In-depth human immune cellular profiling from newborn to frail.

Immune functional decline and remodeling accompany aging and frailty. It is still largely unknown how changes in the immune cellular composition differentiate healthy individuals from those become frail at a relatively early age. Our aim in this exploratory study was to investigate immunological changes from newborn to frailty, and the association between health statute and various immune cell subtypes. The participants analyzed in this study covered human cord blood cells and peripheral blood cells collected from young adults, healthy and frail old individuals. A total of 30 immune cell subsets was performed by flow cytometry based on the surface markers of immune cells. Furthermore, frailty was investigated for its relations with various leukocyte subpopulations. Frail individuals exhibited a higher CD4/CD8 ratio, a higher proportion of CD4+ central memory T (TCM) cells, CD8+ effector memory T cells, CD27- switched memory B (CD27-BSM) cells, CD27+ switched memory B cells, age-associated B cells (ABCs) and CD38-CD24- B cells, and a lower proportion of naïve CD8 + T cells and progenitor B cells. The Frailty index score was found to be associated with naïve T cells, CD4/CD8 ratio, ABCs, CD27-BSM cells, and CD4+ TCM cells. Our findings conducted a relatively comprehensive and extensive atlas of age- and frailty-related changes in peripheral leukocyte subpopulations from newborn to frailty. The immune phenotypes identified in this study can contribute to a deeper understanding of immunosenescence in frailty and may provide a rationale for future interventions and diagnosis.

Global analysis of HLA-A2 restricted MAGE-A3 tumor antigen epitopes and corresponding TCRs in non-small cell lung cancer.

Background: Advanced non-small cell lung cancer (NSCLC) is the most common type of lung cancer with poor prognosis. Adoptive cell therapy using engineered T-cell receptors (TCRs) targeting cancer-testis antigens, such as Melanoma-associated antigen 3 (MAGE-A3), is a potential approach for the treatment of NSCLC. However, systematic analysis of T cell immune responses to MAGE-A3 antigen and corresponding antigen-specific TCR is still lacking. Methods: In this study, we comprehensively screened HLA-A2 restricted MAGE-A3 tumor epitopes and characterized the corresponding TCRs using in vitro artificial antigen presentation cells (APC) system, single-cell transcriptome and TCR V(D)J sequencing, and machine-learning. Furthermore, the tumor-reactive TCRs with killing potency was screened and verified. Results: We identified the HLA-A2 restricted T cell epitopes from MAGE-A3 that could effectively induce the activation and cytotoxicity of CD8+ T cells using artificial APC in vitro. A cohort of HLA-A2+ NSCLC donors demonstrated that the number of epitope specific CD8+ T cells increased in NSCLC than healthy controls when measured with tetramer derived from the candidate MAGE-A3 epitopes, especially epitope Mp4 (MAGE-A3: 160-169, LVFGIELMEV). Statistical and machine-learning based analyses demonstrated that the MAGE-A3-Mp4 epitope-specific CD8+ T cell clones were mostly in effector and proliferating state. Importantly, T cells artificially expressing the MAGE-A3-Mp4 specific TCRs exhibited strong MAGE-A3+ tumor cell recognition and killing effect. Cross-reactivity risk analysis of the candidates TCRs showed high binding stability to MAGE-A3-Mp4 epitope and low risk of cross-reaction. Conclusions: This work identified candidate TCRs potentially suitable for TCR-T design targeting HLA-A2 restricted MAGE-A3 tumor antigen.

Insufficient epitope-specific T cell clones are responsible for impaired cellular immunity to inactivated SARS-CoV-2 vaccine in older adults.

