Early determination of potential critical quality attributes of therapeutic antibodies in developability studies through surface plasmon resonance-based relative binding activity assessment.

Precise measurement of the binding activity changes of therapeutic antibodies is important to determine the potential critical quality attributes (CQAs) in developability assessment at the early stage of antibody development. Here, we report a surface plasmon resonance (SPR)-based relative binding activity method, which incorporates both binding affinity and binding response and allows us to determine relative binding activity of antibodies with high accuracy and precision. We applied the SPR-based relative binding activity method in multiple forced degradation studies of antibody developability assessment. The current developability assessment strategy provided comprehensive, precise characterization of antibody binding activity in the stability studies, enabling us to perform correlation analysis and establish the structure-function relationship between relative binding activity and quality attributes. The impact of a given quality attribute on binding activity could be confidently determined without isolating antibody variants. We identified several potential CQAs, including Asp isomerization, Asn deamidation, and fragmentation. Some potential CQAs affected binding affinity of antibody and resulted in a reduction of binding activity. Certain potential CQAs impaired antibody binding to antigen and led to a loss of binding activity. A few potential CQAs could influence both binding affinity and binding response and cause a substantial decrease in antibody binding activity. Specifically, we identified low abundance Asn33 deamidation in the light chain complementarity-determining region as a potential CQA, in which all the stressed antibody samples showed Asn33 deamidation abundances ranging from 4.2% to 27.5% and a mild binding affinity change from 1.76 nM to 2.16 nM.

Application of individualized nutrition support for adults with Stanford A aortic dissection. / ä¸ªä½“åŒ–è¥å »æ”¯æŒåœ¨æˆäººStanford A型主动脉夹层中的应用.

OBJECTIVES: Aortic dissection is a serious aortic pathological changes. Although the surgical technique for aortic dissection continues to improve, postoperative mortality and surgical complications are still high. This study aims to explore the effect of individualized nutritional support for the adult Stanford A aortic dissection. METHODS: A total of 60 patients with Stanford A aortic dissection, who were treated in the Department of Cardiovascular Surgery at the First Affiliated Hospital of University of Science and Technology of China from January 2019 to February 2020, were selected. The subjects were divided into a control group (n=29) and an observation group (n=31) by random number table method. The control group received routine nutritional support, and the observation group received individualized nutritional support since the 1st day after surgery. The levels of serum nutritional indexes [albumin (Alb), prealbumin (PAB), hemoglobin (Hb), transferrin (TF)], immune function indexes [immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA)], and inflammatory factors [tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), procalcitonin (PCT)] on the first day, the 5th day, and the 10th day after surgery were detected by Roche biochemical analyzer, immunoassay analyzer, and microplate reader, respectively. The acute physiology and chronic health evaluation II (APACHEII) was used to evaluate the prognosis on the first day, the 5th day, and the 10th day after surgery, and the incidence of complications was observed in the 2 groups. RESULTS: There were significant differences between the 2 groups in Alb, PAB, Hb, TF, IgG, IgM, IgA, TNF-α, IL-10, and PCT (all P<0.01), and there were also significant differences in the APACHEII, the time factor and the group factor (all P<0.01). On the 5th day and the 10th day after surgery, the levels of Alb, PAB, Hb, TF, IgG, IgM and IgA were higher, and the levels of TNF-α and IL-10 while the scores of PCT and APACHEII were lower in the observation group than those in the control group (all P<0.01). The incidence of complications in the observation group was significantly lower than that in the control group (P<0.05). CONCLUSIONS: For patients with Stanford A aortic dissection undergoing surgery, the postoperative individualized nutrition support can not only significantly improve nutritional level and immune function, but also effectively reduce levels of postoperative inflammatory factors, which is beneficial to their rapid recovery and has positive clinical significance for reducing postoperative complications and improving prognosis.

An array of 60,000 antibodies for proteome-scale antibody generation and target discovery.

