Proteasome composition in immune cells implies special immune­cell­specific immunoproteasome function.

Immunoproteasomes are a special class of proteasomes, which can be induced with IFN-γ in an inflammatory environment. In recent years, it became evident that certain immune cell types constitutively express high levels of immunoproteasomes. However, information regarding the basal expression of proteolytically active immunoproteasome subunits in different types of immune cells is still rare. Hence, we quantified standard proteasome subunits (ß1c, ß2c, ß5c) and immunoproteasome subunits (LMP2, MECL-1, LMP7) in the major murine (CD4+ T cells, CD8+ T cells, CD19+ B cells, CD11c+ dendritic cells, CD49d+ natural killer cells, Ly-6G+ neutrophils) and human immune cell (CD4+ T cells, CD8+ T cells, CD19+ B cells, CD1c+CD141+ myeloid dendritic cells, CD56+ natural killer cells, granulocytes) subsets. The different human immune cell types were isolated from peripheral blood and the murine immune cell subsets from spleen. We found that proteasomes of most immune cell subsets mainly consist of immunoproteasome subunits. Our data will serve as a reference and guideline for immunoproteasome expression and imply a special role of immunoproteasomes in immune cells.

[Killing effect of anti-MSLN-iCAR-NK cells derived from induced pluripotent stem cells on ovarian epithelial cancer cells].

Objective: To investigate the cytotoxic effects of induced pluripotent stem (iPS) cells of anti-mesothelin (MSLN)-chimeric antigen receptor natural killer (CAR-NK) cells (anti-MSLN-iCAR-NK cells) on ovarian epithelial cancer cells. Methods: Twenty cases of ovarian cancer patients who underwent surgical treatment at Henan Provincial People's Hospital from September 2020 to September 2021 were collected, and 20 cases of normal ovarian tissues resected during the same period due to other benign diseases were also collected. (1) Immunohistochemistry and immunofluorescence were used to verify the expression of MSLN protein in ovarian cancer tissues. (2) Fresh ovarian cancer tissues were extracted and cultured to obtain primary ovarian cancer cells. Recombinant lentiviral vectors targeting anti-MSLN-CAR-CD244 were constructed and co-cultured with iPS cells to obtain anti-MSLN-iCAR cells. These cells were differentiated into anti-MSLN-iCAR-NK cells using cytokine-induced differentiation method. The cell experiments were divided into three groups: anti-MSLN-iCAR-NK cell group, natural killer (NK) cell group, and control group. (3) Flow cytometry and live cell staining experiment were used to detect the apoptosis of ovarian cancer cells in the three groups. (4) Enzyme-linked immunosorbent assay (ELISA) was used to measure the expression levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), granzyme B (GZMB), perforin 1 (PRF1), interleukin (IL)-6, and IL-10 in the three groups of ovarian cancer cells. Results: (1) Immunohistochemistry analysis showed that a positive expression rate of MSLN protein in ovarian cancer tissues of 65% (13/20), while normal ovarian tissues had a positive rate of 30% (6/20). The comparison between the two groups was statistically significant (χ2=4.912, P=0.027). Immunofluorescence analysis revealed that the positive expression rate of MSLN protein in ovarian cancer tissues was 70% (14/20), while normal ovarian tissues had a positive rate of 30% (6/20). The comparison between the two groups was statistically significant (χ2=6.400, P=0.011). (2) Flow cytometry analysis showed that the apoptotic rate of ovarian cancer cells in the anti-MSLN-iCAR-NK cell group was (29.27±0.85)%, while in the NK cell group and control group were (8.44±0.34)% and (6.83±0.26)% respectively. There were statistically significant differences in the comparisons between the three groups (all P<0.01). Live cell staining experiment showed that the ratio of dead cells to live cells in the anti-MSLN-iCAR-NK cell group was (36.3±8.3)%, while in the NK cell group and control group were (5.4±1.4)% and (2.0±1.3)% respectively. There were statistically significant differences in the comparisons between the three groups (all P<0.001). (3) ELISA analysis revealed that the expression levels of IFN-γ, TNF-α, GZMB, PRF1, IL-6, and IL-10 in ovarian cancer cells of the anti-MSLN-iCAR-NK cell group were significantly higher than those in the NK cell group and the control group (all P<0.05). Conclusion: The anti-MSLN-iCAR-NK cells exhibit a strong killing ability against ovarian cancer cells, indicating their potential as a novel immunotherapy approach for ovarian cancer.

[Predictive value of aMAP risk score for early recurrence of small hepatocellular carcinoma after microwave ablation].

