Distinct Gene Expression Patterns Research Articles

Presenilin-1-Derived Circular RNAs: Neglected Epigenetic Regulators with Various Functions in Alzheimer's Disease.

The presenilin-1 (PSEN1) gene is crucial in developing Alzheimer's disease (AD), a progressive neurodegenerative disorder and the most common cause of dementia. Circular RNAs (circRNAs) are non-coding RNA generated through back-splicing, resulting in a covalently closed circular molecule. This study aimed to investigate PSEN1-gene-derived circular RNAs (circPSEN1s) and their potential functions in AD. Our in silico analysis indicated that circPSEN1s (hsa_circ_0008521 and chr14:73614502-73614802) act as sponge molecules for eight specific microRNAs. Surprisingly, two of these miRNAs (has-mir-4668-5p and has-mir-5584-5p) exclusively interact with circPSEN1s rather than mRNA-PSEN1. Furthermore, the analysis of pathways revealed that these two miRNAs predominantly target mRNAs associated with the PI3K-Akt signaling pathway. With sponging these microRNAs, circPSEN1s were found to protect mRNAs commonly targeted by these miRNAs, including QSER1, BACE2, RNF157, PTMA, and GJD3. Furthermore, the miRNAs sequestered by circPSEN1s have a notable preference for targeting the TGF-β and Hippo signaling pathways. We also demonstrated that circPSEN1s potentially interact with FOXA1, ESR1, HNF1B, BRD4, GATA4, EP300, CBX3, PRDM9, and PPARG proteins. These proteins have a prominent preference for targeting the TGF-β and Notch signaling pathways, where EP300 and FOXA1 have the highest number of protein interactions. Molecular docking analysis also confirms the interaction of these hub proteins and Aβ42 with circPSEN1s. Interestingly, circPSEN1s-targeted molecules (miRNAs and proteins) impacted TGF-β, which served as a shared signaling pathway. Finally, the analysis of microarray data unveiled distinct expression patterns of genes influenced by circPSEN1s (WTIP, TGIF, SMAD4, PPP1CB, and BMPR1A) in the brains of AD patients. In summary, our findings suggested that the interaction of circPSEN1s with microRNAs and proteins could affect the fate of specific mRNAs, interrupt the function of unique proteins, and influence cell signaling pathways, generally TGF-β. Further research is necessary to validate these findings and gain a deeper understanding of the precise mechanisms and significance of circPSEN1s in the context of AD.

Growth retardation and suppression of ubiquitin-dependent catabolic processes in the brackish water clam Corbicula japonica in response to salinity changes and bioaccumulation of toxic heavy metals

The brackish water clam (Corbicula japonica) is constantly exposed to stressful salinity gradients and high levels of heavy metals in the freshwater-saltwater interface of estuary environments, which are introduced from upstream regions and land. To identify the key molecular pathways involved in the response to salinity changes and heavy metal bioaccumulation, we obtained the transcriptomes of C. japonica inhabiting different salinities and heavy metal distributions in Gwangyang Bay (Korea) using RNA sequencing. Among a total of 404,486 assembled unigenes, 5534 differentially expressed genes were identified in C. japonica inhabiting different conditions, 1549 of which were significantly upregulated and 1355 were significantly downregulated. Correlation analyses revealed distinct gene expression patterns between the low and high conditions of salinity and heavy metal bioaccumulation. Functional annotation revealed significant downregulation of genes involved in “ubiquitin-dependent protein catabolic process,” “tricarboxylic acid cycle,” and “intracellular protein transport” in C. japonica from the high condition compared to the low condition. Transcription and translation pathways were significantly enriched in the high condition. Additionally, upon comparison of the low and high conditions by qRT-PCR and proteasome enzyme activity analyses, our findings demonstrated that environmental stress could suppress the ubiquitin-proteasome complex (UPC). Additionally, transcriptomic changes under high salinity stress conditions may be related to an increase in cellular protection by defense enzymes, which leads to more energy being required and a disruption of energy homeostasis. Ultimately, this could cause growth retardation in the clam C. japonica. In summary, this study provides the first evidence of UPC suppression induced by a combination of high salinity and heavy metal bioaccumulation stress in C. japonica, which could compromise the survival and growth of estuarine bivalves.

