Immunologic memory to SARS-CoV-2 in convalescent COVID-19 patients at 1 year postinfection.

BACKGROUND: Understanding the complexities of immune memory to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is key to gain insights into the durability of protective immunity against reinfection. OBJECTIVE: We sought to evaluate the immune memory to SARS-CoV-2 in convalescent patients with longer follow-up time. METHODS: SARS-CoV-2-specific humoral and cellular responses were assessed in convalescent patients with coronavirus disease 2019 (COVID-19) at 1 year postinfection. RESULTS: A total of 78 convalescent patients with COVID-19 (26 moderate, 43 severe, and 9 critical) were recruited after 1 year of recovery. The positive rates of both anti-receptor-binding domain and antinucleocapsid antibodies were 100%, whereas we did not observe a statistical difference in antibody levels among different severity groups. Accordingly, the prevalence of neutralizing antibodies (nAbs) reached 93.59% in convalescent patients. Although nAb titers displayed an increasing trend in convalescent patients with increased severity, the difference failed to achieve statistical significance. Notably, there was a significant correlation between nAb titers and anti-receptor-binding domain levels. Interestingly, SARS-CoV-2-specific T cells could be robustly maintained in convalescent patients, and their number was positively correlated with both nAb titers and anti-receptor-binding domain levels. Amplified SARS-CoV-2-specific CD4+ T cells mainly produced a single cytokine, accompanying with increased expression of exhaustion markers including PD-1, Tim-3, TIGIT, CTLA-4, and CD39, while the proportion of multifunctional cells was low. CONCLUSIONS: Robust SARS-CoV-2-specific humoral and cellular responses are maintained in convalescent patients with COVID-19 at 1 year postinfection. However, the dysfunction of SARS-CoV-2-specific CD4+ T cells supports the notion that vaccination is needed in convalescent patients for preventing reinfection.

K562 cell-derived exosomes suppress the adhesive function of bone marrow mesenchymal stem cells via delivery of miR-711.

A failure of bone marrow mesenchymal stem cells (BM-MSCs) to adhere to hematopoietic cells is an essential cause of the progression of chronic myelogenous leukemia and is also a cause of failure of bone marrow (BM) transplantation, but the exact mechanisms of this have not been fully elucidated. Recent studies have indicated that microRNAs (miRNAs) are contained in leukemia-derived exosomes and are involved in modulating the BM microenvironment. In this study, we found that K562 cell-derived exosomes transfer miR-711 to BM-MSCs and suppress the adhesive function of BM-MSCs. Using qRT-PCR, we also confirmed a significantly higher level of miR-711 in exosomes derived from K562 cells than in exosomes derived from parental cells. The BM-MSCs co-cultured with exosomes derived from K562 cells showed a lower adhesion rate than did controls. We further demonstrated that exosomal transfer of miR-711 induced decreased adhesive abilities by inhibiting expression of adhesion molecule CD44 in BM-MSCs. In conclusion, our study reveals that K562 cell-derived exosomal miR-711 can be transferred to BM-MSCs and weaken adhesive abilities by silencing the expression of the adhesion molecule CD44.

Preoperative fibrinogen to prealbumin ratio as a novel predictor for clinical outcome of hepatocellular carcinoma.

AIM: To investigate prognostic value of preoperative inflammatory biomarkers in hepatocellular carcinoma (HCC). PATIENTS & METHODS: Preoperative circulating fibrinogen, prealbumin, fibrinogen to prealbumin ratio (FPR), neutrophil to lymphocyte ratio, derived neutrophil to lymphocyte ratio, lymphocyte to monocyte ratio, platelet to lymphocyte ratio were detected and calculated in 230 HCC patients. X-tile software, Kaplan-Meier curve, Cox regression, time-dependent receiver-operating characteristic were used to explored prognostic roles of them in HCC. RESULTS: Multivariate Cox regression showed that high FPR was significantly associated with decreased recurrence-free survival (p = 0.034) and overall survival (p < 0.001) within HCC patients. FPR generated the largest area under curve of time-dependent receiver-operating characteristic comparing to the other biomarkers. Overall survival of HCC patients receiving chemotherapy was superior to the cases without receiving chemotherapy only in high FPR subgroup (p = 0.028). CONCLUSION: Preoperative FPR was superior to other biomarkers to independently predict survival of HCC patients, and it could identify the patients who could benefit from adjuvant chemotherapy.