Objective: To explore the value of the aMAP risk score (age, male, albumin-bilirubin, and platelets) to predict early recurrence within one year after microwave ablation in patients with small hepatocellular carcinoma. Methods: This was a retrospective study that enrolled 142 patients diagnosed with hepatocellular carcinoma who were treated with microwave ablation in the Department of Hepatology Unit of Nanfang Hospital, Southern Medical University from July 2016 to July 2021. The cohort enrolled 121 male and 21 female patients, including 110 patients that were <60 years old. All the patients were followed-up after microwave ablation to evaluate residual tumor and recurrence of tumor by computed tomography or magnetic resonance imaging. The observation indices mainly included general data and imaging data of patients. Using the X-tile tools, patients were divided into two groups: a high aMAP score group and a low aMAP score group. Multivariate Cox regression analysis was conducted for comparison of independent risk factors. Results: Multivariate Cox regression showed that high aMAP score, maximum tumor diameter >20 mm, and high AFP were the independent risk factors of early recurrence (all P<0.05). Kaplan-Meier survival curves showed that the median recurrence-free survival was 25.5 months in the low aMAP score group and 6.1 months in the high aMAP score group (P=0.001). Conclusions: The aMAP score could predict the early recurrence within 1 year of small hepatocellular carcinoma after microwave ablation. Patients with high aMAP score should undergo rigorous postoperative follow-up evaluations..

[Effects and mechanism of glycine on rat cardiomyocytes pretreated with serum from burned rats].

Objective: To investigate the effect and mechanism of glycine on rat cardiomyocytes pretreated with serum from burned rats (hereinafter referred to as burn serum). Methods: Experimental research methods were adopted. Thirty gender equally balanced Wistar rats aged 7 to 8 weeks were collected, 10 of which were used to prepare normal rat serum (hereinafter referred to as normal serum), and the other 20 were inflicted with full-thickness burn of 30% total body surface area to prepare burn serum. Primary cardiomyocytes were isolated and cultured from the apical tissue of 180 Wistar rats aged 1 to 3 days by either gender for follow-up experiments. Cells were divided into normal serum group and burn serum group treated with corresponding serum according to the random number table (the same grouping method below). Trypanosoma blue staining was performed at post treatment hour (PTH) 1, 3, 6, 9, and 12 to detect the cell survival rate. Cells were divided into burn serum alone group treated with burn serum for 6 h followed by routine culture of 30 min and 0.4 mmol/L glycine group, 0.8 mmol/L glycine group, 1.2 mmol/L glycine group, 1.6 mmol/L glycine group, and 2.0 mmol/L glycine group treated with burn serum for 6 h followed by culture of 30 min with corresponding final molarity of glycine, i.e., at post intervention hour (PIH) 6.5, the cell survival rate was detected as before. Cells were divided into normal serum group, burn serum alone group, 0.8 mmol/L glycine group, 1.2 mmol/L glycine group, and 1.6 mmol/L glycine group, with the same intervention of 6.5 h as before, respectively. The content of adenosine monophosphate (AMP) and adenosine triphosphate (ATP) was detected by high performance liquid chromatography, and the AMP/ATP ratio was calculated. The protein expressions of phosphorylated mammalian target of rapamycin complex 1 (p-mTORC1), phosphorylated p70 ribosomal protein S6 kinase (p-p70 S6K), phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (p-4E-BP1), and phosphorylated AMP-activated protein kinase (p-AMPK) were detected by Western blotting. Cells were divided into normal serum group, burn serum alone group, 0.8 mmol/L glycine group intervened as before and 0.8 mmol/L glycine+25 ng/mL rapamycin group treated with burn serum followed by culture with two reagents. The expressions of heat shock protein 70 (HSP70), metallothionein (MT), and tubulin were detected by immunofluorescence method after 30 min of corresponding culture at PTH 1, 3, and 6, i.e., at PIH 1.5, 3.5, and 6.5, and the microtubule morphology was observed at PIH 6.5. The sample number at each time point was 10. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, least significant difference (LSD)-t test, LSD test, and Bonferroni correction. Results: At PTH 1, 3, 6, 9, and 12, the cell survival rates in burn serum group were significantly lower than those in normal serum group (with t values of 4.96, 16.83, 35.51, 34.33, and 27.88, P<0.05). In burn serum group, the cell survival rate at PTH 3, 6, 9, or 12 was significantly lower than that at PTH 1 (P<0.05), the cell survival rate at PTH 6, 9, or 12 was significantly lower than that at PTH 3 (P<0.05), and the cell survival rate at PTH 6 was similar to that at PTH 9 (P>0.05) but significantly higher than that at PTH 12 (P<0.05). Treatment of 6 h was selected as the follow-up intervention time of burn serum. At PIH 6.5, compared with that in burn serum alone group, the cell survival rate in each glycine group was significantly increased (P<0.05). The cell survival rate in 0.8 mmol/L glycine group was the highest, and 0.8, 1.2, and 1.6 mmol/L were selected as subsequent glycine intervention concentrations. At PIH 6.5, the AMP/ATP ratio of cells in burn serum alone group was significantly higher than that in normal serum group, 1.2 mmol/L glycine group, or 1.6 mmol/L glycine group (P values all <0.05), and the AMP/ATP ratio of cells in 1.6 mmol/L glycine group was significantly lower than that in 0.8 mmol/L glycine group (P<0.05). At PIH 6.5, the protein expressions of p-mTORC1, p-p70 S6K, and p-4E-BP1 of cells in normal serum group, burn serum alone group, 0.8 mmol/L glycine group, 1.2 mmol/L glycine group, and 1.6 mmol/L glycine group were 1.001±0.037, 0.368±0.020, 1.153±0.019, 1.128±0.062, 1.028±0.037, 0.96±0.07, 0.63±0.12, 1.17±0.13, 1.13±0.16, 1.11±0.11, and 0.98±0.06, 0.45±0.08, 1.13±0.05, 0.77±0.12, 0.51±0.13. Compared with those in burn serum alone group, the protein expressions of p-mTORC1, p-p70 S6K, and p-4E-BP1 of cells in normal serum group and each glycine group were significantly increased (P<0.05), while the protein expressions of p-AMPK were significantly decreased (P<0.05). Compared with those in 0.8 mmol/L glycine group, the protein expression of p-4E-BP1 of cells in 1.2 mmol/L glycine group and the protein expressions of p-mTORC1 and p-4E-BP1 of cells in 1.6 mmol/L glycine group were significantly decreased (P<0.05). Compared with those in 1.2 mmol/L glycine group, the protein expressions of p-mTORC1 and p-4E-BP1 of cells in 1.6 mmol/L glycine group were significantly decreased (P<0.05), while the protein expression of p-AMPK was significantly increased (P<0.05). Compared with those in normal serum group, the expression of tubulin of cells in burn serum alone group was significantly decreased at PIH 1.5, 3.5, and 6.5 (P<0.05), while the expression of HSP70 of cells at PIH 1.5 and 3.5 and the expression of MT at PIH 3.5 and 6.5 were significantly increased (P<0.05). The expressions of HSP70 and MT of cells at PIH 1.5, 3.5, and 6.5 and the expression of tubulin at PIH 1.5 and 3.5 in burn serum alone group and 0.8 mmol/L glycine+25 ng/mL rapamycin group were significantly lower than those in 0.8 mmol/L glycine group (P<0.05). At PIH 6.5, compared with that in normal serum group, the cell microtubule structure in burn serum alone group was disordered; the cell boundary in 0.8 mmol/L glycine group was clearer than that in burn serum alone group, and the microtubule structure arranged neatly near the nucleus. Compared with that in 0.8 mmol/L glycine group, 0.8 mmol/L glycine+25 ng/mL rapamycin group had unclear cell boundaries and disordered microtubule structure. Conclusions: Burn serum can cause cardiomyocytes damage in rats. Glycine can significantly up-regulate mammalian target of rapamycin/p70 ribosomal protein S6 kinase/eukaryotic translation initiation factor 4E-binding protein 1 signaling pathway through AMP-activated protein kinase, promote the synthesis of protective proteins HSP70, MT, and tubulin, stabilize the microtubule structure, and exert cardiomyocytes protection function.

