Construction and functional verification of size-reduced plasmids based on TMP resistance gene dfrB10.

IMPORTANCE: Plasmid size is one of the factors affecting transfection efficacy in most of the molecular genetic research studies. One effective approach for reducing plasmid size is to replace relatively large, conventional antibiotic resistance genes with the short-size dfrB10 gene. The successful construct of a series of dfrB10-based tool plasmids and their functional validation, via comparison with original plasmids, suggest that dfrB10 is a potent drug resistance selection marker. The antibiotic trimethoprim offers convenient usage comparable to that of ampicillin or kanamycin. Additionally, fluorescence analysis has demonstrated the compatibility of TMP with protein expression in various host cells. Based on these findings, TMP-dfrB10 could be an alternative choice for future use in molecular genetic research studies that require miniature plasmids to achieve optimal results.

The Comparison of PCR Kits for the Detection of Erythrocytic Parasites on Filter Paper.

Dried blood spot (DBS) based PCR was considered an inexpensive and feasible method for detecting pathogens in the blood. The DBS carrier filter paper and PCR kits are crucial for accurate diagnosis. We evaluated 4 types of filter papers and 20 PCR kits for DBS samples. The PCR detecting Plasmodium results showed that the minimum detection limit of the 4 filter papers was 1 × 102 parasites/µL, and the positive rates of 20 PCR kits ranged from 0% to 100%. PCR results were satisfactory for detecting Plasmodium falciparum (P. falciparum) and Plasmodium. vivax (P. vivax) in archived DBS samples and Babesia gibsoni (B. gibsoni) in fresh pet DBS samples. Our results provided a useful reference for the detection of blood pathogens with DBS samples and direct PCR, especially for screening the cost-efficacy combination of filter paper and PCR kit in resource-limited areas.

Dimeric artesunate-choline conjugate micelles coated with hyaluronic acid as a stable, safe and potent alternative anti-malarial injection of artesunate.

Artesunate (ARS) is the only artemisinin-based intravenous drug approved for treatment of malaria in the clinic. ARS is rapidly metabolized in vivo to short lived (∼30-45 min) but fast acting, dihydroartemisinin (DHA). The short half-life of DHA necessitates multiple dose administration to circumvent the risk of recrudescence and development of artemisinin resistance. In this work, we report a stable, safe and potent alternative artemisinin-based injectable nanocomplex consisting of dimeric artesunate-choline conjugate (dACC) micelles coated with hyaluronic acid (HA). Firstly, dACC was synthesized by one-step esterification of two artesunate molecules with 3-(dimethylamino)-1,2-propanediol followed by quaternization. After that, dACC was self-assembled into cationic nanomicelles and further coated with anionic small molecular weight HA. The HA-coated dACC nanocomplex (dACC/HA nanocomplex) has a narrow size distribution of about 30 nm. Hemolytic toxicity and cytotoxicity studies revealed a favorable bio-safety profile. Finally, in vitro and in vivo studies showed the dACC/HA nanocomplex possess superior safety and antimalarial efficacy compared to ARS. Taken together, the dACC/HA nanocomplex is a promising injectable alternative to the traditional clinically used artesunate.