Exosomes derived from imatinib-resistant chronic myeloid leukemia cells mediate a horizontal transfer of drug-resistant trait by delivering miR-365.

Chronic myeloid leukemia (CML) is a malignant disorder of hematopoietic stem/progenitor cells. Majority of patients can be effectively treated with tyrosine kinase inhibitors (TKIs) such as imatinib, but a portion of patients will develop drug resistance. Accumulated evidences have identified exosomes in cancer as promoters of tumor progression. Herein, we found that exosomes derived from imatinib resistant CML cells can be internalized into sensitive CML cells and confer drug-resistance traits. We also demonstrated a significant higher level of miR-365 in exosomes derived from drug-resistant CML cells compared with those from sensitive ones using microarray and qRT-PCR. The imatinib sensitive CML cells transfected with pre-miR-365 displayed lower chemosensitivity and apoptosis rate compared with controls. We further confirmed that exosomal transfer of miR-365 induced drug resistance by inhibiting expression of pro-apoptosis protein in sensitive CML cells. In conclusion, our study reveals that exosomes mediate a horizontal transfer of drug-resistant trait in chronic myeloid leukemia cell by delivering miR-365.

Circulating fibrinogen to pre-albumin ratio is a promising biomarker for diagnosis of colorectal cancer.

BACKGROUND: Inflammation and nutrition are closely associated with initiation and progression of colorectal cancer (CRC). This study aimed to investigate the diagnostic value of the FAR (FAR = 100*Fibrinogen/Albumin) and FPR (FPR = Fibrinogen/pre-Albumin) in CRC. METHODS: Neutrophil-to-lymphocyte ratio (NLR), FPR, and FAR were calculated in 455 newly diagnosed CRC patients, 455 healthy individuals, and 455 benign controls with colorectal polyp. The diagnostic value of biomarker for CRC was evaluated by receiver operating characteristic curve (ROC). Logistic regression analysis was adopted to assess the risk factors for telling CRC apart from benign disease. Moreover, the combined biomarkers were used for discriminating between CRC and benign disease. RESULTS: Neutrophil-to-lymphocyte ratio, FAR, and FPR were significantly higher in CRC patients compared with the benign or healthy controls (P < 0.05). ROC analysis showed that the diagnostic efficacy of FAR and FPR were better than NLR for CRC. Besides, FPR, NLR, carcinoembryonic antigen (CEA), and carbohydrate antigen 19-9 (CA199) were markedly associated with differentiation of benign disease and CRC in the logistic regression analysis. And the combination of FPR, CEA, and CA199 had the maximum area under the ROC curve (AUC) in separating CRC from benign disease (AUC = 0.845, Sensitivity = 67.9%, Specificity = 85.3%, Positive Predictive Value = 83.5%, Negative Predictive Value = 70.9%). CONCLUSIONS: Fibrinogen/pre-Albumin could be a useful CRC diagnostic biomarker, and the combination of FPR, CEA, and CA199 could significantly improve the diagnostic efficacy in discriminating CRC from the benign colorectal disease.

Simultaneous Optimization of Ultrasound-Assisted Extraction for Flavonoids and Antioxidant Activity of Angelica keiskei Using Response Surface Methodology (RSM).

