Machine learning-derived identification of tumor-infiltrating immune cell-related signature for improving prognosis and immunotherapy responses in patients with skin cutaneous melanoma.

BACKGROUND: Immunoblockade therapy based on the PD-1 checkpoint has greatly improved the survival rate of patients with skin cutaneous melanoma (SKCM). However, existing anti-PD-1 therapeutic efficacy prediction markers often exhibit a poor situation of poor reliability in identifying potential beneficiary patients in clinical applications, and an ideal biomarker for precision medicine is urgently needed. METHODS: 10 multicenter cohorts including 4 SKCM cohorts and 6 immunotherapy cohorts were selected. Through the analysis of WGCNA, survival analysis, consensus clustering, we screened 36 prognostic genes. Then, ten machine learning algorithms were used to construct a machine learning-derived immune signature (MLDIS). Finally, the independent data sets (GSE22153, GSE54467, GSE59455, and in-house cohort) were used as the verification set, and the ROC index standard was used to evaluate the model. RESULTS: Based on computing framework, we found that patients with high MLDIS had poor overall survival and has good prediction performance in all cohorts and in-house cohort. It is worth noting that MLDIS performs better in each data set than almost all models which from 51 prognostic signatures for SKCM. Meanwhile, high MLDIS have a positive prognostic impact on patients treated with anti-PD-1 immunotherapy by driving changes in the level of infiltration of immune cells in the tumor microenvironment. Additionally, patients suffering from SKCM with high MLDIS were more sensitive to immunotherapy. CONCLUSIONS: Our study identified that MLDIS could provide new insights into the prognosis of SKCM and predict the immunotherapy response in patients with SKCM.

Expression Patterns of HOX Gene Family Defines Tumor Microenvironment and Immunotherapy in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) pathophysiology is prevalently related with HOX genes. However, the study on associations of extensive HOX genes with tumor microenvironment and drug sensitivity of HCC remains scarce. The data sets of HCC were downloaded from TCGA, ICGC, and GEO by bioinformatics method and analyzed. Based on a computational frame, HCC samples were divided into a high and a low HOXscore group, and significantly shorter survival time in the high HOXscore was observed relative to low HOXscore group using survival analysis. Gene set enrichment analysis (GSEA) revealed that the high HOXscore group was more likely to be enriched in cancer-specific pathways. Furthermore, the high HOXscore group was involved in the infiltration of inhibitory immune cells. In response to anti-cancer drugs, the high HOXscore group was more sensitive to mitomycin and cisplatin. Importantly, the HOXscore was associated with the therapeutic efficacy of PD-L1 blockade, suggesting that the development of potential drugs targeting these HOX genes to aid the clinical benefits of immunotherapy is needed. In addition, RT-qPCR and immunohistochemistry showed 10 HOX genes mRNA expression was higher in HCC compared to the normal tissues. This study provides a comprehensive analysis of HOX genes family in HCC and revealed the potential function of these HOX genes family in tumor microenvironment (TME) and identified their therapeutic liability in targeted therapy and immunotherapy. Eventually, this work highlights the cross-talk and potential clinical utility of HOX genes family in HCC therapy.

Multi­locus sequence and drug resistance analysis of Salmonella infection in children with diarrhea in Guangdong to identify the dominant ST and cause of antibiotic­resistance.

