Angioimmunoblastic T-cell lymphoma (AITL) is an aggressive subtype of peripheral T-cell lymphoma (PTCL) characterized by its T-follicular helper (TFH) phenotype. Relapsed and refractory disease is common in AITL and often associated with a poor prognosis. The presence of epigenetic abnormalities, immune dysregulation, hyperinflammation and active angiogenesis in AITL offers potential targets for histone deacetylase (HDAC) inhibitors and immunomodulatory drugs (IMiDs). Herein, we present a case of AITL with multiple relapses over a decade. Following intensive chemotherapy and autologous stem cell transplantation (ASCT), the patient relapsed with extensive nodal and extranodal involvement, particularly pulmonary lesions, and subsequently pursued chemo-free treatments. Initially, the patient exhibited a remarkable response to single-agent chidamide, the first oral HDAC inhibitor. Soon after developing resistance to chidamide, continuous treatment with lenalidomide led to an impressive sustained complete remission lasting 64 months, followed by a diminished response for an additional 11 months. Genetic profiling of the patient revealed mutations in KMT2D and ARID1A, along with chromosomal aberrations such as del(5q). Notably, genes commonly mutated in AITL, including RHOA, TET2, DNMT3A, and IDH2, were absent in this case. A review of the literature highlights the heterogeneous genomic landscape of AITL and the diversity of treatment options available, underscoring the importance of tailored approaches to overcome resistance and improve outcomes in this distinct lymphoma subtype.

Multiple myeloma (MM) is an incurable hematological malignancy characterized by relapse and drug resistance. This study investigates the potential of angstrom-scale silver particles (F-Ag & Aring;Ps) to selectively target and eliminate MM cells, focusing on their susceptibility and underlying mechanisms. F-Ag & Aring;Ps are synthesized using a pure physical high-efficiency evaporation-condensation method and coated with fructose, and their cytotoxic effects on MM cell lines are evaluated through cell viability assays and flow cytometry. Reactive oxygen species (ROS) levels are measured to elucidate oxidative stress, while the role of nuclear factor erythroid 2-related factor 2 (NRF2) is examined via western blotting and quantitative PCR. In vivo, the therapeutic potential of F-Ag & Aring;Ps is assessed using a MM xenograft mouse model. F-Ag & Aring;Ps exhibited significant cytotoxicity in MM cells at low concentrations, with higher ROS levels leading to oxidative damage and NRF2 accumulation. This effect is mitigated by the ROS scavenger N-acetyl-l-cysteine. Additionally, the NRF2 activator dimethyl fumarate (DMF) enhanced F-Ag & Aring;Ps-induced cytotoxicity in vitro and suppressed myeloma growth in vivo. These findings suggest that combining DMF with F-Ag & Aring;Ps is a promising therapeutic strategy for MM, effectively targeting ROS-induced oxidative damage through NRF2 activation.

1 案 例 1.1 简要案情 某年7月,本鉴定中心受当事人孔某文的委托,受理了孔某文及其双胞胎孩子孔某萌(女)和孔某祥(男)有无血缘关系的案件.采集孔某文、孔某萌和孔某祥的血样进行STR基因座分型检测.

1 案例 1.1 简要案情 委托人熊某自述其与熊某亮为姑侄关系,因需要更改户口信息,委托本单位证明二人为非母子关系.本中心采集熊某与熊某亮的口腔拭子和血痕,因二联体亲子鉴定未能排除母子关系,本中心又补采了熊某亮亲生父亲熊某明的血痕及头发.

