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1.
Nat Commun ; 12(1): 4902, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385461

RESUMO

Efficient and precise base editors (BEs) for C-to-G transversion are highly desirable. However, the sequence context affecting editing outcome largely remains unclear. Here we report engineered C-to-G BEs of high efficiency and fidelity, with the sequence context predictable via machine-learning methods. By changing the species origin and relative position of uracil-DNA glycosylase and deaminase, together with codon optimization, we obtain optimized C-to-G BEs (OPTI-CGBEs) for efficient C-to-G transversion. The motif preference of OPTI-CGBEs for editing 100 endogenous sites is determined in HEK293T cells. Using a sgRNA library comprising 41,388 sequences, we develop a deep-learning model that accurately predicts the OPTI-CGBE editing outcome for targeted sites with specific sequence context. These OPTI-CGBEs are further shown to be capable of efficient base editing in mouse embryos for generating Tyr-edited offspring. Thus, these engineered CGBEs are useful for efficient and precise base editing, with outcome predictable based on sequence context of targeted sites.


Assuntos
Sistemas CRISPR-Cas , Citidina Desaminase/metabolismo , Edição de Genes/métodos , Aprendizado de Máquina , Uracila-DNA Glicosidase/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Caenorhabditis elegans/genética , Códon/genética , Citidina Desaminase/genética , Escherichia coli/genética , Feminino , Biblioteca Gênica , Células HEK293 , Humanos , Camundongos , Reprodutibilidade dos Testes , Uracila-DNA Glicosidase/genética
2.
J Cell Physiol ; 235(9): 6058-6072, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32020604

RESUMO

Nowadays, abnormal loss of serine proteases appears very frequently in male patients with unexplained sterility. In fact, many testis-specific serine proteases, the largest family among the four protease families implicated in murine spermatogenesis, are indispensable for reproduction. In the present study, we demonstrate that the previously uncharacterized testis-specific serine protease TRYX5 (1700074P13Rik) is required for male fertility in mice. Tryx5-/- male mice are sterile, yet they have normal spermatogenesis and normal sperm parameters. In vivo fertilization experiments showed that the fertilization rate of Tryx5-/- sperm was almost zero. Sperm counting and analysis of paraffin sections of oviducts revealed that Tryx5-/- sperm were unable to migrate into the oviduct, which is likely the cause of the observed infertility of the Tryx5-/- male mice. Importantly, we also found that there was almost no mature ADAM3 present in Tryx5-/- sperm and almost no ADAM3 precursor in Tryx5-/- elongated spermatids of S13-16 stage, even though testes of Tryx5-/- and wild type mice had the same amount of the total precursor ADAM3. Collectively, our results demonstrate that Tryx5 is essential for male fertility in mice and suggest that TRYX5 functions in the stability or localization of ADAM3 precursor in elongated spermatids S13-16 stage, thereby regulating the ability of sperm to migrate from the uterus into the ampulla of the oviduct, the site of fertilization.


Assuntos
Fertilidade/genética , Infertilidade Masculina/genética , Proteínas Serina-Treonina Quinases/genética , Espermatogênese/genética , Animais , Tubas Uterinas/metabolismo , Feminino , Infertilidade Masculina/patologia , Masculino , Camundongos , Camundongos Knockout , Oviductos/citologia , Oviductos/metabolismo , Motilidade Espermática/genética , Espermatozoides/citologia , Espermatozoides/metabolismo , Testículo/crescimento & desenvolvimento , Testículo/metabolismo
3.
Br J Haematol ; 188(2): 321-331, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31418854

RESUMO

Hepcidin (HAMP) synthesis is suppressed by erythropoiesis to increase iron availability for red blood cell production. This effect is thought to result from factors secreted by erythroid precursors. Growth differentiation factor 11 (GDF11) expression was recently shown to increase in erythroid cells of ß-thalassaemia, and decrease with improvement in anaemia. Whether GDF11 regulates hepatic HAMP production has never been experimentally studied. Here, we explore GDF11 function during erythropoiesis-triggered HAMP suppression. Our results confirm that exogenous erythropoietin significantly increases Gdf11 as well as Erfe (erythroferrone) expression, and Gdf11 is also increased, albeit at a lower degree than Erfe, in phlebotomized wild type and ß-thalassaemic mice. GDF11 is expressed predominantly in erythroid burst forming unit- and erythroid colony-forming unit- cells during erythropoiesis. Exogeneous GDF11 administration results in HAMP suppression in vivo and in vitro. Furthermore, exogenous GDF11 decreases BMP-SMAD signalling, enhances SMAD ubiquitin regulatory factor 1 (SMURF1) expression and induces ERK1/2 (MAPK3/1) signalling. ERK1/2 signalling activation is required for GDF11 or SMURF1-mediated suppression in BMP-SMAD signalling and HAMP expression. This research newly characterizes GDF11 in erythropoiesis-mediated HAMP suppression, in addition to ERFE.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Fatores de Diferenciação de Crescimento/metabolismo , Hepcidinas/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/biossíntese , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/farmacologia , Eritropoese/fisiologia , Eritropoetina/farmacologia , Fatores de Diferenciação de Crescimento/biossíntese , Fatores de Diferenciação de Crescimento/genética , Fatores de Diferenciação de Crescimento/farmacologia , Células Hep G2 , Hepatócitos/metabolismo , Hepcidinas/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Hormônios Peptídicos/biossíntese , Hormônios Peptídicos/genética , Proteínas Recombinantes/farmacologia , Proteínas Smad/metabolismo
4.
PLoS One ; 13(4): e0195590, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29649338