Multi-locus sequence typing (MLST) can be used to analyze the homology among the drug resistance gene cassettes in Salmonella and determine the prevalence. Information extracted using this technique can provide a theoretical basis for hospitals to devise protocols to control Salmonella infections. The aim of the present study was to investigate the possible association between drug resistance and integrons in clinical isolates of Salmonella from human fecal samples. Therefore, in the present study, 52 clinical fecal isolates of non-duplicate (i.e., not genome contamination) Salmonella were harvested from children with diarrhea and used for bacterial identification using biochemical tests, drug susceptibility analysis by antibiotic susceptibility testing and serotype identification using an agglutination assay. In total, seven Salmonella housekeeping genes (chorismate synthase, ß sliding clamp of DNA polymerase III, uroporphyrinogen-III synthase, histidinol dehydrogenase, phosphoribosylaminoimidazole carboxylase catalytic subunit, 2-oxoglutarate dehydrogenase E1 component and homoserine dehydrogenase) were amplified and sequenced using MLST, before sequence alignment was performed against the Pub MLST database to determine the sequence-typed (ST) strains and construct genotypic evolutionary diagrams. Subsequently, the 52 Salmonella strains were subdivided into 11 serotypes and 11 sequence types. The dominant subtypes were found to be Salmonella typhimurium ST34 and ST19, which were diversely distributed. However, no new subtypes were found. Although the serotypes, including ST19, ST29, ST34, ST40, ST11, ST27, ST469, ST365, ST1499, ST413 and ST588, were closely associated with the MLST subtype, they did not correspond entirely. The detection rate of class I integrons was 38.46% (20/52), but no class II and III integrons were detected. The variable regions of three of 20 class I integrons were found to be amplified, whereas nine gene cassettes, including dihydrofolate reductase A12, open reading frame F, aminoglycoside-adenylyltransferase (aad)A2, aadA22, aadA23, aadA1, cadmium-translocating P-type ATPase 2, lincosamide and linF, were associated with drug resistance. These data suggest that Class I integrons are important factors underlying drug resistance in Salmonella, which may serve a role in the spread of drug resistance and warrant specific focus. In addition, MLST typing and serotyping should be applied cooperatively in epidemiological research.

A novel 8-genome instability-associated lncRNAs signature predicting prognosis and drug sensitivity in gastric cancer.

BACKGROUND: Genome instability lncRNA (GILnc) is prevalently related with gastric cancer (GC) pathophysiology. However, the study on the relationship GILnc and prognosis and drug sensitivity of GC remains scarce. METHOD: We extracted expression data of 375 GC patients from TCGA cohort and 205 GC patients from GSE26942 cohort. Then, lncRNA was separated from expression data, and systematically characterized the 8 marker lncRNAs using the LASSO method. Next, we constructed a GILnc model (GILnc score) to quantify the GILnc index of each GC patient. Finally, we analyzed the relationship between GILnc score and clinical traits including survival outcomes, TP53, and drug sensitivity of GC. RESULTS: Based on a computational frame, 205 GILncs in GC has been identified. Then, a 8 GILncs was successfully established to predict overall survival in GC patients based on LASSO analysis, divided GC samples into high GILnc score and low GILnc score groups with significantly different outcome and was validated in multiple independent patient cohorts. Furthermore, GILnc model is better than the prediction performance of two recently published lncRNA signatures, and the high GILnc score group was more sensitive to mitomycin. Besides, the GILnc score has greater prognostic significance than TP53 mutation status alone and is capable of identifying intermediate subtype group existing with partial TP53 functionality in TP53 wild-type patients. Finally, GILnc signature as verified in GSE26942. CONCLUSION: We applied bioinformatics approaches to suggest that a 8 GILnc signature could serve as prognostic biomarkers, and provide a novel direction to explore the pathogenesis of GC.

Early Assessment of Response to Radiofrequency Ablation With CT Perfusion Imaging in Rabbit VX2 Liver Tumor Model.

OBJECTIVES: To discriminate viable tumors from benign periablational enhancement (BPE) in early stage after radiofrequency ablation (RFA) is a major confounding problem. The goal of this study is to evaluate quantitative assessment and diagnostic value of CT perfusion between viable tumors and BPE after RFA in the rabbit liver VX2 tumor model, with pathological results as the standard. METHODS: Twenty-eight VX2 liver tumors were treated with RFA, on days 1, 3, 7, and 14, seven rabbits were randomly chosen for CT perfusion and performed pathology examinations immediately. The perfusion parameters along with the profile of time-density curves (TDCs) and pseudo-color images of the parameters were observed in both BPE and viable tumors, then compared with the pathology results. The perfusion parameters included blood flow (BF), blood volume (BV), time to peak (TTP), permeability (P), arterial liver perfusion (ALP), portal venous perfusion (PVP) and hepatic perfusion index (HPI). RESULTS: A total of 26/28 rabbits successfully underwent CT perfusion, while 6/26 lesions were confirmed to be viable tumors. The TDCs of BPE were mainly speed-up platform curves (15/26), while the viable tumors showed mainly speed-up speed-down (3/6) and speed-up platform (2/6) curves. The PVP values were significantly higher, and the HPI values were significantly lower for BPE at all time points than viable tumors (P < 0.05). Both of PVP value and HPI value have high efficiency for the differential diagnosis of the viable tumors and BPE at each time point. These characteristics of CT perfusion parameters were consistent with pathological changes. CONCLUSIONS: The TDCs, PVP and HPI have the potential to indicate BPE and viable tumors effectively early after RFA treatment, the results were highly consistent with pathology. CT perfusion has advantages with great efficacy in monitoring the therapeutic effect early after RFA treatment.