Aging is a critical risk factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine efficacy. The immune responses to inactivated vaccine for older adults, and the underlying mechanisms of potential differences to young adults, are still unclear. Here we show that neutralizing antibody production by older adults took a longer time to reach similar levels in young adults after inactivated SARS-CoV-2 vaccination. We screened SARS-CoV-2 variant strains for epitopes that stimulate specific CD8 T cell response, and older adults exhibited weaker CD8 T-cell-mediated responses to these epitopes. Comparison of lymphocyte transcriptomes from pre-vaccinated and post-vaccinated donors suggested that the older adults had impaired antigen processing and presentation capability. Single-cell sequencing revealed that older adults had less T cell clone expansion specific to SARS-CoV-2, likely due to inadequate immune receptor repertoire size and diversity. Our study provides mechanistic insights for weaker response to inactivated vaccine by older adults and suggests the need for further vaccination optimization for the old population.

The Abnormal Proliferation of Hepatocytes is Associated with MC-LR and C-Terminal Truncated HBX Synergistic Disturbance of the Redox Balance.

Background: Microcystin-LR (MC-LR) and hepatitis B virus (HBV) are associated with hepatocellular carcinoma (HCC). However, the concentrations of MC-LR in drinking water and the synergistic effect of MC-LR and HBV on hepatocellular carcinogenesis through their disturbance of redox balance have not been fully elucidated. Methods: We measured the MC-LR concentrations in 168 drinking water samples of areas with a high incidence of HCC. The relationships between MC-LR and both redox status and liver diseases in 177 local residents were analyzed. The hepatoma cell line HepG2 transfected with C-terminal truncated hepatitis B virus X gene (Ct-HBX) were treated with MC-LR. Reactive oxygen species (ROS), superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA) were measured. Cell proliferation, migration, invasion, and apoptosis were assessed with cell activity assays, scratch and transwell assays, and flow cytometry, respectively. The mRNA and protein expression-related redox status genes were analyzed with qPCR and Western blotting. Results: The average concentration of MC-LR in well water, river water and reservoir water were 57.55 ng/L, 76.74 ng/L and 132.86 ng/L respectively, and the differences were statistically significant (P < 0.05). The MC-LR levels in drinking water were correlated with liver health status, including hepatitis, clonorchiasis, glutamic pyruvic transaminase abnormalities and hepatitis B surface antigen carriage (all P values < 0.05). The serum MDA increased in subjects who drank reservoir water and were infected with HBV (P < 0.05). In the cell experiment, ROS increased when Ct-HBX-transfected HepG2 cells were treated with MC-LR, followed by a decrease in SOD and GSH and an increase in MDA. MC-LR combined with Ct-HBX promoted the proliferation, migration and invasion of HepG2 cells, upregulated the mRNA and protein expression of MAOA gene, and downregulated UCP2 and GPX1 genes. Conclusion: MC-LR and HBV may synergistically affect redox status and play an important role in hepatocarcinoma genesis.

Immunological risk factors for sepsis-associated delirium and mortality in ICU patients.

Background: A major challenge in intervention of critical patients, especially sepsis-associated delirium (SAD) intervention, is the lack of predictive risk factors. As sepsis and SAD are heavily entangled with inflammatory and immunological processes, to identify the risk factors of SAD and mortality in the intensive care unit (ICU) and determine the underlying molecular mechanisms, the peripheral immune profiles of patients in the ICU were characterized. Methods: This study contains a cohort of 52 critical patients who were admitted to the ICU of the First Affiliated Hospital of Jinan University. Comorbidity, including sepsis and SAD, of this cohort was diagnosed and recorded. Furthermore, peripheral blood samples were collected on days 1, 3, and 5 of admission for peripheral immune profiling with blood routine examination, flow cytometry, ELISA, RNA-seq, and qPCR. Results: The patients with SAD had higher mortality during ICU admission and within 28 days of discharge. Compared with survivors, nonsurvivors had higher neutrophilic granulocyte percentage, higher CRP concentration, lower monocyte count, lower monocyte percentage, lower C3 complement level, higher CD14loCD16+ monocytes percentage, and higher levels of IL-6 and TNFα. The CD14hiCD16- monocyte percentage manifested favorable prediction values for the occurrence of SAD. Differentially expressed genes between the nonsurvival and survival groups were mainly associated with immune response and metabolism process. The longitudinal expression pattern of SLC2A1 and STIMATE were different between nonsurvivors and survivors, which were validated by qPCR. Conclusions: Nonsurvival critical patients have a distinct immune profile when compared with survival patients. CD14hiCD16- monocyte prevalence and expression levels of SLC2A1 and STIMATE may be predictors of SAD and 28-day mortality in ICU patients.

