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The coculture of patient-derived tumor organoids (PDOs) and autologous immune cells has been considered as a useful ex vivo surrogate of in vivo tumor-immune environment. However, the immune interactions between PDOs and autologous immune cells, including immune-mediated killing behaviors and immune-related cytokine variations, have yet to be quantitatively evaluated. This study presents a microfluidic chip for quantifying interactions between PDOs and autologous immune cells (IOI-Chip). A baffle-well structure is designed to ensure efficient trapping, long-term coculturing, and in situ fluorescent observation of a limited amount of precious PDOS and autologous immune cells, while a microbeads-based immunofluorescence assay is designed to simultaneously quantify multiple kinds of immune-related cytokines in situ. The PDO apoptosis and 2 main immune-related cytokines, TNF-α and IFN-γ, are simultaneously quantified using samples from a lung cancer patient. This study provides, for the first time, a capability to quantify interactions between PDOs and autologous immune cells at 2 levels, the immune-mediated killing behavior, and multiple immune-related cytokines, laying the technical foundation of ex vivo assessment of patient immune response.
Assuntos
Dispositivos Lab-On-A-Chip , Organoides , Humanos , Organoides/imunologia , Organoides/citologia , Organoides/metabolismo , Interferon gama/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/imunologia , Citocinas/metabolismo , Técnicas de Cocultura , Apoptose , Técnicas Analíticas Microfluídicas/instrumentaçãoRESUMO
Protein glycosylation plays a crucial role in central nervous system, and abnormal glycosylation has major implications for human diseases. This study aims to evaluate an etiological implication of the variation in glycosylation for Parkinson's disease (PD), a neurodegenerative disorder. Based on a PD mouse model constructed by the intraperitoneal injection with 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine, glycosylation variation was accessed using biotinylated lectin of dolichos biflorus agglutinin (DBA) specific for the exposed N-acetylgalactosamine linked to glycoprotein. Consequently, a glycoprotein with a significantly reduced N-acetylgalactosamination was identified as ADP/ATP translocase 1 (ANT1) by lectin affinity chromatography coupled with MALDI-TOF MS/MS (mass spectrometry), and confirmed by the analysis of dual co-immunofluorescence and Western blot. A tissue-specific distribution of de-N-acetylgalactosaminated ANT1 was found to be correlated with high risk of PD. At cellular level, an obvious co-aggregation between ANT1 and DBA was only found in the MPP+-induced PD-like cell model using dual co-immunofluorescence. Thus, we found that ANT1 was a potential glycoprotein with terminal N-acetylgalactosamine moiety, and the variation of glycosylation in ANT1 was associated with PD. This investigation provides an innovative insight in protein glycosylation with PD pathogenesis.
Assuntos
Doença de Parkinson , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Acetilgalactosamina , Translocador 1 do Nucleotídeo Adenina , Difosfato de Adenosina/metabolismo , Animais , Glicoproteínas/metabolismo , Camundongos , Translocases Mitocondriais de ADP e ATP/metabolismo , Doença de Parkinson/metabolismo , Espectrometria de Massas em TandemRESUMO
(1) Background: d-alanine-d-alanine ligase (DdlA), an effective target for drug development to combat against Mycobacterium tuberculosis (Mtb), which threatens human health globally, supplies a substrate of d-alanyl-d-alanine for peptidoglycan crosslinking by catalyzing the dimerization of two d-alanines. To obtain a better understanding of DdlA profiles and develop a colorimetric assay for high-throughput inhibitor screening, we focused on explicating and characterizing Tb-DdlA. (2) Methods and Results: Rv2981c (ddlA) was expressed in Escherichia coli, and the purified Tb-DdlA was identified using (anti)-polyhistidine antibody followed by DdlA activity confirmation by measuring the released orthophosphate via colorimetric assay and the yielded d-alanyl-d-alanine through high performance thin layer chromatography (HP-TLC). The kinetic assays on Tb-DdlA indicated that Tb-DdlA exhibited a higher affinity to ATP (KmATP: 50.327 ± 4.652 µmol/L) than alanine (KmAla: 1.011 ± 0.094 mmol/L). A colorimetric assay for Tb-DdlA activity was developed for high-throughput screening of DdlA inhibitors in this study. In addition, we presented an analysis on Tb-DdlA interaction partners by pull-down assay and MS/MS. Eight putative interaction partners of Tb-DdlA were identified. (3) Conclusions: Our dataset provided a valuable resource for exploring Tb-DdlA biology, and developed an easy colorimetric assay for screening of Tb-DdlA inhibitors.
