Risk of meningomyelocele mediated by the common 22q11.2 deletion.

Meningomyelocele is one of the most severe forms of neural tube defects (NTDs) and the most frequent structural birth defect of the central nervous system. We assembled the Spina Bifida Sequencing Consortium to identify causes. Exome and genome sequencing of 715 parent-offspring trios identified six patients with chromosomal 22q11.2 deletions, suggesting a 23-fold increased risk compared with the general population. Furthermore, analysis of a separate 22q11.2 deletion cohort suggested a 12- to 15-fold increased NTD risk of meningomyelocele. The loss of Crkl, one of several neural tube-expressed genes within the minimal deletion interval, was sufficient to replicate NTDs in mice, where both penetrance and expressivity were exacerbated by maternal folate deficiency. Thus, the common 22q11.2 deletion confers substantial meningomyelocele risk, which is partially alleviated by folate supplementation.

Cell-type-resolved mosaicism reveals clonal dynamics of the human forebrain.

Debate remains around the anatomical origins of specific brain cell subtypes and lineage relationships within the human forebrain1-7. Thus, direct observation in the mature human brain is critical for a complete understanding of its structural organization and cellular origins. Here we utilize brain mosaic variation within specific cell types as distinct indicators for clonal dynamics, denoted as cell-type-specific mosaic variant barcode analysis. From four hemispheres and two different human neurotypical donors, we identified 287 and 780 mosaic variants, respectively, that were used to deconvolve clonal dynamics. Clonal spread and allele fractions within the brain reveal that local hippocampal excitatory neurons are more lineage-restricted than resident neocortical excitatory neurons or resident basal ganglia GABAergic inhibitory neurons. Furthermore, simultaneous genome transcriptome analysis at both a cell-type-specific and a single-cell level suggests a dorsal neocortical origin for a subgroup of DLX1+ inhibitory neurons that disperse radially from an origin shared with excitatory neurons. Finally, the distribution of mosaic variants across 17 locations within one parietal lobe reveals that restriction of clonal spread in the anterior-posterior axis precedes restriction in the dorsal-ventral axis for both excitatory and inhibitory neurons. Thus, cell-type-resolved somatic mosaicism can uncover lineage relationships governing the development of the human forebrain.

Cell-type-resolved somatic mosaicism reveals clonal dynamics of the human forebrain.

Debate remains around anatomic origins of specific brain cell subtypes and lineage relationships within the human forebrain. Thus, direct observation in the mature human brain is critical for a complete understanding of the structural organization and cellular origins. Here, we utilize brain mosaic variation within specific cell types as distinct indicators for clonal dynamics, denoted as cell-type-specific Mosaic Variant Barcode Analysis. From four hemispheres from two different human neurotypical donors, we identified 287 and 780 mosaic variants (MVs), respectively that were used to deconvolve clonal dynamics. Clonal spread and allelic fractions within the brain reveal that local hippocampal excitatory neurons are more lineage-restricted compared with resident neocortical excitatory neurons or resident basal ganglia GABAergic inhibitory neurons. Furthermore, simultaneous genome-transcriptome analysis at both a cell-type-specific and single-cell level suggests a dorsal neocortical origin for a subgroup of DLX1+ inhibitory neurons that disperse radially from an origin shared with excitatory neurons. Finally, the distribution of MVs across 17 locations within one parietal lobe reveals restrictions of clonal spread in the anterior-posterior axis precedes that of the dorsal-ventral axis for both excitatory and inhibitory neurons. Thus cell-type resolved somatic mosaicism can uncover lineage relationships governing the development of the human forebrain.

Variable fragmentation and ionization of amyloid-beta epimers and isomers.

