Multiscale topology classifies and quantifies cell types in subcellular spatial transcriptomics

Spatial transcriptomics has the potential to transform our understanding of RNA expression in tissues. Classical array-based technologies produce multiple-cell-scale measurements requiring deconvolution to recover single cell information. However, rapid advances in subcellular measurement of RNA expression at whole-transcriptome depth necessitate a fundamentally different approach. To integrate single-cell RNA-seq data with nanoscale spatial transcriptomics, we present a topological method for automatic cell type identification (TopACT). Unlike popular decomposition approaches to multicellular resolution data, TopACT is able to pinpoint the spatial locations of individual sparsely dispersed cells without prior knowledge of cell boundaries. Pairing TopACT with multiparameter persistent homology landscapes predicts immune cells forming a peripheral ring structure within kidney glomeruli in a murine model of lupus nephritis, which we experimentally validate with immunofluorescent imaging. The proposed topological data analysis unifies multiple biological scales, from subcellular gene expression to multicellular tissue organization.Comment: Main text: 8 pages, 4 figures. Supplement: 12 pages, 5 figure

B cells require DOCK8 to elicit and integrate T cell help when antigen is limiting

Dedicator of cytokinesis 8 (DOCK8) immunodeficiency syndrome is characterized by a failure of the germinal center response, a process involving the proliferation and positive selection of antigen-specific B cells. Here, we describe how DOCK8-deficient B cells are blocked at a light-zone checkpoint in the germinal centers of immunized mice, where they are unable to respond to T cell–dependent survival and selection signals and consequently differentiate into plasma cells or memory B cells. Although DOCK8-deficient B cells can acquire and present antigen to initiate activation of cognate T cells, integrin up-regulation, B cell–T cell conjugate formation, and costimulation are insufficient for sustained B cell and T cell activation when antigen availability is limited. Our findings provide an explanation for the failure of the humoral response in DOCK8 immunodeficiency syndrome and insight into how the level of available antigen modulates B cell–T cell cross-talk to fine-tune humoral immune responses and immunological memory

Prolidase deficiency causes spontaneous T cell activation and lupus-like autoimmunity

Prolidase deficiency (PD) is a multisystem disorder caused by mutations in the PEPD gene, which encodes a ubiquitously expressed metallopeptidase essential for the hydrolysis of dipeptides containing C-terminal proline or hydroxyproline. PD typically presents in childhood with developmental delay, skin ulcers, recurrent infections, and, in some patients, autoimmune features that can mimic systemic lupus erythematosus. The basis for the autoimmune association is uncertain, but might be due to self-antigen exposure with tissue damage, or indirectly driven by chronic infection and microbial burden. In this study, we address the question of causation and show that Pepd-null mice have increased antinuclear autoantibodies and raised serum IgA, accompanied by kidney immune complex deposition, consistent with a systemic lupus erythematosus–like disease. These features are associated with an accumulation of CD4 and CD8 effector T cells in the spleen and liver. Pepd deficiency leads to spontaneous T cell activation and proliferation into the effector subset, which is cell intrinsic and independent of Ag receptor specificity or antigenic stimulation. However, an increase in KLRG1+ effector CD8 cells is not observed in mixed chimeras, in which the autoimmune phenotype is also absent. Our findings link autoimmune susceptibility in PD to spontaneous T cell dysfunction, likely to be acting in combination with immune activators that lie outside the hemopoietic system but result from the abnormal metabolism or loss of nonenzymatic prolidase function. This knowledge provides insight into the role of prolidase in the maintenance of self-tolerance and highlights the importance of treatment to control T cell activation

Early and Middle Holocene Hunter-Gatherer Occupations in Western Amazonia: The Hidden Shell Middens

