transcriptional profiling
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2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Charlotte A. James ◽  
Yuexin Xu ◽  
Melissa S. Aguilar ◽  
Lichen Jing ◽  
Erik D. Layton ◽  
...  

AbstractT cells recognize mycobacterial glycolipid (mycolipid) antigens presented by CD1b molecules, but the role of CD4 and CD8 co-receptors in mycolipid recognition is unknown. Here we show CD1b-mycolipid tetramers reveal a hierarchy in which circulating T cells expressing CD4 or CD8 co-receptor stain with a higher tetramer mean fluorescence intensity than CD4-CD8- T cells. CD4+ primary T cells transduced with mycolipid-specific T cell receptors bind CD1b-mycolipid tetramer with a higher fluorescence intensity than CD8+ primary T cells. The presence of either CD4 or CD8 also decreases the threshold for interferon-γ secretion. Co-receptor expression increases surface expression of CD3ε, suggesting a mechanism for increased tetramer binding and activation. Targeted transcriptional profiling of mycolipid-specific T cells from individuals with active tuberculosis reveals canonical markers associated with cytotoxicity among CD8+ compared to CD4+ T cells. Thus, expression of co-receptors modulates T cell receptor avidity for mycobacterial lipids, leading to in vivo functional diversity during tuberculosis disease.


Author(s):  
Tingjun Liu ◽  
Ruiqi Yang ◽  
Jiani Zhou ◽  
Xianjun Lu ◽  
Zijian Yuan ◽  
...  

Interspecies coaggregation promotes transcriptional changes in oral bacteria, affecting bacterial pathogenicity. Streptococcus gordonii (S. gordonii) and Fusobacterium nucleatum (F. nucleatum) are common oral inhabitants. The present study investigated the transcriptional profiling of S. gordonii and F. nucleatum subsp. polymorphum in response to the dual-species coaggregation using RNA-seq. Macrophages were infected with both species to explore the influence of bacterial coaggregation on both species’ abilities to survive within macrophages and induce inflammatory responses. Results indicated that, after the 30-min dual-species coaggregation, 116 genes were significantly up-regulated, and 151 genes were significantly down-regulated in S. gordonii; 97 genes were significantly down-regulated, and 114 genes were significantly up-regulated in F. nucleatum subsp. polymorphum. Multiple S. gordonii genes were involved in the biosynthesis and export of cell-wall proteins and carbohydrate metabolism. F. nucleatum subsp. polymorphum genes were mostly associated with translation and protein export. The coaggregation led to decreased expression levels of genes associated with lipopolysaccharide and peptidoglycan biosynthesis. Coaggregation between S. gordonii and F. nucleatum subsp. polymorphum significantly promoted both species’ intracellular survival within macrophages and attenuated the production of pro-inflammatory cytokines IL-6 and IL-1β. Physical interactions between these two species promoted a symbiotic lifestyle and repressed macrophage’s killing and pro-inflammatory responses.


2022 ◽  
Vol 12 ◽  
Author(s):  
Jing Zhang ◽  
Jingjing Zhu ◽  
Genhui Zheng ◽  
Qianyu Wang ◽  
Xiaorui Li ◽  
...  

Chimeric antigen receptor (CAR) T cells targeting CD19 antigen have produced remarkable clinical outcomes for cancer patients. However, identifying measures to enhance effector function remains one of the most challenging issues in CD19-targeted immunotherapy. Here, we report a novel approach in which a microRNA (miRNA) or short-hairpin RNA (shRNA) cassette was integrated into CAR-expressing retroviral vectors. Using this system, we generated anti-CD19 CAR-T cells co-expressing miR155 or LSD1 shRNA and found that anti-CD19 CAR-T cells with miR155 upregulation or LSD1 downregulation exhibited increased anti-tumor functions in vitro and in vivo. Transcriptional profiling analysis by RNA sequencing revealed the targets of miR155 and LSD1 in anti-CD19 CAR-T cells. Our experiments indicated that introduction of miRNA or shRNA expression into anti-CD19 CAR T-cells might be an effective strategy to improve the anti-tumor effects of CAR-T cell therapy.


