Evolution of Brain-Expressed Biogenic Amine Receptors into Olfactory Trace Amine-Associated Receptors.

The family of trace amine-associated receptors (TAARs) is distantly related to G protein-coupled biogenic aminergic receptors. TAARs are found in the brain as well as in the olfactory epithelium where they detect biogenic amines. However, the functional relationship of receptors from distinct TAAR subfamilies and in different species is still uncertain. Here, we perform a thorough phylogenetic analysis of 702 TAAR-like (TARL) and TAAR sequences from 48 species. We show that a clade of Tarl genes has greatly expanded in lampreys, whereas the other Tarl clade consists of only one or two orthologs in jawed vertebrates and is lost in amniotes. We also identify two small clades of Taar genes in sharks related to the remaining Taar genes in bony vertebrates, which are divided into four major clades. We further identify ligands for 61 orphan TARLs and TAARs from sea lamprey, shark, ray-finned fishes, and mammals, as well as novel ligands for two 5-hydroxytryptamine receptor 4 orthologs, a serotonin receptor subtype closely related to TAARs. Our results reveal a pattern of functional convergence and segregation: TARLs from sea lamprey and bony vertebrate olfactory TAARs underwent independent expansions to function as chemosensory receptors, whereas TARLs from jawed vertebrates retain ancestral response profiles and may have similar functions to TAAR1 in the brain. Overall, our data provide a comprehensive understanding of the evolution and ligand recognition profiles of TAARs and TARLs.

[Results of carrier screening for Spinal muscular atrophy among 35 145 reproductive-aged individuals from Dongguan region].

OBJECTIVE: To carry out carrier screening for Spinal muscular atrophy (SMA) in reproductive-aged individuals from Dongguan region and determine the carrier frequency of SMN1 gene mutations. METHODS: Reproductive-aged individuals who underwent SMN1 genetic screening at the Dongguan Maternal and Child Health Care Hospital from March 2020 to August 2022 were selected as the study subjects. Deletions of exon 7 and 8 (E7/E8) of the SMN1 gene were detected by real-time fluorescence quantitative PCR (qPCR), and prenatal diagnosis was provided for carrier couples by multiple ligation-dependent probe amplification (MLPA). RESULTS: Among the 35 145 subjects, 635 were found to be carriers of SMN1 E7 deletion (586 with heterozygous E7/E8 deletion, 2 with heterozygous E7 deletion and homozygous E8 deletion, and 47 with sole heterozygous E7 deletion). The carrier frequency was 1.81% (635/35 145), with 1.59% (29/1 821) in males and 1.82% (606/33 324) in females. There was no significant difference between the two genders (χ² = 0.497, P = 0.481). A 29-year-old woman was found to harbor homozygous deletion of SMN1 E7/E8, and was verified to have a SMN1∶SMN2 ratio of [0∶4], none of her three family members with a [0∶4] genotype had clinical symptoms. Eleven carrier couples had accepted prenatal diagnosis, and one fetus was found to have a [0∶4] genotype, and the pregnancy was terminated. CONCLUSION: This study has determined the SMA carrier frequency in Dongguan region for the first time and provided prenatal diagnosis for carrier couples. The data can provide a reference for genetic counseling and prenatal diagnosis, which has important clinical implications for the prevention and control of birth defects associated with SMA.

Adding colour-realistic video images to audio playbacks increases stimulus engagement but does not enhance vocal learning in zebra finches.

Bird song and human speech are learned early in life and for both cases engagement with live social tutors generally leads to better learning outcomes than passive audio-only exposure. Real-world tutor-tutee relations are normally not uni- but multimodal and observations suggest that visual cues related to sound production might enhance vocal learning. We tested this hypothesis by pairing appropriate, colour-realistic, high frame-rate videos of a singing adult male zebra finch tutor with song playbacks and presenting these stimuli to juvenile zebra finches (Taeniopygia guttata). Juveniles exposed to song playbacks combined with video presentation of a singing bird approached the stimulus more often and spent more time close to it than juveniles exposed to audio playback only or audio playback combined with pixelated and time-reversed videos. However, higher engagement with the realistic audio-visual stimuli was not predictive of better song learning. Thus, although multimodality increased stimulus engagement and biologically relevant video content was more salient than colour and movement equivalent videos, the higher engagement with the realistic audio-visual stimuli did not lead to enhanced vocal learning. Whether the lack of three-dimensionality of a video tutor and/or the lack of meaningful social interaction make them less suitable for facilitating song learning than audio-visual exposure to a live tutor remains to be tested.

