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1.
J Neurosci ; 43(43): 7226-7241, 2023 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-37699718

RESUMO

The insulin/IGF-1 signaling (IIS) regulates a wide range of biological processes, including aging and lifespan, and has also been implicated in the pathogenesis of Alzheimer's disease (AD). We and others have reported that reduced signaling by genetic ablation of the molecules involved in IIS (e.g., insulin receptor substrate 2 [IRS-2]) markedly mitigates amyloid plaque formation in the brains of mouse models of AD, although the molecular underpinnings of the amelioration remain unsolved. Here, we revealed, by a transcriptomic analysis of the male murine cerebral cortices, that the expression of genes encoding extracellular matrix (ECM) was significantly upregulated by the loss of IRS-2. Insulin signaling activity negatively regulated the phosphorylation of Smad2 and Smad3 in the brain, and suppressed TGF-ß/Smad-dependent expression of a subset of ECM genes in brain-derived cells. The ECM proteins inhibited Aß fibril formation in vitro, and IRS-2 deficiency suppressed the aggregation process of Aß in the brains of male APP transgenic mice as revealed by injection of aggregation seeds in vivo Our results propose a novel mechanism in AD pathophysiology whereby IIS modifies Aß aggregation and amyloid pathology by altering the expression of ECM genes in the brain.SIGNIFICANCE STATEMENT The insulin/IGF-1 signaling (IIS) has been recognized as a regulator of aging, a leading risk factor for the onset of Alzheimer's disease (AD). In AD mouse models, genetic deletion of key IIS molecules markedly reduces the amyloid plaque formation in the brain, although the molecular underpinnings of this amelioration remain elusive. We found that the deficiency of insulin receptor substrate 2 leads to an increase in the expression of various extracellular matrices (ECMs) in the brain, potentially through TGF-ß/Smad signaling. Furthermore, some of those ECMs exhibited the potential to inhibit amyloid plaque accumulation by disrupting the formation of Aß fibrils. This study presents a novel mechanism by which IIS regulates Aß accumulation, which may involve altered brain ECM expression.


Assuntos
Doença de Alzheimer , Masculino , Camundongos , Animais , Doença de Alzheimer/metabolismo , Insulina , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Placa Amiloide/patologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Camundongos Transgênicos , Modelos Animais de Doenças , Fator de Crescimento Transformador beta/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo
2.
BMC Genomics ; 25(1): 568, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38840068

RESUMO

BACKGROUND: Transgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called "pronuclear injection-based targeted transgenesis" (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci. PITT was achieved by pre-installing so called landing pad sequences (such as heterotypic LoxP sites or attP sites) to create seed mice and then injecting Cre recombinase or PhiC31 integrase mRNAs along with a compatible donor plasmid into zygotes derived from the seed mice. PITT and its subsequent version, improved PITT (i-PITT), overcome disadvantages of conventional Tg mice such as lack of consistent and reliable expression of the cassettes among different Tg mouse lines, and the PITT approach is superior in terms of cost and labor. One of the limitations of PITT, particularly using Cre-mRNA, is that the approach cannot be used for insertion of conditional expression cassettes using Cre-LoxP site-specific recombination. This is because the LoxP sites in the donor plasmids intended for achieving conditional expression of the transgene will interfere with the PITT recombination reaction with LoxP sites in the landing pad. RESULTS: To enable the i-PITT method to insert a conditional expression cassette, we modified the approach by simultaneously using PhiC31o and FLPo mRNAs. We demonstrate the strategy by creating a model containing a conditional expression cassette at the Rosa26 locus with an efficiency of 13.7%. We also demonstrate that inclusion of FLPo mRNA excludes the insertion of vector backbones in the founder mice. CONCLUSIONS: Simultaneous use of PhiC31 and FLP in i-PITT approach allows insertion of donor plasmids containing Cre-loxP-based conditional expression cassettes.


