Efficient detection of single nucleotide variants in targeted genomic loci.

Single nucleotide variants (SNVs), including single nucleotide polymorphisms, are often associated with morphological and/or physiological abnormalities in various organisms. Targeted genomic DNA can be amplified and directly sequenced to detect these mutations, but this method is relatively time consuming and expensive. We recently established the heteroduplex mobility assay to detect genetic mutations as an easy, low-cost method in genome editing, but detecting such small genetic differences remains difficult. Here, we developed a new, simple method to detect single nucleotide changes in the zebrafish genome by polymerase chain reaction (PCR) and electrophoresis. We first designed a specific single stranded DNA with four tandem guanine nucleotides inserted beside the mutation site, called guanine-inserted primer (GIP). When reannealing, hybridized complexes of GIP and PCR amplicons with or without 1-bp-mutated alleles form different bulge structures, presumably leading to different mobilities on a polyacrylamide gel. This GIP-interacting mobility assay is easy to use; therefore, it could contribute to the detection of SNVs in any organism.

Cooperative contributions of the klf1 and klf17 genes in zebrafish primitive erythropoiesis.

Krüppel-like transcription factors (Klfs), which are characterized by the three conserved C-terminal zinc fingers, are involved in various biological processes, such as haematopoiesis and angiogenesis. However, how the Klf family of transcription factors cooperate in organogenesis remains elusive. During zebrafish embryogenesis, both klf1 and klf17 are expressed in the intermediate cell mass (ICM), where primitive erythroid cells are produced. Using CRISPR-Cas9 genome editing technology, we established klf1-klf17 double mutant zebrafish to investigate the functionally interactive roles of the klf1 and klf17 genes. The klf1-klf17 mutant exhibited a diminished number of circulating primitive erythroid cells at 2 days postfertilization (dpf), while klf1 or klf17 single mutants and wild-type embryos produced comparable numbers of primitive erythroid cells. Circulating erythroid cells from the klf1-klf17 mutant possessed larger nuclei at 2 dpf than wild-type cells, suggesting the impairment of primitive erythroid cell maturation. The expression of the erythroid cell maturation markers band3 and mitoferrin, but not the haematopoietic progenitor markers c-myb and scl, was decreased in the klf1-klf17 mutant at 1 dpf. Thus, these results illustrate the cooperative function of klf1 and klf17 in the maturation processes of zebrafish primitive erythroid cells.

Changes in the skin characteristics associated with dehydration and rehydration.

The present study aimed to test the hypothesis that changes in the dermal tissue dielectric constant (TDC) and biomechanical properties of the skin would be correlated with the indicators related to dehydration. Ten healthy adult men were enrolled in three trials: no fluid intake (DEH), ad libitum fluid intake (AL-HYD), and programmed fluid intake (P-HYD) after exercise in a randomised crossover design. The participants performed a pedalling exercise at 60% heart rate reserve until 2% body mass loss. At 120 min after exercise, an incremental exercise test was carried out. Aerobic capacity, body composition, TDC, biomechanical properties of the skin (pliability, viscoelasticity, and total recovery), and indicators related to dehydration in the serum and urine were measured before and 120 min after exercise. Higher values of the pliability and viscoelasticity, and lower value of the total recovery on the hand were demonstrated in the P-HYD trial compared to the DEH trial (all P < 0.05). Changes in the TDC were significantly correlated with changes in body mass, total body water, serum osmolarity, and hematocrit (all P < 0.05). Changes in the biomechanical properties of the hand were significantly correlated with changes in body mass, hematocrit, and urine specific gravity (all P < 0.05). The present study showed that the changes in skin characteristics correlated with the body water and dehydration-associated indicators in the serum and urine, thus suggesting that skin characteristics may be useful in the assessment of dehydration.HighlightsThis study was the first to investigate the effect of dehydration and rehydration on the TDC and biomechanical properties of the skin upon instrumental measure, and not manual testing.This study confirmed the decline in aerobic capacity by dehydration and immediate recovery with sufficient rehydration.Changes in the TDC and the biomechanical properties of the skin correlated with the body water and dehydration-associated indicators in the serum and urine.Skin characteristics may be useful in the assessment of dehydration.

Effects of Intestinal Bacterial Hydrogen Gas Production on Muscle Recovery following Intense Exercise in Adult Men: A Pilot Study.

