Optimized Design of Hyaluronic Acid-Lipid Conjugate Biomaterial for Augmenting CD44 Recognition of Surface-Engineered NK Cells.

Triple-negative breast cancer (TNBC) presents treatment challenges due to a lack of detectable surface receptors. Natural killer (NK) cell-based adaptive immunotherapy is a promising treatment because of the characteristic anticancer effects of killing malignant cells directly by secreting cytokines and lytic granules. To maximize the cancer recognition ability of NK cells, biomaterial-mediated ex vivo cell surface engineering has been developed for sufficient cell membrane immobilization of tumor-targeting ligands via hydrophobic anchoring. In this study, we optimized amphiphilic balances of NK cell coating materials composed of CD44-targeting hyaluronic acid (HA)-poly(ethylene glycol) (PEG)-lipid to improve TNBC recognition and the anticancer effect. Changes in the modular design of our material by differentiating hydrophilic PEG length and incorporating lipid amount into HA backbones precisely regulated the amphiphilic nature of HA-PEG-lipid conjugates. The optimized biomaterial demonstrated improved anchoring into NK cell membranes and facilitating the surface presentation level of HA onto NK cell surfaces. This led to enhanced cancer targeting via increasing the formation of immune synapse, thereby augmenting the anticancer capability of NK cells specifically toward CD44-positive TNBC cells. Our approach addresses targeting ability of NK cell to solid tumors with a deficiency of surface tumor-specific antigens while offering a valuable material design strategy using amphiphilic balance in immune cell surface engineering techniques.

Networked Cluster Formation via Trigonal Lipid Modules for Augmented Ex Vivo NK Cell Priming.

Current cytokine-based natural killer (NK) cell priming techniques have exhibited limitations such as the deactivation of biological signaling molecules and subsequent insufficient maturation of the cell population during mass cultivation processes. In this study, we developed an amphiphilic trigonal 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE) lipid-polyethylene glycol (PEG) material to assemble NK cell clusters via multiple hydrophobic lipid insertions into cellular membranes. Our lipid conjugate-mediated ex vivo NK cell priming sufficiently augmented the structural modulation of clusters, facilitated diffusional signal exchanges, and finally activated NK cell population with the clusters. Without any inhibition in diffusional signal exchanges and intrinsic proliferative efficacy of NK cells, effectively prime NK cell clusters produced increased interferon-gamma, especially in the early culture periods. In conclusion, the present study demonstrates that our novel lipid conjugates could serve as a promising alternative for future NK cell mass production.

Incidence and Pattern of Aminotransferase Elevation During Anti-Hypertensive Therapy With Angiotensin-II Receptor Blockers.

BACKGROUND: Angiotensin type II receptor blockers (ARBs) are the most widely used anti-hypertensive drugs. This study aimed to elucidate the likelihood and pattern of ARB-induced liver injury in a hospital-based cohort. METHODS: Data of patients receiving fimasartan (n = 5,543), candesartan (n = 6,406), valsartan (n = 6,040), and losartan (n = 9,126) were retrieved from the clinical data warehouse of two tertiary hospitals. Patients with alanine aminotransferase (ALT) levels > 5 times the upper normal limit were assessed according to the Roussel Uclaf Causality Assessment Method (RUCAM). RESULTS: A total of 27,115 patients were enrolled, including 14,630 (54.0%) men, with a mean age of 64.6 years (standard deviation, 13.6). During 31,717 person-years of ARB therapy, serum ALT levels > 120 IU/L were found in 558 (2.1%) person-years, and levels > 200 IU/L were found in 155 (0.6%) person-years. The incidence of ALT elevation > 120 IU/L per 106 cumulative defined daily doses was 6.6, 3.6, 3.9, and 4.0 in the fimasartan, candesartan, valsartan, and losartan groups, respectively (P = 0.002). An ALT level > 200 IU/L with RUCAM score ≥ 6 was found in 20 patients, suggesting probable drug-induced liver injury for 11 (0.2%) patients receiving fimasartan, five (0.1%) receiving candesartan, four (0.1%) receiving valsartan, and none receiving losartan (P < 0.001). CONCLUSION: Approximately 2% of patients receiving ARB therapy had significant ALT elevation (4.24/106 cumulative defined daily doses [cDDDs]), which was associated with probable ARB-related liver injury in 0.07% of patients (0.15/106 cDDDs). Elevation of ALT was more commonly associated with fimasartan than the other ARBs. Clinicians should be aware of the possibility of ARB-related ALT elevation in patients with unexplained chronic abnormal ALT.