Assuntos
Genoma , Integrases , Camundongos Transgênicos , Animais , Camundongos , Integrases/genética , Integrases/metabolismo , Transgenes , Marcação de Genes/métodos , Técnicas de Transferência de Genes , Plasmídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mutagênese Insercional
3.
Mol Biol Rep ; 51(1): 126, 2024 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-38236446

RESUMO

BACKGROUND: Sichuan pepper [Zanthoxylum bungeanum; huajiao (HJ)] is a widely used spice in China and has better antioxidative, anti-glycation, and bile acid-lowering properties than cumin and coriander seeds. HJ affects inflammation-related cytokines and caecal microbiota in mice fed a low-fibre and high-sucrose diet. METHODS AND RESULTS: To determine the ameliorative effect of HJ on inflammatory bowel disease, C57BL/6 mice were divided into three groups and fed distilled water (control) or 3% (w/v) dextran sodium sulphate (DSS) in drinking water with normal chow containing 0% or 5% (w/w) HJ powder for seven days. After 6 days of feeding, diarrhoea, decreased body weight, and blood in faeces were observed in the DSS group. DSS treatment increased the spleen weight and damaged the colon tissue. These inflammatory indices were inhibited by HJ treatment. Amplicon sequencing of the 16S rDNA (V4) gene of the caecal content revealed a decrease in the alpha diversity (Simpson index D) in the DSS treatment group compared to the control group. The abundance of caecal Desulfovibrio, an inflammation-related genus, was higher and the caecal Lachnospiraceae and Bacteroides levels were lower in the DSS-treated mice than those in the control mice. However, HJ suppressed the DSS-induced changes in the caecal microbiota. CONCLUSION: HJ intake contributes to the reduction in inflammation and maintenance of the gut microbiota. However, the strong antioxidant properties of phenolic compounds and fermentability of water-soluble dietary fibres in HJ and their relationship with other functional properties warrant further investigation.


Assuntos
Doenças Inflamatórias Intestinais , Microbiota , Sulfatos , Animais , Camundongos , Camundongos Endogâmicos C57BL , Dextranos , Pós , Inflamação , Antioxidantes , Água
4.
Appl Microbiol Biotechnol ; 107(7-8): 2627-2638, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36922439

RESUMO

Sichuan pepper (Zanthoxylum bungeanum, HJ), a spice widely used in China, has antioxidative, anti-inflammatory, antibacterial, and anti-obesity properties. In this study, to confirm the value of HJ as a functional food, the in vitro antioxidant and bile acid-lowering capacities, as well as the effects on caecal microbiota, were compared with those of cumin (Cuminum cyminum, CM) and coriander (Coriandrum sativum, CR) seeds in Institute of Cancer Research (ICR) mice fed a high-sucrose and low-dietary fibre diet. The total phenolic content, superoxide anion radical-scavenging capacity, and Fe-reducing power of the HJ aqueous solution were higher than those of CM and CR (p < 0.05). The bile acid (taurocholic, glycocholic, and deoxycholic acids)-lowering capacity of the HJ suspension was also higher than those of CM and CR. Compared with mice fed a control diet (no fibre, NF), caecal Lactobacillus gasseri- and Muribaculum intestinale-like bacteria were higher in mice fed a diet containing 5% (w/w) of CM, CR, or HJ for 14 days. Bifidobacterium pseudolongum-, Lactobacillus murinus/animalis-, and Faecalibaculum rodentium-like bacteria were significantly increased, while Desulfovibrio-like bacteria were significantly decreased in the HJ group. In addition, CM and HJ may benefit specific metabolic functions of gut microbiota, such as starch, sucrose, and tyrosine metabolism. The tumour necrosis factor (TNF-α) concentration in the spleen tissue of ICR mice was decreased by the intake of spices. However, there were no changes in interleukin-2 (IL-2) and IL-10 levels in HJ fed mice. These results suggested that HJ has potential as a functional food related to gut microbiota. KEY POINTS: • Bididobacterium and Faecalibaculum in mice gut microbiota are increased by Sichuan pepper (HJ). • Desulfovibrionaceae, an inflammatory LPS producer, in mice gut microbiota is decreased by HJ. • HJ decreases pro-inflammatory TNF both in murine spleen tissue and in vitro macrophages.


Assuntos
Microbioma Gastrointestinal , Sacarose , Camundongos , Animais , Sacarose/farmacologia , Dieta , Bactérias , Antioxidantes/farmacologia , Fibras na Dieta , Ácidos e Sais Biliares , Dieta Hiperlipídica , Camundongos Endogâmicos C57BL
5.
Food Microbiol ; 112: 104245, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-36906313