Effects of percutaneous catheter intervention on pulmonary hemodynamic indexes and safety in elderly patients with acute pulmonary embolism.

OBJECTIVE: To investigate the effects of percutaneous catheter intervention (PCI) on pulmonary hemodynamic indexes and safety in elderly patients with acute pulmonary embolism (APE). METHODS: The retrospective study was performed on 65 elderly APE patients. According to the risk classification of Guidelines for Diagnosis, Treatment and Prevention of Pulmonary Thromboembolism in China, the patients were divided into the medium-high risk group (n=37) and high risk group (n=28). All patients received PCI based on thrombolysis. The clinical efficacy after treatment, and changes of pulmonary hemodynamic indexes and blood gas indexes before and after treatment were compared between the two groups, and the complications and prognosis of patients were recorded. RESULTS: There was no significant difference in the total effective rate between the two groups after treatment (P>0.05). Compared with before operation, mean pulmonary artery pressure (mPAP), ratio of right/left ventricular end diastolic diameter (RVEDD/LVEDD) and pulmonary vascular resistance (PVR) of the two groups were significantly reduced 12 h after operation, and greater changes were observed in the high risk group (all P<0.05). Compared with before operation, arterial oxygen partial pressure (PaO2), blood oxygen saturation (SaO2) and oxygenation index (OI) of the two groups were significantly increased 12 h after operation, and the changes of the high risk group were more obvious (all P<0.05). No significant difference was found in the total incidence of complications between the two groups (P>0.05). The three-month follow-up showed that there was no death in the medium-high risk group and the mortality in the high risk group was 3.08%. CONCLUSION: PCI has obvious effects in the treatment of elderly APE. It can obviously improve pulmonary blood flow dynamics and oxygenation dysfunction, especially for high-risk patients, and it has fewer postoperative complications with certain security.

Renin-angiotensin system inhibitor use and colorectal cancer risk and mortality: A dose-response meta analysis.

OBJECTIVE: This study was undertaken to determine whether use of the renin-angiotensin system (RAS) inhibitors would increase colorectal cancer morbidity and mortality. METHODS: Databases were electronically searched to collect data of RAS use and colorectal cancer morbidity and mortality from inception to October 2018. Stata 12.0 software was used to perform a meta-analysis. RESULTS: A total of 16 publications involving 2,847,597 participants were included. RAS inhibitor use was related to colorectal cancer risk (relative risk (RR): 0.86; 95% confidence interval (CI): 0.78-0.93) and mortality (RR: 0.80; 95% CI: 0.66-0.98) decrement. Subgroup analysis showed angiotensin-converting enzyme inhibitor (ACEI)/angiotensin II receptor blocker (ARB) (RR: 0.82; 95% CI: 0.69-0.96) or ARB (RR: 0.86; 95% CI: 0.73-0.98) or ACEI (RR: 0.81; 95% CI: 0.70-0.92) were related to colorectal cancer risk decrement. Furthermore, RAS inhibitor use was related to colorectal cancer risk decrement in Caucasians (RR: 0.88; 95% CI: 0.80-0.96) and Asians (RR: 0.72; 95% CI: 0.61-0.85). Additionally, dose-response showed that per one year duration of RAS inhibitor use incremental increase was related to 6% colorectal cancer risk decrement (RR: 0.94; 95% CI: 0.90-0.97). CONCLUSION: According to the evidence, RAS inhibitor use was associated with colorectal cancer risk and mortality decrement.

N-glycosylation of serum proteins for the assessment of patients with IgD multiple myeloma.

