Ligand Gene Research Articles

Painful intercellular connections

Pain Chronic pain impairs quality of life and is challenging to treat. Wangzhou et al. measured ligand and receptor gene expression in sensory neurons and various peripheral cell types from humans and mice. Using these data, the authors constructed a connectome of potential cell type–specific interaction points between sensory nerves and innervated tissues, which suggested that blocking heparin-binding epidermal growth factor (HBEGF) signaling may be an effective way to alleviate chronic pain in multiple diseases. This target was validated in a mouse model of mechanical pain. Sci. Signal. 14 , abe1648 (2021).

Protective immune response against P32 oncogenic peptide-pulsed PBMCs in mouse models of breast cancer

High expression of p32 in certain tumors makes it a potential target for immunotherapy. In the present study, the first goal was to design multi-epitope peptides from the P32 protein and the second goal was to compare the prophylactic effects of DCs- and PBMCs- based vaccines by pulsing them with designed peptides. For these purposes, 160 BALB/c mice were vaccinated in 5 different subgroups of each 4 peptides using PBS (F1-4a), F peptides alone (F1-4b), F peptides with CpG-ODN (F1-4c), F peptides with CpGODN and DCs (F1-4d), and F peptides with CpG-ODN and PBMCs (F1-4e). We found a significantly higher interferon-γ (IFN-γ) and granzyme B levels in T cells of F4d and F4e subgroups compared to control (p ≤ 0.05). The result of challenging spleen PBMCs of vaccinated mice with 4T1 cells showed significant up- and down- regulation of Fas ligand (FasL) and forkhead box P3 (Foxp3) gene expression between F4d and F4e subgroups with control, respectively. In addition, a significant change was seen in Caspase3 gene expression of F4d subgroup compared to control (p ≤ 0.05). Supernatant levels of IFN-γ and perforin were significantly increased in F4d and F4e subgroups compared to control. Consequently, significantly lower tumor sizes and prolonged survival time were detected in F4d and F4e subgroups compared to control after challenging mice with 4T1 cells. Accordingly, these results demonstrated that PBMCs pulsed F4 peptide-based vaccine could induce a protective immune response while it is a simple and less expensive vaccine.

Association of RANKL and OPG Gene Polymorphism in Arab Women with and without Osteoporosis.

Receptor activator of the nuclear factor-κB ligand (RANKL) and osteoprotegerin genes (OPG) were identified as susceptible loci for postmenopausal osteoporosis (PMO) in various ethnicities, but neither have been studied in an Arabian population. Hence, the current study aimed to fill this gap. A total of 372 postmenopausal women (174 osteoporosis (OP) and 198 control group (CTRs)) were genotyped for four SNPs: rs2277438A/G and rs9533156T/C (RANKL), and rs2073618C/G and rs3102735T/C (OPG). Anthropometrics, bone mineral density, 25(OH)D and several other bone markers were measured. The frequency distribution of the heterozygous CG genotype of rs2073618 (OPG) was lower in the OP (36.8%) than in CTRs (47%) (OR: 0.6, 95% CI: 0.3–0.97; p = 0.041). No differences in the allelic/genotypic frequencies were detected between the two groups for all other studied SNPs. However, the heterozygous TC genotype of rs3102735 (OPG) was associated significantly with lower BMD at the femoral neck in OP subjects (p = 0.04). The homozygous rare CC genotype of rs9533156 (RANKL) was associated with lower 25(OH)D levels in CTRs (p = 0.032). In contrast, heterozygous AG genotype of rs2277438 (RANKL) is associated with lower 25(OH)D in the OP group (p = 0.02). Our results suggest that RANKL SNPs may impact 25(OH)D levels and that OPG SNP rs2073618A/G is a significant genetic risk factor for PMO Saudi Arabian women.