Simultaneous probing of dual intracellular metabolites (ATP and paramylon) in live microalgae using graphene oxide/aptamer nanocomplex.

The development of an intracellular metabolite imaging platform for live microorganisms has been a challenge in the study of microbes. Herein, we performed metabolite imaging in live microalgal cells using a graphene oxide (GO)/aptamer complex. The properties of the GO were characterized using dynamic light scattering (DLS) and atomic force microscopy (AFM), which were determined to have 140 ± 3 nm in mean diameter. An ATP-specific aptamer was mixed with GO to form a GO/aptamer complex, and the feasibility of the complex was tested in vitro. The high correlation between the fluorescence intensity and concentration of ATP was observed in the range 0-10 mM. Next, the feasibility of the complex was confirmed in vivo. Under both phototrophic and heterotrophic culture conditions, Euglena gracilis internalized the complex, and bright fluorescence was observed as the aptamer was bound to the target metabolite (ATP). The fluorescence intensity of cells was correlated to the ATP concentration in the cells. Imaging of dual intracellular metabolites (ATP and paramylon) was achieved by simply using two different aptamers (ATP-specific aptamer and paramylon-specific aptamer) together, showing the great potential of the complex as a dual-sensing/imaging platform. In addition, the GO/aptamer complex exhibited low cytotoxicity; the proliferation and viability of E. gracilis cells were not significantly affected by the complex. Our results suggested that this new imaging platform can be efficiently used for detecting dual intracellular metabolites in live microalgal cells.

Application of electrical treatment on Euglena gracilis for increasing paramylon production.

Paramylon also called ß-1,3-glucan is a value-added product produced from Euglena gracilis. Recently, researchers have developed various strategies for the enhanced paramylon production, among which electrical treatment for microbial stimulation can be an alternative owing to the applicability to large-scale cultivation. In this study, we applied the electrical treatment for enhanced paramylon production and found the proper treatment conditions. Under the treatment with platinum electrodes at 10 mA, the paramylon production of treated cells was significantly increased about 2.5-fold, compared to those of the untreated cells, although the density of cells was maintained due to considerable stress. The size of treated cells became larger, possibly due to the increased level of paramylon production within the cells. Accordingly, the contents of glucose uptake, glucose-6-phosphate (G6P), glucose-1-phosphate (G1P), and uridine diphosphoglucose (UDPG) were shifted to appropriate states for the process of paramylon synthesis under the treatment. The increased level of transcripts encoding glucan synthase-like 2 (EgGSL2) was also confirmed via droplet digital PCR (ddPCR) under the treatment. Overall, this study makes a major contribution to research on electrical stimulation and provides new insights into E. gracilis metabolism like paramylon synthesis. KEY POINTS: • Electrical treatment induced the paramylon production and morphological change of Euglena gracilis. • The glucose uptake of E. gracilis was increased during the electrical treatment, fueling the paramylon synthesis.

Detection of Particulate Matters with a Field-Portable Microscope Using Side-Illuminated Total Internal Reflection.

