Determination of free acetaldehyde in total blood for investigating the effect of aspartate on metabolism of alcohol in mice

To explore the effect of sodium l-aspartate monohydrate (aspartate) as a NAD⁺ regenerating agent for acetaldehyde in alcohol metabolism, a simple HPLC method has been developed for the measurement of free acetaldehyde in total mice blood digested with alcohol and aspartate. The blood samples were collected in EDTA Vacutainer tubes, and treated with 2,4-dinitrophenylhydrazine (DNP hydrazine) reagent in total blood. Acetaldehyde DNP hydrazone was extracted from total blood and analyzed by HPLC using an Ultrasphere ODS column. The compounds were separated using acetonitrile–water (50:50, v/v) as mobile phase and detected at 356 nm. The detection limit for acetaldehyde DNP hydrazone was 0.1 ppm. A blank determination was carried out for each analysis and subtracted from the results. The amount of acetaldehyde in blood has been determined as a function of time lapse after sole alcohol administration and aspartate ingestion followed by alcohol administration, respectively. This comparative analysis demonstrates that the ingestion of aspartate before the administration of alcohol dramatically decreases the aldehyde level in blood, and aspartate may be utilized as a prospective antagonist for acceleration of ethanol metabolism and prevention of acetaldehyde toxicity.     

Microsatellite DNA analysis reveals lower than expected genetic diversity in the threatened leopard cat (<Emphasis Type="Italic">Prionailurus bengalensis</Emphasis>) in South Korea

To optimize conservation efforts, it is necessary to determine the risk of extinction by collecting reliable population information for a given species. We developed eight novel, polymorphic microsatellite markers and used these markers in conjunction with twelve existing markers to measure genetic diversity of South Korean populations of leopard cat (Prionailurus bengalensis), a species for which population size and habitat area data are unknown in the country, to assess its conservation status. The average number of alleles and the observed heterozygosity of the species were 3.8 and 0.41, respectively, and microsatellite diversity was lower than the average genetic diversity of 57 populations of 12 other felid species, and lower than that of other mammal populations occurring in South Korea, including the raccoon dog (Nyctereutes procyonoides), water deer (Hydropotes inermis), and endangered long-tailed goral (Naemorhedus caudatus). Furthermore, analysis of genetic structure in the national leopard cat population showed no clear genetic differentiation, suggesting that it is not necessary to divide the South Korean leopard cat population into multiple management units for the purposes of conservation. These results indicate that the genetic diversity of the leopard cat in South Korea is unexpectedly low, and that the risk of local extinction is, as a result, substantial. Thus, it is necessary to begin appropriate conservation efforts at a national level to conserve the leopard cat population in South Korea.     

Microsatellite markers for the Ussuri white-toothed shrew (Soricidae: <Emphasis Type="Italic">Crocidura lasiura</Emphasis>) developed by Ion Torrent sequencing and their application to the shrew populations in disturbed forests

Understanding the impacts of forest management practices on genetic diversity is essential for effective animal management and conservation. We characterized novel microsatellite loci in the Ussuri white-toothed shrew (Crocidura lasiura Dobson, 1890) to test the impacts of anthropogenic thinning of forest trees on the shrew populations and their genetic diversity. Using Ion Torrent sequencing technology, we characterized 611 potential microsatellite markers with complete di- to tetra-nucleotide motifs, identifying nine polymorphic loci. The observed and expected heterozygosities across the nine loci were 0.526 and 0.527, respectively. Mean allelic diversity was 5.2 alleles per locus, with the mean polymorphism information content at 0.498. In comparison among shrew populations, which inhabited in the forests thinned in 2004 (CLA; n = 10), 2008 (CLB; n = 9) and 2014 (CLC; n = 3), the observed heterozygosities are similar among the three populations (0.525 at CLA, 0.532 at CLB and 0.519 at CLC), whereas the expected heterozygosities were much lower in population of CLC (0.377) than that of CLA (0.509) and CLB (0.533). The small sample size at CLC limited effective comparison and evaluation of the impact of forest thinning on genetic diversity in this shrew population. Future application of the species-specific microsatellite markers described here to a larger sample size would be valuable in estimating the ecological parameters of shrew populations associated with existing forest management practices.     

