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Research indicated that Paclitaxel (PTX) can induce immunogenic cell death (ICD) through immunogenic modulation. However, the combination of PTX and ICD has not been extensively studied in breast cancer (BRCA). The TCGA-BRCA and GSE20685 datasets were enrolled in this study. Samples from the TCGA-BRCA dataset were consistently clustered based on selected immunogenic cell death-related genes (ICD-RGs). Next, candidate genes were obtained by overlapping differentially expressed genes (DEGs) between BRCA and normal groups, intersecting genes common to DEGs between cluster1 and cluster2 and hub module genes, and target genes of PTX from five databases. The univariate Cox algorithm and the least absolute shrinkage and selection operator (LASSO) were performed to obtain biomarkers and build a risk model. Following observing the immune microenvironment in differential risk subgroups, single-gene gene set enrichment analysis (GSEA) was carried out in all biomarkers. Finally, the expression of biomarkers was analyzed. Enrichment analysis showed that 626 intersecting genes were linked with inflammatory response. Further five biomarkers (CHI3L1, IL18, PAPLN, SH2D2A, and UBE2L6) were identified and a risk model was built. The model's performance was validated using GSE20685 dataset. Furthermore, the biomarkers were enriched with adaptive immune response. Lastly, the experimental results indicated that the alterations in IL18, SH2D2A, and CHI3L1 expression after treatment matched those in the public database. In this study, Five PTX-ICD-related biomarkers (CHI3L1, IL18, PAPLN, SH2D2A, and UBE2L6) were identified to aid in predicting BRCA treatment outcomes.
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BACKGROUND: Cyclic Nucleotide-Binding Domain (CNBD)-family channels display distinct voltage-sensing properties despite sharing sequence and structural similarity. For example, the human Ether-a-go-go Related Gene (hERG) channel and the Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channel share high amino acid sequence similarity and identical domain structures. hERG conducts outward current and is activated by positive membrane potentials (depolarization), whereas HCN conducts inward current and is activated by negative membrane potentials (hyperpolarization). The structural basis for the "opposite" voltage-sensing properties of hERG and HCN remains unknown. RESULTS: We found the voltage-sensing domain (VSD) involves in modulating the gating polarity of hERG. We identified that a long-QT syndrome type 2-related mutation within the VSD, K525N, mediated an inwardly rectifying non-deactivating current, perturbing the channel closure, but sparing the open state and inactivated state. K525N rescued the current of a non-functional mutation in the pore helix region (F627Y) of hERG. K525N&F627Y switched hERG into a hyperpolarization-activated channel. The reactivated inward current induced by hyperpolarization mediated by K525N&F627Y can be inhibited by E-4031 and dofetilide quite well. Moreover, we report an extracellular interaction between the S1 helix and the S5-P region is crucial for modulating the gating polarity. The alanine substitution of several residues in this region (F431A, C566A, I607A, and Y611A) impaired the inward current of K525N&F627Y. CONCLUSIONS: Our data provide evidence that a potential cooperation mechanism in the extracellular vestibule of the VSD and the PD would determine the gating polarity in hERG.
