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1.
Mol Cells ; 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34650007

RESUMO

Although various marine ingredients have been exploited for the development of cosmetic products, no previous study has examined the potential of seaweed extracellular vesicles (EV) in such applications. Our results revealed that EV from Codium fragile and Sargassum fusiforme effectively decreased α-MSH-mediated melanin synthesis in MNT-1 human melanoma cells, associated with downregulation of MITF (microphthalmia-associated transcription factor), tyrosinase and TRP1 (tyrosinase-related proteins 1). The most effective inhibitory concentrations of EV were 250 µg/ml for S. fusiforme and 25 µg/ml for C. fragile, without affecting the viability of MNT-1 cells. Both EV reduced melanin synthesis in the epidermal basal layer of a three-dimensional model of human epidermis. Moreover, the application of the prototype cream containing C. fragile EV (final 5 µg/ml) yielded 1.31% improvement in skin brightness in a clinical trial. Together, these results suggest that EV from C. fragile and S. fusiforme reduce melanin synthesis and may be potential therapeutic and/or supplementary whitening agents.

2.
Endocrinol Metab (Seoul) ; 36(4): 745-756, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34474513

RESUMO

Intermittent fasting has become an increasingly popular strategy in losing weight and associated reduction in obesity-related medical complications. Overwhelming studies support metabolic improvements from intermittent fasting in blood glucose levels, cardiac and brain function, and other health benefits, in addition to weight loss. However, concerns have also been raised on side effects including muscle loss, ketosis, and electrolyte imbalance. Of particular concern, the effect of intermittent fasting on hormonal circadian rhythms has received little attention. Given the known importance of circadian hormonal changes to normal physiology, potential detrimental effects by dysregulation of hormonal changes deserve careful discussions. In this review, we describe the changes in circadian rhythms of hormones caused by intermittent fasting. We covered major hormones commonly pathophysiologically involved in clinical endocrinology, including insulin, thyroid hormones, and glucocorticoids. Given that intermittent fasting could alter both the level and frequency of hormone secretion, decisions on practicing intermittent fasting should take more considerations on potential detrimental consequences versus beneficial effects pertaining to individual health conditions.

3.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204265

RESUMO

Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC50 values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.


Assuntos
ADP Ribose Transferases , Antineoplásicos Imunológicos/farmacologia , Toxinas Bacterianas , Exotoxinas , Imunotoxinas/farmacologia , Proteínas Ligantes de Maltose , Receptor ErbB-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Cadeia Única , Fatores de Virulência , ADP Ribose Transferases/genética , Toxinas Bacterianas/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Relação Dose-Resposta a Droga , Escherichia coli/genética , Escherichia coli/metabolismo , Exotoxinas/genética , Expressão Gênica , Engenharia Genética , Vetores Genéticos/genética , Humanos , Imunotoxinas/genética , Imunotoxinas/isolamento & purificação , Proteínas Ligantes de Maltose/genética , Espectrometria de Massas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Cadeia Única/genética , Fatores de Virulência/genética
4.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198626

RESUMO

Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N-terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a') tags were attached to the N-terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC50 value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.


Assuntos
Fator de Células-Tronco/biossíntese , Sequência de Aminoácidos , Ciclo Celular , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Fator de Células-Tronco/química
5.
Antonie Van Leeuwenhoek ; 114(10): 1585-1593, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34292424

RESUMO

An aerobic, Gram-stain-negative, non-motile, non-spore-forming, rod-shaped, and light pink-colored bacterial strain, designated TS19T, was isolated from a sand sample obtained from a coastal sand dune after exposure to 3 kGy of gamma radiation. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that the isolate was a member of the genus Hymenobacter and was most closely related to H. wooponensis WM78T (98.3% similarity). Strain TS19T and H. wooponensis showed resistance to gamma radiation with D10 values (i.e., the dose required to reduce the bacterial population by tenfold) of 7.3 kGy and 3.5 kGy, respectively. The genome of strain TS19T consists of one contig with 4,879,662 bp and has a G + C content of 56.2%. The genome contains 3,955 protein coding sequences, 44 tRNAs, and 12 rRNAs. The predominant fatty acids of strain TS19T were iso-C15:0, summed feature 4 (iso-C17:1 I and/or anteiso-C17:1 B), summed feature 3 (C16:1 ω6c and/or C16:1 ω7c), and C16:1 ω5c. The major polar lipids were phosphatidylethanolamine, and one unidentified aminophospholipid. The main respiratory quinone was menaquinone-7. Based on the phylogenetic, physiological, and chemotaxonomic characteristics, strain TS19T represents a novel species, for which the name Hymenobacter taeanensis sp. nov. is proposed. The type strain is TS19T (= KCTC 72897T = JCM 34023T).


