MicroRNA expression characterizes oligometastasis(es)

Background

Cancer staging and treatment presumes a division into localized or metastatic disease. We proposed an intermediate state defined by ≤5 cumulative metastasis(es), termed oligometastases. In contrast to widespread polymetastases, oligometastatic patients may benefit from metastasis-directed local treatments. However, many patients who initially present with oligometastases progress to polymetastases. Predictors of progression could improve patient selection for metastasis-directed therapy.

Methods

Here, we identified patterns of microRNA expression of tumor samples from oligometastatic patients treated with high-dose radiotherapy.

Results

Patients who failed to develop polymetastases are characterized by unique prioritized features of a microRNA classifier that includes the microRNA-200 family. We created an oligometastatic-polymetastatic xenograft model in which the patient-derived microRNAs discriminated between the two metastatic outcomes. MicroRNA-200c enhancement in an oligometastatic cell line resulted in polymetastatic progression.

Conclusions

These results demonstrate a biological basis for oligometastases and a potential for using microRNA expression to identify patients most likely to remain oligometastatic after metastasis-directed treatment.     

Redox regulation of PTEN and protein tyrosine phosphatases in H(2)O(2) mediated cell signaling

Protein tyrosine phosphatase (PTP) is a family of enzymes important for regulating cellular phosphorylation state. The oxidation and consequent inactivation of several PTPs by H₂O₂ are well demonstrated. It is also shown that recovery of enzymatic activity depends on the availability of cellular reductants. Among these redox-regulated PTPs, PTEN, Cdc25 and low molecular weight PTP are known to form a disulfide bond between two cysteines, one in the active site and the other nearby, during oxidation by H₂O₂. The disulfide bond likely confers efficiency in the redox regulation of the PTPs and protects cysteine-sulfenic acid of PTPs from further oxidation. In this review, through a comparative analysis of the oxidation process of Yap1 and PTPs, we propose the mechanism of disulfide bond formation in the PTPs.     

Exploiting enzyme specificities in digestions of chondroitin sulfates A and C: production of well-defined hexasaccharides

Interactions between proteins and glycosaminoglycans (GAGs) of the extracellular matrix are important to the regulation of cellular processes including growth, differentiation and migration. Understanding these processes can benefit greatly from the study of protein-GAG interactions using GAG oligosaccharides of well-defined structure. Materials for such studies have, however, been difficult to obtain because of challenges in synthetic approaches and the extreme structural heterogeneity in GAG polymers. Here, it is demonstrated that diversity in structures of oligosaccharides derived by limited enzymatic digestion of materials from natural sources can be greatly curtailed by a proper selection of combinations of source materials and digestive enzymes, a process aided by an improved understanding of the specificities of certain commercial preparations of hydrolases and lyases. Separation of well-defined oligosaccharides can then be accomplished by size-exclusion chromatography followed by strong anion-exchange chromatography. We focus here on two types of chondroitin sulfate (CS) as starting material (CS-A, and CS-C) and the use of three digestive enzymes with varying specificities (testicular hyaluronidase and bacterial chondroitinases ABC and C). Analysis using nuclear magnetic resonance and mass spectrometry focuses on isolated CS disaccharides and hexasaccharides. In all, 15 CS hexasaccharides have been isolated and characterized. These serve as useful contributions to growing libraries of well-defined GAG oligosaccharides that can be used in further biophysical assays.     

High sensitivity of embryonic stem cells to proteasome inhibitors correlates with low expression of heat shock protein and decrease of pluripotent cell marker expression

The ubiquitin-proteasome system is a major proteolytic system for nonlysosomal degradation of cellular proteins. Here, we investigated the response of mouse embryonic stem (ES) cells under proteotoxic stress. Proteasome inhibitors induced expression of heat shock protein 70 (HSP70) in a concentration- and time-dependent manner, and also induced apoptosis of ES cells. Importantly, more apoptotic cells were observed in ES cells compared with other somatic cells. To understand this phenomenon, we further investigated the expression of HSP70 and pluripotent cell markers. HSP70 expression was more significantly increased in somatic cells than in ES cells, and expression levels of pluripotent cell markers such as Oct4 and Nanog were decreased in ES cells. These results suggest that higher sensitivity of ES cells to proteotoxic stress may be related with lower capacity of HSP70 expression and decreased pluripotent cell marker expression, which is essential for the survival of ES cells.     

Application of recombinant hemagglutinin proteins as alternative antigen standards for pandemic influenza vaccines

Objectives

The single radial immunodiffusion (SRID) assay, used to quantify hemagglutinin (HA) in influenza vaccines, requires reference reagents; however, because centralized production of reference reagents may slow the emergency deployment of vaccines, alternatives are needed.

Results

We investigated the production of HA proteins using recombinant DNA technology, rather than a traditional egg-based production process. The HA proteins were then used in an SRID assay as a reference antigen. We found that HA can be quantified in both egg-based and cell-based influenza vaccines when recombinant HAs (rHAs) are used as the reference antigen. Furthermore, we confirmed that rHAs obtained from strains with pandemic potential, such as H5N1, H7N3, H7N9, and H9N2 strains, can be utilized in the SRID assay. The rHA production process takes just one month, in contrast to the traditional process that takes three to four months.