Effectiveness of Booster Doses of the SARS-CoV-2 Inactivated Vaccine KCONVAC against the Mutant Strains.

As the COVID-19 epidemic progresses with the emergence of different SARS-CoV-2 variants, it is important to know the effectiveness of inactivated SARS-CoV-2 vaccines against the variants. To maximize efficiency, a third boost injection of the high-dose SARS-CoV-2 inactivated vaccine KCONVAC was selected for investigation. In addition to the ancestral strain, KCONVAC boost vaccination induced neutralizing antibodies and antigen-specific CD8 T cells to recognize several variants, including B.1.617.2 (Delta), B.1.1.529 (Omicron), B.1.1.7 (Alpha), B.1.351 (Beta), P.3, B.1.526.1 (Lota), B.1.526.2, B.1.618, and B.1.617.3. Both humoral and cellular immunity against variants were lower than those of ancestral variants but continued to increase from day 0 to day 7 to day 50 after boost vaccination. Fifty days post-boost, the KCONVAC-vaccinated CD8 T-cell level reached 1.23-, 2.59-, 2.53-, and 1.01-fold that of convalescents against ancestral, Delta, Omicron and other SARS-CoV-2 variants, respectively. Our data demonstrate the importance of KCONVAC boosters to broaden both humoral and cellular immune responses against SARS-CoV-2 variants.

Phase separation in epigenetics and cancer stem cells.

Accumulating evidence indicates that liquid-liquid phase separation (LLPS) is the basis of the formation of membrane-less compartments in cells. This biomolecular condensate represented by phase separation may influence epigenetics in cancer stem cells (CSCs), a small subpopulation of cancer cells responding to the initiation, maintenance, metastasis, and therapy resistance of cancer. Understanding the underlying biophysical principles and the specific characteristics of biocondensates would provide insights into the precise blocking of potential tumor targets, thereby fundamentally curbing tumor occurrence, recurrence and metastasis. In this review, we summarized the key phenomenon and experimental detection of phase separation and the possibility of regulating the stemness of CSCs through phase separation. We believe that the mechanism of phase separation in CSCs will open up new avenues for the mystery of tumor formation, and modulating phase separation will be a great strategy for CSC-targeted tumor therapy.

Accurate Prediction of Metachronous Liver Metastasis in Stage I-III Colorectal Cancer Patients Using Deep Learning With Digital Pathological Images.