RESUMO

As an important moral emotion, guilt plays a critical role in social interaction. It has been found that people tended to exhibit prosocial behavior under circumstances of guilt. However, all extant studies have predominantly focused on the influence of guilt on macro-level behavior. So far, no study has investigated whether guilt affects people's micro-level perception. The current study closes this gap by examining whether guilt affects one's inclination to perceive approaching motion. We achieved this aim by probing a facing-the-viewer bias (FTV bias). Specifically, when an ambiguous walking biological motion display is presented to participants via the point-light display technique, participants tend to perceive a walking agent approaching them. We hypothesized that guilt modulated FTV bias. To test this hypothesis, we adopted a two-person situation induction task to induce guilt, whereby participants were induced to feel that because of their poor task performance, their partner did not receive a satisfactory payment. We found that when participants were told that the perceived biological motion was motion-captured from their partner, the FTV bias was significantly increased for guilty participants relative to neutral participants. However, when participants were informed that the perceived biological motion was from a third neutral agent, the FTV bias was not modulated by guilt. These results suggest that guilt influences one's inclination to perceive approaching motion, but this effect is constrained to the person towards whom guilt is directed.


Assuntos
Culpa , Comportamento Social , Adolescente , Adulto , Feminino , Humanos , Relações Interpessoais , Masculino , Movimento , Estimulação Luminosa , Adulto Jovem
5.
Asian-Australas J Anim Sci ; 31(11): 1729-1737, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29642674

RESUMO

OBJECTIVE: The aim of the current study is to investigate the relationship between prohibitin (PHB), capping actin protein of muscle Z-line beta subunit (CAPZB), and tektin-2 (TEKT2) and sperm motility in Murrah buffalo. METHODS: We collected the high-motility and low-motility semen samples, testis, ovary, muscle, kidney, liver, brain and pituitary from Murrah buffalo, and analysed the expression of PHB, CAPZB, and TEKT2 in mRNA (message RNA) and protein level. RESULTS: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) result showed that the expression of PHB was higher and CAPZB, TEKT2 were specifically expressed in testis as compared to the other 6 tissues, and that in testis, the expression of TEKT2 was higher than that of CAPZB and PHB. Immunohistochemistry test revealed that all three genes were located on the convoluted seminiferous tubule and enriched in spermatogenic cells. Both qRT-PCR and Western Blot results showed that the expression levels of PHB, CAPZB, and TEKT2 were significantly lower in the low-motility semen group compared to the high-motility semen group (p<0.05). CONCLUSION: The expression levels of PHB, CAPZB, and TEKT2 in Murrah buffalo sperm have a high positive correlation with sperm motility. And the three genes may be potential molecular markers for the decline of buffalo sperm motility.

6.
J Dairy Sci ; 100(3): 1971-1986, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28109598

RESUMO

To study the role of microRNA (miR) in the lactation physiology of water buffalo, 2 multiparous dairy buffaloes (including an 8-yr-old buffalo that had been lactating for 3 mo, as well as a 10-yr-old nonlactating, nonpregnant buffalo) were used for miR library construction. The profile of differentially expressed miR in lactating and nonlactating mammary gland tissues of these water buffalo were investigated using Illumina-Solexa high-throughput sequencing technology (Illumina, San Diego, CA). The data identified 259 miR families, 359 mature miR, 363 pre-miR, 230 novel buffalo miR, and 5 buffalo-specific miR that were expressed in mammary tissues. Some highly significantly differentially expressed miR were explored, including bbu-miR-497, bbu-miR-30a-5p, bbu-miR-148a, bbu-miR-29a, bbu-miR-125a, bbu-miR-125b, and bbu-miR-103. The expression patterns of 18 miR were confirmed by quantitative real-time PCR in both tissues, and the expression of bbu miR-103 and novel miR-57 constituted the largest differences between lactating and nonlactating tissues. Further functional analysis indicated that the overexpression or suppression of miR-103 in buffalo mammary epithelial cells downregulated or upregulated the expression of pantothenate kinase 3, and also significantly increased the transcription factor steroid regulatory element binding protein, followed by the acceleration of de novo synthesis of fatty acids by upregulation of acetyl coenzyme A carboxylase α expression. The expression levels of 34 predicted target genes of novel-miR-57 in lactating and nonlactating mammary gland tissues were all analyzed by quantitative real-time PCR. Finally, only the expression of docking protein 4 could be upregulated or downregulated selectively by bbu-novel-miR-57 in buffalo mammary epithelial cells and the Bcap-37 cell line. This study provides an overview of the miR expression profile of water buffalo and the interaction between some key miR and their target genes, which may improve understanding of the important roles of miR in buffalo milk fat synthesis.


