Ligand Gene Research Articles

Activated hedgehog and insulin ligands by decreased transcriptional factor DAF-16 mediate transgenerational nanoplastic toxicity in Caenorhabditis elegans

In Caenorhabditis elegans, transcriptional factor DAF-16 in insulin signaling pathway played important role in regulating transgenerational nanoplastic toxicity. Activation of insulin signals mediated transgenerational toxicity of polystyrene nanoparticle (PS-NP) by inhibiting DAF-16. Among identified germline ligands, expression of wrt-3 encoding hedgehog ligand was increased by RNAi of daf-16 in PS-NP exposed C. elegans. In PS-NP exposed C. elegans, expressions of 4 other germline hedgehog ligand genes and 10 hedgehog receptor genes were increased by daf-16 RNAi. Among these candidate genes, expressions of hedgehog ligand genes (grl-15, grl-16, qua-1, and wrt-1) and hedgehog receptor genes (ptr-23, scp-1, ptd-2, and ncr-1) could be increased by PS-NP (1–100 μg/L), and their transgenerational expressions were observed after PS-NP exposure. RNAi of grl-15, grl-16, qua-1, wrt-1, ptr-23, scp-1, ptd-2, and ncr-1 caused resistance to transgenerational PS-NP toxicity. In nematodes exposed to PS-NPs, RNAi of wrt-3, grl-15, grl-16, qua-1, and wrt-1 at parental generation (P0-G) inhibited expressions of ptr-23, scp-1, ptd-2, and ncr-1 in their offspring. Moreover, we observed increased expressions of insulin peptides genes (ins-3, ins-39, and daf-28) in PS-NP exposed daf-16(RNAi) nematodes, suggesting formation of feedback loop. We raise the molecular basis for formation of toxicity on multiple generations after nanoplastic exposure at P0-G.

Evaluation of the Frequency of (rs2227981 and rs2282055) Single Nucleotide Polymorphisms in Programmed Cell Death 1 and Its Ligand Genes of Iraqi Patients With Multiple Sclerosis

Abstract Multiple sclerosis (MS) is a central nervous system disease that causes demyelination and persistent inflammation and most of them among young adults. The current study aimed to assess gene polymorphism of PDCD1 and PDLCD1 genes and the frequency of two SNPs (rs2227981 and rs2282055). MS patients (n=100) were divided into two groups; newly diagnosed (42), and patients with ongoing treatments (58). These groups were compared to healthy subjects (n=55); the mean age ±SD was (30±8.46 years), (37±8.06years), and (31±8.73 years) for MS newly patients, patients with ongoing treatments, and healthy subjects, respectively. Studies for gene polymorphism of PDCD1 and PDLCD1 Genes and the Frequency of Two SNPs (rs2227981 and rs2282055) were measured by Real-time PCR by used HRM. The results of this study found that the genotypic frequencies of MS patient were 31% (n=31) normal AA and 48 % (n=48) heterozygous AG. Mutant homozygous was found in GG 21 % (n=21). In controls, the results demonstrate 69% (n=38) wild type AA, 27.27% (n=15) heterozygous AG and mutant homozygous GG 3.6% (n=2). The odds ratio for the GG genotype was 12.8 (0.2-5.9) with p=0.001 indicating that the homomutant genotype In PD1 gene polymorphism rs2227981 GG was a higher risk of MS than the wild-type PD1 gene polymorphism rs2227981 AA, The PD1 gene polymorphism rs2227981 AG genotype has the second risk of MS after PD1 gene polymorphism rs2227981GG with 3.92 fold high risk than the wild type AA (OR =3.92 (1.8-8.2) p=0.0003). The present study concludes that the population-based case-control study, which focused on the three PD-1 SNPs PD-1.3, PD-1.5, and PD-1.9, found that polymorphism variation might actually be a disease-modifying factor as opposed to an MS risk factor. There were substantially more PD-L1 gene SNP loci in MS patients compared to controls, and there were significantly more PD-L1 gene SNP loci in MS patients compared to controls. The pathogenesis of MS might be connected to PD-1 and PD-L1 SNPs. PD-L1 gene SNP locus rs2282055 and PD-1 gene SNP locus rs2227981 might be potential MS candidate polymorphism loci.