MDGA2 is a novel tumour suppressor cooperating with DMAP1 in gastric cancer and is associated with disease outcome.

BACKGROUND: Using the promoter methylation assay, we have shown that MDGA2 (MAM domain containing glycosylphosphatidylinositol anchor 2) is preferentially methylated in gastric cancer. We analysed its biological effects and prognostic significance in gastric cancer. METHODS: MDGA2 methylation status was evaluated by combined bisulfite restriction analysis and bisulfite genomic sequencing. The effects of MDGA2 re-expression or knockdown on cell proliferation, apoptosis and the cell cycle were determined. MDGA2 interacting protein was identified by mass spectrometry and MDGA2-related cancer pathways by reporter activity and PCR array analyses. The clinical impact of MDGA2 was assessed in 218 patients with gastric cancer. RESULTS: MDGA2 was commonly silenced in gastric cancer cells (10/11) and primary gastric cancers due to promoter hypermethylation. MDGA2 significantly inhibited cell proliferation by causing G1-S cell cycle arrest and inducing cell apoptosis in vitro, and suppressed xenograft tumour growth in both subcutaneous and orthotopic xenograft mouse models (both p<0.001). The anti-tumorigenic effect of MDGA2 was mediated through direct stabilising of DNA methyltransferase 1 associated protein 1 (DMAP1), which played a tumour suppressive role in gastric cancer. This interaction activated their downstream key elements of p53/p21 signalling cascades. Moreover, promoter methylation of MDGA2 was detected in 62.4% (136/218) of gastric cancers. Multivariate analysis showed that patients with MDGA2 hypermethylation had a significantly decreased survival (p=0.005). Kaplan-Meier survival curves showed that MDGA2 hypermethylation was significantly associated with shortened survival in patients with early gastric cancer. CONCLUSIONS: MDGA2 is a critical tumour suppressor in gastric carcinogenesis; its hypermethylation is an independent prognostic factor in patients with gastric cancer.

Community-wide changes in intertaxonomic temporal co-occurrence resulting from phenological shifts.

Global climate change is known to affect the assembly of ecological communities by altering species' spatial distribution patterns, but little is known about how climate change may affect community assembly by changing species' temporal co-occurrence patterns, which is highly likely given the widely observed phenological shifts associated with climate change. Here, we analyzed a 29-year phenological data set comprising community-level information on the timing and span of temporal occurrence in 11 seasonally occurring animal taxon groups from 329 local meteorological observatories across China. We show that widespread shifts in phenology have resulted in community-wide changes in the temporal overlap between taxa that are dominated by extensions, and that these changes are largely due to taxa's altered span of temporal occurrence rather than the degree of synchrony in phenological shifts. Importantly, our findings also suggest that climate change may have led to less phenological mismatch than generally presumed, and that the context under which to discuss the ecological consequences of phenological shifts should be expanded beyond asynchronous shifts.

Novel recurrently mutated genes and a prognostic mutation signature in colorectal cancer.

BACKGROUND: Characterisation of colorectal cancer (CRC) genomes by next-generation sequencing has led to the discovery of novel recurrently mutated genes. Nevertheless, genomic data has not yet been used for CRC prognostication. OBJECTIVE: To identify recurrent somatic mutations with prognostic significance in patients with CRC. METHOD: Exome sequencing was performed to identify somatic mutations in tumour tissues of 22 patients with CRC, followed by validation of 187 recurrent and pathway-related genes using targeted capture sequencing in additional 160 cases. RESULTS: Seven significantly mutated genes, including four reported (APC, TP53, KRAS and SMAD4) and three novel recurrently mutated genes (CDH10, FAT4 and DOCK2), exhibited high mutation prevalence (6-14% for novel cancer genes) and higher-than-expected number of non-silent mutations in our CRC cohort. For prognostication, a five-gene-signature (CDH10, COL6A3, SMAD4, TMEM132D, VCAN) was devised, in which mutation(s) in one or more of these genes was significantly associated with better overall survival independent of tumor-node-metastasis (TNM) staging. The median survival time was 80.4 months in the mutant group versus 42.4 months in the wild type group (p=0.0051). The prognostic significance of this signature was successfully verified using the data set from the Cancer Genome Atlas study. CONCLUSIONS: The application of next-generation sequencing has led to the identification of three novel significantly mutated genes in CRC and a mutation signature that predicts survival outcomes for stratifying patients with CRC independent of TNM staging.