BACKGROUND: Because glycosylation is one of the most common post-translational modifications of proteins and because changes in glycosylation have been shown to have a significant correlation with the development of many cancer types, we investigated the serum N-glycome used to diagnose, stage and evaluate the pathological outcomes in IgD multiple myeloma. METHODS: Serum samples were available for 20 patients with IgD multiple myeloma, 41 patients with light chain multiple myeloma and 42 healthy control subjects. Serum N-glycans were released and analysed using DNA sequencer-assisted fluorophore-assisted capillary electrophoresis. RESULTS: Characteristic changes were revealed in the serum N-glycome of IgD myeloma. In particular, three N-glycans (NG1(6)A2F, Peak3; NG1(3)A2F, Peak4; NA2FB, Peak7) showed increased clinical value. The best area under the ROC curve of NG1(6)A2F to diagnose IgD myeloma was 0.981, with a 95.0% sensitivity and 95.2% specificity, and that of NG1(3)A2F was 0.936, with a 95.0% sensitivity and 78.6% specificity. The best area under the ROC curve of NA2FB/NG1(3)A2F to differentially diagnose IgD myeloma versus light chain myeloma was 0.744, with a 95.3% sensitivity and 50.0% specificity. The level of NG1(3)A2F was correlated with the international staging system, while the higher abundance of NA2FB presented in IgD myeloma was predictive of a shorter progression-free survival. CONCLUSIONS: The advent of serum N-glycan signatures may play a role in the diagnosis, staging and prognosis of IgD myeloma and will serve as the foundation for a precision medicine approach to this rare subtype of multiple myeloma.

Serum N-glycans outperform CA19-9 in diagnosis of extrahepatic cholangiocarcinoma.

Extensive efforts have been devoted to improve the diagnosis of extrahepatic cholangiocarcinoma (ECCA) due to its silent clinical character and lack of effective diagnostic biomarkers. Specific alterations in N-glycosylation of glycoproteins are considered a key component in cancer progression, which can serve as a distinct molecular signature for cancer detection. This study aims to find potential serum N-glycan markers for ECCA. In total, 255 serum samples from patients with ECCA (n = 106), benign bile tract disease (BBD, n = 60) and healthy controls (HC, n = 89) were recruited. Only 2 µL of serum from individual patients was used in this assay where the N-glycome of serum glycoproteins was profiled by DNA sequencer-assisted fluorophore-assisted capillary electrophoresis (DSA-FACE) technology. Multi-parameter models were constructed by combining the N-glycans and carbohydrate antigen 19-9 (CA19-9) which is currently used clinically. Quantitative analyses showed that among 13 N-glycan structures, the bifucosylated triantennary N-glycan (peak10, NA3F2) presented the best diagnostic performance for distinguishing ECCA from BBD and HC. Two diagnostic models (Glycotest1 and Glycotest2) performed better than single N-glycan or CA19-9. Additionally, two N-glycan structures (peak9, NA3Fb; peak12, NA4Fb) were tightly related to lymph node metastasis in ECCA patients. In conclusion, sera of ECCA showed relatively specific N-glycome profiling patterns. Serum N-glycan markers and models are novel, valuable and noninvasive alternatives in ECCA diagnosis and progression monitoring.

Statins intake and risk of liver cancer: A dose-response meta analysis of prospective cohort studies.

Previous studies have indicated that statins intake was associated with liver cancer risk, but presented controversial results.Studies in PubMed and EMBASE were searched update to February 2017 to identify and quantify the potential dose-response association between statins intake and liver cancer.Six eligible studies involving a total of 11,8961 participants with 9530 incident cases were included in this meta-analysis. Statistically significant association was observed between increasing statins intake and liver cancer risk reduction (OR = 0.46, 95%CI: 0.24-0.68, P <.001). Furthermore, the summary relative risk of liver cancer for an increase of 50 cumulative defined daily dose per year was 0.86 (95%CI: 0.81-0.90, P <.001). Evidence of a nonlinear dose-response relationship between statins intake and liver cancer risk was found (P for nonlinearity <.01). Subgroups analysis indicated that statins intake was associated with a significantly risk of liver cancer risk reduction in Asia (OR = 0.44, 95%CI: 0.11-0.77, P <.001) and Caucasian (OR = 0.49, 95%CI: 0.36-0.61, P <.001). Subgroup meta-analyses in study design, study quality, number of participants, and number of cases showed consistency with the primary findings.Additional statins intake is associated with liver cancer risk reduction.