Angelica keiskei Koidzumi (A. keiskei), as a Japanese edible herbal plant, enjoys a variety of biological activities due to the presence of numerous active compounds, especially flavonoids. This study aims for the optimization of ultrasound-assisted extraction (UAE) for flavonoids in A. keiskei and their antioxidant activity by using the response surface methodology (RSM). Single-factor experiments and a four-factor three-level Box-Behnken design (BBD) were performed to explore the effects of the following parameters on flavonoid extraction and antioxidant activity evaluation: ultrasonic temperature (X1), ultrasonic time (X2), ethanol concentration (X3) and liquid-solid ratio (X4). The optimum conditions of the combination of total flavonoid content (TFC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity (DPPH-RSC) and ferric-reducing antioxidant power (FRAP) were as follows: X1 = 80 °C, X2 = 4 min, X3 = 78%, X4 = 35 mL/g, respectively. The experimental results provide a theoretical basis for the extensive utilization of A. keiskei and flavonoids extraction from A. keiskei as a potential source of antioxidants.

The predictive and prognostic role of a novel ADS score in esophageal squamous cell carcinoma patients undergoing esophagectomy.

BACKGROUND: Chronic inflammation is deemed to play a significant effect on initiation and progression of esophageal squamous cell carcinoma (ESCC). In current study, we investigated the prognostic and predictive role of albumin (Alb) to fibrinogen (Fib) ratio (AFR) and a novel AFR-Alb-derived neutrophil/lymphocyte ratio (dNLR) score (ADS) in ESCC patients undergoing esophagectomy and compared them with Fib, Alb, neutrophil to lymphocyte ratio (NLR), dNLR, platelet to lymphocyte ratio (PLR) and lymphocyte to monocyte ratio (LMR). MATERIALS AND METHODS: A total of 153 clinical confirmed ESCC patients undergoing esophagectomy between January 2011 and December 2013 were included in present study. We detected preoperative Alb, Fib and neutrophil, monocyte, lymphocyte and platelet count, and obtained overall survival (OS) by 3 years' follow-up in the cases. X-tile software, Kaplan-Meier curve, Cox regression and predicted nomogram were used to evaluate the predictive and prognostic role of them in ESCC patients. RESULTS: The optimal cut-off values of Fib, Alb, AFR, NLR, dNLR, PLR and LMR were 3.2 mg/dL, 38.2 g/L, 9.3, 2.1, 4.3, 145.9 and 2.3, respectively. High levels of Fib [(adjusted hazard ratio (HR) = 2.148, 95% confidential interval (CI) (1.229-3.753)], dNLR (adjusted HR = 2.338, 95% CI 1.626-5.308) and PLR (adjusted HR = 1.964, 95% CI 1.129-3.415) as well as low AFR (adjusted HR = 2.381, 95% CI 1.152-4.926) and Alb (adjusted HR = 2.398, 95% CI 1.342-4.273) were significantly associated with decreased OS in ESCC patients. The survival predictive areas under the time-dependent receiver operating characteristics curve of AFR, dNLR and Alb were higher than Fib and PLR, respectively. High ADS score was significantly associated with short 3 years' OS of ESCC patients (adjusted HR = 2.94, 95% CI 1.70-5.08). Moreover, OS of ESCC patients receiving adjuvant radio-chemotherapy was longer than those without the treatment in high ADS score subgroup (p = 0.001), however, no significant survival difference was observed in the patients with or without treatment radio-chemotherapy (p = 0.297). Additionally, a significant difference was observed in c-index values of the nomograms including or without ADS (0.720 vs. 0.670, p < 0.05). CONCLUSIONS: Preoperative ADS was a prospective biomarker to predict clinical efficacy of adjuvant radio-chemotherapy and clinical prognosis of ESCC patients undergoing esophagectomy, and the score could apparently improve predicted efficacy of the nomogram.

[Screening of small molecule inhibitors for PLK1 PBD and evaluation of antitumor activities].

