Selection of a reference gene for studies on lipid‐related aquatic adaptations of toothed whales (Grampus griseus)

Toothed whales are one group of marine mammals that has developed special adaptations, such as echolocation for predation, to successfully live in a dynamic aquatic environment. Their fat metabolism may differ from that of other mammals because toothed whales have acoustic fats. Gene expression in the metabolic pathways of animals can change with respect to their evolution and environment. A real‐time quantitative polymerase chain reaction (RT‐qPCR) is a reliable technique for studying the relative expressions of genes. However, since the accuracy of RT‐qPCR data is totally dependent on the reference gene, the selection of the reference gene is an essential step. In this study, 10 candidate reference genes (ZC3H10, FTL, LGALS1, RPL27, GAPDH, FTH1, DCN, TCTP, NDUS5, and UBIM) were initially tested for amplification efficiency using RT‐qPCR. After excluding DCN, the remaining nine genes, which are nearly 100% efficient, were selected for the gene stability analysis. Stable reference genes across eight different fat tissue, liver, and muscle samples from Grampus griseus were identified by four algorithms, which were provided in Genorm, NormFinder, BestKeeper, and Delta CT. Finally, a RefFinder comprehensive ranking was performed based on the stability values, and the nine genes were ranked as follows: LGALS1 > FTL > GAPDH > ZC3H10 > FTH1 > NDUS5 > TCTP > RPL27 > UBIM. The LGALS1 and FTL genes were identified as the most stable novel reference genes. The third‐ranked gene, GAPDH, is a well‐known housekeeping gene for mammals. Ultimately, we suggest the use of LGALS1 as a reliable novel reference gene for genomics studies on the lipid‐related aquatic adaptations of toothed whales.     

Viral mutation accelerated by nitric oxide production during infection in vivo.

Nitric oxide (NO), superoxide (O(2)(-)), and their reaction product peroxynitrite (ONOO(-)) are generated in excess during a host's response against viral infection, and contribute to viral pathogenesis by promoting oxidative stress and tissue injury. Here we demonstrate that NO and peroxynitrite greatly accelerates the mutation of Sendai virus (SeV), a nonsegmented negative-strand RNA virus, by using green fluorescent protein (GFP) inserted into and expressed by a recombinant SeV (GFP-SeV) as an indicator for mutation. GFP-SeV mutation frequencies were much higher in the wild-type mice than in those lacking inducible NO synthase, suggesting that mutation of the virus in vivo is NO dependent. High levels of NO and NO-mediated oxidative stress were induced by GFP-SeV infection in the lung of the wild-type mice, but not in the iNOS-deficient mice, as evidenced by electron spin resonance spectroscopy and immunohistochemical analysis for nitrotyrosine formation as well as histopathological examination. Furthermore, peroxynitrite, an NO-derived reactive nitrogen intermediate, enhanced viral mutation in vitro. These results indicate that the oxidative stress induced by NO produced during the natural course of viral infection increases mutation, expands the quasispecies spectrum, and facilitates evolution of RNA viruses.     

Age-Associated Different Transcriptome Profiling in Zebrafish and Rats: an Insight into the Diversity of Vertebrate Aging

Marine Biotechnology - Most mammals, including humans, show obvious aging phenotypes, for example, loss of tissue plasticity and sarcopenia. In this regard, fish can be attractive models to study...     

Low-Carbohydrate Diets and All-Cause Mortality: A Systematic Review and Meta-Analysis of Observational Studies

Objective

Low-carbohydrate diets and their combination with high-protein diets have been gaining widespread popularity to control weight. In addition to weight loss, they may have favorable short-term effects on the risk factors of cardiovascular disease (CVD). Our objective was to elucidate their long-term effects on mortality and CVD incidence.

Data sources

MEDLINE, EMBASE, ISI Web of Science, Cochrane Library, and ClinicalTrials.gov for relevant articles published as of September 2012. Cohort studies of at least one year’s follow-up period were included.

Review methods

Identified articles were systematically reviewed and those with pertinent data were selected for meta-analysis. Pooled risk ratios (RRs) with 95% confidence intervals (CIs) for all-cause mortality, CVD mortality and CVD incidence were calculated using the random-effects model with inverse-variance weighting.

