Discovery of a novel small molecule as CD47/SIRPα and PD-1/PD-L1 dual inhibitor for cancer immunotherapy.

BACKGROUND: Targeting the tumor microenvironment (TME) has emerged as a promising strategy in cancer treatment, particularly through the utilization of immune checkpoint blockade (ICB) agents such as PD-1/PD-L1 inhibitors. Despite partial success, the presence of tumor-associated macrophages (TAMs) contributes to an immunosuppressive TME that fosters tumor progression, and diminishes the therapeutic efficacy of ICB. Blockade of the CD47/SIRPα pathway has proven to be an effective intervention, that restores macrophage phagocytosis and yields substantial antitumor effects, especially when combined with PD-1/PD-L1 blockade. Therefore, the identification of small molecules capable of simultaneously blocking CD47/SIRPα and PD-1/PD-L1 interactions has remained imperative. METHODS: SMC18, a small molecule with the capacity of targeting both SIRPα and PD-L1 was obtained using MST. The efficiency of SMC18 in interrupting CD47/SIRPα and PD-1/PD-L1 interactions was tested by the blocking assay. The function of SMC18 in enhancing the activity of macrophages and T cells was tested using phagocytosis assay and co-culture assay. The antitumor effects and mechanisms of SMC18 were investigated in the MC38-bearing mouse model. RESULTS: SMC18, a small molecule that dual-targets both SIRPα and PD-L1 protein, was identified. SMC18 effectively blocked CD47/SIRPα interaction, thereby restoring macrophage phagocytosis, and disrupted PD-1/PD-L1 interactions, thus activating Jurkat cells, as evidenced by increased secretion of IL-2. SMC18 demonstrated substantial inhibition of MC38 tumor growths through promoting the infiltration of CD8+ T and M1-type macrophages into tumor sites, while also priming the function of CD8+ T cells and macrophages. Moreover, SMC18 in combination with radiotherapy (RT) further improved the therapeutic efficacy. CONCLUSION: Our findings suggested that the small molecule compound SMC18, which dual-targets the CD47/SIRPα and PD-1/PD-L1 pathways, could be a candidate for promoting macrophage- and T-cell-mediated phagocytosis and immune responses in cancer immunotherapy.

Peptide vaccine from cancer-testis antigen ODF2 can potentiate the cytotoxic T lymphocyte infiltration through IL-15 in non-MSI-H colorectal cancer.

About 85% of patients with colorectal cancer (CRC) have the non-microsatellite instability-high (non-MSI-H) subtype, and many cannot benefit from immune checkpoint blockade. A potential reason for this is that most non-MSI-H colorectal cancers are immunologically "cold" due to poor CD8+ T cell infiltration. In the present study, we screened for potential cancer-testis antigens (CTAs) by comparing the bioinformatics of CD8+ T effector memory (Tem) cell infiltration between MSI-H and non-MSI-H CRC. Two ODF2-derived epitope peptides, P433 and P609, displayed immunogenicity and increased the proportion of CD8+ T effector memory (Tem) cells in vitro and in vivo. The adoptive transfer of peptide pool-induced CTLs inhibited tumor growth and enhanced CD8+ T cell infiltration in tumor-bearing NOD/SCID mice. The mechanistic study showed that knockdown of ODF2 in CRC cells promoted interleukin-15 expression, which facilitated CD8+ T cell proliferation. In conclusion, ODF2, a CTA, was negatively correlated with CD8+ T cell infiltration in "cold" non-MSI-H CRC and was selected based on the results of bioinformatics analyses. The corresponding HLA-A2 restricted epitope peptide induced antigen-specific CTLs. Immunotherapy targeting ODF2 could improve CTA infiltration via upregulating IL-15 in non-MSI-H CRC. This tumor antigen screening strategy could be exploited to develop therapeutic vaccines targeting non-MSI-H CRC.

