Korea Research Institute of Bioscience and Biotechnology
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GADD45b regulates hepatic gluconeogenesis via modulating the protein stability of FoxO1
No full textIncreased hepatic gluconeogenesis is one of the main contributors to the development of type 2 diabetes. Recently, it has been reported that growth arrest and DNA damage-inducible 45 beta (GADD45β) is induced under both fasting and high-fat diet (HFD) conditions that stimulate hepatic gluconeogenesis. Here, this study aimed to establish the molecular mechanisms underlying the novel role of GADD45β in hepatic gluconeogenesis. Both whole-body knockout (KO) mice and adenovirus-mediated knockdown (KD) mice of GADD45β exhibited decreased hepatic gluconeogenic gene expression concomitant with reduced blood glucose levels under fasting and HFD conditions, but showed a more pronounced effect in GADD45β KD mice. Further, in primary hepatocytes, GADD45β KD reduced glucose output, whereas GADD45β overexpression increased it. Mechanistically, GADD45β did not affect Akt-mediated forkhead box protein O1 (FoxO1) phosphorylation and forskolin-induced cAMP response element-binding protein (CREB) phosphorylation. Rather it increased FoxO1 transcriptional activity via enhanced protein stability of FoxO1. Further, GADD45β colocalized and physically interacted with FoxO1. Additionally, GADD45β deficiency potentiated insulin-mediated suppression of hepatic gluconeogenic genes, and it were impeded by the restoration of GADD45β expression. Our finding demonstrates GADD45β as a novel and essential regulator of hepatic gluconeogenesis. It will provide a deeper understanding of the FoxO1-mediated gluconeogenesis.
Analysis of genomic pathogenesis according to the revised Bethesda guidelines and additional criteria
No full textPurpose: As few genotype-phenotype correlations are available for nonsyndromic hereditary colorectal cancer (CRC), we implemented genomic analysis on the basis of the revised Bethesda guideline (RBG) and extended (12 items) to verify possible subtypes.
Methods: Patients with sporadic CRC (n = 249) were enrolled, stratified according to the revised Bethesda guidelines (RBG+ and RBG- groups) plus additional criteria. Exome/transcriptome analyses (n = 98) and cell-based functional assays were conducted.
Results: We detected 469 somatic and 830 germline gene mutations differing significantly between the positive and negative groups, associated with 12 RBG items/additional criteria. Twenty-one genes had significantly higher mutation rates in left, relative to right, colon cancer, while USP40, HCFC1, and HSPG2 mutation rates were higher in rectal than colon cancer. FAT4 mutation rates were lower in early-onset CRC, in contrast to increased rates in microsatellite instability (MSI)-positive tumors, potentially defining an early-onset microsatellite-stable subtype. The mutation rates of COL6A5 and MGAM2 were significantly and SETD5 was assumably, associated CRC pedigree with concurrent gastric cancer (GC). The predicted deleterious/damaging germline variants, SH2D4A rs35647122, was associated with synchronous/metachronous CRC with related tumors, while NUP160 rs381660 and KRTAP27-1 rs2244485 were potentially associated with a GC pedigree and less strictly defined hereditary CRC, respectively. SH2D4A and NUP160 acted as oncogenic facilitators.
Conclusion: Our limited genomic analysis for RBG and additional items suggested that specific somatic alterations in the respective items may enlighten relevant pathogenesis along with the knowledge of germline mutations. Further validation is needed to indicate appropriate surveillance in suspected individuals.
Genome insights into the novel species Jejubacter calystegiae, a plant growth-promoting bacterium in saline conditions
No full textJejubacter calystegiae KSNA2T, a moderately halophilic, endophytic bacterium isolated from beach morning glory (Calystegia soldanella), was determined to be a novel species in a new genus in the family Enterobacteriaceae. To gain insights into the genetic basis of the salinity stress response of strain KSNA2T, we sequenced its genome using two complementary sequencing platforms (Illumina HiSeq and PacBio RSII). The genome contains a repertoire of metabolic pathways, such as those for nitrogen, phosphorus, and some amino acid metabolism pathways. Functional annotation of the KSNA2T genome revealed several genes involved in salt tolerance pathways, such as those encoding sodium transporters, potassium transporters, and osmoprotectant enzymes. Plant growth-promoting bacteria-based experiments indicated that strain KSNA2T promotes the germination of vegetable seeds in saline conditions. Overall, the genetic and biological analyses of strain KSNA2T provide valuable insights into bacteria-mediated salt tolerance in agriculture.