Zinc chloride affects chondrogenesis via VEGF signaling

Insulin mimetics, including zinc containing compounds, have previously been shown to influence chondrogenesis as it relates to healing of fractures in various preclinical models. However, the mechanism by which these compounds drive chondrogenic differentiation is yet undefined. Here, via next-generation sequencing (NGS) and in vitro functional validation, we show that Zinc Chloride (ZnCl2) induces expression of both chondrogenic genes (Sox9, Runx1, collagen) as well as genes associated with VEGF-mediated signal transduction, including VEGF receptors 1 and 2 and their ligands; VEGF-A and VEGF-B. Noticeably, although insulin was able to also induce expression of these pro-angiogenic and pro-chondrogenic genes, the impact of insulin on expression of VEGF receptor and ligand genes was marginal when compared to that of ZnCl2. Furthermore, while the VEGFR antagonist, Axitinib, was able to attenuate the pro-chondrogenic effects of both insulin and ZnCl2; a reduction in gene and protein expression was most profoundly observed when the antagonist was applied to cells treated with ZnCl2. Taken together, these data suggest an important role for the VEGF-mediated signal transduction pathways in the positive effects observed when applying zinc-based compounds as adjuvants for chondrogenesis-mediated fracture healing. In this regard, further mechanistic evaluation of ZnCl2 and other zinc-containing insulin mimetics may support rational design of therapies targeted for disease indications associated with impaired fracture healing.

Transcriptional Riboswitches Integrate Timescales for Bacterial Gene Expression Control.

Transcriptional riboswitches involve RNA aptamers that are typically found in the 5′ untranslated regions (UTRs) of bacterial mRNAs and form alternative secondary structures upon binding to cognate ligands. Alteration of the riboswitch's secondary structure results in perturbations of an adjacent expression platform that controls transcription elongation and termination, thus turning downstream gene expression “on” or “off.” Riboswitch ligands are typically small metabolites, divalent cations, anions, signaling molecules, or other RNAs, and can be part of larger signaling cascades. The interconnectedness of ligand binding, RNA folding, RNA transcription, and gene expression empowers riboswitches to integrate cellular processes and environmental conditions across multiple timescales. For a successful response to an environmental cue that may determine a bacterium's chance of survival, a coordinated coupling of timescales from microseconds to minutes must be achieved. This review focuses on recent advances in our understanding of how riboswitches affect such critical gene expression control across time.

Identification of a novel mutation in the KITLG gene in a Chinese family with familial progressive hyper- and hypopigmentation

BackgroundFamilial progressive hyper- and hypopigmentation (FPHH, MIM 145250) is a rare hereditary skin disorder that is predominantly characterized by progressive, diffuse, partly blotchy hyperpigmented lesions intermingled with scattered hypopigmented spots, lentigines and sometimes Cafe-au-lait spots (CALs). Heterozygous mutations of the KIT ligand (KITLG, MIM 184745) gene are responsible for FPHH. To date, only eight KITLG mutations have been reported to be associated with FPHH, and no clear genotype–phenotype correlations have been established. This study aimed to identify the causative mutations in the KITLG gene in two Chinese FPHH patients.MethodsDirect sequencing of the coding regions of KITLG was performed. Pathogenicity prediction was performed using bioinformatics tools, including SIFT, Polyphen2, and SWISS-MODEL, and the results were further evaluated according to the 2015 American College of Medical Genetics and Genomics (ACMG) guidelines.ResultsThe novel mutation c.104A > T (p.Asn35Ile) and the recurrent mutation c.101C > T (p.Thr34Ile) in KITLG were identified. As shown using SIFT and Polyphen-2 software, both mutations identified in this study were predicted to be detrimental variations. Three-dimensional protein structure modeling indicated that the mutant KITLG proteins might affect the affinity of KITLG for its receptor, c-KIT. According to the 2015 ACMG guidelines, the novel mutation c.104A > T was ‘likely pathogenic’.ConclusionsTo date, most of the identified KITLG mutations have been clustered within the conserved VTNNV motif (amino acids 33–37) in exon 2. The known mutations are only involved in 33 V, 34 T, 36 N, and 37 V but not 35 N. We have now identified a novel mutation in KITLG, c.104A > T, that was first reported in FPHH within the conserved 35 N motif. These results strengthen our understanding of FPHH and expand the mutational spectrum of the KITLG gene.

Osteogenic Properties of 3D-Printed Silica-Carbon-Calcite Composite Scaffolds: Novel Approach for Personalized Bone Tissue Regeneration.