Osteoporosis is a frequent complication of chronic inflammatory diseases and increases in the pro-inflammatory cytokines make an important contribution to bone loss by promoting bone resorption and impairing bone formation. Omentin-1 is a newly identified adipocytokine that has anti-inflammatory effects, but little is known about the role of omentin-1 in inflammatory osteoporosis. Here we generated global omentin-1 knockout ( omentin-1 −/− ) mice and demonstrated that depletion of omentin-1 induces inflammatory bone loss-like phenotypes in mice, as defined by abnormally elevated pro-inflammatory cytokines, increased osteoclast formation and bone tissue destruction, as well as impaired osteogenic activities. Using an inflammatory cell model induced by tumor necrosis factor-α (TNF-α), we determined that recombinant omentin-1 reduces the production of pro-inflammatory factors in the TNF-α-activated macrophages, and suppresses their anti-osteoblastic and pro-osteoclastic abilities. In the magnesium silicate-induced inflammatory osteoporosis mouse model, the systemic administration of adenoviral-delivered omentin-1 significantly protects from osteoporotic bone loss and inflammation. Our study suggests that omentin-1 can be used as a promising therapeutic agent for the prevention or treatment of inflammatory bone diseases by downregulating the pro-inflammatory cytokines.

Recently, researchers identified a distinct vessel subtype called type H vessels that couple angiogenesis and osteogenesis. We previously found that type H vessels are reduced in ovariectomy (OVX)-induced osteoporotic mice, and preosteoclasts are able to secrete platelet-derived growth factor-BB (PDGF-BB) to stimulate type H vessel formation and thereby to promote osteogenesis. This study aimed to explore whether harmine, a β-carboline alkaloid, is capable of preventing bone loss in OVX mice by promoting preosteoclast PDGF-BB-induced type H vessel formation. METHODS:The impact of harmine on osteoclastogenesis of RANKL-stimulated RAW264.7 cells was verified by gene expression analysis and tartrate-resistant acid phosphatase (TRAP) staining. Enzyme-linked immunosorbent assay (ELISA) was conducted to test PDGF-BB production by preosteoclasts. A series of angiogenesis-related assays in vitro were performed to assess the pro-angiogenic effects of the conditioned media from RANKL-stimulated RAW264.7 cells treated with or without harmine. Meanwhile, the role of PDGF-BB in this process was determined. In vivo, OVX mice were intragastrically administrated with harmine emulsion or an equal volume of vehicle. 2 months later, bone samples were collected for µCT, histological, immunohistochemical and immunofluorescent analyses to evaluate bone mass, osteogenic and osteoclastic activities, as well as the numbers of type H vessels. Bone marrow PDGF-BB concentrations were assessed by ELISA. RESULTS:Exposure of RANKL-stimulated RAW264.7 cells to harmine enhanced the formation of preosteoclasts and the production of PDGF-BB. Harmine augmented the ability of RANKL-stimulated RAW264.7 cells to promote angiogenesis of endothelial cells, whereas the effect was blocked by PDGF-BB inhibition. In vivo, the oral administration of harmine emulsion to OVX mice resulted in enhanced trabecular bone mass and osteogenic responses, increased numbers of preosteoclasts, as well as reduced numbers of osteoclasts and fat cells. Moreover, OVX mice treated with harmine exhibited higher levels of bone marrow PDGF-BB and much more type H vessels in bone. CONCLUSION:Harmine may exert bone-sparing effects by suppression of osteoclast formation and promotion of preosteoclast PDGF-BB-induced angiogenesis.

Nonbinary single-nucleotide polymorphisms (SNPs) are potential forensic genetic markers because their discrimination power is greater than that of normal binary SNPs, and that they can detect highly degraded samples. We previously developed a nonbinary SNP multiplex typing assay. In this study, we selected additional 20 nonbinary SNPs from the NCBI SNP database and verified them through pyrosequencing. These 20 nonbinary SNPs were analyzed using the fluorescent-labeled SNaPshot multiplex SNP typing method. The allele frequencies and genetic parameters of these 20 nonbinary SNPs were determined among 314 unrelated individuals from Han populations from China. The total power of discrimination was 0.9999999999994, and the cumulative probability of exclusion was 0.9986. Moreover, the result of the combination of this 20 nonbinary SNP assay with the 20 nonbinary SNP assay we previously developed demonstrated that the cumulative probability of exclusion of the 40 nonbinary SNPs was 0.999991 and that no significant linkage disequilibrium was observed in all 40 nonbinary SNPs. Thus, we concluded that this new system consisting of new 20 nonbinary SNPs could provide highly informative polymorphic data which would be further used in forensic application and would serve as a potentially valuable supplement to forensic DNA analysis.