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Canal de Potasio ERG1 , Activación del Canal Iónico , Humanos , Secuencia de Aminoácidos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Activación del Canal Iónico/genética , Mutación , Nucleótidos Cíclicos , Canal de Potasio ERG1/genéticaRESUMEN
BACKGROUND: Targeted estrogen receptor degradation has been approved to effectively treat ER + breast cancers. Due to the poor bioavailability of fulvestrant, the first generation of SERD, many efforts were made to develop oral SERDs. With the approval of Elacestrant, oral SERDs demonstrated superior efficacy than fulvestrant. However, due to the poor ability of known SERDs to penetrate the blood-brain barrier (BBB), breast cancer patients with brain metastasis cannot benefit from clinical SERDs. METHODS: The ER inhibitory effects were evaluated on ERα protein degradation, and target genes downregulation. And anti-proliferation activities were further determined in a panel of ER + breast cancer cell lines. The subcutaneous and intracranial ER + tumor models were used to evaluate the efficacy of anti-tumor effects. Brain penetrability was determined in multiple animal species. RESULTS: SCR-6852 is a novel SERD and currently is under early clinical evaluation. In vitro studies demonstrated that it strongly induced both wildtype and mutant ERα degradation. It potently inhibited cell proliferation in a panel of ER + breast cancer cell lines, including the cell lines containing ESR1 mutations (Y537 and D538). Furthermore, SCR-6852 exhibited pure antagonistic activities on the ERÉ signal axis identified both in vitro and in vivo. Oral administration of SCR-6852 at 10 mg/kg resulted in tumor shrinkage which was superior to Fulvestrant at 250 mg/kg, notably, in the intracranial tumor model, SCR-6852 effectively inhibited tumor growth and significantly prolonged mice survival, which correlated well with the high exposure in brains. In addition to mice, SCR-6852 also exhibited high brain penetrability in rats and dogs. CONCLUSIONS: SCR-6852 is a novel SERD with high potency in inducing ERα protein degradation and pure antagonistic activity on ERÉ signaling in vitro and in vivo. Due to the high brain penetrability, SCR-6852 could be used to treat breast patients with brain metastasis.
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Neoplasias Encefálicas , Receptores de Estrógenos , Ratas , Ratones , Animales , Perros , Receptores de Estrógenos/metabolismo , Fulvestrant/farmacología , Receptor alfa de Estrógeno/metabolismo , Antagonistas de Estrógenos , Encéfalo , Neoplasias Encefálicas/tratamiento farmacológicoRESUMEN
AIMS: Several trials have assessed the antihyperglycemic effects of sodium-glucose cotransporter 2 inhibitors (SGLT2Is) in patients with type 2 diabetes mellitus (T2DM). We conducted a quantitative analysis to assess the effects of SGLT2Is on renal risk factors in patients with abnormal glucose metabolism. MATERIALS AND METHODS: Randomized controlled trials (RCTs) were identified by searching the PubMed, Embase, Scopus, and Web of Science databases published before September 30, 2022. The intervention group received SGLT2Is as monotherapy or add-on treatment, and the control group received placebos, standard care, or active control. Risk of bias assessment was performed using the Cochrane risk of bias assessment tool. Meta-analysis was performed on studies with abnormal glucose metabolism populations and studies using the weighted mean differences (WMDs) as the measure of the effect size. Clinical trials providing changes in serum uric acid (SUA) were included. The mean change of SUA, glycated hemoglobin (HbA1c), body mass index (BMI), and estimated glomerular filtration rate (eGFR) were calculated. RESULTS: After a literature search and detailed evaluation, a total of 11 RCTs were included for quantitative analysis to analyze the differences between the SGLT2I group and the control group. The results showed that SGLT2I significantly reduced SUA (MD = -0.56, 95% CI = -0.66 ~ -0.46, I2 = 0%, P < 0.00001), HbA1c (MD = -0.20, 95% CI = -0.26 ~ -0.13, I2 = 0%, P < 0.00001), and BMI (MD = -1.19, 95% CI = -1.84 ~ -0.55, I2 = 0%, P = 0.0003). There was no significant difference in the reduction of eGFR observed in the SGLT2I group (MD = -1.60, 95% CI = -3.82 ~ 0.63, I2 = 13%, P = 0.16). CONCLUSIONS: These results showed that the SGLT2I group caused greater reductions in SUA, HbA1c, and BMI but had no effect on eGFR. These data suggested that SGLT2Is may have numerous potentially beneficial clinical effects in patients with abnormal glucose metabolism. However, these results need to be consolidated by further studies.