Assuntos
Trifosfato de Adenosina/metabolismo , Alanina/metabolismo , Proteínas de Bactérias/metabolismo , Dipeptídeos/metabolismo , Mycobacterium tuberculosis/enzimologia , Peptídeo Sintases/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Bioensaio , Clonagem Molecular , Inibidores Enzimáticos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Mycobacterium tuberculosis/genética , Peptídeo Sintases/antagonistas & inibidores , Peptídeo Sintases/genética , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Polypeptide N-acetylgalactosamine transferase 9 (GALNT9) catalyzes the initial step of mucin-type O-glycosylation via linking N-acetylgalactosamine (GalNAc) to serine/threonine in a protein. To unravel the association of GALNT9 with Parkinson's disease (PD), a progressive neurodegenerative disorder, GALNT9 levels were evaluated in the patients with PD and mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and statistically analyzed based on the GEO datasets of GSE114918 and GSE216281. Glycoproteins with exposing GalNAc were purified using lectin affinity chromatography and identified by LC-MS/MS. The influence of GALNT9 on cells was evaluated via introducing a GALNT9-specific siRNA into SH-SY5Y cells. Consequently, GALNT9 deficiency was found to occur under PD conditions. GALNT9 silencing contributed to a causative factor in PD pathogenesis via reducing the levels of intracellular dopamine, tyrosine hydroxylase and soluble α-synuclein, and promoting α-synuclein aggregates. MS identification revealed 14 glycoproteins. 5 glycoproteins, including ACO2, ATP5B, CKB, CKMT1A, ALDOC, were associated with energy metabolism. GALNT9 silencing resulted in mitochondrial dysfunctions via increasing ROS accumulation, mitochondrial membrane depolarization, mPTPs opening, Ca2+ releasing and activation of the CytC-related apoptotic pathway. The dysfunctional mitochondria then triggered mitophagy, possibly intermediated by adenine nucleotide translocase 1. Our study suggests that GALNT9 is potentially developed into an auxiliary diagnostic index and therapeutic target of PD.
Assuntos
Doenças Mitocondriais , N-Acetilgalactosaminiltransferases , Neuroblastoma , Doença de Parkinson , Humanos , Camundongos , Animais , Doença de Parkinson/metabolismo , alfa-Sinucleína/química , Acetilgalactosamina/química , Transferases , Cromatografia Líquida , Espectrometria de Massas em Tandem , Peptídeos , Glicoproteínas , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Creatina QuinaseRESUMO
BACKGROUND: The emerging concept is that microRNAs (miRNAs) play a central role in controlling stem cell self-renewal and fate determination by regulating the expression of stem cell regulators. miR-125b, one of neuronal miRNAs, recently was found to be necessary for neural differentiation of neural stem/progenitor cells (NS/PCs). However, the other specific biological role of miR-125b in NS/PCs is little known. We used rat NS/PCs as a model system to study the role of miR-125b in governing the behavior of NS/PCs. RESULTS: We report here the transfection of exogenous miR-125b inhibited proliferation of NS/PCs but promoted differentiation and migration. Whereas anti-miR-125b had the opposite effect. Similar results were observed when Nestin was knocked down by siRNA. Subsequently, we demonstrated that Nestin was a direct functional target of miR-125b. MiR-125b downregulates the expression of luciferase through Nestin 3'untranslated region (3'-UTR), and the regulation was abolished by mutations in the miR-125b binding site. MiR-125b targeted the 3'-UTR of Nestin and reduced the abundance of Nestin at both mRNA and protein levels. CONCLUSION: The results provided new insight into the function by which miR-125b modulates NS/PCs proliferation, differentiation and migration. The data also indicated the regulatory role of miR-125b in NS/PCs might through the suppression of Nestin expression.
