Effects of yogurt containing probiotics on respiratory virus infections: Influenza H1N1 and SARS-CoV-2.

Respiratory virus infections are an escalating issue and have become common worldwide. Influenza and COVID-19 are typical infectious respiratory diseases, and they sometimes lead to various complications. In a situation in which no established drug or treatment exists, consumption of proper food might be beneficial in maintaining health against external infections. We studied the potential effects of mixtures of probiotic strains on various viral infections. The purpose of this study was to assess the ability of yogurt containing probiotics to reduce the risk of respiratory viruses such as influenza H1N1 and SARS-CoV-2 infection. First, we performed in vitro tests using infected Madin-Darby canine kidney (MDCK) and Vero E6 cells, to evaluate the potential effects of yogurt containing high-dose probiotics against influenza H1N1 and SARS-CoV-2 infection. The yogurt significantly reduced plaque formation in the virus-infected cells. We also performed in vivo tests using influenza H1N1-infected C57BL/6 mice and SARS-CoV-2-infected Syrian golden hamsters, to evaluate the potential effects of yogurt. Yogurt was administered orally once daily during the experimental period. Yogurt was also administered orally as pretreatment once daily for 3 wk before viral infection. Regarding influenza H1N1, it was found that yogurt caused an increase in the survival rate, body weight, and IFN-γ, IgG1, and IL-10 levels against viral infection and a decrease in the inflammatory cytokines TNF-α and IL-6. Although the SARS-CoV-2 copy number was not significantly reduced in the lungs of yogurt-treated SARS-CoV-2-infected hamsters, the body weights and histopathological findings of the lungs were improved in the yogurt-treated group. In conclusion, we suggest that consumption of yogurt containing probiotics can lead to beneficial effects to prevent respiratory viral infections.

Facile Green Synthesis of Titanium Dioxide Nanoparticles by Upcycling Mangosteen (Garcinia mangostana) Pericarp Extract.

In the present report, green synthesis of titanium dioxide nanoparticles (TiO2 NPs) was performed by upcycling mangosteen (Garcinia mangostana) pericarp extract (methanol and ethyl acetate extracts). Field emission scanning electron microscopy images revealed an aggregated structure with a highly porous network of TiO2 NPs. TiO2 NPs synthesized with ethyl acetate extract (EtOAc-TiO2 NPs) exhibited more monodispersity and possessed smoother surfaces than the control TiO2 NPs (Con-TiO2 NPs) and TiO2 NPs synthesized with methanol extract (MeOH-TiO2 NPs). High-resolution X-ray diffraction patterns clearly confirmed that TiO2 NPs had a crystalline nature. A mixture of anatase and rutile was observed in Con-TiO2 NPs and MeOH-TiO2 NPs, while EtOAc-TiO2 NPs had only anatase with the smallest size (12.50 ± 1.81 nm). Ethyl acetate extract contained the highest amount of α-mangostin; thus, the surface of TiO2 NPs was functionalized with ethyl acetate extract. The functionalized TiO2 NPs synthesized with ethyl acetate extract (EtOAc-TiO2-αm) showed the highest 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) radical scavenging activity. In vitro cell viability on mouse fibroblast cells (NIH3T3) indicated that the newly synthesized TiO2 NPs did not show any significant cytotoxicity. Therefore, the TiO2 NPs in the present report have the potential to be used in cosmetic applications such as sunscreens.

Purinergic Signaling Involvement in Thymosin ß4-mediated Corneal Epithelial Cell Migration.

