Wolbachia infection in Aedes aegypti does not affect its vectorial capacity for Dirofilaria immitis.

Mosquito-borne diseases such as dengue and filariasis are a growing public health concern in endemic countries. Biological approaches, such as the trans-infection of Wolbachia pipientis in mosquitoes, are an alternative vector control strategy, especially for arthropod-borne viruses such as dengue. In the present study, the effect of Wolbachia (wMel strain) on the vectorial capacity of Aedes aegypti for Dirofilaria immitis was studied. Our results showed that Wolbachia does not affect the phenotype of mosquito survival or the prevalence, number, and molting rate of third-stage larvae in both susceptible and resistant strains of Ae. aegypti. RNA-seq analysis of Malpighian tubules at 2 days post-infection with D. immitis showed the differentially expressed genes (DEGs) with and without wMel infection. No characteristic immune-related gene expression patterns were observed among the DEGs. No significant change in the amount of Wolbachia was observed in the Ae. aegypti after D. immitis infection. Our results suggest that infection of D. immitis in Ae. aegypti populations will not interfere with Wolbachia-based vector control strategies in dengue-endemic areas where cases of D. immitis are present. This study demonstrated the veterinary medical validity of a dengue control program using Wolbachia.

Association of serum syndecan-1 concentrations with albuminuria in type 2 diabetes.

INTRODUCTION: Syndecan (SDC)-1 is a transmembrane heparan sulfate proteoglycan and is a major component of endothelial glycocalyx (EG). This study aimed to investigate the association of serum SDC-1 concentration as a marker of EG degradation with albuminuria in type 2 diabetes. METHODS: We included 370 patients with type 2 diabetes and 219 individuals with no diabetes. The individuals with estimate glomerular filtration rate <30 mL/min/1.73 m2 were excluded. RESULTS: Serum SDC-1 concentration was higher in type 2 diabetes than in no diabetes. The presence of diabetes was independently associated with log [SDC-1] in multivariate analysis. In type 2 diabetes, serum SDC-1 concentration was correlated with log [urinary albumin-to-creatinine ratio (ACR)]. Moreover, log [SDC-1] was an independent determinant of log [ACR] after adjustment for known risk factors of albuminuria. CONCLUSIONS: Serum SDC-1 concentration was higher in patients with type 2 diabetes compared to individuals with no diabetes and an independent determinant of ACR. This study implicates the role of the EG degradation in albuminuria in type 2 diabetes.

A deep learning-based model to estimate pulmonary function from chest x-rays: multi-institutional model development and validation study in Japan.