Circulating lipoprotein (a) and all-cause and cause-specific mortality: a systematic review and dose-response meta-analysis.

AIMS: To investigate the association between circulating lipoprotein(a) (Lp(a)) and risk of all-cause and cause-specific mortality in the general population and in patients with chronic diseases, and to elucidate the dose-response relations. METHODS AND RESULTS: We searched literature to find prospective studies reporting adjusted risk estimates on the association of Lp(a) and mortality outcomes. Forty-three publications, reporting on 75 studies (957,253 participants), were included. The hazard ratios (HRs) and 95% confidence intervals (95%CI ) for the top versus bottom tertile of Lp(a) levels and risk of all-cause mortality were 1.09 (95%CI: 1.01-1.18, I2: 75.34%, n = 19) in the general population and 1.18 (95%CI: 1.04-1.34, I2: 52.5%, n = 12) in patients with cardiovascular diseases (CVD). The HRs for CVD mortality were 1.33 (95%CI: 1.11-1.58, I2: 82.8%, n = 31) in the general population, 1.25 (95%CI: 1.10-1.43, I2: 54.3%, n = 17) in patients with CVD and 2.53 (95%CI: 1.13-5.64, I2: 66%, n = 4) in patients with diabetes mellitus. Linear dose-response analyses revealed that each 50 mg/dL increase in Lp(a) levels was associated with 31% and 15% greater risk of CVD death in the general population and in patients with CVD. No non-linear dose-response association was observed between Lp(a) levels and risk of all-cause or CVD mortality in the general population or in patients with CVD (Pnonlinearity > 0.05). CONCLUSION: This study provides further evidence that higher Lp(a) levels are associated with higher risk of all-cause mortality and CVD-death in the general population and in patients with CVD. These findings support the ESC/EAS Guidelines that recommend Lp(a) should be measured at least once in each adult person's lifetime, since our study suggests those with higher Lp(a) might also have higher risk of mortality.

[Research Progress in Oral Diseases and Oral Microbiota of Organ Transplant Patients].

Organ transplantation is an effective treatment for end-stage organ diseases. However, organ transplant recipients are susceptible to a wide variety of oral diseases, including gingival enlargement, periodontitis, oral mucosal diseases, oral malignant tumors, and dental caries. Oral microbiota may have played an important role in the organ transplant patients' increased susceptibility to oral diseases and is associated with adverse events after organ transplantation, which is gradually gaining more attention among scholars. We, herein, reviewed the common oral diseases, including periodontal tissue diseases, oral mucosal diseases, oral malignant tumors, and dental caries in organ transplantation patients. Furthermore, we discussed the characteristic changes in the oral microbiota of organ transplantation patients and the influencing factors of these changes. In-depth study of oral microbiota of organ transplant patients provides a reference for the prevention and treatment of relevant diseases after organ transplantation and serves an important role in oral and systemic health management of organ transplant patients.

A PD-L1 and VEGFR2 dual targeted peptide and its combination with irradiation for cancer immunotherapy.

Although the blockade of immune checkpoint PD-1/PD-L1 has achieved great success, the lack of tumor-infiltrating immune cells and PD-L1 expression in the tumor microenvironment results in a limited response in certain tumor types. Thus, rational and optimal combination strategies were urgently needed. The combination of PD-1/PD-L1 blockade and anti-angiogenic therapy has been reported to have great potential. Here, a chimeric peptide OGS was designed by conjugating the peptides OPBP-1 (8-12) and DA7R targeting PD-L1 and VEGFR2, respectively. OGS could bind to both human and mouse PD-L1 with high affinity and block the PD-1/PD-L1 interaction, and also inhibit the migration and tube formation of HUVEC cells in wound healing and tube formation assays. To further prolong the half-life of OGS, it was modified by coupling with peptide DSP which has a high binding affinity to both human serum albumin (HSA) and mouse serum albumin (MSA) to form the peptide DSPOGS. DSPOGS could not directly affect the viability, apoptosis, and cell cycle of tumor cells in vitro, while significantly inhibiting the tumor growth in the MC38 mouse model. DSPOGS could elicit a potent anti-tumor immune response and inhibit tumor angiogenesis, with the enhancement of tumor infiltrating CD8+ T cells and the IFN-γ secreting CD8+ T cells in the spleen and tumor-draining lymph node. Further, the combination of radiotherapy with DSPOGS could dramatically improve the therapeutic efficacy. Our study could provide a promising paradigm for the combination of immune checkpoint blockade, anti-angiogenesis, and radiotherapy.