Targeting of YAP1 by microRNA-15a and microRNA-16-1 exerts tumor suppressor function in gastric adenocarcinoma.

BACKGROUND: MicroRNAs (miRNAs) have been reported to play an important role in tumorigenesis. In this study, the role of miR-15a and miR-16-1 in gastric adenocarcinoma (GAC) was investigated. METHODS: The expression of miR-15a and miR-16-1 in cell lines and primary tumors was examined by miRNA qRT-PCR. Proliferative assays, colony formation, cell invasion and migration, flow cytometry analysis and in vivo study were performed by ectopic expression of miR-15a and miR-16-1. The putative target genes of miR-15a and miR-16-1 were explored by TargetScan and further validated. RESULTS: We found that miR-15a and miR-16-1 were down-regulated in GAC cell lines and primary tumor samples compared with normal gastric epithelium. Functional study demonstrated that ectopic expression of miR-15a and miR-16-1 suppressed cell proliferation, monolayer colony formation, invasion and migration, and xenograft formation in vivo. In addition, miR-15a and miR-16-1 induced G0/G1 cell cycle arrest which was further confirmed by Western blot and qRT-PCR of related cell cycle regulators. YAP1 was confirmed to be a functional target of miR-15a and miR-16-1 in GAC. YAP1 re-expression partly abrogated the inhibitory effect of miR-15a and miR-16-1 in GAC cells. In clinical samples, YAP1 protein expression shows negative correlation with miR-15a and miR-16-1 expression. CONCLUSION: In conclusion, targeting YAP1 by tumor suppressor miRNA miR-15a and miR-16-1 plays inhibitory effect and this might have a therapeutic potential in GAC.

Integrative identification of Epstein-Barr virus-associated mutations and epigenetic alterations in gastric cancer.

BACKGROUND & AIMS: The mechanisms by which Epstein-Barr virus (EBV) contributes to the development of gastric cancer are unclear. We investigated EBV-associated genomic and epigenomic variations in gastric cancer cells and tumors. METHODS: We performed whole-genome, transcriptome, and epigenome sequence analyses of a gastric adenocarcinoma cell line (AGS cells), before and after EBV infection. We then looked for alterations in gastric tumor samples, with (n = 34) or without (n = 100) EBV infection, collected from patients at the Prince of Wales Hospital, Chinese University of Hong Kong (from 1998 through 2004), or the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China (from 1999 through 2006). RESULTS: Transcriptome analysis showed that infected cells expressed 9 EBV genes previously detected in EBV-associated gastric tumors and 71 EBV genes not previously reported in gastric tumors. Ten viral genes that had not been reported previously in gastric cancer but were expressed most highly in EBV-infected cells also were expressed in primary EBV-positive gastric tumors. Whole-genome sequence analysis identified 45 EBV-associated nonsynonymous mutations. These mutations, in genes such as AKT2, CCNA1, MAP3K4, and TGFBR1, were associated significantly with EBV-positive gastric tumors, compared with EBV-negative tumors. An activating mutation in AKT2 was associated with reduced survival times of patients with EBV-positive gastric cancer (P = .006); this mutation was found to dysregulate mitogen-activated protein kinase signaling. Integrated epigenome and transcriptome analyses identified 216 genes transcriptionally down-regulated by EBV-associated hypermethylation; methylation of ACSS1, FAM3B, IHH, and TRABD increased significantly in EBV-positive tumors. Overexpression of Indian hedgehog (IHH) and TraB domain containing (TRABD) increased proliferation and colony formation of gastric cancer cells, whereas knockdown of these genes reduced these activities. We found 5 signaling pathways (axon guidance, focal adhesion formation, interactions among cytokines and receptors, mitogen-activated protein kinase signaling, and actin cytoskeleton regulation) to be affected commonly by EBV-associated genomic and epigenomic alterations. CONCLUSIONS: By using genomic, transcriptome, and epigenomic comparisons of EBV infected vs noninfected gastric cancer cells and tumor samples, we identified alterations in genes, gene expression, and methylation that affect different signaling networks. These might be involved in EBV-associated gastric carcinogenesis.