BACKGROUND: Chest x-ray is a basic, cost-effective, and widely available imaging method that is used for static assessments of organic diseases and anatomical abnormalities, but its ability to estimate dynamic measurements such as pulmonary function is unknown. We aimed to estimate two major pulmonary functions from chest x-rays. METHODS: In this retrospective model development and validation study, we trained, validated, and externally tested a deep learning-based artificial intelligence (AI) model to estimate forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1) from chest x-rays. We included consecutively collected results of spirometry and any associated chest x-rays that had been obtained between July 1, 2003, and Dec 31, 2021, from five institutions in Japan (labelled institutions A-E). Eligible x-rays had been acquired within 14 days of spirometry and were labelled with the FVC and FEV1. X-rays from three institutions (A-C) were used for training, validation, and internal testing, with the testing dataset being independent of the training and validation datasets, and then x-rays from the two other institutions (D and E) were used for independent external testing. Performance for estimating FVC and FEV1 was evaluated by calculating the Pearson's correlation coefficient (r), intraclass correlation coefficient (ICC), mean square error (MSE), root mean square error (RMSE), and mean absolute error (MAE) compared with the results of spirometry. FINDINGS: We included 141 734 x-ray and spirometry pairs from 81 902 patients from the five institutions. The training, validation, and internal test datasets included 134 307 x-rays from 75 768 patients (37 718 [50%] female, 38 050 [50%] male; mean age 56 years [SD 18]), and the external test datasets included 2137 x-rays from 1861 patients (742 [40%] female, 1119 [60%] male; mean age 65 years [SD 17]) from institution D and 5290 x-rays from 4273 patients (1972 [46%] female, 2301 [54%] male; mean age 63 years [SD 17]) from institution E. External testing for FVC yielded r values of 0·91 (99% CI 0·90-0·92) for institution D and 0·90 (0·89-0·91) for institution E, ICC of 0·91 (99% CI 0·90-0·92) and 0·89 (0·88-0·90), MSE of 0·17 L2 (99% CI 0·15-0·19) and 0·17 L2 (0·16-0·19), RMSE of 0·41 L (99% CI 0·39-0·43) and 0·41 L (0·39-0·43), and MAE of 0·31 L (99% CI 0·29-0·32) and 0·31 L (0·30-0·32). External testing for FEV1 yielded r values of 0·91 (99% CI 0·90-0·92) for institution D and 0·91 (0·90-0·91) for institution E, ICC of 0·90 (99% CI 0·89-0·91) and 0·90 (0·90-0·91), MSE of 0·13 L2 (99% CI 0·12-0·15) and 0·11 L2 (0·10-0·12), RMSE of 0·37 L (99% CI 0·35-0·38) and 0·33 L (0·32-0·35), and MAE of 0·28 L (99% CI 0·27-0·29) and 0·25 L (0·25-0·26). INTERPRETATION: This deep learning model allowed estimation of FVC and FEV1 from chest x-rays, showing high agreement with spirometry. The model offers an alternative to spirometry for assessing pulmonary function, which is especially useful for patients who are unable to undergo spirometry, and might enhance the customisation of CT imaging protocols based on insights gained from chest x-rays, improving the diagnosis and management of lung diseases. Future studies should investigate the performance of this AI model in combination with clinical information to enable more appropriate and targeted use. FUNDING: None.

Development of a novel rodent model for dog heartworm microfilaremia using the severe-combined immunodeficiency mouse.

Dirofilaria immitis is a mosquito-borne parasitic nematode that causes fatal heartworm disease in canids. The microfilariae are essential for research, including drug screening and mosquito-parasite interactions. However, no reliable methods for maintaining microfilaria long-term are currently available. Therefore, we used severe combined immunodeficiency (SCID) mice to develop a reliable method for maintaining D. immitis microfilaria. SCID mice were injected intravenously with microfilariae isolated from a D. immitis-infected dog. Microfilariae were detected in blood collected from the tail vein 218 days post-inoculation (dpi) and via cardiac puncture 296 dpi. Microfilariae maintained in and extracted from SCID mice showed infectivity and matured into third-stage larvae (L3s) in the vector mosquito Aedes aegypti. L3s can develop into the fourth stage larvae in vitro. Microfilariae from SCID mice respond normally to ivermectin in vitro. The microfilariae in SCID mice displayed periodicity in the peripheral circulation. The SCID mouse model aided in the separation of microfilariae from cryopreserved specimens. The use of SCID mice enabled the isolation and sustained cultivation of microfilariae from clinical samples. These findings highlight the usefulness of the SCID mouse model for studying D. immitis microfilaremia in canine heartworm research.

Far-East Asian Toxoplasma isolates share ancestry with North and South/Central American recombinant lineages.

Toxoplasma gondii is a global protozoan pathogen. Clonal lineages predominate in Europe, North America, Africa, and China, whereas highly recombinant parasites are endemic in South/Central America. Far East Asian T. gondii isolates are not included in current global population genetic structure analyses at WGS resolution. Here we report a genome-wide population study that compared eight Japanese and two Chinese isolates against representative worldwide T. gondii genomes using POPSICLE, a novel population structure analyzing software. Also included were 7 genomes resurrected from non-viable isolates by target enrichment sequencing. Visualization of the genome structure by POPSICLE shows a mixture of Chinese haplogroup (HG) 13 haploblocks introgressed within the genomes of Japanese HG2 and North American HG12. Furthermore, two ancestral lineages were identified in the Japanese strains; one lineage shares a common ancestor with HG11 found in both Japanese strains and North American HG12. The other ancestral lineage, found in T. gondii isolates from a small island in Japan, is admixed with genetically diversified South/Central American strains. Taken together, this study suggests multiple ancestral links between Far East Asian and American T. gondii strains and provides insight into the transmission history of this cosmopolitan organism.