Characterisation of the erythrocyte invasion phenotype of FCB-2: A South American P. falciparum reference strain

The extent of parasite adaptive capability involved in erythrocyte invasion represents a significant challenge for the development of a Plasmodium falciparum vaccine. The parasite's geographical and populational origin may influence such adaptive behaviour; in vitro culture-adapted parasite strains are typically used for such studies. Previous studies have reported invasion phenotypes in strains from Africa and Asia and, to a lesser extent, from Latin America. This study was aimed at expanding the pool of characterised parasite strains from Latin America by describing the invasion phenotype of the P. falciparum Colombia Bogotá 2 (FCB2) strain. The FCB2 genome was sequenced and erythrocyte invasion ligand sequences were analysed and compared to other previously reported ones. RT-PCR was used for assessing Pfeba family erythrocyte invasion ligands and reticulocyte binding homologue (Pfrh) gene transcription. A flow cytometry-based erythrocyte invasion assay (using enzymatically-treated erythrocytes) was used for determining the FCB2 strain's invasion phenotype. The P. falciparum FCB2 genome sequence was analysed, bearing in mind that prolonged in vitro parasite culture may affect its genome sequence and, in some cases, lead to the deletion of certain genes; it was demonstrated that all erythrocyte invasion ligand gene sequences studied here were preserved. Comparative analysis showed that the target genome sequences were conserved whereas transcriptional analysis highlighted Pfebas and Pfrhs gene expression. Erythrocyte invasion analysis demonstrated that the FCB2 strain has a sialic acid-resistant invasion phenotype.

A Saccharomyces boulardii-derived antioxidant protein, thioredoxin, ameliorates intestinal inflammation through transactivating epidermal growth factor receptor

Saccharomyces boulardii (Sb) is a probiotic yeast for the treatment of gastrointestinal disorders, including inflammatory bowel disease (IBD). Little is known about the modulatory capacity of the Sb in IBD. Here, we found that oral gavage of Sb supernatant (SbS) alleviated gut inflammation, protected the intestinal barrier, and reversed DSS-induced down-regulated activation of epidermal growth factor receptor (EGFR) in colitis. Mass spectrum analysis showed that thioredoxin (Trx) is one of the critical secreted soluble proteins participating in EGFR activation detected in SbS. Trx exerted an array of significant effects on anti-inflammatory activity, including alleviating inflammation, protecting gut barrier, suppressing apoptosis, as well as reducing oxidative stress. Mechanistically, Trx promoted EGFR ligand gene expression and transactivated EGFR in a concentration-dependent manner. EGFR kinase inhibitor could block Trx-mediated preventive effects of intestinal epithelial injury. Our data suggested that Sb-derived soluble protein Trx could serve as a potential prophylactic, as a novel postbiotic against colitis, which provides a new strategy for the precision prevention and treatment of IBD.

Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development.

Chromatin domain boundaries delimited by CTCF motifs can restrict the range of enhancer action. However, disruption of domain structure often results in mild gene dysregulation and thus predicting the impact of boundary rearrangements on animal development remains challenging. Here, we tested whether structural perturbation of a chromatin domain with multiple developmental regulators can result in more acute gene dysregulation and severe developmental phenotypes. We targeted clusters of CTCF motifs in a domain of the mouse genome containing three FGF ligand genes - Fgf3, Fgf4, and Fgf15 - that regulate several developmental processes. Deletion of the 23.9kb cluster that defines the centromeric boundary of this domain resulted in ectopic interactions of the FGF genes with enhancers located across the deleted boundary that are active in the developing brain. This caused strong induction of FGF expression and perinatal lethality with encephalocele and orofacial cleft phenotypes. Heterozygous boundary deletion was sufficient to cause these fully penetrant phenotypes, and strikingly, loss of a single CTCF motif within the cluster also recapitulated ectopic FGF expression and caused encephalocele. However, such phenotypic sensitivity to perturbation of domain structure did not extend to all CTCF clusters of this domain, nor to all developmental processes controlled by these three FGF genes - for example, the ability to undergo lineage specification in the blastocyst and pre-implantation development were not affected. By tracing the impact of different chromosomal rearrangements throughout mouse development, we start to uncover the determinants of phenotypic robustness and sensitivity to perturbation of chromatin boundaries. Our data show how small sequence variants at certain domain boundaries can have a surprisingly outsized effect and must be considered as potential sources of gene dysregulation during development and disease.

Comparison of Transgenerational Neurotoxicity between Pristine and Amino-Modified Nanoplastics in C. elegans.