Carbon enriched bioceramic (C-Bio) scaffolds have recently shown exceptional results in terms of their biological and mechanical properties. The present study aims at assessing the ability of the C-Bio scaffolds to affect the commitment of canine adipose-derived mesenchymal stem cells (cAD-MSCs) and investigating the influence of carbon on cell proliferation and osteogenic differentiation of cAD-MSCs in vitro. The commitment of cAD-MSCs to an osteoblastic phenotype has been evaluated by expression of several osteogenic markers using real-time PCR. Biocompatibility analyses through 3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), lactate dehydrogenase (LDH) activity, hemolysis assay, and Ames test demonstrated excellent biocompatibility of both materials. A significant increase in the extracellular alkaline phosphatase (ALP) activity and expression of runt-related transcription factor (RUNX), ALP, osterix (OSX), and receptor activator of nuclear factor kappa-Β ligand (RANKL) genes was observed in C-Bio scaffolds compared to those without carbon (Bio). Scanning electron microscopy (SEM) demonstrated excellent cell attachment on both material surfaces; however, the cellular layer on C-Bio fibers exhibited an apparent secretome activity. Based on our findings, graphene can improve cell adhesion, growth, and osteogenic differentiation of cAD-MSCs in vitro. This study proposed carbon as an additive for a novel three-dimensional (3D)-printable biocompatible scaffold which could become the key structural material for bone tissue reconstruction.

TNF-related apoptosis-inducing ligand (TRAIL), death receptor (DR4) and Fas gene polymorphisms associated with liver cirrhosis in hepatitis C infected patients

Host genetic factors of apoptotic genes could play an important role in the rapid progression of chronic hepatitis C virus (HCV) infection to liver cirrhosis. This study focused on the role of Tumor Necrosis Factor Related Apoptosis-Inducing Ligand (TRAIL) (1525G/A and 1595C/T), TRAIL Death Receptor4 (DR4) (626C/G and 683A/C) and Fas (−670A/G and −1377G/A) genetic polymorphisms in the susceptibility of HCV infected patients to develop liver cirrhosis. Genotyping was performed by restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) in 69 cirrhotic and106 non-cirrhotic patients. Association of selected genes with response to various direct-acting anti-viral drugs was demonstrated. Our data showed an increase in TRAIL (1525AA and 1595TT), DR4 (626CC, 683AA, and 683CC) genotypes, and the DR4 683A allele in cirrhotic patients. However, no statistical significance in their distribution was found indicating the lack of association between these SNPs and liver cirrhosis susceptibility in HCV-infected patients. The genotype frequencies of TRAIL at loci 1525 and 1595 appeared to be in complete linkage disequilibrium (D'= 0.9997, r2= 0.9994). Concerning treatment, a significant elevation in AA genotype and A allele of DR4 (683A/C) was found. The outcome of this study reveals that, in our population group, no association between apoptotic genes polymorphism and susceptibility to HCV-related liver cirrhosis. Studies concerning other apoptosis gene polymorphisms are required to get a better understanding of the development of cirrhosis in HCV-infected patients and to help in limiting HCV complications and improve the management and treatment services.

Reconstruction of Intercellular Signaling Network by Cytokine-Receptor Interactions.

Background: The immune system function depends on the coordination activity of the components of system and communications between them which leads to the formation of a complex communication network between immune cells. In this network, cytokines have an important role in the communication between immune cells through the interaction to their specific receptors. These molecules cause to cellular communications and normal function of a tissue. Reconstruction of such a complex network can be a way to provide a better understanding of cytokines’ function.Objective: Our main goal from reconstructing such a network was investigation of expressed cytokines and cytokines receptors in various lineage and tissues of immune cells and identifying the lineage and tissue with the highest expression of cytokines and their receptors.Materials and Methods: In this study, gene expression data related to part of the Immunological Genome Project (ImmGen) and receptor-ligand interactions dataset were used to reconstruct the immune network in mouse. In next step, the topological properties of reconstructed network, expression specificity of cytokines and their receptors and interactions specificity were analyzed.Results: The results of the network analysis were indicated that non- hematopoietic stromal cells have the highest expression of cytokines and cytokine receptors and interactions specificity is very high. Our results show that chemokine receptor of Ccr1 receives the largest number of signals between receptors and only expressed in three hematopoietic lineages.Conclusions: The most of the network communications belonged to non-hematopoietic stromal and macrophage cells. The relationships between stromal cells and macrophages are necessary to create an appropriate environment for differentiation of immune cells. Studying the cellular expression specificity of receptor and ligand genes reveal the high degree of specificity of these genes that indicate non-random transfer of information between cells in multicellular organisms.