Genome-wide identification of Epstein-Barr virus-driven promoter methylation profiles of human genes in gastric cancer cells.

BACKGROUND: Aberrant methylation of tumor-related genes has been reported in Epstein-Barr virus (EBV)-associated gastric cancers. This study sought to profile EBV-driven hypermethylation in EBV-infected cells. METHODS: The EBV-positive AGS gastric cancer cell line (AGS-EBV) and EBV-negative AGS cells were used in this study. DNA methyltransferase-3b (DNMT3b) activity was assessed by EpiQuick activity assay, and genome-wide DNA methylation profiles were assessed by methyl-DNA immunoprecipitation microarray assay. RESULTS: EBV infection was confirmed in AGS-EBV cells by EBV-encoded RNA in situ hybridization. Expression and activity of DNA methyltransferase-3b (DNMT3b) was significantly increased in AGS-EBV compared to AGS. Ectopic expression of LMP2A (latent membrane protein 2A) in AGS increased activity of DNMT3b. A total of 1065 genes were differentially methylated by EBV infection (fold-changes ≥ 2, P < .05) in AGS-EBV compared to AGS cells. The majority of the differentially methylated genes (83.2%, 886 of 1065 genes) had cytosine-guanine dinucleotide (CpG) hypermethylation in AGS-EBV (fold-changes 2.43∼65.2) versus that found in AGS cells. Gene ontology analysis revealed that hypermethylated genes were enriched in the important cancer pathways (≥ 10 genes each, P ≤ .05) including mitogen-activated protein kinase signaling, cell adhesion molecules, wnt signaling pathway, and so forth. Six novel hypermethylated candidates (IL15RA, REC8, SSTR1, EPHB6, MDGA2, and SCARF2) were further validated. Higher levels of DNA methylation were confirmed for all these genes in AGS-EBV cells by bisulfite genomic sequencing. Furthermore, these candidates were silenced or down-regulated in AGS-EBV cells, but can be restored by demethylation treatment. CONCLUSIONS: EBV infection in AGS cells induced aberrant CpG hypermethylation of 886 genes involving in important cancer-related pathways. Induction of promoter methylation by EBV is regulated by up-regulation of DNMT3b through LMP2A.

DUSP5P1 promotes gastric cancer metastasis and platinum drug resistance.

We elucidated the functional significance and molecular mechanisms of DUSP5P1 lncRNA (dual specificity phosphatase 5 pseudogene 1) in gastric carcinogenesis. We demonstrated that gastric cancer (GC) patients with high DUSP5P1 expression had shortened survival in two independent cohorts. DUSP5P1 promoted GC cell migration and invasion in vitro and metastasis in vivo. Mechanistically, DUSP5P1 activated ARHGAP5 transcription by directly binding to the promoter of ARHGAP5 with a binding motif of TATGTG. RNA-seq revealed that ARHGAP5 activated focal adhesion and MAPK signaling pathways to promote GC metastasis. DUSP5P1 also dysregulated platinum drug resistance pathway. Consistently, DUSP5P1 overexpression in GC cells antagonized cytotoxic effect of Oxaliplatin, and shDUSP5P1 plus Oxaliplatin exerted synergistic effect on inhibiting GC metastasis in vitro and in vivo. DUSP5P1 depletion also suppressed the growth of platinum drug-resistant PDO models. In conclusion, DUSP5P1 promoted GC metastasis by directly modulating ARHGAP5 expression to activate focal adhesion and MAPK pathways, serves as therapeutic target for platinum drug resistant GC, and is an independent prognostic factor in GC.

