• Journal of Microbiology and Biotechnology
• /
• v.29 no.10
• /
• pp.1522-1542
• /
• 2019

To adapt to environmental changes and to maintain cellular homeostasis, microorganisms adjust the intracellular concentrations of biochemical compounds, including metal ions; these are essential for the catalytic function of many enzymes in cells, but excessive amounts of essential metals and heavy metals cause cellular damage. Metal-responsive transcriptional regulators play pivotal roles in metal uptake, pumping out, sequestration, and oxidation or reduction to a less toxic status via regulating the expression of the detoxification-related genes. The sensory and regulatory functions of the metalloregulators have made them as attractive biological parts for synthetic biology, and the exceptional sensitivity and selectivity of metalloregulators toward metal ions have been used in heavy metal biosensors to cope with prevalent heavy metal contamination. Due to their importance, substantial efforts have been made to characterize heavy metal-responsive transcriptional regulators and to develop heavy metal-sensing biosensors. In this review, we summarize the biochemical data for the two major metalloregulator families, SmtB/ArsR and MerR, to describe their metal-binding sites, specific chelating chemistry, and conformational changes. Based on our understanding of the regulatory mechanisms, previously developed metal biosensors are examined to point out their limitations, such as high background noise and a lack of well-characterized biological parts. We discuss several strategies to improve the functionality of the metal biosensors, such as reducing the background noise and amplifying the output signal. From the perspective of making heavy metal biosensors, we suggest that the characterization of novel metalloregulators and the fabrication of exquisitely designed genetic circuits will be required.

• Microbiology and Biotechnology Letters
• /
• v.50 no.1
• /
• pp.126-134
• /
• 2022

Norovirus (NoV) is an important pathogen causing acute gastroenteritis worldwide. The purpose of the present study was the molecular characterization of NoV. A total of 408 stool specimens collected from hospitalized children associated with acute gastroenteritis in Chiang Rai, Thailand, 2015-2017 were investigated for the presence of NoVs by RT-PCR. NoV GII was detected in 32 samples (7.8%). Five distinct genotypes were identified, including GII.4 (13/32, 40.6%), GII.3 (11/32, 34.3%), GII.17 (4/32, 12.5%), GII.2 (2/32, 6.3%), and GII.14 (2/32, 6.3%). NoV infection occurred mostly in young children under 3 years of age (31/32, 96.9%) and showed the main peak in summer months from March to April (18/32, 56.3%). Phylogenetic analysis revealed that all 13 GII.4 strains clustered with GII.4 Sydney 2012 variant. Representative GII.3 strains were analyzed as a recombinant GII.3P[12] strain. Several amino acid differences were found in the antigenic epitopes and antibody binding sites of the VP1 capsid of the GII.3P[12]. Homology modeling of the P domain of the GII.3P[12] strain demonstrated that 10/13 amino acid differences were predicted to be located on the surface-exposed area of the capsid structure. These amino acid changes might affect the infectivity and the antigenicity of the recombinant GII.3P[12]. The prevalence of GII.4 Sydney 2012 and recombinant GII.3P[12] strains indicates the genetic diversity of circulating NoVs in Thailand, emphazing the importance of continuous surveillance to mornitor newly emerging NoV strains in the future.

• Genomics & Informatics
• /
• v.20 no.2
• /
• pp.19.1-19.15
• /
• 2022

Alfalfa (Medicago sativa) is an important food and feed crop which rich in mineral sources. The WUSCHEL-related homeobox (WOX) gene family plays important roles in plant development and identification of putative gene families, their structure, and potential functions is a primary step for not only understanding the genetic mechanisms behind various biological process but also for genetic improvement. A variety of computational tools, including MAFFT, HMMER, hidden Markov models, Pfam, SMART, MEGA, ProtTest, BLASTn, and BRAD, among others, were used. We identified 34 MsWOX genes based on a systematic analysis of the alfalfa plant genome spread in eight chromosomes. This is an expansion of the gene family which we attribute to observed chromosomal duplications. Sequence alignment analysis revealed 61 conserved proteins containing a homeodomain. Phylogenetic study sung reveal five evolutionary clades with 15 motif distributions. Gene structure analysis reveals various exon, intron, and untranslated structures which are consistent in genes from similar clades. Functional analysis prediction of promoter regions reveals various transcription binding sites containing key growth, development, and stress-responsive transcription factor families such as MYB, ERF, AP2, and NAC which are spread across the genes. Most of the genes are predicted to be in the nucleus. Also, there are duplication events in some genes which explain the expansion of the family. The present research provides a clue on the potential roles of MsWOX family genes that will be useful for further understanding their functional roles in alfalfa plants.

