Low-Concentration Arsenic Trioxide Inhibits Skeletal Myoblast Cell Proliferation via a Reactive Oxygen Species-Independent Pathway

Myoblast proliferation and differentiation are essential for skeletal muscle regeneration. Myoblast proliferation is a critical step in the growth and maintenance of skeletal muscle. The precise action of inorganic arsenic on myoblast growth has not been investigated. Here, we investigated the in vitro effect of inorganic arsenic trioxide (As₂O₃) on the growth of C2C12 myoblasts. As₂O₃ decreased myoblast growth at submicromolar concentrations (0.25–1 μM) after 72 h of treatment. Submicromolar concentrations of As₂O₃ did not induce the myoblast apoptosis. Low-concentration As₂O₃ (0.5 and 1 μM) significantly suppressed the myoblast cell proliferative activity, which was accompanied by a small proportion of bromodeoxyuridine (BrdU) incorporation and decreased proliferating cell nuclear antigen (PCNA) protein expression. As₂O₃ (0.5 and 1 μM) increased the intracellular arsenic content but did not affect the reactive oxygen species (ROS) levels in the myoblasts. Cell cycle analysis indicated that low-concentrations of As₂O₃ inhibited cell proliferation via cell cycle arrest in the G1 and G2/M phases. As₂O₃ also decreased the protein expressions of cyclin D1, cyclin E, cyclin B1, cyclin-dependent kinase (CDK) 2, and CDK4, but did not affect the protein expressions of p21 and p27. Furthermore, As₂O₃ inhibited the phosphorylation of Akt. Insulin-like growth factor-1 significantly reversed the inhibitory effect of As₂O₃ on Akt phosphorylation and cell proliferation in the myoblasts. These results suggest that submicromolar concentrations of As₂O₃ alter cell cycle progression and reduce myoblast proliferation, at least in part, through a ROS-independent Akt inhibition pathway.     

Angiopoietin-2 as a Prognostic Biomarker of Major Adverse Cardiovascular Events and All-Cause Mortality in Chronic Kidney Disease

Background

Chronic kidney disease (CKD) patients have higher prevalence of major adverse cardiovascular events (MACE) and all-cause mortality. Endothelial damage and dysfunction have been regarded as early portents of MACE in CKD patients. Angiopoietin-2 (Ang-2) impairs endothelial function and promotes aberrant neovascularization. The aim of the study was to assess the relationship between circulating Ang-2 and MACE or all-cause mortality in a CKD cohort.

Methods

A total of 621 pre-dialysis stage 3–5 CKD patients were enrolled from January 2006 to December 2011 and were followed up till October 2014. Plasma Ang-2 was measured in duplicate using commercial enzyme-linked immunosorbent assays (ELISA). Clinical outcomes included MACE or all-cause mortality

Results

Of all patients, 122 (19.8%) reached MACE or all-cause mortality. Seventy-two had MACE, 79 died, and 29 had both MACE and all-cause mortality during the follow-up period of 41.5±28.3 months. Ang-2 quintile was divided at 1405.0, 1730.0, 2160.9, and 2829.9 pg/ml. The adjusted HR of MACE or all-cause mortality for every single higher log Ang-2 was 5.69 (95% CI: 2.00–16.20, P = 0.001). The adjusted HR of MACE or all-cause mortality was 2.48 (95% CI: 1.25–4.90) for patients of quintile 5 compared with those of quintile 1. A longitudinal association between MACE or all-cause mortality and stepwise increases in Ang-2 levels was found (P-trend = 0.008).

Conclusions

Ang-2 is an independent predictor of MACE or all-cause mortality in CKD patients. Additional study is necessary in order to explore the mechanism of the association of Ang-2 with adverse outcomes in patients with CKD.     

Thyroid Fine-Needle Aspiration Biopsy and Thyroid Cancer Diagnosis: A Nationwide Population-Based Study

Background

Thyroid cancer is the most common endocrine gland malignancy and fine-needle aspiration biopsy is widely used for thyroid nodule evaluation. Repeated aspiration biopsies are needed due to plausible false-negative results. This study aimed to investigate the overall relationship between aspiration biopsy and thyroid cancer diagnosis, and to explore factors related to shorter diagnostic time.

Methods

This nationwide retrospective cohort study retrieved data from the Longitudinal Health Insurance Database in Taiwan. Subjects without known thyroid malignancies and who received the first thyroid aspiration biopsy after 2004 were followed-up from 2004 to 2009 (n = 7700). Chi-square test, Kaplan-Meier survival analysis, and Cox proportional hazards model were used for data analysis.