T1R2-mediated sweet sensing in a lizard.

Sugars are an important class of nutrients found in the flowers and fruits of angiosperms (flowering plants). Although T1R2-T1R3 has been identified as the mammalian sweet receptor, some birds rely on a repurposed T1R1-T1R3 savory receptor to sense sugars. Moreover, as the radiation of flowering plants occurred later than the last common ancestor of amniotes, sugar may not have been an important diet item for amniotes early in evolution, raising the question of whether T1R2-T1R3 is a universal sugar sensor or only a mammalian innovation. Here, using brief-access behavioral tests and functional characterization of taste receptors, we demonstrate that the nectar-taking Madagascar giant day gecko (Phelsuma grandis) can sense sugars through the T1R2-T1R3 receptor. These results reveal the existence of T1R2-based sweet taste in a non-avian reptile, which has important implications for our understanding of the evolutionary history of sugar detection in amniotes.

A single residue confers selective loss of sugar sensing in wrynecks.

Sensory receptors evolve, and changes to their response profiles can directly impact sensory perception and affect diverse behaviors, from mate choice to foraging decisions.1-3 Although receptor sensitivities can be highly contingent on changes occurring early in a lineage's evolutionary history,4 subsequent shifts in a species' behavior and ecology may exert selective pressure to modify and even reverse sensory receptor capabilities.5-7 Neither the extent to which sensory reversion occurs nor the mechanisms underlying such shifts is well understood. Using receptor profiling and behavioral tests, we uncover both an early gain and an unexpected subsequent loss of sugar sensing in woodpeckers, a primarily insectivorous family of landbirds.8,9 Our analyses show that, similar to hummingbirds10 and songbirds,4 the ancestors of woodpeckers repurposed their T1R1-T1R3 savory receptor to detect sugars. Importantly, whereas woodpeckers seem to have broadly retained this ability, our experiments demonstrate that wrynecks (an enigmatic ant-eating group sister to all other woodpeckers) selectively lost sugar sensing through a novel mechanism involving a single amino acid change in the T1R3 transmembrane domain. The identification of this molecular microswitch responsible for a sensory shift in taste receptors provides an example of the molecular basis of a sensory reversion in vertebrates and offers novel insights into structure-function relationships during sensory receptor evolution.

Early origin of sweet perception in the songbird radiation.

Early events in the evolutionary history of a clade can shape the sensory systems of descendant lineages. Although the avian ancestor may not have had a sweet receptor, the widespread incidence of nectar-feeding birds suggests multiple acquisitions of sugar detection. In this study, we identify a single early sensory shift of the umami receptor (the T1R1-T1R3 heterodimer) that conferred sweet-sensing abilities in songbirds, a large evolutionary radiation containing nearly half of all living birds. We demonstrate sugar responses across species with diverse diets, uncover critical sites underlying carbohydrate detection, and identify the molecular basis of sensory convergence between songbirds and nectar-specialist hummingbirds. This early shift shaped the sensory biology of an entire radiation, emphasizing the role of contingency and providing an example of the genetic basis of convergence in avian evolution.

The novel human endogenous retrovirus-related gene, psiTPTE22-HERV, is silenced by DNA methylation in cancers.

The psiTPTE22 gene has been designated as a TPTE pseudogene. Our study found that the 5' part of psiTPTE22 has no sequence similarity to TPTE and contains a 3.8-kb human endogenous retrovirus (HERV) element. Because of the HERV element, the 5' part of psiTPTE22 (psiTPTE22-HERV) expresses independently as a gene. Comparison between the DNA sequences of humans and chimps indicated that psiTPTE22-HERV is human specific. We identified 3 alternatively spliced transcript variants from psiTPTE22-HERV by a PCR-based strategy, which use the transcriptional termination signal contained in the HERV element. A 402-nt ORF was contained in the 2 longer transcripts. Western blotting using antibodies produced with chemically synthesized peptide confirmed that a 15-kDa protein was translated from this ORF. RT-PCR results indicated that the ORF-containing transcripts were mainly expressed in psiTPTE22-HERV-expressing samples. Real-time quantitative RT-PCR results showed that expression of the 402-nt ORF was upregulated in normal tissues of kidney, liver, stomach, and lung but downregulated in corresponding tumor tissues. This gene is located near the centromere of chromosome 22 and has a high GC content around the promoter region. Bisulfite sequencing PCR results indicated that it is silenced in cancers by DNA methylation. The expression of psiTPTE22-HERV can be recovered in cancer cells using DNA methylation and histone deacetylase inhibitors. These results suggest psiTPTE22-HERV is regulated epigenetically by DNA methylation. Our study paved the way for further study on an interesting HERV-related human-specific gene, which is silenced in cancers by DNA methylation.