Assuntos
Cytophagaceae , Areia , Técnicas de Tipagem Bacteriana , Cytophagaceae/genética , DNA Bacteriano/genética , Ácidos Graxos/análise , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2
6.
BMB Rep ; 54(8): 393-402, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34078529

RESUMO

In animals, proper locomotion is crucial to find mates and foods and avoid predators or dangers. Multiple sensory systems detect external and internal cues and integrate them to modulate motor outputs. Proprioception is the internal sense of body position, and proprioceptive control of locomotion is essential to generate and maintain precise patterns of movement or gaits. This proprioceptive feedback system is conserved in many animal species and is mediated by stretch-sensitive receptors called proprioceptors. Recent studies have identified multiple proprioceptive neurons and proprioceptors and their roles in the locomotion of various model organisms. In this review we describe molecular and neuronal mechanisms underlying proprioceptive feedback systems in C. elegans, Drosophila, and mice. [BMB Reports 2021; 54(8): 393-402].

7.
Int J Mol Sci ; 22(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067755

RESUMO

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b'a' domain of protein disulfide isomerase (PDIb'a') greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb'a'-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC50 of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.


Assuntos
Cromatografia em Gel/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/isolamento & purificação , Isomerases de Dissulfetos de Proteínas/metabolismo , Diferenciação Celular , Eletroforese em Gel de Poliacrilamida/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Proteínas Ligantes de Maltose/metabolismo , Células Procarióticas/metabolismo , Isomerases de Dissulfetos de Proteínas/fisiologia , Transporte Proteico , Solubilidade
8.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073063

RESUMO

Conventional human pluripotent stem cell (hPSC) cultures require high concentrations of expensive human fibroblast growth factor 2 (hFGF-2) for hPSC self-renewal and pluripotency in defined media for long-term culture. The thermal instability of the hFGF-2 mandates media change every day, which makes hPSC culture costly and cumbersome. Human DJ-1 (hDJ-1) can bind to and stimulate FGF receptor-1. In this study, for the first time, we have replaced hFGF-2 with hDJ-1 in the essential eight media and maintained the human embryonic stem cells (hESCs), H9, in the defined media at feeder-free condition. After more than ten passages, H9 in both groups still successfully maintained the typical hESC morphology and high protein levels of pluripotency markers, SSEA4, Tra1-60, Oct4, Nanog, and ALP. DNA microarray revealed that more than 97% of the 21,448 tested genes, including the pluripotency markers, Sox2, Nanog, Klf4, Lin28A, Lin28B, and Myc, have similar mRNA levels between the two groups. Karyotyping revealed no chromosome abnormalities in both groups. They also differentiated sufficiently into three germ layers by forming in vitro EBs and in vivo teratomas. There were some variations in the RT-qPCR assay of several pluripotency markers. The proliferation rates and the mitochondria of both groups were also different. Taken together, we conclude that hDJ-1 can replace hFGF-2 in maintaining the self-renewal and the pluripotency of hESCs in feeder-free conditions.


Assuntos
Meios de Cultura/química , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células-Tronco Pluripotentes , Proteína Desglicase DJ-1/metabolismo , Técnicas de Cultura de Células , Proliferação de Células , Humanos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo
9.
Nature ; 593(7857): 114-118, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33790466

RESUMO

Innate social behaviours, such as mating and fighting, are fundamental to animal reproduction and survival1. However, social engagements can also put an individual at risk2. Little is known about the neural mechanisms that enable appropriate risk assessment and the suppression of hazardous social interactions. Here we identify the posteromedial nucleus of the cortical amygdala (COApm) as a locus required for the suppression of male mating when a female mouse is unhealthy. Using anatomical tracing, functional imaging and circuit-level epistatic analyses, we show that suppression of mating with an unhealthy female is mediated by the COApm projections onto the glutamatergic population of the medial amygdalar nucleus (MEA). We further show that the role of the COApm-to-MEA connection in regulating male mating behaviour relies on the neuromodulator thyrotropin-releasing hormone (TRH). TRH is expressed in the COApm, whereas the TRH receptor (TRHR) is found in the postsynaptic MEA glutamatergic neurons. Manipulating neural activity of TRH-expressing neurons in the COApm modulated male mating behaviour. In the MEA, activation of the TRHR pathway by ligand infusion inhibited mating even towards healthy female mice, whereas genetic ablation of TRHR facilitated mating with unhealthy individuals. In summary, we reveal a neural pathway that relies on the neuromodulator TRH to modulate social interactions according to the health status of the reciprocating individual. Individuals must balance the cost of social interactions relative to the benefit, as deficits in the ability to select healthy mates may lead to the spread of disease.