Field-portable observation and analysis of particulate matter (PM) is required to enhance healthy lives. Various types of the PM measurement methods are in use; however, each of these methods has significant limitations in that real time measurement is impossible, the detection system is bulky, or the measurement accuracy is insufficient. In this work, we introduce an optical method to perform a fast and accurate PM analysis with a higher-contrast microscopic image enabled by a side-illuminated total internal reflection (TIR) technique to be implemented in a compact device. The superiority of the proposed method was quantitatively demonstrated by comparing the signal-to-noise ratio of the proposed side-illuminated TIR method with a traditional halogen lamp-based transmission microscope. With the proposed device, signal-to-noise ratios (SNRs) for microbeads (5~20 µm) and ultrafine dust particles (>5 µm) increased 4.5~17 and 4~10 dB, respectively, compared to the conventional transmission microscope. As a proof of concept, the proposed method was also applied to a low-cost commercial smartphone toy microscope enabling field-portable detection of PMs. We believe that the proposed side-illuminated TIR PM detection device holds significant advantages over other commonly used systems due to its sufficient detection capability along with simple and compact configuration as well as low cost.

Optimization of PTFE Coating on PDMS Surfaces for Inhibition of Hydrophobic Molecule Absorption for Increased Optical Detection Sensitivity.

Polydimethylsiloxane (PDMS) is a polymer widely used for fabrication and prototyping of microfluidic chips. The porous matrix structure of PDMS allows small hydrophobic molecules including some fluorescent dyes to be readily absorbed to PDMS and results in high fluorescent background signals, thereby significantly decreasing the optical detection sensitivity. This makes it challenging to accurately detect the fluorescent signals from samples using PDMS devices. Here, we have utilized polytetrafluoroethylene (PTFE) to inhibit absorption of hydrophobic small molecules on PDMS. Nile red was used to analyze the effectiveness of the inhibition and the absorbed fluorescence intensities for 3% and 6% PTFE coating (7.7 ± 1.0 and 6.6 ± 0.2) was twofold lower compared to 1% and 2% PTFE coating results (17.2 ± 0.5 and 15.4 ± 0.5). When compared to the control (55.3 ± 1.6), it was sevenfold lower in background fluorescent intensity. Furthermore, we validated the optimized PTFE coating condition using a PDMS bioreactor capable of locally stimulating cells during culture to quantitatively analyze the lipid production using Chlamydomonas reinhardtii CC-125. Three percent PTFE coating was selected as the optimal concentration as there was no significant difference between 3% and 6% PTFE coating. Intracellular lipid contents of the cells were successfully stained with Nile Red inside the bioreactor and 3% PTFE coating successfully minimized the background fluorescence noise, allowing strong optical lipid signal to be detected within the PDMS bioreactor comparable to that of off-chip, less than 1% difference.

Dynamic m6A modification regulates local translation of mRNA in axons.

N6-methyladenosine (m6A) is a reversible modification in mRNA and has been shown to regulate processing, translation and decay of mRNA. However, the roles of m6A modification in neuronal development are still not known. Here, we found that the m6A eraser FTO is enriched in axons and can be locally translated. Axon-specific inhibition of FTO by rhein, or compartmentalized siRNA knockdown of Fto in axons led to increases of m6A levels. GAP-43 mRNA is modified by m6A and is a substrate of FTO in axons. Loss-of-function of this non-nuclear pool of FTO resulted in increased m6A modification and decreased local translation of axonal GAP-43 mRNA, which eventually repressed axon elongation. Mutation of a predicted m6A site in GAP-43 mRNA eliminated its m6A modification and exempted regulation of its local translation by axonal FTO. This work showed an example of dynamic internal m6A demethylation of non-nuclear localized mRNA by the demethylase FTO. Regulation of m6A modification of axonal mRNA by axonal FTO might be a general mechanism to control their local translation in neuronal development.

Effects of micro-sized biodegradable plastics on Microcystis aeruginosa.