β-Lapachone ameliorates L-DOPA-induced dyskinesia in a 6-OHDA-induced mouse model of Parkinson's disease
No full textThe dopamine precursor 3,4？dihydroxyphenyl？ l？alanine (L？DOPA) is the most widely used symptomatic treatment for Parkinson's disease (PD); however, its prolonged use is associated with L？DOPA？induced dyskinesia in more than half of patients after 10 years of treatment. The present study investigated whether co？treatment with β？Lapachone, a natural compound, and L？DOPA has protective effects in a 6？hydroxydopamine (6？OHDA)？induced mouse model of PD. Unilateral 6？OHDA？lesioned mice were treated with vehicle or β？Lapachone (10 mg/kg/day) and L？DOPA for 11 days. Abnormal involuntary movements (AIMs) were scored on days 5 and 10. β？Lapachone (10 mg/kg) co？treatment with L？DOPA decreased the AIMs score on both days 5 and 10. β？Lapachone was demonstrated to have a beneficial effect on the axial and limb AIMs scores on day 10. There was no significant suppression in dopamine D1 receptor？related and ERK1/2 signaling in the DA？denervated striatum by β？Lapachone？cotreatment with L？DOPA. Notably, β？Lapachone？cotreatment with L？DOPA increased phosphorylation at the Ser9 site of glycogen synthase kinase 3β (GSK？3β), indicating suppression of GSK？3β activity in both the unlesioned and 6？OHDA？lesioned striata. In addition, astrocyte activation was markedly suppressed by β？Lapachone？cotreatment with L？DOPA in the striatum and substantia nigra of the unilateral 6？OHDA model. These findings suggest that β？Lapachone cotreatment with L？DOPA therapy may have therapeutic potential for the suppression or management of the development of L？DOPA？induced dyskinesia in patients with PD.
Structural and biochemical characterization of EFhd1/Swiprosin-2, an actin-binding protein in mitochondria
No full textCa2+ regulates several cellular functions, including signaling events, energy production, and cell survival. These cellular processes are mediated by Ca2+-binding proteins, such as EF-hand superfamily proteins. Among the EF-hand superfamily proteins, allograft inflammatory factor-1 (AIF-1) and swiprosin-1/EF-hand domain-containing protein 2 (EFhd2) are cytosolic actin-binding proteins. AIF-1 modulates the cytoskeleton and increases the migration of immune cells. EFhd2 is also a cytoskeletal protein implicated in immune cell activation and brain cell functions. EFhd1, a mitochondrial fraternal twin of EFhd2, mediates neuronal and pro-/pre-B cell differentiation and mitoflash activation. Although EFhd1 is important for maintaining mitochondrial morphology and energy synthesis, its mechanism of action remains unclear. Here, we report the crystal structure of the EFhd1 core domain comprising a C-terminus of a proline-rich region, two EF-hand domains, and a ligand mimic helix. Structural comparisons of EFhd1, EFhd2, and AIF-1 revealed similarities in their overall structures. In the structure of the EFhd1 core domain, two Zn2+ ions were observed at the interface of the crystal contact, suggesting the possibility of Zn2+-mediated multimerization. In addition, we found that EFhd1 has Ca2+-independent β-actin-binding and Ca2+-dependent β-actin-bundling activities. These findings suggest that EFhd1, an actin-binding and -bundling protein in the mitochondria, may contribute to the Ca2+-dependent regulation of mitochondrial morphology and energy synthesis.
One-step genotyping method in CRISPR based on short inner primer-assisted, tetra primer-paired amplifications
No full textBase editors and prime editors induce precise DNA modifications over one or several nucleotides in eukaryotic cells. The T7E1 assay has been widely adopted for the assessment of genome editing, but it has several limitations in the applications for prime editing and base editing due to low sensitivity, inaccuracy and additional disadvantages. Here, we propose a short inner primer-assisted, tetra primer-paired amplification (SIPATA) method as an alternative to T7E1 analysis. SIPATA is a PCR-based method in which two long outer and two short (15 nt) inner primers are used for the amplification of a specific genotype in the presence of Hot start-Taq. One of the inner primers carries a 3'-terminally wild-type nucleotide sequence, and the other carries a post-editing sequence. Under optimized conditions, SIPATA enabled sensitive and accurate genotyping of single-nucleotide conversions by base editors and prime editors. Furthermore, SIPATA could be applied to trace low levels of DNA modifications achieved by HDR-mediated gene correction or chimerism during the generation of model animals. Multiplexed genotyping was also possible without compromising those multifaceted analytical advantages of SIPATA. Our findings demonstrate that SIPATA offers a robust, fast and sensitive genotyping platform for single-nucleotide variations in a variety of CRISPR applications.