Assuntos
Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/fisiologia , Preferência de Acasalamento Animal/fisiologia , Vias Neurais/fisiologia , Comportamento Social , Animais , Copulação/fisiologia , Complexo Nuclear Corticomedial/citologia , Complexo Nuclear Corticomedial/metabolismo , Feminino , Ácido Glutâmico/metabolismo , Saúde , Ligantes , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Neurônios/metabolismo , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Hormônio Liberador de Tireotropina/metabolismo
10.
J Nanosci Nanotechnol ; 21(7): 3701-3706, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33715677

RESUMO

To improve the surface characteristics of Ti-6Al-4V dental implants and the binding between the bone and implant surface, biocompatible oxide films were formed by plasma electrolytic oxidation (PEO). The PEO treatment was performed using electrolyte solutions containing Ca (calcium acetate monohydrate), P(calcium glycerophosphate), Mn (manganese(II) acetate tetrahydrate), and Si (sodium metasilicate nonahydrate), which are the major constituents of bone, for 3 min at 280 V. The morphology and crystalline phase of the PEO-treated surfaces were characterized using field-emission scanning electron microscopy, energy-dispersive X-ray spectrometry, X-ray diffraction, and Fourier transform infrared spectroscopy. All the obtained PEO-treated samples exhibited a morphology comprising porous structures. Oval and irregular pore structures were observed as the Mn content increased. As the Si content increased, the areas occupied by the pores increased. When both, Si and Mn were used for the PEO treatment, the number of nano- to micro-sized pores gradually decreased with the increasing ratios of the constituents.


Assuntos
Implantes Dentários , Durapatita , Ligas , Materiais Revestidos Biocompatíveis , Propriedades de Superfície , Titânio
11.
J Nanosci Nanotechnol ; 21(7): 4022-4028, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33715737

RESUMO

The purpose of this study was to investigate electrochemical analysis of nano- and micro-sized pore formed Ti-6Al-4V alloys in solution containing Ca, P, Mn and Si ions via plasma eletrolytic oxidation for bio-implant materials. The coatings were produced on Ti-6Al-4V alloy for dental implant using the plasma electrolytic oxidation (PEO) method in electrolytes with the various concentration of 0, 5, and 20% Mn and Si, respectively. Electrochemical potentiodynamic polarization and AC impedance behaviors were carried out in 0.9% NaCl solution at 36.5 ± 1 °C using potentiostat (Potentiostat, EG&G, 362) and electrochemical impedance spectroscope (EIS, EG&G, 1025). The potentiodynamic polarization test with a scan rate of 1.667 mV s-1 was carried out from -1500 mV to 2000 mV. The frequency range used for EIS was 10²-105 Hz. The amplitude of AC signal was 10 mV and 5 points per decade was used. From the potentiodynamic polarization test, PEO treated alloy in electrolyte containing Ca, P, Mn, and Si show a lower corrosion potential than that on the bulk surface. In the case of Mn and Si doped surface, the corrosion resistance increase compared to non-doped surface with Mn and Si elements, and the current density was lower than that of the bulk surface. From the AC impedance test, in the case of Mn and Si doped surface, polarization resistance values were higher than other specimens, and nano- and micro-sized pores were covered with corrosion product consisted Mn and Si elements.


Assuntos
Ligas , Titânio , Corrosão , Íons , Teste de Materiais , Propriedades de Superfície
12.
J Nanosci Nanotechnol ; 21(9): 4807-4812, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-33691870