Antibodies are essential for elucidating gene function. However, affordable technology for proteome-scale antibody generation does not exist. To address this, we developed Proteome Epitope Tag Antibody Library (PETAL) and its array. PETAL consists of 62,208 monoclonal antibodies (mAbs) against 15,199 peptides from diverse proteomes. PETAL harbors binders for a great multitude of proteins in nature due to antibody multispecificity, an intrinsic antibody feature. Distinctive combinations of 10,000 to 20,000 mAbs were found to target specific proteomes by array screening. Phenotype-specific mAb-protein pairs were found for maize and zebrafish samples. Immunofluorescence and flow cytometry mAbs for membrane proteins and chromatin immunoprecipitation-sequencing mAbs for transcription factors were identified from respective proteome-binding PETAL mAbs. Differential screening of cell surface proteomes of tumor and normal tissues identified internalizing tumor antigens for antibody-drug conjugates. By finding high-affinity mAbs at a fraction of current time and cost, PETAL enables proteome-scale antibody generation and target discovery.

HIC1 modulates uveal melanoma progression by activating lncRNA-numb.

Uveal melanoma (UM) is the most common primary intraocular cancer in adults. Although the diagnosis modality of primary UM was improved significantly, there are currently no effective therapies for metastatic UM. Hypermethylated in cancer 1 (HIC1) is frequently deleted or epigenetically silenced in various human cancers. However, the role and mechanism of HIC1 in UM is still unclear. In this study, we found that HIC1 acted as a tumor suppressor and that its expression was downregulated in UM. Functional studies demonstrated that ectopic expression of HIC1 in UM cells inhibited cell proliferation and invasion. Moreover, through long non-coding RNA (lncRNA) microarray and real-time PCR, we found that expression of lncRNA-numb was activated by HIC1 in UM. The results provide evidence that lncRNA-numb is a newly proposed tumor suppressor that is involved in HIC1-induced phenotypes. Taken together, our studies of UM reveal a critical role of HIC1 in the regulation of tumorigenesis, at least partly through its downstream target, lncRNA-numb, and provide a potential therapeutic target for UM.

Higher levels of TIMP-1 expression are associated with a poor prognosis in triple-negative breast cancer.

BACKGROUND: Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a multifunctional protein that can directly regulate apoptosis and metastasis. In this study, we investigated the functional and molecular mechanisms by which TIMP-1 influences triple-negative breast cancer (TNBC). METHODS: The expression level of TIMP-1 in breast cancer tissues was analyzed using the ONCOMINE microarray database. The overall survival of patients with distinct molecular subtypes of breast cancer stratified by TIMP-1 expression levels was evaluated using Kaplan-Meier analysis. Bisulfate sequencing PCR (BSP) was used to analyze the methylation status of the TIMP-1 promoter. Real-time-PCR (RT-PCR), Western blot and ELISA assays were used to evaluate gene and protein expression in cell lines and human tissue specimens. In addition, TIMP-1 function was analyzed using a series of in vitro and in vivo assays with cells in which TIMP-1 was inhibited using RNAi or neutralizing antibodies. RESULTS: We found that serum TIMP-1 levels were strongly enhanced in patients with TNBC and that elevated TIMP-1 levels were associated with a poor prognosis in TNBC. However, TIMP-1 levels were not significantly associated with overall survival in other subtypes of breast cancer or in the overall population of breast cancer patients. We also report the first evidence that the TIMP-1 promoter is hypomethylated in TNBC cell lines compared with non-TNBC cell lines, suggesting that aberrant TIMP-1 expression in TNBC results from reduced DNA methylation. RNAi-mediated silencing of TIMP-1 in TNBC cells induced cell cycle arrest at the G1 phase and reduced cyclin D1 expression. In addition, mechanistic analyses revealed that the p-Akt and p-NF-κB signaling pathways, but not the GSK-3ß and MAPK1/2 pathways, are associated with TIMP-1 overexpression in TNBC cells. Moreover, neutralizing antibodies against TIMP-1 significantly decreased the rate of tumor growth in vivo. CONCLUSIONS: Our findings suggest that TIMP-1 is a biomarker indicative of a poor prognosis in TNBC patients and that targeting TIMP-1 may provide an attractive therapeutic intervention specifically for triple-negative breast cancer patients.