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Diabetes Mellitus Tipo 2 , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Humanos , Glucosa , Hemoglobina Glucada , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Ensayos Clínicos Controlados Aleatorios como Asunto , Hipoglucemiantes , Diabetes Mellitus Tipo 2/metabolismo , Factores de Riesgo , SodioRESUMEN
Mutations in the KCNQ2 gene encoding KV7.2 subunit that mediates neuronal M-current cause a severe form of developmental and epileptic encephalopathy (DEE). Electrophysiological evaluation of KCNQ2 mutations has been proved clinically useful in improving outcome prediction and choosing rational anti-seizure medications (ASMs). In this study we described the clinical characteristics, electrophysiological phenotypes and the in vitro response to KCNQ openers of five KCNQ2 pore mutations (V250A, N258Y, H260P, A265T and G290S) from seven patients diagnosed with KCNQ2-DEE. The KCNQ2 variants were transfected into Chinese hamster ovary (CHO) cells alone, in combination with KCNQ3 (1:1) or with wild-type KCNQ2 (KCNQ2-WT) and KCNQ3 in a ratio of 1:1:2, respectively. Their expression and electrophysiological function were assessed. When transfected alone or in combination with KCNQ3, none of these mutations affected the membrane expression of KCNQ2, but most failed to induce a potassium current except A265T, in which trace currents were observed when co-transfected with KCNQ3. When co-expressed with KCNQ2-WT and KCNQ3 (1:1:2), the currents at 0 mV of these mutations were decreased by 30%-70% compared to the KCNQ2/3 channel, which could be significantly rescued by applying KCNQ openers including the approved antiepileptic drug retigabine (RTG, 10 µM), as well as two candidates subjected to clinical trials, pynegabine (HN37, 1 µM) and XEN1101 (1 µM). These newly identified pathologic variants enrich the KCNQ2-DEE mutation hotspots in the pore-forming domain. This electrophysiological study provides a rational basis for personalized therapy with KCNQ openers in DEE patients carrying loss-of-function (LOF) mutations in KCNQ2.
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Encefalopatías , Canal de Potasio KCNQ2 , Cricetinae , Animales , Canal de Potasio KCNQ2/genética , Canal de Potasio KCNQ2/metabolismo , Canal de Potasio KCNQ3/genética , Canal de Potasio KCNQ3/metabolismo , Células CHO , Cricetulus , Mutación , Encefalopatías/genéticaRESUMEN
Pituitary adenomas have recently become more common and their incidence is increasing yearly. Functional pituitary tumors commonly secrete prolactin, growth hormones, and adrenocorticotropic hormones, which cause diseases such as prolactinoma, acromegaly, and Cushing's disease, but rarely secrete luteinizing, follicle-stimulating, thyroid-stimulating, and melanocyte-stimulating hormones. In addition to the typical clinical manifestations of functional pituitary tumors caused by excessive hormone levels, some pituitary tumors are also accompanied by abnormal glucose metabolism. The effects of these seven hormones on glucose metabolism are important for the treatment of diabetes secondary to pituitary tumors. This review focuses on the effects of hormones on glucose metabolism, providing important clues for the diagnosis and treatment of related diseases.
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In highly fragmented urban areas, plant diversity of remnant vegetation may depend not only on community structure and topographical factors, but also on landscape heterogeneity. Different buffer radius settings can affect the relative importance of these factors to plant diversity. The aim of this study was to examine the relative importance of landscape heterogeneity, community structure, and topographical factors on plant diversity under different buffer radii in biodiversity hotspots. We established 48 plots of remnant vegetation in Guangzhou city, one of the biodiversity hotspots. A buffer radius of 500 m, 1000 m, and 2000 m was established around the center of each sample plot, and 17 landscape heterogeneity indices in each buffer were calculated by FRAGSTATS 4.2 software. Combined with the community structure and topographical factors, the impact factors of plant diversity under different buffer radii were analyzed by multiple regression analysis. We found the following: (1) The combined explanatory power of the three factors accounted for 43% of the species diversity indices and 62% of the richness index at its peak. The three impact factors rarely act independently and usually create comprehensive cumulative effects. (2) Scale does matter in urban landscape studies. At a 500 m buffer radius, community structure combined with road disturbance indices was strongly related to diversity indices in herb and shrub layers. The stand age was negatively correlated with the tree-layer richness index. As the scale increased, the diversity indices and richness index in the three layers decreased or increased under the influence of comprehensive factors. (3) The richness index in the herb layer was more responsive to impact factors than other biodiversity indices. Except for the herb layer, the interpretation of landscape heterogeneity for each plant diversity index was more stable than that for the other two factors. Road disturbance indices, combined with the other six landscape pattern metrics, can well indicate species diversity and richness. We suggest that the vegetation area of remnant patches within a radius of 500-2000 m should be appropriately increased to protect plant diversity, and the negative effects of road disturbance should also be considered.