Increasing evidence has suggested that nanoplastic pollution has become a global concern. More importantly, transgenerational toxicity can be induced by nanoplastics at predicted environmentally relevant doses (ERDs). Considering that amino modification could increase nanoplastic toxicity, we compared transgenerational neurotoxicity between pristine polystyrene nanoparticle (PS-NP) and amino-modified PS-NP (NH2-PS-NP) in Caenorhabditis elegans. At 0.1-10 μg/L, NH2-PS-NP caused more severe transgenerational toxicity on locomotion and neuronal development. Accompanied with a difference in transgenerational neuronal damage, compared to PS-NP (10 μg/L), NH2-PS-NP (10 μg/L) induced more severe transgenerational activation of mec-4, crt-1, itr-1, and tra-3, which are required for the induction of neurodegeneration. Moreover, NH2-PS-NP (10 μg/L) caused more severe transgenerational inhibition in expressions of mpk-1, jnk-1, dbl-1, and daf-7 than PS-NP (10 μg/L), and RNA interference (RNAi) of these genes conferred susceptibility to the toxicity of PS-NP and NH2-PS-NP on locomotion and neuronal development. NH2-PS-NP (10 μg/L) further caused more severe transgenerational activation of germline ligand genes (ins-3, ins-39, daf-28, lin-44, egl-17, efn-3, and lag-2) than PS-NP (10 μg/L), and RNAi of these ligand genes caused resistance to the toxicity of PS-NP and NH2-PS-NP on locomotion and neuronal development. Our results highlighted more severe exposure risk of amino-modified nanoplastics at ERDs in causing transgenerational neurotoxicity in organisms.

Genetic Variants of the Receptor Activator Nuclear of κB Ligand Gene Increase the Risk of Rheumatoid Arthritis in a Mexican Mestizo Population: A Case-Control Study.

To assess the association of rs9533155 (-693C>G) and rs9533156 (-643T>C) genetic variants with RA risk. A case-control study was carried out. A total of 94 patients with RA (RA group) and 134 subjects without any rheumatologic disease (control group) were included. Genetic DNA was extracted from peripheral white blood cells (leukocytes). Genetic variant rs9533155 (-693C>G) was screened by an approach based on Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP), while rs9533156 (-643T>C) was screened using quantitative polymerase chain reaction (qPCR) with TaqMan probes. RANKL serum levels were measured by ELISA. For rs9533155 (-693C>G), the polymorphic homozygous genotype frequencies (CC) were higher in the RA group (p = 0.006). Individuals carrying the risk genotype presented higher levels of serum RANKL. Carriers of the polymorphic homozygous genotype in the dominant model (CC vs. CG + GG) had an increased risk of developing RA (OR: 1.8, 95% CI 1.04 to 3.1). No association between rs9533156 (-643T>C) and the haplotypes with RA risk was observed. The rs9533155 (-693C>G) genetic variant exhibits a potential role in RA risk. The studied population had no association with the rs9533156 (-643T>C) genetic variant.

Transcriptional switches in melanoma T Cells: Facilitating polarizing into regulatory T cells

Melanoma is a malignant skin tumor with a high mortality rate. Regulatory T cells (Tregs) are immune cells with immunosuppressive roles, however, the precise mechanisms governing Treg involvement in melanoma remain enigmatic. Experimental findings unveiled different transcription factor switches between normal and tumor T cell, with heightened FOXP3 and BATF in the latter. These factors induced immunosuppressive molecules and Treg maintenance genes, polarizing tumor T cells into Tregs. Spatial transcriptomics illuminated the preferential settlement of Tregs at the melanoma periphery. Within this context, FOXP3 in Tregs facilitated direct enhancement of specific ligand gene expression, fostering communication with neighboring cells. Novel functional molecules bound to FOXP3 or BATF in Tregs, such as SPOCK2, SH2D2A, and ligand molecules ITGB2, LTA, CLEC2C, CLEC2D, were discovered, which had not been previously reported in melanoma Treg studies. Furthermore, we validated our findings in a large number of clinical samples and identified the Melanoma Treg-Specific Regulatory Tag Set (Mel TregS). ELISA analysis showed that the protein levels of Mel TregS in melanoma Tregs were higher than in normal Tregs. We then utilized SERS technology to measure the signal values of Mel TregS in exosome, and successfully discriminated between healthy individuals and melanoma patients, as well as early and late-stage patients. This approach significantly enhanced detection sensitivity. In sum, our research elucidated fresh insights into the mechanisms governing Treg self-maintenance and communication with surrounding cells in melanoma. We also introduced an innovative method for clinical disease monitoring through SERS technology.