CXCL16/CXCR6 chemokine signaling mediates breast cancer progression by pERK1/2-dependent mechanisms.

Our previous studies demonstrate that CXCL6/CXCR6 chemokine axis induces prostate cancer progression by the AKT/mTOR signaling pathway; however, its role and mechanisms underlying invasiveness and metastasis of breast cancer are yet to be elucidated. In this investigation, CXCR6 protein expression was examined using high-density tissue microarrays and immunohistochemistry. Expression of CXCR6 shows a higher epithelial staining in breast cancer nest site and metastatic lymph node than the normal breast tissue, suggesting that CXCR6 may be involved in breast cancer (BC) development. In vitro and in vivo experiments indicate that overexpression of CXCR6 in BC cells has a marked effect on increasing cell migration, invasion and metastasis. In contrast, reduction of CXCR6 expression by shRNAs in these cells greatly reduce its invasion and metastasis ability. Mechanistic analyses show that CXCL16/CXCR6 chemokine axis is capable of modulating activation of RhoA through activating ERK1/2 signaling pathway, which then inhibits the activity of cofilin, thereby enhancing the stability of F-actin, responsible for invasiveness and metastasis of BC. Taken together, our data shows for the first time that the CXCR6 / ERK1/2/ RhoA / cofilin /F-actin pathway plays a central role in the development of BC. Targeting the signaling pathway may prove beneficial to prevent metastasis and provide a more effective therapeutic strategy for BC.

USP2 promotes cell migration and invasion in triple negative breast cancer cell lines.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is often associated with a poor prognosis. The aim of our study was to identify biomarkers predictive of TNBC progression. Primary TNBC breast tissue samples including four with metastasis and six without metastasis were subjected to Affymetrix GeneChip® analysis (human genome U133). Ubiquitin-specific protease 2 (USP2) was identified as an upregulated gene in the metastatic group, and its expression was analyzed by immunohistochemistry in 121 primary breast cancers, 13 paired normal tissues, and 13 paired metastatic lesions. Survival analysis was performed using the log-rank test and Cox regression hazard model. Matrigel migration and invasion assays in USP2-silenced and USP2-overexpressed breast cancer cell lines were used to investigate the mechanisms of USP2 in vitro. Positive immunostaining for USP2 was detected in breast tumors and was correlated with estrogen receptor (ER) and progesterone receptor (PR) statuses and TNBC subtype. USP2 was overexpressed in distant metastatic lesions compared with primary breast cancers. Survival analyses demonstrated that positive USP2 is a poor prognostic factor for disease-free survival. Silencing of USP2 expression decreased migration and invasion in LM2-4175 and SCP46 cells in association with the downregulation of matrix metalloproteinase-2 (MMP2) expression, whereas overexpression of USP2 in MDA-MB-468 and MDA-MB-231 cells enhanced migration and invasion and upregulated the expression of MMP2. The present study showed that USP2 expression is associated with TNBC cell line's invasiveness and poor survival of breast cancer patients and may serve as a prognostic biomarker and therapeutic target for TNBC.

[Clinical analysis of total aortic arch reconstruction with a novel individualized combined branched stent grafting technique for patients with Stanford A aortic dissection].