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Biodiversidad , Ecosistema , Plantas , CiudadesRESUMEN
The hypothalamus is indispensable in energy regulation and glucose homeostasis. Previous studies have shown that pro-opiomelanocortin neurons receive both central neuronal signals, such as α-melanocyte-stimulating hormone, ß-endorphin, and adrenocorticotropic hormone, as well as sense peripheral signals such as leptin, insulin, adiponectin, glucagon-like peptide-1, and glucagon-like peptide-2, affecting glucose metabolism through their corresponding receptors and related signaling pathways. Abnormalities in these processes can lead to obesity, type 2 diabetes, and other metabolic diseases. However, the mechanisms by which these signal molecules fulfill their role remain unclear. Consequently, in this review, we explored the mechanisms of these hormones and signals on obesity and diabetes to suggest potential therapeutic targets for obesity-related metabolic diseases. Multi-drug combination therapy for obesity and diabetes is becoming a trend and requires further research to help patients to better control their blood glucose and improve their prognosis.
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Owing to the increasing prevalence of type 2 diabetes, the development of novel hypoglycemic drugs has become a research hotspot, with the ultimate goal of developing therapeutic drugs that stimulate glucose-induced insulin secretion without inducing hypoglycemia. Vasoactive intestinal peptide (VIP), a 28-amino-acid peptide, can stimulate glucose-dependent insulin secretion, particularly by binding to VPAC2 receptors. VIP also promotes islet ß-cell proliferation through the forkhead box M1 pathway, but the specific molecular mechanism remains to be studied. The clinical application of VIP is limited because of its short half-life and wide distribution in the human body. Based on the binding properties of VIP and VPAC2 receptors, VPAC2-selective agonists have been developed to serve as novel hypoglycemic drugs. This review summarizes the physiological significance of VIP in glucose homeostasis and the potential therapeutic value of VPAC2-selective agonists in type 2 diabetes.
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Diabetes Mellitus Tipo 2 , Péptido Intestinal Vasoactivo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Glucosa/metabolismo , Humanos , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Secreción de Insulina , Receptores de Tipo II del Péptido Intestinal Vasoactivo/agonistas , Receptores de Tipo II del Péptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/farmacología , Péptido Intestinal Vasoactivo/uso terapéuticoRESUMEN
Objective: This work is aimed at revealing the role and the molecular mechanism of connective tissue growth factor 2 (CTGF) in the osteoblast differentiation of periodontal ligament stem cells (PDLSCs). Methods: The osteogenic differentiation of PDLSCs was induced by osteogenic induction medium (OM), and the expression level of osteogenic related proteins ALP, RUNX2, OCN, and CTGF was estimated using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting analysis. We constructed cell lines with CTGF overexpression or knockdown to verify the role of CTGF in the osteoblast differentiation of PDLSCs. Alkaline phosphatase (ALP) staining was introduced to measure the osteoblasts activity, and alizarin red S (ARS) staining was employed to test matrix mineralization. The interaction between CTGF and bone morphogenetic protein-2 (BMP-2) was determined by endogenous coimmunoprecipitation (Co-IP). Results: The expression level of CTGF was increased during the osteogenic induction of PDLSCs. Additionally, CTGF overexpression effectively maintained the stemness and facilitated the osteoblast differentiation in PDLSCs, and CTGF knockdown exerted opposite effects. Moreover, at molecular mechanism, CTGF increased the activity of BMP-2/Smad signaling pathway. Conclusion: This investigation verified that CTGF promotes the osteoblast differentiation in PDLSCs at least partly by activating BMP-2/Smad cascade signal.
