROUND: Activated phosphoinositide 3-kinase δ syndrome (APDS) is a recently described combined immunodeficiency resulting from gain-of-function mutations in PIK3CD, the gene encoding the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ). OBJECTIVE: We sought to review the clinical, immunologic, histopathologic, and radiologic features of APDS in a large genetically defined international cohort. METHODS: We applied a clinical questionnaire and performed review of medical notes, radiology, histopathology, and laboratory investigations of 53 patients with APDS. RESULTS: Recurrent sinopulmonary infections (98%) and nonneoplastic lymphoproliferation (75%) were common, often from childhood. Other significant complications included herpesvirus infections (49%), autoinflammatory disease (34%), and lymphoma (13%). Unexpectedly, neurodevelopmental delay occurred in 19% of the cohort, suggesting a role for PI3Kδ in the central nervous system; consistent with this, PI3Kδ is broadly expressed in the developing murine central nervous system. Thoracic imaging revealed high rates of mosaic attenuation (90%) and bronchiectasis (60%). Increased IgM levels (78%), IgG deficiency (43%), and CD4 lymphopenia (84%) were significant immunologic features. No immunologic marker reliably predicted clinical severity, which ranged from asymptomatic to death in early childhood. The majority of patients received immunoglobulin replacement and antibiotic prophylaxis, and 5 patients underwent hematopoietic stem cell transplantation. Five patients died from complications of APDS. CONCLUSION: APDS is a combined immunodeficiency with multiple clinical manifestations, many with incomplete penetrance and others with variable expressivity. The severity of complications in some patients supports consideration of hematopoietic stem cell transplantation for severe childhood disease. Clinical trials of selective PI3Kδ inhibitors offer new prospects for APDS treatment.T.C. is supported by National Children’s Research Centre, Our Lady’s Children’s Hospital Crumlin, Dublin, Ireland. A.C. has a Wellcome Trust Postdoctoral Training Fellowship for Clinicians (103413/Z/13/Z). K.O. is supported by funding from BBSRC, MRC, Wellcome Trust and GSK. R.D. and D.S.K are funded by National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre, Cambridge, UK. C.S. and S.E. are supported by the German Federal Ministry of Education and Research (BMBF 01 EO 0803 grant to the Center of Chronic immunodeficiency and BMBF 01GM1111B grant to the PID-NET initiative). S.N.F is supported in part by the Southampton UK National Institute for Health Research (NIHR) Wellcome Trust Clinical Research Facility and NIHR Respiratory Biomedical Research Unit. M.A.A.I. is funded by NHS Innovation London and King’s College Hospital Charitable Trust. A.F., S.L., A.D., F.R-L and S.K. are supported by the European Union’s 7th RTD Framework Programme (ERC advanced grant PID-IMMUNE contract 249816) and a government grant managed by the French Agence Nationale de la Recherche as part of the "Investments for the Future" program (ANR-10-IAHU-01). S.L. is supported by the Agence Nationale de la Recherche (ANR) (ANR-14-CE14-0028-01), the Foundation ARC pour la Recherche sur le Cancer (France), the Rare Diseases Foundation (France) and François Aupetit Association (France). S.L. is a senior scientist and S.K is a researcher at the Centre National de la Recherche Scientifique-CNRS (France). A.D. and S.K. are supported by the “Institut National de la Santé et de la Recherche Médicale". S.K. also supported by the Fondation pour la Recherche Médicale (grant number: ING20130526624), la Ligue Contre le Cancer (Comité de Paris) and the Centre de Référence Déficits Immunitaires Héréditaires (CEREDIH). S.O.B is supported by the Higher Education Funding Council for England. B.V. is supported by the UK Biotechnology and Biological Sciences Research Council [BB/I007806/1], Cancer Research UK [C23338/A15965) and the National Institute for Health Research (NIHR) University College London Hospitals Biomedical Research Centre. B.V. is consultant to Karus Therapeutics (Oxford, UK). S.N. is a Wellcome Trust Senior Research Fellow in Basic Biomedical Science (095198/Z/10/Z). S.N. is also supported by the European Research Council Starting grant 260477, the EU FP7 collaborative grant 261441 (PEVNET project) and the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre, UK. A.M.C. is funded by the Medical Research Council, British Lung Foundation, University of Sheffield and Cambridge NIHR-BRC. Research in A.M.C. laboratory has received non-commercial grant support from GSK, Novartis, and MedImmune.This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.jaci.2016.06.02

