Microsatellite instability evaluation by a novel PCR-based 8-loci test kit in colorectal cancer.

Microsatellite instability (MSI) assessment is strongly recommended for colorectal cancer patients, as MSI status is crucial in determining optimal treatment and predicting prognosis. This study evaluated the reliability and accuracy of a novel polymerase chain reaction (PCR)-based 8-loci MSI test kit, a rapid test kit designed to detect MSI, by comparing its performance with immunohistochemistry (IHC) and the National Cancer Institute (NCI) 2B3D Panel. MSI status was determined in 186 formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissue samples with known mismatch repair (MMR) status by IHC using the novel PCR-based 8-loci MSI test kit. Additionally, the consistency between the NCI 2B3D Panel and the novel PCR-based 8-loci panel was compared using 69 FFPE tumor tissues paired with adjacent non-cancerous tissue. The novel PCR-based 8-loci MSI test kit and IHC demonstrated high concordance (overall agreement: 97.8%). However, four samples displayed discordant results, exhibiting MMR deficiency using IHC and microsatellite stability using the novel PCR-based 8-loci MSI test kit. Of the 69 samples reanalyzed using the NCI 2B3D Panel, high concordance with the novel PCR-based 8-loci MSI test kit was observed in 67 of 69 cases (overall agreement: 97.1%). The novel PCR-based 8-loci MSI test kit is a rapid and reliable tool for accurately detecting MSI status in colorectal cancer.

Characterization and immunogenicity of SARS-CoV-2 spike proteins with varied glycosylation.

The ongoing coronavirus disease-19 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drastically changed our way of life and continues to have an unmitigated socioeconomic impact across the globe. Research into potential vaccine design and production is focused on the spike (S) protein of the virus, which is critical for virus entry into host cells. Yet, whether the degree of glycosylation in the S protein is associated with vaccine efficacy remains unclear. Here, we first optimized the expression of the S protein in mammalian cells. While we found no significant discrepancy in purity, homogeneity, or receptor binding ability among S proteins derived from 293F cells (referred to as 293F S-2P), 293S GnTI- cells (defective in N-acetylglucosaminyl transferase I enzyme; 293S S-2P), or TN-5B1-4 insect cells (Bac S-2P), there was significant variation in the glycosylation patterns and thermal stability of the proteins. Compared with the partially glycosylated 293S S-2P or Bac S-2P, the fully glycosylated 293F S-2P exhibited higher binding reactivity to convalescent sera. In addition, 293F S-2P induced higher IgG and neutralizing antibody titres than 293S or Bac S-2P in mice. Furthermore, a prime-boost-boost regimen, using a combined immunization of S-2P proteins with various degrees of glycosylation, elicited a more robust neutralizing antibody response than a single S-2P alone. Collectively, this study provides insight into ways to design a more effective SARS-CoV-2 immunogen.

Endodomain truncation of the HIV-1 envelope protein improves the packaging efficiency of pseudoviruses.

HIV-1 remains one of the most devastating infectious pathogens without available vaccines. A valid neutralization assay using multiple representative virus strains is prerequisite for antibody response analysis in HIV-1 vaccine development, where HIV pseudoviruses (PsVs) commonly serve as surrogate agents for the authentic HIV, offering a safer manipulation in Biosafety Level 2+. However, PsV production is of low efficiency and is unstable in this field. Here, we optimize PsV production conditions via the use of alternative host cells, packaging ratios and gene truncation. We show that a 153-aa truncation of the endodomain substantially enhances the packaging efficiency of HIV PsVs, providing 4 to 25 times higher infection titers than the full-length Env. Further, we obtained a robust HIV-1 PsV panel covering 12 representative global strains for neutralization assay testing. This work sheds light on how to optimize HIV PsV packaging, and provides functional insight into the cytoplasmic domain of HIV-1.

Role of Slit2 upregulation in recurrent miscarriage through regulation of stromal decidualization.