Objectives: Metachronous liver metastasis (LM) significantly impacts the prognosis of stage I-III colorectal cancer (CRC) patients. An effective biomarker to predict LM after surgery is urgently needed. We aimed to develop deep learning-based models to assist in predicting LM in stage I-III CRC patients using digital pathological images. Methods: Six-hundred eleven patients were retrospectively included in the study and randomly divided into training (428 patients) and validation (183 patients) cohorts according to the 7:3 ratio. Digital HE images from training cohort patients were used to construct the LM risk score based on a 50-layer residual convolutional neural network (ResNet-50). An LM prediction model was established by multivariable Cox analysis and confirmed in the validation cohort. The performance of the integrated nomogram was assessed with respect to its calibration, discrimination, and clinical application value. Results: Patients were divided into low- and high-LM risk score groups according to the cutoff value and significant differences were observed in the LM of the different risk score groups in the training and validation cohorts (P<0.001). Multivariable analysis revealed that the LM risk score, VELIPI, pT stage and pN stage were independent predictors of LM. Then, the prediction model was developed and presented as a nomogram to predict the 1-, 2-, and 3-year probability of LM. The integrated nomogram achieved satisfactory discrimination, with C-indexes of 0.807 (95% CI: 0.787, 0.827) and 0.812 (95% CI: 0.773, 0.850) and AUCs of 0.840 (95% CI: 0.795, 0.885) and 0.848 (95% CI: 0.766, 0.931) in the training and validation cohorts, respectively. Favorable calibration of the nomogram was confirmed in the training and validation cohorts. Integrated discrimination improvement and net reclassification index indicated that the integrated nomogram was superior to the traditional clinicopathological model. Decision curve analysis confirmed that the nomogram has clinical application value. Conclusions: The LM risk score based on ResNet-50 and digital HE images was significantly associated with LM. The integrated nomogram could identify stage I-III CRC patients at high risk of LM after primary colectomy, so it may serve as a potential tool to choose the appropriate treatment to improve the prognosis of stage I-III CRC patients.

Production and purification of infectious diseases-associated tetramer for staining of CD8+ T cells from PBMCs.

Major histocompatibility complex (MHC) tetramers can work as diagnostic tools to identify antigen-specific T cells in immunological research and monitoring. Here, we provide a general protocol for the production of MHC tetramer. We obtain highly pure N-terminal His-tagged HLA-A2 α chain and ß2-microglobulin (ß2m) to fold a monomer with a photocleavable peptide, which can exchange with an HLA-A2 presented peptide derived from influenza A virus. Further those monomers compose tetramer to stain antigen-specific CD8+ T cells. For complete details on the use and execution of this protocol, please refer to Xiao C.C. et al. (2021).

SARS-CoV-2 variant B.1.1.7 caused HLA-A2+ CD8+ T cell epitope mutations for impaired cellular immune response.

Here, we evaluated the immune properties of the HLA-A2 restricted CD8+ T cell epitopes containing mutations from B.1.1.7, and furthermore performed a comprehensive analysis of the SARS-CoV-2 specific CD8+ T cell responses from COVID-19 convalescent patients and SARS-CoV-2 vaccinees recognizing the ancestral Wuhan strain compared to B.1.1.7. First, most of the predicted CD8+ T cell epitopes showed proper binding with HLA-A2, whereas epitopes from B.1.1.7 had lower binding capability than those from the ancestral strain. In addition, these peptides could effectively induce the activation and cytotoxicity of CD8+ T cells. Our results further showed that at least two site mutations in B.1.1.7 resulted in a decrease in CD8+ T cell activation and a possible immune evasion, namely A1708D mutation in ORF1ab1707-1716 and I2230T mutation in ORF1ab2230-2238. Our current analysis provides information that contributes to the understanding of SARS-CoV-2-specific CD8+ T cell responses elicited by infection of mutated strains or vaccination.

Multidimensional single-cell analysis of human peripheral blood reveals characteristic features of the immune system landscape in aging and frailty.

Frailty is an intermediate status of the human aging process, associated with decompensated homeostasis and death. The immune phenotype of frailty and its underlying cellular and molecular processes remain poorly understood. We profiled 114,467 immune cells from cord blood, young adults and healthy and frail old adults using single-cell RNA and TCR sequencing. Here we show an age-dependent accumulation of transcriptome heterogeneity and variability in immune cells. Characteristic transcription factors were identified in given cell types of specific age groups. Trajectory analysis revealed cells from non-frail and frail old adults often fall into distinct paths. Numerous TCR clonotypes were shared among T-cell subtypes in old adults, indicating differential pluripotency and resilience capabilities of aged T cells. A frailty-specific monocyte subset was identified with exclusively high expression of long noncoding RNAs NEAT1 and MALAT1. Our study discovers human frailty-specific immune cell characteristics based on the comprehensive dimensions in the immune landscape of aging and frailty.