We previously reported that Runx2/miR-3960/miR-2861 regulatory feedback loop stimulates osteoblast differentiation. However, the effect of this feedback loop on the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) remains unclear. Our recent study showed that miR-2861 and miR-3960 expression increases significantly during β-glycerophosphate-induced osteogenic transdifferentiation of VSMCs. Overexpression of miR-2861 or miR-3960 in VSMCs enhances β-glycerophosphate-induced osteoblastogenesis, whereas inhibition of miR-2861 or miR-3960 expression attenuates it. MiR-2861 or miR-3960 promotes osteogenic transdifferentiation of VSMCs by targeting histone deacetylase 5 or Homeobox A2, respectively, resulting in increased runt-related transcription factor 2 (Runx2) protein production. Furthermore, overexpression of Runx2 induces miR-2861 and miR-3960 transcription, and knockdown of Runx2 attenuates β-glycerophosphate-induced miR-2861 and miR-3960 transcription in VSMCs. Thus, our data show that Runx2/miR-3960/miR-2861 positive feedback loop plays an important role in osteogenic transdifferentiation of VSMCs and contributes to vascular calcification.

OBJECTIVE:The objective of the present study was to explore the major risk factors of surgical complications using the Clavien-Dindo classification.METHODS:The case-control design was used. A total of 1049 patients who underwent radical gastrectomy in Hunan Cancer Hospital between October 2010 and August 2014 were retrospectively analyzed, including 122 patients (11.6%) with complications and 927 patients (88.4%) with no complications. Risk factors were evaluated.RESULTS:Following radical gastrectomy, 122 patients (11.6%) experienced a total of 151 complications. The incidence of Stages II, IIIa, IIIb, IVa, IVb and V complications was 9.6% (n = 101), 2.5% (n = 26), 1.0% (n = 11), 0.8% (n = 8), 0% (n = 0), and 0.5% (n = 5), respectively. The incidence of severe complications (Stage ≥ IIIa) was 4.8% (n = 50). Multivariate analysis showed that combined resection (Odds Ratio [OR] = 3.36, 95% confidence interval [CI]: 1.71~6.60, P < 0.01), perioperative blood transfusion (OR = 2.13, 95% CI: 1.38-3.29, P < 0.01), and BMI ≥ 25 kg/m(2) (OR = 1.98, 95% CI: 1.16-3.40, P = 0.01) were independent risk factors of complications.CONCLUSIONS:Combined resection, perioperative blood transfusion, and BMI ≥ 25 kg/m(2) are positively correlated with complications.

OBJECTIVE:To examine the expression of Twist1 in cervical cancer and to explore its biological function in the progression of cervical cancer.METHODS:The expressions of Twist1 in 32 cervical cancers and matched normal tissues were examined by immunohistochemistry (IHC). Cell invasive ability and the expression of invasion-related genes were determined in RNAi-based Twist1-silencing HeLa cells. The relationship between Twist1 and microRNA-33a (miR-33a) in cervical cancer was studied by Pearson correlation analysis, and the roles of miR-33a in regulation of Twist1 and cell invasiveness were studied.RESULTS:The positive expression rate of Twist1 was 75.0% (24/32) and 21.9% (7/32) in the cervical cancer and the matched normal tissues, respectively, with significant difference between them (P<0.05). Twist1 shRNA significantly decreased the invasiveness of HeLa cells (P<0.05). Compared with the matched normal tissues, the expression of miR-33a was increased in the cervical cancer tissues, which was negatively correlated with Twist1 (r=-0.661, P<0.05). Overexpression of miR-33a could significantly suppress Twist1 expression as well as cell invasiveness (P<0.05).CONCLUSION:Twist1 is critical for the invasiveness of cervical cancer cells; miR-33a, as a tumor suppressor gene, functions as an upstream regulator of Twist1 and is involved in the invasiveness of cervical cancer cell.