Polylactic acid microplastics cause transgenerational reproductive toxicity associated with activation of insulin and hedgehog ligands in C. elegans

As a member of biodegradable plastics, exposure risk of polylactic acid microplastic (PLA-MP) has received attention recently. Toxicity of PLA-MP at parental generation (P0-G) has been observed in some organisms; however, its possible transgenerational toxicity and underlying mechanisms remain unclear. In Caenorhabditis elegans, 10 and 100 μg/L PLA-MP resulted in transgenerational inhibition in reproductive capacity and transgenerational damage on gonad development. Meanwhile, transgenerational increase in germline apoptosis was detected after PLA-MP exposure at P0-G, which was associated with transgenerational dysregulation in expressions of genes governing apoptosis (ced-3, ced-4, egl-1, and ced-9) and DNA damage related genes (cep-1, mrt-2, hus-1, and clk-2). Among secreted ligand genes, PLA-MP exposure induced transgenerational increase in expression of ins-39 and wrt-3, and RNAi of ins-39 and wrt-3 inhibited germline apoptosis in PLA-MP exposed nematodes. Additionally, PLA-MP caused transgenerational increase in expression of met-2 and set-6 encoding histone methylation transferases, and germline apoptosis induced by PLA-MP could be suppressed by RNAi of met-2 and set-6. Dysregulated expressions of some apoptosis and DNA damage related genes caused by PLA-MP were reversed by RNAi of ins-39, wrt-3, met-2, and set-6. Moreover, in PLA-MP exposed animals, expression of ins-39 and wrt-3 could be further inhibited by RNAi of met-2 and set-6. Therefore, PLA-MP potentially induced reproductive toxicity across multiple generations, which was under the control of MET-2 and SET-6 activated ligands of INS-39 and WRT-3.

Study on the mechanism of heterogeneous tumor-associated macrophages in three subtypes of breast cancer through the integration of single-cell RNA sequencing and in vitro experiments.

Tumor-associated macrophages (TAM) exert a significant influence on the progression and heterogeneity of various subtypes of breast cancer (BRCA). However, the roles of heterogeneous TAM within BRCA subtypes remain unclear. Therefore, this study sought to elucidate the role of TAM across the following three BRCA subtypes: triple-negative breast cancer, luminal, and HER2. This investigation aimed to delineate the variations in marker genes, drug sensitivity, and cellular communication among TAM across the three BRCA subtypes. We identified specific ligand-receptor (L-R) pairs and downstream mechanisms regulated by VEGFA-VEGFR1, SPP1-CD44, and SPP1-ITGB1 L-R pairs. Experimental verification of these pairs was conducted by co-culturing macrophages with three subtypes of BRCA cells. Our findings reveal the heterogeneity of macrophages within the three BRCA subtypes, evidenced by variations in marker gene expression, composition, and functional characteristics. Notably, heterogeneous TAM were found to promote invasive migration and epithelial-mesenchymal transition (EMT) in MDA-MB-231, MCF-7, and SKBR3 cells, activating NF-κB pathway via P38 MAPK, TGF-β1, and AKT, respectively, through distinct VEGFA-VEGFR1, SPP1-CD44, and SPP1-ITGB1 L-R pairs. Inhibition of these specific L-R pairs effectively reversed EMT, migration, and invasion of each cancer cells. Furthermore, we observed a correlation between ligand gene expression and TAM sensitivity to anticancer drugs, suggesting a potential strategy for optimizing personalized treatment guidance. Our study highlights the capacity of heterogeneous TAM to modulate biological functions via distinct pathways mediated by specific L-R pairs within diverse BRCA subtypes. This study might provide insights into precision immunotherapy of different subtypes of BRCA.

Multi-omics analysis of macrophage-associated receptor and ligand reveals a strong prognostic signature and subtypes in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a significant contributor to morbidity and mortality worldwide. The interaction between receptors and ligands is the primary mode of intercellular signaling and plays a vital role in the progression of HCC. This study aimed to identify the macrophage-related receptor ligand marker genes associated with HCC and further explored the molecular immune mechanisms attributed to altered biomarkers. Single-cell RNA sequencing data containing primary and recurrent samples were downloaded from the China National GeneBank. Cell types were first identified to explore differences between immune cells from different sample sources. CellChat analysis was used to infer and analyze intercellular communication networks quantitatively. Three molecular subtypes were constructed based on the screened twenty macrophage-associated receptor ligand genes. Bulk RNA-Seq data were downloaded from The Cancer Genome Atlas and Gene Expression Omnibus databases. After the screening, the minor absolute shrinkage and selection operator (LASSO) regression model was employed to identify key markers. After collecting peripheral blood and clinical information from patients, an enzyme-linked immunosorbent assay (ELISA) was used to detect the correlation between key markers and IL-10, one of the macrophage markers. After developing a new HCC risk adjustment model and conducting analysis, it was found that there were significant differences in immune status and gene mutations between the high-risk and low-risk groups of patients based on macrophage-associated receptor and ligand genes. This study identified SPP1, ANGPT2, and NCL as key biological targets for HCC. The drug-gene interaction network analysis identified wortmannin, ribavirin, and tarnafloxin as potential therapeutic drugs for the three key markers. In a clinical cohort study, patients with immune checkpoint inhibitor (ICI) resistance had significantly higher expression levels of OPN, ANGPT2, NCL, and IL-10 than patients with ICI-responsiveness. These three key markers were positively correlated with the expression level of IL-10. The signature based on macrophage-associated receptor and ligand genes can accurately predict the prognosis of patients with HCC and the sensitivity to immunotherapy. These results may help guide the development of targeted prevention and personalized treatment of HCC.

