RESUMO
The development of green catalysts, specifically biocatalysts, is crucial for building a sustainable society. To enhance the versatility of biocatalysts, the immobilization of enzymes plays a vital role as it improves their recyclability and robustness. As target enzymes to immobilize, glucose dehydrogenases and carboxylases are particularly important among various kinds of enzymes due to their involvement in two significant reactions: regeneration of the reduced form of coenzyme required for various reactions, and carboxylation reactions utilizing CO2 as a substrate, respectively. In this study, we immobilized Thermoplasma acidophilum glucose dehydrogenase (TaGDH) and T. acidophilum isocitrate dehydrogenase (TaIDH) using a previously reported method involving the formation of enzyme-inorganic hybrid nanocrystals, in the course of our continuing study focusing on carboxylation catalyzed by the free form of TaGDH and TaIDH. Subsequently, we investigated the properties of the resulting immobilized enzymes. Our results indicate the successful immobilization of TaGDH and TaIDH through the formation of hybrid nanocrystals utilizing Mn2+. The immobilization process enhanced TaIDH activity, up to 211%, while TaGDH retained 71% of its original activity. Notably, the immobilized TaGDH exhibited higher activity at temperatures exceeding 87 °C than the free TaGDH. Moreover, these immobilized enzymes could be recycled. Finally, we successfully utilized the immobilized enzymes for the carboxylation of 2-ketoglutaric acid under 1 MPa CO2. In conclusion, this study represents the first immobilization of TaGDH and TaIDH using the hybrid nanocrystal forming method. Furthermore, we achieved significant activity enhancement of TaIDH through immobilization and demonstrated the recyclability of the immobilized enzymes.
Assuntos
Glucose 1-Desidrogenase , Nanopartículas , Isocitrato Desidrogenase , Thermoplasma , Enzimas Imobilizadas , Dióxido de CarbonoRESUMO
Cryopyrin-associated periodic syndromes (CAPS) and Schnitzler syndrome (SchS) are autoinflammatory diseases that present with urticaria-like rashes. CAPS is characterized by periodic or persistent systemic inflammation caused by the dysfunction of the NLRP3 gene. With the advent of IL-1-targeted therapies, the prognosis of CAPS has improved remarkably. SchS is considered an acquired form of autoinflammatory syndrome. Patients with SchS are adults of relatively older age. The pathogenesis of SchS remains unknown and is not associated with the NLRP3 gene. Previously, the p.L265P mutation in the MYD88 gene, which is frequently detected in Waldenström macroglobulinemia (WM) with IgM gammopathy, was identified in several cases of SchS. However, because persistent fever and fatigue are symptoms of WM that require therapeutic intervention, it is a challenge to determine whether these patients truly had SchS or whether advanced WM was misidentified as SchS. There are no established treatments for SchS. The treatment algorithm proposed with the diagnostic criteria is to use colchicine as first-line treatment, and systemic administration of steroids is not recommended due to concerns about side effects. In difficult-to-treat cases, treatment targeting IL-1 is recommended. If targeted IL-1 treatment does not improve symptoms, the diagnosis should be reconsidered. We hope that the efficacy of IL-1 therapy in clinical practice will serve as a stepping stone to elucidate the pathogenesis of SchS, focusing on its similarities and differences from CAPS.