Plethora of plastics are being used in current society, generating huge amounts of plastic waste. Non-biodegradability of conventional plastics is one of the main challenges to treat plastic waste. In an effort to increase the efficiency of plastic waste treatment, biodegradable plastics have gained attention. Although the use of biodegradable plastics has been increased, their potential effects on the environments are not fully elucidated yet. In this study, the impacts of micro-sized non-biodegradable plastic (i.e., polystyrene (PS)) and micro-sized biodegradable plastics (i.e., polycaprolactone (PCL) and polylactic acid (PLA)) on Microcystis aeruginosa were investigated. Regardless of microplastic (MP) types, MP treatments inhibited the growth of M. aeruginosa at the beginning (4 days) while significant dose-dependent effect was not observed in the range of 0.1 to 10 mg/L. However, after long-term exposure (12 days), micro-sized biodegradable plastics stimulated the growth of M. aeruginosa (up to 73 % increase compared to the control). The photosynthetic activity showed a similar trend to the cell growth. The MP treatments induced the production of extracellular polymeric substances (EPS). Indeed, micro-sized PCL and PLA stimulated the production of protein compounds in EPS. These might have affected the releases of chemicals from PCL and PLA, suggesting that the chemicals in biodegradable plastic leachates would promote the growth of M. aeruginosa in long-term exposure. The MP treatments also induced cyanotoxin (microcystin-LR) productions. Our results give a new insight into the cyanobacterial blooming and suggest a novel relationship between harmful algal blooms (HABs) and biodegradable plastics.

Azimuthal rotation-controlled nanoinscribing for continuous patterning of period- and shape-tunable asymmetric nanogratings.

We present an azimuthal-rotation-controlled dynamic nanoinscribing (ARC-DNI) process for continuous and scalable fabrication of asymmetric nanograting structures with tunable periods and shape profiles. A sliced edge of a nanograting mold, which typically has a rectangular grating profile, slides over a polymeric substrate to induce its burr-free plastic deformation into a linear nanopattern. During this continuous nanoinscribing process, the "azimuthal angle," that is, the angle between the moving direction of the polymeric substrate and the mold's grating line orientation, can be controlled to tailor the period, geometrical shape, and profile of the inscribed nanopatterns. By modulating the azimuthal angle, along with other important ARC-DNI parameters such as temperature, force, and inscribing speed, we demonstrate that the mold-opening profile and temperature- and time-dependent viscoelastic polymer reflow can be controlled to fabricate asymmetric, blazed, and slanted nanogratings that have diverse geometrical profiles such as trapezoidal, triangular, and parallelogrammatic. Finally, period- and profile-tunable ARC-DNI can be utilized for the practical fabrication of diverse optical devices, as is exemplified by asymmetric diffractive optical elements in this study.

Hollow Microcavity Electrode for Enhancing Light Extraction.

Luminous efficiency is a pivotal factor for assessing the performance of optoelectronic devices, wherein light loss caused by diverse factors is harvested and converted into the radiative mode. In this study, we demonstrate a nanoscale vacuum photonic crystal layer (nVPCL) for light extraction enhancement. A corrugated semi-transparent electrode incorporating a periodic hollow-structure array was designed through a simulation that utilizes finite-difference time-domain computational analysis. The corrugated profile, stemming from the periodic hollow structure, was fabricated using laser interference lithography, which allows the precise engineering of various geometrical parameters by controlling the process conditions. The semi-transparent electrode consisted of a 15 nm thick Ag film, which acted as the exit mirror and induced microcavity resonance. When applied to a conventional green organic light-emitting diode (OLED) structure, the optimized nVPCL-integrated device demonstrated a 21.5% enhancement in external quantum efficiency compared to the reference device. Further, the full width at half maximum exhibited a 27.5% reduction compared to that of the reference device, demonstrating improved color purity. This study presents a novel approach by applying a hybrid thin film electrode design to optoelectronic devices to enhance optical efficiency and color purity.

Surveillance spilled Chironomidae (Diptera) larvae from drinking water treatment plants in South Korea using morphogenetic species analysis and eDNA metabarcoding.