Identification of a novel human endogenous retrovirus and promoter activity of its 5' U3.

Up-regulation of human endogenous retroviruses (HERVs) sometimes occurs in cancer. This study identifies a novel HERV (HERV-HX) in colon tumor tissues, which is an H family HERV (HERV-H) element located on chromosome Xp22.32. The full-length transcript sequence was identified and characterized. Quantitative RT-PCR indicated it is up-regulated in colon tumor samples. Using RT-PCR to analyze the transcription of the env-deleted HERV-HX and other env-intact HERV-H elements, it was demonstrated that transcription of HERV-H in colon cancer is not associated with the env gene. A 17-bp sequence was found within the 5' U3 region of HERV-HX, and only 8-bp sequences existed on its homologous U3 regions in the corresponding loci. Promoter activity assays indicated that replacing the 17-bp sequence with an 8-bp sequence or deleting it reduced its activity, suggesting that the 17-bp sequence is important to the expression of HERV-HX.

C8orf76 Promotes Gastric Tumorigenicity and Metastasis by Directly Inducing lncRNA DUSP5P1 and Associates with Patient Outcomes.

PURPOSE: We identified for the first time that C8orf76 (chromosome 8 open reading frame 76) is preferentially amplified in gastric cancer. We elucidated its role and clinical significance in gastric carcinogenesis. EXPERIMENTAL DESIGN: The clinical impact of C8orf76 was assessed in 592 patients with gastric cancer. The biological function of C8orf76 was studied in vitro, in vivo, and in gastric cancer patient-derived organoid models. C8orf76 downstream effector and pathways were identified by RNA sequencing, chromatin immunoprecipitation sequencing, luciferase reporter, and electrophoretic mobility shift assay. RESULTS: C8orf76 was upregulated in 69.74% and 65.71% of two independent cohorts of gastric cancers and was positively associated with C8orf76 amplification. Multivariate analysis showed that gastric cancer patients with C8orf76 amplification (cohort I, n = 129; cohort II, n = 107) or overexpression (n = 356) had a significantly shortened survival. C8orf76 significantly promoted gastric cancer cell proliferation, cell-cycle transformation, and migration/invasion, but suppressed cell apoptosis. Silencing C8orf76 expression exerted opposite effects in vitro and significantly inhibited xenograft tumor growth, lung metastasis, and liver metastasis in nude mice. Silencing C8orf76 also significantly suppressed the growth of patient-derived organoids. Mechanically, C8orf76 activated MAPK/ERK signaling cascade. C8orf76 directly bound to the promoter region of lncRNA dual specificity phosphatase 5 pseudogene 1 (DUSP5P1) with a binding motif of AGGCTG and activated DUSP5P1 transcription. DUSP5P1 induced MAPK/ERK signaling and promoted gastric tumorigenesis. Knockdown DUSP5P1 abrogated the effect of C8orf76 in activating MAPK/ERK cascade and the tumor-promoting function. CONCLUSIONS: C8orf76 directly binds to oncogenic lncRNA DUSP5P1 to induce its expression and activates MAPK signaling. C8orf76 plays a pivotal oncogenic role in gastric carcinogenesis and is an independent prognostic factor for gastric cancer patients.

Docking protein-1 promotes inflammatory macrophage signaling in gastric cancer.