RESUMO

In this study, nanotube morphology changes of Ti-xTa-Ag-Pt alloys with Ta content for biomaterials were researched using various experimental instruments. Ti-xTa-Ag-Pt alloys were manufactured in an Ar atmosphere using a vacuum arc-melting furnace with Ta contents of 10 and 50, and then heat-treated at 1100 °C for 1 hr. Nanotube formation of Ti-xTa-Ag-Pt (x = 10, 50 wt%) alloys were performed using a DC power of 30 V in 1.0 M H3PO4 + 0.8 wt% NaF electrolyte solution. Surface characteristics were investigated using an optical microscope, X-ray diffractometer, field-emission scanning electron microscope, energy-dispersive X-ray spectroscopy, and Image analyzer (Image J). Ti-10Ta-Ag-Pt alloy had a needle-like structures, and Ti-Ti-50Ta-Ag-Pt showed the mixed structure (equiaxed and needle-like structures). As the Ta content increased, the α-phase decreased and the ß-phase increased. The highly ordered nanotubes were formed on the ß-phase, whereas disordered nanotubes were formed on needle-like structure of α-phase in Ti-10Ta-Ag-Pt alloy. As the Ta content increases, large and small nanotube diameters became smaller in size. Anatase and rutile phases were formed on the alloy surface. Ta, Ag, and Pt elements were uniformly distributed over the entire surface and at the edge or inside of the nanotube.


Assuntos
Ligas , Nanotubos , Materiais Biocompatíveis , Microscopia Eletrônica de Varredura , Prata , Titânio
13.
J Nanosci Nanotechnol ; 21(7): 3753-3758, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33715686

RESUMO

In this study, plasma electrolytic oxidation (PEO) on Ti-xNb-2Ag-2Pt alloys for nano- and micro-pore formation in electrolyte with Ca and P ions for dental implant use was studied using various experimental equipment. The Ti-xNb-2Ag-2Pt alloys were fabricated using a vacuum arc melting furnace, and micro-pores were created through PEO-treatment in an electrolyte containing Ca and P ions. The PEO-treated surface was observed by X-ray diffractometer (XRD), field-emission scanning electron microscopy, and energy dispersive X-ray spectroscopy (EDS). The microstructure of Ti- xNb-2Ag-2Pt alloys showed the transformation of needle-like structure to equiaxed structure, as Nb content increased. Adding small amounts of Ag and Pt to Ti-xNb alloys, microstructure was not observed the significantly difference compared to Ti-xNb. The nano- and micro-pore sizes increased as the Nb content increased after PEO-treatment of Ti-xNb. In the case of Ti-50Nb-2Ag-2Pt, groove structure was observed, also the Ca/P ratio increased as the Nb content increased. The oxide layer thickness of Ti-xNb-2Ag-2Pt alloys was increased, as the Nb content increased.


Assuntos
Ligas , Implantes Dentários , Íons , Oxirredução , Titânio
14.
Antonie Van Leeuwenhoek ; 114(2): 151-159, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33449223

RESUMO

An aerobic, Gram-negative, non-motile, non-spore-forming, rod-shaped, and pale yellow-colored bacterial strain, designated TS118T, was isolated from a sand sample obtained from a coastal sand dune after exposure to 3 kGy of gamma radiation. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate was a member of the genus Spirosoma and most closely related to Spirosoma metallicum PR1014kT (95.1% similarity). The genome of strain TS118T is constituted by one chromosome (5,691,492 bp) and one plasmid (28,440 bp) and has a G + C content of 52.7%. The genome contains 4641 protein coding sequences (CDSs), 38 tRNAs, and 11 rRNAs. The predominant fatty acids of strain TS118T were C16:1 ω5c, iso-C15:0, C16:0, summed feature 3 (C16:1 ω6c and/or C16:1 ω7c), and iso-C17:0 3-OH. The major polar lipids were phosphatidylethanolamine, an unidentified amino lipid and an unidentified aminophospholipid. The main respiratory quinone was menaquinone-7 (MK-7). The novel strain showed resistance to gamma radiation with a D10 value (i.e., the dose required to reduce the bacterial population by tenfold) of 4.3 kGy. Based on the phylogenetic, physiological, and chemotaxonomic characteristics, strain TS118T represents a novel species, for which the name Spirosoma taeanense sp. nov. is proposed. The type strain is TS118T (=KCTC 72898T =JCM 34024T).