Isthmin1, a secreted signaling protein, acts downstream of diverse embryonic patterning centers in development

Extracellular signals play essential roles during embryonic patterning by providing positional information in a concentration-dependent manner, and many such signals, like Wnt, fibroblast growth factor (FGF), Hedgehog (Hh), and retinoic acid, act by being secreted into the extracellular space, thereby triggering receptor-mediated responses in other cells. Isthmin1 (ism1) is a secreted protein whose gene expression pattern coincides with that of early dorsal determinants, nodal ligand genes like sqt and cyc, and with fgf8 during various phases of zebrafish development. Ism1 functions in early embryonic patterning and development are poorly understood; however, it has recently been shown to interact with nodal pathway genes to control organ asymmetry in chicken. Here, we show that misexpression of ism1 deletion constructs disrupts embryonic patterning in zebrafish and exhibits genetic interactions with both Fgf and nodal signaling. Unlike Fgf and nodal pathway mutants, CRISPR/Cas9-engineered ism1 mutants did not show obvious developmental defects. Further, in vivo single molecule fluorescence correlation spectroscopy (FCCS) showed that Ism1 diffuses freely in the extra-cellular space, with a diffusion coefficient similar to that of Fgf8a; however, our measurements do not support direct molecular interactions between Ism1 and either nodal ligands or Fgf8a in the developing zebrafish embryo. Together, data from gain- and loss-of-function experiments suggest that zebrafish Ism1 plays a complex role in regulating extracellular signals during early embryonic development.

Effect of Eight Weeks of Resistance Training and Consumption of Tribulus Terrestris on Androgenic Receptor-1, Fas Ligand Gene Expression, and Lipid Profiles in Rats Exposed to Stanozolol

Background: Protective effect of medicinal plants on the heart has been reported, but the effect of resistance training (RT) and Tribulus terrestris (TT) on the heart exposed to anabolic-androgenic steroids (AAS) abuse is still unknown. Objectives: The present study aimed to investigate the effect of RT and TT on androgen receptor-1 (ar-1), Fas ligand (fasl) gene expression and lipid profiles in rats exposed to stanozolol (S). Methods: Thirty-five male rats were selected and divided into 7 groups as follows: (1) sham (normal saline/Sh), (2) stanozolol (S), (3) S+100 mg/kg TT (S+TT100), (4) S+ 50 mg/kg TT (S+TT50), (5) S+RT+TT, (6) S+RT+TT100, and (7) S+RT+TT50. Over a course of eight-week period, groups 3, 4, 6, and 7 received 50 and 100 mg/kg/d doses of TT peritoneally and groups 5-7 performed three sessions of increasing RT per week. Results: RT decreased plasma cholesterol and low-density lipoprotein cholesterol (LDL-C) levels, as well as ar-1 and fasl gene expression in S-exposed rats (P<0.05). TT50, TT100, SRTT100, and SRTT50 reduced ar-1 and fasl gene expressions (P<0.05). TT50 reduced triglyceride (TG), cholesterol and increased high-density lipoprotein-cholesterol (HDL-C) (P≤0.01), and TT100 decreased LDL-C levels (P<0.05). Additionally, SRTT100 reduced TG, cholesterol, and LDL-C levels and increased HDL-C level (P<0.05), and SRTT50 decreased cholesterol level and increased HDL-C level in S-exposed rats (P<0.05). Conclusion: RT and consumption of TT appear to have protective effects on the improvement of apoptosisdependent androgen receptor-1 and lipid profile in S-exposed rats.

Juvenile idiopathic arthritis fibroblast-like synoviocytes influence chondrocytes to alter BMP antagonist expression demonstrating an interaction between the two prominent cell types involved in endochondral bone formation

