The mediation effect of asprosin on the association between ambient air pollution and diabetes mellitus in the elderly population in Taiyuan, China.

Evidence around the relationship between air pollution and the development of diabetes mellitus (DM) remains limited and inconsistent. To investigate the potential mediation effect of asprosin on the association between fine particulate matter (PM2.5), tropospheric ozone (O3) and blood glucose homeostasis. A case-control study was conducted on a total of 320 individuals aged over 60 years, including both diabetic and non-diabetic individuals, from six communities in Taiyuan, China, from July to September 2021. Generalized linear models (GLMs) suggested that short-term exposure to PM2.5 was associated with elevated fasting blood glucose (FBG), insulin resistance index (HOMA-IR), as well as reduced pancreatic ß-cell function index (HOMA-ß), and short-term exposure to O3 was associated with increased FBG and decreased HOMA-ß in the total population and elderly diabetic patients. Mediation analysis showed that asprosin played a mediating role in the relationship of PM2.5 and O3 with FBG, with mediating ratios of 10.2% and 18.4%, respectively. Our study provides emerging evidence supporting that asprosin mediates the short-term effects of exposure to PM2.5 and O3 on elevated FBG levels in an elderly population. Additionally, the elderly who are diabetic, over 70 years, and BMI over 24 kg/m2 are more vulnerable to air pollutants and need additional protection to reduce their exposure to air pollution.

Antibody-mediated spike activation promotes cell-cell transmission of SARS-CoV-2.

The COVID pandemic fueled by emerging SARS-CoV-2 new variants of concern remains a major global health concern, and the constantly emerging mutations present challenges to current therapeutics. The spike glycoprotein is not only essential for the initial viral entry, but is also responsible for the transmission of SARS-CoV-2 components via syncytia formation. Spike-mediated cell-cell transmission is strongly resistant to extracellular therapeutic and convalescent antibodies via an unknown mechanism. Here, we describe the antibody-mediated spike activation and syncytia formation on cells displaying the viral spike. We found that soluble antibodies against receptor binding motif (RBM) are capable of inducing the proteolytic processing of spike at both the S1/S2 and S2' cleavage sites, hence triggering ACE2-independent cell-cell fusion. Mechanistically, antibody-induced cell-cell fusion requires the shedding of S1 and exposure of the fusion peptide at the cell surface. By inhibiting S1/S2 proteolysis, we demonstrated that cell-cell fusion mediated by spike can be re-sensitized towards antibody neutralization in vitro. Lastly, we showed that cytopathic effect mediated by authentic SARS-CoV-2 infection remain unaffected by the addition of extracellular neutralization antibodies. Hence, these results unveil a novel mode of antibody evasion and provide insights for antibody selection and drug design strategies targeting the SARS-CoV-2 infected cells.

cFLIPS regulates alternative NLRP3 inflammasome activation in human monocytes.

The innate immune responses, including inflammasome activation, are paramount for host defense against pathogen infection. In contrast to canonical and noncanonical inflammasome activation, in this study, heat-killed gram-negative bacteria (HK bacteria) were identified as single-step stimulators of the NLRP3 inflammasome in human monocytes, and they caused a moderate amount of IL-1ß to be released from cells. Time course experiments showed that this alternative inflammasome response was finished within a few hours. Further analysis showed that the intrinsically limited NLRP3 inflammasome activation response was due to the negative regulation of caspase-8 by the short isoform of cFLIP (cFLIPs), which was activated by NF-κB. In contrast, overexpressed cFLIPS, but not overexpressed cFLIPL, inhibited the activation of caspase-8 and the release of IL-1ß in response to HK bacteria infection in human monocytes. Furthermore, we demonstrated that TAK1 activity mediated the expression of cFLIPs and was upstream and essential for the caspase-8 cleavage induced by HK bacteria in human monocytes. The functional specificity of cFLIPs and TAK1 revealed unique responses of human monocytes to a noninvasive pathogen, providing novel insights into an alternative regulatory pathway of NLRP3 inflammasome activation.

Impact of wound complications in obese versus non-obese patients undergoing total hip arthroplasty: A meta-analysis.