Polyester microplastic fibers induce mitochondrial damage, apoptosis and oxidative stress in Daphnia carinata, accompanied by changes in apoptotic and ferroptosis pathway

With the widespread utilization of plastic products, microplastics (MPs) have merged as a newfound environmental contaminant in the United States, and the bulk of these MPs in the environment manifest as fibrous structures. Concerns have also been voiced regarding the potential hazards posed by microplastic fibers (MFs). However, research examining the toxicity of MFs, particularly in relation to planktonic organisms, remains severely limited. Meanwhile, polyester fiber materials find extensive applications across diverse industries. As a result, this investigation delved into the toxicology of polyester microplastic fibers (PET-MFs) with a focus on their impact on Daphnia carinata (D. carinata), a freshwater crustacean. Newly hatched D. carinata were subjected to varying concentrations of PET-MFs (0, 50, and 500 MFs/mL) to scrutinize the accumulation of PET-MFs within these organisms and their resultant toxicity. The outcomes revealed that D. carinata was capable of ingesting PET-MFs, leading to diminished rates of survival and reproduction. These effects were accompanied by mitochondrial impairment, heightened mitochondrial count, apoptosis, escalated generation of reactive oxygen species, augmented activity of antioxidant enzymes, and distinct patterns of gene expression. Interestingly, when comparing the group exposed to 50 MFs/mL with the one exposed to 500 MFs/mL, it was observed that the former triggered a more pronounced degree of mitochondrial damage, apoptosis, and oxidative stress. This phenomenon could be attributed to the fact that brief exposure to 500 MFs/mL resulted in greater mortality, eliminating individuals with lower adaptability. Those that survived managed to regulate elevated in vivo reactive oxygen species levels through an increase in glutathione S-transferase content, thereby establishing an adaptive mechanism. Low concentrations did not induce direct mortality, yet PET-MFs continued to inflict harm within the organism. RNA-seq analysis unveiled significant alterations in 279 and 55 genes in the 50 MFs/mL and 500 MFs/mL exposure groups, respectively. Functional enrichment analysis of the 50 MFs/mL group indicated involvement of the apoptosis pathway and ferroptosis pathway in the toxic effects exerted by PET-MFs on D. carinata. This study imparts valuable insights into the toxicological ramifications of PET-MFs on D. carinata, underscoring their potential risks within aquatic ecosystems.

Deciphering early human pancreas development at the single-cell level

Understanding pancreas development can provide clues for better treatments of pancreatic diseases. However, the molecular heterogeneity and developmental trajectory of the early human pancreas are poorly explored. Here, we performed large-scale single-cell RNA sequencing and single-cell assay for transposase accessible chromatin sequencing of human embryonic pancreas tissue obtained from first-trimester embryos. We unraveled the molecular heterogeneity, developmental trajectories and regulatory networks of the major cell types. The results reveal that dorsal pancreatic multipotent cells in humans exhibit different gene expression patterns than ventral multipotent cells. Pancreato-biliary progenitors that generate ventral multipotent cells in humans were identified. Notch and MAPK signals from mesenchymal cells regulate the differentiation of multipotent cells into trunk and duct cells. Notably, we identified endocrine progenitor subclusters with different differentiation potentials. Although the developmental trajectories are largely conserved between humans and mice, some distinct gene expression patterns have also been identified. Overall, we provide a comprehensive landscape of early human pancreas development to understand its lineage transitions and molecular complexity.

Genome-Wide Identification of Fatty Acyl-CoA Reductase (FAR) Genes in Dendrobium catenatum and Their Response to Drought Stress

Dendrobium catenatum is a high-value medicinal plant that is predominantly found in high mountain areas, thriving amidst cliffs and rock crevices. However, its wild resources face constant threats from adverse environmental conditions, especially drought stress. Fatty acyl-CoA reductase (FAR) is crucial in plant drought resistance, but there is a lack of research on FAR genes in D. catenatum. In this study, the FAR family genes were identified from the D. catenatum genome. Their genomic characteristics were investigated using bioinformatics techniques, and their expression patterns in different tissues and under 20% PEG8000 conditions mimicking drought stress were analyzed using quantitative real-time RT-PCR (RT-qPCR). Seven DcFAR genes were identified from the D. catenatum genome. The encoded amino acids range between 377 and 587 aa, with molecular weights between 43.41 and 66.15 kD and isoelectric points between 5.55 and 9.02. Based on the phylogenetic relationships, the FAR family genes were categorized into three subgroups, each with similar conserved sequences and gene structures. The cis-acting elements of the promoter regions were assessed, and the results reveal that the DcFAR upstream promoter region contains multiple stress-related elements, suggesting its potential involvement in abiotic stress responses. The RT-qPCR results show distinct expression patterns of DcFAR genes in various plant tissues. It was observed that the expression of most DcFAR genes was upregulated under drought stress. Among them, the expression levels of DcFAR2, DcFAR3, DcFAR5, and DcFAR7 genes under drought stress were 544-, 193-, 183-, and 214-fold higher compared to the control, respectively. These results indicate that DcFAR2/3/5/7 might play significant roles in D. catenatum drought tolerance. This research offers insight into the function of DcFAR genes and provides theoretical support for breeding drought-resistant D. catenatum varieties.