High Intake of Dietary Cholesterol Decreases the Risk of All-Cause Dementia and AD Dementia: A Results from Framingham Offspring Cohort.

BACKGROUND: Dietary cholesterol has been confirmed to be associated with high risks of diabetes, hypertension, and stroke, but whether it is detrimental to cognitive health is highly debated. This study aimed to investigate the associations between dietary cholesterol and all-cause dementia and AD dementia. METHODS: This prospective study analyzed Framingham Offspring Study cohort (FOS) participants who were dementia-free at baseline and had detailed information on daily diet (measured by food frequency questionnaires) and demographic characteristics. Surveillance for incident dementia commenced at examination 5 (1991-1995) through 2018 and continued for approximately 30 years. RESULTS: A total of 3249 subjects were included with a mean age of 54.7 years (SD: 9.8). During a median follow-up of 20.2 years (interquartile range: 14.2-24.8), a total of 312 incident dementia events occurred, including 211 (67.7%) cases of AD dementia. After multivariate adjustments for established dementia risk factors, participants with the highest intake of dietary cholesterol had a lower risk of all-cause dementia (HR: 0.70; 95% CI: 0.57-0.93) and AD dementia (HR: 0.68; 95% CI: 0.60-0.88) relative to individuals with the lowest intake. However, the associations were not significant for the group with a medium intake of dietary cholesterol. CONCLUSION: High intake of dietary cholesterol was associated with a decreased risk of all-cause dementia and AD dementia. The findings of this observational study need to be confirmed by other studies to highlight the role of dietary cholesterol in the development of neurodegenerative diseases.