Efficacy evaluation of upper gastrointestinal endoscopy screening for secondary prevention of gastric cancer using the standardized detection ratio during a medical check-up in Japan.

We used standardized detection ratio to evaluate the quality of nasal upper gastrointestinal endoscopy screening for the secondary prevention of gastric cancer, and examined the gastric cancer risk in the era of total Helicobacter pylori (H. pylori) eradication. We performed 21,931 upper gastrointestinal endoscopies, 77 subjects were diagnosed with gastric cancer. Of these, 28 had gastric cancer after H. pylori eradication, 47 had gastric cancer with H. pylori-positive or others, and 2 had H. pylori-negative gastric cancer. The Standardized detection ratios for men and women were 5.33 and 4.82, respectively. Multivariable logistic regression analyses performed exclusively on first endoscopy subjects, excluding H. pylori-negative gastric cancer, revealed that smoking was a risk factor for developing gastric cancer (adjusted odds ratio, 3.31; 95% confidence interval, 1.65-6.64; p = 0.001). A statistically significant interaction was found between daily alcohol consumpption and H. pylori eradication on gastric cancer development (p = 0.005). In conclusion, relatively high standardized detection ratio values suggest that an appropriate endoscopic diagnosis of gastric cancer should be performed during a medical check-up. Smoking is a risk factor for developing gastric cancer, and continued alcohol consumption suggests a possible risk for developing gastric cancer after H. pylori eradication.

Searching for new molecules involved in Anopheles mosquitoes' response to Plasmodium infection.

Plasmodium parasites within mosquitoes are exposed to various physiological processes, such as blood meal digestion activity, the gonotrophic cycle, and host responses preventing the entry of parasites into the midgut wall. However, when in vitro-cultured ookinetes are injected into the hemocoel of mosquitoes, Plasmodium parasites are not affected by the vertebrate host's blood contents and do not pass through the midgut epithelial cells. This infection method might aid in identifying mosquito-derived factors affecting Plasmodium development within mosquitoes. This study investigated novel mosquito-derived molecules related to parasite development in Anopheles mosquitoes. We injected in vitro-cultured Plasmodium berghei (ANKA strain) ookinetes into female and male Anopheles stephensi (STE2 strain) mosquitoes and found that the oocyst number was significantly higher in males than in females, suggesting that male mosquitoes better support the development of parasites. Next, RNA-seq analysis was performed on the injected female and male mosquitoes to identify genes exhibiting changes in expression. Five genes with different expression patterns between sexes and greatest expression changes were identified as being potentially associated with Plasmodium infection. Two of the five genes also showed expression changes with infection by blood-feeding, indicating that these genes could affect the development of Plasmodium parasites in mosquitoes.

Esophageal submucosal hematoma during transnasal endoscopy: A rare case report.

Esophageal submucosal hematoma is a rare, often incidental complication of therapeutic endoscopic procedures marked by disrupted blood vessels beneath the esophageal mucosa, forming a hematoma. We report the unique case of a severely thin and alcoholic 38-year-old woman with a history of reflux esophagitis who developed an esophageal submucosal hematoma during an unsedated transnasal endoscopy for health check-up. During the procedure, the patient experienced strong vomiting reflexes and vomited blood, leading to the initial suspicion of either Mallory-Weiss syndrome or epistaxis. However, subsequent sedated endoscopy revealed an esophageal submucosal tumor-like lesion and a mucosal laceration with blood clots, prompting a dual diagnosis of esophageal submucosal hematoma and Mallory-Weiss syndrome. The bleeding was not severe enough to require hemostatic intervention. The patient opted for conservative treatment with vonoprazan, which resulted in the improvement and healing of the hematoma within 28 days. This is the first report of an esophageal submucosal hematoma during transnasal endoscopy and emphasizes the importance of including an esophageal submucosal hematoma and Mallory-Weiss syndrome in the differential diagnosis of hematemesis encountered in similar scenarios. Factors such as severe thinness, daily alcohol consumption, and reflux esophagitis may have possibly contributed to the development of the esophageal submucosal hematoma in this patient.