This study aimed to examine the effects of hydrogen gas (H2) produced by intestinal microbiota on participant conditioning to prevent intense exercise-induced damage. In this double-blind, randomized, crossover study, participants ingested H2-producing milk that induced intestinal bacterial H2 production or a placebo on the trial day, 4 h before performing an intense exercise at 75% maximal oxygen uptake for 60 min. Blood marker levels and respiratory variables were measured before, during, and after exercise. Visual analog scale scores of general and lower limb muscle soreness evaluated were 3.8- and 2.3-fold higher, respectively, on the morning after treatment than that before treatment during the placebo trial, but not during the test beverage consumption. Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) concentrations and production rates significantly increased with placebo consumption; no changes were observed with test beverage consumption. After exercise, relative blood lactate levels with H2-producing milk consumption were lower than those with placebo consumption. A negative correlation was observed between the variation of 8-OHdG and the area under the curve (AUC) of breath H2 concentrations. Lipid oxidation AUC was 1.3-fold higher significantly with H2-producing milk than with placebo consumption. Conclusively, activating intestinal bacterial H2 production by consuming a specific beverage may be a new strategy for promoting recovery and conditioning in athletes frequently performing intense exercises.

Critical roles of the ddx5 gene in zebrafish sex differentiation and oocyte maturation.

DEAD-box helicase 5 (Ddx5) functions as an ATP-dependent RNA helicase and as a transcriptional coactivator for several transcription factors; however, the developmental function of the ddx5 gene in vertebrates is not fully understood. We found that the zebrafish ddx5 gene was expressed in developing gonads. Using the genome editing technology transcription activator-like effector nuclease, we established a ddx5-disrupted zebrafish and examined the morphological phenotypes of the mutant. We found that the majority of ddx5-deficient mutants developed as fertile males with normal testes and a small number of ddx5-deficient mutants developed as infertile females with small ovaries. Apoptotic cell death at 31 days post fertilization was increased in thick immature gonads (presumptive developing ovaries) of the ddx5-deficient mutant compared to those of heterozygous wild-type fish, while the number of apoptotic cells in thin immature gonads (presumptive developing testes) was comparable between the mutant and wild-type animals. Histological analysis revealed that ovaries of adult ddx5-deficient females had fewer vitellogenic oocytes and a larger number of stage I and II oocytes. The amount of cyclic adenosine monophosphate in the ddx5-deficient ovaries was high compared to that of wild-type ovaries, presumably leading to the mitotic arrest of oocyte maturation. Therefore, the ddx5 gene is dispensable for testis development, but it is essential for female sex differentiation and oocyte maturation in zebrafish.

Characterization of biklf/klf17-deficient zebrafish in posterior lateral line neuromast and hatching gland development.

Krüpple-like factors (Klfs) are highly conserved zinc-finger transcription factors that regulate various developmental processes, such as haematopoiesis and cardiovascular development. In zebrafish, transient knockdown analysis of biklf/klf17 using antisense morpholino suggests the involvement of biklf/klf17 in primitive erythropoiesis and hatching gland development; however, the continuous physiological importance of klf17 remains uncharacterized under the genetic ablation of the klf17 gene among vertebrates. We established the klf17-disrupted zebrafish lines using the CRISPR/Cas9 technology and performed phenotypic analysis throughout early embryogenesis. We found that the klf17-deficient embryos exhibited abnormal lateral line neuromast deposition, whereas the production of primitive erythrocytes and haemoglobin production were observed in the klf17-deficient embryos. The expression of lateral line neuromast genes, klf17 and s100t, in the klf17-deficient embryos was detected in posterior lateral line neuromasts abnormally positioned at short intervals. Furthermore, the klf17-deficient embryos failed to hatch and died without hatching around 15 days post-fertilization (dpf), whereas the dechorionated klf17-deficient embryos and wild-type embryos were alive at 15 dpf. The klf17-deficient embryos abolished hatching gland cells and Ctsl1b protein expression, and eliminated the expression of polster and hatching gland marker genes, he1.1, ctsl1b and cd63. Thus, the klf17 gene plays important roles in posterior lateral line neuromast and hatching gland development.

Involvement of the centrosomal protein 55 (cep55) gene in zebrafish head formation.

Mammalian CEP55 (centrosomal protein 55 kDa) is a coiled-coil protein localized to the centrosome in interphase cells and is required for cytokinesis. A homozygous non-sense mutation in human CEP55 has been recently identified in perinatal lethal MARCH (multinucleated neurons, anhydramnios, renal dysplasia, cerebellar hypoplasia and hydranencephaly) syndrome. We have isolated zebrafish cep55 mutants defective in head morphology. The zebrafish cep55 gene was expressed in the head including the retina and the pectoral fin at 1 day post-fertilization (dpf), and extensive cell death was widely observed in the head and tail of the cep55 mutant. In the cep55 mutant, the anterior-posterior distance of the ventral pharyngeal arches was short, and retinal lamination was disorganized. Neural cells, such as islet1-positive cells and pax2-positive cells, and fli1b-positive vascular cells were reduced in the head of the cep55 mutant. Thus, we propose that the zebrafish cep55 mutant is a model organism for human MARCH syndrome.