Cervical cancer, the second most common type of cancer in women worldwide, is responsible for >275,100 mortalities each year and is associated with high-risk human papilloma virus (HR-HPV). HPVs have two important oncogenes, E6 and E7, which have crucial roles in malignant transformation in cervical cancer. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA originally identified in non-small cell lung cancer. Previous studies have revealed that MALAT1 is expressed in numerous tissue types, and is significant in maintaining the normal function of the body. However, it also appeared to be notably upregulated in numerous carcinoma types compared with adjacent non-cancerous tissues. In the present study, it was identified that MALAT1 expression was upregulated in cervical cancer cell lines compared with normal cervical squamous cell samples. Further study into the effect of MALAT1 on cellular phenotype revealed that MALAT1 was able to promote cell migration and proliferation. Of note, it was revealed that the expression of MALAT1 was decreased with the knockdown of HPV16 E6/E7 in CaSki cells. Furthermore, the investigations in clinical samples also revealed that MALAT1 was expressed in HPV-positive cervical squamous cells, but not in HPV-negative normal cervical squamous cells. These results indicate that HPV correlates with MALAT1 deregulation in cervical cancer.

To construct an improved biological missile, an immunoconjugate ADM–Dex–ScFv-SA3 was synthesized, which was composed of a hepatocellular carcinoma-specific, single-chain Fv antibody (ScFv-SA3) and a highly potent cytotoxic drug, adriamycin (ADM), as the warhead. Oxidized Dextran T10 (Dex-T10) was used as a linker to connect these two moieties. The 40 KD soluble anti-hepatoma human Trx-ScFv-SA3 protein was expressed in E. coli BL21 (DE3), using a prokaryotic expression vector, pET21a (+)-Trx-ScFv-SA3-His. It was purified using a His-Tag Ni-Agarose column and identified by western blot. The activity of Trx-ScFv-SA3 was verified by enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry to confirm that it specifically binds to the hepatocellular carcinoma cell line HepG2. To prepare ADM–Dex–ScFv-SA3, ADM was conjugated to the antibody at a molar ratio of 14.21:1. The antitumor effect of the conjugate was tested by MTT assay, plate colony formation assay and xenografts in a nude mice experimental model. In vitro experiments revealed that ADM–Dex–ScFv-SA3 could bind to tumor cells selectively and inhibit the proliferation and the colony formation ability of HepG2 cells. In vivo experiments showed that ADM–Dex–ScFv-SA3 suppressed the tumor growth and prolonged the median survival time in tumor-bearing mice. Tumor histology slides indicated a significantly slower tumor tissue proliferation in the ADM–Dex–ScFv-SA3 group. These data indicate that the targeted drug, ADM–Dex–ScFv-SA3, may be a highly potent and selective therapy for the treatment of hepatoma.

BACKGROUND:Paired box 6 (PAX6), a highly conserved transcriptional factor, has been implicated in tumorigenesis.AIM:We aimed to explore the roles and molecular mechanisms of PAX6 and microRNA (miR-7) in colorectal cancer cells.METHODS:Tissue microarray immunohistochemistry and Western blot were applied to examine the PAX6 expression. Real-time RT-PCR and Western blot were performed to determine the expression of miR-7 and PAX6. Luciferase reporter assay was used to determine whether PAX6 was a target of miR-7. Effects of miR-7 and PAX6 on colorectal cell proliferation, cell cycle progression, colony formation and invasion were then investigated. Western blot was used to determine the activities of the ERK and PI3K signal pathways, as well as the protein expression of MMP2 and MMP9.RESULTS:The protein levels of PAX6 were gradually increased, while the expression of miR-7 was gradually reduced with malignancy of colorectal cancer. PAX6 was further identified as a target of miR-7, and its protein expression was negatively regulated by miR-7 in human colorectal cancer cells. Overexpression of PAX6 in Caco-2 and SW480 cells enhanced cellular proliferation, cell cycle progression, colony formation, and invasion, while miR-7 upregulation repressed these biological processes. Furthermore, the activities of ERK and PI3K signal pathways, as well as the protein levels of MMP2 and MMP9, were upregulated in PAX6-overexpressed Caco-2 and SW480 cells but deregulated in miR-7-overexpressed Caco-2 and SW480 cells.CONCLUSIONS:Our study suggests that as a novel target of miR-7, PAX6 may serve as a promising therapeutic target for colorectal cancer.