Results

We included 17 studies for a systematic review, followed by a meta-analysis using pertinent data. Of the 272,216 people in 4 cohort studies using the low-carbohydrate score, 15,981 (5.9%) cases of death from all-cause were reported. The risk of all-cause mortality among those with high low-carbohydrate score was significantly elevated: the pooled RR (95% CI) was 1.31 (1.07–1.59). A total of 3,214 (1.3%) cases of CVD death among 249,272 subjects in 3 cohort studies and 5,081 (2.3%) incident CVD cases among 220,691 people in different 4 cohort studies were reported. The risks of CVD mortality and incidence were not statistically increased: the pooled RRs (95% CIs) were 1.10 (0.98–1.24) and 0.98 (0.78–1.24), respectively. Analyses using low-carbohydrate/high-protein score yielded similar results.

Conclusion

Low-carbohydrate diets were associated with a significantly higher risk of all-cause mortality and they were not significantly associated with a risk of CVD mortality and incidence. However, this analysis is based on limited observational studies and large-scale trials on the complex interactions between low-carbohydrate diets and long-term outcomes are needed.     

The ciliary protein Nek8/Nphp9 acts downstream of Inv/Nphp2 during pronephros morphogenesis and left-right establishment in zebrafish

Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease. Among 12 reported Nphp gene products, Inv/Nphp2, Nphp3 and Nek8/Nphp9 are localized to the proximal segment in the primary cilium. However, the functional relationships are unknown. This study focused on phenotype analysis of nek8 knockdown embryos and the genetic relationship between nek8 and inv in zebrafish. Knockdown of nek8 produced both pronephric cysts and abnormal cardiac looping. Simultaneous knockdown of nek8 and inv synergistically increased the incidence of these defects. Interestingly, nek8 mRNA rescued inv morphant phenotypes, although inv mRNA could not rescue nek8 morphant phenotypes. These results suggest that Nek8 acts downstream of Inv function.     

Comparison of conventional and liquid-based cytology, and human papillomavirus testing using SurePath preparation in Japan

We compared the detection rate of cervical neoplasias between a liquid-based cytology (LBC) method using SurePath and the conventional method. We also studied the feasibility of human papillomavirus (HPV) typing by linear array assay. Cytological specimens from 1551 Japanese women were prepared using the conventional and SurePath methods; the cytological and histological results from biopsy samples were compared. HPV typing using an HPV linear array assay was carried out on residual specimens using the SurePath method. The cytodiagnostic results showed a concordance rate of 85.3% (Κ= 0.46) between the two methods. The sensitivity of lesions histopathologically diagnosed as CIN1 or above was not significantly different between the two methods (P = 0.575-1.000). The receiver operating characteristic curve analysis of the detectability in CIN2 or above revealed no significant difference between the two methods (P = 0.096). Among the 44 patients who underwent HPV typing using a linear array assay, 33 samples were eligible for HPV testing and were stored at ambient temperature. In conclusion, the SurePath and conventional methods have equivalent abilities for detecting cervical lesions. After preparation for cytological diagnosis, use of the remaining cells from the SurePath specimens to perform HPV typing using the linear array method could be feasible.     

Inactivation and sedimentation of mercury from organomercurials by Klebsiella pneumoniae

Abstract A susceptibility of 63 clinical isolates of Klebsiella pneumoniae to inorganic and organic mercuric compounds was determined. 18 of them were found to be resistant to fluorescein mercuric acetate (FMA) and merbromin (MB). Moreover, all the resistant strains inactivate the antibacterial effect of FMA. The changes in the amount of organic mercury at the time of inactivation of the drug and the structures of the end products were examined in detail with the plasmid-bearing strain JK9 and its transconjugants of Escherichia coli .
The results showed that FMA was inactivated by an intracellular enzyme produced inducively and was degraded to fluorescein (sodium salt, uranine), which led to the sedimentation of metallic mercury. The discovery of the genes conferring inducible organic mercury-inactivating enzymes determined by plasmids was the next step and their application in the recovery of metallic mercury from organomercurials is now imminent.     

Secretion of human superoxide dismutase in Escherichia coli using the condensed single-protein-production system

A secretion vector, pColdV for the Single-Protein-Production (SPP) system was constructed using the E. coli OmpA signal peptide. Using this vector, human superoxide dismutase (hSOD) was co-expressed with MazF, an ACA-specific mRNA interferase, allowing E. coli cells to produce only hSOD, which was secreted into the periplasmic space with a yield of ～20% of total cellular proteins. The signal peptide was properly cleaved. Using cells overproducing DsbA protein, two S-S bridges were also properly formed to yield enzymatically active SOD. A well resolved heteronuclear single quantum coherence (HSQC) spectrum of hSOD isotope-labeled in the condensed SPP (cSPP) system was obtained by simply isolating the periplasmic fraction. These results indicate that human secretory proteins can be expressed well in the cSPP system using pColdV.