This meta-analysis examined the post-operative wound effect of both obese and non-obese in total hip arthroplasty (THA) patients. To gather as complete an overview as possible, the researchers took advantage of 4 databases-PubMed, Embase, Cochrane Library and Web of Science-to conduct a critical assessment. Following the development of inclusion and exclusion criteria, the researchers evaluated the quality of each document. A total of 9 related trials were conducted to determine the 95% CI (CI) and OR using a fixed-effect model. The final meta-analyses were conducted with RevMan 5.3. Our findings indicate that there is no statistically significant benefit in terms of post-operative wound complications among obese and non-obese patients. Obese subjects had a significantly higher risk of injury than those without obesity (OR, 1.43; 95% CI, 1.04, 1.95, p = 0.03); obesity was also associated with a significantly higher risk of operative site infection than in non-obese subjects (OR, 1.96; 95% CI, 1.76, 2.18, p < 0.0001); and after surgery, there was also a significant increase in the risk of post-operative wound infections among obese subjects than in non-obese subjects (OR, 1.57; 95% CI, 1.34, 1.84, p < 0.0001). However, due to the small size of the cohort study in this meta-study, caution is required in the analysis. More randomized, controlled studies will be needed to validate these results.

TRAF6-TAK1-IKKß pathway mediates TLR2 agonists activating "one-step" NLRP3 inflammasome in human monocytes.

Gram-positive bacterial infection causes high morbidity and mortality worldwide, while the underlying mechanism for host sensing bacterial components and initiating immune responses remains elusive. The NLRP3 inflammasome is a cytosolic multi-protein complex sensing a broad spectrum of endogenous danger signals and environmental irritants. In contrast to canonical NLRP3 inflammasome activation that needs both priming and activation signals, Lipopolysaccharide (LPS) from gram-negative bacteria activates the "one-step" NLRP3 inflammasome in human monocytes, which relies on the TLR4-TRIF-Caspase-8 signaling. Here, we show that in human monocytes, TLR2 agonists such as heat-killed gram-positive bacteria, peptidoglycan (PGN) or synthetic bacterial lipoprotein analog Pam3CysSerLys4 (Pam3CSK4) are able to induce the "one-step" NLRP3 inflammasome activation. Using genetic targeting and pharmacological inhibition approaches, it was found that TLR2 propagates signal through TRAF6, TAK1 and IKKß, ultimately activated NLRP3 independent of RelA. In addition, IKKß interacts with NLRP3 directly and affects NLRP3 inflammasome activation. These results reveal the signaling cascade downstream of TLR2 upon sensing gram-positive bacterial infection and activating the "one-step" NLRP3 inflammasome in human monocytes, which provides clue for controlling gram-positive bacterial infection-related inflammation.

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.

Enzyme-Free Autocatalysis-Driven Feedback DNA Circuits for Amplified Aptasensing of Living Cells.

Aptasensors with high specificity have emerged as powerful tools for understanding various biological processes, thus providing tremendous opportunities for clinical diagnosis and prognosis. However, their applications in intracellular molecular imaging are largely impeded due to the low anti-interference capacity in biological environments and the moderate sensitivity to targets. Herein, a robust enzyme-free autocatalysis-driven feedback DNA circuit is devised for amplified aptasensing, for example, adenosine triphosphate (ATP) and thrombin, with a significantly improved sensitivity in living cells. This initiator-replicated hybridization chain reaction (ID-HCR) circuit was acquired by integrating the HCR circuit with the DNAzyme biocatalysis. Also, the autocatalysis-driven aptasensor consists of a recognition element and an amplification element. The recognition unit can specifically identify ATP or thrombin via a versatile conformational transformation, resulting in the exposure of the initiator to the autocatalysis-driven circuit. The ID-HCR element integrates the charming self-assembly characteristics of the HCR and the remarkable catalytic cleavage capacity of DNAzyme for realizing the continuously self-sustained regeneration or replication of trigger strands and for achieving an exponential signal gain. The autocatalysis-driven aptasensor has been validated for quantitative analysis of ATP and thrombin in vitro and for monitoring the corresponding aptamer substrates with various expressions in live cells. More importantly, the autocatalysis-driven aptasensor, as a versatile amplification strategy, holds enormous potential for analysis of other less abundant biomarkers by changing only the recognition element of the system.

Real-Time Investigation of Intracellular Polynucleotide Kinase Using a Cascaded Amplification Circuit.