With the method of fluorescence polarization (FP), we screened small molecule inhibitors for PLK1 PBD to identify the lead compounds for antitumor drugs. FP led to the identification of a potent hit, F083-0063, whose inhibition rate was (99.7 ± 0.4) % at 10 µg·mL−1. The IC50 was calculated to be 1.9 ± 0.1 µmol·L−1 using Graphpad Prism 5. The effect of the compound on cells' multiplication was measured by MTT assay which showed that F083-0063 inhibited the proliferation of many tumor cell lines. Flow cytometry analysis indicated that the F083-0063 promoted cell apoptosis and induced cell G2/M arrest. Migration abilities of cells, evaluated using scratch test, increased significantly in the presence of F083-0063 with the migration rate as low as (37.6 ± 0.7) % at 20 µmol·L−1. Molecular linkage technique found F083-0063 had good affinity with PLK1 PBD. The results of Western blotting showed that the expression of cyclin-dependent proteins was increased after treatment with F083-0063. In summary, F083-0063 has an antitumor activity and is expected to be an antitumor lead compound targeting PLK1 PBD.

Rituximab-induced gut microbiota changes in Chinese neuromyelitis optica spectrum disorders.

BACKGROUND: Recent evidence shows that immunosuppressive agents can affect the gut microbiota in autoimmune diseases. However, the relationship between the gut microbiome and B-cell depletion immunotherapy in neuromyelitis optica spectrum disorder (NMOSD) remains poorly understood. OBJECTIVES: To evaluate the distinct intestinal microbial patterns and serum cytokine levels after short-term rituximab treatment (three months) in patients with NMOSD. METHODS: Firstly, we conducted a cross-sectional study involving 46 treatment-naïve NMOSD patients and 48 matched healthy controls. We collected fecal specimens, which were then analyzed using next-generation sequencing, and quantified serum cytokines. Subsequently, fecal and serum samples were re-collected and re-evaluated in 31 of the 46 treatment-naïve NMOSD patients after RTX treatment. RESULTS: Comparing the gut microbiome of treatment-naïve NMOSD patients to that of healthy controls revealed low α-diversity and distinct microbial compositions in the former. The microbial composition in NMOSD patients underwent changes following three months of RTX treatment. Specifically, the levels of IL-17F and IL-6 decreased, while those of IL-10 and TNFα increased after RTX treatment. LEfSe analysis identified 27 KEGG categories with significantly differential abundances between NMOSD patients and RTX treatment group. CONCLUSIONS: Our study provides a comprehensive understanding of the gut microbiota landscape in the context of B-cell depletion immunotherapy. We observed dysbiosis in the gut microbiome of NMOSD patients, which was partially alleviated by three months of RTX treatment. This suggests that B-cell depletion may play a crucial role in driving changes in the gastrointestinal environment.

High-free Fatty Acid Treatment Induced Anti-inflammatory Changes in a Natural Killer (NK) Cell Line.

Background: Natural killer (NK) cells play a role in the pathogenesis of various metabolic diseases related to obesity. While our initial findings have indicated a potential involvement of NK cells in the pathogenesis of type 2 diabetes mellitus, the precise mechanism underlying NK cell-mediated development of this form of diabetes remains inadequately comprehended. Objective: To investigate the impact and the underlying mechanism of high glucose and elevated levels of free fatty acids (FFAs) on immune and inflammatory responses and oxidative stress in NK92 cells. Methods: In this experiment, the CCK8 cytotoxicity assay was used to select the 44.4 mM and 1.5 mM concentrations of high glucose and high FFAs, respectively, to treat NK92 cells for 4 days. The concentrations of superoxide dismutase (SOD) and glutathione (GSH) were determined using a biochemical analyzer. Intracellular reactive oxygen species (ROS) levels, cytokines concentrations (TNF-α, IFN-γ, IL-6, and IL-10), and the expression levels of intracellular molecules (perforin and granzyme B) were assessed by flow cytometry. Results: The number of NK92 cell clumps was significantly reduced in the high-FFA (HF) group. In addition, the production of ROS and levels of cytokines (TNF-α, IFN-γ, IL-6, and IL-10) significantly decreased in the HF group but showed no significant change in the high-glucose (HG) group. This observation was consistent with the expression levels of perforin and granzyme B that decreased in the HF group. Conclusion: High FFAs induced morphological changes and serious damage to oxidative stress and inflammatory response in NK92 cells.

Three-dimensional graphene aerogel mitigated the toxic impact of chloramphenicol wastewater on microorganisms in an EGSB reactor.

