Imaged capillary isoelectric focusing tandem high-resolution mass spectrometry using nano electrospray ionization (ESI) for protein heterogeneity characterization.

Recombinant monoclonal antibodies (mAbs) have been spurring the rapid growth of commercial biotherapeutics. During production their charge heterogeneity must be assessed as a critical quality attribute to ensure safety, efficacy, and potency. Although imaged capillary isoelectric focusing (icIEF) is a powerful tool for this process, it could be improved further with tandem high-resolution mass spectrometry (HRMS). In this work, a nano-electrospray ionization (nano-ESI) apparatus was constructed to directly couple icIEF to HRMS. The system was evaluated with the standard NISTmAb, as well as more complex mAb, bi-specific antibody, and fusion protein samples. NISTmAb concentrations as low as 0.25 mg/ml demonstrated excellent sensitivity. There were good repeatabilities at 1 mg/ml with 7.58% and 8.01% RSDs for intention time and MS intensity, respectively, and the HRMS signal showed a strong linearity (R = 0.9983) across different concentrations. Meanwhile, the fingerprinting of the complex samples illustrated the versatility and potential of icIEF-HRMS. icIEF-HRMS developed can provide a comprehensive understanding of the underlying structural modifications that impact protein charge heterogeneity. Compared to the traditional ESI, nano-ESI can significantly improve sensitivity while maintaining a reasonable repeatability and throughput. Furthermore, the interface is much easier to connect, and is compatible with many commercial HRMS instruments.

Study of oral microorganisms contributing to non-carious cervical lesions via bacterial interaction and pH regulation.

There is a lack of evidence about the relationship between microorganisms and non-carious cervical lesions (NCCLs) due to limited technologies. A group of 78 patients was enrolled for microbial 16S rRNA sequencing of dental plaques on normal and defective cervical surfaces. Parallel data from 39 patients were analysed with paired t tests, and Fusobacteriales exhibited significantly less distribution on NCCLs than on normal surfaces. As a result, Fusobacterium nucleatum, the most common oral bacterial strain belonging to the order Fusobacteriales, was selected for further research. From a scanning electron microscopy (SEM) scan, the tooth surface with Fusobacterium nucleatum and Streptococcus mutans culture was more intact than that without Fusobacterium nucleatum. Furthermore, the calcium contents in groups with Fusobacterium nucleatum were significantly higher than that without it. In further mechanistic research, Fusobacterium nucleatum was demonstrated to adhere to and disturb other organisms as well as producing alkaline secretions to neutralize the deleterious acidic environment, protecting the tooth structure. In conclusion, microorganisms and NCCLs were confirmed directly related through adherent bacterial interactions and pH regulation. The research provides a new perspective and experimental evidence for the relation between microorganisms and NCCLs, which guides clinical treatment and preventive dentistry in the future.

Establishment of urban waterlogging pre-warning system based on coupling RBF-NARX neural networks.

The optimal layout of low-impact development (LID) facilities satisfying annual runoff control for low rainfall expectation is not effective under extreme rainfall conditions and urban waterlogging may occur. In order to avoid the losses of urban waterlogging, it is particularly significant to establish a waterlogging early warning system. In this study, based on coupling RBF-NARX neural networks, we establish an early warning system that can predict the whole rainfall process according to the rainfall curve of the first 20 minutes. Using the predicted rainfall process curve as rainfall input to the rainfall-runoff calculation engine, the area at risk of waterlogging can be located. The results indicate that the coupled neural networks perform well in the prediction of the hypothetical verification rainfall process. Under the studied extreme rainfall conditions, the location of 25 flooding areas and flooding duration are well predicted by the early warning system. The maximum of average flooding depth and flooding duration is 16.5 cm and 99 minutes, respectively. By predicting the risk area and the corresponding flooding time, the early warning system is quite effective in avoiding and reducing the losses from waterlogging.

The oncogenic effects of HES1 on salivary adenoid cystic carcinoma cell growth and metastasis.