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Síndromes Periódicas Associadas à Criopirina , Exantema , Síndrome de Schnitzler , Urticária , Adulto , Humanos , Síndromes Periódicas Associadas à Criopirina/diagnóstico , Síndromes Periódicas Associadas à Criopirina/genética , Síndromes Periódicas Associadas à Criopirina/tratamento farmacológico , Síndrome de Schnitzler/diagnóstico , Síndrome de Schnitzler/genética , Síndrome de Schnitzler/terapia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Urticária/diagnóstico , Urticária/genética , Interleucina-1/uso terapêuticoRESUMO
We present a new water-dependent molecular mechanism for the widely-used protein stabilizing osmolyte, trimethylamine N-oxide (TMAO), whose mode of action has remained controversial. Classical interpretations, such as osmolyte exclusion from the vicinity of protein, cannot adequately explain the behavior of this osmolyte and were challenged by recent data showing the direct interactions of TMAO with proteins, mainly via hydrophobic binding. Solvent effect theories also fail to propose a straightforward mechanism. To explore the role of water and the hydrophobic association, we disabled osmolyte-protein hydrophobic interactions by replacing water with hexane and using lipase enzyme as an anhydrous-stable protein. Biocatalysis experiments showed that under this non-aqueous condition, TMAO does not act as a stabilizer, but strongly deactivates the enzyme. Molecular dynamics (MD) simulations reveal that TMAO accumulates near the enzyme and makes many hydrogen bonds with it, like denaturing osmolytes. Some TMAO molecules even reach the active site and interact strongly with the catalystic traid. In aqueous solvent, the enzyme functions well: the extent of TMAO interactions is reduced and can be divided into both polar and non-polar terms. Structural analysis shows that in water, some TMAO molecules bind to the enzyme surface like a surfactant. We show that these interactions limit water-protein hydrogen bonds and unfavorable water-hydrophobic surface contacts. Moreover, a more hydrophobic environment is formed in the solvation layer, which reduces water dynamics and subsequently, rigidifies the backbone in aqueous solution. We show that osmolyte amphiphilicity and protein surface heterogeneity can address the weaknesses of exclusion and solvent effect theories about the TMAO mechanism.
Assuntos
Metilaminas , Proteínas , Interações Hidrofóbicas e Hidrofílicas , Metilaminas/química , Proteínas/química , Solventes/química , Ureia/química , Água/químicaRESUMO
OBJECTIVE: A novel biocatalyst for Baeyer-Villiger oxidations is necessary for pharmaceutical and chemical industries, so this study aims to find a Baeyer-Villiger monooxygenase (BVMO) and to improve its stability by immobilization. RESULTS: Acetone, the simplest ketone, was selected as the only carbon source for the screening of microorganisms with a BVMO. A eukaryote, Fusarium sp. NBRC 109816, with a BVMO (FBVMO), was isolated from a soil sample. FBVMO was overexpressed in E. coli and successfully immobilized by the organic-inorganic nanocrystal formation method. The immobilization improved the thermostability of FBVMO. Substrate specificity investigation revealed that both free and immobilized FBVMO were found to show catalytic activities not only for Baeyer-Villiger oxidation of ketones to esters but also for oxidation of sulfides to sulfoxides. Furthermore, a preparative scale reaction using immobilized FBVMO was successfully conducted. CONCLUSIONS: FBVMO was discovered from an environmental sample, overexpressed in E. coli, and immobilized by the organic-inorganic nanocrystal formation method. The immobilization successfully improved its thermostability.
Assuntos
Fusarium , Oxigenases de Função Mista , Acetona , Escherichia coli/genética , Escherichia coli/metabolismo , Fusarium/metabolismo , Cetonas/química , Oxigenases de Função Mista/química , Oxirredução , Especificidade por SubstratoRESUMO
Cell-to-cell variability in cell populations arises from a combination of intrinsic factors and extrinsic factors related to the milieu. However, the heterogeneity of high cell density suspension cultures for therapeutic protein production remains unknown. Here, we illustrate the increasing heterogeneity in the cellular transcriptome of serum-free adapted CHO K1 cells during high cell density suspension culture over time without concomitant changes in the genomic sequence. Cell cycle-dependent subpopulations and cell clusters, which typically appear in other single-cell transcriptome analyses, were not found in these suspension cultures. Our results indicate that cell division changes the intracellular microenvironment and leads to cell cycle-dependent heterogeneity. Whole mitochondrial single-cell genome sequencing showed cell-to-cell mitochondrial genome variation and heteroplasmy within cells. The mitochondrial genome sequencing method developed here is potentially useful for the validation of cell clonality. The culture time-dependent increase in cellular heterogeneity observed in this study did not show any attenuation in this increasing heterogeneity. Future advances in bioengineering such as culture upscaling, prolonged culturing, and complex culture systems will be confronted with the need to assess and control cellular heterogeneity, and the method described here may prove useful for this purpose.