We report on previously unknown early archaeological sites in the Bolivian lowlands, demonstrating for the first time early and middle Holocene human presence in western Amazonia. Multidisciplinary research in forest islands situated in seasonally-inundated savannahs has revealed stratified shell middens produced by human foragers as early as 10,000 years ago, making them the oldest archaeological sites in the region. The absence of stone resources and partial burial by recent alluvial sediments has meant that these kinds of deposits have, until now, remained unidentified. We conducted core sampling, archaeological excavations and an interdisciplinary study of the stratigraphy and recovered materials from three shell midden mounds. Based on multiple lines of evidence, including radiocarbon dating, sedimentary proxies (elements, steroids and black carbon), micromorphology and faunal analysis, we demonstrate the anthropogenic origin and antiquity of these sites. In a tropical and geomorphologically active landscape often considered challenging both for early human occupation and for the preservation of hunter-gatherer sites, the newly discovered shell middens provide evidence for early to middle Holocene occupation and illustrate the potential for identifying and interpreting early open-air archaeological sites in western Amazonia. The existence of early hunter-gatherer sites in the Bolivian lowlands sheds new light on the region's past and offers a new context within which the late Holocene "Earthmovers" of the Llanos de Moxos could have emerged. © 2013 Lombardo et al

Unlocking the bottleneck in forward genetics using whole-genome sequencing and identity by descent to isolate causative mutations

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis.The High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics is funded by Wellcome Trust grant reference 090532/Z/09/Z and MRC Hub grant G0900747 91070. This study was supported by Wellcome Trust Strategic Award 082030 (CCG), Wellcome Trust Studentship 094446/Z/10/Z (KRB), the Oxford NIHR Biomedical Research Centre, and the MRC Human Immunology Unit (RJC). AJR and GL were supported by Wellcome Trust grant 090532/Z/ 09/Z, CCG and AE by a Major initiative Award from the Clive and Vera Ramaciotti Foundation, and AE by an NHMRC Career Development Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

B cell survival, surface BCR and BAFFR expression, CD74 metabolism, and CD8-dendritic cells require the intramembrane endopeptidase SPPL2A

Druggable proteins required for B lymphocyte survival and immune responses are an emerging source of new treatments for autoimmunity and lymphoid malignancy. In this study, we show that mice with an inactivating mutation in the intramembrane protease signal peptide peptidase-like 2A (SPPL2A) unexpectedly exhibit profound humoral immunodeficiency and lack mature B cell subsets, mirroring deficiency of the cytokine B cell-activating factor (BAFF). Accumulation of Sppl2a-deficient B cells was rescued by overexpression of the BAFF-induced survival protein B cell lymphoma 2 (BCL2) but not BAFF and was distinguished by low surface BAFF receptor and IgM and IgD B cell receptors. CD8-negative dendritic cells were also greatly decreased. SPPL2A deficiency blocked the proteolytic processing of CD74 MHC II invariant chain in both cell types, causing dramatic build-up of the p8 product of Cathepsin S and interfering with earlier steps in CD74 endosomal retention and processing. The findings illuminate an important role for the final step in the CD74-MHC II pathway and a new target for protease inhibitor treatment of B cell diseases.R01 AI052127/AI/NIAID NIH HHS/United States U19 AI100627/AI/NIAID NIH HHS/United States Medical Research Council/United Kingdom Wellcome Trust/United Kingdo

VEGF regulates local inhibitory complement proteins in the eye and kidney

10.1172/JCI86418Journal of Clinical Investigation1271199-21

Effect of educational intervention on medication timing in Parkinson's disease: a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Medicine usage in Parkinson's disease patients is often imperfect, in particular irregular timing of medication. The effect of informing Parkinson's disease patients about the continuous dopaminergic hypothesis (to encourage regular medicine intake) on medication adherence and motor control was tested.</p> <p>Methods</p> <p>Patients were randomised either to the active group (receiving the intervention) or control group (no extra information). Antiparkinson medicine usage was monitored for 3 months before and after the intervention using electronic pill bottles which record the date and time of opening (MEMS^®, Aardex, Switzerland) and data used to calculate the percentage of doses taken at correct time intervals.</p> <p>Results</p> <p>43 patients (52%) were randomised to active counselling, and 40 (48%) were controls (standard management). The intervention effect (difference in timing adherence pre- to post-intervention between the 2 groups) was 13.4% (CI 5.1 to 21.7), p = 0.002. Parkinson motor scores did not change significantly (active group 0.1, CI -3.4 to 3.7) versus controls (4.5, CI 1.6 to 7.1), p = 0.06.</p> <p>Conclusion</p> <p>Timing adherence, but not motor scores, improves by providing patients with extra information. Therapy timing is of potential importance in Parkinson's disease management.</p> <p>Trial registration number</p> <p>NCT00361205</p

