Biology subjects

Klein, J. A.

Publications and source records attributed to Klein, J. A..

8 recordsLinked to original sources

bioRxiv2026

Aberrant chromatin remodeling influences human neural cell fate change in Trisomy 21

Correct neural progenitor cell (NPC) fate specification is essential to produce the full complement of neurons and glia needed for proper brain structure and function. Neurodevelopmental disorders, including the autosomal aneuploidy Down syndrome (DS), or Trisomy 21 (T21), are frequently associated with impaired cell fate decisions which ultimately drive differences in overall brain size and cell type composition through unknown mechanisms. To uncover mechanisms driving altered NPC fate in T21, we leverage paired single-nuclei transcriptomic and epigenomic analyses of human induced pluripotent stem cell (iPSC)-derived NPCs and their differentiated progeny coupled with in depth clonal cell fate, cell cycle, and proteomic analyses. Here we show that T21 NPCs fail to activate an orchestrated neurogenic program during the earliest stages of fate specification, instead maintaining a repressive chromatin structure over neurogenic loci, leading to reduced neurogenesis and continued NPC proliferation. We identify novel enrichment of the repressive histone mark H3K27me3 at fate instructive genes dysregulated across diverse cell and tissue types in T21, with corresponding genome-wide changes in H3K27me3 binding in T21 NPCs. Moreover, pharmacological treatment with an inhibitor of the Polycomb repressive complex 2 (PRC2) which catalyzes H3K27 methylation, is sufficient to partially restore neurogenesis in T21 cells. Collectively, our analyses reveal a chromatin mechanism influencing neurogenic defects in T21.

neuroscience↗

bioRxiv2024

Pathogenic diversification of the gut commensal Providencia alcalifaciens via acquisition of a second type III secretion system

Providencia alcalifaciens is a Gram-negative bacterium found in a wide variety of water and land environments and organisms. It has been isolated as part of the gut microbiome of animals and insects, as well as from stool samples of patients with diarrhea. Specific P. alcalifaciens strains encode gene homologs of virulence factors found in other pathogenic members of the same Enterobacterales order, such as Salmonella enterica serovar Typhimurium and Shigella flexneri. Whether these genes are also pathogenic determinants in P. alcalifaciens is not known. Here we have used P. alcalifaciens 205/92, a clinical isolate, with in vitro and in vivo infection models to investigate P. alcalifaciens-host interactions at the cellular level. Our particular focus was the role of two type III secretion systems (T3SS) belonging to the Inv-Mxi/Spa family. T3SS1b is widespread in Providencia spp. and encoded on the chromosome. T3SS1a is encoded on a large plasmid that is present in a subset of P. alcalifaciens strains, which are primarily isolates from diarrheal patients. Using a combination of electron and fluorescence microscopy and gentamicin protection assays we show that P. alcalifaciens 205/92 is internalized into eukaryotic cells, rapidly lyses its internalization vacuole and proliferates in the cytosol. This triggers caspase-4 dependent inflammasome responses in gut epithelial cells. The requirement for the T3SS1a in entry, vacuole lysis and cytosolic proliferation is host-cell type specific, playing a more prominent role in human intestinal epithelial cells as compared to macrophages. In a bovine ligated intestinal loop model, P. alcalifaciens colonizes the intestinal mucosa, inducing mild epithelial damage with negligible fluid accumulation. No overt role for T3SS1a or T3SS1b was seen in the calf infection model. However, T3SS1b was required for the rapid killing of Drosophila melanogaster. We propose that the acquisition of two T3SS by horizontal gene transfer has allowed P. alcalifaciens to diversify its host range, from a highly virulent pathogen of insects to an opportunistic gastrointestinal pathogen of animals.

microbiology↗

bioRxiv2022

Asynchronous excitatory neuron development in an isogenic cortical spheroid model of Down syndrome

The intellectual disability (ID) in Down syndrome (DS) is thought to result from a variety of developmental deficits such as alterations in neural progenitor division, neurogenesis, gliogenesis, cortical architecture, and reduced cortical volume. However, the molecular processes underlying these neurodevelopmental changes are still elusive, preventing an understanding of the mechanistic basis of ID in DS. In this study, we used a pair of isogenic (trisomic and euploid) induced pluripotent stem cell (iPSC) lines to generate cortical spheroids (CS) that model the impact of trisomy 21 on brain development. CS contain neurons, astrocytes, and oligodendrocytes and they are widely used to approximate early neurodevelopment. Using single cell RNA sequencing (scRNA-seq), we uncovered cell type-specific transcriptomic changes in the trisomic CS. In particular, we found that excitatory neuron populations were most affected and that a specific population of cells with a transcriptomic profile resembling layer IV cortical neurons displayed the most profound divergence in developmental trajectory between trisomic and euploid genotypes. We also identified candidate genes potentially driving the developmental asynchrony between trisomic and euploid excitatory neurons. Direct comparison between the current isogenic CS scRNA-seq data and previously published datasets revealed several recurring differentially expressed genes between DS and control samples. Altogether, our study highlights the power and importance of cell type-specific analyses within a defined genetic background, coupled with broader examination of mixed samples, to comprehensively evaluate cellular phenotypes in the context of DS.

neuroscience↗

bioRxiv2021

Sonic Hedgehog Pathway Modulation Normalizes Expression of Olig2 in Rostrally Patterned NPCs with Trisomy 21