The diagnostic value of serum fucosylated fetuin A in hepatitis B virus-related liver diseases.

BACKGROUND: Some changes of glycoproteins have been identified in the serum of patients with different liver diseases, which provided potential glycan biomarkers for diagnosis, prognosis and monitoring of disease progression. METHODS: We established a lectin-antibody sandwich ELISA method to detect fucosylated fetuin A (fuc-fetuin A) level in serum, in which biotinylated Aleuria aurantia lectin (AAL) was used for specific recognition. Then serum fuc-fetuin A level was detected in 108 healthy controls and 548 hepatitis B virus (HBV)-infected patients, including 232 hepatocellular carcinoma (HCC) patients, 114 liver cirrhosis (LC) patients, 86 liver fibrosis (LF) patients, and 116 asymptomatic HBV carriers, to assess its diagnostic and prognostic value in HBV-related liver diseases. RESULTS: Serum fetuin A level decreased in LC patients as compared to HCC patients or healthy controls, while it decreased further according to the increasing Child-Pugh grades. The fuc-fetuin A level was in a decreasing order in LC, HCC, LF, HBV-carriers and healthy controls. For distinguishing LC and HCC patients from LF, HBV-carriers and healthy controls, the area under the receiver operating characteristic (ROC) curve is 0.871, with a sensitivity of 0.818 and specificity of 0.819. The survival analysis revealed that higher fuc-fetuin A level was significantly associated with worse recurrence-free survival in HCC patients (p=0.018). CONCLUSIONS: The results indicated that the serum fuc-fetuin A might serve as a potential glycan biomarker for distinguishing LC and HCC from LF, HBV-carriers and healthy controls. Furthermore, the preoperative fuc-fetuin A level could be a useful prognostic biomarker for HCC patients.

Elevated core-fucosylated IgG is a new marker for hepatitis B virus-related hepatocellular carcinoma.

Immunoglubulin G (IgG) and its abnormal glycosylations are associated with carcinogenesis. The present study investigates the relationship between cancer-derived IgG and clinicopathological characteristics in hepatocellular carcinoma (HCC) and assesses the value of serum N-glycosylated IgG in diagnosing and monitoring hepatitis B virus (HBV)-related HCC. Tissue microarray analysis of 90 HCC tissues showed that HCC patients with IgG immunopositivity had higher levels of core-fucosylated α fetoprotein (AFP-L3), larger tumors, and a higher incidence of portal vein tumor thrombus. HCC-derived IgG stimulated the growth of liver cancer cells in vitro. HCC patients presented a significantly increased fraction of Lens culinaris agglutinin binding IgG (core-fucosylated IgG, IgG-L3) among total serum IgG. The clinical diagnostic performance of serum IgG-L3% was evaluated in 3 case-control studies (1 training set and 2 validation cohorts), including 293 patients with HCC, 131 with liver cirrhosis, 132 HBV carriers, and 151 healthy controls. IgG-L3% had better general diagnostic performance than AFP in the training set and validation cohort 1 (accuracy: 81.33-85.11% versus 63.33-78.61%). In validation cohort 2, where we aimed to assess the efficiency of IgG-L3% in patients with AFP-negative HCC, the diagnostic accuracy of IgG-L3% was 72.54-73.60%. Finally, a longitudinal evaluation based on 31 HCC patients demonstrated that IgG-L3% decreased in 24 patients after curative surgery. The remaining 7 patients showed elevated IgG-L3% and post-operative recurrence. HCC patients with higher IgG-L3% had poor survival during a 3-year follow up. We conclude that HCC-derived IgG is correlated with progressive behavior of HCC. Therefore, elevated core-fucosylated IgG is a new diagnostic and prognostic marker in HBV-related HCC.

Factors affecting the interactions between beta-lactoglobulin and fatty acids as revealed in molecular dynamics simulations.