Optimization of antigen-specific CD8+ T cell activation conditions for infectious diseases including COVID-19.

Here, we describe the use of the artificial antigen-presenting cell (aAPC) system for the verification of T-cell epitopes. We purify and activate CD8+ T cells from blood samples from HLA-A2 that are negative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). CD8+ T cells are combined with peptide-loaded T2-A2 cells, which are then stained with a SARS-CoV-2-specific MHC-1 tetramer to identify specific HLA-A2-restricted T-cell epitopes. The use of aAPC and healthy donors means that only BSL2 lab conditions are needed. For details of the use and implementation of this protocol, please refer to Deng et al. (2021).

C-terminal-truncated hepatitis B virus X protein promotes hepatocarcinogenesis by activating the MAPK pathway.

PURPOSE: C-terminally truncated hepatitis B virus X (ctHBx) is frequently detected in hepatocellular carcinoma (HCC) patients with hepatitis B virus (HBV) integrated into their genomes, but the molecular mechanisms of ctHBx-related oncogenic signaling remain unclear. In this study, the effects of ctHBx on HepG2 cells were investigated by measuring ctHBx-induced changes in the cell cycle-related target proteins cell division cycle 25C (cdc25C) and p53 downstream of the mitogen-activated protein kinase (MAPK) pathway. MATERIALS AND METHODS: ctHBx lentiviruses were constructed and transfected into HepG2 cells. Then, we investigated HepG2 cell line function by conducting the Cell Counting Kit-8 (CCK8) assay, clone formation assay, scratch wound testing, Transwell assays and flow cytometry to examine cell cycle and apoptosis. Western blotting (WB) was performed to detect proteins related to and downstream of the extracellular signal-regulated kinase(ERK)/c-Jun N-terminal kinase(JNK)/p38 MAPK pathway, including cdc25C and p53. RESULTS: ctHBx significantly enhanced the proliferation, migration, invasion and colony-forming capability of HepG2 cells. In addition, ctHBx activated the ERK/JNK/p38 MAPK signaling pathway to regulate cell viability by affecting the expression of cyclin-related proteins, including cdc25C and p53. CONCLUSION: The present study demonstrates that ctHBx promote the formation and development of HCC via regulating MAPK/cdc25C and p53 axis. ctHBx should be the driving factor of HBV-induced hepatocarcinogenesis.

Capsaicin Inhibits Proliferation and Induces Apoptosis in Breast Cancer by Down-Regulating FBI-1-Mediated NF-κB Pathway.

BACKGROUND: As a natural compound extracted from a variety of hot peppers, capsaicin has drawn increasing attention to its anti-cancer effects against multiple human cancers including breast cancer. FBI-1 is a major proto-oncogene negatively regulating the transcription of many tumor suppressor genes, and plays a vital role in tumorigenesis and progression. However, whether FBI-1 is involved in capsaicin-induced breast cancer suppression has yet to be ascertained. This study aimed to investigate the effects of capsaicin on proliferation and apoptosis and its association with FBI-1 expression in breast cancer. METHODS: CCK-8 and morphological observation assay were employed to detect cell proliferation. Flow cytometry and TUNEL assay were conducted to detect cell apoptosis. RNA interference technique was used to overexpress or silence FBI-1 expression. qRT-PCR and/or Western blot analysis were applied to detect the protein expression of FBI-1, Ki-67, Bcl-2, Bax, cleaved-Caspase 3, Survivin and NF-κB p65. Xenograft model in nude mice was established to assess the in vivo effects. RESULTS: Capsaicin significantly inhibited proliferation and induced apoptosis in breast cancer in vitro and in vivo, along with decreased FBI-1, Ki-67, Bcl-2 and Survivin protein expression, increased Bax protein expression and activated Caspase 3. Furthermore, FBI-1 overexpression obviously attenuated the capsaicin-induced anti-proliferation and pro-apoptosis effect, accompanied with the above-mentioned proteins reversed, whereas FBI-1 silencing generated exactly the opposite response. In addition, as a target gene of FBI-1, NF-κB was inactivated by p65 nuclear translocation suppressed with capsaicin treatment, which was perceptibly weakened with FBI-1 overexpression or enhanced with FBI-1 silencing. CONCLUSION: This study reveals that FBI-1 is closely involved in capsaicin-induced anti-proliferation and pro-apoptosis of breast cancer. The underlying mechanism may be related to down-regulation of FBI-1-mediated NF-κB pathway. Targeting FBI-1 with capsaicin may be a promising therapeutic strategy in patients with breast cancer.