Assuntos
Búfalos/genética , Glândulas Mamárias Humanas , Animais , Humanos , Lactação/genética , Glândulas Mamárias Animais/metabolismo , MicroRNAs/genética , Áreas Alagadas
7.
Cognition ; 154: 95-101, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27253863

RESUMO

We report on how visual working memory (VWM) forms intact perceptual representations of visual objects using sub-object elements. Specifically, when objects were divided into fragments and sequentially encoded into VWM, the fragments were involuntarily integrated into objects in VWM, as evidenced by the occurrence of both positive and negative object-based attention effects: In Experiment 1, when subjects' attention was cued to a location occupied by the VWM object, the target presented at the location of that object was perceived as occurring earlier than that presented at the location of a different object. In Experiment 2, responses to a target were significantly slower when a distractor was presented at the same location as the cued object (Experiment 2). These results suggest that object fragments can be integrated into objects within VWM in a manner similar to that of visual perception.


Assuntos
Atenção , Memória de Curto Prazo , Reconhecimento Visual de Modelos , Sinais (Psicologia) , Humanos
8.
Theriogenology ; 85(5): 999-1012, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26778140

RESUMO

BMP1/TLD-related metalloproteinases play a key role in morphogenesis via the proteolytic maturation of a number of extracellular matrix proteins and the activation of a subset of growth factors of the transforming growth factor beta superfamily. Recent data indicated that BMP1 is expressed in sheep ovarian follicles and showed a protease activity. The aim of the present study was to characterize the function of the buffalo BMP1 gene in folliculogenesis. A 3195-bp buffalo BMP1 mRNA fragment was firstly cloned and sequenced, which contained a whole 2967-bp codon sequence. The multialigned results suggested that BMP1 is highly conserved among different species both at the nucleic acid and the amino acid level. BMP1 is located in the oogonium of the fetal buffalo ovary and in the granulosa cells (GCs) and the oocytes of adult ovary from the primordial to the large antral follicles. Further study showed that BMP1 promoted cell cycle and proliferation and inhibited apoptosis in IVC GCs. Adding BMP1 recombinant protein to the culture medium of the GCs increased the expression of the key cell cycle regulators such as cyclin D1 and cyclin D2 and downregulated the expression of cell apoptosis pathway genes such as Cytochrome C, Fas, FasL, and Chop, both at the mRNA and at the protein levels. It also upregulated the expression of PAPP-A, IGF system, and VEGF, and so forth, which play important roles in the selection and dominance of growth follicles. The opposite results were observed by adding BMP1 antibody to the investigation groups. This study suggests that BMP1 regulates the proliferation and apoptosis of IVC GCs by changing the expression pattern of related genes and may potentially promote the selection and dominance of the buffalo follicles.


Assuntos
Apoptose , Proteína Morfogenética Óssea 1/fisiologia , Búfalos , Proliferação de Células , Células da Granulosa/fisiologia , Oogênese/genética , Folículo Ovariano/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína Morfogenética Óssea 1/genética , Proteína Morfogenética Óssea 1/farmacologia , Búfalos/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Células da Granulosa/citologia , Células da Granulosa/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Ovulação/efeitos dos fármacos , Ovulação/genética , Proteínas Recombinantes/farmacologia
9.
Cognition ; 141: 26-35, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25912893

RESUMO

Visual working memory (VWM) has been traditionally viewed as a mental structure subsequent to visual perception that stores the final output of perceptual processing. However, VWM has recently been emphasized as a critical component of online perception, providing storage for the intermediate perceptual representations produced during visual processing. This interactive view holds the core assumption that VWM is not the terminus of perceptual processing; the stored visual information rather continues to undergo perceptual processing if necessary. The current study tests this assumption, demonstrating an example of involuntary integration of the VWM content, by creating the Ponzo illusion in VWM: when the Ponzo illusion figure was divided into its individual components and sequentially encoded into VWM, the temporally separated components were involuntarily integrated, leading to the distorted length perception of the two horizontal lines. This VWM Ponzo illusion was replicated when the figure components were presented in different combinations and presentation order. The magnitude of the illusion was significantly correlated between VWM and perceptual versions of the Ponzo illusion. These results suggest that the information integration underling the VWM Ponzo illusion is constrained by the laws of visual perception and similarly affected by the common individual factors that govern its perception. Thus, our findings provide compelling evidence that VWM functions as a buffer serving perceptual processes at early stages.


Assuntos
Memória de Curto Prazo/fisiologia , Ilusões Ópticas/fisiologia , Reconhecimento Visual de Modelos/fisiologia , Percepção Espacial/fisiologia , Adulto , Feminino , Humanos , Masculino , Adulto Jovem
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