[Short-term outcome of patients after transcatheter aortic valve replacement receiving different anticoagulants].

Objective: To compare the safety and efficacy of different anticoagulants in patients with indications for anticoagulation after transcatheter aortic valve replacement (TAVR). Methods: This is a retrospective study. Patients who underwent TAVR from April 2016 to February 2022 in Guangdong Provincial People's Hospital and had indications for anticoagulation were included and divided into two groups according to the type of anticoagulants, i.e. non-vitamin K antagonist oral anticoagulant (NOAC) and warfarin, and patients were followed up for 30 days. The primary endpoint was the combination of death, stroke, myocardial infarction, valve thrombosis, intracardiac thrombosis and major bleeding. The incidence of endpoints was compared between two groups, and multivariate logistic regression analysis was applied to adjust the bias of potential confounders. Results: A total of 80 patients were included. Mean age was (74.4±7.1) years, 43 (53.8%) were male. Forty-nine (61.3%) patients used NOAC, 31 used warfarin, and major indication for anticoagulants was atrial fibrillation (76/80, 95.0%). The adjusted risks of the primary endpoint (OR=0.23, 95%CI 0.06-0.94, P=0.040) of NOAC were lower than that of warfarin, mainly driven by a lower risk of major bleeding (OR=0.19, 95%CI 0.04-0.92, P=0.039). Conclusions: The short-term outcome of NOAC is better than that of warfarin in patients with indications for anticoagulation after TAVR. Randomized controlled trials of large sample size with long-term follow-up are needed to further testify this finding.

Zetomipzomib (KZR-616) attenuates lupus in mice via modulation of innate and adaptive immune responses.

Zetomipzomib (KZR-616) is a selective inhibitor of the immunoproteasome currently undergoing clinical investigation in autoimmune disorders. Here, we characterized KZR-616 in vitro and in vivo using multiplexed cytokine analysis, lymphocyte activation and differentiation, and differential gene expression analysis. KZR-616 blocked production of >30 pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs), polarization of T helper (Th) cells, and formation of plasmablasts. In the NZB/W F1 mouse model of lupus nephritis (LN), KZR-616 treatment resulted in complete resolution of proteinuria that was maintained at least 8 weeks after the cessation of dosing and was mediated in part by alterations in T and B cell activation, including reduced numbers of short and long-lived plasma cells. Gene expression analysis of human PBMCs and tissues from diseased mice revealed a consistent and broad response focused on inhibition of T, B, and plasma cell function and the Type I interferon pathway and promotion of hematopoietic cell lineages and tissue remodeling. In healthy volunteers, KZR-616 administration resulted in selective inhibition of the immunoproteasome and blockade of cytokine production following ex vivo stimulation. These data support the ongoing development of KZR-616 in autoimmune disorders such as systemic lupus erythematosus (SLE)/LN.

Effect of avian influenza virus subtype H9N2 on the expression of complement-associated genes in chicken erythrocytes.

1. The H9N2 subtype avian influenza virus can infect both chickens and humans. Previous studies have reported a role for erythrocytes in immunity. However, the role of H9N2 against chicken erythrocytes and the presence of complement-related genes in erythrocytes has not been studied. This research investigated the effect of H9N2 on complement-associated gene expression in chicken erythrocytes.2. The expression of complement-associated genes (C1s, C1q, C2, C3, C3ar1, C4, C4a, C5, C5ar1, C7, CD93 and CFD) was detected by reverse transcription-polymerase chain reaction (RT-PCR). Quantitative Real-Time PCR (qRT-PCR) was used to analyse the differential expression of complement-associated genes in chicken erythrocytes at 0 h, 2 h, 6 h and 10 h after the interaction between H9N2 virus and chicken erythrocytes in vitro and 3, 7 and 14 d after H9N2 virus nasal infection of chicks.3. Expression levels of C1q, C4, C1s, C2, C3, C5, C7 and CD93 were significantly up-regulated at 2 h and significantly down-regulated at 10 h. Gene expression levels of C1q, C3ar1, C4a, CFD and C5ar1 were seen to be different at each time point. The expression levels of C1q, C4, C1s, C2, C3, C5, C7, CFD, C3ar1, C4a and C5ar1 were significantly up-regulated at 7 d and the gene expression of levels of C3, CD93 and C5ar1 were seen to be different at each time point.4. The results confirmed that all the complement-associated genes were expressed in chicken erythrocytes and showed the H9N2 virus interaction with chicken erythrocytes and subsequent regulation of chicken erythrocyte complement-associated genes expression. This study reported, for the first time, the relationship between H9N2 and complement system of chicken erythrocytes, which will provide a foundation for further research into the prevention and control of H9N2 infection.

Promotion strategies for environmentally friendly packaging: a stochastic differential game perspective.