Results

Of 7700 newly-aspirated patients, 276 eventually developed thyroid cancer (malignancy rate 3.6%). Among the 276 patients with thyroid cancer, 61.6% underwent only one aspiration biopsy and 81.2% were found within the first year after the initial aspiration. Cox proportional hazards model revealed that aspiration frequency (HR 1.07, 95% CI 1.06–1.08), ultrasound frequency (HR 1.02, 95% CI 1.01–1.03), older age, male sex, and aspiration biopsies arranged by surgery, endocrinology or otolaryngology subspecialties were all associated with shorter time to thyroid cancer diagnosis.

Conclusions

About 17.4% of thyroid cancer cases received more than two aspiration biopsies and 18.8% were diagnosed one year after the first biopsy. Regular follow-up with repeated aspiration or ultrasound may be required for patients with clinically significant thyroid nodules.     

Neutrophil Responses to Sterile Implant Materials

In vivo implantation of sterile materials and devices results in a foreign body immune response leading to fibrosis of implanted material. Neutrophils, one of the first immune cells to be recruited to implantation sites, have been suggested to contribute to the establishment of the inflammatory microenvironment that initiates the fibrotic response. However, the precise numbers and roles of neutrophils in response to implanted devices remains unclear. Using a mouse model of peritoneal microcapsule implantation, we show 30–500 fold increased neutrophil presence in the peritoneal exudates in response to implants. We demonstrate that these neutrophils secrete increased amounts of a variety of inflammatory cytokines and chemokines. Further, we observe that they participate in the foreign body response through the formation of neutrophil extracellular traps (NETs) on implant surfaces. Our results provide new insight into neutrophil function during a foreign body response to peritoneal implants which has implications for the development of biologically compatible medical devices.     

Algal chloroplast produced camelid VHH antitoxins are capable of neutralizing botulinum neurotoxin

We have produced three antitoxins consisting of the variable domains of camelid heavy chain‐only antibodies (V_HH) by expressing the genes in the chloroplast of green algae. These antitoxins accumulate as soluble proteins capable of binding and neutralizing botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble protein, sufficient expression to easily produce these antitoxins at scale from algae. The genes for the three different antitoxins were transformed into Chlamydomonas reinhardtii chloroplasts and their products purified from algae lysates and assayed for in vitro biological activity using toxin protection assays. The produced antibody domains bind to botulinum neurotoxin serotype A (BoNT/A) with similar affinities as camelid antibodies produced in Escherichia coli, and they are similarly able to protect primary rat neurons from intoxication by BoNT/A. Furthermore, the camelid antibodies were produced in algae without the use of solubilization tags commonly employed in E. coli. These camelid antibody domains are potent antigen‐binding proteins and the heterodimer fusion protein containing two V_HH domains was capable of neutralizing BoNT/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (GI) tract of mice treated orally with antitoxin‐producing microalgae. These findings support the use of orally delivered antitoxins produced in green algae as a novel treatment for botulism.     

Multiple Structural and Epigenetic Defects in the Human Leukocyte Antigen Class I Antigen Presentation Pathway in a Recurrent Metastatic Melanoma Following Immunotherapy

Scant information is available about the molecular basis of multiple HLA class I antigen-processing machinery defects in malignant cells, although this information contributes to our understanding of the molecular immunoescape mechanisms utilized by tumor cells and may suggest strategies to counteract them. In the present study we reveal a combination of IFN-γ-irreversible structural and epigenetic defects in HLA class I antigen-processing machinery in a recurrent melanoma metastasis after immunotherapy. These defects include loss of tapasin and one HLA haplotype as well as selective silencing of HLA-A3 gene responsiveness to IFN-γ. Tapasin loss is caused by a germ-line frameshift mutation in exon 3 (TAPBP^684delA) along with a somatic loss of the other gene copy. Selective silencing of HLA-A3 gene and its IFN-γ responsiveness is associated with promoter CpG methylation nearby site-α and TATA box, reversible after DNA methyltransferase 1 depletion. This treatment combined with tapasin reconstitution and IFN-γ stimulation restored the highest level of HLA class I expression and its ability to elicit cytotoxic T cell responses. These results represent a novel tumor immune evasion mechanism through impairing multiple components at various levels in the HLA class I antigen presentation pathway. These findings may suggest a rational design of combinatorial cancer immunotherapy harnessing DNA demethylation and IFN-γ response.