Assuntos
Areia , Microbiologia do Solo , Técnicas de Tipagem Bacteriana , Composição de Bases , Cytophagaceae , DNA Bacteriano/genética , Ácidos Graxos/análise , Fosfolipídeos/análise , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2
15.
Sci Rep ; 11(1): 2575, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33510438

RESUMO

The mammalian molecular clock is based on a transcription-translation feedback loop (TTFL) comprising the Period1, 2 (Per1, 2), Cryptochrome1, 2 (Cry1, 2), and Brain and Muscle ARNT-Like 1 (Bmal1) genes. The robustness of the TTFL is attributed to genetic redundancy among some essential clock genes, deterring genetic studies on molecular clocks using genome editing targeting single genes. To manipulate multiple clock genes in a streamlined and efficient manner, we developed a CRISPR-Cas9-based single adeno-associated viral (AAV) system targeting the circadian clock (CSAC) for essential clock genes including Pers, Crys, or Bmal1. First, we tested several single guide RNAs (sgRNAs) targeting individual clock genes in silico and validated their efficiency in Neuro2a cells. To target multiple genes, multiplex sgRNA plasmids were constructed using Golden Gate assembly and packaged into AAVs. CSAC efficiency was evident through protein downregulation in vitro and ablated molecular oscillation ex vivo. We also measured the efficiency of CSAC in vivo by assessing circadian rhythms after injecting CSAC into the suprachiasmatic nuclei of Cas9-expressing knock-in mice. Circadian locomotor activity and body temperature rhythms were severely disrupted in these mice, indicating that our CSAC is a simple yet powerful tool for investigating the molecular clock in vivo.


Assuntos
Sistemas CRISPR-Cas/fisiologia , Animais , Temperatura Corporal , Sistemas CRISPR-Cas/genética , Relógios Circadianos/genética , Relógios Circadianos/fisiologia , Ritmo Circadiano/genética , Ritmo Circadiano/fisiologia , Dependovirus/genética , Locomoção/genética , Locomoção/fisiologia , Camundongos , Neurociências
16.
Curr Opin Cell Biol ; 68: 55-63, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33049465

RESUMO

The origin of the eukaryotic cell is one of the greatest mysteries in modern biology. Eukaryotic-wide specific biological processes arose in the lost ancestors of eukaryotes. These distinctive features, such as the actin cytoskeleton, define what it is to be a eukaryote. Recent sequencing, characterization, and isolation of Asgard archaea have opened an intriguing window into the pre-eukaryotic cell. Firstly, sequencing of anaerobic sediments identified a group of uncultured organisms, Asgard archaea, which contain genes with homology to eukaryotic signature genes. Secondly, characterization of the products of these genes at the protein level demonstrated that Asgard archaea have related biological processes to eukaryotes. Finally, the isolation of an Asgard archaeon has produced a model organism in which the morphological consequences of the eukaryotic-like processes can be studied. Here, we consider the consequences for the Asgard actin cytoskeleton and for the evolution of a regulated actin system in the archaea-to-eukaryotic transition.

17.
Curr Microbiol ; 77(12): 4167-4173, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33025184

RESUMO

An aerobic, Gram-stain-negative, non-motile, non-spore-forming, rod-shaped and pink-colored bacterial strain, designated BRD72T, was isolated from a crater lake (Baengnokdam) at the top of Mt. Hallasan in the Republic of Korea. Cells were catalase-positive and oxidase-negative. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that the isolate was a member of the genus Hymenobacter and most closely related to Hymenobacter marinus KJ035T (96.2% similarity). The isolate was found to produce carotenoid pigment, but not flexirubin-type pigment. The predominant fatty acids of strain BRD72T were summed feature 3 (C16:1 ω7c and/or C16:1 ω6c, 21.6%), iso-C15:0 (17.9%), anteiso-C15:0 (13.3%) and summed feature 4 (iso-C17:1 I and/or anteiso-C17:1 B, 11.3%). The major polar lipids were phosphatidylethanolamine, an unidentified amino lipid, and two unidentified aminophospholipids. The main respiratory quinone was menaquinone-7 (MK-7), and the main polyamine was homospermidine. The DNA G+C content was 59.8 mol%. Based on the phylogenetic, physiological, and chemotaxonomic characteristics, strain BRD72T represents a novel species, for which the name Hymenobacter baengnokdamensis sp. nov. is proposed. The type strain is BRD72T (= KCTC 72649T = JCM 33837T).