Polynucleotide kinase (PNK) shows an in-depth correlationship with DNA repair and metabolism processes. The in situ visualization of intracellular PNK revealed an extremely biological significance in supplementing reliable and quantitative information on its spatiotemporal distribution in live cells. Herein, we developed a versatile cascaded DNA amplification circuit through the integration of catalytic DNA assembly and hybridization chain reaction circuits and realized the accurate evaluation of intracellular PNK activity via the Förster resonance energy transfer (FRET) principle. Initially, without PNK, trigger T was firmly caged in the PNK-recognizing hairpin HT, resulting in no disturbance of the concatenated circuit. However, with the introduction of PNK, the 5'-OH terminal of PNK-addressing HT was phosphorylated, then the phosphorylated HT could be subsequently digested by λ exonuclease (λ Exo) to produce trigger T of the cascaded DNA circuit. As a result, the integrated circuit was stimulated to produce an amplified FRET signal for quantitatively monitoring the activity of PNK. Due to the λ Exo-specific digestion of 5'-phosphate DNA and the high signal gain of the cascade circuit, our proposed strategy enables the sensitive analysis of PNK activity in vitro and in complex biological samples. Furthermore, our PNK-sensing platform was extensively explored in HeLa cells for realizing reliable intracellular PNK imaging and thus showed high potential in the future diagnosis and treatment of kinase-related diseases.

An Optimal Geometry Configuration Algorithm of Hybrid Semi-Passive Location System Based on Mayfly Optimization Algorithm.

In view of the demand of location awareness in a special complex environment, for an unmanned aerial vehicle (UAV) airborne multi base-station semi-passive positioning system, the hybrid positioning solutions and optimized site layout in the positioning system can effectively improve the positioning accuracy for a specific region. In this paper, the geometric dilution of precision (GDOP) formula of a time difference of arrival (TDOA) and angles of arrival (AOA) hybrid location algorithm is deduced. Mayfly optimization algorithm (MOA) which is a new swarm intelligence optimization algorithm is introduced, and a method to find the optimal station of the UAV airborne multiple base station's semi-passive positioning system using MOA is proposed. The simulation and analysis of the optimization of the different number of base stations, compared with other station layout methods, such as particle swarm optimization (PSO), genetic algorithm (GA), and artificial bee colony (ABC) algorithm. MOA is less likely to fall into local optimum, and the error of regional target positioning is reduced. By simulating the deployment of four base stations and five base stations in various situations, MOA can achieve a better deployment effect. The dynamic station configuration capability of the multi-station semi-passive positioning system has been improved with the UAV.

Alum Pickering Emulsion as Effective Adjuvant to Improve Malaria Vaccine Efficacy.

Malaria is a life-threatening global epidemic disease and has caused more than 400,000 deaths in 2019. To control and prevent malaria, the development of a vaccine is a potential method. An effective malaria vaccine should either combine antigens from all stages of the malaria parasite's life cycle, or epitopes of multiple key antigens due to the complexity of the Plasmodium parasite. Malaria's random constructed antigen-1 (M.RCAg-1) is one of the recombinant vaccines, which was selected from a DNA library containing thousands of diverse multi-epitope chimeric antigen genes. Moreover, besides selecting an antigen, using an adjuvant is another important procedure for most vaccine development procedures. Freund's adjuvant is considered an effective vaccine adjuvant for malaria vaccine, but it cannot be used in clinical settings because of its serious side effects. Traditional adjuvants, such as alum adjuvant, are limited by their unsatisfactory immune effects in malaria vaccines, hence there is an urgent need to develop a novel, safe and efficient adjuvant. In recent years, Pickering emulsions have attracted increasing attention as novel adjuvant. In contrast to classical emulsions, Pickering emulsions are stabilized by solid particles instead of surfactant, having pliability and lateral mobility. In this study, we selected aluminum hydroxide gel (termed as "alum") as a stabilizer to prepare alum-stabilized Pickering emulsions (ALPE) as a malaria vaccine adjuvant. In addition, monophosphoryl lipid A (MPLA) as an immunostimulant was incorporated into the Pickering emulsion (ALMPE) to further enhance the immune response. In vitro tests showed that, compared with alum, ALPE and ALMPE showed higher antigen load rates and could be effectively endocytosed by J774a.1 cells. In vivo studies indicated that ALMPE could induce as high antibody titers as Freund's adjuvant. The biocompatibility study also proved ALMPE with excellent biocompatibility. These results suggest that ALMPE is a potential adjuvant for a malaria vaccine.

Bioorthogonal regulation of DNA circuits for smart intracellular microRNA imaging.

Catalytic DNA circuits represent a versatile toolbox for tracking intracellular biomarkers yet are constrained with low anti-interference capacity originating from their severe off-site activation. Herein, by introducing an unprecedented endogenous DNA repairing enzyme-powered pre-selection strategy, we develop a sequential and specific on-site activated catalytic DNA circuit for achieving the cancer cell-selective imaging of microRNA with high anti-interference capacity. Initially, the circuitry reactant is firmly caged by an elongated stabilizing duplex segment with a recognition/cleavage site of a cell-specific DNA repairing enzyme, which can prevent undesired signal leakage prior to its exposure to target cells. Then, the intrinsic DNA repairing enzyme of target cells can liberate the DNA probe for efficient intracellular microRNA imaging via the multiply guaranteed molecular recognition/activation procedures. This bioorthogonal regulated DNA circuit presents a modular and programmable amplification strategy for highly reliable assays of intracellular biomarkers, and provides a pivotal molecular toolbox for living systems.

