The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel, and this complex is believed to consist of a pore-forming subunit, MCU, and its regulatory subunits. As yeast cells lack orthologues of the mammalian proteins, the yeast expression system for the mammalian calcium uniporter subunits is useful for investigating their functions. We here established a yeast expression system for the native-form mouse MCU and 4 other subunits. This expression system enabled us to precisely reconstitute the properties of the mammalian MCU complex in yeast mitochondria. Using this expression system, we analyzed the essential MCU regulator (EMRE), which is a key subunit for Ca(2+) uptake but whose functions and structure remain unclear. The topology of EMRE was revealed: its N- and C-termini projected into the matrix and the inter membrane space, respectively. The expression of EMRE alone was insufficient for Ca(2+) uptake; and co-expression of MCU with EMRE was necessary. EMRE was independent of the protein levels of other subunits, indicating that EMRE was not a protein-stabilizing factor. Deletion of acidic amino acids conserved in EMRE did not significantly affect Ca(2+) uptake; thus, EMRE did not have basic properties of ion channels such as ion-selectivity filtration and ion concentration. Meanwhile, EMRE closely interacted with the MCU on both sides of the inner membrane, and this interaction was essential for Ca(2+) uptake. This close interaction suggested that EMRE might be a structural factor for opening of the MCU-forming pore.
BackgroundFive species of Plasmodium are known to infect humans. For proper treatment of malaria, accurate identification of the parasite species is crucial. The current gold standard for malaria diagnosis is microscopic examination of Giemsa-stained blood smears. Since the parasite species are identified by microscopists who manually search for the parasite-infected red blood cells (RBCs), misdiagnosis due to human error tends to occur in case of low parasitaemia or mixed infection. Then, molecular methods, such as polymerase chain reaction or loop-mediated isothermal amplification (LAMP), are required for conclusive identification of the parasite species. However, since molecular methods are highly sensitive, false-positive results tend to occur due to contamination (carry over) or the target gene products may be detected even after clearance of the parasites from the patient’s blood. Therefore, accurate detection of parasites themselves by microscopic examination is essential for the definitive diagnosis. Thus, the method of in situ LAMP for the parasites was developed.ResultsRed blood cell suspensions, including cultured Plasmodium falciparum, strain 3D7, infected-RBCs, were dispersed on cyclic olefin copolymer (COC) plate surfaces rendered hydrophilic by reactive ion-etching treatment using a SAMCO RIE system (hydrophilic-treated), followed by standing for 10 min to allow the RBCs to settle down on the plate surface. By rinsing the plate with RPMI 1640 medium, monolayers of RBCs formed on almost the entire plate surface. The plate was then dried with a hair drier. The RBCs were fixed with formalin, followed by permeabilization with Triton X-100. Then, amplification of the P. falciparum 18S rRNA gene by the LAMP reaction with digoxigenin (DIG)-labelled dUTP and a specific primer set was performed. Infected RBCs as fluorescence-positive cells with anti-DIG antibodies conjugated with fluorescein using fluorescent microscopy could be detected.ConclusionsThe present work shows that the potential of in situ LAMP for the identification of Plasmodium species at the single cell level on hydrophilic-treated COC palates, allowing highly sensitive and accurate malaria diagnosis. The findings will improve the efficacy of the gold standard method for malaria diagnosis.
There is an urgent need to develop an automated malaria diagnostic system that can easily and rapidly detect malaria parasites and determine the proportion of malaria-infected erythrocytes in the clinical blood samples. In this study, we developed a quantitative, mobile, and fully automated malaria diagnostic system equipped with an on-disc SiO 2 nanofiber filter and blue-ray devices. The filter removes the leukocytes and platelets from the blood samples, which interfere with the accurate detection of malaria by the blue-ray devices. We confirmed that the filter, which can be operated automatically by centrifugal force due to the rotation of the disc, achieved a high removal rate of leukocytes (99.7%) and platelets (90.2%) in just 30 s. The automated system exhibited a higher sensitivity (100%) and specificity (92.8%) for detecting Plasmodium falciparum from the blood of 274 asymptomatic individuals in Kenya when compared to the common rapid diagnosis test (sensitivity = 98.1% and specificity = 54.8%). This indicated that this system can be a potential alternative to conventional methods used at local health facilities, which lack basic infrastructure. Globally, malaria is one of the "big three" infectious diseases with an incidence rate of 57 cases per 1000 individuals 1. Malaria is a vector-borne disease, which is caused by infection from Plasmodium spp. and is transmitted by Anopheles mosquitoes. The United Nations Sustainable Development Goals had proposed to end the epidemic of malaria by 2030 2. Although the annual fatality rate has decreased since 2016, about 405,000 malaria-related deaths were reported in 2018. Similarly, new malaria cases have increased slightly since 2014 with 231 million recorded cases in 2017 and 228 million recorded cases in 2018 3. Several factors have stagnated global progress in eradicating malaria 4. Particularly, most tools to tackle the current malaria infection were developed before 2000. Thus, there is a need to develop new tools using novel technologies to accelerate the efforts toward malaria elimination 4 .
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