OBJECTIVE: To compare the clinical efficacy between total aortic arch reconstruction with a individualized combined branched stent grafting technique and total aortic arch replacement combined with stented elephant trunk implantation for patients with Stanford A aortic dissection. METHODS: Totally 44 patients with Stanford A aortic dissection treated with surgical treatment from January 2007 to July 2013 were included in this study. The patients were divided into two groups. Group A (n = 22) patients were treated by total arch replacement with stented elephant trunk procedure. Group B (n = 22) patients received individualized combined branched stent grafting technique. Age, gender and disease severity were similar between the two groups (all P > 0.05). Echocardiography and aortic CT angiography were performed pre-operation and at 1 month after operation. RESULTS: Operation was successful in all 44 patients. Cardiopulmonary bypass time, aortic cross clamp time, circulation arrest time and duration of ventilator assisted breathing were significantly longer, postoperative drainage volume and blood transfusion volume were significantly larger and hospitalization cost was significantly higher in group A patients compared those in group B patients (t = 2.791 to 43.465, all P < 0.05). One month after operation, the maximum internal diameter of aorta was smaller than pre-operation in both group A ((33 ± 1) mm vs. (45 ± 6) mm, t = 10.076, P = 0.000) and group B ((33 ± 2) mm vs. (45 ± 8) mm, t = 5.979, P = 0.000) . Left ventricular ejection fraction had no significant difference before and 1 month after operation in both groups (P > 0.05). CONCLUSION: The total aortic arch reconstruction with individualized combined branched stent grafting technique is technically easier, shortens the operation time, reduces the blood transfusion volume compared to the classical aortic arch operation.

HIC1 silencing in triple-negative breast cancer drives progression through misregulation of LCN2.

The tumor suppressor gene HIC1 is frequently deleted or epigenetically silenced in human cancer, where its restoration may improve cancer prognosis. Here, we report results illuminating how HIC1 silencing alters effect or signals in triple-negative breast cancer (TNBC), which are crucial for its pathogenesis. HIC1 expression was silenced only in TNBC compared with other molecular subtypes of breast cancer. Restoring HIC1 expression in TNBC cells reduced cell migration, invasion, and metastasis, whereas RNAi-mediated silencing of HIC1 in untransformed human breast cells increased their invasive capabilities. Mechanistic investigations identified the small-secreted protein lipocalin-2 (LCN2), as a critical downstream target of HIC1 in TNBC cells. Elevating LCN2 expression in cells expressing HIC1 partially rescued its suppression of cell invasion and metastasis. Notably, autocrine secretion of LCN2 induced by loss of HIC1 activated the AKT pathway through the neutrophil gelatinase-associated lipocalin receptor, which is associated with TNBC progression. Taken together, our findings revealed that the HIC1-LCN2 axis may serve as a subtype-specific prognostic biomarker, providing an appealing candidate target for TNBC therapy.

A combined branched stent grafting technique to reconstruct total aortic arch in the treatment of stanford A aortic dissection.

Traditional total arch replacement technology and artificial blood vessels are designed for patients with Stanford A aortic dissection who have 3 branches (brachiocephalic trunk, left common carotid artery, and left subclavian artery) arising from the arch of the aorta. However, if there is anatomical variation of the aortic arch branches, the operation will be very difficult. The number of primary branches of the aortic arch can be reduced to 1 or 2 or increased to 4 to 6. Also, anastomoses of the graft to the left subclavian artery and descending aorta are usually very difficult because of the deep surgical field. Moreover, once bleeding occurs after the anastomoses, hemostasis in the deep field is difficult. Therefore, we applied a "combined branched" stent grafting technique for total arch reconstruction to reduce such problems.

HIC1 modulates prostate cancer progression by epigenetic modification.