Docking protein-1 (DOK1) is a tumor suppressor frequently lost in malignant cells, however, it retains the ability to control activities of immune receptors in adjacent stroma cells of the tumor microenvironment. We therefore hypothesized that addressing DOK1 may be useful for cancer immunotherapy. DOK1 mRNA and DOK1 protein expression were downregulated in tumor cells of gastric cancer patients (n = 249). Conversely, its expression was up-regulated in cases positive for Epstein Barr Virus (EBV+) together with genes related to macrophage biology and targets of clinical immunotherapy such as programmed-cell-death-ligand-1 (PD-L1). Notably, high DOK1 positivity in stroma cells conferred poor prognosis in patients and correlated with high levels of inducible nitric oxide synthase in CD68+ tumor-associated macrophages. In macrophages derived from human monocytic leukemia cell lines, DOK1 (i) was inducible by agonists of the anti-diabetic transcription factor peroxisome proliferator-activated receptor-gamma (PPARγ), (ii) increased polarization towards an inflammatory phenotype, (iii) augmented nuclear factor-κB-dependent transcription of pro-inflammatory cytokines and (iv) reduced PD-L1 expression. These properties empowered DOK1+ macrophages to decrease the viability of human gastric cancer cells in contact-dependent co-cultures. DOK1 also reduced PD-L1 expression in human primary blood monocytes. Our data propose that the drugability of DOK1 may be exploited to reprogram myeloid cells and enforce the innate immune response against EBV+ human gastric cancer.

Characterization and validation of somatic mutation spectrum to reveal heterogeneity in gastric cancer by single cell sequencing.

Gastric cancer (GC) is a highly heterogeneous disease with multiple cellular types and poor prognosis. However, the cellular evolution and molecular basis of GC at the individual intra-tumor level has not been well demonstrated. We performed single-cell whole exome sequencing to detect somatic single-nucleotide variants (SNVs) and significantly mutated genes (SMGs) among 34 tumor cells and 9 normal cells from a patient with GC. The Complete Prediction for Protein Conformation (CPPC) approach directly predicting the folding conformation of the protein 3D structure with Protein Folding Shape Code, combined with functional experiments were used to confirm the characterization of mutated SMGs in GC cells. We identified 201 somatic SNVs, including 117 non-synonymous mutations in GC cells. Further analysis identified 24 significant mutated genes (SMGs) in single cells, for which a single amino acid change might affect protein conformation. Among them, two genes (CDC27 and FLG) that were mutated only in single cells but not in the corresponding tumor tissue, were recurrently present in another GC tissue cohort, and may play a potential role to promote carcinogenesis, as confirmed by functional characterization. Our findings showed a mutational landscape of GC at intra-tumor level for the first time and provided opportunities for understanding the heterogeneity and individualized target therapy for this disease.

Differentiated tumor immune microenvironment of Epstein-Barr virus-associated and negative gastric cancer: implication in prognosis and immunotherapy.

Epstein-Barr virus-associated gastric cancer (EBVaGC) has been proposed to be a distinct subtype with a specific immune microenvironment. Here, we evaluated tumor-infiltrating T-cell subsets and the expression of programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) in 571 gastric cancers (GCs). Tissue microarrays were stained using EBER in situ hybridization for EBV and using immunohistochemistry for CD4, CD8, Foxp3, PD-1 and PD-L1. GCs were categorized into four types based on CD8+ infiltration and PD-L1 expression. The 5-year overall survival (OS) was evaluated according to EBV infection, T-cell subsets, PD-1 and PD-L1 expression and immune types. Thirty-two (5.3%) EBVaGCs were identified, which were more prevalent for CD8+ (p<0.001) and Foxp3+ (p=0.020) cell infiltration than EBV-negative GCs (EBVnGCs), suggesting a better 5-year OS (p=0.003). CD8+ (p=0.001) and Foxp3+ (p=0.018) cell infiltration was associated with better 5-year OS, whereas PD-L1 expression correlated with a poor 5-year OS (p=0.002). EBVaGC and EBVnGC had heterogeneous immune microenvironment, with CD8+ PD-L1- GC exhibiting the best 5-year OS (p<0.001). GC was an immune ignorant dominant tumor and poor to no T-cell infiltration. An immune type classification algorithm can provide prognostic information and a rational basis for immunotherapy.