RESUMO

In this study, we aimed to investigate how microbial contamination progresses on the carcass surface during the slaughter process. Cattle carcasses were tracked during a series of slaughter processes (five steps), and carcass surfaces (four parts) and equipment (nine types) were swabbed to investigate the bacterial contamination. Results showed that the outer surface (near the rear region of the flank [Top round] and [Top sirloin butt]) had significantly higher total viable counts (TVCs) than inner surface (p < 0.01) and that TVCs gradually decreased along the process. Enterobacteriaceae (EB) counts were high on the splitting saw and in top round region, and EB was detected on the inner surface of the carcasses. Furthermore, in some carcasses, Yersinia spp., Serratia spp., and Clostridium spp. present on top round and top sirloin butt immediately after skinning and remained on the carcass surface after the final process. These bacterial groups are detrimental to beef quality as they can grow in the package during cold distribution. Our results show that the skinning process is the most prone to microbial contamination, including psychrotolerant microorganisms. Moreover, this study provides information for understanding the dynamics of microbial contamination in the cattle slaughter process.


Assuntos
Microbiologia de Alimentos , Carne , Bovinos , Animais , Carne/microbiologia , Contagem de Colônia Microbiana , Matadouros , Enterobacteriaceae
6.
Biol Reprod ; 107(2): 605-618, 2022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-35368067

RESUMO

The litter size of mouse strains is determined by the number of oocytes naturally ovulated. Many attempts have been made to increase litter sizes by conventional superovulation regimens (e.g., using equine or human gonadotropins, eCG/hCG but had limited success because of unexpected decreases in the numbers of embryos surviving to term. Here, we examined whether rat-derived anti-inhibin monoclonal antibodies (AIMAs) could be used for this purpose. When C57BL/6 female mice were treated with an AIMA and mated, the number of healthy offspring per mouse increased by 1.4-fold (11.9 vs. 8.6 in controls). By contrast, treatment with eCG/hCG or anti-inhibin serum resulted in fewer offspring than in nontreated controls. The overall efficiency of production based on all females treated (including nonpregnant ones) was improved 2.4 times with AIMA compared with nontreated controls. The AIMA treatment was also effective in ICR mice, increasing the litter size from 15.3 to 21.2 pups. We then applied this technique to an in vivo genome-editing method (improved genome-editing via oviductal nucleic acid delivery, i-GONAD) to produce C57BL/6 mice deficient for tyrosinase. The mean litter size following i-GONAD increased from 4.8 to 7.3 after the AIMA treatment and genetic modifications were confirmed in 80/88 (91%) of the offspring. Thus, AIMA treatment is a promising method for increasing the litter size of mice and may be applied for the easy proliferation of mouse colonies as well as in vivo genetic manipulation, especially when the mouse strains are sensitive to handling.


Assuntos
Gonadotropina Coriônica , Inibinas , Animais , Anticorpos Monoclonais , Feminino , Edição de Genes , Cavalos , Humanos , Tamanho da Ninhada de Vivíparos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Gravidez , Ratos , Superovulação , Tecnologia
7.
J Gastroenterol Hepatol ; 37(4): 678-684, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34911147

RESUMO

BACKGROUND AND AIM: Recently, multimodal representation learning for images and other information such as numbers or language has gained much attention. The aim of the current study was to analyze the diagnostic performance of deep multimodal representation model-based integration of tumor image, patient background, and blood biomarkers for the differentiation of liver tumors observed using B-mode ultrasonography (US). METHOD: First, we applied supervised learning with a convolutional neural network (CNN) to 972 liver nodules in the training and development sets to develop a predictive model using segmented B-mode tumor images. Additionally, we also applied a deep multimodal representation model to integrate information about patient background or blood biomarkers to B-mode images. We then investigated the performance of the models in an independent test set of 108 liver nodules. RESULTS: Using only the segmented B-mode images, the diagnostic accuracy and area under the curve (AUC) values were 68.52% and 0.721, respectively. As the information about patient background and blood biomarkers was integrated, the diagnostic performance increased in a stepwise manner. The diagnostic accuracy and AUC value of the multimodal DL model (which integrated B-mode tumor image, patient age, sex, aspartate aminotransferase, alanine aminotransferase, platelet count, and albumin data) reached 96.30% and 0.994, respectively. CONCLUSION: Integration of patient background and blood biomarkers in addition to US image using multimodal representation learning outperformed the CNN model using US images. We expect that the deep multimodal representation model could be a feasible and acceptable tool for the definitive diagnosis of liver tumors using B-mode US.