Assuntos
Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células , Proteínas de Filamentos Intermediários/metabolismo , MicroRNAs/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Bromodesoxiuridina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Hipocampo/citologia , MicroRNAs/antagonistas & inibidores , Mutação/genética , Proteínas do Tecido Nervoso/genética , Nestina , Células-Tronco Neurais/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , TransfecçãoRESUMO
Biochemistry, a complicated basic course in health sciences, plays a fundamental role in describing pathogenic mechanism of illness in molecular terms, and is required to be learned for all medical students. Due to various obstacles to biochemistry learning and teaching, there is a pressing issue of curriculum reform to arouse the student's enthusiasm in biochemistry learning. In this study, an integrated topic-based biochemistry training program (TBBTP) combining the traditional lectures, question-based learning and experimental practices, was introduced in biochemistry teaching. Its effectiveness was evaluated through examination and questionnaire analyses. Consequently, we found that TBBTP promoted the dissemination of biochemical knowledge via traditional lecture, designated learning issues and experimental practices, and acquisition of various skills through practical operation, presentation, and independent learning. It motivated students to study biochemistry with intense interest and enthusiasm. This study explored the feasibility of the topic-based biochemistry teaching as a supplement to biochemistry curriculum in medical education and as a mainstream pedagogy in biochemistry lab.
Assuntos
Educação de Graduação em Medicina , Estudantes de Medicina , Humanos , Currículo , Bioquímica/educação , Aprendizagem , EnsinoRESUMO
BACKGROUND: ADP/ATP translocase 1 (ANT1) is involved in the exchange of cytosolic ADP and mitochondrial ATP, and its defection plays an important role in mitochondrial pathogenesis. To reveal an etiological implication of ANT1 for Parkinson's disease (PD), a neurodegenerative disorder, a mouse model treated with 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine and neuroblastoma cell model induced by 1-methyl-4-pehny1-pyridine were utilized in this study. RESULTS: The tissue-specific abundance in ANT1 in mouse brains was accessed using the analysis of Western blot and immunohistochemistry. Down-regulated soluble ANT1 was found to be correlated with PD, and ANT1 was associated with PD pathogenesis via forming protein aggregates with α-synuclein. This finding was confirmed at cellular level using neuroblastoma cell models. ANT1 supplement in neuronal cells revealed the protective roles of ANT1 against cytotoxicity caused by MPP+. Protein interaction assay, coupled with the analysis of LC-MS/MS, silver-stained SDS-PAGE and Western blot against anti-ANT1 antibody respectively, illustrated the interaction of ANT1 with α-synuclein using the expressed α-synuclein as a bite. Additionally, a significant increasing ROSs was detected in the MPP+-treated cells. CONCLUSIONS: This study indicated that ANT1 was a potentially causative factor of PD, and led to neuropathogenic injury via promoting the formation of protein aggregates with α-synuclein. This investigation potentially promotes an innovative understanding of ANT1 on the etiology of PD and provides valuable information on developing potential drug targets in PD treatment or reliable biomarkers in PD prognostication.