• Korean journal of applied entomology
• /
• v.61 no.1
• /
• pp.117-128
• /
• 2022

In this study, we have examined pyrethroid actions on sodium channels in the pest insect Heliothis virescens. The synthetic pyrethroid permethrin increased steady-state sodium current in H. virescens central neurons and prolonged tail currents (I_Na-tail) due to extreme slowing of sodium channel deactivation. Prolongation of I_Na-tail was evident at permethrin concentrations as low as 60 nM, which modified ~1.7% of sodium channels and 10 μM permethrin modified about 30% of channels. The average time constant (τ₁) of tail current decay was ~335 ms for permethrin-modified channels. These modified channels activated at more negative potentials and showed slower activation kinetics, and failed to inactivate. Permethrin modification of sodium channels was dramatically potentiated by the α scorpion toxin LqhαIT, showing positive cooperativity between two binding sites. The amplitude of the tail current induced by 0.3 μM permethrin was enhanced ~8-fold by LqhαIT (200 pM). Positive cooperativity was also observed between permethrin and the insect-specific scorpion toxin AaIT as 10 nM permethrin potentiated the shift of voltage dependence caused by AaIT (~2-fold).

• BMB Reports
• /
• v.55 no.6
• /
• pp.281-286
• /
• 2022

Hepatocellular carcinoma is a major health burden, and though various treatments through much research are available, difficulties in early diagnosis and drug resistance to chemotherapy-based treatments render several ineffective. Cancer stem cell model has been used to explain formation of heterogeneous cell population within tumor mass, which is one of the underlying causes of high recurrence rate and acquired chemoresistance, highlighting the importance of CSC identification and understanding the molecular mechanisms of CSC drivers. Extracellular CSC-markers such as CD133, CD90 and EpCAM have been used successfully in CSC isolation, but studies have indicated that increasingly complex combinations are required for accurate identification. Pseudogene-derived long non-coding RNAs are useful candidates as intracellular CSC markers - factors that regulate pluripotency and self-renewal - given their cancer-specific expression and versatile regulation across several levels. Here, we present the use of microarray data to identify stemness-associated factors in liver cancer, and selection of sole pseudogene-derived lncRNA ZNF204P for experimental validation. ZNF204P knockdown impairs cell proliferation and migration/invasion. As the cytosolic ZNF204P shares miRNA binding sites with OCT4 and SOX2, well-known drivers of pluripotency and self-renewal, we propose that ZNF204P promotes tumorigenesis through the miRNA-145-5p/OCT4, SOX2 axis.

• Biomolecules & Therapeutics
• /
• v.31 no.6
• /
• pp.661-673
• /
• 2023

Treatment of colorectal cancer (CRC) has always been challenged by the development of resistance. We investigated the antiproliferative activity of licochalcone H (LCH), a regioisomer of licochalcone C derived from the root of Glycyrrhiza inflata, in oxaliplatin (Ox)-sensitive and -resistant CRC cells. LCH significantly inhibited cell viability and colony growth in both Ox-sensitive and Ox-resistant CRC cells. We found that LCH decreased epidermal growth factor receptor (EGFR) and AKT kinase activities and related activating signaling proteins including pEGFR and pAKT. A computational docking model indicated that LCH may interact with EGFR, AKT1, and AKT2 at the ATP-binding sites. LCH induced ROS generation and increased the expression of the ER stress markers. LCH treatment of CRC cells induced depolarization of MMP. Multi-caspase activity was induced by LCH treatment and confirmed by Z-VAD-FMK treatment. LCH increased the number of sub-G1 cells and arrested the cell cycle at the G1 phase. Taken together LCH inhibits the growth of Ox-sensitive and Ox-resistant CRC cells by targeting EGFR and AKT, and inducing ROS generation and ER stress-mediated apoptosis. Therefore, LCH could be a potential therapeutic agent for improving not only Ox-sensitive but also Ox-resistant CRC treatment.

