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1.
Am J Rhinol Allergy ; 35(6): 817-829, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33736454

RESUMO

BACKGROUND: Exposure to airborne urban particulate matter (UPM) has been closely related to the development and aggravation of respiratory disease, including sinonasal disorders. OBJECTIVE: The aims of this study were to investigate the effect of UPM on nasal epithelial tight junctions (TJs) and mucosal barrier function and delineate the underlying mechanism by using both in vitro and in vivo models. METHODS: In this study, human nasal epithelial cells (hNECs) and BALB/c mice were exposed to UPMs. UPM 1648a and 1649 b were employed. TJ and endoplasmic reticulum (ER) stress marker expression was measured using western blot analysis and immunofluorescence. TJ integrity and nasal epithelial barrier function were evaluated by transepithelial electric resistance (TER) and paracellular flux. In addition, the effects of N-acetyl-L-cysteine (NAC) on UPM-induced nasal epithelial cells were investigated. RESULTS: UPM significantly impaired the nasal epithelial barrier, as demonstrated by decreased protein expression of TJ and ER stress markers in human nasal epithelial cells. This finding was in parallel to reduced transepithelial electrical resistance and increased fluorescein isothiocyanate-dextran permeability. Pretreatment with NAC decreased the degree of UPM-mediated ER stress and restored nasal epithelial barrier disruption in human nasal epithelial cells (hNEC) and the nasal mucosa of experimental animals. CONCLUSION: These data suggest that UPMs may induce nasal epithelial barrier dysfunction by targeting TJs and ER stress could be related in this process. Based on these results, we suggest that suppression of this process with an inhibitor targeting ER stress responses could represent a novel promising therapeutic target in UPM-induced sinonasal disease.


Assuntos
Material Particulado , Junções Íntimas , Animais , Estresse do Retículo Endoplasmático , Células Epiteliais , Camundongos , Camundongos Endogâmicos BALB C , Material Particulado/toxicidade
2.
Allergy Asthma Immunol Res ; 12(6): 994-1011, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32935491

RESUMO

PURPOSE: The Toll-like receptor 9 (TLR9) signaling pathway is involved in the pathogenesis of chronic rhinosinusitis (CRS) with nasal polyposis. The aim of this study was to assess the therapeutic potential of the TLR9 pathway inhibitor chloroquine in CRS mice. METHODS: The expression of type I interferons (IFNs) in human CRS tissues was evaluated using quantitative polymerase chain reaction (qPCR), western blotting, and immunofluorescence. Mice were divided into 4 treatment groups: the control, nasal polyp (NP), chloroquine treatment (NP + Chlq), and dexamethasone treatment (NP + Dexa) groups. The effects of chloroquine on polyp formation and mucosal inflammation were examined by hematoxylin and eosin staining. The expression levels of type I IFN, B-cell activating factor (BAFF), TLR9, high mobility group box 1 (HMGB1), and proinflammatory cytokine expression levels were assessed using qPCR, western blot, or enzyme-linked immunosorbent assay. RESULTS: IFN-α and IFN-ß mRNA levels were significantly higher in patients with eosinophilic NPs (EPs) than in healthy individuals or non-EP patients. The polyp score, epithelial thickness, mucosal thickness, and the number of eosinophils in nasal mucosa were significantly higher in the NP group compared with the control, NP + Chlq, and NP + Dexa groups. NP + Chlq or NP + Dexa significantly suppressed the induction of type I IFN and BAFF expression in the NP group; these treatments also significantly suppressed the induction of TLR9, HMGB1, interferon regulatory factors, interleukin (IL)-6, IL-1ß, tumor necrosis factor-α, and Th cytokine expression in the NP group. The secreted levels of anti-dsDNA Immunoglobulin G (IgG) were significantly higher in the NP group than in the control, NP + Chlq, and NP + Dexa groups. There were significant positive correlations between BAFF and mRNA levels of IFN-α/ß/the protein levels of anti-dsDNA IgG. CONCLUSIONS: Chloroquine may be used for the treatment of patients with eosinophilic CRS.