INTRODUCTION: Knockout mouse model has shown a relationship between Slit2/Robo1 signalling and altered fertility. Altered expression by endometrial epithelium and trophoblast and is associated with the pathogenesis of pregnancy complications but few studies have investigated the expression of decidual Slit2 in miscarriage. METHODS: Expression profiles of Slit2 and Robo1 were measured in human endometrial tissues during the menstrual cycle phases (n = 30), in decidua tissues from recurrent miscarriage (n = 20) and healthy control (n = 20) at 6-8 weeks of gestation. The hormonal regulation of Slit2/Robo1 expression and the role of Slit2/Robo1 signalling in decidualization was investigated in vitro, along with its effects on ß-catenin and MET expression. RESULTS: In human endometrium, Slit2 and Robo1 protein expression in stromal cells were decreased between the late-proliferative and early-secretory phase. In recurrent miscarriage patients, decidual expression Slit2 was increased and associated with lower expression of E-cadherin and higher level vimentin compared to controls. In vitro, the expression of Slit2 was downregulated by cAMP and progesterone in hESCs. Upregulation of Slit2 resulted in inhibition of cell decidualization and ß-catenin translocation to nucleus. DISCUSSION: This study indicates a functional role for Slit2 in endometrial stromal cell decidualization and the pathogenesis of recurrent miscarriage. Aberrant Increase in Slit2 expression may impairs decidualization of endometrial stromal cells leading to recurrent in recurrent miscarriage.

The Prognostic Role of Expression of Nectin-4 in Esophageal Cancer.

BACKGROUND Nectin-4 is overexpressed in several human malignant tumors. This study aimed to investigate the expression of Nectin-4 in esophageal cancer tissues compared with adjacent normal esophageal tissue and its association with clinicopathological parameters and prognosis. MATERIAL AND METHODS Nectin-4 expression in esophageal cancer tissues was compared with adjacent normal esophageal tissue from 94 patients using immunohistochemistry, Western blot, and quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The chi-squared (χ²) test and Fisher's exact test compared categorical variables. The log-rank test and Kaplan-Meier survival analysis assessed the relationship between Nectin-4 expression and overall survival (OS). Univariate and multivariate Cox proportional risk models compared Nectin-4 expression, patient prognosis, and clinicopathological parameters. RESULTS Nectin-4 expression was significantly increased in esophageal cancer tissue compared with normal tissue (P<0.001), tumor size ≥4.5 cm, and tumor invasion in T3/T4 compared with T1/T2 stage. Kaplan-Meier survival analysis showed that the OS of patients with increased Nectin-4 expression was significantly reduced compared with patients with low levels of Nectin-4 expression. Patient prognosis in men was less than women, tumor diameter ≥4.5 cm, lymph node involvement, and depth of invasion were associated with poor prognosis. Independent prognostic factors were Nectin-4 expression, lymph node involvement, and depth of invasion. CONCLUSIONS In patients with esophageal cancer, the expression levels of Nectin-4, lymph node involvement, and depth of tumor invasion were independent prognostic factors. Further studies should be performed to evaluate the diagnostic and prognostic roles of Nectin-4 and its potential role as a therapeutic target.

HIV-1 Membrane-Proximal External Region Fused to Diphtheria Toxin Domain-A Elicits 4E10-Like Antibodies in Mice.

The production of broadly neutralizing antibodies (bNAbs) is a major goal in the development of an HIV-1 vaccine. The membrane-proximal external region (MPER) of gp41, which plays a critical role in the virus membrane fusion process, is highly conserved and targeted by bNAbs 2F5, 4E10, and 10E8. As such, MPER could be a promising epitope for vaccine design. In this study, diphtheria toxin domain A (CRM197, amino acids 1-191) was used as a scaffold to display the 2F5 and 4E10 epitopes of MPER, named CRM197-A-2F5 and CRM197-A-4E10. Modest neutralizing activities were detected against HIV-1 clade B and D viruses in the sera from mice immunized with CRM197-A-4E10. Monoclonal antibodies raised from CRM197-A-4E10 could neutralize several HIV-1 strains, and epitope-mapping analysis indicated that some antibodies recognized the same amino acids as 4E10. Collectively, we show that 4E10-like antibodies can be induced by displaying MPER epitopes using an appropriate scaffold. These results provide insights for HIV-1 MPER-based immunogens design.