2021 ◽  
Author(s):  
Hannah E Jones ◽  
Kelsey A Abrams ◽  
Julie A Siegenthaler

Fibroblasts are found associated with blood vessels in various locations across the CNS: in the meninges, the choroid plexus, and in the parenchyma within perivascular spaces. CNS fibroblasts have been characterized using transcriptional profiling and a Col1a1-GFP mouse line used to identify CNS fibroblasts in vivo. However, current methods for visualizing CNS fibroblasts are lacking and, in particular, prevent adequate assessment of fibroblast-vessel interactions. Here, we describe methods for whole mount visualization of meningeal and choroid plexus fibroblasts, and optical tissue clearing methods for visualization of parenchymal vessel-associated fibroblasts. Importantly, these techniques can be combined with immunohistochemistry methods for labeling different cell types in the meninges and blood vasculature as well as EdU-based cell proliferation assays. These methods are ideal for visualization of vessel-fibroblast interactions in these CNS structures and provide significant improvement over traditional sectioning and staining methods. We expect these methods will advance studies of CNS fibroblast development and functions in homeostasis, injury, and disease.


Life ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 30
Author(s):  
Konstantina Athanasopoulou ◽  
Michaela A. Boti ◽  
Panagiotis G. Adamopoulos ◽  
Paraskevi C. Skourou ◽  
Andreas Scorilas

Although next-generation sequencing (NGS) technology revolutionized sequencing, offering a tremendous sequencing capacity with groundbreaking depth and accuracy, it continues to demonstrate serious limitations. In the early 2010s, the introduction of a novel set of sequencing methodologies, presented by two platforms, Pacific Biosciences (PacBio) and Oxford Nanopore Sequencing (ONT), gave birth to third-generation sequencing (TGS). The innovative long-read technologies turn genome sequencing into an ease-of-handle procedure by greatly reducing the average time of library construction workflows and simplifying the process of de novo genome assembly due to the generation of long reads. Long sequencing reads produced by both TGS methodologies have already facilitated the decipherment of transcriptional profiling since they enable the identification of full-length transcripts without the need for assembly or the use of sophisticated bioinformatics tools. Long-read technologies have also provided new insights into the field of epitranscriptomics, by allowing the direct detection of RNA modifications on native RNA molecules. This review highlights the advantageous features of the newly introduced TGS technologies, discusses their limitations and provides an in-depth comparison regarding their scientific background and available protocols as well as their potential utility in research and clinical applications.


2021 ◽  
Author(s):  
Jeong Hyun Lee ◽  
Henry J. Sutton ◽  
Christopher A. Cottrell ◽  
Ivy Phung ◽  
Gabriel Ozorowski ◽  
...  

Germinal centers (GCs) are the engines of antibody evolution. Using HIV Env protein immunogen priming in rhesus monkeys (RM) followed by a long period without further immunization, we demonstrate GC B cells (BGC) lasted at least 6 months (29 weeks), all the while maintaining rapid proliferation. A 186-fold BGC cell increase was present by week 10 compared to a conventional immunization. Single cell transcriptional profiling revealed that both light zone and dark zone GC states were sustained throughout the 6 months. Antibody somatic hypermutation (SHM) of BGC cells continued to accumulate throughout the 29 week priming period, with evidence of selective pressure. Additionally, Env-binding BGC cells were still 49-fold above baseline 29 weeks after immunization, suggesting that they could be active for significantly longer periods of time. High titers of HIV neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing significant immunodominance challenges for B cells, among other difficulties. Memory B cells (BMem) generated under these long priming conditions had higher levels of SHM, and both BMem cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous BGC cell lineage phylogenies spanning the >6-month GC period were identified, demonstrating continuous GC activity and selection for at least 191 days, with no additional antigen exposure. A long prime, adjuvanted, slow delivery (12-day) immunization approach holds promise for difficult vaccine targets, and suggests that patience can have great value for tuning GCs to maximize antibody responses.


2021 ◽  
Vol 17 (12) ◽  
pp. e1010140
Author(s):  
Aracely A. Romero ◽  
Sarah A. Cobb ◽  
Julie N. R. Collins ◽  
Steven A. Kliewer ◽  
David J. Mangelsdorf ◽  
...  