Assuntos
Lagos , Solo , Técnicas de Tipagem Bacteriana , Bacteroidetes , DNA Bacteriano/genética , Ácidos Graxos/análise , Filogenia , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNA , Vitamina K 2
18.
Exp Mol Med ; 52(9): 1614-1626, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32968200

RESUMO

Circadian clock controls an organism's biological rhythm and regulates its physiological processes in response to external time cues. Most living organisms have their own time-keeping mechanism that is maintained by transcriptional-translational autoregulatory feedback loops involving several core clock genes, such as Period. Recent studies have found the relevance between the modulation of circadian oscillation and posttranscriptional modifications by microRNAs (miRNAs). However, there are limited studies on candidate miRNAs that regulate circadian oscillation. Here, we characterize the functions of novel miRNA-25 regulating circadian Period2 (Per2) expression. Using several in silico algorithms, we identified novel miR-25-3p that, together with miR-24-3p, targets the Per2 gene. Luciferase reporter assays validated that miR-25-3p and miR-24-3p repressed Per2 expression and confirmed their predicted binding sites in the 3'-untranslated region (UTR) of Per2 mRNA. Real-time bioluminescence analyses using Per2::Luc mouse embryonic fibroblasts confirmed that PER2 protein oscillation patterns were responsive to miR-25-3p and miR-24-3. The overexpression of miR-25-3p or miR-24-3p resulted in the dampening and period shortening of the PER2::LUC oscillation, while inhibition of either miRNA increased the relative amplitude of the PER2::LUC oscillation. Notably, endogenous miR-25-3p expression in the suprachiasmatic nucleus (SCN) showed no circadian rhythmicity, but the expression levels differed in various brain regions and peripheral tissues. These results suggest that the posttranscriptional regulation of miR-25-3p and miR-24-3p may differ according to Per2 gene expression in different tissue regions. In summary, we found that novel miR-25-3p was involved in fine-tuning circadian rhythmicity by regulating Per2 oscillation at the posttranscriptional level and that it functioned synergistically with miR-24-3p to affect Per2 oscillation.

19.
Mol Cells ; 43(7): 600-606, 2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32489185

RESUMO

Numerous physiological processes in nature have multiple oscillations within 24 h, that is, ultradian rhythms. Compared to the circadian rhythm, which has a period of approximately one day, these short oscillations range from seconds to hours, and the mechanisms underlying ultradian rhythms remain largely unknown. This review aims to explore and emphasize the implications of ultradian rhythms and their underlying regulations. Reproduction and developmental processes show ultradian rhythms, and these physiological systems can be regulated by short biological rhythms. Specifically, we recently uncovered synchronized calcium oscillations in the organotypic culture of hypothalamic arcuate nucleus (ARN) kisspeptin neurons that regulate reproduction. Synchronized calcium oscillations were dependent on voltage-gated ion channel-mediated action potentials and were repressed by chemogenetic inhibition, suggesting that the network within the ARN and between the kisspeptin population mediates the oscillation. This minireview describes that ultradian rhythms are a general theme that underlies biological features, with special reference to calcium oscillations in the hypothalamic ARN from a developmental perspective. We expect that more attention to these oscillations might provide insight into physiological or developmental mechanisms, since many oscillatory features in nature still remain to be explored.


Assuntos
Núcleo Arqueado do Hipotálamo/metabolismo , Sinalização do Cálcio , Kisspeptinas/metabolismo , Neurônios/metabolismo , Ritmo Ultradiano , Animais , Núcleo Arqueado do Hipotálamo/crescimento & desenvolvimento , Núcleo Arqueado do Hipotálamo/fisiologia , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Humanos , Recém-Nascido , Kisspeptinas/genética , Neurônios/citologia , Ritmo Ultradiano/genética , Ritmo Ultradiano/fisiologia
20.
J Nanosci Nanotechnol ; 20(9): 5618-5624, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32331146

RESUMO

Ti alloy for implant materials is not good biocompatibility between metal implants and natural bone without surface modification. In order to improve the bone attachment ability, implant surface modification is required, and corrosion resistance is also important when a surface modified implant is inserted into the body. In this study, corrosion behaviors of anodic oxide film formed on Ti-6Al-4V alloy with concentration of Mn and Zn ions were studied using various experimental techniques. Ti-6Al-4V ELI disk were used as the substrate for PEO treatment. The sample was used a anodic electrode and PEO treatment was carried out by varying the content of each of the electrolytic solutions containing Zn, Mn, Si, Ca and P, using a Pt bar as a cathodic electrode. Pulsed DC power was used at 280 V for 3 min. For the corrosion behaviors of the specimens, the potentiodynamic polarization and AC impedance test were carried out in 0.9% NaCl solution. From the result of experiments, as Zn ion content increases, the number of pores and the pore size increase. As the Mn ion increases, the number of pores decreases, while the pore size increases. As the ions of Mn increase, the corrosion potential decreases. As Zn ion content increased, the polarization resistance increased.

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