Phenotypic Characterization of EIF2AK4 Mutation Carriers in a Large Cohort of Patients Diagnosed Clinically With Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare disease with an emerging genetic basis. Heterozygous mutations in the gene encoding the bone morphogenetic protein receptor type 2 (BMPR2) are the commonest genetic cause of PAH, whereas biallelic mutations in the eukaryotic translation initiation factor 2 alpha kinase 4 gene (EIF2AK4) are described in pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Here, we determine the frequency of these mutations and define the genotype-phenotype characteristics in a large cohort of patients diagnosed clinically with PAH. METHODS: Whole-genome sequencing was performed on DNA from patients with idiopathic and heritable PAH and with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis recruited to the National Institute of Health Research BioResource-Rare Diseases study. Heterozygous variants in BMPR2 and biallelic EIF2AK4 variants with a minor allele frequency of <1:10 000 in control data sets and predicted to be deleterious (by combined annotation-dependent depletion, PolyPhen-2, and sorting intolerant from tolerant predictions) were identified as potentially causal. Phenotype data from the time of diagnosis were also captured. RESULTS: Eight hundred sixty-four patients with idiopathic or heritable PAH and 16 with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis were recruited. Mutations in BMPR2 were identified in 130 patients (14.8%). Biallelic mutations in EIF2AK4 were identified in 5 patients with a clinical diagnosis of pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Furthermore, 9 patients with a clinical diagnosis of PAH carried biallelic EIF2AK4 mutations. These patients had a reduced transfer coefficient for carbon monoxide (Kco; 33% [interquartile range, 30%-35%] predicted) and younger age at diagnosis (29 years; interquartile range, 23-38 years) and more interlobular septal thickening and mediastinal lymphadenopathy on computed tomography of the chest compared with patients with PAH without EIF2AK4 mutations. However, radiological assessment alone could not accurately identify biallelic EIF2AK4 mutation carriers. Patients with PAH with biallelic EIF2AK4 mutations had a shorter survival. CONCLUSIONS: Biallelic EIF2AK4 mutations are found in patients classified clinically as having idiopathic and heritable PAH. These patients cannot be identified reliably by computed tomography, but a low Kco and a young age at diagnosis suggests the underlying molecular diagnosis. Genetic testing can identify these misclassified patients, allowing appropriate management and early referral for lung transplantation

Telomerecat: A ploidy-agnostic method for estimating telomere length from whole genome sequencing data.

Telomere length is a risk factor in disease and the dynamics of telomere length are crucial to our understanding of cell replication and vitality. The proliferation of whole genome sequencing represents an unprecedented opportunity to glean new insights into telomere biology on a previously unimaginable scale. To this end, a number of approaches for estimating telomere length from whole-genome sequencing data have been proposed. Here we present Telomerecat, a novel approach to the estimation of telomere length. Previous methods have been dependent on the number of telomeres present in a cell being known, which may be problematic when analysing aneuploid cancer data and non-human samples. Telomerecat is designed to be agnostic to the number of telomeres present, making it suited for the purpose of estimating telomere length in cancer studies. Telomerecat also accounts for interstitial telomeric reads and presents a novel approach to dealing with sequencing errors. We show that Telomerecat performs well at telomere length estimation when compared to leading experimental and computational methods. Furthermore, we show that it detects expected patterns in longitudinal data, repeated measurements, and cross-species comparisons. We also apply the method to a cancer cell data, uncovering an interesting relationship with the underlying telomerase genotype

The Gene Ontology knowledgebase in 2023

The Gene Ontology (GO) knowledgebase (http://geneontology.org) is a comprehensive resource concerning the functions of genes and gene products (proteins and noncoding RNAs). GO annotations cover genes from organisms across the tree of life as well as viruses, though most gene function knowledge currently derives from experiments carried out in a relatively small number of model organisms. Here, we provide an updated overview of the GO knowledgebase, as well as the efforts of the broad, international consortium of scientists that develops, maintains, and updates the GO knowledgebase. The GO knowledgebase consists of three components: (1) the GO-a computational knowledge structure describing the functional characteristics of genes; (2) GO annotations-evidence-supported statements asserting that a specific gene product has a particular functional characteristic; and (3) GO Causal Activity Models (GO-CAMs)-mechanistic models of molecular "pathways" (GO biological processes) created by linking multiple GO annotations using defined relations. Each of these components is continually expanded, revised, and updated in response to newly published discoveries and receives extensive QA checks, reviews, and user feedback. For each of these components, we provide a description of the current contents, recent developments to keep the knowledgebase up to date with new discoveries, and guidance on how users can best make use of the data that we provide. We conclude with future directions for the project