HTA is a novel hepatoma associated gene screened by bioinformatic strategies in our previous study. In the present investigation, the full-length sequence of the HTA gene was cloned by 3'-rapid amplification of cDNA ends (RACE) and 5'-RACE, which was 1414 bp and the open reading frame (ORF) was constituted with 3 exons and 2 introns. There are two types of splicing of the mRNA of HTA. Northern blot analysis showed that the 1.4 kb HTA mRNA and 1.7 kb HTA mRNA transcripts were present in the hepatocellular carcinoma (HCC) cell lines HepG2 and QGY-7703 but not in the normal cell line L02 and the human umbilical vein endothelial cells (HUVECs). Overexpression of the HTA gene in the hepatic cell line QSG-7701 via stable transfection can promote its proliferation rate and colony forming ability and change the cell cycle distribution of the cell lines. These results showed that the HTA gene is a potential therapeutic target in HCC and the clarification of its gene structure and sequence information provide an essential tool for future research.

Golgi phosphoprotein 2 (Golph2) is a type II Golgi‑specific membrane protein, which has been found to be overexpressed in hepatocellular carcinoma (HCC) patients. The sensitivity of diagnosis of HCC using Golph2 (76%) was markedly elevated compared with alpha‑fetoprotein (AFP) (70%), and Golph2 is expected to be a novel and effective serum biomarker for the diagnosis of HCC. The aim of this study was to prepare monoclonal antibodies against Golph2 and to establish double-antibody sandwich enzyme-linked immunosorbent assay (s-ELISA), which will be used in diagnostics, therapeutics and as a tool in understanding the role of Golph2 in the pathogenesis of liver diseases and cancer. In this study, fusion protein TRX-Golph2 was expressed and purified using an Escherichia coli system. BALB/c mice were immunized with TRX-Golph2 recombinant protein. The hybridoma technique was used for the production of anti-Golph2 monoclonal antibody. Hybridoma clones were screened using indirect ELISA and anti-Golph2 monoclonal antibody was produced in the ascites of BALB/c mice. The specificity of anti-Golph2 monoclonal antibody was detected by western blot analysis and immunocytochemistry. s-ELISA was established using horseradish peroxidase (HRP)‑labeled anti-Golph2 monoclonal antibody and used to detect the antigen in the serum of HCC patients. As a result, five stable hybridoma cell clones (5C6D5, 5B7F5, 7F5F3, 8A7B4, 8C9E8) producing anti-Golph2 monoclonal antibody were established. The highest titer of anti-Golph2 monoclonal antibody (5C6D5) was 1:51,200. Western blot analysis revealed that anti-Golph2 monoclonal antibody had a high specificity for Golph2 protein. Anti-Golph2 monoclonal antibody was HRP-labeled and the optimal working concentration was found to be 1:500. The levels of antigen in a proportion of HCC patients were shown to be significantly higher compared to those found in healthy controls.

Conclusion: The selected scFv antibody could specifically recognize and target nasopharyngeal carcinoma (NPC), and could be applied to clinical diagnosis and therapy. Objective: The aim was to construct and screen fully human anti-NPC single chain Fv fusion phage libraries, and to identify the specificity of the scFv antibody. Methods: Peripheral blood mononuclear cells of patients with NPC were immunized in vitro by NPC cells and transformed by Epstein-Barr virus. The total RNAwas used to construct the scFv libraries. By means of ELISA and immunochemistry, the positively bound scFv was selected and identified. The positive scFv was fused to EGFP, and was then expressed in E. coli strain BL21 (DE3) and purified. Furthermore, we observed the binding bioactivity. Results: The fusion protein has the biological activity of binding the NPC cells and emitting green fluorescence. In targeting experiments in vivo, the results showed that the fusion protein can successfully target the NPC.