• Asian-Australasian Journal of Animal Sciences
• /
• v.33 no.2
• /
• pp.219-229
• /
• 2020

Objective: Considering the physiological and clinical importance of leptin receptor (LEPR) in regulating obesity and the fact that porcine LEPR expression is not known to be controlled by lncRNAs and miRNAs, we aim to characterize this gene as a potential target of SSC-miR-323 and the lncRNA TCONS_00010987. Methods: Bioinformatics analyses revealed that lncRNA TCONS_00010987 and LEPR have SSC-miR-323-binding sites and that LEPR might be a target of lncRNA TCONS_00010987 based on cis prediction. Wild-type and mutant TCONS_00010987-target sequence fragments and wild-type and mutant LEPR 3'-UTR fragments were generated and cloned into pmiRRB-REPORT^TM-Control vectors to construct respective recombinant plasmids. HEK293T cells were co-transfected with the SSC-miR-323 mimics or a negative control with constructs harboring the corresponding binding sites and relative luciferase activities were determined. Tissue expression patterns of lncRNA TCONS_00010987, SSC-miR-323, and LEPR in Anqing six-end-white (AQ, the obese breed) and Large White (LW, the lean breed) pigs were detected by real-time quantitative polymerase chain reaction; backfat expression of LEPR protein was detected by western blotting. Results: Target gene fragments were successfully cloned, and the four recombinant vectors were constructed. Compared to the negative control, SSC-miR-323 mimics significantly inhibited luciferase activity from the wild-type TCONS_00010987-target sequence and wild-type LEPR-3'-UTR (p<0.01 for both) but not from the mutant TCONS_00010987-target sequence and mutant LEPR-3'-UTR (p>0.05 for both). Backfat expression levels of TCONS_00010987 and LEPR in AQ pigs were significantly higher than those in LW pigs (p<0.01), whereas levels of SSC-miR-323 in AQ pigs were significantly lower than those in LW pigs (p<0.05). LEPR protein levels in the backfat tissues of AQ pigs were markedly higher than those in LW pigs (p<0.01). Conclusion: LEPR is a potential target of SSC-miR-323, and TCONS_00010987 might act as a sponge for SSC-miR-323 to regulate LEPR expression.

• Proceedings of the Korean Environmental Sciences Society Conference
• /
• 1998.10a
• /
• pp.2-4
• /
• 1998

Proliferation of Nocardia amarae cells in activated sludge has often been associated with the generation of nuisance foams. Despite intense research activities in recent years to examine the causes and control of Nocardia foaming in activated sludge, the foaming continued to persist throughout the activated sludge treatment plants in United States. In addition to causing various operational problems to treatment processes, the presence of Nocardia may have secondary effects on the fate of heavy metals that are not well known. For example, for treatment plants facing more stringent metal removal requirements, potential metal removal by Nocardia cells in foaming activated sludge would be a welcome secondary effect. In contrast, with new viosolid disposal regulations in place (Code o( Federal Regulation No. 503), higher concentration of metals in biosolids from foaming activated sludge could create management problems. The goal of this research was to investigate the metal sorption property of Nocardia amarae cells grown in batch reactors and in chemostat reactors. Specific surface area and metal sorption characteristics of N. amarae cells harvested at various growth stages were compared. Three metals examined in this study were copper, cadmium and nickel. Nocardia amarae strain (SRWTP isolate) used in this study was obtained from the University of California at Berkeley. The pure culture was grown in 4L batch reactor containing mineral salt medium with sodium acetate as the sole carbon source. In order to quantify the sorption of heavy metal ions to N amarae cell surfaces, cells from the batch reactor were harvested, washed, and suspended in 30mL centrifuge tubes. Metal sorption studies were conducted at pH 7.0 and ionlc strength of 10-2M. The sorption Isotherm showed that the cells harvested from the stationary and endogenous growth phase exhibited significantly higher metal sorption capacity than the cells from the exponential phase. The sequence of preferential uptake of metals by N. amarae cells was Cu>Cd>Ni. The specific surFace area of Nocardia cells was determined by a dye adsorption method. N.amarae cells growing at ewponential phase had significantly less specific surface area than that of stationary phase, indicating that the lower metal sorption capacity of Nocardia cells growing at exponential phase may be due to the lower specific surface area. The growth conditions of Nocardia cells in continuous culture affect their cell surface properties, thereby governing the adsorption capacity of heavy metal. The comparison of dye sorption isotherms for Nocardia cells growing at various growth rates revealed that the cell surface area increased with increasing sludge age, indicating that the cell surface area is highly dependent on the steady-state growth rate. The highest specific surface area of 199m21g was obtained from N.amarae cell harvested at 0.33 day-1 of growth rate. This result suggests that growth condition not only alters the structure of Nocardia cell wall but also affects the surface area, thus yielding more binding sites of metal removal. After reaching the steady-state condition at dilution rate, metal adsorption isotherms were used to determine the equilibrium distributions of metals between aqueous and Nocardia cell surfaces. The metal sorption capacity of Nocardia biomass harvested from 0.33 day-1 of growth rate was significantly higher than that of cells harvested from 0.5- and 1-day-1 operation, indicatng that N.amarae cells with a lower growth rate have higher sorpion capacity. This result was in close agreement with the trend observed from the batch study. To evaluate the effect of Nocardia cells on the metal binding capacity of activated sludge, specific surface area and metal sorption capacity of the mixture of Nocardia pure cultures and activated sludge biomass were determined by a series of batch experiments. The higher levels of Nocardia cells in the Nocardia-activated sludge samples resulted in the higher specific surface area, explaining the higher metal sorption sites by the mixed luquor samples containing greater amounts on Nocardia cells. The effect of Nocardia cells on the metal sorption capacity of activated sludge was evaluated by spiking an activated sludge sample with various amounts of pre culture Nocardia cells. The results of the Langmuir isotherm model fitted to the metal sorption by various mixtures of Nocardia and activated sludge indicated that the mixture containing higher Nocardia levels had higher metal adsorption capacity than the mixture containing lower Nocardia levels. At Nocardia levels above 100mg/g VSS, the metal sorption capacity of activate sludge increased proportionally with the amount of Noeardia cells present in the mixed liquor, indicating that the presence of Nocardia may increase the viosorption capacity of activated sludge.