Challenges for Precise Subtyping and Sequencing of a H5N1 Clade 2.3.4.4b Highly Pathogenic Avian Influenza Virus Isolated in Japan in the 2022-2023 Season Using Classical Serological and Molecular Methods.

The continuous evolution of H5Nx highly pathogenic avian influenza viruses (HPAIVs) is a major concern for accurate diagnosis. We encountered some challenges in subtyping and sequencing a recently isolated H5N1 HPAIV strain using classical diagnostic methods. Oropharyngeal, conjunctival, and cloacal swabs collected from a dead white-tailed eagle (Haliaeetus albicilla albicilla) were screened via real-time RT-PCR targeting the influenza A virus matrix (M) gene, followed by virus isolation. The hemagglutination inhibition test was applied in order to subtype and antigenically characterize the isolate using anti-A/duck/Hong Kong/820/80 (H5N3) reference serum or anti-H5N1 cross-clade monoclonal antibodies (mAbs). Sequencing using previously reported universal primers was attempted in order to analyze the full-length hemagglutinin (HA) gene. Oropharyngeal and conjunctival samples were positive for the M gene, and high hemagglutination titers were detected in inoculated eggs. However, its hemagglutination activity was not inhibited by the reference serum or mAbs. The antiserum to a recently isolated H5N1 clade 2.3.4.4b strain inhibited our isolate but not older strains. A homologous sequence in the previously reported forward primer and HA2 region in our isolate led to partial HA gene amplification. Finally, next-generation sequencing confirmed the isolate as H5N1 clade 2.3.4.4b HPAIV, with genetic similarity to H5N1 strains circulating in Japan since November 2021.

Traction suture fixation technique using a capsular tension ring fragment for severe lens displacement during cataract surgery in dogs.

OBJECTIVES: To describe the traction suture fixation technique for severely displaced lenses (≥180°) using a capsular tension ring (CTR) fragment during cataract surgery in dogs, and to retrospectively investigate its clinical outcomes. ANIMALS STUDIED: Eight dogs (nine eyes). PROCEDURES: The medical records of dogs with severe lens displacement (≥180°) due to cataracts that underwent traction suture fixation during cataract surgery using a CTR fragment at the Grand Animal Hospital (Hyogo, Japan) between November 2019 and September 2022 were retrospectively reviewed. The retrieved data included the signalment, type of CTR, and postoperative outcomes. RESULTS: A single CTR fragment was used to fix the capsular bag to the sclera in six eyes, and two CTR fragments were used at two sites in three eyes. The lengths of the CTR fragments used were 1 one-fifth fragment, 3 one-fourth fragments, and 8 one-third fragments. Postoperative complications included uveitis (nine eyes), glaucoma (one eye), posterior iris adhesion (one eye), corneal ulcer (one eye), and intracorneal stromal hemorrhage (one eye). Vision was maintained in all dogs during the follow-up period (2 months to 2 years and 8 months) without recurrent capsular bag displacement. CONCLUSIONS: This technique can correct and stabilize capsular bag displacement relatively easily through a small incision during cataract surgery in dogs with severe lens displacement. Intraocular lens implantation was also possible.

Association of plasma xanthine oxidoreductase activity with vascular endothelial function independent of serum uric acid level: MedCity21 health examination registry.