Epigenomic profiling nominates master transcription factors (TFs) driving sarcomatoid differentiation (SD) of renal cell carcinoma (RCC)

Abstract Background Sarcomatoid differentiation of RCC (sRCC) is associated with poor survival. Recent studies showed marked response of sRCC to immune checkpoint blockade (ICB). While distinctive patterns of gene expression in sRCC have been identified, the gene regulatory programs and TFs that drive SD remain unknown. The aim of this study is to nominate TFs responsible for SD and to investigate their association with the clinical outcomes of patients with RCC. Methods Chromatin immunoprecipitation and sequencing (ChIP-seq) for H3K27ac – a histone modification associated with active regulatory elements – was performed on pathologically reviewed sRCC and non-sRCC samples collected at the Dana-Farber Cancer Institute. Regulatory elements that were differentially active between the two groups were identified based on levels of H3K27ac (Benjamini-Hochberg q<0.01, log-fold change (LFC) threshold=1). Enrichment of specific TF binding motifs at activated regulatory elements in sRCC was assessed using HOMER. Differential gene expression analysis of TFs was performed using DESeq2 on RNA-seq data from TCGA. A Mann-Whitney U test was performed on RNA-seq data from the IMmotion151 and Javelin Renal 101 clinical trials to compare mean expression level of TFs in transcript per million (TPM) in these trials. Patients with non-sRCC enrolled in the IMmotion151 trials were divided into quartiles based on gene expression levels of candidate TFs. Progression-free survival (PFS) was compared between non-sRCC patients stratified by expression quartiles as well as patients with sRCC using a multivariable Cox proportional-hazards model accounting for age and IMDC risk score. To validate these findings, a similar analysis was performed in the Javelin Renal 101 trial. Results We obtained high-quality H3K27ac ChIP-seq profiles for 9 sRCC and 17 non-sRCC samples. We identified 278 candidate regulatory elements with increased H3K27ac levels in sRCC vs. non-sRCC. These regulatory elements were enriched for nucleotide motifs bound by the TFs FOSL1 and E2F7. Differential expression analysis between 48 sRCC vs. 493 non-sRCC samples showed that FOSL1 and E2F7 were significantly upregulated in sRCC vs. non-sRCC (LFC=1.7, q=5e-11; LFC=1.8, q=1.3e-20; resp.). Mean TPMs of FOSL1 and E2F7 were significantly increased in sRCC vs. non-sRCC in IMmotion151 cohort and Javelin Renal 101 (all p<0.001). Among patients who received sunitinib, those with the highest quartile of FOSL1 and E2F7 expression showed significantly shorter PFS in IMmotion151 patients compared to patients with the lowest quartile of expression (HR=1.6, 95%CI=1.3-2.2, p=0.008 & HR=2.6, 95%CI=1.8-3.7, p<0.001; resp.). Furthermore, patients with highest quartile of expression showed similar PFS compared to patients with sRCC (p=0.56 and p=0.64; resp.). These results were validated in the sunitinib arm of the Javelin Renal 101 cohort (Figure). Figure: Kaplan-Meier curves of progression-free survival (PFS) in patients with in the sunitinib arm of Javelin Renal 101 by sarcomatoid differentiation and FOSL1 expression levels. Conclusions This is the first study to characterize the epigenomic landscape of sRCC by integrating ChIP-seq and RNA-seq data. Our findings implicated FOSL1 and E2F7 as transcriptional regulators of SD with prognostic relevance. These TFs seems to be associated with aggressive behavior in non-sRCC as well. Further studies are underway to functionally validate these results.

Jasmonates, gibberellins, and powdery mildew modify cell cycle progression and evoke differential spatiotemporal responses along the barley leaf.

Barley (Hordeum vulgare) is an important cereal crop, and its development, defence, and stress responses are modulated by different hormones including jasmonates (JAs) and the antagonistic gibberellins (GAs). Barley productivity is severely affected by the foliar biotrophic fungal pathogen Blumeria hordei. In this study, primary leaves were used to examine the molecular processes regulating responses to methyl-jasmonate (MeJA) and GA to B. hordei infection along the leaf axis. Flow cytometry, microscopy, and spatiotemporal expression patterns of genes associated with JA, GA, defence, and the cell cycle provided insights on cell cycle progression and on the gradient of susceptibility to B. hordei observed along the leaf. Notably, the combination of B. hordei with MeJA or GA pre-treatment had a different effect on the expression patterns of the analysed genes compared to individual treatments. MeJA reduced susceptibility to B. hordei in the proximal part of the leaf blade. Overall, distinctive spatiotemporal gene expression patterns correlated with different degrees of cell proliferation, growth capacity, responses to hormones, and B. hordei infection along the leaf. Our results highlight the need to further investigate differential spatial and temporal responses to pathogens at the organ, tissue, and cell levels in order to devise effective disease control strategies in crops.