Chironomid larvae (Diptera: Chironomidae) are tremendous indicator species that can tolerate a broad range of environmental conditions, from polluted to unimpaired water ecosystems. These species are ubiquitously observed in all bioregions and can even be found in drinking water treatment plants (DWTPs). Detection of chironomid larvae in DWTPs is a critical issue because their presence may be indicative of the water quality in the supply of tap water for human consumption. Therefore, the aim of the present study was to identify the chironomid communities that reflect the water quality of DWTPs and develop a biomonitoring tool to detect biological contamination of the chironomids in DWTPs. To do so, we investigated the identity and distribution of chironomid larvae in seven DWTP areas using morphological identification, DNA barcoding, and sediment environmental DNA (eDNA) analysis. A total of 7924 chironomid individuals encompassing three subfamilies and 25 species of 19 genera were identified in 33 sites within the DWTPs. The Gongchon and Bupyeong DWTPs were dominated by Chironomus spp. larvae, which were correlated with low levels of dissolved oxygen in the water. In the Samgye DWTP and Hwajeong DWTP, Chironomus spp. were almost absent, and instead, Tanytarsus spp. were abundant. Additionally, the Gangjeong DWTP was dominated by a Microtendipes sp., and two species of Orthocladiinae (a Parametriocnemus sp. and a Paratrichocladius sp.) were found only in the Jeju DWTP. We also identified the eight most abundant Chironomidae larvae found in the DWTPs. Furthermore, eDNA metabarcoding of DWTP sediment indicated the presence of different eukaryotic fauna and confirmed the presence of chironomids in DWTPs. These data provide useful morphological and genetic information regarding chironomid larvae that can be used for the water quality biomonitoring of DWTPs to support the supply of clean drinking water.

Monodisperse Micro-Droplet Generation in Microfluidic Channel with Asymmetric Cross-Sectional Shape.

Micro-droplets are widely used in the fields of chemical and biological research, such as drug delivery, material synthesis, point-of-care diagnostics, and digital PCR. Droplet-based microfluidics has many advantages, such as small reagent consumption, fast reaction time, and independent control of each droplet. Therefore, various micro-droplet generation methods have been proposed, including T-junction breakup, capillary flow-focusing, planar flow-focusing, step emulsification, and high aspect (height-to-width) ratio confinement. In this study, we propose a microfluidic device for generating monodisperse micro-droplets, the microfluidic channel of which has an asymmetric cross-sectional shape and high hypotenuse-to-width ratio (HTWR). It was fabricated using basic MEMS processes, such as photolithography, anisotropic wet etching of Si, and polydimethylsiloxane (PDMS) molding. Due to the geometric similarity of a Si channel and a PDMS mold, both of which were created through the anisotropic etching process of a single crystal Si, the microfluidic channel with the asymmetric cross-sectional shape and high HTWR was easily realized. The effects of HTWR of channels on the size and uniformity of generated micro-droplets were investigated. The monodisperse micro-droplets were generated as the HTWR of the asymmetric channel was over 3.5. In addition, it was found that the flow direction of the oil solution (continuous phase) affected the size of micro-droplets due to the asymmetric channel structures. Two kinds of monodisperse droplets with different sizes were successfully generated for a wider range of flow rates using the asymmetric channel structure in the developed microfluidic device.

The cytotoxicity of nano- and micro-sized graphene oxides on microalgae depends on the characteristics of cell wall and flagella.