Background: Xanthine oxidoreductase (XOR) inhibitor administration, known to reduce uric acid and reactive oxygen species (ROS) production, also improves vascular endothelial function (VEF). This cross-sectional study examined our hypothesis that XOR contributes to impaired VEF through ROS but not uric acid production. Methods: In 395 subjects (196 males, 199 females) without urate-lowering agent administration who underwent a health examination, plasma XOR activity was determined using our highly sensitive assay based on [13C2,15N2] xanthine and liquid chromatography/triple quadrupole mass spectrometry. For VEF evaluation, flow-mediated dilatation (FMD) in the brachial artery was determined by ultrasound, with physical and laboratory measurements also obtained. Results: The median values for plasma XOR activity, serum uric acid, and FMD were 26.6 pmol/h/mL, 5.4 mg/dL, and 6.2%, respectively. Simple regression analysis showed weak correlations of both log plasma XOR activity and serum uric acid level with FMD (r = -0.213, p < 0.001 and r = -0.139, p = 0.006, respectively). However, multivariable linear regression analyses revealed that log plasma XOR activity but not serum uric acid level remained associated with FMD (ß = -0.116, p = 0.037 and ß = 0.041, p = 0.549, respectively) after adjustments for various clinical parameters, with no remarkable inconsistencies for the association observed in subgroups divided based on sex or uric acid level. Finally, a series of mediation analyses showed that serum uric acid level did not meet the criteria for mediator of the association of plasma XOR activity with FMD (p = 0.538). Conclusions: These findings suggest the possibility that XOR contributes to the pathophysiology of impaired VEF through ROS but not uric acid production.

Formation of free oocysts in Anopheles mosquitoes injected with Plasmodium ookinetes.

Malaria needs new strategies for its control. Plasmodium spp., the causative agent of malaria, is transmitted by mosquitoes. These parasites develop into oocysts and sporozoites in the body of the mosquitoes. A deeper understanding of oocysts that produce the infectious form of the parasite, sporozoites, can facilitate the development of novel countermeasures. However, the isolation of Plasmodium oocysts is challenging as these are formed between midgut epithelial cells and basal lamina after gametocytes enter the mosquito's body through blood feeding. Further research on oocysts has been impeded by issues related to oocyst isolation. Therefore, in this study, we injected Plasmodium into mosquitoes-an artificial and unique method-and aimed to clarify how oocysts were formed in mosquitoes after Plasmodium injection and whether free oocysts were formed from the mosquito tissue. Plasmodium berghei (ANKA strain) ookinetes cultured in vitro were injected into the thoracic body cavity (hemocoel) of female and male Anopheles stephensi mosquitoes. Oocysts were formed in the body of female and male mosquitoes at 14 days post injection. In addition, oocysts formed as a result of injection developed into sporozoites, which were infectious to mice. These findings suggest that P. berghei can complete its developmental stage in mosquitoes by injection. Some of the oocysts formed were free from mosquito tissue, and it was possible to collect oocysts with minimal contamination of mosquito tissue. These free oocysts can be used for investigating oocyst proteins and metabolism.

Upper gastrointestinal endoscopic findings in functional constipation and irritable bowel syndrome diagnosed using the Rome IV criteria: a cross-sectional survey during a medical check-up in Japan.