BackgroundTo examine critical interactions between juvenile idiopathic arthritis synovial fibroblasts (JFLS) and chondrocytes (Ch), and their role in bony overgrowth seen in patients with juvenile idiopathic arthritis (JIA).MethodsControl (CFLS) and JFLS were cultured in synoviocyte media containing recombinant BMP4. Ch were cultured in either CFLS or JFLS conditioned-media without stimulation. Media supernatants were analyzed by ELISA. RNA from conditioned media experiment was analyzed by ClariomS microarray.ResultsAs expected, genes expressed in untreated JFLS and CFLS cultured in synoviocyte media were similar to each other and this expression differed from untreated Ch cultured in chondrocyte media. JFLS favor BMP ligand gene expression while downregulating TGFβ receptors’ expression. Noggin and chordin, antagonists with high affinity for BMP4, are JFLS- but not Ch-preferred regulators of BMP signaling. Compared to Ch, JFLS overexpress collagen X (COLX), a marker of chondrocyte hypertrophy. Exogenous BMP4 causes JFLS to significantly decrease expression of noggin and collagen II (COL2), a marker of chondrocyte proliferation, and causes overexpression of COLX and alkaline-phosphatase (ALP). Chondrocytes cultured in JFLS-conditioned media (Ch-JFLS) express BMP genes and favor chordin protein expression over other antagonists. Ch-JFLS have significantly increased expression of COL2 and significantly decreased expression of COLX.ConclusionsThese data suggest JFLS, in the presence of BMP4, undergo hypertrophy and that JFLS-conditioned media influence chondrocytes to become highly proliferative. To the authors’ knowledge, no prior study has shown that JFLS and chondrocytes play a direct role in the bony overgrowth in joints of patients with JIA and that BMPs or regulation of these growth factors influence the interaction between two prominent synovial cell types.

Establishment of immune prognostic signature and analysis of prospective molecular mechanisms in childhood osteosarcoma patients.

In pediatric tumors, immunotherapy exhibits less toxicity than chemotherapy and radiation. The current study aims to identify potential immune targets in immune-related genes of C-C motif chemokine ligand genes (CCLs) and C-C motif chemokine receptors (CCRs) in childhood osteosarcoma (OS) and to explore the underlying molecular mechanisms of childhood OS. Firstly, we identified immune-related genes in CCLs and CCRs, these genes were used for functional annotation and interaction analysis. Then, the prognostic value of these genes was evaluated using Kaplan-Meier analysis and multivariate COX regression model. And the potential relationship between risk score and immune infiltrating cells was identified. Finally, gene set enrichment analysis was used to determine the underlying molecular mechanism of OS. Immune-related genes in CCLs and CCRs are inextricably linked. The results of survival analysis of these genes show that CCL5, CCL8, CCR4, and CCR5 are significantly associated with the prognosis of childhood OS. The combined effect survival analysis shows that the co-high expression of these 4 genes has a good prognosis for childhood OS. A prognostic signature model was constructed based on the 4 genes mentioned above, and the result of time-dependent receiver operating characteristic curves showed that this model was a good predictor of childhood OS 3- and 5-year prognosis. In addition, the risk score of the constructed prognostic signature model was closely related to immune infiltration. We also found that CCL5, CCL8, and CCR5 may affect the prognosis of OS through complex regulation among Toll-like receptor signaling pathway, mitogen-activated protein kinase (MAPK) family signaling cascade, and nuclear factor-kappaB pathway, whereas CCR4 affects the prognosis of OS by regulating eukaryotic translation. CCL5, CCL8, CCR4, and CCR5 are potential prognostic markers for the prognosis of childhood OS, and the underlying molecular mechanisms of childhood OS have been identified.

The Polycomb group protein Ring1 regulates dorsoventral patterning of the mouse telencephalon

Dorsal-ventral patterning of the mammalian telencephalon is fundamental to the formation of distinct functional regions including the neocortex and ganglionic eminence. While Bone morphogenetic protein (BMP), Wnt, and Sonic hedgehog (Shh) signaling are known to determine regional identity along the dorsoventral axis, how the region-specific expression of these morphogens is established remains unclear. Here we show that the Polycomb group (PcG) protein Ring1 contributes to the ventralization of the mouse telencephalon. Deletion of Ring1b or both Ring1a and Ring1b in neuroepithelial cells induces ectopic expression of dorsal genes, including those for BMP and Wnt ligands, as well as attenuated expression of the gene for Shh, a key morphogen for ventralization, in the ventral telencephalon. We observe PcG protein–mediated trimethylation of histone 3 at lysine-27 and binding of Ring1B at BMP and Wnt ligand genes specifically in the ventral region. Furthermore, forced activation of BMP or Wnt signaling represses Shh expression. Our results thus indicate that PcG proteins suppress BMP and Wnt signaling in a region-specific manner and thereby allow proper Shh expression and development of the ventral telencephalon.