3.
Int Forum Allergy Rhinol ; 10(7): 884-892, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32479710

RESUMO

BACKGROUND: Povidone-iodine (PVP-I) is well known as an antiseptic and exhibits extensive activity against various pathogens. However, due to its uniquely unpleasant nature, it cannot be used locally to deactivate various sinonasal pathogens. Therefore, we developed a PVP-I composite that blocks the unpleasant odor of PVP-I for use as a local antiseptic in the sinonasal cavity and evaluated its effect on bacterial biofilm's formation and elimination in in vivo and in vitro models. METHODS: MTT, lactate dehydrogenase, and live/dead staining assay were performed to examine the cellular toxicity of PVP-I composites on the primary human nasal epithelial and RPMI 2650 cells. Crystal violet assay was performed to quantify bacterial biofilm after treating with various agents, including PVP-I and antibiotics. Hematoxylin-and-eosin staining, live/dead staining assay, and scanning electron microscopy were conducted to evaluate the effect of PVP-I on biofilm formation in a mice biofilm model. RESULTS: It was observed that the PVP-I composite did not have any significant toxic effect on the nasal epithelial cells. Furthermore, the PVP-I composite effectively inhibited the formation of bacterial biomass within a dose-dependent manner after 48 hours of incubation with Pseudomonas aeruginosa and Staphylococcus aureus. In mice, it effectively eliminated biofilm from the mucosa of the nasal cavity and maxillary sinus at the tested concentrations. CONCLUSION: The results of this study indicate that the PVP-I composite is a promising compound that could be used locally to prevent the formation of biofilms and to eliminate them from the sinonasal cavity.


Assuntos
Anti-Infecciosos Locais , Infecções Estafilocócicas , Animais , Biofilmes , Camundongos , Povidona-Iodo , Staphylococcus aureus
4.
Allergy Asthma Immunol Res ; 12(4): 653-668, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32400131

RESUMO

PURPOSE: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a complex inflammatory disease of the nasal and paranasal sinus mucosa. The disease is associated with mitochondrial dysfunction, structural changes in the mitochondria, and reactive oxygen species (ROS) generation. This study investigated whether there are functional and morphological changes in the mitochondria in the epithelial cells of nasal polyps (NPs) and Staphylococcus aureus enterotoxin B (SEB)-stimulated nasal epithelial cells. METHODS: In all, 30 patients with CRSwNP and 15 healthy subjects were enrolled. Mitochondrial ROS (mtROS) and changes in mitochondrial functions and structures were investigated in the uncinate tissue (UT) of healthy controls, the UT or NPs of CRSwNP patients, and human nasal epithelial cells with or without SEB stimulation. RESULTS: Oxidative phosphorylation complexes showed various responses following SEB stimulation in the nasal epithelial cells, and their expressions were significantly higher in the NPs of patients with CRSwNP than in the UT of controls. Generation of mtROS was increased following SEB exposure in nasal epithelial cells and was reduced by pretreatment with MitoTEMPO, which is used as an mtROS scavenger. In the tissues, mtROS was significantly increased in the NPs of CRSwNP patients compared to the UT of controls or CRSwNP patients. The expressions of fusion- and fission-related molecules were also significantly higher in SEB-exposed nasal epithelial cells than in non-exposed cells. In tissues, the expression of fission (fission mediator protein 1)- and fusion (membrane and mitofusin-1, and optic atrophy protein 1)-related molecules was significantly higher in the NPs of CRSwNP patients than in UT of controls or CRSwNP patients. Transmission electron microscopy revealed elongated mitochondria in SEB-exposed nasal epithelial cells and epithelial cells of NPs. CONCLUSIONS: Production of mtROS, disrupted mitochondrial function, and structural changes in nasal epithelial cells might be involved in the pathogenesis of CRSwNP.