Assuntos
Neoplasias Hepáticas , Área Sob a Curva , Humanos , Neoplasias Hepáticas/diagnóstico por imagem , Redes Neurais de Computação , Ultrassonografia/métodos
8.
Biosci Biotechnol Biochem ; 86(8): 1106-1113, 2022 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-35830523

RESUMO

Primal cuts of Australian beef transported by sea were stored under different chilled temperatures (0, 2, and 4 °C) for 6 weeks in different packaging conditions (aerobic or anaerobic packaging). The number of microorganisms and the transition of the microbiota were investigated using culture methods and amplicon sequencing. After 6 weeks of storage, the beef tended to show a high total viable count under aerobic packaging conditions and a high lactic acid bacteria count under anaerobic packaging conditions. The result of amplicon sequencing analysis showed that different beef samples had different predominant bacterial groups. Moreover, at high storage temperatures, Serratia sp. having high putrefactive activity showed increased abundance, while at low storage temperatures, Lactobacillus sp. showed increased abundance. Thus, differences in the packaging conditions and distribution temperatures after import affect the number of bacteria and the type of microorganisms in the Australian beef primal cuts, which may affect their quality.


Assuntos
Embalagem de Alimentos , Microbiota , Animais , Austrália , Bactérias/genética , Bovinos , Contagem de Colônia Microbiana , Microbiologia de Alimentos , Embalagem de Alimentos/métodos , Armazenamento de Alimentos/métodos , Temperatura
9.
Chem Pharm Bull (Tokyo) ; 70(10): 679-683, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36184450

RESUMO

The liver X receptor is a nuclear hormone receptor that regulates lipid metabolism. Previously, we had demonstrated the antiviral properties of a liver X receptor antagonist associated with the hepatitis C virus and severe acute respiratory syndrome coronavirus 2. In this study, we screened a chemical library and identified two potential liver X receptor antagonists. Spectroscopic analysis revealed that the structures of both antagonists (compounds 1 and 2) were cyclic dimer and trimer of esters, respectively, that consisted of phthalate and 1,6-hexane diol. This study is the first to report the structure of the cyclic trimer of phthalate ester. Further experiments revealed that the compounds were impurities of solvents used for purification, although their source could not be traced. Both phthalate esters exhibited anti-hepatitis C virus activity, whereas the cyclic dimer showed anti-severe acute respiratory syndrome coronavirus 2 activity. Cyclic phthalate derivatives may constitute a novel class of liver X receptor antagonists and broad-spectrum antivirals.


Assuntos
COVID-19 , Ésteres , Antivirais/farmacologia , Ésteres/farmacologia , Hepacivirus , Hexanos , Humanos , Receptores X do Fígado , Ácidos Ftálicos , Receptores Citoplasmáticos e Nucleares , SARS-CoV-2 , Solventes
10.
Food Microbiol ; 100: 103849, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34416953

RESUMO

In this study, changes in the microbiota of Japanese Black beef carcasses, which are expected to be transported for a long time in chilled temperatures, were investigated. Three Japanese Black beef samples (carcasses A, B, and C) immediately after slaughter were stored at 0 °C for 15 weeks under aerobic and vacuum conditions. The initial bacterial counts were 50 CFU/g for carcass A and less than the reliable quantitative detection limit for carcasses B and C. Under aerobic storage conditions, the bacterial count increased to 8.0 log CFU/g or higher, which is a measure of putrefaction, at 6-9 weeks. Under anaerobic storage conditions, the bacterial counts of carcasses A and C reached 3.5-6.5 log CFU/g, but carcass B showed no bacterial growth during the 15-week storage period. The predominant group was Pseudomonas spp. under aerobic conditions and Serratia spp. under anaerobic conditions. To the best of our knowledge, there is no previous study investigating the transition of microbiota when Japanese Black beef is stored at low temperatures for a long period of time, and the results of this study are considered very important findings for the expansion of international trade of Japanese Beef in the future.