Characterizing dysregulations via cell-cell communications in Alzheimer’s brains using single-cell transcriptomes

BackgroundAlzheimer’s disease (AD) is a devastating neurodegenerative disorder affecting 44 million people worldwide, leading to cognitive decline, memory loss, and significant impairment in daily functioning. The recent single-cell sequencing technology has revolutionized genetic and genomic resolution by enabling scientists to explore the diversity of gene expression patterns at the finest resolution. Most existing studies have solely focused on molecular perturbations within each cell, but cells live in microenvironments rather than in isolated entities. Here, we leveraged the large-scale and publicly available single-nucleus RNA sequencing in the human prefrontal cortex to investigate cell-to-cell communication in healthy brains and their perturbations in AD. We uniformly processed the snRNA-seq with strict QCs and labeled canonical cell types consistent with the definitions from the BRAIN Initiative Cell Census Network. From ligand and receptor gene expression, we built a high-confidence cell-to-cell communication network to investigate signaling differences between AD and healthy brains.ResultsSpecifically, we first performed broad communication pattern analyses to highlight that biologically related cell types in normal brains rely on largely overlapping signaling networks and that the AD brain exhibits the irregular inter-mixing of cell types and signaling pathways. Secondly, we performed a more focused cell-type-centric analysis and found that excitatory neurons in AD have significantly increased their communications to inhibitory neurons, while inhibitory neurons and other non-neuronal cells globally decreased theirs to all cells. Then, we delved deeper with a signaling-centric view, showing that canonical signaling pathways CSF, TGFβ, and CX3C are significantly dysregulated in their signaling to the cell type microglia/PVM and from endothelial to neuronal cells for the WNT pathway. Finally, after extracting 23 known AD risk genes, our intracellular communication analysis revealed a strong connection of extracellular ligand genes APP, APOE, and PSEN1 to intracellular AD risk genes TREM2, ABCA1, and APP in the communication from astrocytes and microglia to neurons.ConclusionsIn summary, with the novel advances in single-cell sequencing technologies, we show that cellular signaling is regulated in a cell-type-specific manner and that improper regulation of extracellular signaling genes is linked to intracellular risk genes, giving the mechanistic intra- and inter-cellular picture of AD.

Single-cell insights into mouse testicular toxicity under peripubertal exposure to di(2-ethylhexyl) phthalate.

Male fertility depends on normal pubertal development. Di-(2-ethylhexyl) phthalate (DEHP) is a potent antiandrogen chemical, and exposure to DEHP during peripuberty can damage the developing male reproductive system, especially the testis. However, the specific cellular targets and differentiation processes affected by DEHP, which lead to testicular toxicity, remain poorly defined. Herein, we presented the first single-cell transcriptomic profile of the pubertal mouse testis following DEHP exposure. To carry out the experiment, 2 groups (n = 8 each) of 3-week-old male mice were orally administered 0.5% carboxymethylcellulose sodium salt or 100 mg/kg body weight DEHP daily from postnatal day 21-48, respectively. Using single-cell RNA sequencing, a total of 31 distinct cell populations were identified, notably, Sertoli and Leydig cells emerged as important targets of DEHP. DEHP exposure significantly decreased the proportions of Sertoli cell clusters expressing mature Sertoli markers (Sox9 and Ar), and selectively reduced the expression of testosterone synthesis genes in fetal Leydig cells. Through cell-cell interaction analyses, we observed changed numbers of interactions in Sertoli cells 1 (SCs1), Leydig cells 1 (LCs1), and interstitial macrophages, and we also identified cell-specific ligand gene expressions in these clusters, such as Inha, Fyn, Vcam1, and Apoe. Complementary in vitro assays confirmed that DEHP directly reduced the expression of genes related to Sertoli cell adhesion and intercellular communication. In conclusion, peripubertal DEHP exposure reduced the number of mature Sertoli cells and may disrupt testicular steroidogenesis by affecting the testosterone synthesis genes in fetal Leydig cells rather than adult Leydig cells.