Purpose: This study aimed to determine the effect of thymosin beta 4 (Tß4) on human corneal epithelial cell migration and the downstream signaling pathways. Tß4 has a role in tissue development, cell migration, inflammation, and wound healing. A previous study showed that Tß4 directly binds to F0-F1 ATP synthase. Other studies reported the role of extracellular ATP and purinergic receptors in cell migration with several cell types. Despite advancing to the clinical stage for treatment of eye disorders, the effect of Tß4 on human corneal epithelial cell (HCEC) migration and proliferation and the precise downstream signaling pathway(s) have not been identified. Methods: Various concentrations of Tß4 were tested in vitro on human corneal epithelial cell proliferation using the CCK-8 Kit and on cell migration using the gap closure migration assay. Additionally, ATP levels at various time points were determined using the ATP Lite One-Step Kit. The Fluo 8 NO Wash Calcium Assay Kit was used to measure the intracellular Ca2+ concentration after treatment with various concentrations of Tß4. P2X7 inhibitors were tested on ATP signaling and migration. Total- and phospho-ERK1/2 levels were determined in western blot. Results: Tß4 enhanced HCEC proliferation and migration in a dose- and time-dependent manner. Moreover, these functions were related to increased extracellular ATP levels, intracellular Ca2+ influx, and ERK1/2 phosphorylation. Tß4-mediated HCEC migration was inhibited by specific P2X7 purinergic receptor antagonists suggesting the role of this receptor in Tß4-mediated human corneal epithelial cell migration. Conclusions: These results suggest that Tß4-mediated HCEC proliferation and migration are associated with increased ATP levels, P2X7 R-mediated Ca2+ influx, and the ERK1/2 signaling pathway. This study begins to describe the mechanisms for Tß4-mediated corneal healing and regeneration.

Author Correction: B-cell translocation gene 2 enhances fibroblast growth factor 21 production by inducing Kruppel-like factor 15.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Development of p-Coumaric Acid Analysis in Human Plasma and Its Clinical Application to PK/PD Study.

It has been recognized that p-Coumaric acid (p-CA) has protective effects as an antioxidant, anti-inflammatory agent. A sensitive and efficient Liquid Chromatography-Mass Spectrometry (LC-MS) method for maximum determination of p-CA in human plasma has been established using Ultra-performance liquid Chromatography-tandem mass Spectrometry (UPLC-MS/MS). This study provides the developed analysis of p-CA extracted from Bambusae Caulis in Taeniam (BC) to examine the improvement of the treatment p-CA, IGF-1 and Osteocalcin level in human children which are important factors on the growth of children's height through Pharmacokinetics/Pharmacodynamics (PK/PD) model. p-CA and internal standard in a plasma sample were detected by the Multiple Reaction Monitoring (MRM) scan mode with positive ion detection. The sample participating in the study was made of 34 subjects (placebo = 18, treatment = 16). The subjects were enrolled to be randomized to the control group and BC group. Randomized subjects took tested treatment twice a day, three capsules with oral administration (258 mg/capsule) each time after a meal. Standard calibration curves (reproducibility) were constructed and the lower limit of quantitation (LLOQ) for p-CA was found to be 0.2 ng/mL on injection of the sample into the UPLC-MS/MS system. Accuracy and precision were evaluated and the intra-accuracy was 99.2-103.8% with precision of 1.0-5.6% and inter-accuracy was 99.6-108.4% and precision of 1.3-6.4% for p-CA. The method has been successfully applied to PK/PD studies of p-CA in human plasma. The p-CA, BC in Taeniam extract increased the level of IGF-1 and Osteocalcin, and changed the height from baseline, which suggested that the p-CA could play an important role in longitudinal bone growth. Therefore, the p-CA extracted from BC in Taeniam might be a good alternative medicine to growth hormone (GH) therapy.

Electrospining of Nanofibrous Meshes Composed of Hypromellose and Poly(vinyl alcohol) for One-Day Release of Cationic Peptide.