BACKGROUND: Our previous study demonstrated a close relationship between NOTCH signaling pathway and salivary adenoid cystic carcinoma (SACC). HES1 is a well-known target gene of NOTCH signaling pathway. The purpose of the present study was to further explore the molecular mechanism of HES1 in SACC. METHODS: Comparative transcriptome analyses by RNA-Sequencing (RNA-Seq) were employed to reveal NOTCH1 downstream gene in SACC cells. Immunohistochemical staining was used to detect the expression of HES1 in clinical samples. After HES1-siRNA transfected into SACC LM cells, the cell proliferation and cell apoptosis were tested by suitable methods; animal model was established to detect the change of growth ability of tumor. Transwell and wound healing assays were used to evaluate cell metastasis and invasion. RESULTS: We found that HES1 was strongly linked to NOTCH signaling pathway in SACC cells. The immunohistochemical results implied the high expression of HES1 in cancerous tissues. The growth of SACC LM cells transfected with HES1-siRNAs was significantly suppressed in vitro and tumorigenicity in vivo by inducing cell apoptosis. After HES1 expression was silenced, the SACC LM cell metastasis and invasion ability was suppressed. CONCLUSIONS: The results of this study demonstrate that HES1 is a specific downstream gene of NOTCH1 and that it contributes to SACC proliferation, apoptosis and metastasis. Our findings serve as evidence indicating that HES1 may be useful as a clinical target in the treatment of SACC.

Identification of Treg-related prognostic molecular subtypes and individualized characteristics in clear cell renal cell carcinoma through single-cell transcriptomes and bulk RNA sequencing.

BACKGROUND: In clear cell renal cell carcinoma (ccRCC), the role of Regulatory T cells (Treg cells) as prognostic and immunotherapy response predictors is not fully explored. METHODS: Analyzing renal clear cell carcinoma datasets from TISCH, TCGA, and GEO, we focused on 8 prognostic Treg genes to study patient subtypes in ccRCC. We assessed Treg subtypes in relation to patient prognosis, tumor microenvironment, metabolism. Using Cox regression and principal component analysis, we devised Treg scores for individual patient characterization and explored the molecular role of C1QL1, a critical gene in the Treg model, through in vivo and in vitro studies. RESULTS: Eight Treg-associated prognostic genes were identified, classifying ccRCC patients into cluster A and B. Cluster A patients showed poorer prognosis with distinct clinical and molecular profiles, potentially benefiting more from immunotherapy. Low Treg scores correlated with worse outcomes and clinical progression. Low scores also suggested that patients might respond better to immunotherapy and targeted therapies. In ccRCC, C1QL1 knockdown reduced tumor proliferation and invasion via NF-kb-EMT pathways and decreased Treg cell infiltration, enhancing immune efficacy. CONCLUSIONS: The molecular subtype and Treg score in ccRCC, based on Treg cell marker genes, are crucial in personalizing ccRCC treatment and underscore C1QL1's potential as a tumor biomarker and target for immunotherapy.

FZD2 inhibits the cell growth and migration of salivary adenoid cystic carcinomas.

Several studies have reported that FZD2 regulates tumor biology in a complex manner. The aim of the present study was to identify the role of FZD2 in the cell growth and metastasis of salivary adenoid cystic carcinomas (SACCs). The expression of FZD2 in ACC-83 and ACC-LM cells were measured with real-time PCR. Immunohistochemical staining was used to detect the expression of FZD2 in clinical SACC samples with or without metastasis. Cell proliferation and Transwell assays were performed to explore the effects of FZD2 on cell growth and migration following the silencing of FZD2 with small interference RNAs and the overexpression of FZD2 with plasmid. Our data showed that FZD2 was downregulated in ACC-LM cells, which are an adenoid cystic carcinoma cell line with high metastatic potential, compared to ACC-83 cells, which have low metastatic potential. Additionally, the expression of FZD2 was lower in SACC tissues with metastasis compared to SACC tissues without metastasis (P<0.05). Cell proliferation and migration of ACC-83 cells were increased after the knockdown of FZD2 and decreased following overexpression of FZD2. Knockdown of FZD2 downregulated the expression of PAI-1. Our results suggest that FZD2 may be a tumor suppressor gene in SACCs that inhibits cell growth and migration.

CDH4 suppresses the progression of salivary adenoid cystic carcinoma via E-cadherin co-expression.