Schistosomes infect over 200 million of the world’s poorest people, but unfortunately treatment relies on a single drug. Nuclear hormone receptors are ligand-activated transcription factors that regulate diverse processes in metazoans, yet few have been functionally characterized in schistosomes. During a systematic analysis of nuclear receptor function, we found that an FTZ-F1-like receptor was essential for parasite survival. Using a combination of transcriptional profiling and chromatin immunoprecipitation (ChIP), we discovered that the micro-exon gene meg-8.3 is a transcriptional target of SmFTZ-F1. We found that both Smftz-f1 and meg-8.3 are required for esophageal gland maintenance as well as integrity of the worm’s head. Together, these studies define a new role for micro-exon gene function in the parasite and suggest that factors associated with the esophageal gland could represent viable therapeutic targets.


2021 ◽  
Author(s):  
Hazem F. M. Abdelaal ◽  
Tyler C. Thacker ◽  
Bishoy Wadie ◽  
Mitchell V. Palmer ◽  
Adel M. Talaat

Bovine tuberculosis, caused by Mycobacterium tuberculosis var. bovis ( M. bovis ), is an important enzootic disease affecting mainly cattle, worldwide. Despite the implementation of national campaigns to eliminate the disease, bovine tuberculosis remains recalcitrant to eradication in several countries. Characterizing the host response to M. bovis infection is crucial for understanding the immunopathogenesis of the disease and for developing better control strategies. To profile the host responses to M. bovis infection, we analyzed the transcriptome of whole blood cells collected from experimentally infected calves with a virulent strain of M. bovis using RNA transcriptome sequencing (RNAseq). Comparative analysis of calf transcriptomes at early (8 weeks) vs. late (20 weeks) aerosol infection with M. bovis revealed divergent and unique profile for each stage of infection. Notably, at the early time point, transcriptional upregulation was observed among several of the top-ranking canonical pathways involved in T-cell chemotaxis. At the late time point, enrichment in the cell mediated cytotoxicity (e.g. Granzyme B) was the predominant host response. These results showed significant change in bovine transcriptional profiles and identified networks of chemokine receptors and monocyte chemoattractant protein (CCL) co-regulated genes that underline the host-mycobacterial interactions during progression of bovine tuberculosis in cattle. Further analysis of the transcriptomic profiles identified potential biomarker targets for early and late phases of tuberculosis in cattle. Overall, the identified profiles better characterized identified novel immunomodulatory mechanisms and provided a list of targets for further development of potential diagnostics for tuberculosis in cattle.


2021 ◽  
Vol 2021 ◽  
pp. 1-14
Author(s):  
Feng Li ◽  
Jin-Yu Sun ◽  
Li-Da Wu ◽  
Qiang Qu ◽  
Zhen-Ye Zhang ◽  
...  

Background. There are few biomarkers with an excellent predictive value for postacute myocardial infarction (MI) patients who developed heart failure (HF). This study aimed to screen candidate biomarkers to predict post-MI HF. Methods. This is a secondary analysis of a single-center cohort study including nine post-MI HF patients and eight post-MI patients who remained HF-free over a 6-month follow-up. Transcriptional profiling was analyzed using the whole blood samples collected at admission, discharge, and 1-month follow-up. We screened differentially expressed genes and identified key modules using weighted gene coexpression network analysis. We confirmed the candidate biomarkers using the developed external datasets on post-MI HF. The receiver operating characteristic curves were created to evaluate the predictive value of these candidate biomarkers. Results. A total of 6,778, 1,136, and 1,974 genes (dataset 1) were differently expressed at admission, discharge, and 1-month follow-up, respectively. The white and royal blue modules were most significantly correlated with post-MI HF (dataset 2). After overlapping dataset 1, dataset 2, and external datasets (dataset 3), we identified five candidate biomarkers, including FCGR2A, GSDMB, MIR330, MED1, and SQSTM1. When GSDMB and SQSTM1 were combined, the area under the curve achieved 1.00, 0.85, and 0.89 in admission, discharge, and 1-month follow-up, respectively. Conclusions. This study demonstrates that FCGR2A, GSDMB, MIR330, MED1, and SQSTM1 are the candidate predictive biomarker genes for post-MI HF, and the combination of GSDMB and SQSTM1 has a high predictive value.


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