CD8+ T-Cell Epitope Variations Suggest a Potential Antigen HLA-A2 Binding Deficiency for Spike Protein of SARS-CoV-2.

We identified SARS-CoV-2 specific antigen epitopes by HLA-A2 binding affinity analysis and characterized their ability to activate T cells. As the pandemic continues, variations in SARS-CoV-2 virus strains have been found in many countries. In this study, we directly assess the immune response to SARS-CoV-2 epitope variants. We first predicted potential HLA-A*02:01-restricted CD8+ T-cell epitopes of SARS-CoV-2. Using the T2 cell model, HLA-A*02:01-restricted T-cell epitopes were screened for their binding affinity and ability to activate T cells. Subsequently, we examined the identified epitope variations and analyzed their impact on immune response. Here, we identified specific HLA-A2-restricted T-cell epitopes in the spike protein of SARS-CoV-2. Seven epitope peptides were confirmed to bind with HLA-A*02:01 and potentially be presented by antigen-presenting cells to induce host immune responses. Tetramers containing these peptides could interact with specific CD8+ T cells from convalescent COVID-19 patients, and one dominant epitope (n-Sp1) was defined. These epitopes could activate and generate epitope-specific T cells in vitro, and those activated T cells showed cytolytic activity toward target cells. Meanwhile, n-Sp1 epitope variant 5L>F significantly decreased the proportion of specific T-cell activation; n-Sp1 epitope 8L>V variant showed significantly reduced binding to HLA-A*02:01 and decreased proportion of n-Sp1-specific CD8+ T cell, which potentially contributes to the immune escape of SARS-CoV-2. Our data indicate that the variation of a dominant epitope will cause the deficiency of HLA-A*02:01 binding and T-cell activation, which subsequently requires the formation of a new CD8+ T-cell immune response in COVID-19 patients.

Synergistic Effect of MC-LR and C-Terminal Truncated HBx on HepG2 Cells and Their Effects on PP2A Mediated Downstream Target of MAPK Signaling Pathway.

C-terminally truncated hepatitis B virus (HBV) X (ctHBx) infection and exposure to microcystins-LR (MC-LR) can lead to human hepatitis and liver cancer, but the mechanism associated with their synergistically effects not been fully elucidated. The ctHBx (HBxΔ4 and HBxΔ32) lentivirus were constructed and transfected into the HepG2 cells. Then we investigated the function of MC-LR and ctHBx using the molecular biology approaches, including enzyme-linked immunosorbent assay, clone formation assay, scratch wound testing, transwell assays, carried out flow cytometry respectively to examine cell cycle and apoptosis in each group, and detected the related proteins of HBx, MEK/ERK/JNK/p38 in mitogen-activated protein kinase (MAPK) pathway and the downstream proteins such as cdc2, cdc25C, and p53 by western blotting. We found that the protein phosphorylase 2A (PP2A) enzyme activity in MC-LR and HBxΔ32/HBxΔ4 groups decreased more than in MC-LR and HBx group at the same time point and MC-LR concentration (P < 0.05). Meanwhile the proliferation, migration, invasion and colony formation capability of HepG2 cells were significantly enhanced in MC-LR and ctHBx groups (P < 0.05). In addition the proportion of S stage cells in the MC-LR-treated HBxΔ32/HBxΔ4 groups was significantly greater than that in the untreated groups (P < 0.05). Furthermore, the protein expression of MAPK signaling pathway including phospho-MEK1/2, ERKl/2, p38, and JNK were up-regulated by MC-LR and HBxΔ32, and the expression of cyclin-related proteins, including p53, cdc25C, and cdc2 were also activated (P < 0.05). Taken together, our findings revealed the essential significance of the MC-LR and ctHBx on the PP2A/MAPK/p53, cdc25C and cdc2 axis in the formation and development of HCC and identified MC-LR and ctHBx as potential causal cofactors of hepatocarcinogenesis.