Circulating Osteoprogenitor Cells Have a Mixed Immune and Mesenchymal Progenitor Function in Humans.

Circulating osteoprogenitors (COP) are a population of cells in the peripheral circulation that possess functional and phenotypical characteristics of multipotent stromal cells (MSCs). This population has a solid potential to become an abundant, accessible, and replenishable source of MSCs with multiple potential clinical applications. However, a comprehensive functional characterization of COP cells is still required to test and fully develop their use in clinical settings. This study characterized COP cells by comparing them to bone marrow-derived MSCs (BM-MSCs) and adipose-derived MSCs (ASCs) through detailed transcriptomic and proteomic analyses. We demonstrate that COP cells have a distinct gene and protein expression pattern with a significantly stronger immune footprint, likely owing to their hematopoietic lineage. In addition, regarding progenitor cell differentiation and proliferation pathways, COP cells have a similar expression pattern to BM-MSCs and ASCs. COP cells are a unique but functionally similar population to BM-MSCs and ASCs, sharing their proliferation and differentiation capacity, thus presenting an accessible source of MSCs with strong potential for translational regenerative medicine strategies.

Droplet-based high-throughput single microbe RNA sequencing by smRandom-seq

Bacteria colonize almost all parts of the human body and can differ significantly. However, the population level transcriptomics measurements can only describe the average bacteria population behaviors, ignoring the heterogeneity among bacteria. Here, we report a droplet-based high-throughput single-microbe RNA-seq assay (smRandom-seq), using random primers for in situ cDNA generation, droplets for single-microbe barcoding, and CRISPR-based rRNA depletion for mRNA enrichment. smRandom-seq showed a high species specificity (99%), a minor doublet rate (1.6%), a reduced rRNA percentage (32%), and a sensitive gene detection (a median of ~1000 genes per single E. coli). Furthermore, smRandom-seq successfully captured transcriptome changes of thousands of individual E. coli and discovered a few antibiotic resistant subpopulations displaying distinct gene expression patterns of SOS response and metabolic pathways in E. coli population upon antibiotic stress. smRandom-seq provides a high-throughput single-microbe transcriptome profiling tool that will facilitate future discoveries in microbial resistance, persistence, microbe-host interaction, and microbiome research.

ClpP protease modulates bacterial growth, stress response, and bacterial virulence in Brucella abortus

The process of intracellular proteolysis through ATP-dependent proteases is a biologically conserved phenomenon. The stress responses and bacterial virulence of various pathogenic bacteria are associated with the ATP-dependent Clp protease. In this study, a Brucella abortus 2308 strain, ΔclpP, was constructed to characterize the function of ClpP peptidase. The growth of the ΔclpP mutant strain was significantly impaired in the TSB medium. The results showed that the ΔclpP mutant was sensitive to acidic pH stress, oxidative stress, high temperature, detergents, high osmotic environment, and iron deficient environment. Additionally, the deletion of clpP significantly affected Brucella virulence in macrophage and mouse infection models. Integrated transcriptomic and proteomic analyses of the ΔclpP strain showed that 1965 genes were significantly affected at the mRNA and/or protein levels. The RNA-seq analysis indicated that the ΔclpP strain exhibited distinct gene expression patterns related to energy production and conversion, cell wall/membrane/envelope biogenesis, carbohydrate transport, and metabolism. The iTRAQ analysis revealed that the differentially expressed proteins primarily participated in amino acid transport and metabolism, energy production and conversion, and secondary metabolites biosynthesis, transport and catabolism. This study provided insights into the preliminary molecular mechanism between Clp protease to bacterial growth, stress response, and bacterial virulence in Brucella strains.