Assuntos
Bactérias/crescimento & desenvolvimento , Carne/microbiologia , Microbiota , Animais , Bactérias/classificação , Bactérias/isolamento & purificação , Carga Bacteriana , Bovinos , Contaminação de Alimentos/análise , Armazenamento de Alimentos
11.
Int J Clin Oncol ; 23(2): 375-381, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29063983

RESUMO

BACKGROUND: Follicular lymphoma (FL) is the most common type of non-Hodgkin lymphoma (NHL), with indolent progression. Several treatment options are selected, based not only on disease status, quality of life (QOL), and age of patient, but also on recent increasing medical costs. We retrospectively analysed the first-line treatment of FL with regard to treatment outcomes and medical economics, and discuss the appropriate strategies for FL. METHODS: Data on a total of 69 newly-diagnosed patients with FL was retrospectively collected from 2001 to 2015. RESULTS: The median age of the patients was 60 years and the median follow-up was 58 months. A total of 25 cases with FL were treated with R monotherapy, and 28 cases were treated with R-CHOP as first-line treatment. The factors affecting the decision of physicians to use R or R-CHOP treatment were serum level of lactate dehydrogenase (LDH) and disease stage. The first-line treatment-associated survival did not show any statistical differences between R and R-CHOP. The average hospitalization and average of all medical costs during the first-line treatment were 4.1 days (R) versus 55.7 days (R-CHOP), and JPY 1,707,693 (USD 15,324) (R) versus JPY 2,136,117 (USD 19,170) (R-CHOP), respectively. CONCLUSION: R monotherapy for patients whose diseases show low tumor burden and who are not candidates for local treatment has benefits as a first-line treatment compared to R-CHOP, based on the patients' QOL and medical economics.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/economia , Linfoma Folicular/tratamento farmacológico , Linfoma Folicular/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Murinos/economia , Anticorpos Monoclonais Murinos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Custos e Análise de Custo , Ciclofosfamida/economia , Ciclofosfamida/uso terapêutico , Doxorrubicina/economia , Doxorrubicina/uso terapêutico , Custos de Medicamentos , Feminino , Humanos , Imunoterapia/economia , Imunoterapia/métodos , Linfoma Folicular/economia , Masculino , Pessoa de Meia-Idade , Prednisona/economia , Prednisona/uso terapêutico , Qualidade de Vida , Estudos Retrospectivos , Rituximab/administração & dosagem , Rituximab/economia , Resultado do Tratamento , Vincristina/economia , Vincristina/uso terapêutico
12.
J Plant Res ; 131(3): 409, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29679361

RESUMO

The caption of Figure 5 was published incorrectly in the original publication of the article.

13.
J Plant Res ; 131(3): 395-407, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29549525

RESUMO

Monocots are remarkably homogeneous in sharing a common trimerous pentacyclic floral Bauplan. A major factor affecting monocot evolution is the unique origin of the clade from basal angiosperms. The origin of the floral Bauplan of monocots remains controversial, as no immediate sister groups with similar structure can be identified among basal angiosperms, and there are several possibilities for an ancestral floral structure, including more complex flowers with higher stamen and carpel numbers, or strongly reduced flowers. Additionally, a stable Bauplan is only established beyond the divergence of Alismatales. Here, we observed the floral development of five members of the three 'petaloid' Alismatales families Butomaceae, Hydrocharitaceae, and Alismataceae. Outer stamen pairs can be recognized in mature flowers of Alismataceae and Butomaceae. Paired stamens always arise independently, and are either shifted opposite the sepals or close to the petals. The position of stamen pairs is related to the early development of the petals. In Butomaceae, the perianth is not differentiated and the development of the inner tepals is not delayed; the larger inner tepals (petals) only permit the initiation of stamens in antesepalous pairs. Alismataceae has delayed petals and the stamens are shifted close to the petals, leading to an association of stamen pairs with petals in so-called stamen-petal complexes. In the studied Hydrocharitaceae species, which have the monocot floral Bauplan, paired stamens are replaced by larger single stamens and the petals are not delayed. These results indicate that the origin of the floral Bauplan, at least in petaloid Alismatales, is closely linked to the position of stamen pairs and the rate of petal development. Although the petaloid Alismatales are not immediately at the base of monocot divergence, the floral evolution inferred from the results should be a key to elucidate the origin of the floral Bauplan of monocots.


Assuntos
Alismatales/crescimento & desenvolvimento , Flores/crescimento & desenvolvimento , Alismatales/genética , Alismatales/ultraestrutura , Flores/genética , Flores/ultraestrutura , Hydrocharitaceae/genética , Hydrocharitaceae/crescimento & desenvolvimento , Hydrocharitaceae/ultraestrutura , Microscopia Eletrônica de Varredura
15.
3 Biotech ; 14(4): 118, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38524237