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Técnicas de Cultura de Células , Divisão Celular , Perfilação da Expressão Gênica , Genoma Mitocondrial , Análise de Célula Única , Animais , Células CHO , CricetulusRESUMO
OBJECTIVES: To collect clinical information and NOD2 mutation data on patients with Blau syndrome and to evaluate their prognosis. METHODS: Fifty patients with NOD2 mutations were analysed. The activity of each NOD2 mutant was evaluated in HEK293 cells by reporter assay. Clinical information was collected from medical records through the attending physicians. RESULTS: The study population comprised 26 males and 24 females aged 0-61 years. Thirty-two cases were sporadic, and 18 were familial from 9 unrelated families. Fifteen different mutations in NOD2 were identified, including 2 novel mutations (p.W490S and D512V); all showed spontaneous nuclear factor kappa B activation, and the most common mutation was p.R334W. Twenty-six patients had fever at relatively early timepoints in the disease course. Forty-three of 47 patients had a skin rash. The onset of disease in 9 patients was recognised after BCG vaccination. Forty-five of 49 patients had joint lesions. Thirty-eight of 50 patients had ocular symptoms, 7 of which resulted in blindness. After the diagnosis of Blau syndrome, 26 patients were treated with biologics; all were antitumour necrosis factor agents. Only 3 patients were treated with biologics alone; the others received a biologic in combination with methotrexate and/or prednisolone. None of the patients who became blind received biologic treatment. CONCLUSIONS: In patients with Blau syndrome, severe joint contractures and blindness may occur if diagnosis and appropriate treatment are delayed. Early treatment with a biologic agent may improve the prognosis.
Assuntos
Artrite/tratamento farmacológico , Artrite/genética , Artrite/patologia , Proteína Adaptadora de Sinalização NOD2/genética , Sarcoidose/tratamento farmacológico , Sarcoidose/genética , Sarcoidose/patologia , Sinovite/tratamento farmacológico , Sinovite/genética , Sinovite/patologia , Uveíte/tratamento farmacológico , Uveíte/genética , Uveíte/patologia , Adolescente , Adulto , Idade de Início , Antirreumáticos/uso terapêutico , Cegueira/epidemiologia , Cegueira/etiologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Japão , Masculino , Metotrexato/uso terapêutico , Pessoa de Meia-Idade , Mutação , Adulto JovemRESUMO
Alcohol dehydrogenases (ADHs) catalyze the reversible reduction of a carbonyl group to its corresponding alcohol. ADHs are widely employed for organic synthesis due to their lack of harm to the environment, broad substrate acceptance, and high enantioselectivity. This review focuses on the impact and relevance of ADH enantioselectivities on their biotechnological application. Stereoselective ADHs are beneficial to reduce challenging ketones such as ketones owning two bulky substituents or similar-sized substituents to the carbonyl carbon. Meanwhile, in cascade reactions, non-stereoselective ADHs can be utilized for the quantitative oxidation of racemic alcohol to ketone and dynamic kinetic resolution.
Assuntos
Álcool Desidrogenase/química , Álcool Desidrogenase/metabolismo , Biotecnologia , Álcoois/química , Álcoois/metabolismo , Catálise , Cetonas/metabolismo , Cinética , Oxirredução , Engenharia de Proteínas , Estereoisomerismo , Especificidade por SubstratoRESUMO
Enzyme engineering has been widely employed to tailor the substrate specificity and enantioselectivity of enzymes. In this study, we mutated Trp288, an unconserved residue in the small binding pocket of an acetophenone reductase from Geotrichum candidum NBRC 4597 (GcAPRD). Trp288 mutants showed substrate specificity expansion towards bulky-bulky ketones and enantioselectivity alteration which was highly dependent on the substrate substituent length. In aliphatic ketone reduction, enantioselectivity inverted from (S) to (R) when one of the substituents to the carbonyl carbon was elongated from propyl to butyl or pentyl. The best (R)-selective mutant, Trp288Val, achieved the reduction of 3-heptanone to its corresponding (R)-alcohol with 97% ee. Our docking simulation suggested that when enantioselectivity inverted to (R), only pro-R binding poses were productive. Gly94 played an important role to stabilize the butyl or pentyl group for their productive pro-R poses. Interestingly, when the substituent was further elongated, the enantioselectivity inverted back to the (S) form.