Cytotoxic effects of emerging contaminants in aquatic environments have been widely studied using diverse microalgal species. However, the role of microalgal characteristics such as presence/absence of cell wall or flagella on cytotoxicity of contaminants was not elucidated yet. In this study, four different Chlamydomonas reinhardtii strains that have different characteristics were used to confirm how these characteristics affect toxicity of contaminants, nano-/micro-sized graphene oxide (GO). The nano-sized GO inhibited the growth of cell wall-deficient strains and reduced the photosynthetic activity. The micro-sized GO inhibited the growth of all strains, but the inhibition efficiency was higher in flagella-deficient strains, indicating that cell wall and flagella have different roles in response to contaminant exposure. The electron microscopy analysis demonstrated that nano-sized GO caused the cell rupture in cell wall-deficient strains. In flagella-deficient strains, the nano- and micro-sized GOs were parallelly attached on the surface of cells, covering the cells. The wrapping of flagella-deficient cells by GO led to the increase of reactive oxygen species (ROS) contents. These results indicate main cytotoxic mechanism of nano-sized GO was the membrane damage of cells, and the presence of cell wall can protect the cells from the attack of nano-sized GO. On the one hand, the presence of flagella might help to avoid the attachment of GO while the cell proliferation and photosynthesis were inhibited in flagella-deficient cells due to the GO wrapping. Overall, given that different microalgal species have different characteristics and these characteristics might affect the cytotoxic effect of the contaminants, it is of great importance to consider the characteristics of test microalgal species when evaluating the cytotoxic mechanism of the nano-/micro-sized pollutants.

Recent Advancements in Technologies to Detect Enterohaemorrhagic Escherichia coli Shiga Toxins.

Shiga toxin (Stxs)-producing enterohaemorrhagic Escherichia coli (EHEC) and Shigella dysenteriae serotype 1 are major causative agents of severe bloody diarrhea (known as hemorrhagic colitis) and hemolytic uremic syndrome (HUS) associated with extraintestinal complications such as acute renal failure and neurologic impairment in infected patients under 9 years of age. Extreme nephrotoxicity of Stxs in HUS patients is associated with severe outcomes, highlighting the need to develop technologies to detect low levels of the toxin in environmental or food samples. Currently, the conventional polymerase chain reaction (PCR) or immunoassay is the most broadly used assay to detect the toxin. However, these assays are laborious, time-consuming, and costly. More recently, numerous studies have described novel, highly sensitive, and portable methods for detecting Stxs from EHEC. To contextualize newly emerging Stxs detection methods, we briefly explain the basic principles of these methods, including lateral flow assays, optical detection, and electrical detection. We subsequently describe existing and newly emerging rapid detection technologies to identify and measure Stxs.

Ultrasonic Treatment Enhanced Astaxanthin Production of Haematococcus pluvialis.

In this study, effects of ultrasonic treatment on Haematococcus pluvialis (H. pluvialis) were investigated. It has been confirmed that the ultrasonic stimulation acted as stress resources in the red cyst stage H. pluvialis cells containing astaxanthin, resulting in additional astaxanthin production. With the increase in production of astaxanthin, the average diameter of H. pluvialis cells increased accordingly. In addition, to determine how ultrasonic stimulation had an effect on the further biosynthesis of astaxanthin, genes related to astaxanthin synthesis and cellular ROS level were measured. As a result, it was confirmed that astaxanthin biosynthesis related genes and cellular ROS levels were increased, and thus ultrasonic stimulation acts as an oxidative stimulus. These results support the notion on the effect of the ultrasonic treatment, and we believe our novel approach based on the ultrasonic treatment would help to enhance the astaxanthin production from H. pluvialis.

Volatile fatty acid-treated mixotrophic cultivation of lipid/carbohydrate-rich cyanobacterial species, Pseudanabaena mucicola GO0704, for the enhancement of biofuel production.

Cyanobacteria-derived biofuels can be helpful in achieving a circular bioeconomy. To increase the production of biodiesel/bioethanol from cyanobacterium, Pseudanabaena mucicola GO0704, mixotrophic cultivation using volatile fatty acid (VFA), a cheap organic carbon source, was performed. The treatment of butyric acid or acetic acid enhanced the cell growth, particularly, the dry weight of the butyric acid-treated cells was 2.30-fold higher than the control. The enhancement of the growth led to the increase of metabolite (i.e., lipid and carbohydrate) productions, resulting in high amount of biodiesel and bioethanol to be produced. Butyric acid was more effective compared to acetic acid and the productions of biodiesel (52.2 mg/L) and bioethanol (132.6 mg/L) from the butyric acid-treated P. mucicola GO0704 were 2.34- and 2.17-fold higher compared to the control, respectively. This study will provide a foundation to commercialize the cyanobacteria-based carbon-neutral fuels, and ultimately, achieve a circular bioeconomy.

