descSPIM: an affordable and easy-to-build light-sheet microscope optimized for tissue clearing techniques.

Despite widespread adoption of tissue clearing techniques in recent years, poor access to suitable light-sheet fluorescence microscopes remains a major obstacle for biomedical end-users. Here, we present descSPIM (desktop-equipped SPIM for cleared specimens), a low-cost ($20,000-50,000), low-expertise (one-day installation by a non-expert), yet practical do-it-yourself light-sheet microscope as a solution for this bottleneck. Even the most fundamental configuration of descSPIM enables multi-color imaging of whole mouse brains and a cancer cell line-derived xenograft tumor mass for the visualization of neurocircuitry, assessment of drug distribution, and pathological examination by false-colored hematoxylin and eosin staining in a three-dimensional manner. Academically open-sourced ( https://github.com/dbsb-juntendo/descSPIM ), descSPIM allows routine three-dimensional imaging of cleared samples in minutes. Thus, the dissemination of descSPIM will accelerate biomedical discoveries driven by tissue clearing technologies.

Acto3D: an open-source user-friendly volume rendering software for high-resolution 3D fluorescence imaging in biology.

Advances in fluorescence microscopy and tissue-clearing have revolutionised 3D imaging of fluorescently labelled tissues, organs and embryos. However, the complexity and high cost of existing software and computing solutions limit their widespread adoption, especially by researchers with limited resources. Here, we present Acto3D, an open-source software, designed to streamline the generation and analysis of high-resolution 3D images of targets labelled with multiple fluorescent probes. Acto3D provides an intuitive interface for easy 3D data import and visualisation. Although Acto3D offers straightforward 3D viewing, it performs all computations explicitly, giving users detailed control over the displayed images. Leveraging an integrated graphics processing unit, Acto3D deploys all pixel data to system memory, reducing visualisation latency. This approach facilitates accurate image reconstruction and efficient data processing in 3D, eliminating the need for expensive high-performance computers and dedicated graphics processing units. We have also introduced a method for efficiently extracting lumen structures in 3D. We have validated Acto3D by imaging mouse embryonic structures and by performing 3D reconstruction of pharyngeal arch arteries while preserving fluorescence information. Acto3D is a cost-effective and efficient platform for biological research.

Prevalence of common aneuploidy in twin pregnancies.

The incidence of chromosomal abnormalities in twin pregnancies is not well-studied. In this retrospective study, we investigated the frequency of chromosomal abnormalities in twin pregnancies and compared the incidence of chromosomal abnormalities in dichorionic diamniotic (DD) and monochorionic diamniotic (MD) twins. We used data from 57 clinical facilities across Japan. Twin pregnancies of more than 12 weeks of gestation managed between January 2016 and December 2018 were included in the study. A total of 2899 and 1908 cases of DD and MD twins, respectively, were reported, and the incidence of chromosomal abnormalities in one or both fetuses was 0.9% (25/2899) and 0.2% (4/1908) in each group (p = 0.004). In this study, the most common chromosomal abnormality was trisomy 21 (51.7% [15/29]), followed by trisomy 18 (13.8% [4/29]) and trisomy 13 (6.9% [2/29]). The incidence of trisomy 21 in MD twins was lower than that in DD twins (0.05% vs. 0.5%, p = 0.007). Trisomy 21 was less common in MD twins, even when compared with the expected incidence in singletons (0.05% vs. 0.3%, RR 0.15 [95% CI 0.04-0.68]). The risk of chromosomal abnormality decreases in twin pregnancies, especially in MD twins.

Unique Terminal Regions and Specific Deletions of the Segmented Double-Stranded RNA Genome of Alternaria Alternata Virus 1, in the Proposed Family Alternaviridae.