Anaerobic treatment of chloramphenicol wastewater holds significant promise due to its potential for bioenergy generation. However, the high concentration of organic matter and residual toxic substances in the wastewater severely inhibit the activity of microorganisms. In this study, a three-dimensional graphene aerogel (GA), as a conductive material with high specific surface area (114.942 m2 g-1) and pore volume (0.352 cm3 g-1), was synthesized and its role in the efficiency and related mechanism for EGSB reactor to treat chloramphenicol wastewater was verified. The results indicated that synergy effects of GA for Chemical Oxygen Demand (COD) removal (increased by 8.17 %), chloramphenicol (CAP) removal (increased by 4.43 %) and methane production (increased by 70.29 %). Furthermore, GA increased the average particle size of anaerobic granular sludge (AGS) and promoted AGS to secrete more redox active substances. Microbial community analysis revealed that GA increased the relative abundance of functional bacteria and archaea, specifically Syntrophomonas, Geobacter, Methanothrix, and Methanolinea. These microbial species can participate in direct interspecific electron transfer (DIET). This research serves as a theoretical foundation for the application of GA in mitigating the toxic impact of refractory organic substances, such as antibiotics, on microorganisms during anaerobic treatment processes.

Exosomes as Potential Functional Nanomaterials for Tissue Engineering.

Exosomes are cell-derived extracellular vesicles of 40-160 nm diameter, which carry numerous biomolecules and transmit information between cells. They are used as functional nanomaterials with great potential in biomedical areas, such as active agents and delivery systems for advanced drug delivery and disease therapy. In recent years, potential applications of exosomes in tissue engineering have attracted significant attention, and some critical progress has been made. This review gives a complete picture of exosomes and their applications in the regeneration of various tissues, such as the central nervous systems, kidney, bone, cartilage, heart, and endodontium. Approaches employed for modifying exosomes to equip them with excellent targeting capacity are summarized. Furthermore, current concerns and future outlook of exosomes in tissue engineering are discussed.

Redistribution and Activation of CD16brightCD56dim NK Cell Subset to Fight against Omicron Subvariant BA.2 after COVID-19 Vaccination.

With the alarming surge in COVID-19 cases globally, vaccination must be prioritised to achieve herd immunity. Immune dysfunction is detected in the majority of patients with COVID-19; however, it remains unclear whether the immune responses elicited by COVID-19 vaccination function against the Omicron subvariant BA.2. Of the 508 enrolled patients infected with Omicron BA.2, 102 were unvaccinated controls, and 406 were vaccinated. Despite the presence of clinical symptoms in both groups, vaccination led to a significant decline in nausea or vomiting, abdominal pain, headache, pulmonary infection, and overall clinical symptoms and a moderate rise in body temperature. The individuals infected with Omicron BA.2 were also characterised by a mild increase in both serum pro- and anti-inflammatory cytokine levels after vaccination. There were no significant differences or trend changes between T- and B-lymphocyte subsets; however, a significant expansion of NK lymphocytes in COVID-19-vaccinated patients was observed. Moreover, the most effective CD16brightCD56dim subsets of NK cells showed increased functional capacities, as evidenced by a significantly greater IFN-γ secretion and a stronger cytotoxic potential in the patients infected with Omicron BA.2 after vaccination. Collectively, these results suggest that COVID-19 vaccination interventions promote the redistribution and activation of CD16brightCD56dim NK cell subsets against viral infections and that they could facilitate the clinical management of patients infected with Omicron BA.2.

Identification of AGT and CD44 in methotrexate-resistant colorectal cancer and reversal of methotrexate-resistance.