PURPOSE: Prostate cancer is the second leading cause of cancer deaths among men in Western counties, which has also occurred in Chinese male with markedly increasing incidence in recent years. Although the mechanism underlying its progression still remains unclear, epigenetic modifications are important ethological parameters. The purpose of this study is to determine the methylation status and function of hypermethylatioted in cancer 1 (HIC1) in prostate cancer progression. EXPERIMENTAL DESIGN: The methylation status of HIC1 promoter was assayed in cell lines, tissues, and plasma of patients with prostate cancer by using methylation-specific PCR and bisulfate sequencing PCR. The ability of HIC1 to regulate proliferation, migration, and invasion was assessed by MTT, scratch-healing assay, and reconstituted extracellular matrices in porous culture chambers. Tumorigenesis, metastases, and bone destruction were analyzed in mice bearing prostate cancer cells restoring HIC1 by using Xenogen IVIS with radiographic system and small-animal positron emission tomography computed tomographic images. Microarrays were searched for genes that had correlated expression with HIC1 mRNA. Reporter gene assays were used to determine whether HIC1 affected the expression of CXCR7, and chromatin immunoprecipitation was used to determine whether HIC1 bound to CXCR7 promoters. All P values were determined using 2-sided tests. RESULTS: The methylation status of 11 CpG sites within HIC1 promoter was abundantly methylated in cell lines, tissues, and plasma of patients with prostate cancer compared with those of respective normal controls. Restoring HIC1 expression in prostate cancer cells markedly inhibited proliferation, migration, and invasion and induced the apoptosis in these cells. Moreover, mice bearing prostate cancer-restoring HIC1 cells had a marked effect on reducing tumor growth, multiple tissue metastases, and bone destruction. Notably, we also identified that the chemokine receptor CXCR7 is a direct downstream target gene of HIC1. Finally, we showed that CXCR7 promoter in prostate cancer cells is negatively regulated by HIC1, which may be responsible for prostate cancer progression. CONCLUSIONS: Our data show for the first time that hypermethylation of HIC1 promoter results in loss of its repressive function, responsible for prostate cancer progression and invasion. These findings suggest that therapies targeting epigenetic events regulating HIC1 expression may provide a more effective strategy for prostate cancer treatment.

[Efficacy comparison between total aortic arch reconstruction with open placement of triple-branched stent graft and total aortic replacement combined with stented elephant trunk implantation for patients with Stanford A aortic dissection].

OBJECTIVE: To compare the clinical efficacy between total aortic arch reconstruction with open triple-branched stent graft placement and total aortic replacement combined with stented elephant trunk implantation for patients with Stanford A aortic dissection. METHODS: Patients with Stanford A aortic dissection treated with surgical treatment from January 2006 to May 2011 were included in this study. The patients were divided into two groups. Group I (n = 20) patients were treated by total arch replacement with stented elephant trunk procedure. Group II (n = 8) patients received open triple-branched stent graft placement. Echocardiography and aortic CT angiography were performed before and at 1 month after operation. RESULTS: Age, gender and disease severity were similar between the 2 groups (all P > 0.05). Operation was successful in all 28 patients. Cardiopulmonary bypass time, aortic cross clamp time, circulation arrest time and duration of ventilator assisted breathing were significantly longer; postoperative drainage volume and blood transfusion volume were significantly larger and hospitalization cost was significantly higher in group I patients compared those in group II patients (all P < 0.05). One month after operation, the maximum internal diameter of aorta was smaller than pre-operation in both group I [(30.2 ± 3.1) mm vs. (42.5 ± 6.5) mm, P < 0.05] and group II [(31.5 ± 2.5) mm vs. (44.1 ± 7.3) mm, P < 0.05]. CONCLUSIONS: Short-term procedural success rate was similar between the two groups. The total aortic arch reconstruction with open triple-branched stent graft placement procedure is simpler, shortens the operation time, reduces the blood transfusion volume and is more cost-effective compared to the classical aortic arch operation.

Early and midterm results of repair of Ebstein's anomaly with autologous pericardium.