Influence of acute high-intensity exercise on salivary nitric oxide levels.

This study, employing an exercise versus control crossover design, was conducted to investigate the influence of acute high-intensity exercise on salivary nitric oxide (NO) levels. Nine healthy males (aged 23.8 ± 1.4 years) performed ergometer exercise at 80%VO2peak for 60 min, whereas controls sat at rest for 60 min. Saliva samples were collected before (Pre: 0800 h) and after (Post 0-h: 0900 h, Post 1-h: 1000 h, Post 2-h: 1100 h, Post 3-h: 1200 h) the interventions. Salivary NO levels were determined by colorimetric assay. It was found that the salivary NO levels in controls were decreased (P < 0.05) at Post 0-h (-94 ± 15), Post 1-h (-80 ± 20), Post 2-h (-92 ± 34) and Post 3-h (-145 ± 39) relative to the Pre values. Under exercise conditions, salivary NO levels did not change after high-intensity ergometer exercise relative to the Pre values. Thus, the response of salivary NO levels appeared to differ between high-intensity ergometer exercise and inactivity, that exercise-related stress induces the production of salivary NO.

Assessment of Salivary Nitric Oxide Levels in Elite University Athletes in Japan: Findings From a Cross Sectional Study Design.

BACKGROUND: High-intensity exercise affects the level of salivary nitric oxide (NO) with an impact on oxidative stress such as a reactive nitrogen-oxide species. However, in athletes with high-intensity training, the relationship between salivary NO levels and oxidative stress is yet to be clear. Additionally, the association of salivary NO levels and the common health disorders of athletes is unknown. Thus, the aim of this cross-sectional study was to clarify the relationship between salivary NO levels and oxidative stress, and the health/medical disorders existing in elite class university athletes. METHODS: In 250 athletes (males, 151 and females, 99) from undergraduate levels of Japanese University, we investigated the relationship between levels of salivary NO and oxidative stress markers: derived reactive oxygen species (d-ROMs) and biological antioxidant potential (BAP), and also examined that whether salivary NO levels are associated with diseases. RESULTS: There were no significant association between the levels of salivary NO and oxidative stress markers (such as d-ROM and BAP). From the questionnaire, asthma was the most prevalent as evident from medical history of the athletes. Additionally, the salivary NO levels were higher (520 ± 43 µmol/L vs. 375 ± 13 µmol/L, P < 0.05) in the asthma group (n = 9) than in the non-asthma group (n = 241). We determined the optimal cut-off value (P = 0.019) of the salivary NO levels for asthma was 425 µmol/L, with a sensitivity of 88.9% and specificity of 61.8% (area under the curve (AUC), 0.73). CONCLUSIONS: Our results suggest that the high levels of salivary NO in trained university athletes in Japan may potentially predict asthma. And this salivary NO level is not associated with markers of oxidative stress and existing diseases in athletes studied here.

Ontogenetic and temperature-dependent changes in tolerance to hypoxia and hydrogen sulfide during the early life stages of the Manila clam Ruditapes philippinarum.

Wind-induced upwelling of hypoxic waters containing hydrogen sulfide (H2S) sometimes causes mass mortalities of aquatic organisms inhabiting coastal areas, including the hypoxia-tolerant Manila clam Ruditapes philippinarum. We examined the tolerance of Manila clam to H2S under controlled laboratory conditions. Larvae and juveniles obtained by artificial fertilization or from a wild population were exposed to normoxic or to hypoxic water with or without un-ionized H2S (concentrations, 0.2-52.2 mg/L). Twenty-four-hour exposure experiments revealed ontogenetic changes in the clam's tolerance to H2S exposure: tolerance was enhanced from the larval stages to juveniles just after settlement but was attenuated as juveniles grew. Tolerance of larvae and juveniles to H2S exposure weakened as the water temperature rose from 20 to 28 °C. Prolonged 48-h exposure to H2S attenuated the tolerance of juveniles to H2S. Temporary suspension of H2S exposure by 24-h reoxygenation improved the ability of juveniles to withstand repeated H2S exposure.