BACKGROUND: The Rome IV criteria have been established as an international standard for diagnosing disorders of gut-brain interaction. In this study, we aimed to examine the upper gastrointestinal (GI) endoscopic findings and symptoms of subjects with functional constipation (FC) and irritable bowel syndrome (IBS) of individuals undergoing a medical check-up. METHODS: A total of 13,729 subjects underwent a medical check-up at Osaka City University-affiliated clinic, MedCity21, between April 2018 and March 2019. Among the 5,840 subjects who underwent screening upper GI endoscopy and completed a questionnaire based on the Rome IV criteria, 5,402 subjects were consecutively enrolled after excluding subjects with a large amount of gastric residue (n = 6), those who had previously undergone partial or total gastrectomy (n = 40), or those with daily use of low-dose aspirin (n = 82), nonsteroidal anti-inflammatory drugs (n = 63), or acid secretion inhibitors (n = 308). RESULTS: Robust Poisson regression analyses adjusted for age, sex, Helicobacter pylori infection status, alcohol intake, and smoking habits showed a significant association between FC and corpus erosion (adjusted prevalence ratio [aPR], 2.93; 95% confidence interval [CI], 1.51-5.67; p < 0.01) and red streaks (aPR, 3.83; 95% CI, 2.53-5.79; p < 0.01), whereas IBS was significantly associated with erosive gastritis (aPR, 8.46; 95% CI, 4.89-14.67; p < 0.01) and duodenitis (aPR, 7.28; 95% CI, 3.64-14.59; p < 0.01). Red streaks tended to be associated with IBS (aPR, 1.96; 95% CI, 1.00-3.83; p = 0.05). Subjects with IBS were the most to complain of both upper and lower GI symptoms and psychological symptoms, followed by those with FC and controls. IBS subjects with erosive gastritis or duodenitis had significantly more complaints of stomachache and feeling stressed than those without erosive gastritis or duodenitis (54.5% vs. 18.8%; p = 0.03 and 66.7% vs. 25.0%; p = 0.01). CONCLUSIONS: Subjects with FC and IBS had a variety of upper GI and psychological symptoms. In the upper GI endoscopic findings, corpus erosion and red streaks were associated with FC, and erosive gastritis, duodenitis, and possibly red streaks were associated with IBS.

Seasonal Variation and Factors Affecting Trypanosoma theileri Infection in Wild Sika Deer (Ezo Sika Deer Cervus nippon yesoensis) in Eastern Hokkaido.

Trypanosoma (Megatrypanum) spp. are isolated from domestic and wild ruminants, including deer, worldwide. The prevalence of trypanosomes in mammals is influenced by a number of factors such as host age and vector abundance. However, the seasonal variation of and factors affecting trypanosome infection in the wild deer population remain elusive. In this study, we analyzed the seasonal variation in trypanosome prevalence and the factors that affect Trypanosoma theileri Laveran, 1902, infection in wild sika deer (Ezo sika deer) Cervus nippon yesoensis (Heude, 1884) in Eastern Hokkaido through a two-year survey. Seasonal variation in the prevalence of trypanosome infection in the deer population ranged from 0 to 41% as per hematocrit concentration and 17 to 89% as per PCR results. In general, the prevalence of T. theileri by PCR in 2020 was higher than that in 2019. Moreover, the prevalence was significantly higher in the aged population than among the younger population. These findings may explain why individual conditions and sampling season were associated with trypanosome prevalence. This is the first study to investigate the seasonal variation in and risk factors affecting trypanosome infection in wild deer.

Seasonal changes of the prostate gland in the raccoon (Procyon lotor) inhabiting Hokkaido, Japan.

In the prostate gland of the raccoon (Procyon lotor), the morphological appearance of the epithelial cells, such as basal and luminal cells, and the expressions of p63, androgen receptor (AR), and proliferating cell nuclear antigen (PCNA) were examined histologically and immunohistochemically to clarify their seasonal dynamics throughout the year. In this study, the regression with luminal cell defluxion and the regeneration process of the prostatic glandular epithelium was revealed in the seasons with declined spermatogenesis (June to August). The expression of p63 was observed only in the basal cells. AR immunoreactivity in the luminal cells was shown in the developed and regenerating (close to developed) prostates, whereas the basal cells exhibited AR immunoreactivity all year round. PCNA expression was rare in epithelial cells of the developed prostate gland. In the regressed gland, the basal cells demonstrated proliferative ability, whereas PCNA of the luminal cells appeared for the first time in the regenerating phase. This study is the first to clarify the regression with luminal cell defluxion and restoration and the seasonal dynamics of AR expression and proliferative activity in the prostate gland of seasonal breeders.

Serum phosphate as an independent factor associated with cholesterol metabolism in patients undergoing hemodialysis: a cross-sectional analysis of the DREAM cohort.