With the evolution of the e-commerce and express delivery industry, the consumption of packaging materials is increasing rapidly. Many members of society encourage using environmentally friendly packaging. However, due to the attitude-behavior gap, i.e., expressing concerns about environmental issues does not necessarily lead to green consumption, promoting the use of green packaging remains a challenge. This paper considers a stochastic differential game between green packaging manufacturers and e-commerce platforms. The optimal promotion strategies are derived for scenarios involving cooperation as well as non-cooperation. In addition, a welfare allocation mechanism for attaining stable cooperation is also discussed under the bargaining model. Numerical simulations and a sensitivity analysis were conducted to demonstrate the results. This paper finds that the cooperation between manufacturers and platforms can expand the actual market demand and promote the consumption of green packaging. The proposed model provides an effective tool for manufacturers and platforms to devise optimal strategies for promoting the use of green packaging.

Comparison of Pulmonary and Extrapulmonary Models of Sepsis-Associated Acute Lung Injury.

To compare different rat models of sepsis at different time points, based on pulmonary or extrapulmonary injury mechanisms, to identify a model which is more stable and reproducible to cause sepsis-associated acute lung injury (ALI). Adult male Sprague-Dawley rats were subjected to (1) cecal ligation and puncture (CLP) with single (CLP1 group) or two repeated through-and-through punctures (CLP2 group); (2) tail vein injection with lipopolysaccharide (LPS) of 10mg/kg (IV-LPS10 group) or 20 mg/kg (IV-LPS20 group); (3) intratracheal instillation with LPS of 10mg/kg (IT-LPS10 group) or 20mg/kg (IT-LPS20 group). Each of the model groups had a sham group. 7-day survival rates of each group were observed (n=15 for each group). Moreover, three time points were set for additional experimental studying in each model group: 4 hours, 24 hours and 48 hours after modeling (every time point, n=8 for each group). Rats were sacrificed to collect BALF and lung tissue samples at different time points for detection of IL-6, TNF-alpha, total protein concentration in BALF and MPO activity, HMGB1 protein expression in lung tissues, as well as the histopathological changes of lung tissues. More than 50 % of the rats died within 7 days in each model group, except for the IT-LPS10 group. In contrast, the mortality rates in the two IV-LPS groups as well as the IT-LPS20 group were significantly higher than that in IT-LPS10 group. Rats received LPS by intratracheal instillation exhibited evident histopathological changes and inflammatory exudation in the lung, but there was no evidence of lung injury in CLP and IV-LPS groups. Rat model of intratracheal instillation with LPS proved to be a more stable and reproducible animal model to cause sepsis-associated ALI than the extrapulmonary models of sepsis.

Observation of a Strongly Isospin-Mixed Doublet in

ß decay of proton-rich nuclei plays an important role in exploring isospin mixing. The ß decay of ^{26}P at the proton drip line is studied using double-sided silicon strip detectors operating in conjunction with high-purity germanium detectors. The T=2 isobaric analog state (IAS) at 13 055 keV and two new high-lying states at 13 380 and 11 912 keV in ^{26}Si are unambiguously identified through ß-delayed two-proton emission (ß2p). Angular correlations of two protons emitted from ^{26}Si excited states populated by ^{26}P ß decay are measured, which suggests that the two protons are emitted mainly sequentially. We report the first observation of a strongly isospin-mixed doublet that deexcites mainly via two-proton decay. The isospin mixing matrix element between the ^{26}Si IAS and the nearby 13 380-keV state is determined to be 130(21) keV, and this result represents the strongest mixing, highest excitation energy, and largest level spacing of a doublet ever observed in ß-decay experiments.

[Application of temporalis muscle flap in repair and reconstruction after the resection of tumor or necrotic foci following radiotherapy of nasopharyngeal carcinoma].

Objective: To summarize and popularize the application of temporalis muscle flap in repair and reconstruction after the resection of tumor or necrotic foci following radiotherapy of nasopharyngeal carcinoma (NPC). Methods: A retrospective analysis was made on the patients treated in the Department of Otorhinolaryngology Head and Neck Surgery of Xiangya Hospital between January 2019 and March 2021 who underwent surgical resection of tumor or necrosis of NPC after radiotherapy and temporalis muscle flap repair. The effect of the repair and the patients' postoperative conditions were analyzed. Results: A total 29 patients, 19 males and 10 females, aged from 33 to 65 years old, were included in the study, and were followed up for 6-35 months. Except for 2 patients who were not followed due to bleeding or special bacterial infection, the others' temporalis muscle flap healed well and no cerebrospinal fluid rhinorrhea or massive hemorrhage occurred. After the operation, all patients had no nasopharyngeal reflux or new open rhinolalia, and in some patients, the open rhinolalia even got relieved. Except for one case of depressed temporal fossa caused by infection and followed debridement and another one case of shallowed forehead wrinkles, the appearances of the other patients were basically symmetrical. Some patients had temporary mouth opening limitation after operation, and all of them recovered after rehabilitation exercises. Conclusions: The temporalis muscle flap can protect the skull base and internal carotid artery, and improve the quality of life of patients after the resection of NPC or necrotic foci. It is a reliable pedicled flap for repairing skull base defect with simple operation procedures and relatively few complications.