AIM To investigate the function of gamma-aminobutyric acid (GABA) and gamma-aminobutyric acid A receptor θ subunit (GABRQ) in hepatocellular carcinoma (HCC). METHODS Semiquantitative polymerase chain reaction was used for detecting the expression of GABRQ receptor among HCC cell line HepG2, normal liver cell line L-02, non-malignant Chang's liver cells, 8 samples of HCC tissues and paired non-cancerous tissues. HepG2 cells were treated with GABA at serial concentrations (0, 1, 10, 20, 40 and 60 μmol/L), and their proliferating abilities were analyzed with the methyl thiazolyl tetrazolium assay, cell cycle analysis and tumor implanted in nude mice. Small interfering RNA was used for knocking down the endogenous GABRQ in HepG2. Proliferating abilities of these cells treated with or without GABA were analyzed. RESULTS We identified the overexpression of GABRQ in HCC cell lines and half of the tested HCC tissues. Knockdown of endogenous GABRQ expression in HepG2 attenuated HCC cell growth, suggesting its role in HCC cell viability. We studied the effect of GABA in the proliferation of GABRQ-positive cell lines in vitro and in vivo, and found that GABA increased HCC growth in a dose-dependent manner. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRQ-expressing HepG2 cells, but not GABRQ-knockdown HepG2 cells, which means that GABA stimulates HepG2 cell growth through GABRQ. CONCLUSION GABRQ play important roles in HCC development and progression and could be a promising molecular target for the development of new diagnostic and therapeutic strategies of HCC.

We present a detailed method for constructing a mammalian cell-based full-length antibody display library for targeting hepatocellular carcinoma. Two novel mammalian library vectors pcDNA3-CHm and pcDNA3-CLm were constructed that contained restriction enzyme sites NheI, ClaI and antibody constant domain. Mammalian expression vector pcDNA3-CHm contains IgG heavy-chain (HC) constant region and glycosylphosphatidylinositol anchor (GPI) that could be anchored full-length antibodies on the surface of mammalian cells. GOLPH2 prokaryotic expression vector was carried out in Escherichia coli and purified by immobilized metal affinity chromatography. Variable domain of heavy-chain and variable domain of light-chain genes were respectively inserted into the vector pcDNA3-CHm and pcDNA3-CLm by ligation, and antibody libraries are displayed as whole IgG molecules on the cell surface by co-transfecting this HC-GPI with a light chain. By screening the cell library using magnetic beads and cell ELISA, the cell clone that displayed GOLPH2-specific antibodies on cell surfaces was identified. The mammalian cell-based antibody display library is a great potential application for displaying full-length functional antibodies of targeting hepatocellular carcinoma on the surface of mammalian cells. Anti-GOLPH2 display antibody was successfully isolated from the library.

Epidermal growth factor-like domain 7 (EGFL7) has been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. The advent of antibody display technology (phage, bacteria, and yeast) led to an enormous revival in the use of antibodies as diagnostic and therapeutic tools for fighting cancer. However, problems with protein folding, posttranslational modification, and codon usage still limit the number of improved antibodies that can be obtained. We describe here the isolation of an EGFL7-specific antibody from a mammalian cell-based full-length antibody display library generated from peripheral blood mononuclear cells of patients with hepatocellular carcinoma. Using a novel vector, contained glycosylphosphatidylinositol anchor and restriction enzyme sites NheI and ClaI, antibody libraries are displayed as whole IgG molecules on the cell surface and screened for specific antigen binding by a combination of magnetic beads and measured by cell ELISA. Anti-EGFL7 antibody was successfully isolated from the library. The mammalian cell-based full-length antibody display library is a great potential application for rapid identification and cloning of human mAbs of targeting hepatocellular carcinoma.