Abstract A029: Epigenomic profiling nominates master transcription factors (TFs) driving sarcomatoid differentiation of renal cell carcinoma (RCC)

Abstract Introduction Sarcomatoid RCC (sRCC) is associated with poor prognosis. Recent studies showed promising response rates of sRCC to immune checkpoint blockade (ICB). Although distinct patterns of gene expression in sRCC have been reported, the gene regulatory programs that drive SD remain unknown. This study aims to nominate TFs driving SD and to evaluate their impact on the clinical outcomes of patients (pts) with RCC. Methods Chromatin immunoprecipitation and sequencing (ChIP-seq) for H3K27ac was performed on histologically annotated areas of sRCC and non-sRCC fresh frozen tissue samples collected at Dana-Farber Cancer Institute. Regulatory elements that were differentially active between the two groups were identified (cutoff p<0.01, log-fold change (LFC) threshold=1). Enrichment of specific TF binding motifs at activated regulatory elements in sRCC was assessed using HOMER. Differential gene expression analysis of TFs was performed using DESeq2 on RNA-seq data from TCGA (Benjamini-Hochberg q <0.01, LFC threshold=1). An unpaired student’s t-test was performed to compare mean gene expression levels in transcript per million (TPM) on baseline sRCC and non-sRCC tissue samples from the IMmotion151 trial (sunitinib vs. atezolizumab-bevacizumab in metastatic RCC). Pts enrolled in the IMmotion151 trial were divided into quartiles based on gene expression levels of candidate TFs. Progression-free survival (PFS) was compared between the quartiles using a multivariable Cox proportional-hazards model accounting for age and IMDC risk score. The association of TF expression with the objective radiographic response (ORR) was analyzed using a logistic regression model. Results ChIP-seq was performed on 9 sRCC & 17 non-sRCC samples. We identified 278 candidate regulatory elements with increased H3K27ac levels in sRCC vs. non-sRCC that were enriched for nucleotide motifs bound by the TFs FOSL1 and E2F7. Differential expression analysis between 48 sRCC vs. 493 non-sRCC samples showed that FOSL1 & E2F7 were significantly upregulated in sRCC vs. non-sRCC (LFC=1.7, q=5e-11; LFC=1.8, q=1.3e-20; resp.). Mean TPMs of FOSL1 & E2F7 were significantly increased in sRCC vs. non-sRCC in IMmotion151 cohort (2.5 vs. 1.5 TPM for FOSL1, and 2.9 vs. 1.9 TPM for E2F7, all p<0.001). Pts in the highest quartile of FOSL1 and E2F7 expression showed significantly shorter PFS in IMmotion151 pts receiving sunitinib alone (HR=1.6, 95%CI=1.3-2.2, p=0.008 & HR=2.6, 95%CI=1.8-3.7, p<0.001; resp.). We observed similar results even after controlling for the presence of SD. Increased expression of E2F7 was associated with worse ORR in IMmotion151 pts who received sunitinib alone, even after controlling for the presence of SD (OR=0.24±0.4, p<0.001). Conclusion This is the first study to analyze the epigenomic landscape of sRCC by integrating ChIP-seq and RNA-seq data. Our findings implicated FOSL1 & E2F7 as transcriptional regulators of SD with prognostic relevance. Further studies are underway to validate these results. Citation Format: Karl Semaan, Talal El Zarif, Marc Eid, Valisha Shah, Brad Fortunato, Renee Maria Saliby, Amin H. Nassar, Sarah Abou Alaiwi, Ji-Heui Seo, Chris Labaki, Ziad Bakouny, Sayed Matar, Nourhan El Ahmar, Yasmin Nabil Laimon, Gwo-Shu Mary Lee, Mark Pomerantz, Jacob Berchuck, Sabina Signoretti, Eliezer Van Allen, Toni Choueiri, David Braun, Matthew Freedman, Sylvan Baca. Epigenomic profiling nominates master transcription factors (TFs) driving sarcomatoid differentiation of renal cell carcinoma (RCC) [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A029.

Dynamic Transcriptional Landscape of Mycobacterium smegmatis under Cold Stress.

Bacterial adaptation to cold stress requires wide transcriptional reprogramming. However, the knowledge of molecular mechanisms underlying the cold stress response of mycobacteria is limited. We conducted comparative transcriptomic analysis of Mycobacterium smegmatis subjected to cold shock. The growth of M. smegmatis cultivated at 37 °C was arrested just after exposure to cold (acclimation phase) but later (by 24 h) was resumed at a much slower rate (adaptation phase). Transcriptomic analyses revealed distinct gene expression patterns corresponding to the two phases. During the acclimation phase, differential expression was observed for genes associated with cell wall remodeling, starvation response, and osmotic pressure stress, in parallel with global changes in the expression of transcription factors and the downregulation of ribosomal genes, suggesting an energy-saving strategy to support survival. At the adaptation phase, the expression profiles were recovered, indicating restoration of the processes repressed earlier. Comparison of transcriptional responses in M. smegmatis with those in other bacteria revealed unique adaptation strategies developed by mycobacteria. Our findings shed light on the molecular mechanisms underlying M. smegmatis survival under cold stress. Further research should clarify whether the discovered transcriptional mechanisms exist in other mycobacterial species, including pathogenic Mycobacterium tuberculosis, which could be important for transmission control.