The tick Ixodes scapularis has five different GPCRs specifically activated by ACP (adipokinetic hormone/corazonin-related peptide)

Insects have about 50 neuropeptide genes and about 70 genes, coding for neuropeptide G protein-coupled receptors (GPCRs). An important, but small family of evolutionarily related insect neuropeptides consists of adipokinetic hormone (AKH), corazonin, and AKH/corazonin-related peptide (ACP). Normally, insects have one specific GPCR for each of these neuropeptides. The tick Ixodes scapularis is not an insect, but belongs to the subphylum Chelicerata, which comprises ticks, scorpions, mites, spiders, and horseshoe crabs. Many of the neuropeptides and neuropeptide GPCRs occurring in insects, also occur in chelicerates, illustrating that insects and chelicerates are evolutionarily closely related. The tick I. scapularis is an ectoparasite and health risk for humans, because it infects its human host with dangerous pathogens during a blood meal. Understanding the biology of ticks will help researchers to prevent tick-borne diseases. By annotating the I. scapularis genome sequence, we previously found that ticks contain as many as five genes, coding for presumed ACP receptors. In the current paper, we cloned these receptors and expressed each of them in Chinese Hamster Ovary (CHO) cells. Each expressed receptor was activated by nanomolar concentrations of ACP, demonstrating that all five receptors were functional ACP receptors. Phylogenetic tree analyses showed that the cloned tick ACP receptors were mostly related to insect ACP receptors and, next, to insect AKH receptors, suggesting that ACP receptor genes and AKH receptor genes originated by gene duplications from a common ancestor. Similar duplications have probably occurred for the ligand genes, during a process of ligand/receptor co-evolution. Interestingly, chelicerates, in contrast to all other arthropods, do not have AKH or AKH receptor genes. Therefore, the ancestor of chelicerates might have lost AKH and AKH receptor genes and functionally replaced them by ACP and ACP receptor genes. For the small family of AKH, ACP, and corazonin receptors and their ligands, gene losses and gene gains occur frequently between the various ecdysozoan clades. Tardigrades, for example, which are well known for their survival in extreme environments, have as many as ten corazonin receptor genes and six corazonin peptide genes, while insects only have one of each, or none.

Cloning Sheep Cytokin Genes and Preliminary Transfect to HT29 Cell Line

The study was carried out to clone sheep cytokine genes for protein expression in mammalian cell culture. Once expressed it is hypothesized that these proteins when added to a primary mast cell culture will allow the growth of these cells. Mast cells are used by sheep to combat gastro-intestinal nematode (GIN) by helping to establish the host immune response and to eliminate of GIN. Kit ligand and IL3 proteins are growth factors of mast cells that were derived from extraction of total RNA from lung and thymus tissues, respectively. The IL3 and kit ligand genes were cloned into bacterial expression pGEM – T easy vector to determine correct sequences before being sub-cloned into the mammalian expression pWPI vector. The Pac 1 enzyme was used to cut restriction sites of pGEM – T easy vector, pWPI vector, IL3 and kit ligand genes in cloning strategies. The study also established 24 different conditions for transient transfection IL3 and Kit ligand genes into HT29 cells and screen positive cells on the flow cytometry. Results showed that clones of IL3 and kit ligand with the correct sequences were created for using transient transfection. The transfection efficiency of IL3 and kit ligand DNA/ pWPI plasmid into HT29 cells were extremely low with approximately 9% for the best condition. This low transfection efficiency leads to shortage of IL3 and kit ligand for growing mast cells. Therefore, stable transfection will be required to have highest efficiencies that produce the large quantities of IL3 and kit ligand proteins required for culture of sheep mast cell lines.

The role of FASL gene polymorphism (rs: 763110), its serum level and IL21 level with incidence of psoriasis in Iraqi patients

Psoriasis is a chronic, immune-mediated skin disease with systemic pro-inflammatory can defined by keratinocyte hyper proliferation. FASL gene cell surface death receptor which is important in apoptosis mediated by cytotoxic T cells and natural killer cells. The goals of current study is design to investigate genotyping of the FASL gene (Fas ligand gene) -844 T>C (rs:763110), evaluate its serum level and to estimate level of IL-21correlated with incidence of psoriasis in Iraqi patients. 100 blood samples collected from December 2022 to February 2023, blood samples split into two groups first 50 psoriasis patients (30 males and 20 females) with age range (17-66years) and second group 50 Apparently healthy as control group (24 males 26 females) with age range (20-60 years) visit the Al-Yarmouk Teaching Hospital’s Department of Dermatology in Iraq/Baghdad. (HRM–PCR) Technique was use for the investigation of gene polymorphism of the FASL gene (rs763110). While EIISA technique used to estimate serum level of each FASL gene and IL-21 in serum. The result of FASL gene polymorphism revealed that there are three genotype (TT),(TC),(CC). There was no significant difference, between (TC) and (CC) genotype In the FASLG -844 and between the psoriasis and control groups. Moreover there was correlation between genotyping and serum level for FASL (rs763110) gene in that (TC) genotype serum level show significant different from (CC) genotype serum level with p-value (P≤0.05) as well as the FASL serum level in patient which represent high significantly different between psoriasis patients when compared to the healthy controls with p-value (P≤0.01). While result of IL21 serum level in psoriasis patients revealed high significant different with p-value (P≤0.01) vs healthy control with high significant different in sever psoriatic patient vs mild and healthy control with p value (P≤0.01).