Various biocompatible polymers have been developed using electrospun nanofibers for local drug delivery matrices, but many of them are non-FDA-approved polymers or chemicals. Material safety should be considered in biomedical devices, but the effectiveness of electrospun nanofibers is limited with only the approved chemicals. Therefore, we considered the material in FDA-approved polymers and solvents and developed nanofibers using the general additives in the pharmaceutical industry, such as hypromellose, poly(vinyl alcohol) (PVA), and Gellan. The nanofiber, mainly composed of hypromellose and PVA, was applied to a local peptide drug delivery system. Electrostatically complexed Gellan and peptide were loaded in the nanofiber by co-electrospinning. The morphology of the nanofibers with different PVA blending ratios was visualized by scanning electron microscope. The nanofiber composed of only hydrophilic polymers quickly wetted in water and became a transparent gel-like lump. A drug release test of peptide-loaded nanofiber was performed resulted in 37% of initial burst release suppression with the gellan and peptide loaded nanofiber compared with solely peptide loaded hypromellose nanofiber. In addition, higher PVA blending to hypromellose was slightly effective for sustained release of peptide compared with pure peptide-loaded hypromellose nanofiber. Therefore, we suggest the potential application of hypromellose/PVA nanofiber-loaded Gellan/peptide complex to a mucosal layer drug delivery patch.

B-cell translocation gene 2 enhances fibroblast growth factor 21 production by inducing Kruppel-like factor 15.

Fibroblast growth factor 21 (FGF21) is a hormone that is vital for the regulation of metabolic homeostasis. In the present study, we report that Kruppel-like factor 15 (KLF15) is a novel mediator of b-cell translocation gene 2 (BTG2)-induced FGF21 biosynthesis. The expression levels of hepatic Fgf21, Btg2, and Klf15, and the production of serum FGF21 increased significantly in fasted and forskolin (FSK)-treated mice. The overexpression of Btg2 using an adenoviral delivery system elevated FGF21 production by upregulating Klf15 transcription. Interaction studies indicated that BTG2 was co-immunoprecipitated with KLF15 and recruited by the Fgf21 promoter. The disruption of hepatic Btg2 and Klf15 genes markedly attenuated the induction of Fgf21 expression and FGF21 biosynthesis in fasted mice. Similarly, the FSK-mediated induction of Fgf21 promoter activity was strikingly ablated by silencing of Btg2 and Klf15. Taken together, these findings suggest that KLF15 and BTG2 are mediators of fasting-induced hepatic FGF21 expression. Therefore, targeting BTG2 and KLF15 might be a therapeutically important strategy for combat metabolic dysfunction.

OKN-007 Increases temozolomide (TMZ) Sensitivity and Suppresses TMZ-Resistant Glioblastoma (GBM) Tumor Growth.

Treatment of glioblastoma (GBM) remains a challenge using conventional chemotherapy, such as temozolomide (TMZ), and is often ineffective as a result of drug resistance. We have assessed a novel nitrone-based agent, OKN-007, and found it to be effective in decreasing tumor volumes and increasing survival in orthotopic GBM xenografts by decreasing cell proliferation and angiogenesis and increasing apoptosis. In this study, we assessed combining OKN-007 with TMZ in vivo in a human G55 GBM orthotopic xenograft model and in vitro in TMZ-resistant and TMZ-sensitive human GBM cell lines. For the in vivo studies, magnetic resonance imaging was used to assess tumor growth and vascular alterations. Percent animal survival was also determined. For the in vitro studies, cell growth, IC50 values, RNA-seq, RT-PCR, and ELISA were used to assess growth inhibition, possible mechanism-of actions (MOAs) associated with combined OKN-007 + TMZ versus TMZ alone, and gene and protein expression levels, respectively. Microarray analysis of OKN-007-treated rat F98 glioma tumors was also carried out to determine possible MOAs of OKN-007 in glioma-bearing animals either treated or not treated with OKN-007. OKN-007 seems to elicit its effect on GBM tumors via inhibition of tumorigenic TGF-ß1, which affects the extracellular matrix. When combined with TMZ, OKN-007 significantly increases percent survival, decreases tumor volumes, and normalizes tumor blood vasculature in vivo compared to untreated tumors and seems to affect TMZ-resistant GBM cells possibly via IDO-1, SUMO2, and PFN1 in vitro. Combined OKN-007 + TMZ may be a potentially potent treatment strategy for GBM patients.

Real-Time Lossless Compression Algorithm for Ultrasound Data Using BL Universal Code.