Sodium Butyrate Combined with Docetaxel for the Treatment of Lung Adenocarcinoma A549 Cells by Targeting Gli1.

PURPOSE: This study is aimed to investigate the combined treating efficacy of sodium butyrate and docetaxel on proliferation and apoptosis of the lung adenocarcinoma A549 cell line based on Gli1 regulation in vitro and in vivo. MATERIALS AND METHODS: RNA interference method was used to overexpress Gli1 in A549 cells. Cells were treated with varying concentrations of sodium butyrate, docetaxel or both in combination. CCK-8, colony formation assay, Hoechst 33258 staining, flow cytometry and TUNEL assay were employed to detect proliferation, cell cycle and apoptosis. qRT-PCR and Western blot analysis were applied to detect the mRNA and protein expression of Gli1. In vivo tumorigenicity was detected by tumor transplantation in nude mice. Downstream protein levels of Gli1 were detected using Western blot assay. RESULTS: It was found that sodium butyrate or docetaxel alone, respectively, inhibited proliferation and promoted apoptosis of A549 cells in vitro and in vivo, while the combination of the two generated significantly higher responses, which were also effective in another lung adenocarcinoma cell line H1299. Furthermore, the combined therapy had an additive effect in suppressing Gli1 expression and regulating the expression of its downstream proteins that involve in proliferation, cell cycle and apoptosis of A549 cells in vitro and in vivo, including decreased protein expression of Ki-67, CDK1, CDK2, Cyclin D1, Bcl-2 and Survivin, and increased protein expression of Cyclin A, p21, Bax and cleaved-Caspase 3. On the other hand, Gli1 overexpression perceptibly reversed the above-mentioned additive effect in vitro and in vivo. CONCLUSION: This study demonstrates that the combined therapy of sodium butyrate and docetaxel additively inhibits proliferation and promotes apoptosis of A549 lung adenocarcinoma cells via suppressing Gli1 expression in vitro and in vivo. Targeting Gli1 by the combined therapy may provide new insights into the therapeutic management of patients with lung adenocarcinoma.

Novel nomogram with microvascular density in the surgical margins can accurately predict the risk for anastomotic leakage after anterior resection for rectal cancer.

BACKGROUND: Anastomotic leakage (AL) is a serious complication after anterior resection. The purpose of this study was to determine the role of microvascular density (MVD) in AL and to develop a nomogram to accurately predict AL. METHODS: This study retrospectively enrolled 477 consecutive patients who underwent anterior resection for rectal cancer from January 2011 to January 2019. Tissue samples of the resection margins were assessed for MVD. Univariate and multivariate regression analyses were used to identify the risk factors for AL. RESULTS: The incidence of clinical AL was 6.7%. MVD in the distal margin was associated with AL (P < .001). Univariate and multivariate regression analysis identified the following variables as independent risk factors for AL: preoperative albumin ≤35 g/L (odds ratio [OR] = 2.511), neoadjuvant treatment (OR = 3.560), location of tumor ≤7 cm (OR = 3.381), blood loss ≥100 mL (OR = 2.717), and MVD in the distal margin ≤20 (OR = 4.265). Then, a nomogram including these predictors was developed. The nomogram showed good discrimination (AUC = 0.816) and calibration (concordance index = 0.816). The decision curve analysis demonstrated that the nomogram was clinically useful. CONCLUSIONS: MVD in the distal margin is closely associated with AL. The nomogram can be used for individualized prediction of AL after anterior resection for patients with rectal cancer.