BACKGROUND: The surgical management of Ebstein's anomaly represents a major challenge. METHODS: Thirty consecutive patients with Ebstein's anomaly received surgical treatment in the period from April 2002 to October 2009. Operations included annuloplasty, repair of the tricuspid valve using autologous pericardium and tricuspid valve replacement. In most cases, repair of the tricuspid valve was done using autologous pericardium instead of an annuloplasty with the anterior leaflet alone. Associated congenital malformations were also repaired during the operation. Follow-up ranged from 6 to 82 months. RESULTS: Intraoperative transesophageal echocardiography revealed no or only minimal tricuspid incompetence after cardiopulmonary bypass in 25 patients. Mild or moderate incompetence was observed in 3 and 2 patients, respectively. At the last follow-up echocardiography, 5 patients with mild and 2 patients with moderate tricuspid incompetence were detected. There was no sign of pericardial degeneration, tricuspid valve stenosis, or calcification of the pericardial patch in any patient during follow-up. CONCLUSIONS: Although further study is required to assess the long-term function of the reconstructed tricuspid valve, our early and midterm results from this current study indicate that repairing the tricuspid valve with autologous pericardium achieves reasonable outcomes in the majority of patients with Ebstein's anomaly, including pediatric and adult patients.

In-vitro seeding of human umbilical cord vein endothelial cells on hydroxyapatite for mechanical heart valve applications.

BACKGROUND AND AIM OF THE STUDY: Although heart valve replacement with either a mechanical or biological prosthesis is an effective method to treat valvular heart disease, both approaches have limitations, including thrombus formation, thromboembolism and degeneration problems. The study aim was to demonstrate the in-vitro endothelialization of hydroxyapatite (HAp) to be used as a biomaterial in heart valve prostheses. METHODS: The HAp samples were characterized using X-ray diffractometry to identify the crystalline phase, while the surface morphology of HAp discs was examined using scanning electron microscopy (SEM). Human umbilical vein endothelial cells (HUVECs) were cultured on HAp discs for 1, 3, 5, and 7 days, and on pyrolytic carbon discs for 7 days; cytotoxicity was assessed using the methyl thiazolyl tetrazolium (MTT) assay. The cells were incubated in three groups: (i) an experimental group (cultured with HAp extract); (ii) a negative control (cultured with high-density polyethylene chaff); and (iii) a positive control (culture medium containing 0.1% phenol solution). RESULTS: A morphological examination of the HAp discs revealed the presence of micropores on the disc surface, together with cultured HUVECs. After seven days of culture, the HUVECs began to form a confluent endothelial cell layer covering the HAp discs. There were no visible cells attached to the pyrolytic carbon surface. The MTT assay indicated that HAp did not exert any cytotoxic effect on HUVECs, and low optical density values were obtained in the positive controls. CONCLUSION: The study results showed that HUVECs were able to grow well on HAp discs, and that HAP possessed a good in-vitro bioactivity and biocompatibility towards these cells. Consequently, HAp might be used as a film on mechanical heart valve prostheses, and serve as a promising biomaterial for heart valve replacement.

CXCL12 / CXCR4 / CXCR7 chemokine axis and cancer progression.

Chemokines, small pro-inflammatory chemoattractant cytokines that bind to specific G-protein-coupled seven-span transmembrane receptors, are major regulators of cell trafficking and adhesion. The chemokine CXCL12 (also called stromal-derived factor-1) is an important α-chemokine that binds primarily to its cognate receptor CXCR4 and thus regulates the trafficking of normal and malignant cells. For many years, it was believed that CXCR4 was the only receptor for CXCL12. Yet, recent work has demonstrated that CXCL12 also binds to another seven-transmembrane span receptor called CXCR7. Our group and others have established critical roles for CXCR4 and CXCR7 on mediating tumor metastasis in several types of cancers, in addition to their contributions as biomarkers of tumor behavior as well as potential therapeutic targets. Here, we review the current concepts regarding the role of CXCL12 / CXCR4 / CXCR7 axis activation, which regulates the pattern of tumor growth and metastatic spread to organs expressing high levels of CXCL12 to develop secondary tumors. We also summarize recent therapeutic approaches to target these receptors and/or their ligands.