Software-based ultrasound imaging systems provide high flexibility that allows easy and fast adoption of newly developed algorithms. However, the extremely high data rate required for data transfer from sensors (e.g., transducers) to the ultrasound imaging systems is a major bottleneck in the software-based architecture, especially in the context of real-time imaging. To overcome this limitation, in this paper, we present a Binary cLuster (BL) code, which yields an improved compression ratio compared to the exponential Golomb code. Owing to the real-time encoding/decoding features without overheads, the universal code is a good solution to reduce the data transfer rate for software-based ultrasound imaging. The performance of the proposed method was evaluated using in vitro and in vivo data sets. It was demonstrated that the BL-beta code has a good stable lossless compression performance of 20%~30% while requiring no auxiliary memory or storage.

RGN-259 (thymosin ß4) improves clinically important dry eye efficacies in comparison with prescription drugs in a dry eye model.

This study evaluated the clinical activity of RGN-259 (thymosin ß4) in comparison with cyclosporine A (CsA), diquafosol (DQS), and lifitegrast (LFA) in a murine model of dry eye. The model was NOD.B10-H2b mice in a 30-40% humidified environment together with daily scopolamine hydrobromide injections for 10 days. After desiccation stress, all drugs were evaluated after 10 treatment days. RGN-259 increased tear production similar to that in the DQS- and LFA-treated mice while CsA was inactive. RGN-259 improved corneal smoothness and decreased fluorescein staining similar to that of LFA group while CsA and DQS were inactive. Corneal epithelial detachment was reduced by RGN-259, and DQS and LFA showed similar activity but the CsA was inactive. RGN-259 increased conjunctival goblet cells and mucin production comparable to that seen with CsA, while DQS and LFA were inactive. RGN-259 reduced the over-expression of inflammatory factors comparable to that with CsA and LFA, while DQS was inactive. RGN-259 increased mucin production comparable to that observed with CsA, while DQS and LFA were inactive. In conclusion, RGN-259 promoted recovery of mucins and goblet cells, improved corneal integrity, and reduced inflammation in a dry eye mouse model and was equal to or more effective than prescription treatments.

Removing Control of Cyclodextrin-Drug Complexes Using High Affinity Molecule.

Nanostructured supramolecular assemblies with hydrophobic cavities are used for improving the solubility, bioavailability, and stability of poorly water soluble drugs. In particular, host-guest inclusion using 2-hydroxypropyl-beta-cyclodextrin (HP-ß-CD) is a typical approach in the pharmaceutical field. In this study, celecoxib (CXB), a cyclooxygenase-2 selective nonsteroidal anti-inflammatory drug (NSAID), was used as the model drug (guest material) and effectively incorporated into HP-ß-CD (host material). After forming a complete complex of HP-ß-CD and CXB, 1-adamantylamine (ADA) was used to allow CXB to be released from the HP-ß-CD in a concentration-dependent manner. This was revealed from Fourier-transform infrared spectroscopy and drug dissolution studies. Notably, the use of ADA, which is a high-affinity guest molecule, with cyclodextrin accelerated the removal of CXB from the host material through the exchange of guest molecules. Taken together, the host-guest based approach using a second guest molecule is useful for regulating on-demand drug release and could therefore be a potential tool for biomedical applications.

Improved Dissolution and Oral Bioavailability of Celecoxib by a Dry Elixir System.

The purpose of this study was to develop and evaluate a dry elixir (DE) system for enhancing the dissolution rate and oral bioavailability of celecoxib. DE system has been used for improving solubility, oral bioavailability of poorly water-soluble drugs. The encapsulated drugs or solubilized drugs in the matrix are rapidly dissolved due to the co-solvent effect, resting in both an enhanced dissolution and bioavailability. DEs containing celecoxib were prepared by spray-drying method and characterized by morphology, drug/ethanol content, drug crystallinity, dissolution rate and oral bioavailability. The ethanol content and drug content in DE system could be easily altered by controlling the spraydrying conditions. The dissolution profile of celecoxib from DE proved to be much higher than that of celecoxib powder due to the nano-structured matrix, amorphous state and encapsulated ethanol. The bioavailability of celecoxib from DEs was compared with celecoxib powder alone and commercial product (Celebrex®) in rats. In particular, blood concentrations of celecoxib form DE formulation were much greater than those of native celecoxib and market product. The data demonstrate that the DE system could provide an useful solid dosage form to enhance the solubility, dissolution rate and oral bioavailability of celecoxib.