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During early embryogenesis, mammary glands are derived from surface ectoderm and their morphogenesis is controlled by mammary stem cells (MaSCs) and epithelial-mesenchymal transition (EMT). Mammary anlagen stage (E13.5-15.5) is an important stage for fetal mice to achieve EMT dependent mammary morphogenesis. And the characteristics of mammary anlagen repopulating cell population (MaRC) should be identified for understanding its stemness at earlier embryonic stage. Here we quantify and characterize MaSCs proportion at mammary anlagen stage. Compared with adult mouse mammary gland, our data revealed that E14.5 mammary anlagen exhibit higher stem cell activities. Then we purified mammary anlagen cell populations depending on the expression levels of CD24 and CD49f in mouse mammary anlagen, and identified an unique MaRC population (Lin-CD24medCD49f+) by real-time PCR, transplantation and mammosphere forming assays. In addition, by comparing with adult MaSC (Lin-CD24+CD29hi) and differentiated mammary anlagen cells, we find that E14.5 mouse MaRC population exhibit gene expression programs related to mesenchymal properties. To further identify the cell types of E14.5 mouse MaRC population, the expressions of K8, K14, K18, e-cadherin, n-cadherin and vimentin in mammary anlagen Lin-CD24medCD49f + cells were detected by immunofluorescence assay. These findings verified that the undifferentiated E14.5 mouse MaRC population is a heterogeneous population with mesenchymal property, which is associated with cell stemness and mammary duct morphogenesis.
Assuntos
Autorrenovação Celular , Glândulas Mamárias Humanas/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Embrionárias Murinas/citologia , Animais , Antígeno CD24/genética , Antígeno CD24/metabolismo , Caderinas/genética , Caderinas/metabolismo , Células Cultivadas , Transição Epitelial-Mesenquimal , Feminino , Humanos , Integrina alfa6/genética , Integrina alfa6/metabolismo , Glândulas Mamárias Humanas/embriologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/fisiologiaRESUMO
Mycobacterium tuberculosis, a causative pathogen of tuberculosis (TB), still threatens human health worldwide. To find a novel drug to eradicate this pathogen, we tested taurine-5- bromosalicylaldehyde Schiff base (TBSSB) as an innovative anti-mycobacterial drug using Mycobacterium smegmatis as a surrogate model for M. tuberculosis. We investigated the antimicrobial activity of TBSSB against M. smegmatis by plotting growth curves, examined the effect of TBSSB on biofilm formation, observed morphological changes by scanning electron microscopy and transmission electron microscopy, and detected differentially expressed proteins using two-dimensional gel electrophoresis coupled with mass spectrometry. TBSSB inhibited mycobacterial growth and biofilm formation, altered cell ultrastructure and intracellular content, and inhibited cell division. Furthermore, M. smegmatis adapted itself to TBSSB inhibition by regulating the metabolic pathways and enzymatic activities of the identified proteins. NDMA-dependent methanol dehydrogenase, NAD(P)H nitroreductase, and amidohydrolase AmiB1 appear to be pivotal factors to regulate the M. smegmatis survival under TBSSB. Our dataset reinforced the idea that Schiff base-taurine compounds have the potential to be developed as novel anti-mycobacterial drugs.
Assuntos
Aldeídos/farmacologia , Antibacterianos/farmacologia , Mycobacterium smegmatis/efeitos dos fármacos , Proteômica , Bases de Schiff/farmacologia , Taurina/análogos & derivados , Proteínas de Bactérias/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Parede Celular/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/ultraestrutura , Mycobacterium tuberculosis , Taurina/farmacologiaRESUMO
Functionally uncharacterized gene Rv3627c is predicted to encode a carboxypeptidase in the pathogen of Mycobacterium tuberculosis (M. tuberculosis), which remains a major threat to human health. Here, we sought to reveal the function of Rv3627c and to elucidate its effects on mycobacterial growth. Rv3627c was purified from E. coli using Ni2+-NTA affinity chromatography, and its identity was confirmed with a monoclonal anti-polyhistidine antibody. An enzyme activity assay involving a d-amino acid oxidase-peroxidase coupled colorimetric reaction and high-performance thin layer chromatography was performed. A pull-down assay and MS-MS were also employed to identify putative interaction partners of Rv3627c. Scanning electron microscopy and transmission electron microscopy were performed to observe any morphological alterations to Mycobacterium smegmatis (M. smegmatis). We successfully obtained soluble expressed Rv3627c and identified it as carboxypeptidase using prepared peptidoglycan. Four proteins were identified as potential interaction partners with Rv3627c based on results obtained from both a pull-down assay and MS/MS analysis. Rv3627c over-expression induced M. smegmatis cells to become elongated, and promoted the formation of increased numbers of Z-rings. Rv3627c, a novel carboxypeptidase in M. tuberculosis identified in this study, exerts important effects on mycobacterial cell morphology and cell division. This functional information provides a promising insight into anti-mycobacterial target designs.