The colonized microbiota composition in the peritoneal fluid in women with endometriosis.

PURPOSE: The imbalance of microbiome in vivo is believed to be involved in the pathogenicity of endometriosis. This study aimed to investigate and analyze the composition of bacterial communities in the peritoneal fluid of women with endometriosis. METHODS: To collect peritoneal fluid samples from women with (N = 36) and without (N = 25) endometriosis in a generalized hospital in Hunan, China during January to December of 2019. Genomic DNA was extracted from peritoneal fluid samples, and targeted amplified for the V4 region of 16S ribosomal RNA gene followed by amplicon sequencing. Non-parametric Wilcoxon rank-sum test and chi-squared test were used to compare and analysis the difference between groups. RESULTS: Analysis showed that microbiota diversity was similar in the peritoneal fluid of women with or without endometriosis. Ralstonia mainly dominated in the peritoneal fluid of patients in both groups, with an overall relative abundance of 11.15% (95% CI: 10.51-11.80%) in endometriosis patients, followed by Acinetobacter, Pseudomonas, Asticcacaulis, and Methyloversatilis, with no significant difference between endometriosis patients and the control group. Nevertheless, there were microbes with different abundance in peritoneal fluid of the two groups, and the relative abundance was less than 0.5%. Acidovorax (P = 0.01), Devosia (P = 0.03), Methylobacterium (P = 0.03), Phascolarctobacterium (P = 0.03), and Streptococcus (P = 0.04) were more abundant in the peritoneal fluid of endometriosis patients than the controls, while Brevundimonas (P = 0.01) and Stenotrophomonas (P = 0.04) were less abundant. CONCLUSION: The composition of minority microbiota including Acidovorax, Devosia, Methylobacterium, Phascolarctobacterium, and Streptococcus in peritoneal fluid were found to change among women with endometriosis. Further research is needed to explore the mechanisms of these microorganisms in the pathophysiology of endometriosis.

Serum exosomal miRNA from endometriosis patients correlates with disease severity.

PURPOSE: Exosomes are vesicles secreted by cells that contain a wide variety of biomolecules, including proteins or nucleic acids. MicroRNAs (miRNAs), which are commonly found in exosomes, are known to play important roles in the pathophysiology of endometriosis. METHODS: This study investigated the miRNA expression profile of serum exosomes from women with endometriosis in comparison with normal controls as well as the possible role of identified miRNAs in the pathogenesis of endometriosis. Exosomes with a diameter between 60 and 100 nm were identified by their expression of exosomal marker proteins CD9 and CD63. RESULTS: Microarray miRNA expression profiling analysis revealed that 26 genes were significantly up-regulated and 19 genes were significantly down-regulated in serum exosomes from endometriosis patients compared with normal controls. These differentially expressed miRNAs were mainly enriched in the regulation of cellular development, metabolism, and involved in the regulation of the MAPK and PI3k-Akt pathways. qRT-PCR analysis verified the differential expression of three miRNAs, miR-26b-5p, miR-215-5p, and miR-6795-3p. CONCLUSION: Further analysis indicated that these differentially expressed miRNAs in serum exosomes may be involved in the pathogenesis of endometriosis and are related to the severity and certain symptoms of endometriosis.

Engineered Nanovaccine Targeting Clec9a+ Dendritic Cells Remarkably Enhances the Cancer Immunotherapy Effects of STING Agonist.