5.
BMC Complement Altern Med ; 19(1): 285, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31660945

RESUMO

BACKGROUND: Electroacupuncture (EA) is often used in clinical settings due to its analgesic effect, but its safety has not been verified due to the lack of clear criteria. This study examined the critical range of the corrosion of stainless steel types STS304 and STS316, which have been used clinically, and the relationship between needle corrosion and cell necrosis. METHOD: The critical point of corrosion for STS304 and STS316 was identified by varying the time, frequency, and stimulation intensity. In a tissue necrosis experiment, EA stimulation was applied to rats using STS316 needles with different thicknesses at maximum intensity for 60 min, and the presence of corrosion and tissue necrosis was determined. A cytotoxicity experiment was also conducted and assessed the needles and tissue necrosis. RESULTS: The results showed that STS316 was more stable than STS304 and that only coated needles corroded. Furthermore, tissue necrosis was observed regardless of corrosion, and slight cell necrosis was associated with needles with corrosion. CONCLUSIONS: This study demonstrated that non-coated STS316 was the most stable for EA stimulation and that corrosion byproducts and cell necrosis were not directly related.


Assuntos
Eletroacupuntura/instrumentação , Agulhas/efeitos adversos , Pontos de Acupuntura , Animais , Eletroacupuntura/efeitos adversos , Eletroacupuntura/métodos , Masculino , Necrose/etiologia , Necrose/patologia , Ratos , Ratos Sprague-Dawley , Aço Inoxidável/efeitos adversos , Aço Inoxidável/análise
6.
Clin Immunol ; 197: 19-26, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30056130

RESUMO

B-cell activating factor (BAFF) has been proposed to play a crucial role in the pathogenesis of chronic rhinosinusitis with nasal polyp (CRSwNP). The aim of this study was to evaluate the role of toll-like receptor (TLR) 9-mediated BAFF activation on the pathogenesis of CRSwNP. NP and uncinate tissue (UT) were obtained from patients with CRSwNP or CRS without NP, and control subjects. The expression of TLR9, high mobility group box-1 protein (HMGB1), type I interferon (IFN), BAFF, and anti-double stranded DNA (dsDNA) antibody were examined in the tissues and the cultured dispersed NP cells (DNPCs). The expression of TLR9, HMGB1, type I IFN, BAFF, and anti-dsDNA antibody were elevated in NP tissue compared to the UTs. Exposure to TLR9 agonist increased the type I IFN expression in vitro, which further increased BAFF production. In conclusion, we provided a novel therapeutic potential of TLR9 agonist in CRSwNP.


Assuntos
Fator Ativador de Células B/genética , Proteína HMGB1/genética , Pólipos Nasais/metabolismo , Rinite/metabolismo , Sinusite/metabolismo , Receptor Toll-Like 9/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Fator Ativador de Células B/efeitos dos fármacos , Fator Ativador de Células B/metabolismo , Doença Crônica , Feminino , Seio Frontal/metabolismo , Proteína HMGB1/metabolismo , Humanos , Técnicas In Vitro , Interferon-alfa/efeitos dos fármacos , Interferon-alfa/genética , Interferon-alfa/metabolismo , Interferon beta/efeitos dos fármacos , Interferon beta/genética , Interferon beta/metabolismo , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Receptor Toll-Like 9/agonistas
7.
Int Forum Allergy Rhinol ; 8(9): 1001-1012, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29901280