Beta-lactoglobulin (BLG), a bovine dairy protein, is a promiscuously interacting protein that can bind multiple hydrophobic ligands. Fatty acids (FAs), common hydrophobic molecules bound to BLG, are important sources of fuel for life because they yield large quantities of ATP when metabolized. The binding affinity increases with the length of the ligands, indicating the importance of the van der Waals (vdW) interactions between the hydrocarbon tail and the hydrophobic calyx of BLG. An exception to this rule is caprylic acid (OCA) which is two-carbon shorter but has a stronger binding affinity than capric acid. Theoretical calculations in the current literature are not accurate enough to shed light on the underlying physics of this exception. The computed affinity values are greater for longer fatty acids without respect for the caprylic exception and those values are generally several orders of magnitude away from the experimental data. In this work, we used hybrid steered molecular dynamics to accurately compute the binding free energies between BLG and the five saturated FAs of 8 to 16 carbon atoms. The computed binding free energies agree well with experimental data not only in rank but also in absolute values. We gained insights into the exceptional behavior of caprylic acid in the computed values of entropy and electrostatic interactions. We found that the electrostatic interaction between the carboxyl group of caprylic acid and the two amino groups of K60/69 in BLG is much stronger than the vdW force between the OCA's hydrophobic tail and the BLG calyx. This pulls OCA to the top of the beta barrel where it is easier to fluctuate, giving rise to greater entropy of OCA at the binding site.

Serum N-Glycans: A New Diagnostic Biomarker for Light Chain Multiple Myeloma.

The aim of this study was to evaluate the diagnostic and differential diagnostic power of serum N-glycans for light chain multiple myeloma (LCMM). A total of 167 cases of subjects, including 42 LCMM, 42 IgG myeloma, 41 IgA myeloma, and 42 healthy controls were recruited in this study. DNA sequencer-assisted fluorophore-assisted capillary electrophoresis (DSA-FACE) was applied to determine the quantitive abundance of serum N-glycans. The core fucosylated, bisecting and sialylated modifications were analyzed in serum of LCMM patients (n=20) and healthy controls (n=20) randomly selected from the same cohort by lectin blot. Moreover, serum sialic acid (SA) level was measured by enzymatic method. We found two N-glycan structures (NG1A2F, Peak3; NA2FB, Peak7) showed the optimum diagnostic efficacy with area under the ROC curve (AUC) 0.939 and 0.940 between LCMM and healthy control. The sensitivity and specificity of Peak3 were 88.1% and 92.9%, while Peak7 were 92.9% and 97.6%, respectively. The abundance of Peak3 could differentiate LCMM from IgG myeloma with AUC 0.899, sensitivity 100% and specificity 76.2%, and Peak7 could be used to differentiate LCMM from IgA myeloma with AUC 0.922, sensitivity 92.9% and specificity 82.9%. Serum SA level was significantly higher in patients with LCMM than that in healthy controls. Moreover, the decreased core fucosylation, bisecting and increased sialylation characters of serum glycoproteins were observed in patients with LCMM. We concluded that serum N-glycan could provide a simple, reliable and non-invasive biomarker for LCMM diagnosis and abnormal glycosylation might imply a new potential therapeutic target in LCMM.

Revealing origin of decrease in potency of darunavir and amprenavir against HIV-2 relative to HIV-1 protease by molecular dynamics simulations.

Clinical inhibitors Darunavir (DRV) and Amprenavir (APV) are less effective on HIV-2 protease (PR2) than on HIV-1 protease (PR1). To identify molecular basis associated with the lower inhibition, molecular dynamics (MD) simulations and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations were performed to investigate the effectiveness of the PR1 inhibitors DRV and APV against PR1/PR2. The rank of predicted binding free energies agrees with the experimental determined one. Moreover, our results show that two inhibitors bind less strongly to PR2 than to PR1, again in agreement with the experimental findings. The decrease in binding free energies for PR2 relative to PR1 is found to arise from the reduction of the van der Waals interactions induced by the structural adjustment of the triple mutant V32I, I47V and V82I. This result is further supported by the difference between the van der Waals interactions of inhibitors with each residue in PR2 and in PR1. The results from the principle component analysis suggest that inhibitor binding tends to make the flaps of PR2 close and the one of PR1 open. We expect that this study can theoretically provide significant guidance and dynamics information for the design of potent dual inhibitors targeting PR1/PR2.