Factors associated with use of ultrasonography screening for hepatocellular carcinoma among hepatitis B or C carriers

Objectives: Chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are important risk factors for hepatocellular carcinoma (HCC). Yet, there have been few studies on adherence to screening recommendations for groups at high risk for HCC. We assessed whether demographic factors or medical conditions affected screening participation among HBV/HCV carriers. Methods: The study population consisted of 15 565 men and women who visited the National Cancer Center, Korea between August 2002 and July 2009. A self-administered questionnaire was used to collect information on demographic characteristics, medical history, including chronic HBV and HCV infection, and health check-up history. HBV surface antigen and HCV antibody levels were measured in serum. Results: Among 781 HBV carriers, 596 (76.3%) were aware of their infection and 451 (57.8%) had ever been tested by ultrasonography. Among HCV carriers, 49 of 127 (36.6%) were aware of their infection and 61 (48.0%) had ever been tested by ultrasonography. Among HBV carriers, male sex (OR, 1.68; 95% CI, 1.22–2.31), family history of liver disease (OR, 2.04; 95% CI, 1.43–2.90), medical history of hyperlipidemia (OR, 2.70; 95% CI, 1.36–5.33), and awareness of infection status (OR, 4.30; 95% CI, 2.99–6.17) were associated with being tested. Among HCV carriers, awareness of infection (OR, 3.77; 95% CI, 1.72–8.26) was significantly associated with being tested by ultrasonography. Conclusion: Male sex, family history of liver disease, medical history of hyperlipidemia, and awareness of high risk status were associated with being tested by ultrasonography.     

Evolutionary rates of mitochondrial genomes correspond to diversification rates and to contemporary species richness in birds and reptiles

Rates of biological diversification should ultimately correspond to rates of genome evolution. Recent studies have compared diversification rates with phylogenetic branch lengths, but incomplete phylogenies hamper such analyses for many taxa. Herein, we use pairwise comparisons of confamilial sauropsid (bird and reptile) mitochondrial DNA (mtDNA) genome sequences to estimate substitution rates. These molecular evolutionary rates are considered in light of the age and species richness of each taxonomic family, using a random-walk speciation–extinction process to estimate rates of diversification. We find the molecular clock ticks at disparate rates in different families and at different genes. For example, evolutionary rates are relatively fast in snakes and lizards, intermediate in crocodilians and slow in turtles and birds. There was also rate variation across genes, where non-synonymous substitution rates were fastest at ATP8 and slowest at CO3. Family-by-gene interactions were significant, indicating that local clocks vary substantially among sauropsids. Most importantly, we find evidence that mitochondrial genome evolutionary rates are positively correlated with speciation rates and with contemporary species richness. Nuclear sequences are poorly represented among reptiles, but the correlation between rates of molecular evolution and species diversification also extends to 18 avian nuclear genes we tested. Thus, the nuclear data buttress our mtDNA findings.     

Distinct Features of Brain-Resident Macrophages: Microglia and Non-Parenchymal Brain Macrophages

Tissue-resident macrophages play an important role in maintaining tissue homeostasis and innate immune defense against invading microbial pathogens. Brain-resident macrophages can be classified into microglia in the brain parenchyma and non-parenchymal brain macrophages, also known as central nervous system-associated or border-associated macrophages, in the brain-circulation interface. Microglia and non-parenchymal brain macrophages, including meningeal, perivascular, and choroid plexus macrophages, are mostly produced during embryonic development, and maintained their population by self-renewal. Microglia have gained much attention for their dual roles in the maintenance of brain homeostasis and the induction of neuroinflammation. In particular, diverse phenotypes of microglia have been increasingly identified under pathological conditions. Single-cell phenotypic analysis revealed that microglia are highly heterogenous and plastic, thus it is difficult to define the status of microglia as M1/M2 or resting/activated state due to complex nature of microglia. Meanwhile, physiological function of non-parenchymal brain macrophages remain to be fully demonstrated. In this review, we have summarized the origin and signatures of brain-resident macrophages and discussed the unique features of microglia, particularly, their phenotypic polarization, diversity of subtypes, and inflammasome responses related to neurodegenerative diseases.     