RESUMO

BACKGROUND: Nasal polyposis is characterized by persistent inflammation and remodeling in sinonasal mucosa. Toll-like receptor 9 (TLR9) is a DNA receptor of the innate immune system that plays a pivotal role in fibrosis and inflammatory responses. The aim of this study is to explore the expression, activity, and potential pathogenic role of TLR9 signaling in tissue remodeling in nasal polyp-derived fibroblasts (NPDFs). METHODS: Fibrotic and inflammatory responses elicited by type A CpG oligonucleotides were examined in the NPDFs by a combination of real-time quantitative polymerase chain reaction, Western blot analysis, enzyme-linked immunosorbent assay, and immunofluorescence staining. For these experiments, the NPDFs were stimulated with different TLR9 agonists (CpG A and B) and blocked with inhibitors (MyD88 inhibitor and chloroquine). RESULTS: TLR9 expression was significantly higher in nasal polyposis (NP) tissues compared to control or chronic rhinosinusitis (CRS) mucosa. In the NPDFs, TLR9 showed intracellular localization and expression of TLR9 was increased after treatment with CpG A. CpG A increased production of α-smooth muscle actin (α-SMA), fibronectin, and matrix metalloproteinases (MMPs) (MMP1, MMP2, and MMP9) in the NPDFs, while MyD88 inhibitor and chloroquine, which are known to block the TLR9 signaling pathway, inhibited their production. CpG A also produced type I interferons (IFN-α and IFN-ß), which were inhibited by MyD88 inhibitor. CONCLUSION: Our data indicates that CpG A-induced fibroblast activation and cytokine production were mediated via TLR9 stimulation in NPDFs. Disrupting this process with an inhibitor targeting TLR9 or its downstream signaling pathways could represent a novel approach to CRS with NP (CRSwNP) therapy.


Assuntos
Fibroblastos/patologia , Pólipos Nasais/fisiopatologia , Transdução de Sinais/fisiologia , Sinusite/fisiopatologia , Receptor Toll-Like 9/metabolismo , Adulto , Células Cultivadas , Cloroquina/farmacologia , Citoplasma/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Pólipos Nasais/metabolismo , Pólipos Nasais/patologia , Oligonucleotídeos/farmacologia , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinusite/metabolismo , Sinusite/patologia , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/antagonistas & inibidores , Receptor Toll-Like 9/genética
8.
PLoS One ; 12(8): e0181806, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28771607

RESUMO

BACKGROUND AND OBJECTIVE: Interleukin (IL)-25 has been shown to play an important role in the pathogenesis of chronic rhinosinusitis with nasal polyps. Nasal polyps are associated with chronic inflammation of the mucous membranes in the paranasal sinuses and are involved in extracellular matrix (ECM) accumulation. The aim of this study is to evaluate the effects of IL-25 on myofibroblast differentiation, ECM production and the expression of matrix metalloproteinases in nasal polyp derived fibroblasts (NPDFs) and to determine the molecular mechanism underlying these processes. MATERIALS AND METHODS: A total of 40 patients were enrolled in this study for Immunofluorescence studies. Expression of IL17 receptor B was evaluated by real time reverse transcription polymerase chain reaction (PCR) in NPDFs. NPDFs were stimulated with IL-25 for 48 h in the presence or absence of mitogen-activated protein kinase (MAPK) and NF-κB inhibitors or small interfering RNAs (siRNA). The protein levels of fibrosis active mediators were examined using western blotting. Fibroblast migration was evaluated with a scratch assay. The total collagen amount was analyzed with the Sircol collagen assay. RESULTS: IL-25 induced α-SMA, fibronectin, and MMP-1 and -13, which were dependent on IL-17RB. IL-25 also induced activation of NF-κB and mitogen-activated protein kinase (MAPKs). By using the specific inhibitor of ERK, p38, JNK and NF-κB (U, SB, SP and Bay), we found that IL-25-induced expressions of α-SMA, fibronectin, and MMPs was regulated by the signaling pathways of MAPKs and NF-κB. IL-25 also induces α-SMA, fibronectin, and MMPs expression through IL-17RB-dependent pathways in NPDFs. The increased migration ability induced by IL-25 was suppressed by the specific inhibitors of MAPKs and NF-κB. CONCLUSION: Our data indicate that IL-25 induced myofibroblast differentiation, fibronectin production, and MMP-1 and -13 expressions through the signaling pathways of MAPKs and NF-κB. in NPDFs and increased expression of IL-25 were also involved in the pathogenesis of nasal polyposis by affecting nasal fibroblasts in chronic rhinosinusitis with nasal polyps.