Assuntos
Proteínas de Bactérias/metabolismo , Carboxipeptidases/metabolismo , Divisão Celular , Mycobacterium smegmatis/citologia , Mycobacterium smegmatis/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Carboxipeptidases/genética , Clonagem Molecular , Dipeptídeos/metabolismo , Humanos , Mapas de Interação de Proteínas , Homologia de SequênciaRESUMO
D-Alanyl-D-alanine ligase A (DdlA) catalyses the dimerization of two D-alanines yielding D-alanyl-D-alanine required for mycobacterial peptidoglycan biosynthesis, and is a promising antimycobacterial drug target. To better understand the roles of DdlA in mycobacteria in vivo, we established a cell model in which DdlA expression was specifically downregulated by ddlA antisense RNA by introducing a 380 bp ddlA fragment into pMind followed by transforming the construct into nonpathogenic Mycobacterium smegmatis. The M. smegmatis cell model was verified by plotting the growth inhibition curves and quantifying endogenous DdlA expression using a polyclonal anti-DdlA antibody produced from the expressed DdlA. Scanning electron microscopy and transmission electron microscopy were used to investigate mycobacterial morphology. Bidimensional gel electrophoresis and mass spectrometry were used to analyze differentially expressed proteins. Consequently, the successful construction of the M. smegmatis cell model was verified. The morphological investigation of the model indicated that DdlA deficiency led to an increased number of Z rings and a rearrangement of intracellular content, including a clear nucleoid and visible filamentous DNA. Proteomic techniques identified six upregulated and 14 downregulated proteins that interacted with each other to permit cell survival by forming a regulatory network under DdlA deficiency. Finally, our data revealed that DdlA deficiency inhibited cell division in mycobacteria and attenuated the process of carbohydrate catabolism and the pathway of fatty acid anabolism, while maintaining active protein degradation and synthesis. N-Nitrosodimethylamine (NDMA)-dependent methanol dehydrogenase (MSMEG_6242) and fumonisin (MSMEG_1419) were identified as potential antimycobacterial drug targets.
Assuntos
Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/crescimento & desenvolvimento , Peptídeo Sintases/deficiência , Proteoma/análise , Eletroforese em Gel Bidimensional , Técnicas de Silenciamento de Genes , Espectrometria de Massas , Microscopia Eletroquímica de Varredura , Microscopia Eletrônica de Transmissão , Mycobacterium smegmatis/química , Mycobacterium smegmatis/ultraestrutura , Peptídeo Sintases/metabolismo , VirulênciaRESUMO
Aberrant glycosylation of proteins has major implications for human diseases. To determine whether protein glycosylation contributes to the pathogenesis of Parkinson's disease (PD), a mouse model of PD was established by injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Induction of PD-like features was verified by assessing motor impairment and confirming reductions in biological markers, including dopamine, 5-hydroxytryptamine and tyrosine hydroxylase, as well as the aggregation of α-synuclein. Altered glycosylation was detected using biotinylated agaracus bisporus lectin, which specifically binds exposed Gal-(ß-1,3)-GalNAc linked to glycoproteins. Subsequent lectin affinity chromatography coupled with mass spectrometry revealed enhanced glycosylation of microtubule-associated protein 6 (MAP6) in PD mice as compared to healthy controls. In situ dual co-immunofluorescence analysis and immunoblotting confirmed that MAP6 is glycosylated with Gal-(ß-1,3)-GalNAc oligosaccharides, which in turn alters the distribution and structure of MAP6 complexes within neurons. This is the first study to described MAP6 as a glycoprotein containing Gal-(ß-1,3)-GalNAc oligosaccharides and to show that hyperglycosylation of MAP6 is strongly associated with the pathogenesis of PD. These findings provide potentially valuable information for developing new therapeutic targets for the treatment of PD as well as reliably prognostic biomarkers.