Transcriptomic Features and Deregulation of Genes Involved in Monopoiesis Influence Outcomes of Patients with Chronic Myelomonocytic Leukemia

INTRODUCTION: Chronic myelomonocytic leukemia (CMML) is characterized by TET2, SRSF2, ASXL1 and RAS pathway mutations known to induce myelomonocytic bias. We have previously shown that upregulation of KDM6B, a histone demethylase that acts as an innate immune signal activator, leads to myeloid bias and expansion of immature myelomonocytic precursors and LSK cells in mice, resulting in rapid development of a myeloproliferative CMML (MP-CMML) phenotype particularly in cooperation with TET2 deletion. However, the role of genes involved in innate immunity regulation and monocyte differentiation in CMML phenotype and outcomes remains unclear. METHODS: We evaluated bone marrow aspirates from 19 patients with CMML and compared their transcriptomic features to those of healthy individuals obtained from AllCells (Emeryville, CA). CD34+ cells were isolated using the CD34 MicroBead Kit and RNA was isolated using the PicoPure RNA isolation kit. Fastq files were mapped to the human genome (build GRCh38) in TopHat2 using the default options. Differential gene expression analysis was conducted using DESeq2 in R version 3.4.2. Pathway enrichment analysis was performed using gene set enrichment analysis, with the fgsea library in R. Genes were ranked according to their Spearman correlation with the gene of interest, and this ranking was used as the input to fgsea. 10 000 gene permutations were used to calculate statistical significance, and a false discovery corrected p-value of 0.05 was required for statistical significance of a gene set. Cox regression and time ROC curves were used to study the relationship between gene expression and survival. We implemented Kaplan-Meier estimator along with optimum cutoff method to show the survival behavior in high versus low predicted model groups. RESULTS: Compared to healthy controls, a total of 1495 genes had significantly differential expression in CMML (q<0.05, FC>2) including 1271 genes which were significantly upregulated, and 224 which were significantly downregulated in CMML (Figure 1A). Gene set enrichment analysis identified 162 gene sets with differential expression in CMML compared to control (q<0.05). Top upregulated genes were associated with interferon (IFN) alpha and beta signaling, chemokine receptors, IFN-gamma, GPC receptor ligand signaling and genes involved in immunomodulatory interactions between lymphoid and non-lymphoid cells (Figure 1B). Unsupervised clustering of gene expression profiles did not discriminate MP-CMML from myelodysplastic (MD-CMML). However, 20 genes were significantly overexpressed and 16 were significantly downregulated in patients with MP-CMML compared to MD-CMML (q<0.05, FC>2). In addition, 6 gene sets were differentially upregulated and 139 were significantly downregulated in pts with MP-CMML compared to MD-CMML (Figure 1C). To evaluate aberrant monopoiesis in CMML, we compared the expression of genes reported to be involved in regulation of monopoiesis among healthy controls and patients with CMML. A total of 23 genes involved in regulation of monopoiesis were found to be upregulated in CMML (Figure 1D). No significant differences in expression of these genes was found between MP-CMML and MD-CMML. To determine if aberrant expression of genes involved in monopoiesis influenced outcomes of pts with CMML, we developed a prediction model using Cox regression including 18 of these genes. Use of this model with optimum cutoffs allowed segregation of pts into two prognostic subsets with distinct survival outcomes (Figure 1E). Use of ROC curves identified high AUC particularly in pts with prolonged survival (>40 months). CONCLUSIONS: CMML is characterized by upregulation of IFN and chemokine receptor signaling which could represent potential therapeutic targets. Aberrant expression of genes involved in regulation of monopoiesis may influence prognosis in CMML. Figure Disclosures Sasaki: Otsuka: Honoraria; Pfizer Japan: Consultancy; Daiichi Sankyo: Consultancy; Novartis: Consultancy, Research Funding. Kantarjian:Sanofi: Research Funding; Pfizer: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; BMS: Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Immunogen: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ascentage: Research Funding; Adaptive biotechnologies: Honoraria; Aptitute Health: Honoraria; BioAscend: Honoraria; Delta Fly: Honoraria; Janssen: Honoraria; Oxford Biomedical: Honoraria; Jazz: Research Funding. Garcia-Manero:AbbVie: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Onconova: Research Funding; Acceleron Pharmaceuticals: Consultancy, Honoraria; H3 Biomedicine: Research Funding; Jazz Pharmaceuticals: Consultancy; Merck: Research Funding; Amphivena Therapeutics: Research Funding; Helsinn Therapeutics: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