Assuntos
Acetofenonas/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Geotrichum/enzimologia , Cetonas/química , Sítios de Ligação , Biocatálise , Geotrichum/genética , Cinética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Estereoisomerismo , Especificidade por SubstratoRESUMO
The original version of this article contains error for some of the authors corrections were not included during correction stage.
RESUMO
Aliphatic ketones, such as 2-butanone and 3-hexanone, with only one carbon difference among side chains adjacent to the carbonyl carbon are difficult to be reduced enantioselectively. In this study, we utilized an acetophenone reductase from Geotrichum candidum NBRC 4597 (GcAPRD) to reduce challenging aliphatic ketones such as 2-butanone (methyl ethyl ketone) and 3-hexanone (ethyl propyl ketone) to their corresponding (S)-alcohols with 94% ee and > 99% ee, respectively. Through crystallographic structure determination, it was suggested that residue Trp288 limit the size of the small binding pocket. Docking simulations imply that Trp288 plays an important role to form a C-Hâ¯π interaction for proper orientation of ketones in the pro-S binding pose in order to produce (S)-alcohols. The excellent (S)-enantioselectivity is due to a non-productive pro-R binding pose, consistent with the observation that the (R)-alcohol acts as an inhibitor of (S)-alcohol oxidation.
Assuntos
Oxirredutases do Álcool/química , Carbono/química , Cetonas/química , Oxirredutases/química , Sítios de Ligação , Cristalografia , Geotrichum/enzimologia , Cinética , Simulação de Acoplamento Molecular , Oxirredução , Conformação Proteica , Estereoisomerismo , Especificidade por SubstratoRESUMO
Chinese ripe pu-erh tea is produced by aerobic microbial fermentation of green tea. To clarify the microbial degradation of tea polyphenols, Japanese commercial green tea was mixed with Chinese ripe pu-erh tea, which retains microorganisms, and fermented for 5 d. Chromatographic separation yielded a novel water-soluble yellow pigment termed theagalloflavic acid. Spectroscopic and chemical evidence suggested that this pigment was produced by oxidative ring cleavage of hexahydroxydiphenoyl esters. In addition, two new oxygenated lignin metabolites, (+)-5,5'-dihydroxypinoresinol and 5-hydroxydihydrodehydrodiconiferyl alcohol, were also isolated together with known degradation products of quercetin and tea catechins.
Assuntos
Taninos Hidrolisáveis/metabolismo , Lignanas/metabolismo , Fenóis/metabolismo , Chá/química , Aerobiose , Fermentação , Taninos Hidrolisáveis/química , Lignanas/química , Estrutura Molecular , Oxirredução , Fenóis/química , Chá/metabolismoRESUMO
OBJECTIVE: Approximately 19,000 people die in Japan each year from accidents that occur while bathing. Yamagata Prefectural Shounai public health center had educated residents about proper bathing practice. We evaluated residents' knowledge about safe bathing practices after an educational campaign in the area. METHODS: We distributed a questionnaire to 3,078 people to gauge their awareness of bathing accidents in general and the effectiveness of a safety campaign from the public health center prevention. Participants included people who had attended a meeting, workshop, lecture, or event at the public health center. RESULTS: A total of 2,697 people responded (87.6% response rate). A large majority (92%) knew about safe bathing practices in general. However, only 43% knew that bathing in hot water less than 41 degrees Celsius could help to reduce bathing accidents. Only 56% bathed in water below 41 degrees Celsius. Similarly, 81% knew about the preheating before bathing in dressing room and bathroom, but practiced only 50% of those. About recognition of the actual situation of the bathing accident and recognition of the prophylaxis, a student attending a lecture was higher than a lecture non-student attending a lecture more than 10%. CONCLUSION: Recognitions and practices of safe bathing practices had big difference, and development of an effective communication method will be important.