Intelligent demethylase-driven DNAzyme sensor for highly reliable metal-ion imaging in living cells.

The accurate intracellular imaging of metal ions requires an exquisite site-specific activation of metal-ion sensors, for which the pervasive epigenetic regulation strategy can serve as an ideal alternative thanks to its orthogonal control feature and endogenous cell/tissue-specific expression pattern. Herein, a simple yet versatile demethylation strategy was proposed for on-site repairing-to-activating the metal-ion-targeting DNAzyme and for achieving the accurate site-specific imaging of metal ions in live cells. This endogenous epigenetic demethylation-regulating DNAzyme system was prepared by modifying the DNAzyme with an m6A methylation group that incapacitates the DNAzyme probe, thus eliminating possible off-site signal leakage, while the cell-specific demethylase-mediated removal of methylation modification could efficiently restore the initial catalytic DNAzyme for sensing metal ions, thus allowing a high-contrast bioimaging in live cells. This epigenetic repair-to-activate DNAzyme strategy may facilitate the robust visualization of disease-specific biomarkers for in-depth exploration of their biological functions.

Application of Firefly Luciferase (Luc) as a Reporter Gene for the Chemoautotrophic and Acidophilic Acidithiobacillus spp.

Acidithiobacillus spp. are the most active bacteria in bioleaching and bioremediation, because of their remarkable extreme environmental adaptabilities and unique metabolic characteristics. The researches on regulatory mechanisms of energy metabolism and stress resistance are critical for the understanding and application of Acidithiobacillus spp. However, the lack of an ideal reporter gene has become an obstacle for studying genes expression and regulatory mechanism in these chemoautotrophic bacteria. In this study, we reported the firefly luciferase as a reporter gene for Acidithiobacillus caldus (A. caldus) and created a firefly luciferase (Luc) reporter system. The Luc system was applied for the quantitative analysis of the transcription strength of the promoters of tetH gene and the feoA gene in A. caldus. Moreover, the regulating effect of ferric uptake regulator (Fur) on the feoP gene in A. caldus was determined using the Luc system. The Luc reporter system is not only used in the study of regulatory mechanism of A. caldus, but also applied in the researches of other Acidithiobacillus species. Therefore, this study provides a new useful tool for the studies on the molecular biological mechanism and synthetic biological modification of these chemoautotrophic bacteria, which would promote the industrial application of Acidithiobacillus spp.

DNA Amplifier-Functionalized Metal-Organic Frameworks for Multiplexed Detection and Imaging of Intracellular mRNA.

DNA amplification is a useful technique for low-abundance biomarker detection and environmental monitoring because of its high signal-amplifying ability. However, intracellular application of DNA amplifiers remains challenging due to poor delivery efficiency and stability. Herein, we report an entropy-driven DNA amplifier-functionalized metal-organic framework (DNA amplifier-MOF) for the detection and imaging of multiple intracellular messenger RNAs (mRNAs). The DNA amplifier-MOF conjugate exhibits high cellular uptake, enhanced enzymatic stability, and good biocompatibility. Importantly, in the presence of phosphate ions, a surface-functionalized DNA amplifier can be released in cells with high efficiency, which facilitates the imaging of mRNA. This method is rapid and of high sensitivity/specificity, as validated in HepG2 and HL7702 cells for the imaging of TK1 and survivin mRNA, respectively. With further optimization, the strategy can become a powerful biotechnology tool for the detection of cancers at early stages and for elucidating biological processes.

Mobile Phone Passive Positioning through the Detection of Uplink Signals for Search and Rescue.

To satisfy the service requirements of high accuracy and efficient life detection and location for search and rescue (SAR) missions after a disaster, we developed a passive positioning method to locate mobile phones by capturing the random access preamble, which can be applied to fourth-generation (4G) and even fifth-generation (5G) communication systems. We analyzed the characteristics of the random access procedure of a communication system and established a way to detect mobile phones by combining the time-difference-of-arrival (TDOA) estimation to determine the location. Then, we performed an experiment and a simulation of preamble sequence acquisition, and the results proved that the method is feasible and has high detection accuracy in high-noise conditions.

Comprehensive Analysis of the Relationship Between RAS and RAF Mutations and MSI Status of Colorectal Cancer in Northeastern China.