Alternaria alternata virus 1 (AaV1) has been identified in the saprophytic fungus Alternaria alternata strain EGS 35-193. AaV1 has four genomic double-stranded (ds)RNA segments (dsRNA1-4) packaged in isometric particles. The 3' end of each coding strand is polyadenylated (36-50nt), but the presence of a cap structure at each 5' end has not previously been investigated. Here, we have characterized the AaV1 genome and found that it has unique features among the mycoviruses. We confirmed the existence of cap structures on the 5' ends of the AaV1 genomic dsRNAs using RNA dot blots with anti-cap antibodies and the oligo-capping method. Polyclonal antibodies against purified AaV1 particles specifically bound to an 82kDa protein, suggesting that this protein is the major capsid component. Subsequent Edman degradation indicated that the AaV1 dsRNA3 segment encodes the major coat protein. Two kinds of defective AaV1 dsRNA2, which is 2,794bp (844 aa) in length when intact, appeared in EGS 35-193 during subculturing, as confirmed by RT-PCR and northern hybridization. Sequence analysis revealed that one of the two defective dsRNA2s contained a 231bp deletion, while the other carried both the 231bp deletion and an additional 465bp deletion in the open reading frame. Both deletions occurred in-frame, resulting in predicted proteins of 767 aa and 612 aa. The fungal isolates carrying virions with the defective dsRNA2s showed impaired growth and abnormal pigmentation. To our best knowledge, AaV1 is the first dsRNA virus to be identified with both 5' cap and 3'poly(A) structures on its genomic segments, as well as the specific deletions of dsRNA2.

A mutant equipped with a regenerated disulphide for the missing His loop of a serine protease zymogen in the horseshoe crab coagulation cascade.

The lipopolysaccharide (LPS)-triggered coagulation cascade in horseshoe crabs is composed of three zymogens belonging to the trypsinogen family: prochelicerase C, prochelicerase B (proB) and the proclotting enzyme (proCE). Trypsinogen-family members contain three conserved disulphides located around the active site. While it is known that proB evolutionarily lost one of the disulphides, the His-loop disulphide, the roles of the missing His-loop disulphide in proB remain unknown. Here, we prepared a proB mutant, named proB-murasame, equipped with a regenerated His-loop disulphide. The activation rate by upstream α-chelicerase C for proB-murasame was indistinguishable from that for wild-type (WT) proB. The resulting protease chelicerase B-murasame exhibited an 8-fold higher kcat value for downstream proCE than WT chelicerase B, whereas the Km value of chelicerase B-murasame was equivalent to that of WT chelicerase B. WT serpins-1, -2 and -3, identified as scavengers for the cascade, had no reactivity against WT chelicerase B, whereas chelicerase B-murasame was inhibited by WT serpin-2, suggesting that WT chelicerae B may trigger as-yet-unsolved phenomena after performing its duty in the cascade. The reconstituted LPS-triggered cascade containing proB-murasame exhibited ∼5-fold higher CE production than that containing WT proB. ProB-murasame might be used as a high value-adding reagent for LPS detection.

Female detrusor underactivity: Two uterine leiomyoma cases.

INTRODUCTION: Female urinary retention is rare. CASE PRESENTATION: Case 1, a 35-year-old nulliparous woman, and case 2, a 47-year-old nulliparous woman, had transient urinary retention. A urodynamics revealed increased bladder sensation in case 1 and detrusor underactivity with a large post-void residual in cases 1 and 2. Both women had a uterine leiomyoma of >10 cm in diameter. Soon after extraction of the tumor, retention episodes disappeared completely in case 1. CONCLUSION: Although rare, uterine leiomyoma should be listed as a cause of female detrusor underactivity.

Noninvasive prenatal testing aids identification of tetrasomy 18p: A case report.

Tetrasomy 18p syndrome (Online Mendelian Inheritance in Man 614290) is a rare chromosomal disorder that is seen in approximately 1 in every 180,000 live births. It is caused by the presence of isochromosome 18p, which is a supernumerary marker composed of two copies of the short arms of chromosome 18. Isochromosome 18p is one of the most commonly observed isochromosomes. We report tetrasomy 18p syndrome diagnosed prenatally after noninvasive prenatal testing (NIPT) was positive for trisomy 18. Tetrasomy 18p was finally diagnosed by G-banding and fluorescence in situ hybridization of chromosome 18p, before invasive confirmatory testing the karyotype findings by NIPT showed an increase in the DNA fragments from chromosome 18p, indicating duplication of chromosome 18p. NIPT can detect not only trisomy 13, 18, and 21, but also structural chromosomal anomalies, such as deletions and duplications. An NIPT report "positive for trisomy 18" indicates the possibility of tetrasomy 18p, and detailed analysis of NIPT data can reveal subchromosomal copy number variations, to a certain extent, before definitive diagnostic testing.