Assuntos
Acidentes Domésticos , Banhos , Acidentes Domésticos/prevenção & controle , Idoso , Banhos/efeitos adversos , Banhos/métodos , Feminino , Temperatura Alta , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Inquéritos e QuestionáriosRESUMO
OBJECTIVE: This article is based on our previously reported results of irregular bowel movement and disturbances of the intestinal microbiota/environment in gastrectomized patients. A placebo-controlled, double-blind comparative study was carried out to evaluate the effects of a fermented milk beverage containing Lactobacillus casei strain Shirota (LcS) in such patients. The major evaluated factors of this article were "bowel movement" and "quality of life." The secondary evaluated factors were "fecal microbiota" and "enteric environment." METHODS: Of the 190 gastrectomized subjects who participated in our previously reported defecation survey, 134 subjects judged as having abnormal defecation gave consent to participate in this study. These subjects continuously ingested the test beverage containing 40 billion LcS or placebo (one bottle per day, 4 weeks). RESULTS: In the LcS-ingested group, among the 118 subjects who completed the tests, the assessments of the subjects were based on their division into groups based on their symptoms with our scoring system for constipation/diarrhea; although there was no significant ingestion effect in total, in the constipation group, LcS reduced the degree of constipation compared with that in the placebo group. In the diarrhea group, LcS ingestion improved diarrhea compared with that in the preingestion state. Fecal Staphylococcus level was decreased. CONCLUSIONS: The results suggest the possibility that the continuous consumption of LcS-fermented milk relieves irregular bowel movement in gastrectomized patients.
Assuntos
Constipação Intestinal/terapia , Diarreia/terapia , Fezes/química , Fezes/microbiologia , Gastrectomia , Lacticaseibacillus casei , Probióticos/administração & dosagem , Idoso , Amônia/análise , Constipação Intestinal/etiologia , Cresóis/análise , Defecação , Diarreia/etiologia , Método Duplo-Cego , Feminino , Gastrectomia/efeitos adversos , Humanos , Indóis/análise , Masculino , Pessoa de Meia-Idade , Leite Humano , Fenol/análise , Qualidade de VidaRESUMO
CO2 fixation technology has gained attention as a method to effectively utilize the abundant CO2 in the atmosphere by converting it into useful chemicals. However, since CO2 is a highly stable molecule, many of the currently developed methods for chemical CO2 fixation require harsh conditions and reactive reagents. The establishment of efficient and sustainable processes is eagerly awaited. In this study, we investigated a biocatalytic process and achieved a carboxylation reaction under mild conditions (37 °C, 0.1 MPa CO2) using a biocatalyst, Thermoplasma acidophilum NADP+-malic enzyme (TaME), and gaseous CO2 by coupling enzymatic coenzyme regeneration. We also demonstrated for the first time that the carboxylation reaction by ME proceeds not only with pyruvate, a natural substrate, but also with 2-ketoglutarate.
RESUMO
The gene encoding acetophenone reductase (APRD), a useful biocatalyst for producing optically pure alcohols, was cloned from the cDNA of Geotrichum candidum NBRC 4597. The gene contained an open reading frame that consisted of 1,029 nucleotides corresponding to 342 amino acid residues. The subunit molecular weight was calculated to be 36.7 kDa. The predicted amino acid sequence did not have significant similarity to those of the acetophenone reductase reported previously. The gene was inserted into the pET-21b(+) expression vector and expressed in Escherichia coli Rosetta™(DE3)pLysS by induction with 1 mM of isopropyl-ß-D-thiogalactopyranoside. E. coli cell-free extract gave 21.9 U/mg APRD activity, which was 81 times that of the G. candidum cell-free extract. The enzyme was purified with a HisTrap FF crude column. The enzyme exhibited the highest activity at 60 °C, and optimum reducing and oxidizing activity were observed in a pH range around 7.0-8.0 and 8.5, respectively. The enzyme was most stable at 60 °C and pH 6.5-7.5. The Vmax and the apparent Km value of the reductase were 67.6 µmol/min per milligram of protein and 0.146 mM for acetophenone, respectively. From 4 % (v/v) 4-phenyl-2-butanone, (S)-4-phenyl-2-butanol was obtained with a yield >80 % and an enantiomeric excess >99 % in a 20 h reaction recycling NADH with 15 % (v/v) 2-propanol.
Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Inibidores Enzimáticos/metabolismo , Geotrichum/enzimologia , Compostos Orgânicos/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/isolamento & purificação , Cromatografia de Afinidade , Estabilidade Enzimática , Escherichia coli/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Geotrichum/genética , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peso Molecular , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
BACKGROUND: We conducted a survey regarding irregular bowel movement in gastrectomized patients. Their defecation frequency, intestinal microflora, and intestinal environment were studied and compared with those of healthy controls. METHODS: As a first step, a questionnaire survey on bowel movement, involving 769 patients and 312 healthy controls (total: 1,081 subjects), was carried out. As a second step, the defecation frequency (scoring of the survey results conducted to evaluate the state of constipation/diarrhea), intestinal microflora, and intestinal environment were evaluated in 190 gastrectomized patients with irregular bowel movement and 31 controls identified in the first survey. RESULTS: First step: Of the 769 patients, 58% complained of irregular bowel movements (constipation, diarrhea, or their alternate occurrence), and their frequency of complaints was significantly higher (p < 0.01) than that in the healthy controls (33%). Second step: The levels of the most predominant obligate anaerobe and harmful bacteria in the feces were lower and higher, respectively, the fecal pH was lower, the fecal water content was lower, and the level of putrefactive metabolites in the feces was higher in the gastrectomized patients than in the healthy controls. The intestinal flora and environment were more disrupted in the totally gastrectomized than in the partially gastrectomized patients. CONCLUSIONS: Many gastrectomized patients with irregular bowel movements exhibited significant changes showing impaired intestinal microflora and metabolite levels.
Assuntos
Defecação/fisiologia , Fezes/microbiologia , Gastrectomia/efeitos adversos , Adulto , Idoso , Amônia/análise , Amônia/metabolismo , Estudos de Casos e Controles , Clostridium , Constipação Intestinal/microbiologia , Diarreia/microbiologia , Humanos , Concentração de Íons de Hidrogênio , Pessoa de Meia-Idade , Fenóis/análise , Fenóis/metabolismo , Valores de Referência , Inquéritos e QuestionáriosRESUMO
Blau syndrome is a systemic autoinflammatory granulomatous disease caused by mutations in the nucleotide-binding oligomerization domain 2 (NOD2) gene. NOD2 is an intracellular pathogen recognition receptor. Upon binding to muramyl dipeptide (MDP), NOD2 activates the NF-κB pathway, leading to the upregulation of proinflammatory cytokines. Clinical manifestations of Blau syndrome appear in patients before the age of four. Skin manifestations resolve spontaneously in some cases; however, joint and eye manifestations are progressive, and lead to serious complications, such as joint contracture and blindness. Currently, there is no specific curative treatment for the disease. Administration of high-dose oral steroids can improve clinical manifestations; however, treatments is difficult to maintain due to the severity of the side effects, especially in children. While several new therapies have been reported, including JAK inhibitors, anti-IL-6 and anti-IL-1 therapies, anti-TNF therapy plays a central role in the treatment of Blau syndrome. We recently performed an ex vivo study, using peripheral blood and induced pluripotent stem cells from patients. This study demonstrated that abnormal cytokine expression in macrophages from untreated patients requires IFNγ stimulation, and that anti-TNF treatment corrects the abnormalities associated with Blau syndrome, even in the presence of IFNγ. Therefore, although the molecular mechanisms by which the genetic mutations in NOD2 lead to granuloma formation remain unclear, it is possible that prior exposure to TNFα combined with IFNγ stimulation may provide the impetus for the clinical manifestations of Blau syndrome.