A priori and a posteriori approaches for finding genes of evolutionary interest in non-model species: Osmoregulatory genes in the kidney transcriptome of the desert rodent Dipodomys spectabilis (banner-tailed kangaroo rat)

One common goal in evolutionary biology is the identification of genes underlying adaptive traits of evolutionary interest. Recently next-generation sequencing techniques have greatly facilitated such evolutionary studies in species otherwise depauperate of genomic resources. Kangaroo rats (Dipodomys sp.) serve as exemplars of adaptation in that they inhabit extremely arid environments, yet require no drinking water because of ultra-efficient kidney function and osmoregulation. As a basis for identifying water conservation genes in kangaroo rats, we conducted a priori bioinformatics searches in model rodents (Mus musculus and Rattus norvegicus) to identify candidate genes with known or suspected osmoregulatory function. We then obtained 446,758 reads via 454 pyrosequencing to characterize genes expressed in the kidney of banner-tailed kangaroo rats (Dipodomys spectabilis). We also determined candidates a posteriori by identifying genes that were overexpressed in the kidney. The kangaroo rat sequences revealed nine different a priori candidate genes predicted from our Mus and Rattus searches, as well as 32 a posteriori candidate genes that were overexpressed in kidney. Mutations in two of these genes, Slc12a1 and Slc12a3, cause human renal diseases that result in the inability to concentrate urine. These genes are likely key determinants of physiological water conservation in desert rodents.     

A machine learning based method for the prediction of G protein-coupled receptor-binding PDZ domain proteins

G protein-coupled receptors (GPCRs) are part of multi-protein networks called ‘receptosomes’. These GPCR interacting proteins (GIPs) in the receptosomes control the targeting, trafficking and signaling of GPCRs. PDZ domain proteins constitute the largest protein family among the GIPs, and the predominant function of the PDZ domain proteins is to assemble signaling pathway components into close proximity by recognition of the last four C-terminal amino acids of GPCRs. We present here a machine learning based approach for the identification of GPCR-binding PDZ domain proteins. In order to characterize the network of interactions between amino acid residues that contribute to the stability of the PDZ domain-ligand complex and to encode the complex into a feature vector, amino acid contact matrices and physicochemical distance matrix were constructed and adopted. This novel machine learning based method displayed high performance for the identification of PDZ domain-ligand interactions and allowed the identification of novel GPCR-PDZ domain protein interactions.     

Physicochemical and biological characterization of SB2, a biosimilar of Remicade® (infliximab)

A biosimilar is a biological medicinal product that contains a version of the active substance of an already authorized original biological medicinal product. Biosimilarity to the reference product (RP) in terms of quality characteristics, such as physicochemical and biological properties, safety, and efficacy, based on a comprehensive comparability exercise needs to be established. SB2 (Flixabi® and Renflexis®) is a biosimilar to Remicade® (infliximab). The development of SB2 was performed in accordance with relevant guidelines of the International Conference on Harmonisation, the European Medicines Agency, and the United States Food and Drug Administration. To determine whether critical quality attributes meet quality standards, an extensive characterization test was performed with more than 80 lots of EU- and US-sourced RP. The physicochemical characterization study results revealed that SB2 was similar to the RP. Although a few differences in physicochemical attributes were observed, the evidence from the related literature, structure-activity relationship studies, and comparative biological assays showed that these differences were unlikely to be clinically meaningful. The biological characterization results showed that SB2 was similar to the RP in terms of tumor necrosis factor–α (TNF-α) binding and TNF-α neutralization activities as a main mode of action. SB2 was also similar in Fc-related biological activities including antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, neonatal Fc receptor binding, C1q binding, and Fc gamma receptor binding activities. These analytical findings support that SB2 is similar to the RP and also provide confidence of biosimilarity in terms of clinical safety and efficacy.