[PCGF1 is highly expressed in rectal adenocarcinoma and silencing PCGF1 inhibits proliferation of rectal adenocarcinoma cells in vitro].

OBJECTIVE: To investigate the expression of PCGF1 in rectal adenocarcinoma (READ) and the effect of PCGF1 silencing on proliferation READ cells in vitro. METHODS: The UALCAN and ENCORI online databases were used to analyze the expression level of PCGF1 in READ tissues and normal tissues and its association with the clinicopathological parameters and survival outcomes of patients with READ. The expression levels of PCGF1 were detected in two READ cell lines and a normal rectal epithelial cell line (HcoEpiC cells) using qPCR and Western blotting. Lentiviral vectors were used to construct PCGF1-overexpressing and PCGF1-silenced cell lines, and the proliferative activity of the cells was assessed using CCK-8 assay. The effect of PCGF1 silencing on tumor proliferation in vivo was also evaluated by observing tumorigenicity of the cells in nude mice. RESULTS: PCGF1 was highly expressed in READ tissue (P < 0.001), and its expression levels was correlated with READ stage, differentiation and lymph node metastasis (P < 0.001). A high PCGF1 expression level was associated with a poor survival outcome of READ patients (P < 0.05). In SW837 and SW1463 cells, PCGF1 silencing significantly lowered the proliferative activity of the cells both in vitro (P < 0.05) and in nude mice (P < 0.01). CONCLUSION: PCGF1 is highly expressed in READ tissue and may potentially serve as a prognostic biomarker as well as a therapeutic target for READ.

[Multi-omics research contributes to early screening, diagnosis and treatment of liver cancer].

In 2016, the World Health Organization set an ambitious goal of reducing viral hepatitis-related deaths by 65% by 2030. The key to this goal is to reduce viral hepatitis-related HCC deaths. Liver cancer is the fourth most common malignant tumor and the second leading cause of cancer death in China. The onset of HCC is insidious, and most patients are already in the middle and late stage when diagnosed. Despite the great progress on management of HCC, the therapeutic effect and prognosis of HCC are still unsatisfactory. Therefore, multi-dimensional and comprehensive analysis of the mechanism of liver cancer, improving the early screening, diagnosis and treatment rate of liver cancer are the key points of reducing the harm of liver cancer in China. In recent years, multi-omics studies have been widely applied in the field of liver cancer, providing a basis for the pathogenesis of liver cancer, early detection and diagnosis, development of individual treatment strategies and prognosis assessment. This issue will focus on the application of genomics, proteomics, metabolomics and imaging omics in early screening, diagnosis and treatment of liver cancer.

[Research advances of metabolomics in early diagnosis of hepatocellular carcinoma].

Primary liver cancer is the second leading cause of death from malignant tumors in China, and hepatocellular carcinoma (HCC) is the main type. The disease stage at the time of HCC diagnosis largely determines the efficacy of subsequent treatment. Due to the HCC screening among high-risk population has not yet popularized, and the current diagnose method of early HCC is not satisfactory, the early HCC diagnosis rate is less than 30% in China. Metabolomics research emerging in recent years has promoted the research progress of HCC in many fields, such as elaborating the mechanism of occurrence and development, early prevention and diagnosis, exploring drug treatment targets. At the same time, a large number of serum metabolites with excellent sensitivity and specificity were discovered, which made up for the deficiency of traditional serological indicators and helped the early screening and early diagnosis of HCC. This review will summarize the studies on serum metabolomic markers of HCC in recent 5 years, explore the role of metabolomics in the early prediction and diagnosis of HCC and its application prospect.

[Clinical characteristics of 272 437 patients with different histopathological subtypes of primary esophageal malignant tumors].