Assuntos
Galectinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Oligossacarídeos/metabolismo , Doença de Parkinson/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Animais , Cromatografia de Afinidade , Modelos Animais de Doenças , Imunofluorescência , Glicoproteínas/metabolismo , Glicosilação , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doença de Parkinson/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The complete mitogenome of the smalleye pygmy shark Squaliolus aliae (Squaliformes: Dalatiidae) is presented in this study firstly. It is 16,717 bp with a nucleotide base composition of 30.7% A, 26.2% C, 14.7% G and 28.5% T, containing 37 genes and a control region with typical order as vertebrate. There are 23bp short intergenic spaces and 28bp overlaps locating in gene junctions. 2 rRNA genes are located in heavy strand. Twenty-two tRNA genes range from 67bp (tRNA-Cys) to 75 bp (tRNA-Leu1). Two initial codons (ATG and GTG) and two terminal codons (TAA and T) are found in protein-coding genes. The phylogenetic result shows that S. aliae in this study is clustered to the (Somniosus pacificus + Squalidae).
RESUMO
In this study, the complete mitochondrial genome of the Borneo leg skate Sinobatis borneensis (Rajiformes, Anacanthobatidae) was determined. It had circular molecules (16,701 bp), consisting of 37 genes with a typical gene order in vertebrate mitogenome. In the whole mitogenome, there were 28 bp short intergenic and 31 bp overlaps, respectively, located in 12 and 7 gene junctions. The nucleotide composition was 31.1% A, 26.0% C, 13.9% G and 29.1% T. Two start codons (GTG and ATG) and two stop codons (TAG, TAA/T) were used in the protein-coding genes. The 22 tRNA genes ranged from 66 bp (tRNA-Cys) to 75 bp (tRNA-Leu1 and tRNA-Lys). The phylogenetic result showed that S. borneensis was clustered with the Atlantoraja castelnaui and Pavoraja nitida.
RESUMO
In this study, we presented the complete mitochondrial genome of the Sharpnose stingray Himantura gerrardi for the first time, which was 17,685 bp in length and contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a putative control region. The overall nucleotide composition was 30.1% A, 27.5% C, 14.5% G and 28.0% T. A total of 22 bp overlaps and 83 bp short intergenic spaces were found in the mitogenome. Two start codons (ATG and GTG) and two stop codons (TAG and TAA/T) were used in the protein-coding genes. The origin of L-strand replication (OL) sequence formed a hairpin structure between the tRNA-Asn and tRNA-Cys genes. The termination associated sequence (TAS) formed a hairpin structure near the tRNA-Pro in the control region.
Assuntos
Ordem dos Genes , Genes Mitocondriais , Genoma Mitocondrial , Rajidae/genética , Animais , Composição de Bases , Códon de Iniciação , Códon de Terminação , Evolução Molecular , Tamanho do Genoma , Análise de Sequência de DNARESUMO
In this study, the mitogenome of the blackspotted catshark Halaelurus burgeri was first determined. This circle molecular was rich in A (30.7%)+T (30.4%), poor in C (25.0%)+G (13.8%) and contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a longest control region (3481 bp with lots of repeated motifs) in sharks. Two start codons (ATG and GTG) and two stop codons (TAG and TAA/T) were found in the protein-coding genes. The 22 tRNA genes ranged from 66 bp (tRNA-Ser2) to 75 bp (tRNA-Leu1). The phylogenetic result showed that H. burgeri did not cluster to the two species of Scyliorhinidae (Cephaloscyllium umbratile and Scyliorhinus canicula).