Conclusions

The use of rHAs may reduce the time required to produce reference reagents and facilitate timely introduction of vaccines during emergencies.

     

MUC1-C influences cell survival in lung adenocarcinoma Calu-3 cells after SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces coronavirus disease 2019 (COVID-19) and may increase the risk of adverse outcomes in lung cancer patients. In this study, we investigated the expression and function of mucin 1 (MUC1) after SARS-CoV-2 infection in the lung epithelial cancer cell line Calu-3. MUC1 is a major constituent of the mucus layer in the respiratory tract and contributes to pathogen defense. SARS-CoV-2 infection induced MUC1 C-terminal subunit (MUC1-C) expression in a STAT3 activation-dependent manner. Inhibition of MUC1-C signaling increased apoptosis-related protein levels and reduced proliferation-related protein levels; however, SARS-CoV-2 replication was not affected. Together, these results suggest that increased MUC1-C expression in response to SARS-CoV-2 infection may trigger the growth of lung cancer cells, and COVID-19 may be a risk factor for lung cancer patients.     

A biochemical and physicochemical comparison of two recombinant enzymes used for enzyme replacement therapies of hunter syndrome

Mucopolysaccharidosis II (MPS II, Hunter syndrome; OMIM 309900) is an X-linked lysosomal storage disease caused by a deficiency in the enzyme iduronate-2-sulfatase (IDS), leading to accumulation of glycosaminoglycans (GAGs). For enzyme replacement therapy (ERT) of Hunter syndrome, two recombinant enzymes, idursulfase (Elaprase®, Shire Human Genetic Therapies, Lexington, MA) and idursulfase beta (Hunterase®, Green Cross Corporation, Yongin, Korea), are currently available in Korea. To compare the biochemical and physicochemical differences between idursulfase and idursulfase beta, we examined the formylglycine (FGly) content, specific enzyme activity, mannose-6-phosphate (M6P) content, sialic acid content, and in vitro cell uptake activity of normal human fibroblasts of these two enzymes. The FGly content, which determines the enzyme activity, of idursulfase beta was significantly higher than that of idursulfase (79.4?±?0.9 vs. 68.1?±?2.2 %, P?<?0.001). In accordance with the FGly content, the specific enzyme activity of idursulfase beta was significantly higher than that of idursulfase (42.6?±?1.1 vs. 27.8?±?0.9 nmol/min/μg protein, P?<?0.001). The levels of M6P and sialic acid were not significantly different (2.4?±?0.1 vs 2.4?±?0.3 mol/mol protein for M6P and 12.3?±?0.7 vs. 12.4?±?0.4 mol/mol protein for sialic acid). However, the cellular uptake activity of the normal human fibroblasts in vitro showed a significant difference (K_uptake, 5.09?±?0.96 vs. 6.50?±?1.28 nM protein, P?=?0.017). In conclusion, idursulfase beta exhibited significantly higher specific enzyme activity than idursulfase, resulting from higher FGly content. These biochemical differences may be partly attributed to clinical efficacy. However, long-term clinical evaluations of Hunter syndrome patients treated with these two enzymes will be needed to demonstrate the clinical implications of significant difference of the enzyme activity and the FGly content.     

Identification of a functionally relevant signal peptide of mouse ficolin A

Mouse ficolin A is a plasma protein with lectin activity, and plays a role in host defense by binding carbohydrates, especially GlcNAc, on microorganisms. The ficolin A subunit consists of an N-terminal signal peptide, a collagen-like domain, and a C-terminal fibrinogen-like domain. In this study, we show that ficolin A can be synthesized and oligomerized in a cell and secreted into culture medium. We also identify a functionally relevant signal peptide of ficolin A by using MS/MS analysis to determine the N-terminal sequence of secreted ficolin A. When the signal peptide of mouse ficolin A was fused with enhanced green fluorescent protein (EGFP), EGFP was released into HEK 293 cell medium, suggesting that the signal peptide can efficiently direct ficolin A secretion. Moreover, our results suggest that the signal peptide of ficolin A has potential application for the production of useful secretory proteins.     

Production of a Monoclonal Antibody Targeting the M Protein of MERS-CoV for Detection of MERS-CoV Using a Synthetic Peptide Epitope Formulated with a CpG–DNA–Liposome Complex

The Middle East respiratory syndrome-related coronavirus (MERS-CoV) contains four major structural proteins, the spike glycoprotein, nucleocapsid phosphoprotein, membrane (M) glycoprotein and small envelope glycoprotein. The M protein of MERS-CoV has a role in the morphogenesis or assembly of the virus and inhibits type I interferon expression in infected cells. Here, we produced a monoclonal antibody specific against the M protein of MERS-CoV by injecting BALB/c mice with a complex containing the epitope peptide and CpG–DNA encapsulated with a phosphatidyl-β-oleoyl-γ-palmitoyl ethanolamine (DOPE):cholesterol hemisuccinate (CHEMS). The monoclonal antibody was reactive to the epitope peptide of the M protein of MERS-CoV which was confirmed by western blotting and immunoprecipitations. Indirect immunofluorescence assay and confocal image analysis showed that the monoclonal antibody binds specifically to the M protein of MERS-CoV in the virus-infected cells. Further studies using this monoclonal antibody may provide important information on the function of the M protein and its future application in diagnostics.