Characterization of native-like HIV-1 gp140 glycoprotein expressed in insect cells.

The trimeric HIV-1 envelope glycoprotein (Env) is critical for vaccine development aimed at achieving broadly-neutralizing antibody responses. The use of various recombinant expression systems and construct designs are associated with the resultant nature of produced proteins, especially in terms of glycosylation, antigenicity, and immunogenicity of the glycoprotein. Here, we explored an otherwise baculovirus cassette than classical one designed to express HIV-1 Env protein, including SOSIP mutation and Foldon moiety involvement. This improved design increased the ratio of the Env trimer fraction from ∼40% to ∼60% with respect to that of prototypical design, as indicated by high-performance size-exclusion chromatography and sedimentation velocity analysis. In addition, the design prolonged cell viability and enhanced the final yield (approximately 13-15 mg/L) after affinity purification. gp140 produced from insect cells mimicked the native-like trimer and mainly adopted glycosylation pattern of oligomannose glycans. The native-like Env proteins conferred cross-clade neutralizing antibody production in BALB/c mice. In summary, the expression of Env in insect cells by optimizing the baculovirus vector provides an alternative strategy for HIV-1 immunogen production and may benefit future Env-based HIV vaccine design.

T = 4 Icosahedral HIV-1 Capsid As an Immunogenic Vector for HIV-1 V3 Loop Epitope Display.

The HIV-1 mature capsid (CA) assumes an amorphous, fullerene conical configuration due to its high flexibility. How native CA self-assembles is still unclear despite having well-defined structures of its pentamer and hexamer building blocks. Here we explored the self-assembly of an engineered capsid protein built through artificial disulfide bonding (CA N21C/A22C) and determined the structure of one fraction of the globular particles. CA N21C/A22C was found to self-assemble into particles in relatively high ionic solutions. These particles contained disulfide-bonding hexamers as determined via non-reducing SDS-PAGE, and exhibited two major components of 57.3 S and 80.5 S in the sedimentation velocity assay. Particles had a globular morphology, approximately 40 nm in diameter, in negative-staining TEM. Through cryo-EM 3-D reconstruction, we determined a novel T = 4 icosahedral structure of CA, comprising 12 pentamers and 30 hexamers at 25 Å resolution. We engineered the HIV-1 V3 loop to the CA particles, and found the resultant particles resembled the morphology of their parental particles in TEM, had a positive reaction with V3-specific neutralizing antibodies, and conferred neutralization immunogenicity in mice. Our results shed light on HIV CA assembly and provide a particulate CA for epitope display.

[Expression and self-assembly of HIV-1 CAP2NC protein].

We constructed the CAP2NC prokaryotic expression vector of HIV-1 NL4-3 strain and obtained relatively pure CAP2NC protein by optimizing its purification conditions to explore its in vitro self-assembly conditions. Primers were designed according to the CAP2NC DNA sequence of HIV-1 NL4-3 strain. The target gene was amplified by PCR and cloned into prokaryotic expression vector pTO-T7. Then the recombinant strain was transformed into Escherichia coli BL21 (DE3). IPTG induced protein expression, then the protein was purified by hydrophobic chromatography. SDS-PAGE and Western blotting were performed to analyze the target protein, and the biological activity of the antigen was identified through ELISA. The self-assembly of CAP2NC protein was analyzed by transmission electron microscopy and gel filtration chromatography. The protein had good reaction with the specific antibodies of p24 and formed different structures in various conditions. When 10% yeast RNA was added to the protein complex, the recombinant protein only formed into a tubular structure, which was similar to the self-assembled structure of the HIV-1 virus capsid. The results showed that the HIV-1 CAP2NC protein had in vitro self-assembly activity, and the RNA affected the structure of CAP2NC protein assembly. The protein can be used as a simple and effective molecular model to study its structure, and then it can provide a reference for the study of HIV immature virus particles.

Characterization and epitope mapping of a panel of monoclonal antibodies against HIV-1 matrix protein.