Objective: To characterize the histopathological subtypes and their clinicopathological parameters of gender and onset age by common, rare and sparse primary esophageal malignant tumors (PEMT). Methods: A total of 272 437 patients with PEMT were enrolled in this study, and all of the patients were received radical surgery. The clinicopathological information of the patients was obtained from the database established by the State Key Laboratory of Esophageal Cancer Prevention & Treatment from September 1973 to December 2020, which included the clinical treatment, pathological diagnosis and follow-up information of esophagus and gastric cardia cancers. All patients were diagnosed and classified by the criteria of esophageal tumor histopathological diagnosis and classification (2019) of the World Health Organization (WHO). The esophageal tumors, which were not included in the WHO classification, were analyzed separately according to the postoperative pathological diagnosis. The χ2 test was performed by the SPSS 25.0 software on count data, and the test standard α=0.05. Results: A total of 32 histopathological types were identified in the enrolled PEMT patients, of which 10 subtypes were not included in the WHO classification. According to the frequency, PEMT were divided into common (esophageal squamous cell carcinoma, ESCC, accounting for 97.1%), rare (esophageal adenocarcinoma, EAC, accounting for 2.3%) and sparse (mainly esophageal small cell carcinoma, malignant melanoma, etc., accounting for 0.6%). All the common, rare, and sparse types occurred predominantly in male patients, and the gender difference of rare type was most significant (EAC, male∶ female, 2.67∶1), followed with common type (ESCC, male∶ female, 1.78∶1) and sparse type (male∶ female, 1.71∶1). The common type (ESCC) mainly occurred in the middle thoracic segment (65.2%), while the rare type (EAC) mainly occurred in the lower thoracic segment (56.8%). Among the sparse type, malignant melanoma and malignant fibrous histiocytoma were both predominantly located in the lower thoracic segment (51.7%, 66.7%), and the others were mainly in the middle thoracic segment. Conclusion: ESCC is the most common type among the 32 histopathological types of PEMT, followed by EAC as the rare type, and esophageal small cell carcinoma and malignant melanoma as the major sparse type, and all of which are mainly occur in male patients. The common type of ESCC mainly occur in the middle thoracic segment, while the rare type of EAC mainly in the lower thoracic segment. The mainly sparse type of malignant melanoma and malignant fibrous histiocytoma predominately occur in the lower thoracic segment, and the remaining sparse types mainly occur in the middle thoracic segment.

[Expert consensus on measurement and clinical application of serum HBV RNA in patients with chronic HBV infection].

Since the discovery of circulating hepatitis B virus (HBV) RNA in the peripheral blood of patients with chronic hepatitis B in 1996, a growing number of studies have focused on clarifying the biological characteristics and clinical application value of serum HBV RNA. This consensus mainly summarizes the research progress of serum HBV RNA existing profiles, quantitative detection methods, and current clinical applications. In order to better apply this indicator for the clinical management of patients with chronic HBV infection, recommendations on quantitative detection target regions, detection results, and clinical applications are put forward.

[Effects and cell signaling mechanism of glutamine on rat cardiomyocytes intervened with serum from burned rat].