While the existence of D-amino acids in peptides and proteins has recently been accepted in higher forms of life, their roles and importance are yet to be understood. The lack of analytical methods present for such epimeric and/or isomeric analyses often limits developments in the field. Studies have shown the elevated presence of epimeric and isomeric modifications to amyloid-beta (Aß) peptides extracted from Alzheimer's disease patients. These modifications most frequently occur through aspartic acid and serine residues. Because such peptides are indistinguishable by mass alone, selective liquid chromatography tandem mass spectrometry analysis is required to differentiate such peptides. Herein, we examine MS/MS of tryptic fragments of Aß peptides containing D-Asp, L-iso-Asp, D-iso-Asp, and/or D-Ser modifications. Peptide ionizability and fragmentation are explored through selected reaction monitoring, selected ion monitoring, and product ion scan. The results show the variability of ionization and fragmentation for many "identical mass peptides" and how these differences can affect the analysis of isomeric and epimeric peptides.

Antibody binding of amyloid beta peptide epimers/isomers and ramifications for immunotherapies and drug development.

Extracellular deposition of amyloid beta (Aß) peptide is a contributing factor of Alzheimer's disease (AD). Considerable effort has been expended to create effective antibodies, or immunotherapies, targeting Aß peptides. A few immunotherapies are thought to provide some benefit. It is possible that a contributing factor to the responses of such therapies may be the presence of modified, or aberrant, Aß peptides found in AD patients. These aberrations include the isomerization and epimerization of L-Asp and L-Ser residues to form D-Asp, L/D-isoAsp, and D-Ser residues, respectively. An effective methodology is essential to isolate all Aß peptides and then to quantify and locate the aberrant amino acids. Modifications to Aß peptides may elevate the deposition of Aß plaques and/or contribute to the neurodegeneration in AD patients, and may alter the binding affinity to antibodies. Herein, we used immunoprecipitation to examine the binding affinity of four antibodies against 18 epimeric and/or isomeric Aß peptides compared to wild type (all L) Aß peptide. Tandem mass spectrometry was used as a detection method, which also was found to produce highly variable results for epimeric and/or isomeric Aß.

One-Pot Synthesis of Novel 2-Imino-5-Arylidine-Thiazolidine Analogues and Evaluation of Their Anti-Proliferative Activity against MCF7 Breast Cancer Cell Line.

An efficient surface-mediated synthetic method to facilitate access to a novel class of thiazolidines is described. The rationale behind the design of the targeted thiazolidines was to prepare stable thiazolidine analogues and evaluate their anti-proliferative activity against a breast cancer cell line (MCF7). Most of the synthesized analogues exhibited increased potency ranging from 2-15-fold higher compared to the standard reference, cisplatin. The most active thiazolidines contain a halogenated or electron withdrawing group attached to the N-phenyl ring of exocyclic 2-imino group. However, combination of the two substituents did not enhance the activity. The anti-proliferative activity was measured in terms of IC50 values using an MTT assay.

The human liver microenvironment shapes the homing and function of CD4+ T-cell populations.

OBJECTIVE: Tissue-resident memory T cells (TRM) are vital immune sentinels that provide protective immunity. While hepatic CD8+ TRM have been well described, little is known about the location, phenotype and function of CD4+ TRM. DESIGN: We used multiparametric flow cytometry, histological assessment and novel human tissue coculture systems to interrogate the ex vivo phenotype, function and generation of the intrahepatic CD4+ T-cell compartment. We also used leukocytes isolated from human leukocyte antigen (HLA)-disparate liver allografts to assess long-term retention. RESULTS: Hepatic CD4+ T cells were delineated into three distinct populations based on CD69 expression: CD69-, CD69INT and CD69HI. CD69HICD4+ cells were identified as tissue-resident CD4+ T cells on the basis of their exclusion from the circulation, phenotypical profile (CXCR6+CD49a+S1PR1-PD-1+) and long-term persistence within the pool of donor-derived leukcoocytes in HLA-disparate liver allografts. CD69HICD4+ T cells produced robust type 1 polyfunctional cytokine responses on stimulation. Conversely, CD69INTCD4+ T cells represented a more heterogenous population containing cells with a more activated phenotype, a distinct chemokine receptor profile (CX3CR1+CXCR3+CXCR1+) and a bias towards interleukin-4 production. While CD69INTCD4+ T cells could be found in the circulation and lymph nodes, these cells also formed part of the long-term resident pool, persisting in HLA-mismatched allografts. Notably, frequencies of CD69INTCD4+ T cells correlated with necroinflammatory scores in chronic hepatitis B infection. Finally, we demonstrated that interaction with hepatic epithelia was sufficient to generate CD69INTCD4+ T cells, while additional signals from the liver microenvironment were required to generate liver-resident CD69HICD4+ T cells. CONCLUSIONS: High and intermediate CD69 expressions mark human hepatic CD4+ TRM and a novel functionally distinct recirculating population, respectively, both shaped by the liver microenvironment to achieve diverse immunosurveillance.