Facilitation of axonal transcriptome analysis with quantitative microfluidic devices.

Microfluidic chambers are powerful tools for studying axonal mRNA localization and translation in neurons. In addition to specific manipulation and measurements of axons, microfluidic chambers are used for collecting axonal materials to perform axonal transcriptome analysis. However, traditional bipartite and tripartite chambers have limitations either in purity or quantity of collected axons. Here, we improved the design of traditional chambers. Moreover, we developed two new quantitative chambers, multi-compartmental quantitative bipartite chamber (MQBC) and long quantitative tripartite chamber (LQTC). Compared with the traditional chambers, MQBC and LQTC could dramatically increase the efficiency in collecting axonal RNA. Finally, we applied these chambers to do comparative axon transcriptome analysis of different types of neurons. Thus, our newly designed quantitative chambers significantly improve axon collection efficiency and facilitate axonal transcriptome analysis.

Genetic polymorphism and natural selection in the C-terminal 42 kDa region of merozoite surface protein-1 among Plasmodium vivax Korean isolates.

BACKGROUND: The carboxy-terminal 42 kDa region of Plasmodium vivax merozoite surface protein-1 (PvMSP-142) is a leading candidate antigen for blood stage vaccine development. However, this region has been observed to be highly polymorphic among filed isolates of P. vivax. Therefore it is important to analyse the existing diversity of this antigen in the field isolates of P. vivax. In this study, the genetic diversity and natural selection in PvMSP-142 among P. vivax Korean isolates were analysed. METHODS: A total of 149 P. vivax-infected blood samples collected from patients in Korea were used. The region flanking PvMSP-142 was amplified by PCR, cloned into Escherichia coli, and then sequenced. The polymorphic characteristic and natural selection of PvMSP-142 were analysed using the DNASTAR, MEGA4 and DnaSP programs. RESULTS: A total of 11 distinct haplotypes of PvMSP-142 with 40 amino acid changes, as compared to the reference Sal I sequence, were identified in the Korean P. vivax isolates. Most of the mutations were concentrated in the 33 kDa fragment (PvMSP-133), but a novel mutation was found in the 19 kDa fragment (PvMSP-119). PvMSP-142 of Korean isolates appeared to be under balancing selection. Recombination may also play a role in the resulting genetic diversity of PvMSP-142. CONCLUSIONS: PvMSP-142 of Korean P. vivax isolates displayed allelic polymorphisms caused by mutation, recombination and balancing selection. These results will be useful for understanding the nature of the P. vivax population in Korea and for development of a PvMSP-142 based vaccine against P. vivax.

Quantitative determination of Plasmodium parasitemia by flow cytometry and microscopy.

The traditional light microscopy has limitations for precise growth assays of malaria parasites in culture or for assessment of new compounds for antimalarial activity; the speed and high reproducibility of flow cytometry can overcome these limitations. A flow cytometric method using PicoGreen, a DNA-binding fluorochrome, was developed with optimal precision suitable for performing growth assays of low-parasitemia field isolates. In addition, intra- and inter-person reproducibility of the flow cytometric and the microscopic method were compared in order to quantitatively demonstrate the improved precision. RNase treatment contributed to the precision of the flow cytometric measurements by enhancing the signal-to-noise ratios. Coefficients of variation of the method were smaller than 10% for 0.1% or higher parasitemia samples. The intra- and inter-person coefficients of variation of the flow cytometric method were three to six times smaller than those of the microscopic method. The flow cytometric method developed in this study yielded substantially more precise results than the microscopic method, allowing determination of parasitemia levels of 0.1% or higher, with coefficients of variation smaller than 10%. Thus, the PicoGreen method could be a reliable high sensitivity assay for analysis of low parasitemia samples and might be applied to a high throughput system testing antimalarial drug activity.