Agonists of the stimulator of interferon gene (STING) are considered as promising therapeutics for cancer immunotherapy. However, drug-delivery barriers and adverse effects limit the clinical application of STING agonists. Therefore, it is an urgent need to develop an ideal delivery system to deliver STING agonists and avoid side effects. Here, we discovered that STING agonists significantly stimulated type I interferon (IFN) secretion in Clec9a+ dendritic cells (DCs). Then, we designed an engineered peptide-expressed biomimetic cancer cell membrane (EPBM)-coated nanovaccine drug-delivery system (PLGA/STING@EPBM) to deliver STING agonists and tumor antigens to Clec9a+ DCs. The PLGA/STING@EPBM nanovaccine significantly enhanced IFN-stimulated expression of genes and antigen cross-presentation of Clec9a+ DCs, thus eliciting strong antitumor effects in both anti-PD-1-responsive and -resistant tumor models without obvious cytotoxicity. Moreover, the PLGA/STING@EPBM nanovaccine combined with radiotherapy exhibited remarkable synergistic antitumor effects. Our work highlights the great potential of a EPBM-coated nanovaccine for systemic STING agonist delivery as an attractive tool for cancer immunotherapy.

Metformin leads to accumulation of reactive oxygen species by inhibiting the NFE2L1 expression in human hepatocellular carcinoma cells.

Metformin, as the first-line drug for the treatment of type 2 diabetes mellitus, has been shown to possess a capability to activate or inhibit the production of reactive oxygen species (ROS) in different ways. However, the detailed mechanisms of the opposite effect are poorly understood. Here we provide evidence that metformin induces accumulation of ROS by inhibiting the expression of a core antioxidant transcription factor nuclear factor erythroid 2 like 1 (NFE2L1/Nrf1) in human hepatocellular carcinoma HepG2 cells. In the present study, we originally found that the increased ROS induced by metformin was blunted in NFE2L1 knockdown cell line. Furtherly by examining the effects of metformin on endogenous and exogenous NFE2L1, we also found metformin could not only inhibit the transcription of NFE2L1 gene, but also promote the degradation of NFE2L1 protein at the post-transcriptional level, whereas this effect can be reversed by high glucose. The inhibitory effect of metformin on NFE2L1 was investigated to occur through the N-terminal domain (NTD) of NFE2L1 protein, and its downregulation by metformin was in an AMP-activated protein kinase (AMPK)-independent manner. But the activation of AMPK signaling pathway by metformin in NFE2L1 knockdown HepG2 cells is reversed, indicating that NFE2L1 may be an important regulator of AMPK signal. Altogether, this work provides a better understanding of the relationship between metformin and oxidative stress, and hence contributes to translational study of metformin through its hypoglycemic and tumor suppressive effects.

The Role of E3s in Regulating Pluripotency of Embryonic Stem Cells and Induced Pluripotent Stem Cells.

Pluripotent embryonic stem cells (ESCs) are derived from early embryos and can differentiate into any type of cells in living organisms. Induced pluripotent stem cells (iPSCs) resemble ESCs, both of which serve as excellent sources to study early embryonic development and realize cell replacement therapies for age-related degenerative diseases and other cell dysfunction-related illnesses. To achieve these valuable applications, comprehensively understanding of the mechanisms underlying pluripotency maintenance and acquisition is critical. Ubiquitination modifies proteins with Ubiquitin (Ub) at the post-translational level to monitor protein stability and activity. It is extensively involved in pluripotency-specific regulatory networks in ESCs and iPSCs. Ubiquitination is achieved by sequential actions of the Ub-activating enzyme E1, Ub-conjugating enzyme E2, and Ub ligase E3. Compared with E1s and E2s, E3s are most abundant, responsible for substrate selectivity and functional diversity. In this review, we focus on E3 ligases to discuss recent progresses in understanding how they regulate pluripotency and somatic cell reprogramming through ubiquitinating core ESC regulators.