RESUMO

Kanpyo (KP) is an edible dried product produced by peeling the fruit of the gourd Lagenaria siceraria var. hispida; it is used in the traditional Japanese cuisine. The health functionality of KP due to its rich dietary fibre is expected to include a possible combined effect of KP-responsive indigenous gut bacteria (KP-RIB). However, its effect on the gut microbiota is unclear. To determine the effects of the KP on the gut microbiota and their host, Institute of Cancer Research mice were fed a high-sucrose diet containing no fibre (NF) or 5% (w/w) KP for 14 days, and their caecal microbiota was analysed by 16S rRNA (V4) amplicon sequencing. Higher faecal frequency and weight and lower spleen weight and spleen tumour necrosis factor-α levels were observed in KP-fed mice than in NF-fed mice (p < 0.05). KP increased and decreased the abundance of short-chain fatty acid producer Lachnospiraceae and obesity-inflammation related Allobaculum species, respectively. In the case of human faecal cultures, stool samples from five healthy volunteers were inoculated and incubated at 37 °C for 24 h anaerobically; 3.2% (w/v) KP suppressed putrefactive compounds (indole, phenol, and ammonia). KP increased butyrate-producer Faecalibacterium, acetate/lactate-producer Bifidobacterium, and Lachnospira. Furthermore, KP cultures showed high antioxidant and RAW264.7 macrophage cell activation capacities. These results suggest that KP-RIB and KP intake may synergistically affect host health. However, further studies are required to clarify the synergistic effects of KP and KP-RIB.

16.
Methods Mol Biol ; 2631: 325-340, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36995675

RESUMO

The clustered regularly interspaced short palindromic repeats (CRISPR) technology has made it possible to produce genome-edited (GE) animals more easily and rapidly than before. In most cases, GE mice are produced by microinjection (MI) or by in vitro electroporation (EP) of CRISPR reagents into fertilized eggs (zygotes). Both of these approaches require ex vivo handling of isolated embryos and their subsequent transfer into another set of mice (called recipient or pseudopregnant mice). Such experiments are performed by highly skilled technicians (especially for MI). We recently developed a novel genome editing method, called "GONAD (Genome-editing via Oviductal Nucleic Acids Delivery)," which can completely eliminate the ex vivo handling of embryos. We also made improvements to the GONAD method, termed "improved-GONAD (i-GONAD)." The i-GONAD method involves injection of CRISPR reagents into the oviduct of an anesthetized pregnant female using a mouthpiece-controlled glass micropipette under a dissecting microscope, followed by EP of the entire oviduct allowing the CRISPR reagents to enter into the zygotes present inside the oviduct, in situ. After the i-GONAD procedure, the mouse recovered from anesthesia is allowed to continue the pregnancy to full term to deliver its pups. The i-GONAD method does not require pseudopregnant female animals for embryo transfer, unlike the methods relying on ex vivo handling of zygotes. Therefore, the i-GONAD method can reduce the number of animals used, compared to the traditional methods. In this chapter, we describe some newer technical tips about the i-GONAD method. Additionally, even though the detailed protocols of GONAD and i-GONAD have been published elsewhere (Gurumurthy et al., Curr Protoc Hum Genet 88:15.8.1-15.8.12, 2016 Nat Protoc 14:2452-2482, 2019), we provide all the protocol steps of i-GONAD in this chapter so that the reader can find most of the information, needed for performing i-GONAD experiments, in one place.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Humanos , Gravidez , Feminino , Camundongos , Animais , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , Tubas Uterinas , Oviductos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Eletroporação/métodos , Gônadas
17.
bioRxiv ; 2023 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-36747769

RESUMO

mRNAs produced in a cell are almost always translated within the same cell. Some mRNAs are transported to other cells of the organism through processes involving membrane nanotubes or extracellular vesicles. A recent report describes a surprising new phenomenon of encapsulating mRNAs inside virus-like particles (VLPs) to deliver them to other cells in a process that was named SEND (Selective Endogenous eNcapsidation for cellular Delivery). Although the seminal work demonstrates the SEND process in cultured cells, it is unknown whether this phenomenon occurs in vivo . Here, we demonstrate the SEND process in living organisms using specially designed genetically engineered mouse models. Our proof of principle study lays a foundation for the SEND-VLP system to potentially be used as a gene therapy tool to deliver therapeutically important mRNAs to tissues.