BACKGROUND: Hyperphosphatemia is a risk factor for cardiovascular outcomes in patients with chronic kidney disease. In an experimental model, hyperphosphatemia promoted atherosclerosis by activating sterol regulatory element-binding protein 2, which controls cholesterol homeostasis. In the present study, we hypothesized that serum phosphate level is associated with cholesterol metabolism in patients with kidney failure. METHODS: We conducted a single-center cross-sectional study including 492 patients undergoing hemodialysis and 100 healthy controls not on statin or ezetimibe treatment. Serum lathosterol and campesterol levels were measured as a marker of cholesterol synthesis and absorption, respectively. As compared with the control group, the hemodialysis patients had higher median phosphate {5.8 mg/dL [interquartile range (IQR 5.0-6.6) versus 3.3 (3.0-3.6); P < .001], lower lathosterol [1.2 µg/mL (IQR 0.8-1.7) versus 2.6 (1.9-3.4); P < .001] and higher campesterol levels [4.5 µg/mL (IQR 3.6-6.0) versus 4.1 (3.2-5.4); P = .02]. Serum phosphate correlated positively to campesterol in the control group (Spearman's r = 0.21, P = .03) and in hemodialysis patients (Spearman's r = 0.19, P < .001). The positive association between phosphate and campesterol levels in the hemodialysis group remained significant in multivariable-adjusted linear regression analysis. There was no significant association between phosphate and lathosterol in either group. CONCLUSIONS: An independent association was found between phosphate and campesterol levels in patients with kidney failure. This study suggests a novel relationship between phosphate and cholesterol metabolism, both of which could affect cardiovascular outcomes in this population.

New PCR primers targeting the cytochrome b gene reveal diversity of Leucocytozoon lineages in an individual host.

Avian haemosporidian parasites have received considerable attention in ecology and evolution as a result of their wide distribution and ease of detection. However, conventional PCR-based detection methods may sometimes underestimate haemosporidian mixed infections, which are frequent in natural populations. This underestimation is due to differences in PCR sensitivity for detection of lineages within the mixed infections. Therefore, we designed new primers to amplify sequences that were not detected by the conventional primers and examined if our primers were useful for accurate detection of mixed infections. Blood samples were collected from 32 wild birds captured in Hokkaido, and 16 of these were positive for Leucocytozoon using the conventional primers, while 15 were positive using our primers. All positively amplified samples were sequenced, and we found that the conventional primers detected 16% (5/32) of multiple infections and none of them was a novel lineage, whereas our primers detected 44% (14/32) of multiple infections and ten of them were novel lineages. A phylogenetic analysis showed that the new primers can detect a wide range of Leucocytozoon lineages compared with that detected by the conventional primers. The results indicate that our primers are particularly suitable for revealing unique strains from multiple infections. Highly variable multiple infections in the same population of birds at the same location were found for the first time. We revealed a higher diversity of Leucocytozoon lineages in nature than expected, which would provide more information to better understand parasite diversity and host-vector interactions in wildlife.

Visual and quantitative evaluation of microcalcifications in mammograms with deep learning-based super-resolution.

PURPOSE: To evaluate visually and quantitatively the performance of a deep-learning-based super-resolution (SR) model for microcalcifications in digital mammography. METHOD: Mammograms were consecutively collected from 5080 patients who underwent breast cancer screening from January 2015 to March 2017. Of these, 93 patients (136 breasts, mean age, 50 ± 7 years) had microcalcifications in their breasts on mammograms. We applied an artificial intelligence model known as a fast SR convolutional neural network to the mammograms. SR and original mammograms were visually evaluated by four breast radiologists using a 5-point scale (1: original mammograms are strongly preferred, 5: SR mammograms are strongly preferred) for the detection, diagnostic quality, contrast, sharpness, and noise of microcalcifications. Mammograms were quantitatively evaluated using a perception-based image-quality evaluator (PIQE). RESULTS: All radiologists rated the SR mammograms better than the original ones in terms of detection, diagnostic quality, contrast, and sharpness of microcalcifications. These ratings were significantly different according to the Wilcoxon signed-rank test (p <.001), while the noise score of the three radiologists was significantly lower (p <.001). According to PIQE, SR mammograms were rated better than the original mammograms, showing a significant difference by paired t-test (p <.001). CONCLUSION: An SR model based on deep learning can improve the visibility of microcalcifications in mammography and help detect and diagnose them in mammograms.