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Osteogénesis , Ligamento Periodontal , Fosfatasa Alcalina/metabolismo , Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular , Células Cultivadas , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Humanos , Osteoblastos/metabolismo , Transducción de Señal , Células Madre/metabolismoRESUMEN
Capsid assembly modulators (CpAMs) represent a new class of antivirals targeting hepatitis B virus (HBV) core protein to disrupt the assembly process. In this work, a novel chemotype featuring a fused heterocycle amide was discovered through pharmacophore exploration. Lead optimization resulted in compound 8 with an EC50 value of 511 nM, and then methyl substitution on the piperazine was found to improve the in vitro potency remarkably. Further SAR studies established the key compound SHR5133, which showed high in vitro antiviral potency, favorable pharmacokinetic profiles across species, and robust in vivo efficacy.
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Human cytomegalovirus (HCMV), a ubiquitous pathogen, can cause severe illness in immunocompromised individuals. Typically, glioma is one of the most common malignant primary brain tumors and originates in the central nervous system. The IE86 gene of HCMV exerts a major role in regulating virus replication. By using coimmunoprecipitation combined with mass spectrometry, the components of the IE86 complex were identified, and the heterogeneous ribonucleoprotein A2/B1 (hnRNP A2/B1) was recognized as one of the IE86 complex components. hnRNP A2/B1 is highly expressed in U251 cells, and the data suggest that IE86 can promote hnRNP A2/B1 expression. Furthermore, the knockdown of hnRNP A2/B1 significantly attenuates IE86-mediated apoptosis and cell proliferation. Importantly, IE86 can also inhibit the alternative splicing of Bcl-x by decreasing the Bcl-xS/Bcl-xL ratio, which is closely related to apoptosis. Meanwhile, the knockdown of hnRNP A2/B1 can mitigate the inhibitory effect of IE86 on the alternative splicing of Bcl-x. In conclusion, the inhibition of apoptosis and enhancement of cell proliferation by IE86 may be related to the hnRNP A2/B1-mediated alternative splicing of Bcl-x.
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Recently, targeting the G protein-biased signaling has emerged as an attractive therapeutic strategy for treating severe acute pain with the potential to reduce the side effect of the traditional opioid drug. Herein, we describe the discovery of a highly potent G protein-biased µ-opioid receptor (MOR) agonist, SHR9352. This novel molecule exhibited excellent MOR activity and limited ß-arrestin recruitment, as well as a high selectivity over κ-opioid receptor and δ-opioid receptor demonstrated robust in vivo efficacy and displayed favorable pharmacokinetic properties across species.
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The Candida parapsilosis complex consists of C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis. Recently, many studies described the prevalence of this species complex mainly in invasive candidiasis. Additionally, data showed that these three species are different in virulence and in vitro drug susceptibility. However, to our knowledge, the prevalence and distribution of the species complex in superficial candidiasis is not very clear to date. In this study, 2,128 Candida isolates from specimens of superficial candidiasis were collected over a 1-year period. Combination of routine and molecular tools, a total of 214 samples were identified to be positive for the C. parapsilosis complex (10.1%), of which 198 (92.5%) were monofungal and 16 (7.5%) were polyfungal. Among the 198 monofungal isolates, 191 (96.5%) were identified as C. parapsilosis sensu stricto, 5 (2.5%) as C. metapsilosis, and 2 (1.0%) as C. orthopsilosis species based on the molecular method. All C. parapsilosis complex isolates from the 16 polyfungal populations were found to be C. parapsilosis sensu stricto. Further analysis showed that the distribution profiles of the C. parapsilosis complex in adult patients were different from that in pediatric patients, and the prevalence rate of it varied greatly by sites of isolation. This study provides insight into the epidemiology of the species complex in superficial candidiasis.