Dissecting the epigenomic dynamics of human fetal germ cell development at single-cell resolution

Proper development of fetal germ cells (FGCs) is vital for the precise transmission of genetic and epigenetic information through generations. The transcriptional landscapes of human FGC development have been revealed; however, the epigenetic reprogramming process of FGCs remains elusive. Here, we profiled the genome-wide DNA methylation and chromatin accessibility of human FGCs at different phases as well as gonadal niche cells at single-cell resolution. First, we found that DNA methylation levels of FGCs changed in a temporal manner, whereas FGCs at different phases in the same embryo exhibited comparable DNA methylation levels and patterns. Second, we revealed the phase-specific chromatin accessibility signatures at the promoter regions of a large set of critical transcription factors and signaling pathway genes. We also identified potential distal regulatory elements including enhancers in FGCs. Third, compared with other hominid-specific retrotransposons, SVA_D might have a broad spectrum of binding capacity for transcription factors, including SOX15 and SOX17. Finally, using an in vitro culture system of human FGCs, we showed that the BMP signaling pathway promoted the cell proliferation of FGCs, and regulated the WNT signaling pathway by orchestrating the chromatin accessibility of its ligand genes. Our single-cell epigenomic atlas and functional assays provide valuable insights for understanding the strongly heterogeneous, unsynchronized, yet highly robust nature of human germ cell development.

Deletion of Mediator 1 suppresses TGFβ signaling leading to changes in epidermal lineages and regeneration.

Epidermal lineages and injury induced regeneration are controlled by transcriptional programs coordinating cellular signaling and epigenetic regulators, but the mechanism remains unclear. Previous studies showed that conditional deletion of the transcriptional coactivator Mediator 1 (Med1) changes epidermal lineages and accelerates wound re-epithelialization. Here, we studied a molecular mechanism by which Med1 facilitates these processes, in particular, by focusing on TGFβ signaling through genome wide transcriptome analysis. The expression of the TGF ligands (Tgfβ1/β2) and their downstream target genes is decreased in both normal and wounded Med1 null skin. Med1 silencing in cultured keratinocytes likewise reduces the expression of the ligands (TGFβ1/β2) and diminishes activity of TGFβ signaling as shown by decreased p-Smad2/3. Silencing Med1 increases keratinocyte proliferation and migration in vitro. Epigenetic studies using chromatin immuno-precipitation and next generation DNA sequencing reveals that Med1 regulates transcription of TGFβ components by forming large clusters of enhancers called super-enhancers at the regulatory regions of the TGFβ ligand and SMAD3 genes. These results demonstrate that Med1 is required for the maintenance of the TGFβ signaling pathway. Finally, we show that pharmacological inhibition of TGFβ signaling enhances epidermal lineages and accelerates wound re-epithelialization in skin similar to that seen in the Med1 null mice, providing new insights into epidermal regeneration.

ALTERNATIVE SPLICING OF mRNA TRAIL REGULATES APOPTOSIS IN THE GLIOBLASTOMA MULTIFORME T-98G CELL LINE

Objective: This is an in vitro experimental study designed to analyze the role of alternative splicing of mRNA in the apoptotic process of the cancer cells. Here we induced apoptosis in the glioblastoma multiforme (GBM) T-98G cell line to obtain a better understanding in the regulation of mRNA expression of the soluble Tumor Necrosis factor-related Apoptosis-Inducing Ligand (sTRAIL) gene.
 Methods: Cells were induced to undergo apoptosis by treatment with rotenone at 10, 20 and 40 µM for 6 h. Dimethylsulphoxide (DMSO) was used to dissolve rotenone and as a negative control. The morphology of the GBM-T98G cells was viewed with an inverted microscope. DNA, RNA and protein extractions were performed to analyse apoptotic DNA fragmentation by a DNA laddering assay, a quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) for TRAIL mRNA expression and ELISA for caspase-9 protein expression. Electrophoresis was also performed on TRAIL complementary DNA (cDNA) produced from TRAIL qRT-PCR mRNA.
 Results: Nucleosomal DNA degradation was confirmed by DNA laddering, whereas the TRAIL melting curve and the cDNA electrophoresis showed a shift in the balance of the TRAIL mRNA isoform to the pro-apoptotic mRNA isoform, in conjunction with a significant increase in expression of TRAIL mRNA and caspase-9 protein.
 Conclusion: These findings indicate the regulation of apoptotic events at the level of TRAIL mRNA expression, as indicated by the shift in the balance of mRNA expression of the TRAIL isoform towards the pro-apoptotic isoform.