• Journal of Life Science
• /
• v.17 no.2 s.82
• /
• pp.167-173
• /
• 2007

To study the transcriptional mechanisms by which expression of the murine glial cell-derived neurotrophic factor inducible transcription factor (mGIF) gene is regulated, a murine genomic clone was iso-lated using a mGIF cDNA as probe. A 13-kb genomic fragment, which comprises 4-kb upstream of the transcription initiation site was sequenced. The promoter region lacks a TATA box and CAAT box, is rich in G+C content, and has multiple putative binding sites for the transcription factor Spl. The mGIF gene also has consensus sequences for AP2 binding sites. The transcriptional activity of five deletion mutants of a 2.1-kb fragment was analyzed by modulating transcription of the heterologous luciferase gene in the promoterless plasmid pGL2-Basic. All mutants showed significant transcriptional activity in the murine neuroblastoma cell line NB41A3. Transient expression assays suggested the presence of a positive regulator between -213 and -129 while a negative regulator was found in the region between -806 and -214. Relatively strong transcriptional activity was observed in neuronal NB41A3, glial C6 cells and hepatic HepG2, but very weak activity in skeletal muscle C2C12 cells. These findings confirm the tissue-specific activity of the mGIF promoter and suggest that this gene shares structural and functional similarities with the dopamine receptor genes that it regulates.

• Journal of Life Science
• /
• v.27 no.2
• /
• pp.149-154
• /
• 2017

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-hodgkin lymphoma. Advances in the chemotherapeutic treatment of this disease have improved the outcomes of DLBCL; nonetheless, many patients still die of DLBCL, and therefore, a better understanding of this disease and identification of novel therapeutic targets are urgently required. In a recent gene expression profiling study, PDE (phosphodiesterase) 4B was found to be overexpressed in chemotherapy-resistant tumors. The major function of PDE4B is to inactivate the second messenger cyclic 3',5' monophosphate (cAMP) by catalyzing the hydrolysis of cAMP to 5'AMP. It is known that cAMP induces cell cycle arrest and/or apoptosis in B cells, and PDE4B abolishes cAMP's effect on B cells. However, the mechanism by which PDE4B is overexpressed remains unclear. Here, we show that the aberrant expression of miRNA may be associated with the overexpression of this gene. The PDE4B 3' untranslated region (UTR) has three functional binding sites of miR-23b, as confirmed by luciferase reporter assays. Interestingly, miR-23b-binding sites were evolutionarily conserved from humans to lizards, implying the critical role of PDE4B-miR-23b interaction in cellular physiology. The ectopic expression of miR-2 3b repressed PDE4B mRNA levels and enhanced intracellular cAMP concentrations. Additionally, miR-23b expression inhibited cell proliferation and survival of DLBCL cells only in the presence of forskolin, an activator of adenylyl cyclase, suggesting that miR-23b's effect is via the downregulation of PDE4B. These results together suggest that miR-23b could be a therapeutic target for overcoming drug resistance by repressing PDE4B in DLBCL.