[Evaluation of transfection effectiveness using fluorescein-labelled oligonucleotides and entraster-R siRNA transfection into Plasmodium falciparum].

The cultured Plasmodium falciparum parasites were synchronized twice by 5% sorbitol treatment twice (8-hour window), and then incubated at 37 degrees C for 16 h. Parasites were transfected with fluorescein-labelled oligonucleotides (group A) or fluorescein-labelled oligonucleotides+Entranster-R siRNA transfection reagent (group B). After 5 h a part of parasites was evaluated by fluorescence microscopy and flow cytometry. The rest of parasites were washed with RPMI 1640 medium, and then incubated with 500 microl new medium containing 2% fresh erythrocytes for another 12 h, and detected by flow cytometry. The fluorescein-labelled oligonucleotides were localized in erythrocytes in group B, but nearly no fluorescence was observed for group A. Flow cytometry analysis indicated that the transfection efficiency of group B [(47.40 +/- 3.39)%] was higher than that of group A [(0.60 +/- 0.27)%]. In the second cell cycle, the transfection efficiency in group B was (26.85 +/- 2.90)%, while that of group A was nearly zero. The results indicated that Entranster-R siRNA transfection reagent may increase the oligonucleotides transfection efficiency.

PROTACS: A technology with a gold rush-like atmosphere.

Abnormally expressed or malfunctioning proteins may affect or even damage cells, leading to the onset of diseases. Proteolysis targeting chimera (PROTAC) technology has been proven to be a fresh therapeutic strategy, superior to conventional small molecule inhibitors for the treatment of diseases caused by pathogenic proteins. Unlike conventional small molecule inhibitors that are occupancy-driven, PROTACs are heterobifunctional small molecules with catalytic properties. They combine with E3 ligases and target proteins to form a ternary complex, rendering the target protein ubiquitous and subsequently degraded by the proteasome. This paper focuses first on significant events in the development of PROTAC technology from 2001 to 2022, followed by a brief overview of various PROTACs categorized by target proteins. In addition, the applications of PROTACs in the treatment of diseases and fundamental biology are also under discussion.

Identification of a vaccine candidate antigen, PfMAg-1, from Plasmodium falciparum with monoclonal antibody M26-32.

Monoclonal antibody M26-32 has been shown to strongly inhibit the growth of Plasmodium falciparum in vitro. To identify the target antigen of M26-32, a P. falciparum Dd2 asexual stage cDNA expression library was screened with this antibody, and a full open reading frame cDNA was obtained. This gene, named pfmag-1, encodes a polypeptide of 589 amino acids. The protein PfMAg-1 was characterized as a membrane-associated protein that expressed on the surface of merozoite during erythrocytic stage. Remarkably, at the C terminus of PfMAg-1, there are 14 copies of a deca-peptide sequence of QTDEIKND (H/N) I. This tandem repeat domain was identified to harbor the epitope of the protective M26-32 monoclonal antibody, and was also recognized by sera of patients infected with P. falciparum. Rabbit antibody elicited against this deca-peptide repeat domain effectively inhibited P. falciparum invasion in vitro. Our work suggests that PfMAg-1 is a promising malaria vaccine candidate.

[Cloning and optimized prokaryotic expression of a pbmag-1 cDNA fragment from Plasmodium berghei ANKA].

OBJECTIVE: To clone and express a novel gene cDNA fragment, pbmag-1, from Plasmodium berghei ANKA strain. METHODS: The cDNA sequence of pbmag-1 was obtained from the GenBank of P. berghei ANKA genomic databases, with which a pair of primers was designed and RT-PCR was used to get a cDNA fragment of the gene from the parasite. The expanded cDNA 3' fragment of the gene was obtained by 3'-RACE using the oligo dT primer and a set of specific primers. The intact cDNA 3' fragment was cloned into a prokaryotic expressional vector and transformed into the BL21-(DE3)-RIL strain of Escherichia coli. The recombinant protein of PbMAg-1 was expressed with an optimized strategy and used to immunize mice. RESULTS: The pbmag-1 cDNA fragment obtained was 1341 bp in length, A/T rich (73%) and with a correct 3' end sequence. By Western blot, the anti-serum of mice immunized with the recombinant protein of PbMAg-1/GST, which was expressed as inclusion bodies, specifically recognized a band with Mr 64,000 molecule from the protein extracts of P. berghei-infected mouse erythrocytes. CONCLUSION: The pbmag-l cDNA sequence with intact 3' has been obtained, which will be used for further study on its role in the immune response of P. berghei infection.