The cadherin-4 gene (CDH4) of the cadherin family encodes non-epithelial R-cadherin (R-cad); however, the function of this gene in different types of cancer remains controversial. In this study, we found higher expression of CDH4 mRNA in a salivary adenoid cystic carcinoma (SACC) cell line with low metastatic potential (SACC-83) than in a cell line with high metastatic potential (SACC-LM). By analyzing 67 samples of SACC tissues and 40 samples of paraneoplastic normal tissues, we found R-cad highly expressed in 100% of normal paraneoplastic tissue but only expressed in 64% of SACC tumor tissues (P<0.001). Knockdown of CDH4 expression in vitro promoted the growth, mobility and invasion of SACC cells, and in vivo experiments showed that decreased CDH4 expression enhanced SACC tumorigenicity. Furthermore, CDH4 suppression resulted in down-regulation of E-cadherin (E-cad), which is encoded by CDH1 gene and is a well-known tumor suppressor gene by inhibition of cell proliferation and migration. These results indicate that CDH4 may play a negative role in the growth and metastasis of SACC via co-expression with E-cadherin.

NOTCH1 signaling contributes to cell growth, anti-apoptosis and metastasis in salivary adenoid cystic carcinoma.

BACKGROUND: Numerous studies have reported both the tumor-suppressive and oncogenic roles of the Notch pathway, indicating that Notch activity regulates tumor biology in a complex, context-dependent manner. The aim of the present study was to identify the role of NOTCH1 in the cell growth and metastasis of SACC. METHODS: We analyzed the expression of NOTCH1 in clinical SACC samples using immunohistochemical staining. We silenced the expression of NOTCH1 and overexpressed activated NOTCH1 to elucidate the effects of NOTCH1 on proliferation, migration and invasion. NOTCH1 target genes were validated by real-time PCR. RESULTS: Our results showed that NOTCH1 was upregulated in SACC tissues when compared with normal tissues, and this upregulation was further enhanced in SACC tissues with metastasis and recurrence when compared with SACC tissues without metastasis. Overexpression of NOTCH1 in SACC cells promoted cell growth, migration and invasion, and knockdown of NOTCH1 inhibited cell proliferation in vitro and tumorigenicity in vivo by inducing cell apoptosis. CONCLUSIONS: The results of this study suggest that NOTCH1 plays a key role in the cell growth, anti-apoptosis, and metastasis of SACC. NOTCH1 inhibitors might therefore have potential therapeutic applications in treating SACC patients by inhibiting cancer cell growth and metastasis.

CDH12 promotes the invasion of salivary adenoid cystic carcinoma.

Cadherins are found in almost all living organisms. In addition to their role in the formation and maintenance of normal tissue architecture, cadherins seem to play a crucial role in the cell-cell interactions of cancer cells in tumorigenesis, invasion and metastasis. The aim of the present study was to identify the role of CDH12 in the invasion and metastasis of salivary adenoid cystic carcinoma (SACC). Real-time PCR results showed that CDH12 is abnormally expressed in the highly metastatic SACC cell line ACC-M, compared to ACC-2, a SACC cell line with low metastatic ability. CDH12 expression was significantly higher in clinical samples with metastasis and recurrence than in those without metastasis and recurrence (P<0.05), as demonstrated by immunohistochemical analysis. Overexpression of the CDH12 protein in ACC-M cells infected with an adenovirus vector containing CDH12 enhanced the invasive and migratory ability of ACC-M cells in vitro compared to ACC-M cells infected with empty vector. Likewise, knockdown of CDH12 by small interfering RNA efficiently inhibited the invasion and migration of ACC-M cells in vitro. These results indicate that CDH12 may play an important role in the invasion and metastasis of SACC.

Notch-4 contributes to the metastasis of salivary adenoid cystic carcinoma.

The Notch signaling pathway is important for cell-cell communication; it is involved in gene regulation mechanisms that control multiple cell differentiation processes during embryonic and adult life. Notch is present in all metazoans, and vertebrates possess four different Notch receptors: Notch-1, Notch-2, Notch-3, and Notch-4. The aim of the present study was to identify the role of Notch protein in the metastasis of salivary adenoid cystic carcinoma (SACC). Real-time PCR results showed that Notch-1, Notch-2, and Notch-4 were upregulated in the highly metastatic SACC cell line ACC-M, compared to ACC-2, a SACC cell line with low metastatic ability. Knockdown of Notch-4 by small interfering RNA efficiently inhibited the invasion of ACC-M cells. Notch-4 expression was significantly higher in the clinical samples with metastasis and recurrence compared to that in control (p<0.05), shown by immunohistochemistry analysis. These results indicate that Notch-4 may play an important role in SACC metastasis.