Multiple approaches revealed MGc80-3 as a somatic hybrid with HeLa cells rather than a gastric cancer cell line.

The short-tandem-repeats (STR) profiles of MGc80-3 and HeLa partially overlap, raising suspicion of contamination in the MGc80-3 cell line. However, there has not been any relevant study demonstrating whether MGc80-3 was fully replaced by HeLa cells, just mixed with HeLa cells (co-existing), or was a somatic hybrid with HeLa cells. In addition to STR profiling, various approaches, including single nucleotide polymorphisms genotyping, polymerase chain reaction, screening for human papillomaviruses type 18 (HPV-18) fragment, chromosome karyotyping, pathological examination of xenografts, tissue-specific-90-gene expression signature and high-throughput RNA sequencing were used to determine the nature of MGc80-3. Our study found that the abnormal STR profile, partially overlapping with that of HeLa cells (64.62% to 71.64%), could not verify MGc80-3 as a HeLa cell line. However, the STR 13.3 repeat allele in the D13S317 locus that seemed to be unique to HeLa cells was detected in MGc80-3. Almost all the MGc80-3 cells exhibited HPV-18 fragments in the genome as well as certain HeLa marker chromosomes, such as M7 and M12. The molecular assay of the 90-gene expression signature still considered MGc80-3 as a stomach cancer using an algorithmic analysis. The expression pattern of multiple genes in MGc80-3 was quite different from that in HeLa cells, which showed that certain characteristics belonged to gastric cancer cell lines. High throughput RNA sequencing showed the distinct patterns of gene expression in MGc80-3. In conclusion, MGc80-3 cell line is a somatic hybrid with HeLa cells rather than a pure gastric cancer cell line.

Abstract P1040: An Engineered Human Cardiac Tissue Model Reveals The Potential Contribution Of Systemic Lupus Erythematosus Autoantibodies To Myocardial Injury

A considerable number of patients with systemic lupus erythematosus (SLE) develop myocardial injury. SLE patients have highly heterogenous populations of autoantibodies that may contribute to cardiac disease pathogenesis and heterogeneity; however, little is known about their direct role in myocardial injury. Here, we engineered a model of matured human cardiac tissue and used it to study SLE induced myocardial injury. To this end, we cultured tissues with immunoglobulin G (IgG) from SLE patients with and without myocardial injury. We found that IgG binding levels differed between patient groups and correlated with measures of myocardial inflammation and systolic function. Interestingly, IgG from a subset of patients with systolic dysfunction exerted functional effects on our model system in the absence of immune cells, suggesting a direct contribution to myocardial injury. This was demonstrated by altered calcium handling and cellular composition, impaired cellular respiration, and distinctive patterns of gene expression determined by RNA sequencing. We further harnessed this model to identify potential disease-contributing autoantibodies. We propose that this model can be used to improve risk stratification and therapeutic strategies for SLE patients with myocardial injury.

Distinct patterns of gene expression in the medial preoptic area are related to gregarious singing behavior in European starlings (Sturnus vulgaris)

BackgroundSong performed in flocks by European starlings (Sturnus vulgaris), referred to here as gregarious song, is a non-sexual, social behavior performed by adult birds. Gregarious song is thought to be an intrinsically reinforced behavior facilitated by a low-stress, positive affective state that increases social cohesion within a flock. The medial preoptic area (mPOA) is a region known to have a role in the production of gregarious song. However, the neurochemical systems that potentially act within this region to regulate song remain largely unexplored. In this study, we used RNA sequencing to characterize patterns of gene expression in the mPOA of male and female starlings singing gregarious song to identify possibly novel neurotransmitter, neuromodulator, and hormonal pathways that may be involved in the production of gregarious song.ResultsDifferential gene expression analysis and rank rank hypergeometric analysis indicated that dopaminergic, cholinergic, and GABAergic systems were associated with the production of gregarious song, with multiple receptor genes (e.g., DRD2, DRD5, CHRM4, GABRD) upregulated in the mPOA of starlings who sang at high rates. Additionally, co-expression network analyses identified co-expressing gene clusters of glutamate signaling-related genes associated with song. One of these clusters contained five glutamate receptor genes and two glutamate scaffolding genes and was significantly enriched for genetic pathways involved in neurodevelopmental disorders associated with social deficits in humans. Two of these genes, GRIN1 and SHANK2, were positively correlated with performance of gregarious song.ConclusionsThis work provides new insights into the role of the mPOA in non-sexual, gregarious song in starlings and highlights candidate genes that may play a role in gregarious social interactions across vertebrates. The provided data will also allow other researchers to compare across species to identify conserved systems that regulate social behavior.