Abstract 6212: A differential ligand-receptor network inference method to identify alterations in communication between myeloid cells and CD8 T cells in response to PancVAX neo-epitope peptide vaccine, anti-CTLA-4 and anti-PD-1 antibodies in a murine model of pancreatic ductal adenocarcinoma

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly cancer with a low tumor mutational burden that leads to the generation of few neo-epitopes derived from mutated genes that can be recognized by cytotoxic T cells. The immunosuppressive microenvironment of PDAC comprises suppressive cell populations that include myeloid cells, cancer associated fibroblast subpopulations, and regulatory T cells that reduce T cell infiltration and cytotoxic function. We have developed a murine personalized cancer vaccine to enhance cytotoxic T cell function against PDAC, PancVAX (Kinkead et al, JCI Insight 2018). Survival benefit with PancVAX was dependent on the addition of immune checkpoint inhibitor (ICI) therapy to increase the number of cytotoxic CD8 T cells infiltrating tumors and maintain their cytotoxic function (Huff et al, JCI Insight 2023). Yet, the impact of cell-cell communication on effector T cell function arising from the immunosuppressive microenvironment of PDAC remains unknown. We previously developed a ligand-receptor inference method called Domino that infers inter- and intra-cellular signaling networks in single-cell RNA-seq data (Cherry et al, Nat Biomed Eng 2021). However, this technique is limited to data from single conditions. Understanding how this combination immunotherapy alters cellular signaling networks requires new computational tools that identify changes in regulatory networks between treatment conditions. We developed capabilities for assessment of differential signaling with Domino founded on a basis of differential signal receipt. Active receptors are identified by receptor expression in recipient cells that is correlated with downstream transcription factors in the intracellular signaling cascade. Identification of causative ligands for receptor activation is assessed by a probability of interaction for ligand-receptor interactions between cell types. This probability metric considers mean expression of ligand and receptor genes as well as the number of sender cells for comparison across treatment groups. Comparing communication networks in PancVAX alone to PancVAX with ICI, PancVAX with ICI enhances cell-cell adhesion and interferon gamma signaling in cytotoxic CD8 T cells towards exhausted CD8 T cells. Macrophages in PancVAX with ICI enhance expression of immune checkpoint ligands towards CD8 T cells, despite the presence of blocking ICI antibodies. Intracellular signaling downstream of PD-1 in exhausted T cells shows diminished numbers of transcription factors linked to PD-1 signaling. This software informs prioritization of inhibitory signals for modulation in future immunotherapy regimens and can be applied generally to other tumor subtypes and treatment contexts. Citation Format: Jacob T. Mitchell, Atul Deshpande, Amanda L. Huff, Chris Cherry, Sushma Nagaraj, Kavita Krishnan, Dmitrijs Lvovs, Jennifer H. Elisseeff, Elizabeth M. Jaffee, Neeha Zaidi, Elana J. Fertig. A differential ligand-receptor network inference method to identify alterations in communication between myeloid cells and CD8 T cells in response to PancVAX neo-epitope peptide vaccine, anti-CTLA-4 and anti-PD-1 antibodies in a murine model of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6212.

A division-of-labor mode contributes to the cardioprotective potential of mesenchymal stem/stromal cells in heart failure post myocardial infarction.