Platycodon saponins from Platycodi Radix (Platycodon grandiflorum) for the Green Synthesis of Gold and Silver Nanoparticles.

A green synthesis of gold and silver nanoparticles is described in the present report using platycodon saponins from Platycodi Radix (Platycodon grandiflorum) as reducing agents. Platycodin D (PD), a major triterpenoidal platycodon saponin, was enriched by an enzymatic transformation of an aqueous extract of Platycodi Radix. This PD-enriched fraction was utilized for processing reduction reactions of gold and silver salts to synthesize gold nanoparticles (PD-AuNPs) and silver nanoparticles (PD-AgNPs), respectively. No other chemicals were introduced during the reduction reactions, providing an entirely green, eco-friendly, and sustainable method. UV-visible spectra showed the surface plasmon resonance bands of PD-AuNPs at 536 nm and PD-AgNPs at 427 nm. Spherically shaped nanoparticles were observed from high-resolution transmission electron microscopy with average diameters of 14.94 ± 2.14 nm for PD-AuNPs and 18.40 ± 3.20 nm for PD-AgNPs. Minor triangular and other polygonal shapes were also observed for PD-AuNPs along with spherical ones. Atomic force microscopy (AFM) images also demonstrated that both nanoparticles were mostly spherical in shape. Curvature-dependent evolution was employed to enhance the AFM images and precisely measure the sizes of the nanoparticles. The sizes were measured as 19.14 nm for PD-AuNPs and 29.93 nm for PD-AgNPs from the enhanced AFM images. Face-centered cubic structures for both nanoparticles were confirmed by strong diffraction patterns from high-resolution X-ray diffraction analyses. Fourier transform infrared spectra revealed the contribution of -OH, aromatic C=C, C-O, and C-H functional groups to the synthesis. Furthermore, the catalytic activity of PD-AuNPs was assessed with a reduction reaction of 4-nitrophenol to 4-aminophenol in the presence of sodium borohydride. The catalytic activity results suggest the potential application of these gold nanoparticles as catalysts in the future. The green strategy reported in this study using saponins as reducing agents will pave new roads to develop novel nanomaterials with versatile applications.

Safety and potential efficacy of gemfibrozil as a supportive treatment for children with late infantile neuronal ceroid lipofuscinosis and other lipid storage disorders.

Neuronal Ceroid Lipofuscinosis (NCL), also known as Batten disease, is a group of genetically distinct lysosomal disorders that mainly affect the central nervous system, resulting in progressive motor and cognitive decline primarily in children. Multiple distinct genes involved in the metabolism of lipids have been identified to date with various mutations in this family of diseases. There is no cure for these diseases but some new therapeutic approaches have been tested that offer more hope than the standard palliative care. Many of the therapeutic advances require invasive procedures but some progress in slowing the disease has been found and more options can be expected in the future. We also review the literature on children with disease/conditions other than NCL for the non-invasive use, safety, and tolerability of a lipid-lowering drug, gemfibrozil, as a potential treatment for NCLs. Gemfibrozil has shown efficacy in an animal model of NCL known as CLN2 (late infantile classic juvenile) and has been shown to be safe for lowering lipids in children. Among the 200 non-NCL children found in the published literature who were treated with gemfibrozil for NCL-related problems, only 3 experienced adverse events, including 2 with muscle pain and 1 with localized linear IgA bullous dermatitis. We conclude that gemfibrozil is safe for long-term use in children, causes minimal adverse events, is well tolerated, and may delay the progression of NCLs. Gemfibrozil may potentially be an alternative to more invasive therapeutic approaches currently under investigation and has the potential to be used in combination with other therapeutic approaches.