18.
Vet Res ; 43: 34, 2012 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-22520572

RESUMO

The relationship between stress and disease is thought to be unambiguous: chronic stress induces immunosuppression, which likely increases the risk of infection. However, this link has not been firmly established in wild animals, particularly whether stress hormones affect host responses to zoonotic pathogens, which can be transmitted to domesticated animal, wildlife and human populations. Due to the dynamic effects of stress hormones on immune functions, stress hormones may make hosts better or poorer amplifying hosts for a pathogen contingent on context and the host species evaluated. Using an important zoonotic pathogen, West Nile virus (WNV) and a competent host, the Northern Cardinal (Cardinalis cardinalis), we tested the effects of exogenous corticosterone on response to WNV infection. Corticosterone was administered at levels that individuals enduring chronic stressors (i.e., long-term inclement weather, food shortage, anthropogenic pollution) might experience in the wild. Corticosterone greatly impacted mortality: half of the corticosterone-implanted cardinals died between five - 11 days post-inoculation whereas only one of nine empty-implanted (control) birds died. No differences were found in viral titer between corticosterone- and empty-implanted birds. However, cardinals that survived infections had significantly higher average body temperatures during peak infection than individuals that died. In sum, this study indicates that elevated corticosterone could affect the survival of WNV-infected wild birds, suggesting that populations may be disproportionately at-risk to disease in stressful environments.


Assuntos
Doenças das Aves/virologia , Corticosterona/metabolismo , Tolerância Imunológica , Aves Canoras , Febre do Nilo Ocidental/veterinária , Vírus do Nilo Ocidental/isolamento & purificação , Animais , Doenças das Aves/imunologia , Doenças das Aves/mortalidade , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Técnicas Imunoenzimáticas/veterinária , Masculino , Michigan , Viremia/imunologia , Viremia/mortalidade , Viremia/veterinária , Viremia/virologia , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/mortalidade , Febre do Nilo Ocidental/virologia
19.
J Food Prot ; 85(10): 1404-1409, 2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-35749697

RESUMO

ABSTRACT: Although essential oils exhibit antimicrobial properties, their application is limited, owing to their strong volatility and poor water solubility. Emulsification is a valid strategy for improving chemical stability. In this study, we prepared a mustard oil (MO) emulsion with egg yolk lecithin and evaluated its antimicrobial activity against Listeria monocytogenes in vitro and in cheese curd. The particle size of the MO emulsion was approximately 0.19 µm and remained stable for 30 days of storage. The MO emulsion showed strong antimicrobial activity against L. monocytogenes in vitro. Moreover, 40 ppm of MO was sufficient to inhibit the growth of L. monocytogenes in culture, and the addition of 160 ppm of MO decreased the population of L. monocytogenes. When 50 ppm of emulsified MO was added to milk during cheese curd production and it was stored at 10°C for 10 days, the growth of L. monocytogenes was suppressed. When the cheese curd with MO emulsion was stored at 4°C, the bacterial count was significantly decreased (P < 0.05), and no bacterial growth was observed after 14 days of storage. Furthermore, the sensory characteristics of cheese curd with the MO emulsion were acceptable. These results indicate MO emulsions may be useful in controlling the growth of L. monocytogenes in fresh cheese.


Assuntos
Queijo , Listeria monocytogenes , Queijo/microbiologia , Emulsões , Microbiologia de Alimentos , Mostardeira , Óleos de Plantas
20.
Curr Res Food Sci ; 5: 992-997, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35734141

RESUMO

Plant-based milk-like products from soybeans and other legumes and nuts have been explored worldwide, owing to their nutritional and functional characteristics. This study was conducted to develop new functional food materials from peanut (Arachis hypogaea) milk (PM) with desirable health functions to mitigate lifestyle and age-related diseases. The antioxidant, anti-glycation and bile acid-lowering properties of PM fermented with lactic acid bacteria Lactiplantibacillus plantarum Kinko-SU4 (FPM) were determined in vitro. L. plantarum Kinko-SU4 lowered the pH level from 6.4 to 4.3, 3.9, and 3.7 at 10, 24, and 48 h, respectively. The lactic acid concentration was 4.4 mg/mL after 48 h of incubation. The starter degraded the dissolved proteins in PM, including Ara h 1, one of the peanut allergens. Although the total phenolic content was 36% lower in FPM than in unfermented PM, O2 - radical-scavenging capacity was high in FPM. Anti-glycation in a bovine serum albumin-fructose model and the bile acid-lowering capacities of PM were distinctly increased following fermentation. The result of this study infers that PM fermented with L. plantarum Kinko-SU4 can be considered a desirable food material to prevent and ameliorate chronic lifestyle diseases, particularly in the elderly.

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