Effectiveness of plasma lyso-Gb3 as a biomarker for selecting high-risk patients with Fabry disease from multispecialty clinics for genetic analysis.

PURPOSE: Plasma globotriaosylsphingosine (lyso-Gb3) is a promising secondary screening biomarker for Fabry disease. Here, we examined its applicability as a primary screening biomarker for classic and late-onset Fabry disease in males and females. METHODS: Between 1 July 2014 and 31 December 2015, we screened 2,359 patients (1,324 males) referred from 168 Japanese specialty clinics (cardiology, nephrology, neurology, and pediatrics), based on clinical symptoms suggestive of Fabry disease. We used the plasma lyso-Gb3 concentration, α-galactosidase A (α-Gal A) activity, and analysis of the α-Gal A gene (GLA) for primary and secondary screens, respectively. RESULTS: Of 8 males with elevated lyso-Gb3 levels (≥2.0 ng ml-1) and low α-Gal A activity (≤4.0 nmol h-1 ml-1), 7 presented a GLA mutation (2 classic and 5 late-onset). Of 14 females with elevated lyso-Gb3, 7 displayed low α-Gal A activity (5 with GLA mutations; 4 classic and 1 late-onset) and 7 exhibited normal α-Gal A activity (1 with a classic GLA mutation and 3 with genetic variants of uncertain significance). CONCLUSION: Plasma lyso-Gb3 is a potential primary screening biomarker for classic and late-onset Fabry disease probands.

Production of zebrafish offspring from cultured spermatogonial stem cells.

Germ-line stem cells have the potential to be a very powerful tool for modifying the genetic information of individual animals. As a first step to use spermatogonial stem cells (SSCs) to enable genetic modification, we here describe effective long-term culture conditions for propagating zebrafish SSCs and for the production of offspring from these cultured SSCs after their differentiation into sperm in transplanted testicular cell aggregates. Dissociated testicular cells were cultured in specific medium with some modified supplements, including several mammalian growth factors. The spermatogonia actively proliferated and retained the expression of exogenous green fluorescent protein under the control of vas and sox17 promoters and also of promyelocytic leukemia zinc finger (Plzf), a marker of undifferentiated spermatogonia, after 1 month in culture. This is a longer period than the entire natural spermatogenic cycle (from SSCs to sperm). The use of subcutaneously grafted aggregates of these cultured spermatogonia and freshly dissociated testicular cells showed that these SSCs could undergo self-renewal and differentiation into sperm. Artificial insemination of these grafted aggregates successfully produced offspring. This culture method will facilitate the identification of new factors for the maintenance of SSCs and enable the future enrichment of genetically modified SSCs that will produce offspring in zebrafish.

Circadian variation in coronary flow velocity reserve and its relation to α1-sympathetic activity in humans.

BACKGROUND: The circadian change in coronary microvascular function has not been directly assessed in human beings. Recent advances in transthoracic Doppler echocardiography (TTDE) provide noninvasive, physiological assessment of coronary flow velocity reserve (CFVR). METHODS: This study consisted of 20 young healthy subjects (24 ± 2 years, 20 men) who underwent CFVR examinations at 3 different times; early morning (6AM), late morning (11AM) and late evening (10PM). The flow velocity in the distal portion of the left anterior descending coronary artery was measured with TTDE at baseline and during adenosine infusion to calculate CFVR. These examinations were repeated with the intake of α1-blocker (prazosin 1mg) on the other day. RESULTS: CFVR showed a circadian variation with an increase from the early morning to the late morning, following a decrease to the late evening thereafter (4.4 ± 0.9 at 6AM; 5.2 ± 1.3 at 11AM; 4.2 ± 1.1 at 10PM, p<0.001). In the study with α1-blocker, CFVR was comparable between the early morning and the late morning, whereas CFVR in the late evening was lower than those in other 2 time points (5.0 ± 1.1 at 6AM; 4.9 ± 0.9 at 11AM; 4.3 ± 0.9 at 10PM, p<0.001). CONCLUSIONS: This study demonstrates that CFVR has a circadian variation in humans, with an increase from the late evening to the late morning. Adding α1-blocker ameliorated CFVR only in the early morning, indicating that α1-sympathetic activity plays a heterogeneous and important role in the circadian change of CFVR in humans.

Evaluation of Sycp3, Plzf and Cyclin B3 expression and suitability as spermatogonia and spermatocyte markers in zebrafish.

Recent studies in mammals have revealed the heterogeneity of spermatogonial populations which contain differentiated and undifferentiated cells that further divide into actual stem cells and potential stem cells. In fish however, there are no functional definitions, and very few molecular markers, for germ cells. In our present study, specific antibodies were raised against Sycp3, Plzf and Cyclin B3 in zebrafish and then used to determine the localization of these proteins in the testis. We wished to confirm whether these molecules were potential markers for spermatocytes and spermatogonia. Immunohistochemical observations revealed that Sycp3 is specifically localized in spermatocytes in typical nuclear patterns at each meiotic stage. Plzf was found to be localized in the nucleus of both type A and type B spermatogonia until the 8-cell clone, similar to the pattern in Plzf-positive A(single)-A(aligned) undifferentiated spermatogonia in rodents. In addition to Plzf, the localization of Cyclin B3 was predominantly detected in the nuclei of type A and early type B spermatogonia until the 16-cell clone. Additionally, Cyclin B3 protein signals were detected in germ cells in large cysts, possibly corresponding to spermatocytes at the preleptotene stage. Our present data thus show that these molecules have properties that will enable their use as markers of spermatocytes and early spermatogonia in zebrafish.