This study aims to screen out hub genes in 2 methotrexate-resistant colorectal cancer (CRC) cells (HT29 and Caco2), compared with parental CRC cells and reverse methotrexate-resistance in methotrexate-resistant CRC. GEO database and R software were utilized to analyze the gene expression profiles GSE11440 and GSE16066. Venn diagram was used to identify intersection differentially expressed genes (DEGs) between GSE11440 and GSE16066. Protein-protein interaction (PPI) was utilized to screen out central node genes. Hub genes were determined by volcano graphs, heatmaps and box plots. The functional enrichment analysis was exhibited with DAVID. The GEPIA was used to obtain survival curves to analyze association between patient prognosis and hub genes. Western blotting was used to detect the expressions of hub genes. CCK-8 assay was used to show MTX-resistant CRC cell viability following CD44 inhibitor (THIQ) and AGT inhibitor (O6-BG) treatments. In our results, there were 180 intersection DEGs between GSE11440 and GSE16066. CD44 and AGT were screened out as hub genes by PPI, heatmaps, volcano and box plots. In the 2 MTX-resistant CRC cells, the expressions of CD44 and AGT were up-regulated compared with parental CRC cells. The results of western blotting showed that CD44 and AGT were up-regulated in MTX-resistant HT29 and Caco2 cells compared with parental CRC cells. CCK-8 assay results showed that the combination of MTX with O6-BG or THIQ could significantly reduce the activity of MTX-resistant CRC cells. This research screened out CD44 and AGT in MTX-resistant CRC cells by bioinformatics and suggested that the combination of MTX with O6-BG or THIQ could enhance the sensitivity of MTX-resistant CRC cells to MTX. This research provides a new strategy for overcoming MTX-resistance in CRC.

Horizontal gene transfer via OMVs co-carrying virulence and antimicrobial-resistant genes is a novel way for the dissemination of carbapenem-resistant hypervirulent Klebsiella pneumoniae.

Introduction: The rapidly increased isolation rate of CR-HvKP worldwide has brought great difficulties in controlling clinical infection. Moreover, it has been demonstrated that the transmission of drug-resistant genes among bacteria can be mediated by outer membrane vesicles (OMVs), which is a new way of horizontal gene transfer (HGT). The transmission of virulence genes among bacteria has also been well studied; however, it remains unclear whether virulence and drug-resistant genes can be co-transmitted simultaneously. Co-transmission of virulence and drug-resistant genes is essential for the formation and prevalence of CR-HvKP. Methods: First, we isolated OMVs from CR-HvKP by cushioned-density gradient ultracentrifugation (C-DGUC). TEM and DLS were used to examine the morphology and size of bacterial OMVs. OMV-mediated gene transfer in liquid cultures and the acquisition of the carbapenem gene and virulence gene was confirmed using colony-PCR. Antimicrobial susceptibility testing, mCIM and eCIM were conducted for the resistance of transformant. Serum killing assay, assessment of the anti-biofilm effect and galleria mellonella infection model, mucoviscosity assay, extraction and quantification of capsules were verified the virulence of transformant. Pulsed-field gel electrophoresis (PFGE), S1 nuclease-pulsed-field gel electrophoresis (S1-PFGE), Southern blotting hybridization confirmed the plasmid of transformant. Results: Firstly, OMVs were isolated from CR-HvKP NUHL30457 (K2, ST86). TEM and DLS analyses revealed the spherical morphology of the vesicles. Secondly, our study demonstrated that CR-HvKP delivered genetic material, incorporated DNA within the OMVs, and protected it from degradation by extracellular exonucleases. Thirdly, the vesicular lumen DNA was delivered to the recipient cells after determining the presence of virulence and carbapenem-resistant genes in the CR-HvKP OMVs. Importantly, S1-PFGE and Southern hybridization analysis of the 700603 transformant strain showed that the transformant contained both drug-resistant and virulence plasmids. Discussion: In the present study, we aimed to clarify the role of CRHvKP-OMVs in transmitting CR-HvKP among K. pneumoniae. Collectively, our findings provided valuable insights into the evolution of CR-HvKP.

Autophagy: Mechanisms and Therapeutic Potential of Flavonoids in Cancer.

