Enhancing malaria detection in resource-limited areas: A high-performance colorimetric LAMP assay for Plasmodium falciparum screening.

Malaria eradication efforts in resource-limited areas require a rapid, economical, and accurate tool for detecting of the low parasitemia. The malaria rapid diagnostic test (mRDT) is the most suitable for on-site detection of the deadliest form of malaria, Plasmodium falciparum. However, the deletions of histidine rich protein 2 and 3 genes are known to compromise the effectiveness of mRDT. One of the approaches that have been explored intensively for on-site diagnostics is the loop-mediated isothermal amplification (LAMP). LAMP is a one-step amplification that allows the detection of Plasmodium species in less than an hour. Thus, this study aims to present a new primer set to enhance the performance of a colorimetric LAMP (cLAMP) for field application. The primer binding regions were selected within the A-type of P. falciparum 18S rRNA genes, which presents a dual gene locus in the genome. The test result of the newly designed primer indicates that the optimal reaction condition for cLAMP was 30 minutes incubation at 65°C, a shorter incubation time compared to previous LAMP detection methods that typically takes 45 to 60 minutes. The limit of detection (LoD) for the cLAMP using our designed primers and laboratory-grown P. falciparum (3D7) was estimated to be 0.21 parasites/µL which was 1,000-fold higher than referencing primers. Under optimal reaction condition, the new primer sets showed the sensitivity (100%, 95% CI: 80.49-100%) and specificity (100%, 95% CI: 94.64-100%) with 100% (95% CI: 95.70-100%) accuracy on the detection of dried blood spots from Malawi (n = 84). Briefly, the newly designed primer set for P. falciparum detection exhibited high sensitivity and specificity compared to referenced primers. One great advantage of this tool is its ability to be detected by the naked eye, enhancing field approaches. Thus, this tool has the potential to be effective for accurate early parasite detection in resource-limited endemic areas.

Gonadal Changes during the Annual Reproductive Cycle of the Ascidian Halocynthia aurantium (Pallas).

The ascidian Halocynthia aurantium (sea peach), a marine invertebrate, belongs to the same genus of the phylum Chordata along with the ascidian Halocynthia roretzi (sea pineapple), which is one of the model animals in the field of developmental biology. The characteristics of development and reproduction of H. aurantium are not yet known in detail. In order to find out the spawning period of H. aurantium, we investigated development of the gonads during the annual reproductive cycle. Testis and ovary were both in the bisexual gonads (ovotestes) of H. aurantium, which is a hermaphrodite like H. roretzi. In H. aurantium, the right gonad was longer and slightly larger than the left gonad throughout the year. In each gonad, the number of the testis gonoducts was slightly higher than that of the ovary gonoducts. These features were similarly observed in H. roretzi. However, the number of the testis gonoducts and the ovary gonoducts in each gonad of H. aurantium was about half that of H. roretzi. The gonads of H. aurantium contracted during the winter and summer seasons. The gonads decreased to the smallest size around February, and then started to increase again in March. The gonads were most developed in September of the year. Therefore, it is estimated that the spawning of H. aurantium begins around this period.

Mitochondria-Specific Monoclonal Antibodies in Eggs and Embryos of the Ascidian Halocynthia roretzi.

Ascidian embryos have become an important model for embryological studies, offering a simple example for mechanisms of cytoplasmic components segregation. It is a well-known example that the asymmetric segregation of mitochondria into muscle lineage cells occurs during ascidian embryogenesis. However, it is still unclear which signaling pathway is involved in this process. To obtain molecular markers for studying mechanisms involved in the asymmetric distribution of mitochondria, we have produced monoclonal antibodies, Mito-1, Mito-2 and Mito-3, that specifically recognize mitochondriarich cytoplasm in cells of the ascidian Halocynthia roretzi embryos. These antibodies stained cytoplasm like reticular structure in epidermis cells, except for nuclei, at the early tailbud stage. Similar pattern was observed in vital staining of mitochondria with DiOC2, a fluorescent probe of mitochondria. Immunostaining with these antibodies showed that mitochondria are evenly distributed in the animal hemisphere blastomeres at cleavage stages, whereas not in the vegetal hemisphere blastomeres. Mitochondria were transferred to the presumptive muscle and nerve cord lineage cells of the marginal zone in the vegetal hemisphere more than to the presumptive mesenchyme, notochord and endoderm lineage of the central zone. Therefore, it is suggested that these antibodies will be useful markers for studying mechanisms involved in the polarized distribution of mitochondria during ascidian embryogenesis.