Regeneration of spermatogenesis and production of functional sperm by grafting of testicular cell aggregates in Zebrafish (Danio rerio).

The self-renewal and differentiation of spermatogonial stem cells (SSCs) is essential for the continuous production of sperm throughout life in male vertebrates. The development of a functional assay to analyze these properties in isolated SSCs remains necessary. In our current study, we have developed a transplantation method for testicular cell aggregates in zebrafish (Danio rerio) in which allogeneic SSCs can undergo self-renewal and differentiation. The immature testes from juveniles are dissociated, aggregated by cultivation, and then transplanted under the abdominal skin of the recipient fish. The grafted aggregates reconstitute the appropriate testicular structures, including the lobule structure, consisting of basement membrane and interstitial steroid-producing cells on the outside, and the cysts, which comprise germ cell clusters and surrounding Sertoli cells. Bromodeoxyuridine incorporation analysis indicated that continuous spermatogenesis is maintained for at least 6 mo in the reconstituted testis. Moreover, when the sperm generated from the aggregates at 3 mo postgrafting were used for artificial insemination, fertilized eggs were obtained that developed sexually mature fish. These results suggest that self-renewal of SSCs takes place in reconstituted testes under the abdominal skin and that their differentiating progeny can develop into functional sperm. Furthermore, allogeneic spermatogonia were also found to proliferate and differentiate into sperm in these grafts. Our method of grafting testicular cell aggregates should thus prove useful not only analyzing the stem cell ability of an individual SSC but also for the production of progeny from cultured SSCs or SSCs of sterile mutants with somatic cell defects.

"Passive exercise" using whole body periodic acceleration: effects on coronary microcirculation.

BACKGROUND: The whole body periodic acceleration (WBPA) system has recently been developed as a "passive exercise" device by providing increased pulsatile shear stress for improvement of endothelial function. This study aimed to investigate the short-term effect of WBPA on coronary flow reserve (CFR) through transthoracic Doppler echocardiography (TTDE) in healthy subjects and patients with coronary artery disease (CAD). METHODS: This study consisted of 15 healthy subjects and 20 patients with CAD who underwent CFR examination before and immediately after WBPA. The flow velocity in the distal portion of the left anterior descending coronary artery (LAD) was measured with TTDE at baseline and during adenosine infusion. Coronary flow reserve was calculated as the ratio of hyperemic to basal mean diastolic flow velocity. RESULTS: The WBPA treatment was completed in all 35 subjects without complications. There were no significant differences in heart rate and systolic blood pressure before and after WBPA. Whole body periodic acceleration increased CFR from 3.3 +/- 1.0 to 3.7 +/- 1.1 in the 35 subjects (P < .001). Coronary angiography showed significant LAD narrowing in 8 of the 20 CAD patients, but WBPA increased CFR from 2.4 +/- 0.4 to 2.7 +/- 0.5 in them as well (P < .01). CONCLUSIONS: This study demonstrates that WBPA improves CFR in healthy subjects and patients with CAD.

Introduction of a foreign gene into zebrafish and medaka cells using adenoviral vectors.

Viral vectors represent a tractable system that can efficiently introduce an exogenous gene into different target cells and are thus a potentially powerful genetic manipulation tool. In our current study, we investigated the infection efficiency of mammalian virus vectors, adenoviruses (Ads), adeno-associated viruses, and lentiviruses to the Sertoli cell line and the newly established cell line from a single embryo in zebrafish. Among the viral vectors tested, Ads showed the highest infection efficiency of 10(7)-10(8) green fluorescent protein-transducing units (gtu)/mL in zebrafish cells. In addition, the adenoviral vector was also infected at 10(5) gtu/mL in the medaka testicular somatic cell line that was established from the testes of p53-deficient mutant. Further, we found that Ads could successfully infect cultured male zebrafish germ cells. Our results thus indicate that the adenoviral vector could be used as a chromosomally nonintegrating vector system in zebrafish.

Functional demonstration of the ability of a primary spermatogonium as a stem cell by tracing a single cell destiny in Xenopus laevis.

In Xenopus, although primary spermatogonium (PG), the largest cell in the testis, is believed to be spermatogonial stem cell by histological observations, functional evidence has never been obtained. In the present study, we first indicated that culture of juvenile testis in a medium supplemented with follicle stimulating hormone resulted in no proliferation of PG. In this culture system, early secondary spermatogonia could undergo mitotic divisions with a concomitant decrease in their size, so that they became distinguishable in size from PG. Because the subcutaneous environment of juveniles permitted aggregates of the dissociated testicular cells to reconstruct the normal testis structure, we next inserted a genetically marked PG isolated from cultured testes into the aggregate and transplanted it subcutaneously. In this system, 73.9% of the aggregates contained a marked PG. When we observed the aggregates 12 weeks after transplantation, most aggregates (70.0%) contained marked PG that had self-renewed. Among these, fully growing aggregates contained many spermatogenic cells at the later developmental stage. These results suggested that isolated PG from the cultured testes had the ability as stem cells, and that purification of the spermatogenic stem cells became reliable in Xenopus.