Detection of Rhodococcus fascians, the causative agent of lily fasciation in South Korea
No full textRhodococcus fascians is an important pathogen that infects various herbaceous perennials and reduces their economic value. In this study, we examined R. fascians isolates carrying a virulence gene from symptomatic lily plants grown in South Korea. Phylogenetic analysis using the nucleotide sequences of 16S rRNA, vicA, and fasD led to the classification of the isolates into four different strains of R. fascians. Inoculation of Nicotiana benthamiana with these isolates slowed root growth and resulted in symptoms of leafy gall. These findings elucidate the diversification of domestic pathogenic R. fascians and may lead to an accurate causal diagnosis to help reduce economic losses in the bulb market.
Biomass quantification and 3-D topography reconstruction of microalgal biofilms using digital image processing
No full textAn accurate and non-invasive technique for online biomass quantification of microbial attached growth is needed. In this research, image processing through Red-Green-Blue (RGB) analysis is used to assess biomass thickness from simple macroscopic images captured from microalgal biofilms by a digital camera. The results show that the green (G) vector in images of an Ettlia sp. biofilm can estimate the biomass concentration with R2 = 0.994 through an exponential correlation. Moreover, the R2 coefficient for the biofilm thickness measurement using the G vector is 0.973, which shows the high potential of this method. Furthermore, using the mathematical correlation between the G index and the biofilm thickness, it is possible to reconstruct the 3-D topography of a microalgal biofilm and to calculate the quantitative parameters, such as biomass yield and thickness, at every specific point of the biofilm. RGB analysis can easily determine the biofilm concentration and 3-D topography with satisfactory accuracy. This is promising technique for biofilm quantification and can be used in different applications, such as wastewater treatment by moving a bed biofilm reactor (MBBR), which was formerly possible only through sophisticated techniques.
A new surface charge neutralizing nano-adjuvant to potentiate polymyxins in killing Mcr-1 mediated drug-resistant Escherichia coli
No full textResistance to polymyxins when treating multidrug-resistant (MDR) Gram-negative bacterial infections limit therapeutic options. Here, we report the synthesis of a nickel (Ni) doped Zinc oxide (NZO) combined with black phosphorus (BP) (NZB) nanocomposite and its synergistic action with polymyxin B (PolB) against polymyxin-resistant Escherichia coli harboring mobilized colistin resistance (mcr-1) gene. NZB and PolB combination therapy expressed a specific and strong synergy against Mcr-1 expressing E. coli cells. The underlying mechanism of the synergy is the charge neutralization of the E. coli cell surface by NZB, resulting in a more feasible incorporation of PolB to E. coli. The synergistic concentration of NZB with PolB was proved biocompatible. Thus, the NZB is the first biocompatible nano-adjuvant to polymyxins against polymyxin-resistant E. coli cells, recognizing the physical status of bacteria instead of known adjuvants targeting cellular gene products. Therefore, NZB has the potential to revive polymyxins as leading last-resort antibiotics to combat polymyxin-resistant Gram-negative bacterial infections.
Characteristics of the gut microbiome of healthy young male soldiers in South Korea: the effects of smoking
No full textBackground/Aims: South Korean soldiers are exposed to similar environmental factors. In this study, we sought to evaluate the gut microbiome of healthy young male soldiers (HYMS) and to identify the primary factors influencing the microbiome composition.
Methods: We prospectively collected stool from 100 HYMS and performed next-generation sequencing of the 16S rRNA genes of fecal bacteria. Clinical data, including data relating to the diet, smoking, drinking, and exercise, were collected.
Results: The relative abundances of the bacterial phyla Firmicutes, Actinobacteria, Bacteroidetes, and Proteobacteria were 72.3%, 14.5%, 8.9%, and 4.0%, respectively. Fifteen species, most of which belonged to Firmicutes (87%), were detected in all examined subjects. Using cluster analysis, we found that the subjects could be divided into the two enterotypes based on the gut microbiome bacterial composition. Compared with enterotype 2 subjects, subjects classified as enterotype 1 tended to be characterized by higher frequencies of potentially harmful lifestyle habits (current smoker: 55.6% vs 36.6%, p=0.222; heavy drinker: 16.7% vs 3.7%, p=0.120; insufficient physical activity: 27.8% vs 14.6%, p=0.318). We identified a significant difference in the microbiome compositions of current and noncurrent smokers (p=0.008); the former differed from the latter mainly in a relatively lower abundance of Bifidobacterium species and a higher abundance of Negativicutes.
Conclusions: A high abundance of Actinobacteria and low abundance of Bacteroidetes were the main features distinguishing the gut microbiomes of HYMS, and current smokers could be differentiated from noncurrent smokers by their lower abundance of Bifidobacterium and higher abundance of Negativicutes.