BACKGROUND/AIMS: Colorectal cancer (CRC) is mainly caused by chromosomal instability (CIN) and microsatellite instability (MSI). The RAS and RAF genes are essential components of the CIN pathway, and several studies have found that RAS and RAF mutations are associated with MSI status in CRC. Here, we examined these three factors in CRC in Northeast China and aimed to reveal new details of the relationship between these mutations and MSI status. METHODS: This study involved 290 patients with CRC who had RAS or RAF gene mutation detected using fluorescence-based allele-specific polymerase chain reaction or Sanger sequencing. The majority of the identified patients were found to harbor MSI (MSI status). Accurate molecular detection was carried out using formalin-fixed paraffin-embedded tissue or blood samples. RESULTS: The rates of RAS and RAF mutations were 58.5% and 4.1%, respectively. The prevalence of RAS mutation in CRC was clearly higher and that of RAF mutation was lower in Northeast China compared with previously reported cohorts in other locations. High MSI level (MSI-H status) was more complex, at around 10%. This was consistent with previous data from China. However, compared with data reported from other continents, MSI-H was higher than that of Japan or South Korea in Asia, and lower than that of Europe or the United States. CONCLUSION: RAS/RAF mutations and MSI status in CRC are closely associated with tumor location and ethnicity. Further studies investigating the relationship between these three factors can help in the development of treatment strategies for patients with CRC.

Prompt and Robust Humoral Immunity Elicited by a Conjugated Chimeric Malaria Antigen with a Truncated Flagellin.

As one of the pathogen-associated molecular patterns (PAMPs), flagellin is recently utilized as a potent adjuvant for many subunit vaccines. In this study, a truncated flagellin (tFL) with deletion of the hypervariable regions was adopted as a carrier-adjuvant by chemical conjugation with a chimeric malaria antigen M.RCAg-1 (M312) via a heterobifunctional polyethylene glycol (PEG) linker. After booster immunization in mice without any extra adjuvants, the M312-PEG-tFL conjugates elicited M312-specific antibody titers 100-1000 times higher than M312 and 10-100 times higher than the physical mixture of M312 and tFL. The elicited specific antibodies could recognize the native parasites, and the immunofluorescence assay (IFA) titer was 2100 for M312-P5k-tFL, which was about 7 times higher than M312. Furthermore, the IFA titers of the conjugates were comparable to the positive control of complete Freund's adjuvant (CFA). Compared to M312, the M312-PEG-tFL conjugates enhanced the proliferation index, lymphocyte activation, and memory T-cell generation. IgG subclasses of sera and cytokines analysis of splenocytes showed that conjugation with tFL could slightly trigger the Th1 polarization, while the antigen alone predominantly induced a Th2-biased immune response. Furthermore, a more-efficient innate immune response was provoked by the M312-PEG-tFL conjugates, as determined by the detection of antigen-specific TNF-α secretion by splenocytes. Our results indicated that tFL mainly retained the function as an agonist of TLR5. Conjugation of antigen to tFL could induce strong humoral and moderate cellular immune responses. Thus, conjugation of antigen to tFL as a potent carrier-adjuvant is an effective strategy for developing a promising protein-based vaccine.

Red blood cells release microparticles containing human argonaute 2 and miRNAs to target genes of Plasmodium falciparum.

Red blood cells (RBCs) are known to function as a refuge for providing food resources and as a shelter against the host's immune system after malaria parasite (Plasmodium) infection. Recent studies have reported significant production of extracellular vesicles (microparticles, MPs) in the circulation of malaria patients. However, it is unclear how these extracellular vesicles are generated and what their biological functions are. In this study, we isolated the MPs from a culture medium of normal RBCs and malaria parasite-infected RBCs (iRBCs), compared their quantity and origins, and profiled their miRNAs by deep sequencing. We found a much larger number of MPs released in the culture of iRBCs than in the culture of normal RBCs. Further investigation indicated that, in these MPs, human argonaute 2 (hAgo2) was found to bind to hundreds of miRNAs. These hAgo2-miRNA complexes were transferred into the parasites, and the expression of an essential malaria antigen, PfEMP1, was downregulated by miR-451/140 through its binding to the A and B subgroups of var genes, a family of genes encoding PfEMP1. Our data suggest for the first time that, through the release of MPs, mature RBCs present an innate resistance to malaria infection. These studies also shed new light on the reason why RBCs' genetic mutation occurs mainly in populations living in intensive malaria endemic areas and on the possibility of using miRNAs as novel medicines for malaria patients.

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.

[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.