Endometrial stromal cells treated by tumor necrosis factor-α stimulate macrophages polarized toward M2 via interleukin-6 and monocyte chemoattractant protein-1.

AIM: This study aimed to investigate the effects of endometrial stromal cells (ESC)-derived interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1 on macrophage polarization in endometriosis. METHODS: Macrophage polarization was measured in eutopic endometrium of control participants ('normal endometrium'), eutopic endometrium of patients with endometriosis ('eutopic endometrium') and ectopic endometrium of endometriosis patients ('ectopic endometrium') by immunohistochemical staining. Expression of IL-6 and MCP-1 were measured in the eutopic and ectopic endometrium through enzyme-linked immunosorbent assays. Expression of CD163 was measured in human acute monocytic leukemia (THP-1) cell-derived macrophages that were treated with conditional medium induced by tumor necrosis factor (TNF)-α, TNF-α + anti-IL-6 or TNF-α + anti-MCP-1 via flow cytometry. RESULTS: The ratio of CD163+/CD68+ macrophages in the normal endometrium was higher than that in the eutopic endometrium, while differences between the eutopic and ectopic endometrium were not statistically significant. IL-6 and MCP-1 exhibited enhanced expression in the ectopic endometrium group and decreased expression in the eutopic endometrium group. TNF-α could promote the expression of ESC-derived IL-6 and MCP-1. Intervention with TNF-α-induced conditioned medium resulted in the upregulation of CD163 in THP-1 cells, while conditional medium induced with IL-6 and MCP-1 neutralizing antibodies decreased the proportion of CD163+ macrophages significantly. CONCLUSION: In endometriosis patients, the macrophages of the eutopic endometrium polarize toward M1 compared with the normal endometrium, and those of the ectopic endometrium were mainly M2-polarized. Under the action of TNF-α, ESC-derived IL-6 and MCP-1 could stimulate peritoneal macrophages toward M2-polarization, which could modulate endometriosis.

A Novel d-Peptide Identified by Mirror-Image Phage Display Blocks TIGIT/PVR for Cancer Immunotherapy.

The low response rate and adaptive resistance of PD-1/PD-L1 blockade demands the studies on novel therapeutic targets for cancer immunotherapy. We discovered that a novel immune checkpoint TIGIT expressed higher than PD-1 in many tumors especially anti-PD-1 resistant tumors. Here, mirror-image phage display bio-panning was performed using the d-enantiomer of TIGIT synthesized by hydrazide-based native chemical ligation. d-peptide D TBP-3 was identified, which could occupy the binding interface and effectively block the interaction of TIGIT with its ligand PVR. D TBP-3 showed proteolytic resistance, tumor tissue penetrating ability, and significant tumor suppressing effects in a CD8+ T cell dependent manner. More importantly, D TBP-3 could inhibit tumor growth and metastasis in anti-PD-1 resistant tumor model. This is the first d-peptide targeting TIGIT, which could serve as a potential candidate for cancer immunotherapy.

Proteomic analysis by iTRAQ-PRM provides integrated insight into mechanisms of resistance in pepper to Bemisia tabaci (Gennadius).