The HIV-1 Gag precursor protein (p55) is the main structural protein comprising the matrix (MA/p17), capsid (CA/p24), and nucleocapsid (NC/p7) proteins, and is uniquely responsible for virion assembly within the virus life cycle. The MA protein plays a critical role in plasma membrane targeting and envelope glycoprotein (Env) uptake during virion assembly. Yet, when viral infection occurs, the MA protein may also be involved in virion uncoating, dissociating from the plasma membrane, and participating in the nuclear importation process. Thus, the MA protein contains a reversibly membrane-binding signal and varied conformation to govern its subcellular localization and biological functions. However, these purported different conformations of the MA protein during assembly are poorly understood, especially in terms of its function as a component of the precursor protein. In this study, we characterized a panel of monoclonal antibodies against MA that showed discrete reactivity to p55, an intermediate (p41), and the final p17 mature form. We suggest that these antibodies could be used to track the different conformations of MA during the HIV-1 life cycle, particularly during HIV-1 assembly and maturation, and contribute to structure determination of MA or MA precursors. These antibodies would also have clinical value, including serving for therapeutic strategy to interfere AIDS progression, reagent in diagnostic kit for the detection of virion-free p17 or p17 derived from virion lysate.

Expression, Purification and Characterization of Hiv-1 Capsid Precursor Protein p41.

Human immunodeficiency virus type 1 (HIV-1) has been a global epidemic since 1983; yet, the virology and immunology related to HIV-1 remain elusive. Furthermore, as there is still no effective chemoprophylaxis or vaccine to treat patients with HIV-1, most research focuses on strategies to prevent HIV-1 infection, such as with antiviral drugs, novel therapeutics, or improved diagnostic kits. The HIV-1 Gag precursor protein (p55)-comprising the matrix (MA/p17), capsid (CA/p24), and nucleocapsid (NC/p7) protein domains-is the main structural HIV-1 protein, and is uniquely responsible for virion assembly within the virus life cycle. Recently, the immature and mature capsid structures were solved; however, the precursor protein structure is still unknown. Here, we expressed two subtypes of HIV-1 MA-CA stretch of the Gag protein, referred to as p41, in a bacterial expression system. We characterized the purified p41 protein, and showed its superior antigenicity over that of p24, highlighting the potential influence of the p17 domain on p24 structure. We further showed that p41 has good immunogenicity to induce an antibody response in mice. These results will aid future investigations into the HIV-1 capsid precursor structure, and potentially contribute to improving the design of diagnostic kits.

Molecular insights into the inhibition of HIV-1 infection using a CD4 domain-1-specific monoclonal antibody.

An HIV-1 infection in a host cell occurs through an ordered process that involves HIV-1 attachment to the host's cellular CD4 receptor, co-receptor binding to CCR5 or CXCR4, and the subsequent fusion with the cellular membrane. The natural viral entry pathway into a host cell provides an opportunity to develop agents for the treatment of HIV-1 infections. Several engineered monoclonal antibodies specifically targeting CD4 have shown antiviral activities in clinical trials. Here, we report on an anti-CD4 mAb (15A7) that displays a unique binding specificity for domain 1 of CD4, whose epitope partially overlaps with the gp120 binding region. Moreover, 15A7 displays a much stronger binding affinity to CD4(+) cell lines after HIV infection. 15A7 is able to block and neutralize a broad range of primary HIV-1 isolates and T cell-line passage strains. Notably, the bivalent F(ab')2 form of 15A7 is more effective than the Fab form in blocking HIV-1 infection, which is further supported by molecular docking analyses. Together, these results suggest that this novel antibody may exert its antiviral activity by blocking gp120 targeting to the CD4 receptor or competing with gp120 for CD4 receptor binding and might present post-attachment neutralization activity. This antibody could provide a new candidate to efficiently block HIV-1 infection or provide new starting materials for HIV treatment, especially when HIV-1-resistant strains against the current CD4 mAb treatments have already been identified.

Repression of engrailed 2 inhibits the proliferation and invasion of human bladder cancer in vitro and in vivo.