Assuntos
Sinovite , Uveíte , Artrite , Criança , Citocinas/metabolismo , Humanos , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Sarcoidose , Sinovite/tratamento farmacológico , Sinovite/genética , Sinovite/metabolismo , Inibidores do Fator de Necrose Tumoral , Uveíte/tratamento farmacológico , Uveíte/genética , Uveíte/metabolismoRESUMO
We succeeded in achieving visible-light responsiveness on a tubular TiO(2) sample through the treatment of a tubular TiO(2) that has a large surface area with an aqueous solution of ammonia or triethylamine at room temperature and subsequent calcination at 623 K, which produced a nitrided tubular TiO(2) sample. It was found that the ease of nitridation is dependent on the surface states; washing the tubular TiO(2) sample with an aqueous acidic solution is very effective and indispensable. This treatment causes the appearance of acidic sites on the tubular TiO(2), which was proved by the following experiments: NH(3) temperature-programmed desorption and two types of organic reactions exploiting the acid properties. The prepared samples, TiO(2-δ)N(δ), efficiently absorb light in the visible region, and they exhibit a prominent feature for the decomposition of methylene blue in an aqueous solution at 300 K under irradiation with visible light, indicating the achievement of visible-light responsiveness on the tubular TiO(2) sample. This type of tubular TiO(2-δ)N(δ) sample has merit in the sense that it has a large surface area and a characteristic high transparency for enabling photocatalytic reactions because it has a tubular structure and is composed of thin walls.
Assuntos
Luz , Nitrogênio/química , Processos Fotoquímicos , Titânio/química , Catálise , Concentração de Íons de Hidrogênio , Azul de Metileno/química , Fotólise , Propriedades de Superfície , TemperaturaRESUMO
Large volumes of high-throughput sequencing data have been submitted to the Sequencing Read Archive (SRA). The lack of experimental metadata associated with the data makes reuse and understanding data quality very difficult. In the case of RNA sequencing (RNA-Seq), which reveals the presence and quantity of RNA in a biological sample at any moment, it is necessary to consider that gene expression responds over a short time interval (several seconds to a few minutes) in many organisms. Therefore, to isolate RNA that accurately reflects the transcriptome at the point of harvest, raw biological samples should be processed by freezing in liquid nitrogen, immersing in RNA stabilization reagent or lysing and homogenizing in RNA lysis buffer containing guanidine thiocyanate as soon as possible. As the number of samples handled simultaneously increases, the time until the RNA is protected can increase. Here, to evaluate the effect of different lag times in RNA protection on RNA-Seq data, we harvested CHO-S cells after 3, 5, 6, and 7 days of cultivation, added RNA lysis buffer in a time course of 15, 30, 45, and 60 min after harvest, and conducted RNA-Seq. These RNA samples showed high RNA integrity number (RIN) values indicating non-degraded RNA, and sequence data from libraries prepared with these RNA samples was of high quality according to FastQC. We observed that, at the same cultivation day, global trends of gene expression were similar across the time course of addition of RNA lysis buffer; however, the expression of some genes was significantly different between the time-course samples of the same cultivation day; most of these differentially expressed genes were related to apoptosis. We conclude that the time lag between sample harvest and RNA protection influences gene expression of specific genes. It is, therefore, necessary to know not only RIN values of RNA and the quality of the sequence data but also how the experiment was performed when acquiring RNA-Seq data from the database.
RESUMO
A novel Geotrichum candidum aldehyde dehydrogenase (GcALDH) effectively oxidized aldehydes to carboxylic acids under mild conditions. Nonetheless, the free form of GcALDH still had a limitation in stability and recyclability. Herein, to overcome these limitations, this study achieved the immobilization of the GcALDH by the organic-inorganic nanocrystal formation. The GcALDH nanocrystal exhibited 261 ± 40 % of activity comparing to the free enzyme. It also exhibited improved thermostability and recyclability as well as remained excellent substrate promiscuity, which suggested its potential use in green industries. To the best of our knowledge, this is the first time to report the ALDH-inorganic nanocrystal formation with superior properties.