Resolving the polygenic aetiology of a late onset combined immune deficiency caused by NFKB1 haploinsufficiency and modified by PIK3R1 and TNFRSF13B variants.

Genetic variants in PIK3CD, PIK3R1 and NFKB1 cause the primary immune deficiencies, activated PI3Kδ syndrome (APDS) 1, APDS2 and NFκB1 haploinsufficiency, respectively. We have identified a family with known or potentially pathogenic variants NFKB1, TNFRSF13B and PIK3R1. The study's aim was to describe their associated immune and cellular phenotypes and compare with individuals with monogenic disease. NFκB1 pathway function was measured by immunoblotting and PI3Kδ pathway activity by phospho-flow cytometry. p105/p50 expression was absent in two individuals but elevated pS6 only in the index case. Transfection of primary T cells demonstrated increased basal pS6 signalling due to mutant PIK3R1, but not mutant NFKB1 or their wildtype forms. We report on the presence of pathogenic variant NFKB1, with likely modifying variants in TNFRSF13B and PIK3R1 in a family. We describe immune features of both NFκB1 haploinsufficiency and APDS2, and the inhibition of excessive PI3K signalling by rapamycin in vitro.

The Role of B Cells in Adult and Paediatric Liver Injury.

B lymphocytes are multitasking cells that direct the immune response by producing pro- or anti-inflammatory cytokines, by presenting processed antigen for T cell activation and co-stimulation, and by turning into antibody-secreting cells. These functions are important to control infection in the liver but can also exacerbate tissue damage and fibrosis as part of persistent inflammation that can lead to end stage disease requiring a transplant. In transplantation, immunosuppression increases the incidence of lymphoma and often this is of B cell origin. In this review we bring together information on liver B cell biology from different liver diseases, including alcohol-related and metabolic fatty liver disease, autoimmune hepatitis, primary biliary and primary sclerosing cholangitis, viral hepatitis and, in infants, biliary atresia. We also discuss the impact of B cell depletion therapy in the liver setting. Taken together, our analysis shows that B cells are important in the pathogenesis of liver diseases and that further research is necessary to fully characterise the human liver B cell compartment.

Decreased ATM Function Causes Delayed DNA Repair and Apoptosis in Common Variable Immunodeficiency Disorders.

PURPOSE: Common variable immunodeficiency disorders (CVID) is characterized by low/absent serum immunoglobulins and susceptibility to bacterial infection. Patients can develop an infections-only phenotype or a complex disease course with inflammatory, autoimmune, and/or malignant complications. We hypothesized that deficient DNA repair mechanisms may be responsible for the antibody deficiency and susceptibility to inflammation and cancer in some patients. METHODS: Germline variants were identified following targeted sequencing of n = 252 genes related to DNA repair in n = 38 patients. NanoString nCounter PlexSet assay measured gene expression in n = 20 CVID patients and n = 7 controls. DNA damage and apoptosis were assessed by flow cytometry in n = 34 CVID patients and n = 11 controls. RESULTS: Targeted sequencing supported enrichment of rare genetic variants in genes related to DNA repair pathways with novel and rare likely pathogenic variants identified and an altered gene expression signature that distinguished patients from controls and complex patients from those with an infections-only phenotype. Consistent with this, flow cytometric analyses of lymphocytes following DNA damage revealed a subset of CVID patients whose immune cells have downregulated ATM, impairing the recruitment of other repair factors, delaying repair and promoting apoptosis. CONCLUSION: These data suggest that germline genetics and altered gene expression predispose a subset of CVID patients to increased sensitivity to DNA damage and reduced DNA repair capacity.