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Candida/aislamiento & purificación , Candidiasis/epidemiología , Infección Hospitalaria/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Candida/clasificación , Candida/genética , Candidiasis/microbiología , Niño , Preescolar , China/epidemiología , Infección Hospitalaria/microbiología , Femenino , Hospitales Universitarios/estadística & datos numéricos , Humanos , Masculino , Persona de Mediana Edad , Prevalencia , Adulto JovenRESUMEN
Combining results from previous studies, a multi-epitope antigen PCXZ against the hepatitis C virus was synthesized in this study. The antigenic specificity of PCXZ was determined by recognizing antibodies in serum samples from hepatitis C virus patients, but not from healthy subjects or subjects who had the hepatitis B virus. The characteristics of PCXZ immunogenicity were evaluated in BALB/c mice. Strong antibody responses were generated in mice immunized with either naked PCXZ or PCXZ in Freund's adjuvant. As for the T-cell responses, Freund's adjuvant significantly increased interferon-γ secretion and enhanced the lytic activity of cytotoxic T lymphocytes. The epitope Pa, one component of PCXZ, made the most significant contribution to specific CTL lysis; this epitope was also a B-cell epitope and was able to induce high IgG titers. In summary, PCXZ was found to be highly immunogenic, and elicited both humoral and cellular immune responses in mice.
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Antígenos Virales/inmunología , Linfocitos B/inmunología , Epítopos/inmunología , Hepacivirus/inmunología , Linfocitos T/inmunología , Vacunas Virales/inmunología , Animales , Citotoxicidad Inmunológica , Anticuerpos contra la Hepatitis C/sangre , Humanos , Interferón gamma/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Vacunas Sintéticas/inmunologíaRESUMEN
The left/right asymmetry of adult flatfishes (Pleuronectiformes) is remarkable given the external body symmetry of the larval fish. The best-known change is the migration of their eyes: one eye migrates from one side to the other. Two extinct primitive pleuronectiformes with incomplete orbital migration have again attracted public attention to the mechanism of eye migration, a subject of speculation and research for over a century. Cranial asymmetry is currently believed to be responsible for eye migration. Contrary to that hypothesis, we show here that the initial migration of the eye is caused by cell proliferation in the suborbital tissue of the blind side and that the twist of frontal bone is dependent on eye migration. The inhibition of cell proliferation in the suborbital area of the blind side by microinjected colchicine was able to prevent eye migration and, thereafter, cranial asymmetry in juvenile Solea senegalensis (right sideness, Soleidae), Cynoglossus semilaevis (left sideness, Cynoglossidae), and Paralichthys olivaceus (left sideness, Paralichthyidae) with a bottom-dwelling lifestyle. Our results correct the current misunderstanding that eye migration is driven by the cranial asymmetry and simplify the explanation for broken left/right eye-symmetry. Our findings should help to focus the search on eye migration-related genes associated with cell proliferation. Finally, a novel model is proposed in this research which provides a reasonable explanation for differences in the migrating eye between, and sometimes within, different species of flatfish and which should aid in our overall understanding of eye migration in the ontogenesis and evolution of Pleuronectiformes.
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Proliferación Celular , Ojo/crecimiento & desarrollo , Asimetría Facial/embriología , Peces Planos/crecimiento & desarrollo , Metamorfosis Biológica/fisiología , Órbita/citología , Animales , Ojo/citología , Peces Planos/anatomía & histología , Larva/crecimiento & desarrolloRESUMEN
Grass carp, Ctenopharyngodon idellus (Valenciennes, 1844), is an economically important species widely cultured in the world, but its genome research resources are largely lacking. The objectives of this study were to construct normalized cDNA libraries for efficient EST analysis, to generate ESTs from these libraries, and to identify EST-related molecular markers such as microsatellites and single nucleotide polymorphisms (SNPs) for genetic analysis of this species. A total of 6,269 ESTs were generated representing 4,815 unique sequences, from which 105 putative microsatellites and 5,228 SNPs were identified. These genome resources provide the material basis for future genetic and functional analyses in this species.