Comparative study on secretome and transmembranome of immature and mature metacestodes of Echinococcus multilocularis.

Alveolar echinococcosis (AE) is a worldwide zoonosis caused by E. multilocularis. Humans become infected through oral ingestion of the eggs. Host of E. multilocularis produces immune responses that help to either reject and/or limit the growth of this parasite, and in response the parasite produces molecules against this immune attack. This study identifies candidate key molecules in the early infection phase and the chronic stage of the parasite infestation, through comparison of gene expression of 4- and 16-week metacestodes. First, RNA was isolated from 4- and 16-weeks metacestodes of E. multilocularis (Nemuro strain). Thereafter, clean reads with lengths of 50bp or longer were compared against a reference genome using TopHat. Functional annotation of transcripts of E. multilocularis were investigated using multi-step bioinformatics tools. At the gene ontology (GO) level, 356 and 1774 transmembrane (TM) predicted proteins of the E. multilocularis were mapped to an enhanced 'hydrolase activity' and increased 'transmembrane transporter activity', respectively. In addition, comparison of gene expression level between 4- and 16-week metacestode revealed 168 different expression (DE) genes. This study has demonstrated that, the expression levels of predicted ES and TM proteins in E. multilocularis change in the transformation from one stage to another. Genes that are highly expressed in immature or mature metacestode could be explored as novel candidates for diagnostic antigens and vaccine targets.

Monitoring and risk assessment of polychlorinated biphenyls (PCBs) in agricultural soil from two industrialized areas.

For monitoring and risk assessment, levels and distributions of Σ29 PCBs in paddy soil samples collected from Gwangyang (10 sites) and Ulsan (20 sites), heavily industrialized cities in Korea, were investigated using high-resolution gas chromatography/high-resolution mass spectrometry. Overall, total concentrations of Σ29 PCBs in Gwangyang (216.4-978.6 pg g-1 dw) and Ulsan (273.8-1824.1 pg g-1 dw) were higher than those (106.6-222.6 pg g-1 dw) in agricultural soil from Anseong in Korea. The TEQ (toxic equivalency) values from Gwangyang (0.06-0.40 ng TEQ kg-1 dw) and Ulsan (0.06-0.22 ng TEQ kg-1 dw) were higher than those (0.04-0.11 ng TEQ kg-1 dw) in Anseong but lower than the WHO threshold level (20 ng TEQ kg-1). However, one of the most toxic congeners, PCB 126, gave the highest concentration, possibly posing a risk to the biota. Seven indicator PCB congeners contributed to 50-80% of the total concentration of Σ29 PCBs, indicating the 7 PCBs can be used as valuable indicators for monitoring. The principal component analysis and cluster analysis for the homologue profiles of PCBs indicated that all the samples from both cities had the similar PCB contamination patterns, and the major sources of the PCB contamination were most likely from the usage of Aroclor 1254 than those of Aroclors 1242 and 1260. These PCB technical mixtures were possibly significantly used by various industries including iron and steel industries in Gwangyang and petrochemical and shipbuilding industries in Ulsan.

Catalytic reduction of 4-nitrophenol with gold nanoparticles synthesized by caffeic acid.

In this study, various concentrations of caffeic acid (CA) were used to synthesize gold nanoparticles (CA-AuNPs) in order to evaluate their catalytic activity in the 4-nitrophenol reduction reaction. To facilitate catalytic activity, caffeic acid was removed by centrifugation after synthesizing CA-AuNPs. The catalytic activity of CA-AuNPs was compared with that of centrifuged CA-AuNPs (cf-CA-AuNPs). Notably, cf-CA-AuNPs exhibited up to 6.41-fold higher catalytic activity compared with CA-AuNPs. The catalytic activity was dependent on the caffeic acid concentration, and the lowest concentration (0.08 mM) produced CA-AuNPs with the highest catalytic activity. The catalytic activities of both CA-AuNPs and cf-CA-AuNPs decreased with increasing caffeic acid concentration. Furthermore, a conversion yield of 4-nitrophenol to 4-aminophenol in the reaction mixture was determined to be 99.8% using reverse-phase high-performance liquid chromatography. The product, 4-aminophenol, was purified from the reaction mixture, and its structure was confirmed by 1H-NMR. It can be concluded that the removal of the reducing agent, caffeic acid in the present study, significantly enhanced the catalytic activity of CA-AuNPs in the 4-nitrophenol reduction reaction.