Rural and urban exposures shape early life immune development in South African children with atopic dermatitis and nonallergic children.

Immunological traits and functions have been consistently associated with environmental exposures and are thought to shape allergic disease susceptibility and protection. In particular, specific exposures in early life may have more significant effects on the developing immune system, with potentially long-term impacts. We performed RNA-Seq on peripheral blood mononuclear cells (PBMCs) from 150 children with atopic dermatitis and healthy nonallergic children in rural and urban settings from the same ethnolinguistic AmaXhosa background in South Africa. We measured environmental exposures using questionnaires. A distinct PBMC gene expression pattern was observed in those children with atopic dermatitis (132 differentially expressed genes [DEGs]). However, the predominant influences on the immune cell transcriptome were related to early life exposures including animals, time outdoors, and types of cooking and heating fuels. Sample clustering revealed two rural groups (Rural_1 and Rural_2) that separated from the urban group (3413 and 2647 DEGs, respectively). The most significantly regulated pathways in Rural_1 children were related to innate activation of the immune system (e.g., TLR and cytokine signaling), changes in lymphocyte polarization (e.g., TH17 cells), and immune cell metabolism (i.e., oxidative phosphorylation). The Rural_2 group displayed evidence for ongoing lymphocyte activation (e.g., T cell receptor signaling), with changes in immune cell survival and proliferation (e.g., mTOR signaling, insulin signaling). This study highlights the importance of the exposome on immune development in early life and identifies potentially protective (e.g., animal) exposures and potentially detrimental (e.g., pollutant) exposures that impact key immunological pathways.

Transcription factor TCF-1 regulates the functions, but not the development, of lymphoid tissue inducer subsets in different tissues

Lymphoid tissue inducer (LTi) cells, a subset of innate lymphoid cells (ILCs), play an essential role in the formation of secondary lymphoid tissues. However, the regulation of the development and functions of this ILC subset is still elusive. In this study, we report that the transcription factor Tcell factor 1 (TCF-1), just as GATA3, is indispensable for the development of non-LTi ILC subsets. While LTi cells are still present in TCF-1-deficient mice, the organogenesis of Peyer's patches (PPs), but not of lymph nodes, is impaired in these mice. LTi cells from different tissues have distinct gene expression patterns, and TCF-1 regulates the expression of lymphotoxin specifically in PP LTi cells. Mechanistically, TCF-1 may directly and/or indirectly regulate Lta, including through promoting the expression of GATA3. Thus, the TCF-1-GATA3 axis, which plays an important role during Tcell development, also critically regulates the development of non-LTi cells and tissue-specific functions of LTi cells.

Molecular characterization, gene expression and functional analysis of goldfish (Carassius auratus L.) macrophage colony stimulating factor 2.

Macrophage colony-stimulating factor 2 (MCSF-2) is an important cytokine that controls how cells of the monocyte/macrophage lineage proliferate, differentiate, and survive in vertebrates. Two isoforms of MCSF have been identified in fish, each exhibiting distinct gene organization and expression patterns. In this study, we investigated a goldfish MCSF-2 gene in terms of its immunomodulatory and functional properties. In this study, goldfish were acclimated for 3 weeks and sedated with TMS prior to handling. Two groups of fish were used for infection experiments, and tissues from healthy goldfish were collected for RNA isolation. cDNA synthesis was performed, and primers were designed based on transcriptome database sequences. Analysis of gfMCSF-2 sequences, including nucleotide and amino acid analysis, molecular mass prediction, and signal peptide prediction, was conducted. Real-time quantitative PCR (qPCR) was used to analyze gene expression levels, while goldfish head kidney leukocytes (HKLs) were isolated using standard protocols. The expression of gfMCSF-2 in activated HKLs was investigated, and recombinant goldfish MCSF-2 was expressed and purified. Western blot analysis, cell proliferation assays, and flow cytometric analysis of HKLs were performed. Gene expression analysis of transcription factors and pro-inflammatory cytokines in goldfish head kidney leukocytes exposed to rgMCSF-2 was conducted. Statistical analysis using one-way ANOVA and Dunnett's post hoc test was applied. We performed a comparative analysis of MCSF-1 and MCSF-2 at the protein and nucleotide levels using the Needleman-Wunsch algorithm. The results revealed significant differences between the two sequences, supporting the notion that they represent distinct genes rather than isoforms of the same gene. Sequence alignment demonstrated high sequence identity with MCSF-2 homologs from fish species, particularly C. carpio, which was supported by phylogenetic analysis. Expression analysis in various goldfish tissues demonstrated differential expression levels, with the spleen exhibiting the highest expression. In goldfish head kidney leukocytes, gfMCSF-2 expression was modulated by chemical stimuli and bacterial infection, with upregulation observed in response to lipopolysaccharide (LPS) and live Aeromonas hydrophila. Recombinant gfMCSF-2 (rgMCSF-2) was successfully expressed and purified, showing the ability to stimulate cell proliferation in HKLs. Flow cytometric analysis revealed that rgMCSF-2 induced differentiation of sorted leukocytes at a specific concentration. Moreover, rgMCSF-2 treatment upregulated TNFα and IL-1β mRNA levels and influenced the expression of transcription factors, such as MafB, GATA2, and cMyb, in a time-dependent manner. Collectively, by elucidating the effects of rgMCSF-2 on cell proliferation, differentiation, and the modulation of pro-inflammatory cytokines and transcription factors, our findings provided a comprehensive understanding of the potential mechanisms underlying gfMCSF-2-mediated immune regulation. These results contribute to the fundamental knowledge of MCSF-2 in teleosts and establish a foundation for further investigations on the role of gfMCSF-2 in fish immune responses.