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Carpas/genética , Etiquetas de Secuencia Expresada , Biblioteca de Genes , Animales , Secuencia de Bases , Clonación Molecular , Marcadores Genéticos , Repeticiones de Microsatélite , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , ARN/química , ARN/aislamiento & purificación , Especificidad de la EspecieRESUMEN
Attenuated Salmonella typhimurium possess the ability to stimulate innate immune responses and preferentially allocate within the solid tumor. These two main characteristics make attenuated Salmonella one of the most attractive vehicles for development of vaccine and also targeted cancer therapies. However, location of Salmonella prevents the process of antigen presentation. Salmonella Type III secretion system can be utilized to circumvent this problem because this system secretes the protein it encoded outside the cells. Heat shock protein 70 (Hsp70) is referred to as an "immunochaperone" for its capacity to elicit tumor-specific adaptive immune responses in the form of Hsp70-TAA (tumor associated antigen) complex. Hsp70 facilitates the cross-presentation of exogenous antigens through its receptor on antigen-presenting cells and therefore activates an antigen-specific cytotoxic T lymphocyte (CTL) response, which can directly contribute to potent anti-tumor immunity. Here, we designed a novel therapeutic vaccine utilizing the type III secretion system and Hsp70 to deliver and present the tumor-specific antigen. This live recombinant bacteria vaccine, when administrated orally, successfully broke the immune tolerance, induced a specific CTL response against tumor cells, and therefore revealed protective and therapeutic effects against generation and growth of B16F10 melanoma in C57BL/6J mice.
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Antígenos de Neoplasias/inmunología , Proteínas Bacterianas/uso terapéutico , Vacunas contra el Cáncer/inmunología , Proteínas HSP70 de Choque Térmico/uso terapéutico , Melanoma Experimental/tratamiento farmacológico , Proteínas de la Membrana/uso terapéutico , Fragmentos de Péptidos/uso terapéutico , Animales , Presentación de Antígeno/inmunología , Proteínas Bacterianas/inmunología , Sistemas de Secreción Bacterianos/inmunología , Separación Celular , Reactividad Cruzada/inmunología , Citometría de Flujo , Vectores Genéticos , Proteínas HSP70 de Choque Térmico/inmunología , Activación de Linfocitos/inmunología , Masculino , Melanoma Experimental/inmunología , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/inmunología , Salmonella typhimurium/inmunología , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/uso terapéuticoRESUMEN
Genes encoding purine nucleoside phosphorylase (deo D), uridine phosphorylase (udp) and thimidine phosphorylase (deo A) from Escherichia coli BL21 were cloned and overexpressed in E. coli DH5alpha. The recombinant strains were employed to synthesize 2'-deoxyadenosine (dAR) and 6-methylpurine-2'-deoxyriboside (MePdR). Experimental parameters such as strains, temperature, pH, reagent concentration and cell mass were optimized. Under the optimal situation, 96% adenine was converted to dAR and 95% 6-methylpurine (MeP) was converted to MePdR in an hour, using 0.2 per thousand (dry wt./v) cell paste as biocatalyst.
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Desoxiadenosinas/biosíntesis , Escherichia coli/fisiología , Pentosiltransferasa/metabolismo , Ingeniería de Proteínas/métodos , Nucleósidos de Purina/biosíntesis , Pentosiltransferasa/genética , Proteínas Recombinantes/metabolismoRESUMEN
Puffer fish (Takifugu obscurus) from the Yangtze River of China were screened for tetrodotoxin-producing bacteria. An Aeromonas strain was isolated from the ovary of the puffer fish and was shown to produce tetrodotoxin; this strain was denoted Ne-1. The identity of tetrodotoxin produced by strain Ne-1 was confirmed by mouse bioassay, high performance liquid chromatography coupled with mass chromatography (LC-MS), and ELISA. Strain Ne-1 was characterized morphologically, biochemically, and by 16S rDNA phylogenetic analysis; these analyses suggested that strain Ne-1 is closely related to Aeromonas molluscorum. Given that strain Ne-1 was isolated from the ovary of T. obscurus, we propose that the TTX-producing Aeromonas sp. is a parasite or symbiotic bacterium rather than a sample contaminant. Collectively, our studies suggest that Aeromonas sp. strain Ne-1 produces tetrodotoxin in T. obscurus.