Multi-epitope chimeric antigen used as a serological marker to estimate Plasmodium falciparum transmission intensity in the border area of China-Myanmar.

BACKGROUND: Following the decline of malaria transmission in many countries and regions, serological parameters have become particularly useful for estimating malaria transmission in low-intensity areas. This study evaluated a novel serological marker, Malaria Random Constructed Antigen-1 (M.RCAg-1), which contains 11 epitopes from eight Plasmodium falciparum antigens, as a tool for assessing malaria transmission intensity along the border area of China-Myanmar. METHOD: Serum from Plasmodium falciparum and P. vivax patients was used to detect the properties of M.RCAg-1 and antibody responses. Cross-sectional surveys were conducted at the China-Myanmar border and in Hainan province in 2012 and 2013 using cluster sampling. Filter blood spot papers were collected from all participants. Antibodies against M.RCAg-1 were detected using indirect ELISA. The Mann-Whitney test and Spearman's rank correlation test were performed to analyze antibody data. P. falciparum malaria transmission intensity was estimated using a catalytic conversion model based on the maximum likelihood of generating a community seroconversion rate (SCR). RESULTS: M.RCAg-1 was well-recognized by the naturally acquired anti-malaria antibodies in P. falciparum patients and had very limited cross-reactivity with P. vivax infection. The total amount of IgG antibodies was decreased with the decrease in parasitemia after taking medication and lasted several weeks. In a population survey, the antibody levels were higher in residents living close to the China-Myanmar border than those living in non-epidemic areas (P < 0.0001), but no significant difference was observed between residents from Hainan and non-epidemic areas. The calculated SCR was 0.0128 for Jieyangka, 0.004 for Susuzhai, 0.0047 for Qiushan, and 0.043 for Kayahe. The estimated exposure rate obtained from the anti-M.RCAg-1 antibody level correlated with traditional measures of transmission intensity derived from altitude. CONCLUSION: Our study demonstrates that M.RCAg-1 is potentially useful as a serological indicator of exposure to P. falciparum malaria, especially for malaria surveillance in low transmission areas.

Dynamin like protein 1 participated in the hemoglobin uptake pathway of Plasmodium falciparum.

BACKGROUND: During the blood stage of malaria infection, parasites internalize in the host red blood cells and degrade massive amounts of hemoglobin for their development. Although the morphology of the parasite's hemoglobin uptake pathway has been clearly observed, little has been known about its molecular mechanisms. METHODS: The recombinant proteins from Plasmodium falciparum, dynamin like protein 1 (PfDYN1) and 2 (PfDYN2) GTPase domain, were expressed in E.coli and showed GTPase activity. By using a dynamin inhibitor, dynasore, we demonstrated the involvement of PfDYN1 in the hemoglobin uptake pathway. RESULTS: The GTPase activity of the two recombinant proteins was inhibited by dynasore in vitro. Treatment of parasite cultures with 80 micromol/L dynasore at the ring and early trophozoite stage resulted in substantial inhibition of parasite growth and in an obvious decline of hemoglobin quantum. Furthermore, reduced intracellular hemozoin accumulation and decreased uptake of the FITC-dextran were also observed, together with distinctive changes in the ultrastructure of parasites after the dynasore treatment. CONCLUSIONS: Our results show that PfDYN1 plays an important role in the hemoglobin uptake pathway of P. falciparum and suggest its possibility of being a novel target for malaria chemotherapy.

[Beta diversity of successional series in mash communities of Sanjiang Plain, China].

Four typical successional series in the marsh communities of Sanjiang Plain were selected to investigate the changes of their beta diversity. The results indicated that from the 'starting community' to the 'end community', which had the most and the least moisture level in the series, respectively, the similarity coefficient between each community and 'starting' community decreased with decreasing moisture gradient and increasing horizontal distance. The species turnover speed among the communities in each series had an increasing trend from lowland to upland. In the process of succession, the general trend was that the total number of plant species increased while the common species decreased, with the beta diversity between communities increased gradually. The analysis of succession mechanism supported the 'initial floristics composition hypothesis', and the plant Calamagrostis angustifolia would intensify its dominance in the mash of Sanjiang Plain.