Development and optimization of a DNA aptamer to delay ß-bungarotoxin-induced lethality in a rodent model.

Current treatment of snakebite relies on immunoglobulin-rich antivenoms. However, production of these antivenoms is complicated and costly. Aptamers - single-stranded DNAs or RNAs with specific folding structures that bind to specific target molecules - represent excellent alternatives or complements to antibody-based therapeutics. However, no studies have systematically assessed the feasibility of using aptamers to mitigate venom-induced toxicity in vivo. ß-bungarotoxin is the predominant protein responsible for the toxicity of the venom of Bungarus multicinctus, a prominent venomous snake inhabiting Taiwan. In this study, we reported the screening and optimization of a DNA aptamer against ß-bungarotoxin and tested its utility in a mouse model. After 14 rounds of directed evolution of ligands by exponential enrichment, an aptamer, called BB3, displaying remarkable binding affinity and specificity for ß-bungarotoxin was obtained. Following structural prediction and point-modification experiments, BB3 underwent truncation and was modified with 2'-O-methylation and a 3'-inverted dT. This optimized aptamer showed sustained, high-affinity binding for ß-bungarotoxin and exhibited remarkable nuclease resistance in plasma. Importantly, administration of this optimized aptamer extended the survival time of mice treated with a lethal dose of ß-bungarotoxin. Collectively, our data provide a compelling illustration of the potential of aptamers as promising candidates for development of recombinant antivenom therapies.

Thrombin-Activated Platelets Protect Vascular Endothelium against Tumor Cell Extravasation by Targeting Endothelial VCAM-1.

When activated by thrombin, the platelets release their granular store of factors. These thrombin-activated platelets (TAPLT) have been shown to be capable of ameliorating pro-inflammatory processes. In this study, we tested if TAPLT could also protect the endothelium against tumor-related pro-inflammatory changes that promote angiogenesis and metastasis. Using endothelial cell (EC) models in vitro, we demonstrated that TAPLT protected EC against tumor conditioned medium (TCM)-induced increases of reactive oxygen species (ROS) production, EC permeability and angiogenesis, and inhibited transendothelial migration that was critical for cancer cell extravasation and metastasis. In vivo observations of TAPLT-mediated inhibition of angiogenesis and pulmonary colonization in a BALB/c nude mouse model were consistent with the in vitro findings. Neutralization of vascular cell adhesion molecule-1 (VCAM-1) binding significantly inhibited the ability of TAPLT to interact with EC and abrogated the TAPLT-mediated protection of EC against tumor angiogenesis and metastasis. Taken together, these findings suggest that VCAM-1-mediated linkage to EC is required for TAPLT to confer protection of EC against tumor-induced permeation and angiogenesis, thereby resisting tumor extravasation and metastasis.

Association of urinary ketamine and APOA1 levels with bladder dysfunction in ketamine abusers revealed via proteomics and targeted metabolite analyses.

Chronic ketamine abuse is associated with bladder dysfunction and cystitis. However, the effects of ketamine abuse on the urinary proteome profile and the correlations among urinary proteins, urinary ketamine (and metabolites) and clinicopathological features of ketamine-induced bladder dysfunction remain to be established. Here, we recruited 56 ketamine abusers (KA) and 40 age-matched healthy controls (HC) and applied the iTRAQ-based proteomics approach to unravel quantitative changes in the urine proteome profile between the two groups. Many of the differentially regulated proteins are involved in the complement and coagulation cascades and/or fibrotic disease. Among them, a significant increase in APOA1 levels in KA relative to control samples (392.1 ± 59.9 ng/ml vs. 13.7 ± 32.6 ng/ml, p < 0.0001) was detected via ELISA. Moreover, urinary ketamine, norketamine and dehydronorketamine contents (measured via LC-SRM-MS) were found to be positively correlated with overactive bladder syndrome score (OABSS) and APOA1 levels with urinary RBC, WBC, OABSS and numeric pain rating scale in KA. Collectively, our results may aid in developing new molecular tool(s) for management of ketamine-induced bladder dysfunction. Moreover, information regarding the differentially regulated proteins in urine of KA provides valuable clues to establish the molecular mechanisms underlying ketamine-induced cystitis.

Rapid and Efficient Enrichment of Snake Venoms from Human Plasma Using a Strong Cation Exchange Tip Column to Improve Snakebite Diagnosis.

Snake envenomation is a serious public health issue in many tropical and subtropical countries. Accurate diagnosis and immediate antivenom treatment are critical for effective management. However, the venom concentration in the victims' plasma is usually low, representing one of the bottlenecks in developing clinically applicable assays for venom detection and snakebite diagnosis. In this study, we attempted to develop a simple method for rapid enrichment of venom proteins from human plasma to facilitate detection. Our experiments showed that several major protein components of both Naja atra (N. atra) and Bungarus multicinctus (B. multicinctus) venoms have higher isoelectric point (pI) values relative to high-abundance human plasma proteins and could be separated via strong cation exchange-high-performance liquid chromatography (SCX-HPLC). Based on this principle, we developed an SCX tip column-based protocol for rapid enrichment of N. atra and B. multicinctus venom proteins from human plasma. Application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) led to the identification of cytotoxin and beta-bungarotoxin as the major proteins enriched by the SCX tip column in each venom sample. The entire process of venom enrichment could be completed within 10-15 min. Combination of this method with our previously developed lateral flow strip assays (rapid test) significantly enhanced the sensitivity of the rapid test, mainly via depletion of the plasma protein background, as well as increase in venom protein concentration. Notably, the SCX tip column-based enrichment method has the potential to efficiently enrich other Elapidae snake venoms containing proteins with higher pI values, thereby facilitating venom detection with other assays. This simple and rapid sample preparation method should aid in improving the clinical utility of diagnostic assays for snakebite.

Basic fibroblast growth factor antagonizes activin A-mediated growth inhibition and hemoglobin synthesis in K562 cells by activating ERK1/2 and deactivating p38 MAP kinase.

Activin A can induce erythroid differentiation, whereas basic fibroblast growth factor (bFGF) can maintain the undifferentiated status of erythroid progenitors. How these two factors together can affect the regulation of erythroid differentiation in hematopoietic cells has not been elucidated. This study demonstrates that bFGF antagonizes activin A-mediated growth inhibition and hemoglobin (Hb) synthesis in K562 cells. Analyses of mitogen-activated protein kinases revealed that activin A-induced p38 phosphorylation and inhibited ERK1/2 phosphorylation. In contrast, bFGF worked antagonistically to induce ERK1/2 phosphorylation and inhibited p38 phosphorylation in K562 cells. Furthermore, co-treatment of cells with activin A and bFGF decreased p38 phosphorylation and increased ERK1/2 phosphorylation. SB203580 inhibition of p38 activity eliminated activin A-mediated growth inhibition and Hb synthesis, whereas U0126 inhibition of ERK1/2 activity augmented the effects of activin A on K562 cells. These results suggest that bFGF can negatively modulate p38 and positively modulate ERK1/2 to antagonize activin A-mediated growth inhibition and Hb synthesis in K562 cells.