Function characterization of a glyco-engineered anti-EGFR monoclonal antibody cetuximab in vitro.

AIM: To evaluate the biochemical features and activities of a glyco-engineered form of the anti-human epidermal growth factor receptor monoclonal antibody (EGFR mAb) cetuximab in vitro. METHODS: The genes encoding the Chinese hamster bisecting glycosylation enzyme (GnTIII) and anti-human EGFR mAb were cloned and coexpressed in CHO DG44 cells. The bisecting-glycosylated recombinant EGFR mAb (bisec-EGFR mAb) produced by these cells was characterized with regard to its glycan profile, antiproliferative activity, Fc receptor binding affinity and cell lysis capability. The content of galactose-α-1,3-galactose (α-Gal) in the bisec-EGFR mAb was measured using HPAEC-PAD. RESULTS: The bisec-EGFR mAb had a higher content of bisecting N-acetylglucosamine residues. Compared to the wild type EGFR mAb, the bisec-EGFR mAb exhibited 3-fold higher cell lysis capability in the antibody-dependent cellular cytotoxicity assay, and 1.36-fold higher antiproliferative activity against the human epidermoid carcinoma line A431. Furthermore, the bisec-EGFR mAb had a higher binding affinity for human FcγRIa and FcγRIIIa-158F than the wild type EGFR mAb. Moreover, α-Gal, which was responsible for cetuximab-induced hypersensitivity reactions, was not detected in the bisec-EGFR mAb. CONCLUSION: The glyco-engineered EGFR mAb with more bisecting modifications and lower α-Gal content than the approved therapeutic antibody Erbitux shows improved functionality in vitro, and requires in vivo validations.

Decreased core-fucosylation contributes to malignancy in gastric cancer.

The object of the study is to identify N-glycan profiling changes associated with gastric cancer and explore the impact of core-fucosylation on biological behaviors of human gastric cancer cells. A total of 244 subjects including gastric cancer, gastric ulcer and healthy control were recruited. N-glycan profiling from serum and total proteins in gastric tissues was analyzed by DNA sequencer-assisted fluorophore-assisted capillary electrophoresis. The abundance of total core-fucosylated residues and the expression of enzymes involved in core-fucosylation were analyzed with lectin blot, quantitative reverse transcription-polymerase chain reaction, western blot, Immunohistochemical staining and lectin-histochemical staining. The recombinant plasmids of GDP-fucose transporter and α-1,6-fucosyltransferase (Fut8) were constructed and transfected into gastric cancer cell lines BGC-823 and SGC-7901. CCK-8 and wound healing assay were used to assess the functional impact of core-fucosylation modulation on cell proliferation and migration. Characteristic serum N-glycan profiles were found in gastric cancer. Compared with the healthy control, a trianntenary structure abundance, peak 9 (NA3Fb), was increased significantly in gastric cancer, while the total abundance of core-fucosylated residues (sumfuc) was decreased. Core-fucosylated structures, peak6(NA2F) and peak7(NA2FB) were deceased in gastric tumor tissues when compared with that in adjacent non-tumor tissues. Consistently, lens culinaris agglutinin (LCA)-binding proteins were decreased significantly in sera of gastric cancer, and protein level of Fut8 was decreased significantly in gastric tumor tissues compared with that in adjacent non-tumor tissues. Upregulation of GDP-Tr and Fut8 could inhibit proliferation, but had no significant influence on migration of BGC-823 and SGC-7901 cells. Core-fucosylation is down regulated in gastric cancer. Upregulation of core-fucosylation could inhibit proliferation of the human gastric cancer cells.

Expression of alpha 1,6-fucosyltransferase 8 in hepatitis B virus-related hepatocellular carcinoma influences tumour progression.