Therapeutic Efficacy of Immune Stimulatory Thymidine Kinase and fms-like Tyrosine Kinase 3 Ligand (TK/Flt3L) Gene Therapy in a Mouse Model of High-Grade Brainstem Glioma.

Diffuse intrinsic pontine glioma (DIPG) bears a dismal prognosis. A genetically engineered brainstem glioma model harboring the recurrent DIPG mutation, Activin A receptor type I (ACVR1)-G328V (mACVR1), was developed for testing an immune-stimulatory gene therapy. We utilized the Sleeping Beauty transposase system to generate an endogenous mouse model of mACVR1 brainstem glioma. Histology was used to characterize and validate the model. We performed RNA-sequencing analysis on neurospheres harboring mACVR1. mACVR1 neurospheres were implanted into the pons of immune-competent mice to test the therapeutic efficacy and toxicity of immune-stimulatory gene therapy using adenoviruses expressing thymidine kinase (TK) and fms-like tyrosine kinase 3 ligand (Flt3L). mACVR1 neurospheres expressing the surrogate tumor antigen ovalbumin were generated to investigate whether TK/Flt3L treatment induces the recruitment of tumor antigen-specific T cells. Histologic analysis of mACVR1 tumors indicates that they are localized in the brainstem and have increased downstream signaling of bone morphogenetic pathway as demonstrated by increased phospho-smad1/5 and Id1 levels. Transcriptome analysis of mACVR1 neurosphere identified an increase in the TGFβ signaling pathway and the regulation of cell differentiation. Adenoviral delivery of TK/Flt3L in mice bearing brainstem gliomas resulted in antitumor immunity, recruitment of antitumor-specific T cells, and increased median survival (MS). This study provides insights into the phenotype and function of the tumor immune microenvironment in a mouse model of brainstem glioma harboring mACVR1. Immune-stimulatory gene therapy targeting the hosts' antitumor immune response inhibits tumor progression and increases MS of mice bearing mACVR1 tumors.

Soft tissue healing around platform-switching and platform-matching single implants: A randomized clinical trial.

Implants with platform-switching (PS) design have been demonstrated to reduce marginal bone loss. However, the influence on peri-implant soft tissue healing is unclear. This study was designed to investigate its effect on peri-implant soft tissue healing after implant uncovery. Non-smokers needing two implants in different quadrants were recruited in this study. For each individual, one PS and one platform-matching (PM) implants were placed using two-stage protocol. Following 2 to 8 months of healing, all implants were uncovered and connected to the corresponding healing abutments. Clinical measurements and peri-implant crevicular fluid (PICF) were taken at 1-, 2-, 4-, and 6-week after 2nd stage surgery. The cytokine concentrations in PICF were analyzed. Peri-implant mucosa (1 × 2 × 2mm) was harvested around the healing abutment for the analysis of gene expression at uncovery and 6-week post-uncovery. Eighteen participants (nine males; 51.7 ± 14.9 years) were recruited. Compared to PM, PS showed significantly lower probing depth (PD) at 1- and 2-week as well as modified sulcus bleeding index (mSBI) at 1-, 4-, and 6-week (P<0.05). Over time, a decrease in osteoprotegerin and interleukin-1β concentrations in PICF along with an increase in receptor activator of unclear factor kappa-B ligand, periostin, and peroxidasin gene expressions in peri-implant mucosa were noted within both groups (P<0.05) without significant intergroup differences. Within the limits, implants with PS design rendered significant benefits over PM design in PD and mSBI reduction during a 6-week healing. However, molecular changes within PICF and peri-implant mucosa as a response to PM and PS appear negligible.