Demultiplexing of single-cell RNA-sequencing data using interindividual variation in gene expression

Motivation: Pooled designs for single-cell RNA sequencing, where many cells from distinct samples are processed jointly, offer increased throughput and reduced batch variation. This study describes expression-aware demultiplexing (EAD), a computational method that employs differential co-expression patterns between individuals to demultiplex pooled samples without any extra experimental steps. Results: We use synthetic sample pools and show that the top interindividual differentially co-expressed genes provide a distinct cluster of cells per individual, significantly enriching the regulation of metabolism. Our application of EAD to samples of six isogenic inbred mice demonstrated that controlling genetic and environmental effects can solve interindividual variations related to metabolic pathways. We utilized 30 samples from both sepsis and healthy individuals in six batches to assess the performance of classification approaches. The results indicate that combining genetic and EAD results can enhance the accuracy of assignments (Min. 0.94, Mean 0.98, Max. 1). The results were enhanced by an average of 1.4% when EAD and barcoding techniques were combined (Min. 1.25%, Median 1.33%, Max. 1.74%). Furthermore, we demonstrate that interindividual differential co-expression analysis within the same cell type can be used to identify cells from the same donor in different activation states. By analysing single-nuclei transcriptome profiles from the brain, we demonstrate that our method can be applied to nonimmune cells. Availability and implementation: EAD workflow is available at https://isarnassiri.github.io/scDIV/ as an R package called scDIV (acronym for single-cell RNA-sequencing data demultiplexing using interindividual variations)

Structural and non-coding variants increase the diagnostic yield of clinical whole genome sequencing for rare diseases

Background Whole genome sequencing is increasingly being used for the diagnosis of patients with rare diseases. However, the diagnostic yields of many studies, particularly those conducted in a healthcare setting, are often disappointingly low, at 25–30%. This is in part because although entire genomes are sequenced, analysis is often confined to in silico gene panels or coding regions of the genome. Methods We undertook WGS on a cohort of 122 unrelated rare disease patients and their relatives (300 genomes) who had been pre-screened by gene panels or arrays. Patients were recruited from a broad spectrum of clinical specialties. We applied a bioinformatics pipeline that would allow comprehensive analysis of all variant types. We combined established bioinformatics tools for phenotypic and genomic analysis with our novel algorithms (SVRare, ALTSPLICE and GREEN-DB) to detect and annotate structural, splice site and non-coding variants. Results Our diagnostic yield was 43/122 cases (35%), although 47/122 cases (39%) were considered solved when considering novel candidate genes with supporting functional data into account. Structural, splice site and deep intronic variants contributed to 20/47 (43%) of our solved cases. Five genes that are novel, or were novel at the time of discovery, were identified, whilst a further three genes are putative novel disease genes with evidence of causality. We identified variants of uncertain significance in a further fourteen candidate genes. The phenotypic spectrum associated with RMND1 was expanded to include polymicrogyria. Two patients with secondary findings in FBN1 and KCNQ1 were confirmed to have previously unidentified Marfan and long QT syndromes, respectively, and were referred for further clinical interventions. Clinical diagnoses were changed in six patients and treatment adjustments made for eight individuals, which for five patients was considered life-saving. Conclusions Genome sequencing is increasingly being considered as a first-line genetic test in routine clinical settings and can make a substantial contribution to rapidly identifying a causal aetiology for many patients, shortening their diagnostic odyssey. We have demonstrated that structural, splice site and intronic variants make a significant contribution to diagnostic yield and that comprehensive analysis of the entire genome is essential to maximise the value of clinical genome sequencing