The intellectual disability found in people with Down syndrome (DS) is associated with a decrease in white matter in the central nervous system. To study the mechanism of this myelination deficit, we differentiated two isogenic lines of induced pluripotent stem cells (iPSCs) derived from people with DS into brain-like and spinal cord-like neural progenitor cells (NPCs) and promoted a transition towards oligodendroglial fate by activating the Sonic hedgehog (SHH) pathway. In the spinal cord-like trisomic cells, we found no difference in expression of OLIG2 or NKX2.2, two transcription factors essential for commitment to the oligodendrocyte (OL) lineage. However, in the brain-like trisomic NPCs, OLIG2 is significantly upregulated and is associated with reduced expression of NKX2.2. We found that this gene dysregulation and block in NPC transition can be normalized by increasing the concentration of a SHH pathway agonist (SAG) during differentiation. These results underscore the importance of regional and cell type differences in gene expression in DS and demonstrate that modulation of SHH signaling in trisomic cells can rescue an early perturbed step in neural lineage specification in DS.

neuroscience↗

bioRxiv2021

Gut regulatory T cells mediate immunological tolerance in Salmonella Typhimurium-infected superspreader hosts by suppressing cytotoxic activity of T cells.

Superspreader hosts carry out most pathogen transmission events and are often disease tolerant since they remain asymptomatic despite high pathogen burdens. Here we describe the superspreader immune state that allows for disease tolerance. In a model of Salmonella infection, superspreader mice develop colitis with robust CD4+ and CD8+ T-cell responses, however, they remain asymptomatic. We found that superspreaders have significantly more regulatory T cells (Tregs) in the distal gut compared to non-superspreader infected hosts. Surprisingly, the depletion of Tregs did not induce pathogen clearance but rather exacerbated weight loss, increased gut inflammation, and compromised epithelial intestinal barrier. This loss of tolerance correlated with dramatic increases in cytotoxic CD4+ and CD8+ T cells. Interestingly, CD4 neutralization in Tregs-depleted superspreaders was sufficient to rescue tolerance. Our results indicate that Tregs play a crucial role in maintaining immunologic tolerance in the guts of superspreader mice by suppressing cytotoxic CD4+ and CD8+ T-cell activities. AUTHOR SUMMARYSuperspreader hosts are the main cause of disease transmission and a very important public health concern. Here, we evaluated the immunological tolerance of the Salmonella infected superspreaders in a mouse model. By manipulating Tregs, we demonstrated the immunological mechanism from the host to maintain health status and high pathogen burden. Tregs depletion in the superspreaders led to severe disease, with damage of the intestinal epithelia, and high morbidity without having any effect on shedding and systemic Salmonella burden. Furthermore, we demonstrated that the damage of the intestinal epithelia was related to cytotoxic activity of T cells. When Tregs were depleted, CD8+ T cells produced high levels of granzyme B and perforin. CD8+ T cells neutralization in Tregs depleted mice led to increased cytotoxic CD4+ T cells. Interestingly, neutralization of CD4+ T cells in the Tregs depleted mice led to a reduction in the CD8+ T cells producing granzyme B and it was sufficient to rescue host tolerance in this model. We demonstrate for the first time that cytotoxic CD4+ T cells damage the epithelial intestinal barrier and contribute to loss of tolerance in the context of a superspreader host. These findings open new perspectives to understand mechanisms of tolerance in the intestine of a superspreader host.

immunology↗

bioRxiv2021

Expanding N-Glycopeptide Identifications by Fragmentation Prediction and Glycome Network Smoothing

Accurate glycopeptide identification in mass spectrometry-based glycoproteomics is a challenging problem at scale. Recent innovation has been made in increasing the scope and accuracy of glycopeptide identifications, with more precise uncertainty estimates for each part of the structure. We present a layered approach to glycopeptide fragmentation modeling that improves N-glycopeptide identification in samples without compromising identification quality, and a site-specific method to increase the depth of the glycoproteome confidently identifiable even further. We demonstrate our techniques on a pair of previously published datasets, showing the performance gains at each stage of optimization, as well as its flexibility in glycome definition and search space complexity. These techniques are provided in the open-source glycomics and glycoproteomics platform GlycReSoft available at https://github.com/mobiusklein/glycresoft.

bioinformatics↗

bioRxiv2020

Assignment of coronavirus spike protein site-specific glycosylation using GlycReSoft

Widely-available LC-MS instruments and methods allow users to acquire glycoproteomics data. Complex glycans, however, add a dimension of complexity to the data analysis workflow. In a sense, complex glycans are post-translationally modified post-translational modifications, reflecting a series of biosynthetic reactions in the secretory pathway that are spatially and temporally regulated. One problem is that complex glycan is micro-heterogeneous, multiplying the complexity of the proteome. Another is that glycopeptide glycans undergo dissociation during tandem MS that must be considered for tandem MS interpretation algorithms and quantitative tools. Fortunately, there are a number of algorithmic tools available for analysis of glycoproteomics LC-MS data. We summarize the principles for glycopeptide data analysis and show use of our GlycReSoft tool to analyze SARS-CoV-2 spike protein site-specific glycosylation.

bioinformatics↗

bioRxiv2020

Protocol for analysis of glycoproteomics LC-MS data using GlycReSoft

Summary/AbstractThe GlycReSoft software tool allows users to process glycoproteomics LC-MS data sets. The tool accepts proteomics database search results or a user-defined list of proteins in the sample. GlycReSoft processes LC-MS data to yield deconvoluted exact mass values. The user has the option to import a list of theoretical glycans from an external database, a curated glycan list, or a measured glycome. The tool assembles a list of theoretical glycopeptides from the lists of theoretical glycans and proteins, respectively. The program then scores the tandem mass spectra in the LC-MS data files and provides graphical views of the identified glycopeptides for each protein in the sample, and the set of glycoforms identified for each peptide sequence.

biochemistry↗