Qualitative investigation of the factors that generate ambivalent feelings in women who give birth after receiving negative results from non-invasive prenatal testing.

BACKGROUND: Women who receive negative results from non-invasive prenatal genetic testing (NIPT) may find that they later have mixed or ambivalent feelings, for example, feelings of accepting NIPT and regretting undergoing the test. This study aimed to investigate the factors generating ambivalent feelings among women who gave birth after having received negative results from NIPT. METHODS: A questionnaire was sent to women who received a negative NIPT result, and a contents analysis was conducted focusing on ambivalent expressions for those 1562 women who responded the questionnaire. The qualitative data gathered from the questionnaire were analyzed using the N-Vivo software package. RESULTS: Environmental factors, genetic counseling-related factors, and increased anticipatory anxiety, affected the feeling of ambivalence among pregnant women. Furthermore, pregnant women desired more information regarding the detailed prognosis for individuals with Down syndrome and living with them and/or termination, assuming the possibility that they were positive. CONCLUSIONS: Three major interrelated factors affected the feeling of ambivalence in women. Highlighting and discussing such factors during genetic counseling may resolve some of these ambivalences, thereby enhancing the quality of decisions made by pregnant women.

Deficiency of ascorbic acid decreases the contents of tetrahydrobiopterin in the liver and the brain of ODS rats.

Tetrahydrobiopterin (BH4) is a cofactor for tyrosine hydroxylase and tryptophan hydroxylase, which are essential enzymes for the biosynthesis of dopamine, norepinephrine, and serotonin. It has been known that BH4 is a labile molecule and easily oxidized. As ascorbic acid (AsA) is an antioxidant that is rich in the brain, alteration in the AsA concentration in the brain may affect the proper metabolism of BH4. Here, we examined the effect of AsA deficiency on the concentration of BH4 using ODS rats, which are defective in the gene for AsA synthesis. Intake of an AsA-deficient diet for 2 weeks in ODS rats resulted in great reductions in the AsA levels up to 7 % in the liver and up to 55 % in the brain compared to animals fed a basal diet containing an adequate amount of AsA. The BH4 concentrations in ODS rats fed an AsA-free diet were decreased to 71 % in the liver and 88 % in the brain of those fed a basal diet. We found that the levels of dopamine, norepinephrine, and serotonin were also decreased compared with the ODS rats fed a basal diet. Our data showed that AsA deficiency can affect the BH4 concentrations in the liver and brain, resulting in decreases in the monoamine levels in the brain. These results suggest the importance of AsA in the pathophysiology of neuropsychiatric and cardiovascular disorders through alteration in the BH4 metabolism.

Classification of factors involved in nonreportable results of noninvasive prenatal testing (NIPT) and prediction of success rate of second NIPT.

OBJECTIVE: To evaluate the reasons for nonreportable cell-free DNA (cfDNA) results in noninvasive prenatal testing (NIPT), we retrospectively studied maternal characteristics and other details associated with the results. METHODS: A multicenter retrospective cohort study in pregnant women undergoing NIPT by massively parallel sequencing (MPS) with failed cfDNA tests was performed between April 2013 and March 2017. The women's data and MPS results were analyzed in terms of maternal characteristics, test performance, fetal fraction (FF), z scores, anticoagulation therapy, and other details of the nonreportable cases. RESULTS: Overall, 110 (0.32%) of 34 626 pregnant women had nonreportable cfDNA test results after an initial blood sampling; 22 (20.0%) cases had a low FF (<4%), and 18 (16.4%) cases including those with a maternal malignancy, were found to have altered genomic profile. Approximately half of the cases with nonreportable results had borderline z score. Among the women with nonreportable results because of altered genomic profile, the success rate of retesting using a second blood sampling was relatively low (25.0%-33.3%). Thirteen (11.8%) of the women with nonreportable results had required hypodermic heparin injection. CONCLUSIONS: The classification of nonreportable results using cfDNA analysis is important to provide women with precise information and to reduce anxiety during pregnancy.