Analysis on Gene Expression Profile in Oncospheres and Early Stage Metacestodes from Echinococcus multilocularis.

Alveolar echinococcosis is a worldwide zoonosis of great public health concern. Analysis of genome data for Echinococcus multilocularis has identified antigen families that can be used in diagnostic assays and vaccine development. However, little gene expression data is available for antigens of the egg and early larval stages. To address this information gap, we used a Next-Generation Sequencing approach to investigate three different stages (non-activated and activated oncospheres, and early stage metacestodes) of E. multilocularis (Nemuro strain). Transcriptome data analysis revealed that some diagnostic antigen gp50 isoforms and the antigen Eg95 family dominated in activated oncospheres, and the antigen B family dominated in early stage metacestodes. Furthermore, heat shock proteins and antigen II/3 are constantly expressed in the three stages. The expression pattern of various known antigens in E. multilocularis may give fundamental information for choosing candidate genes used in diagnosis and vaccine development.

Sporogony and sporozoite rates of avian malaria parasites in wild Culex pipiens pallens and C. inatomii in Japan.

BACKGROUND: Malaria infection in mosquitoes is traditionally detected by microscopic examination for Plasmodium oocysts and sporozoites. Although PCR is now widely used, the presence of parasite DNA in a mosquito does not prove that sporogony is achieved. Thus, detection of sporozoites by microscopy is still required to definitively identify vector mosquitoes. The aim of this study was to confirm sporogony of avian Plasmodium spp. in Culex pipiens pallens and C. inatomii caught from the wild. FINDINGS: Mosquitoes collected at two sites in Japan were dissected and examined by microscopy for Plasmodium oocysts and sporozoites. DNA was extracted from the midgut and salivary gland of infected mosquitoes, and the infecting Plasmodium species was identified by sequencing 478 bp of cytochrome b. Oocysts, or both oocysts and sporozoites, were found in 3.94 and 0.46% of C. p. pallens and C. inatomii, respectively. Four (CXPIP09, GRW4, GRW11 and SGS1) and three cytochrome b lineages (CXINA01, CXINA02 and CXQUI01) were confirmed to achieve sporogony in C. p. pallens and C. inatomii, respectively. One mosquito each of C. p. pallens and C. inatomii was co-infected with two different Plasmodium lineages. CONCLUSIONS: These findings demonstrate that C. p. pallens and C. inatomii are natural vectors of four and three lineages of avian Plasmodium spp., respectively. The data indicate that a systematic procedure combining microscopy and PCR is a feasible and reliable approach to identify natural vectors of wildlife malaria.

First record of avian Plasmodium DNA from mosquitoes collected in the Yaeyama Archipelago, southwestern border of Japan.

We studied the prevalence of avian Plasmodium in 509 mosquitoes of 9 species collected from the Ishigaki and Iriomote islands in the Yaeyama Archipelago, located southwest from the mainland of Japan. Two identical avian Plasmodium lineages were detected from Culex (Culiciomyia) nigropunctatus. Detected lineages were phylogenetically classified into different clade to avian Plasmodium lineages from birds and mosquitoes in the mainland of Japan but identical to a lineage detected from a resident bird, White-breasted Waterken (Amaurornis phoenicurus). This is the first detection of avian Plasmodium DNA from mosquitoes in the Yaeyama Archipelago and suggested that resident birds might have been infected with an avian Plasmodium lineage specific to the studied area and C. nigropunctatus could be the candidate vector mosquito species.