Effect of Inflammatory Microenvironment on the Regenerative Capacity of Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells are increasingly being used in regenerative medicine as cell therapy targets, including in the treatment of burns and ulcers. The regenerative potential of AD-MSCs and some of their immunological properties are known from in vitro studies; however, in clinical applications, cells are used in non-ideal conditions and can behave differently in inflammatory environments, affecting the efficacy and outcome of therapy. Our aim was to investigate and map the pathways that the inflammatory microenvironment can induce in these cells. High-throughput gene expression assays were performed on AD-MSCs activated with LPS and TNFα. Analysis of RNA-Seq data showed that control, LPS-treated and TNFα-treated samples exhibited distinct gene expression patterns. LPS treatment increased the expression of 926 genes and decreased the expression of 770 genes involved in cell division, DNA repair, the cell cycle, and several metabolic processes. TNFα treatment increased the expression of 174 genes and decreased the expression of 383 genes, which are related to cell division, the immune response, cell proliferation, and differentiation. We also map the biological pathways by further investigating the most altered genes using the Gene Ontology and KEGG databases. Secreted cytokines, which are important in the immunological response, were also examined at the protein level, and a functional assay was performed to assess wound healing. Activated AD-MSC increased the secretion of IL-6, IL-8 and CXCL-10, and also the closure of wounds. AD-MSCs presented accelerated wound healing under inflammation conditions, suggesting that we could use this cell in clinical application.

Prenyltransferase gene expression reveals an essential role of prenylation for the inflammatory response in human gingival fibroblasts.

Prenyltrasferases (PTases) are a class of enzymes known to be responsible for promoting posttranslational modification at the carboxyl terminus of proteins containing a so-called CaaX-motif. The process is responsible for proper membrane localization and the appropriate function of several intracellular signaling proteins. Current research demonstrating the pathomechanistic importance of prenylation in inflammatory illnesses emphasizes the requirement to ascertain the differential expression of PT genes under inflammatory settings, particularly in periodontal disease. Telomerase-immortalized human gingival fibroblasts (HGF-hTert) were cultured and treated with either inhibitors of prenylation (PTI) lonafarnib, tipifarnib, zoledronic acid, or atorvastatin at concentrations of 10 μM in combination with or without 10μg Porphyromonas gingivalis lipopolysaccharide (LPS) for 24h. Prenyltransferase genes FNTB, FNTA, PGGT1B, RABGGTA, RABGGTB, and PTAR1 as well as inflammatory marker genes MMP1 and IL1B were detected using quantitative real-time polymerase chain reaction (RT-qPCR). Immunoblot and protein immunoassay were used to confirm the results on the protein level. RT-qPCR experiments revealed significant upregulation of IL1B, MMP1, FNTA, and PGGT1B upon LPS treatment. PTase inhibitors caused significant downregulation of the inflammatory cytokine expression. Interestingly, FNTB expression was significantly upregulated in response to any PTase inhibitor in combination with LPS, but not upon LPS treatment only, indicating a vital role of protein farnesyltransferase in the proinflammatory signaling cascade. In this study, distinct PTase gene expression patterns in pro-inflammatory signaling were discovered. Moreover, PTase inhibiting drugs ameliorated inflammatory mediator expression by a significant margin, indicating that prenylation is a major pre-requisite for innate immunity in periodontal cells.