Engrailed 2 (EN2) is a member of the homeobox gene family. Many studies suggest that overexpression of EN2 protein may be associated with tumor development, including bladder cancer (BC). However, to date, the mechanisms of how EN2 functions to promote BC progression remain elusive. The present study introduced RNAi to silence the expression of EN2 in BC cell lines. In vitro invasion and migration assays and in vivo experiments were carried out to examine the functions of EN2 in BC invasion and metastasis. The results of the present study indicated that EN2 was significantly expressed in BC cells. Ectopic expression of EN2 in normal urothelial cells significantly enhanced cellular proliferation and invasion, but inhibited cellular apoptosis. EN2 knockdown significantly promoted cell cycle arrest and apoptosis of BC cells with inhibition of proliferation and invasion in vitro as well as EN2 knockdown decreased the tumor growth of BC. The tumor growth was decreased by regulation of the cell cycle, apoptosis and epithelial-mesenchymal transition-related proteins, with inhibition of metastasis to the liver and lung in vivo. Furthermore, EN2 knockdown significantly decreased the levels of pAkt-473, pAkt-308 and phosphatidylinositol 3-kinase (PI3K), whereas EN2 knockdown increased the expression of PTEN in vitro. Taken together, EN2 may be a candidate oncogene in BC by activating the PI3K/Akt pathway and inhibiting PTEN, and may be a potential therapeutic target for the treatment of BC.

The Lin28/let-7a/c-Myc pathway plays a role in non-muscle invasive bladder cancer.

We investigate the role of the Lin28/let-7a/c-Myc pathway in non-muscle invasive bladder cancer (NMIBC). Using RT-PCR, western blot and immunohistochemistry techniques, the levels of pre-let-7a, let-7a, Lin28 and c-Myc RNA and/or proteins were determined in samples of normal bladder tissue and bladder cancer. Expression of pre-let-7a was found to be negatively correlated with the pathological grade of bladder cancer, while let-7a showed a positive correlation with bladder cancer pathological grade. Expression of Lin28 RNA and protein was not significantly different between normal bladder tissue and low-grade transitional cell carcinoma of bladder (TCC) but the expression levels in high-grade TCC were remarkably increased. Expression of c-Myc RNA and protein was significantly higher in bladder cancer samples in comparison to normal bladder tissue without correlation with cancer differentiation. Expression of all the above RNAs and proteins showed no significant difference in Ta and T1 stages. The Lin28/let-7a/c-Myc pathway plays an important role in NMIBC. In particular, expression levels of let-7a correlate with the degree of cancer differentiation but not cancer stage.

[Expression of Engrailed-2 and ß-catenin in bladder urothelial carcinoma and their significance].

OBJECTIVE: To investigate the expressions of Engrailed-2 (EN2) and ß-catenin in bladder urothelial carcinoma and explore their significance. METHODS: Sixty bladder urothelial carcinoma samples of different grades and stages and 10 normal bladder mucosal tissues were examined for expressions of EN2 and ß-catenin proteins and mRNA using immunochemistry, Western blotting and RT-PCR. RESULTS Compared to normal bladder mucosa, bladder urothelial carcinoma tissues showed significantly increased expressions of EN2 and ß-catenin proteins (P<0.05), and the high-grade carcinoma tissues exhibited significantly stronger expressions than the low-grade ones (P<0.05); the expressions of the proteins increased also significantly with advanced pathological stages of bladder urothelial carcinoma (P<0.05). The expressions of EN2 and ß-catenin mRNAs showed a consistent pattern of changes with their protein expressions. CONCLUSION: The expressions of EN2 and ß-catenin are significantly increased in bladder urothelial carcinoma. EN2 may contribute to the development and progression of bladder urothelial carcinoma by activating Wnt/ß-catenin signal pathway.

[Construction of a novel gene therapy lentiviral vector for drug resistant selection and detection in vivo].

Lentiviral vectors were powerful gene delivery tools for gene therapy. We developed a new lentiviral vector pBobi-MIL that constitutively expressed O6-methylguanine-DNAmethyltransferase (MGMT) and Luciferase, linked by the internal ribosomal entry site (IRES), to realize drug tolerance and real time monitoring in vivo. All results from RT-PCR, drug treating clones forming, immunofluorometric assay and chemiluminescence detection showed that cells infected by recombinant lentivirus L-MIL simultaneously expressed these two genes. This lays the foundation for the further research in gene therapy and can also help identify lentivirus titer.