Assuntos
Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Interleucina-17/farmacologia , Pólipos Nasais/complicações , Nariz/patologia , Sinusite/patologia , Actinas/metabolismo , Adulto , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Fibronectinas/biossíntese , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina-17/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinusite/genética , Sinusite/metabolismo
9.
Acupunct Med ; 35(2): 93-99, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27515415

RESUMO

BACKGROUND: Moxibustion therapy has been used historically for thousands of years and there are many clinical trials supporting its efficacy and effectiveness for various conditions. Moxa smoke has been a major reason for avoiding moxibustion due to its smell and potential risks to the human body. METHODS: 10 units of commercial indirect moxa (CIM) from six manufacturers (A-F) were burnt in a 2.5×2.5×2.5 m chamber without ventilation, and concentrations of carbon oxides (CO and CO2), nitrogen oxides (NOx), and volatile organic compounds (VOCs) from the indoor air samples were measured. RESULTS: For brands A, B, C, D, E, and F, respectively, relative to baseline values, we observed an increase in CO (from 0.002 to 0.007, 0.006, 0.005, 0.006, 0.005, and 0.006 parts per billion (ppb)), NOx (from 0.009 to 0.051, 0.025, 0.015, 0.050, 0.019, and 0.020 ppb), and total VOCs (TVOC; from 48.06 to 288.83, 227.93, 140.82, 223.22, 260.15, and 161.35 µg/m3), while the concentration of CO2 was not elevated. Each CIM brand demonstrated different VOC emission characteristics, which could be divided into three groups. On average, we estimated that 20 units of CIM or 2.41 g moxa floss would need to be combusted in order to exceed the safe levels set by Korean environmental law. This limit is likely to be greater in the case of a larger room or use of ventilation. CONCLUSIONS: Despite increased CO/NOx/VOC concentrations, overall levels remained within safe limits. These findings may help clinicians to maintain safe moxibustion treatment conditions to help keep both patients and clinicians safe from the pollutants generated by moxa combustion.


Assuntos
Moxibustão/instrumentação , Poluentes Atmosféricos/análise , Humanos , Moxibustão/economia , Moxibustão/métodos , Moxibustão/normas , Segurança , Fumaça/análise
10.
Integr Med Res ; 5(1): 56-62, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28462098

RESUMO

BACKGROUND: The novel threadlike structure is called the primo structure, and studies are conducted through many different approaches. Although various ways of differentiation are currently used, a standard for differentiation is deemed necessary in order to identify the primo structure based on the overall form of the structure. This study aims to explore the differentiation standard through the histological common point of the threadlike structure of rat and swine by using the hematoxylin and eosin (H&E) staining method commonly used for histological research in various structures. METHODS: An 8-week old Sprague-Dawley rat and a Yorkshire pig weighing 30-40 kg were used. A total of 65 pieces of rat threadlike structure and 100 pieces of swine threadlike structure were collected after the abdomen was cut and opened. The following three different characteristics were confirmed using the H&E staining method for the collected structures: (1) bright cell availability; (2) cavity availability; and (3) nuclei density. RESULTS: For the rat threadlike structure, the bright cell (70.5%) and nuclei density (92.6%) were mainly observed; in the swine threadlike structure, however, the bright cell (60.6%) and cavity (67.2%) were mainly observed. The bright cell was confirmed to have been observed in the threadlike structures of both rat and swine. CONCLUSION: The bright cell is determined to be the common point in the primo structure. However, further research is deemed necessary in the future as to the functions performed by the characteristic shown by the Primo structure.