RESUMO
We determined the complete mitochondrial genome of the Jenkins whipray Himantura jenkinsii. The total length of the mitogenome was 17, 670 bp, consisted of 37 genes with typical gene order in vertebrate mitogenome. The nucleotide composition was: 30.5% A, 29.1% T, 26.5% C and 13.9% G. It had 70 bp short intergenic spaces and 22 bp overlaps. Two start codons (GTG and ATG) and two stop codons (TAG and TAA/T) were used in the protein-coding genes. The 22 tRNA genes were ranged from 67 bp (tRNA-Ser2) to 75 bp (tRNA-Leu1). The phylogenetic result showed that H. jenkinsii was clustered with the Hortle's whipray H. hortlei.
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We first determined the complete mitochondrial genome of the White-spotted guitarfish Rhynchobatus australiae (Rajiformes, Rhinobatidae). The complete mitogenome was 16,804 bp, with a base composition of 32.3% A, 27.5% T, 26.9% C and 13.3% G, containing 13 protein-coding genes, two rRNAs, 22 tRNAs and a control region (D-loop). It had 35 bp short intergenic spaces and 39 bp overlaps between genes. The gene order and composition of R. australiae was similar to most other fishes. The codon usage followed the typical vertebrate mitochondrial pattern (ATG or GTG for start codon and TAA or T for stop codon). The phylogenetic result showed that R. australiae was clustered with the Rhinobatos.
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Previous studies have demonstrated that several mechanisms, including numerous inhibitory molecules, weak neurotrophic stimulation and deficient intrinsic regenerative responses, collectively contribute to the failure of mature spinal cord axon regeneration. Thus, combinatorial therapies targeting multiple mechanisms have attracted much attention. In the present study, a porous collagen scaffold was used to support neuronal attachment and bridge axonal regeneration. The scaffold was specifically functionalized using neutralizing proteins (CBD-EphA4LBD, CBD-PlexinB1LBD and NEP1-40) and collagen-binding neurotrophic factors (CBD-BDNF and CBD-NT3) to simultaneously antagonize myelin inhibitory molecules (ephrinB3, Sema4D and Nogo) and exert neurotrophic protection and stimulation. Cerebellar granular neurons cultured on the functionalized collagen scaffold promoted neurite outgrowth in the presence of myelin. Furthermore, a full combinatorial treatment comprising functionalized scaffold implantation and cAMP administration was developed to evaluate the synergistic repair ability in a rat T10 complete removal spinal cord injury model. The results showed that full combinatorial therapy exhibited the greatest advantage in reducing the volume of cavitation, facilitating axonal regeneration, and promoting neuronal generation. The newborn neurons generated in the lesion area could form the neuronal relay and enhance the locomotion recovery after severe spinal cord injury. STATEMENT OF SIGNIFICANCE: A porous collagen scaffold was specifically functionalized with neutralizing proteins and neurotrophic factors to antagonize the myelin inhibitory molecules and exert neurotrophic protection and stimulation for spinal cord regeneration. Cerebellar granular neurons seeded on the functionalized collagen scaffold showed enhanced neurite outgrowth ability in vitro. The functionalized scaffold implantation combined with cAMP administration exhibited synergistic repair ability for rat T10 complete spinal cord transection injury.
Assuntos
Colágeno , Traumatismos da Medula Espinal/terapia , Regeneração da Medula Espinal/efeitos dos fármacos , Alicerces Teciduais/química , Animais , Colágeno/química , Colágeno/farmacologia , AMP Cíclico/química , AMP Cíclico/farmacologia , Modelos Animais de Doenças , Humanos , Ratos , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
The conserved sequences of the mouse histamine H3 receptor at the potential alternative splice junctions suggest that the splice isoforms found in guinea pig, rat, human, and hamster may also be present in the mouse. However, the trace amount isoforms are hard to be detected by the regular PCR approach. In this paper, we report a method in which the unspliced long isoform is cut by restriction endonuclease so that the short isoforms can be amplified to detectable levels to confirm the existence of the splice isoforms of H3 receptor mRNA in the mouse. This method is applicable to the detection of trace amounts of splice isoforms that coexist with the long, more abundant isoforms.