Plasmodium berghei Brca2 is required for normal development and differentiation in mice and mosquitoes.

BACKGROUND: Malaria is a major global parasitic disease caused by species of the genus Plasmodium. Zygotes of Plasmodium spp. undergo meiosis and develop into tetraploid ookinetes, which differentiate into oocysts that undergo sporogony. Homologous recombination (HR) occurs during meiosis and introduces genetic variation. However, the mechanisms of HR in Plasmodium are unclear. In humans, the recombinases DNA repair protein Rad51 homolog 1 (Rad51) and DNA meiotic recombinase 1 (Dmc1) are required for HR and are regulated by breast cancer susceptibility protein 2 (BRCA2). Most eukaryotes harbor BRCA2 homologs. Nevertheless, these have not been reported for Plasmodium. METHODS: A Brca2 candidate was salvaged from a database to identify Brca2 homologs in Plasmodium. To confirm that the candidate protein was Brca2, interaction activity between Plasmodium berghei (Pb) Brca2 (PbBrca2) and Rad51 (PbRad51) was investigated using a mammalian two-hybrid assay. To elucidate the functions of PbBrca2, PbBrca2 was knocked out and parasite proliferation and differentiation were assessed in mice and mosquitoes. Transmission electron microscopy was used to identify sporogony. RESULTS: The candidate protein was conserved among Plasmodium species, and it was indicated that it harbors critical BRCA2 domains including BRC repeats, tower, and oligonucleotide/oligosaccharide-binding-fold domains. The P. berghei BRC repeats interacted with PbRad51. Hence, the candidate was considered a Brca2 homolog. PbBrca2 knockout parasites were associated with reduced parasitemia with increased ring stage and decreased trophozoite stage counts, gametocytemia, female gametocyte ratio, oocyst number, and ookinete development in both mice and mosquitoes. Nevertheless, the morphology of the blood stages in mice and the ookinete stage was comparable to those of the wild type parasites. Transmission electron microscopy results showed that sporogony never progressed in Brca2-knockout parasites. CONCLUSIONS: Brca2 is implicated in nearly all Plasmodium life cycle stages, and especially in sporogony. PbBrca2 contributes to HR during meiosis.

Possible role of insulin resistance in activation of plasma xanthine oxidoreductase in health check-up examinees.

We previously found an association of insulin resistance (IR) with plasma xanthine oxidoreductase (XOR) activity in a cross-sectional study. However, whether IR induces increased XOR activity has not been elucidated. This retrospective longitudinal observational study included 347 participants (173 males, 174 females) who underwent annual health examinations and were medication naïve. Homeostasis model assessment of IR (HOMA-IR) index, and physical and laboratory measurements were determined at the baseline. At baseline and 12-month follow-up examinations, plasma XOR activity was determined using our novel assay based on [13C2,15N2] xanthine and liquid chromatography/triple quadrupole mass spectrometry. Subjects with IR, defined as HOMA-IR index ≥ 1.7 (n = 92), exhibited significantly (p < 0.001) higher plasma XOR activity levels than those without IR (n = 255), with an increase in that activity seen in 180 (51.9%) after 12 months. Multivariable linear and logistic regression analyses showed that IR, but not BMI or waist circumference, at baseline was significantly associated with plasma XOR activity (ß = 0.094, p = 0.033) and increased plasma XOR activity over the 12-month period (odds ratio, 1.986; 95% confidence interval, 1.048-3.761; p = 0.035), after adjustments for various clinical parameters, including plasma XOR activity at baseline. These results suggest that IR induces increased plasma XOR activity in a manner independent of adiposity.