11.
Artigo em Inglês | MEDLINE | ID: mdl-23861710

RESUMO

In a previous study, we found that Primo vessels generate different action potentials in smooth muscles, but this study compared the pulse shape to distinguish the two tissues. Thus, a more sophisticated extracellular experiment was performed in this study using an acetylcholine injection; we then observed changes in the amplitude, FWHM (full width at half maximum), and period to explore Primo vessel function. A third type of pulse was recorded for Primo vessels. We observed fast depolarizing and repolarizing phases for this pulse. Further, its FWHM was 30 ms between smooth muscles and neurons. Acetylcholine affected only the period. The amplitude and FWHM were consistent after injection. Primo-vessels generated action potentials at twice the frequency after injection. From the results, we speculate that Primo-vessels perform a role in transferring signals in a different manner, which may be relevant for acupuncture treatment.

12.
Integr Med Res ; 2(3): 99-105, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28664060

RESUMO

BACKGROUND: The primo vascular system has been suggested as the third circulatory system. Although primo tissues have been observed in various tissues isolated from a number of animal species, including mice, rats, and rabbits, very few reports on the histological characteristics of primo tissues from different body parts are available. This study was designed to compare the histological characteristics of primo tissues isolated from different body parts of rats. METHODS: Rats were anesthetized and operated on, to locate primo tissues. The primo tissues were searched at the all organs' surface and in lymphatic vessels using suitable finding methods. The tissues found were then separated and observed by histological test methods. RESULTS: This histological study revealed that there was no difference between the histological characteristics of the organ-surface primo tissues, the primo tissues inside lymphatic vessels, and the primo tissues on the falciform ligament. Moreover, primo tissues could be differentiated from those with a similar structure, such as lymphatic vessels and blood vessels, by immunostaining against a-LYVE-1 and a-CD31, and can be observed by specific immunostaining against the Von Willebrand factor (vWF). CONCLUSION: The results of this study support the fact that primo tissues could possibly constitute a third circulatory system in the whole-body network.

13.
J Microbiol ; 43(1): 1-10, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15765050

RESUMO

We isolated and cultured bacteria inhabiting solar saltern ponds in Taean-Gun, Chungnam Province, Korea. All of the isolated 64 strains were found to be moderately halophilic bacteria, growing in a salt range of 2-20 %, with an optimal concentration of 5% salt. Bacterial diversity among the isolated halophiles was evaluated via RFLP analyses of PCR-amplified 16S rDNAs, followed by phylogenetic analysis of the partial 16S rDNA sequences. The combination of restriction enzyme digestions with HaeIII, CfoI, MspI and RsaI generated 54 distinct patterns. A neighbor-joining tree of the partial 16S rDNA sequences resulted in the division of the 64 strains into 2 major groups, 45 strains of gamma-Proteobacteria (70.3%) and 19 strains of Firmicutes (29.7%). The alpha-Proteobacteria and Cytophaga-Flavobacterium-Bacterioides groups, which were repeatedly found to exist in thalassohaline environments, were not represented in our isolates. The gamma-Proteobacteria group consisted of several subgroups of the Vibrionaceae (37.5%), Pseudoalteromonadaceae (10.9%), Halomonadaceae (7.8%), Alteromonadaceae (7.8%), and Idiomarinaceae (6.3%). Members of Salinivibrio costicola (29.7%) were the most predominant species among all of the isolates, followed by Halobacillus treperi (12.5%). Additionally, three new species candidates were found, based on similarities of the 16S rDNA sequences to those of previously published species.


Assuntos
Bactérias/isolamento & purificação , Microbiologia da Água , Bacillaceae/classificação , Bacillaceae/genética , Bacillaceae/crescimento & desenvolvimento , Bacillaceae/isolamento & purificação , Bactérias/classificação , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Sequência de Bases , DNA Bacteriano/genética , DNA Ribossômico/genética , Gammaproteobacteria/classificação , Gammaproteobacteria/genética , Gammaproteobacteria/crescimento & desenvolvimento , Gammaproteobacteria/isolamento & purificação , Coreia (Geográfico) , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Cloreto de Sódio
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