Autophagy, which is a conserved biological process and essential mechanism in maintaining homeostasis and metabolic balance, enables cells to degrade cytoplasmic constituents through lysosomes, recycle nutrients, and survive during starvation. Autophagy exerts an anticarcinogenic role in normal cells and inhibits the malignant transformation of cells. On the other hand, aberrations in autophagy are involved in gene derangements, cell metabolism, the process of tumor immune surveillance, invasion and metastasis, and tumor drug-resistance. Therefore, autophagy-targeted drugs may function as anti-tumor agents. Accumulating evidence suggests that flavonoids have anticarcinogenic properties, including those relating to cellular proliferation inhibition, the induction of apoptosis, autophagy, necrosis, cell cycle arrest, senescence, the impairment of cell migration, invasion, tumor angiogenesis, and the reduction of multidrug resistance in tumor cells. Flavonoids, which are a group of natural polyphenolic compounds characterized by multiple targets that participate in multiple pathways, have been widely studied in different models for autophagy modulation. However, flavonoid-induced autophagy commonly interacts with other mechanisms, comprehensively influencing the anticancer effect. Accordingly, targeted autophagy may become the core mechanism of flavonoids in the treatment of tumors. This paper reviews the flavonoid-induced autophagy of tumor cells and their interaction with other mechanisms, so as to provide a comprehensive and in-depth account on how flavonoids exert tumor-suppressive effects through autophagy.

4-Hydroxyderricin Promotes Apoptosis and Cell Cycle Arrest through Regulating PI3K/AKT/mTOR Pathway in Hepatocellular Cells.

4-hydroxyderricin (4-HD), as a natural flavonoid compound derived from Angelica keiskei, has largely unknown inhibition and mechanisms on liver cancer. Herein, we investigated the inhibitory effects of 4-HD on hepatocellular carcinoma (HCC) cells and clarified the potential mechanisms by exploring apoptosis and cell cycle arrest mediated via the PI3K/AKT/mTOR signaling pathway. Our results show that 4-HD treatment dramatically decreased the survival rate and activities of HepG2 and Huh7 cells. The protein expressions of apoptosis-related genes significantly increased, while those related to the cell cycle were decreased by 4-HD. 4-HD also down-regulated PI3K, p-PI3K, p-AKT, and p-mTOR protein expression. Moreover, PI3K inhibitor (LY294002) enhanced the promoting effect of 4-HD on apoptosis and cell cycle arrest in HCC cells. Consequently, we demonstrate that 4-HD can suppress the proliferation of HCC cells by promoting the PI3K/AKT/mTOR signaling pathway mediated apoptosis and cell cycle arrest.

HLA-DR Expression Level in CD8+ T Cells Correlates With the Severity of Children With Acute Infectious Mononucleosis.

Background: This study aimed to assess the host immune signatures associated with EBV infection and its clinical value in indicating the severity of children with acute infectious mononucleosis (IM). Methods: Twenty-eight pediatric patients with IM aged 3-8 years were enrolled. The immune phenotypes and cytokine secretion capability of T cells were detected. Results: The percentages and absolute numbers of CD3+ and CD8+ T cells were significantly increased in IM patients compared with HCs. The percentages of Naïve CD4+ and CD8+ T cells were decreased but with increased percentages of memory CD4+ and CD8+ T subsets. Our results showed the upregulation of active marker HLA-DR, TCR-αß, and inhibitory receptors PD-1, TIGIT in CD8+ T cells from IM patients, which suggested that effective cytotoxic T cells were highly against EBV infection. However, EBV exposure impaired the cytokine (IFN-γ, IL-2, and TNF-α) secretion capability of CD4+ and CD8+ T cells after stimulation with PMA/ionomycin in vitro. Multivariate analysis revealed that the percentage of HLA-DR+ CD8+ T cells was an independent prognostic marker for IM. The percentage of HLA-DR+ CD8+ T cells was significantly correlated with high viral load and abnormal liver function results. Conclusion: Robust expansion and upregulation of HLA-DR in CD8+ T cells, accompanied with impaired cytokine secretion, were typical characteristics of children with acute IM. The percentage of HLA-DR+ CD8+ T cells might be used as a prominent marker not only for the early diagnosis but also for indicating the severity of IM.