Treatment of heart failure post myocardial infarction (post-MI HF) with mesenchymal stem/stromal cells (MSCs) holds great promise. Nevertheless, 2-dimensional (2D) GMP-grade MSCs from different labs and donor sources have different therapeutic efficacy and still in a low yield. Therefore, it is crucial to increase the production and find novel ways to assess the therapeutic efficacy of MSCs. hUC-MSCs were cultured in 3-dimensional (3D) expansion system for obtaining enough cells for clinical use, named as 3D MSCs. A post-MI HF mouse model was employed to conduct in vivo and in vitro experiments. Single-cell and bulk RNA-seq analyses were performed on 3D MSCs. A total of 125 combination algorithms were leveraged to screen for core ligand genes. Shinyapp and shinycell workflows were used for deploying web-server. 3D GMP-grade MSCs can significantly and stably reduce the extent of post-MI HF. To understand the stable potential cardioprotective mechanism, scRNA-seq revealed the heterogeneity and division-of-labor mode of 3D MSCs at the cellular level. Specifically, scissor phenotypic analysis identified a reported wound-healing CD142+ MSCs subpopulation that is also associated with cardiac protection ability and CD142- MSCs that is in proliferative state, contributing to the cardioprotective function and self-renewal, respectively. Differential expression analysis was conducted on CD142+ MSCs and CD142- MSCs and the differentially expressed ligand-related model was achieved by employing 125 combination algorithms. The present study developed a machine learning predictive model based on 13 ligands. Further analysis using CellChat demonstrated that CD142+ MSCs have a stronger secretion capacity compared to CD142- MSCs and Flow cytometry sorting of the CD142+ MSCs and qRT-PCR validation confirmed the significant upregulation of these 13 ligand factors in CD142+ MSCs. Clinical GMP-grade 3D MSCs could serve as a stable cardioprotective cell product. Using scissor analysis on scRNA-seq data, we have clarified the potential functional and proliferative subpopulation, which cooperatively contributed to self-renewal and functional maintenance for 3D MSCs, named as "division of labor" mode of MSCs. Moreover, a ligand model was robustly developed for predicting the secretory efficacy of MSCs. A user-friendly web-server and a predictive model were constructed and available (https://wangxc.shinyapps.io/3D_MSCs/).

Malignant cell receptor-ligand subtypes guide the prediction of prognosis and personalized immunotherapy of liver cancer.

Liver cancer is a prevalent disease with a dismal prognosis. The aim of the research is to identify subgroups based on malignant cell receptor ligand gene from single-cell RNA, which might lead to customized immunotherapy for patients with liver cancer. Based on scRNA-seq data, we identified the receptor-ligand genes associated with prognosis and classify patients into molecular subtypes by univariate Cox regression and consensus clustering. LASSO regression was performed to construct a prognostic model, which was validated in TCGA and ICGC datasets. Immune infiltration and prediction of immunotherapy response were analyzed using ssGSEA, ESTIMATE, TIDE, and TRS score calculation. Finally, qPCR and Western blot validation of key genes and protein levels in cell lines. A risk model using 16-gene expression levels predicted liver cancer patients' prognosis. The RiskScore associated significantly with tumor clinical characteristics and immunity, integrated with clinicopathological features for survival prediction. Differential expression of SRXN1 was verified in hepatocellular carcinoma and normal liver cells. Our study utilizes single-cell analysis to investigate the communication between malignant cells and other cell types, identifying molecular subtypes based on malignant cell receptor ligand genes, offering new insights for the development of personalized immunotherapy and prognostic prediction models.

Sex differences of NF-κB-targeted therapy for mitigating osteoporosis associated with chronic inflammation of bone.

Transcription factor nuclear factor kappa B (NF-κB) plays a major role in the pathogenesis of chronic inflammatory diseases in all organ systems. Despite its importance, NF-κB targeted drug therapy to mitigate chronic inflammation has had limited success in preclinical studies. We hypothesized that sex differences affect the response to NF-κB treatment during chronic inflammation in bone. This study investigated the therapeutic effects of NF-κB decoy oligodeoxynucleotides (ODN) during chronic inflammation in male and female mice. We used a murine model of chronic inflammation induced by continuous intramedullary delivery of lipopolysaccharide-contaminated polyethylene particles (cPE) using an osmotic pump. Specimens were evaluated using micro-CT and histomorphometric analyses. Sex-specific osteogenic and osteoclastic differentiation potentials were also investigated in vitro, including alkaline phosphatase, Alizarin Red, tartrate-resistant acid phosphatase staining, and gene expression using reverse transcription polymerase chain reaction (RT-PCR). Local delivery of NF-κB decoy ODN in vivo increased osteogenesis in males, but not females, in the presence of chronic inflammation induced by cPE. Bone resorption activity was decreased in both sexes. In vitro osteogenic and osteoclastic differentiation assays during inflammatory conditions did not reveal differences among the groups. Receptor activator of nuclear factor kappa Β ligand (Rankl) gene expression by osteoblasts was significantly decreased only in males when treated with ODN. We demonstrated that NF-κB decoy ODN increased osteogenesis in male mice and decreased bone resorption activity in both sexes in preclinical models of chronic inflammation. NF-κB signalling could be a therapeutic target for chronic inflammatory diseases involving bone, especially in males.