Objective: To investigate the effects and cell signaling mechanism of glutamine on rat cardiomyocytes intervened with serum from burned rat (hereinafter referred to as burn serum). Methods: The experimental research method was applied. Ten gender equally distributed Wistar rats aged 7-8 months were taken to prepare normal rat serum (hereinafter referred to as normal serum), another twenty gender equally distributed Wistar rats aged 7-8 months were taken to prepare burn serum after full- thickness burn injury of 30% total body surface area, and primary cardiomyocytes were isolated and cultured from 180 Wistar rats aged 1-3 days by either gender and used in the following experiments. The cells were divided into normal serum group and burn serum group according to the random number table (the same grouping method below) and cultured with the corresponding serum. At post culture hour (PCH) 1, 3, 6, 9, and 12, trypanosoma blue test was used to detect the cell survival rate. The cells were divided into burn serum alone group, burn serum+4 mmol/L glutamine group, burn serum+8 mmol/L glutamine group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group, which were treated with burn serum alone or burn serum added with the corresponding final molarity of glutamine and cultured for the time screened in the experiment before, and then the cell survival rate was detected as before. The cells were divided into normal serum group, burn serum alone group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group and treated the same as before. After 30 min of culture, phosphorylation levels of mammalian target of rapamycin complex 1 (mTORC1), p70 ribosomal protein S6 kinase (p70 S6K), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) were detected by Western blotting. Cells were divided into normal serum group, burn serum alone group, burn serum+12 mmol/L glutamine group, burn serum+12 mmol/L glutamine+25 ng/mL rapamycin group, and treated correspondingly. At PCH 1, 3, and 6, the expressions of heat shock protein 70 (HSP70) and metallothionein (MT), and the morphology of microtubule were determined with immunofluorescence method. The sample numbers in each index at each time point in each group were all 10. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, least significant difference t test, least significant difference test, and Bonferroni correction. Results: At PCH 1, 3, 6, 9, and 12, the cell survival rates in burn serum group were significantly lower than those in normal serum group (t=4.950, 16.752, 35.484, 34.428, 27.781, P<0.01). Compared within the group at PCH 1, the cell survival rate was significantly decreased in burn serum group at PCH 3, 6, 9, and 12 (P<0.05). Compared within the group at PCH 3, the cell survival rate was significantly decreased in burn serum group at PCH 6, 9, and 12 (P<0.05). Compared within the group at PCH 6 and 9, the cell survival rate was significantly decreased in burn serum group at PCH 12 (P<0.05). There were no statistically significant differences in the cell survival rates in burn serum group between PCH 6 and 9 (P>0.05). Thus PCH 6 was selected as the subsequent intervention time of burn serum. At PCH 6, compared with burn serum alone group, the cell survival rates in burn serum+4 mmol/L glutamine group, burn serum+8 mmol/L glutamine group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group were significantly increased (P<0.01). There were no statistically significant differences in cell survival rates between burn serum+12 mmol/L glutamine group and burn serum+16 mmol/L glutamine group (P>0.05). There were no statistically significant differences in cell survival rates in burn serum+16 mmol/L glutamine group and burn serum+20 mmol/L glutamine group (P>0.05). Thus 12, 16, and 20 mmol/L were selected as the subsequent intervention concentrations of glutamine. After 30 min of culture, the phosphorylation levels of mTORC1, p70 S6K, and 4E-BP1 of cells were respectively 1.001±0.042, 0.510±0.024, 0.876±0.022, 0.836±0.074, 0.856±0.041, 1.00±0.11, 0.38±0.09, 0.95±0.13, 0.96±0.13, 0.89±0.24, 1.00±0.07, 0.29±0.08, 0.87±0.27, 0.68±0.08, 0.60±0.21 in normal serum group, burn serum alone group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group. Compared with normal serum group, the phosphorylation levels of mTORC1, p70 S6K, and 4E-BP1 of cells were significantly decreased in the other 4 burn serum groups (P<0.01). Compared with those of burn serum alone group, the phosphorylation levels of mTORC1, p70 S6K, and 4E-BP1 of cells were significantly increased in the other 3 burn serum groups (P<0.01). The phosphorylation level of 4E-BP1 of cells in burn serum+12 mmol/L glutamine group was significantly higher than the levels in burn serum+16 mmol/L glutamine group and burn serum+20 mmol/L glutamine group (P<0.05). The expression of MT of cells in burn serum alone group was significantly lower than that in normal serum group at PCH 1 (P<0.05), while the expressions of MT of cells in burn serum alone group were significantly higher than those in normal serum group at the other time points (P<0.05). At PCH 1, 3, and 6, the expressions of HSP70 of cells in burn serum alone group were significantly higher than those in normal serum group (P<0.05), the expressions of HSP70 and MT of cells in burn serum+12 mmol/L glutamine group were significantly higher than those in burn serum alone group (P<0.05), and the expressions of HSP70 and MT of cells in burn serum+12 mmol/L glutamine+25 ng/mL rapamycin group were significantly lower than those in burn serum+12 mmol/L glutamine group (P<0.01). The microtubular structures were intact, displaying grid alinement and uniform staining in cells of normal serum group at PCH 1, 3, and 6. In burn serum alone group, some microtubules showed fracture and irregular grid arrangement at PCH 1; the microtubular structures near the nucleus were clear, while the microtubules at the distal end of the nucleus were blurry at PCH 3; the microtubular structures were blurry at PCH 6. The microtubular damage of cells was alleviated in burn serum+12 mmol/L glutamine group as compared with that in burn serum alone group at each time point of culture. The morphology of microtubules of cells in burn serum+12 mmol/L glutamine+25 ng/mL rapamycin group at each time point of culture was similar to that of burn serum alone group. Conclusions: The burn serum can lead to damages to cardiomyocytes and significant decrease of cell survival rate in rats. Glutamine can exert cell protective function through the regulation of mTOR/p70 S6K/4E-BP1 signaling pathway, thus promoting the expressions of HSP70 and MT and stabilizing the microtubule structures.

Proteasome Inhibitors and Their Pharmacokinetics, Pharmacodynamics, and Metabolism.

The proteasome is responsible for mediating intracellular protein degradation and regulating cellular function with impact on tumor and immune effector cell biology. The proteasome is found predominantly in two forms, the constitutive proteasome and the immunoproteasome. It has been validated as a therapeutic drug target through regulatory approval with 2 distinct chemical classes of small molecular inhibitors (boronic acid derivatives and peptide epoxyketones), including 3 compounds, bortezomib (VELCADE), carfilzomib (KYPROLIS), and ixazomib (NINLARO), for use in the treatment of the plasma cell neoplasm, multiple myeloma. Additionally, a selective inhibitor of immunoproteasome (KZR-616) is being developed for the treatment of autoimmune diseases. Here, we compare and contrast the pharmacokinetics (PK), pharmacodynamics (PD), and metabolism of these 2 classes of compounds in preclinical models and clinical studies. The distinct metabolism of peptide epoxyketones, which is primarily mediated by microsomal epoxide hydrolase, is highlighted and postulated as a favorable property for the development of this class of compound in chronic conditions.