BACKGROUND: Alpha 1,6-fucosyltransferase 8 expression was demonstrated to be enhanced during hepatocarcinogenesis. AIMS: Our study aimed to find out the clinical significance and biological function of alpha 1,6-fucosyltransferase 8 in hepatitis B virus-related hepatocellular carcinoma. METHODS: Alpha 1,6-fucosyltransferase 8 expression levels were determined in 52 pairs of tissues to compare its expression between tumour tissues [with/without portal vein tumour thrombus] and adjacent noncancerous liver tissues. Relationship between alpha 1,6-fucosyltransferase 8 expression and clinical indicators was also investigated. An alpha 1,6-fucosyltransferase 8-knockdown (by RNAi) cell line MHCC97-H/siFUT8 was established to reveal functional impact of alpha 1,6-fucosyltransferase 8 on cell growth, migration and invasion in hepatocellular carcinoma, respectively using Cell Counting Kit-8, wound healing migration assay, transwell assay and gelatin zymography. RESULTS: We observed a higher alpha 1,6-fucosyltransferase 8 expression level in tumour tissues than adjacent noncancerous liver tissues. In portal vein tumour thrombus group, alpha 1,6-fucosyltransferase 8 protein expressed more in portal vein tumour thrombus tissues than that in adjacent noncancerous liver tissues. The expression level in tumour tissues was highly correlated with tumour size and presence of satellite nodules (P<0.05). Furthermore, alpha 1,6-fucosyltransferase 8-knockdown suppressed the tumour proliferation, migration and invasion of MHCC97-H cells. CONCLUSION: These findings suggest that alpha 1,6-fucosyltransferase 8 expression might be a good indicator of poor prognosis in hepatocellular carcinoma. High alpha 1,6-fucosyltransferase 8 expression may play an important role in hepatitis B virus-related hepatocellular carcinoma progression.

Transforming growth factor beta1 gene variation Leu10Pro affects secretion and function in hepatic cells.

BACKGROUND: Our previous work revealed transforming growth factor beta1 (TGFß1) gene polymorphisms are associated with susceptibility to hepatocellular carcinoma and liver cirrhosis. However, no further study of functional substitution in hepatic cells has yet been reported. AIMS: This study was designed to uncover the functional mechanisms of TGFß1 gene polymorphisms in the pathogenesis of liver diseases. METHODS: Two recombinant TGFß1 expression plasmids containing TGFß1 codon 10 Leu/Pro variation were constructed with CMV promoter and transfected into human hepatoma cell lines (HepG2 and SMMU 7721), hepatic stellate cells (LX-2), and immortalized hepatocytes (L02). The secretion capacities of TGFß1 protein in the transfected cells were determined by ELISA. Apoptosis, proliferative activity, and expression of CD 105, CD83, and CD80 were also measured by use of flow cytometry. RESULTS: The ELISA results showed that cells transfected with CMV-Pro10 were more capable of TGFß1 secretion than those transfected with CMV-Leu10. Functionally, CMV-Pro10 was more apoptosis-protective and induced more proliferation than CMV-Leu10 in transfected hepatic cells. Pro10 up-regulated expression of CD105 and down-regulated expression of CD83. CONCLUSIONS: TGFß1 gene Leu10Pro variation in signal peptide has significant effects on TGFß1 secretion and functions in hepatic cells.

Insights into the functional role of protonation states in the HIV-1 protease-BEA369 complex: molecular dynamics simulations and free energy calculations.

The molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method combined with molecular dynamics (MD) simulations were used to investigate the functional role of protonation in human immunodeficiency virus type 1 (HIV-1) protease complexed with the inhibitor BEA369. Our results demonstrate that protonation of two aspartic acids (Asp25/Asp25') has a strong influence on the dynamics behavior of the complex, the binding free energy of BEA369, and inhibitor-residue interactions. Relative binding free energies calculated using the MM-PBSA method show that protonation of Asp25 results in the strongest binding of BEA369 to HIV-1 protease. Inhibitor-residue interactions computed by the theory of free energy decomposition also indicate that protonation of Asp25 has the most favorable effect on binding of BEA369. In addition, hydrogen-bond analysis based on the trajectories of the MD simulations shows that protonation of Asp25 strongly influences the water-mediated link of a conserved water molecule, Wat301. We expect that the results of this study will contribute significantly to binding calculations for BEA369, and to the design of high affinity inhibitors.