BACKGROUND: The Bemisia tabaci is a major leaf feeding insect pest to pepper (Capsicum annuum), causing serious damage to pepper growth and yield. It is particularly important to study the mechanism of pepper resistance to B. tabaci, and to breed and promote the varieties of pepper resistant to B. tabaci. However, very limited molecular mechanism is available about how plants perceive and defend themselves from the destructive pest. Proteome technologies have provided an idea method for studying plant physiological processes in response to B. tabaci. RESULTS: Here, a highly resistant genotype and a highly susceptible genotype were exposed to B. tabaci feeding for 48 h to explore the defense mechanisms of pepper resistance to B. tabaci. The proteomic differences between both genotypes were compared using isobaric tag for relative and absolute quantification (iTRAQ). The quantitative data were validated by parallel reaction monitoring (PRM). The results showed that 37 differential abundance proteins (DAPs) were identified in the RG (resistant genotype), while 17 DAPs were identified in the SG (susceptible genotype) at 48 h after B. tabaci feeding. 77 DAPs were identified when comparing RG with SG without feeding. The DAP functions were determined for the classification of the pathways, mainly involved in redox regulation, stress response, protein metabolism, lipid metabolism and carbon metabolism. Some candidate DAPs are closely related to B. tabaci resistance such as annexin D4-like (ANN4), calreticulin-3 (CRT3), heme-binding protein 2-like (HBP1), acidic endochitinase pcht28-like (PR3) and lipoxygenase 2 (LOX2). CONCLUSIONS: Taken together, this study indicates complex resistance-related events in B. tabaci interaction, provides novel insights into the molecular mechanism underlying the response of plant to B. tabaci, and identifies some candidate proteins against B. tabaci attack.

Eutopic endometrium from patients with endometriosis modulates the expression of CD36 and SIRP-α in peritoneal macrophages.

AIM: This study aimed to investigate the in vitro alterations of the expression of signal regulatory protein-α (SIRP-α) and CD36 in macrophages in the endometriosis condition. METHODS: The expression of SIRP-α and CD36 was measured in peritoneal macrophages and peripheral blood mononuclear cells of endometriosis patients and control participants. The expressions of SIRP-α and CD36 were measured in human acute monocytic leukemia (THP-1) cell-derived macrophages that were treated with interleukin-6 (IL-6)-induced conditioned medium, eutopic versus normal endometrial homogenate, or lipopolysaccharide in the presence or absence of nuclear factor kappa-B (NF-κB) or transforming growth factor (TGF-ß) inhibitors, respectively. RESULTS: Peritoneal macrophages that were isolated from women with endometriosis exhibited an enhanced expression of SIRP-α and a decreased expression of CD36 compared to control participants. Women with endometriosis had significantly higher levels of SIRP-α and CD36 in peripheral circulating mononuclear cells than in control participants. SIRP-α expression was significantly increased, whereas the CD36 expression was decreased in THP-1 cell-derived macrophages after treatment with eutopic endometrial homogenate. Intervention with IL-6-induced conditioned medium resulted in the downregulation of SIRP-α but the upregulation of CD36 in THP-1 cells. Incubation with the NF-κBp50 inhibitor decreased the expression of CD36 and SIRP-α in macrophages that were treated with normal endometrial homogenate, whereas the TGF-ß inhibitor enhanced the CD36 expression of THP-1 cell-derived macrophages treated with eutopic endometrial homogenate. CONCLUSION: The eutopic endometrium could reduce the phagocytic ability of peritoneal macrophages in women with endometriosis through the modulation of SIRP-α and CD36 expression. Inhibition of the TGF-ß signal pathway may be a potential therapeutic target for the treatment of endometriosis.

[Expression of PROK 1 and its receptor PROKR 1 in endometriosis and its clinical significance].

OBJECTIVE: To investigate the role of prokineticin (PROK) 1 and prokineticin-receptor (PROKR) 1 in the pathogenesis of endometriosis and its clinical signifaicance.
 Methods: Quantitative real-time PCR (qPCR) and Western bloting were used to detect the expression of PROK 1 and PROKR 1 in eutopic and ectopic endometrium of endometriosis (n=22) and normal control endometrium (n=18). Endometrial stromal cells were isolated and cultured in 6 normal controls. The expression of PROK 1 mRNA was detected by qPCR after treated with estradiol (E2) or TNF-α.
 Results: PROK 1 and PROKR 1 mRNA were expressed in eutopic and ectopic endometrium of endometriosis and normal control endometrium, and the expression level gradually declined (P<0.05). The expression of PROKR-1 protein in eutopic and ectopic endometrium of endometriosis and normal control endometrium gradually declined (P<0.05). The expression of PROK-1 protein in normal control endometrial cells and eutopic endometrium cell was higher in secretory phase than in proliferative phase (P<0. 05). E2 did not change the expression of PROK 1, whereas TNF-α up-regulated the expression of PROK 1.
 Conclusion: PROK-1 and its receptors are involved in the pathogenesis and development of endometriosis. TNF-α can promote angiogenesis via up-regulating the expression of PROK 1.