Triptolide Suppresses Growth of Breast Cancer by Targeting HMGB1 in Vitro and in Vivo.

Triptolide has been indicated potent anti-cancer effect involving multiple molecular targets and signaling pathways. High-mobility group box 1 (HMGB1) is a highly conserved DNA-binding protein taking part in breast cancer development. The therapeutic effect of triptolide on HMGB1 has not been reported. Thus, our study aims to clarify the role of HMGB1 in triptolide-induced anti-growth effect on breast cancer in vitro and in vivo. We demonstrated that triptolide significantly suppressed growth of breast cancer cells by inhibition of cell viability, clonogenic ability. Further studies evidenced that triptolide treatment not only inhibited HMGB1 mRNA expression, but also decreased supernatant level of HMGB1 in vitro. In line with these observations, exogenous recombinant HMGB1 (rHMGB1) promoted cell proliferation of breast cancer, and triptolide reversed the rHMGB1-promoted proliferative effect. As well, triptolide enhanced the anti-proliferative activity of ethyl pyruvate (EP) (HMGB1 inhibitor). Furthermore, downstream correlation factors (Toll-like receptor 4 (TLR4) and phosphorylated-nuclear factor-kappaB (NF-κB) p65) of HMGB1 were significantly decreased in vitro after triptolide treatment. Consistantly, we confirmed that tumor growth was significantly inhibited after triptolide treatment in vivo. Meanwhile, immunohistochemical analyses showed that triptolide treatment significantly decreased the level of cytoplasmic HMGB1 and TLR4 expression, whereas the expression of NF-κB p65 was relatively higher in cytoplasm, and conversely lower in nucleus as compared to the control group. Collectively, these results demonstrate that triptolide suppresses the growth of breast cancer cells via reduction of HMGB1 expression in vitro and in vivo, which may provide new insights into the treament of breast cancer.

Analysis of HBV X gene quasispecies characteristics by next-generation sequencing and cloning-based sequencing and its association with hepatocellular carcinoma progression.

OBJECTIVES: This study aimed to describe the differences between next-generation sequencing (NGS) and cloning-based sequencing (CBS) in HBX quasispecies research and primitively investigate the relationship between the dominant HBX quasispecies and hepatocellular carcinoma (HCC). METHODS: A total of 12 serum samples were collected. Serum hepatitis B virus (HBV) DNA was extracted, and the HBV X-region (HBX) was amplified by nested polymerase chain reaction (PCR). The PCR products were simultaneously tested with NGS and CBS to detect quasispecies of the HBX. RESULTS: A total of 9348 eligible quasispecies sequences were obtained by NGS, which were much larger than the 98 of that by CBS. By the phylogenetic tree, the dominant quasispecies sequence of each sample could be found, although they had several nucleotides differences between the dominant quasispecies sequences found by CBS and NGS. By comparing the quasispecies heterogeneity, it was found that the quasispecies complexity value of HBV X-region obtained by NGS was higher than CBS (P < 0.05). The diversity values, including d, dS, dN, an d d N/ dS obtained by NGS were lower than by CBS (all of P < 0.01). The relativity of Spearman(rs) in d, dS, and dN were statistically significant (rs_ d = 0.865, P = 0.001; rs_ dS = 0.722, P = 0.014; and rs_ dN = 0.738, P = 0.011, respectively). There were 21 different bases between the HBX quasispecies of case A and control B. CONCLUSION: The results of this can be used as guidance when researchers plan to choose a suitable method to study quasispecies, especially the HBV X gene quasispecies. Some high-risk mutations of HBX quasispecies were also found in this study and their relationship with HCC need deeper exploration.