Optimization of urinary small extracellular vesicle isolation protocols: implications in early diagnosis, stratification, treatment and prognosis of diseases in the era of personalized medicine.

Extracellular vesicles isolation from urine was severely interfered by polymeric Tamm-Harsefall protein due to its ability to entrap exosome. Studies had been reported to optimize the extraction of urine extracellular vesicles by using reducing agents, surfactants, salt precipitation or ultrafiltration, but rarely based on highly specific purification methods. We optimized the density gradient centrifugation method for the isolation of urinary small extracellular vesicles (sEV) and compared seven differential centrifugation protocols to obtain the high-yield and high-purity sEV isolation procedures. Our study showed Tris sucrose gradient centrifugation at 25°C had more concentrated distribution of exosomal marker in the gradient compared to Tris sucrose gradient centrifugation at 4°C and PBS sucrose gradient centrifugation. Dissolving the 16000 g pellet using Tris, Nonidet™ P 40 or Dithiothreitol then pooling the supernatants did not increase the exosomal markers and number of nanoparticles in sEV preparation compared to the control and PBS groups. Differential centrifugation at room temperature without ultrafiltration recovered more exosome-like vesicles, exosomal markers and nanoparticles than that at 4°C or combining ultrafiltration. Differential centrifugation at RT without ultrafiltration and salt precipitation recovered the highest number of nanoparticles than other protocols. However, differential centrifugation at RT combining 100 kd ultrafiltration obtained the highest purity of sEV calculated by Nanoparticle number/Total protein. In conclusion, we had established two urinary sEV isolation procedures that can recovered higher yield of sEV and more pure preparation of sEV. It is not recommended to treating 16000 g pellet with reducing agents or surfactants to increase the yield of sEV.

Chemotherapy plus bevacizumab as an optimal first-line therapeutic treatment for patients with right-sided metastatic colon cancer: a meta-analysis of first-line clinical trials.

BACKGROUND: Monoclonal antibodies of anti-epidermal growth factor receptor (EGFR) have been recommended as first-line therapy for patients with left-sided metastatic colorectal cancer (mCRC) with wild-type RAS. The effect of tumour laterality on antivascular endothelial growth factor antibody and how to optimise targeted therapies for the right-sided cases remain controversial. PATIENTS AND METHODS: A comprehensive meta-analysis enrolling 16 first-line clinical trials was performed to evaluate the efficacy of chemotherapy alone and chemotherapy plus targeted therapies for patients with mCRC with right primary tumour site, and we validated the results in metastatic setting (14 trials containing 4306 patients with unresectable mCRC). RESULTS: Here, we found that progression-free survival (PFS) (combined HR 1.30, 95% CI 1.17 to 1.44) and overall survival (OS) (combined HR 1.46, 95% CI 1.32 to 1.62) of the right-sided patients were significantly inferior to the left-sided individuals receiving chemotherapy alone in overall population, regardless of race. Similar results were also observed in metastatic setting. OS of patients with left-sided mCRC receiving chemotherapy plus bevacizumab was superior to the right-sided individuals (combined median survival ratio (MSR)=1.23, 95% CI 1.08 to 1.39 for overall population; combined MSR=1.23, 95% CI 1.05 to 1.45 for metastatic setting), especially for wild-type RAS and mixed population. Moreover, the right-sided patients benefited more from chemotherapy plus bevacizumab comparing with chemotherapy alone in both overall population and metastatic setting. Importantly, the RAS-wild right-sided patients achieved longer PFS (combined HR 0.67, 95% CI 0.52 to 0.88) and OS (combined HR 0.74, 95% CI 0.56 to 0.98) from chemotherapy plus bevacizumab comparing with chemotherapy associated with anti-EGFR agents. CONCLUSIONS: Patients with right-sided mCRC show impaired chemosensitivity, and chemotherapy plus bevacizumab can be an optimal first-line therapeutic regimen for the RAS-wild patients with right-sided mCRC.