The design of high affinity human PD-1 mutants by using molecular dynamics simulations (MD).

BACKGROUND: Programmed cell death protein 1 (PD-1), a negative co-stimulatory molecule, plays crucial roles in immune escape. Blockade of the interaction between PD-1 and PD-L1 shows exciting clinical responses in a fraction of cancer patients and the success makes PD-1 as a valuable target in immune checkpoint therapy. For the rational design of PD-1 targeting modulators, the ligand binding mechanism of PD-1 should be well understood in prior. METHODS: In this study, we applied 50 ns molecular dynamics simulations to observe the structural properties of PD-1 molecule in both apo and ligand bound states, and we studied the structural features of PD-1 in human and mouse respectively. RESULTS: The results showed that the apo hPD-1 was more flexible than that in PD-L1 bound state. We unexpectedly found that K135 was important for binding energy although it was not at the binding interface. Moreover, the residues which stabilized the interactions with PD-L1 were distinguished. Taking the dynamic features of these residues into account, we identified several residual sites where mutations may gain the function of ligand binding. The in vitro binding experiments revealed the mutants M70I, S87 W, A129L, A132L, and K135 M were better in ligand binding than the wild type PD-1. CONCLUSIONS: The structural information from MD simulation combined with in silico mutagenesis provides guidance to design engineered PD-1 mutants to modulate the PD-1/PD-L1 pathway.

Establishment and characterization of a kidney cell line from kelp grouper Epinephelus moara.

A novel cell line, Epinephelus moara kidney cell line (EMK), was established from kidneys of kelp grouper E. moara. Cells were cultured at 24 °C in Leibovitz's L-15 medium (L15) supplemented with antibiotics, basic fibroblast growth factor (bFGF), foetal bovine serum (FBS) and 2-mercaptoethanol (2-ME). EMK cells, fibroblastic in morphology, proliferated to 100% confluency in 3-4 days and were subcultured for over 50 passages. The cells could grow from 18 to 30 °C, with optimal growth at 24 °C. Chromosome analysis indicated that the modal chromosome number was 48 in the cells at passage 42. Green fluorescent signals could be observed in EMK cells when the cells were transfected with pEGFP-N3 plasmid. Moreover, a significant cytopathic effect (CPE) was observed in the cells after infection with Singapore grouper iridovirus (SGIV) or nervous necrosis virus (NNV), and viral replication was confirmed by quantitative real-time PCR (qPCR). These results suggested the potential of the EMK cell line for studies of transgene and pathogenesis of SGIV and NNV.

WWP1 as a potential tumor oncogene regulates PTEN-Akt signaling pathway in human gastric carcinoma.

Whelming evidence has demonstrated that WW domain containing E3 ubiquitin protein ligase 1 (WWP1) participates in a wide variety of biological processes and is tightly related to the initiation and progression of many tumors. Currently, although mounting evidence supports a role of WWP1 in tumor promotion and tumorigenesis, the potential roles of WWP1 and its biological functions in gastric carcinoma are not fully understood. Here, we found that WWP1 messenger RNA (mRNA) and protein were highly expressed in gastric carcinoma tissues and cells. High WWP1 mRNA and protein levels were tightly related to differentiation status, TNM stage, invasive depth, lymph node metastasis, and poor prognosis in gastric carcinoma. Furthermore, WWP1 siRNA significantly decreased WWP1 protein level in MKN-45 and AGS cells; meanwhile, WWP1 depletion markedly inhibited tumor proliferation in vitro and in vivo, arrested cell cycle at G0/G1 phase, and induced cell apoptosis in MKN-45 and AGS cells. Most notably, WWP1 downregulation both inactivated PTEN-Akt signaling pathway in MKN-45 and AGS cells. Taken altogether, our findings suggest that WWP1 acts as an oncogenic factor and should be considered as a novel interfering molecular target for gastric carcinoma.

Epidural dexmedetomidine or esketamine versus fentanyl to decrease ropivacaine use for labor analgesia: A randomized non-inferiority study.

Background: Epidural nonopioid adjuvants also reduce local anesthetic use. We aimed to test the hypothesis that, compared with the present standard fentanyl, the hourly consumption of local anesthetic was at least as good when dexmedetomidine or esketamine was combined with local anesthetic for patient-controlled epidural analgesia (PCEA). Methods: A total of 120 laboring nulliparous subjects requiring labor analgesia were recruited for the final statistical analysis. Subjects were randomized to receive 0.075 % ropivacaine added with one of three equivalent adjuvants: 0.4 µg/mL fentanyl, 0.4 µg/mL dexmedetomidine, or 1.0 mg/mL esketamine. The primary outcome was hourly ropivacaine consumption. Compared with the fentanyl group, a 20 % difference in hourly local anesthetic consumption between the dexmedetomidine and esketamine groups was considered a clinical difference (non-inferiority margin). Results: The hourly ropivacaine consumption of the fentanyl group was 12.4 (95 % confidence interval CI 11.2 to 13.6) ml/h, so the prespecified non-inferiority limit was 2.5 ml/h. The hourly ropivacaine consumption of the fentanyl group was not inferior to that of the dexmedetomidine group (12.4 ml/h vs. 11.9 ml/h, risk difference, 0.5; 95 % confidence interval CI, -1.0 to 2.0, meeting criteria for non-inferiority). However, the hourly ropivacaine consumption of the esketamine group was 14.3 ml/h, and that of the fentanyl group was 12.4 ml/h (risk difference, 1.9, 95 % CI, 0.2 to 3.6), failing to confirm non-inferiority with a non-inferiority margin of 20 %. The incidence of pruritus was highest in the fentanyl group, whereas the occurrence of mild dizziness was highest in the esketamine group. Conclusions: In setting of the conditions of this study, epidural dexmedetomidine was non-inferior compared with epidural fentanyl in combination with ropivacaine for PCEA during labor. Meanwhile, we failed to establish the non-inferiority of epidural esketamine compared with epidural fentanyl in combination with ropivacaine for labor analgesia.

Dual targeting of TIGIT and PD-1 with a novel small molecule for cancer immunotherapy.

Immune checkpoint inhibitors have unveiled promising clinical prospects in cancer treatment. Nonetheless, their effectiveness remains restricted, marked by consistently low response rates and affecting only a subset of patients. The co-blockade of TIGIT with PD-1 has exhibited substantial anti-tumor effects. Notably, there is a dearth of reports on small-molecule inhibitors concurrently targeting both TIGIT and PD-1. In this study, we employed Microscale Thermophoresis (MST) to screen our laboratory's existing repository of small molecules. Our findings illuminated Gln(TrT) 's affinity for both TIGIT and PD-1, affirming its potential to effectively inhibit TIGIT/PVR and PD-1/PD-L1 pathways. In vitro co-culture experiments substantiated Gln(TrT)'s proficiency in restoring Jurkat T-cell functionality by blocking both TIGIT/PVR and PD-1/PD-L1 interactions. In the MC38 murine tumor model, Gln(TrT) emerges as a pivotal modulator, promoting the intratumoral infiltration and functional competence of CD8+ T cells. Furthermore, whether used as a monotherapy or in conjunction with radiotherapy, Gln(TrT) substantially impedes MC38 tumor progression, significantly extending the survival of murine subjects.