RESUMO
COVID-19 has spread worldwide since 2019 and is now a severe threat to public health. We previously identified the causative agent as a novel SARS-related coronavirus (SARS-CoV-2) that uses human angiotensin-converting enzyme 2 (hACE2) as the entry receptor. Here, we successfully developed a SARS-CoV-2 hACE2 transgenic mouse (HFH4-hACE2 in C3B6 mice) infection model. The infected mice generated typical interstitial pneumonia and pathology that were similar to those of COVID-19 patients. Viral quantification revealed the lungs as the major site of infection, although viral RNA could also be found in the eye, heart, and brain in some mice. Virus identical to SARS-CoV-2 in full-genome sequences was isolated from the infected lung and brain tissues. Last, we showed that pre-exposure to SARS-CoV-2 could protect mice from severe pneumonia. Our results show that the hACE2 mouse would be a valuable tool for testing potential vaccines and therapeutics.
Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/patologia , Modelos Animais de Doenças , Camundongos Transgênicos , Pneumonia Viral/patologia , Enzima de Conversão de Angiotensina 2 , Animais , COVID-19 , Feminino , Humanos , Doenças Pulmonares Intersticiais/patologia , Doenças Pulmonares Intersticiais/virologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/genética , Pandemias , Peptidil Dipeptidase A/genética , SARS-CoV-2 , Tropismo Viral , Redução de PesoRESUMO
Since the outbreak of severe acute respiratory syndrome (SARS) 18 years ago, a large number of SARS-related coronaviruses (SARSr-CoVs) have been discovered in their natural reservoir host, bats1-4. Previous studies have shown that some bat SARSr-CoVs have the potential to infect humans5-7. Here we report the identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China. The epidemic, which started on 12 December 2019, had caused 2,794 laboratory-confirmed infections including 80 deaths by 26 January 2020. Full-length genome sequences were obtained from five patients at an early stage of the outbreak. The sequences are almost identical and share 79.6% sequence identity to SARS-CoV. Furthermore, we show that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus. Pairwise protein sequence analysis of seven conserved non-structural proteins domains show that this virus belongs to the species of SARSr-CoV. In addition, 2019-nCoV virus isolated from the bronchoalveolar lavage fluid of a critically ill patient could be neutralized by sera from several patients. Notably, we confirmed that 2019-nCoV uses the same cell entry receptor-angiotensin converting enzyme II (ACE2)-as SARS-CoV.
Assuntos
Betacoronavirus/classificação , Betacoronavirus/genética , Quirópteros/virologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Surtos de Doenças , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Antivirais/sangue , Betacoronavirus/metabolismo , Betacoronavirus/ultraestrutura , COVID-19 , Linhagem Celular , China/epidemiologia , Chlorocebus aethiops , Feminino , Genoma Viral/genética , Humanos , Masculino , Peptidil Dipeptidase A/metabolismo , Filogenia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/classificação , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , SARS-CoV-2 , Homologia de Sequência do Ácido Nucleico , Síndrome Respiratória Aguda Grave , Células VeroRESUMO
Electrochemistry that empowers innovative nanoscopic analysis has long been pursued. Here, the concept of aggregation-enabled electrochemistry (AEE) in a confined nanopore is proposed and devised by reactive oxygen species (ROS)-responsive aggregation of CdS quantum dots (QDs) within a functional nanopipette. Complementary Faradaic and non-Faradaic operations of the CdS QDs aggregate could be conducted to simultaneously induce the signal-on of the photocurrents and the signal-off of the ionic signals. Such a rationale permits the cross-checking of the mutually corroborated signals and thus delivers more reliable results for single-cell ROS analysis. Combined with the rich biomatter-light interplay, the concept of AEE can be extended to other stimuli-responsive aggregations for electrochemical innovations.
RESUMO
While the spike proteins from severe acute respiratory syndrome coronaviruses-1 and 2 (SARS-CoV and SARS-CoV-2) bind to host angiotensin-converting enzyme 2 (ACE2) to infect cells, the majority of bat sarbecoviruses cannot use ACE2 from any species. Despite their discovery almost 20 years ago, ACE2-independent sarbecoviruses have never been isolated from field samples, leading to the assumption these viruses pose little risk to humans. We have previously shown how spike proteins from a small group of ACE2-independent bat sarbecoviruses may possess the ability to infect human cells in the presence of exogenous trypsin. Here, we adapted our earlier findings into a virus isolation protocol and recovered two new ACE2-dependent viruses, RsYN2012 and RsYN2016A, as well as an ACE2-independent virus, RsHuB2019A. Although our stocks of RsHuB2019A rapidly acquired a tissue-culture adaption that rendered the spike protein resistant to trypsin, trypsin was still required for viral entry, suggesting limitations on the exogenous entry factors that support bat sarbecoviruses. Electron microscopy revealed that ACE2-independent sarbecoviruses have a prominent spike corona and share similar morphology to other coronaviruses. Our findings demonstrate a broader zoonotic threat posed by sarbecoviruses and shed light on the intricacies of coronavirus isolation and propagation in vitro. IMPORTANCE Several coronaviruses have been transmitted from animals to people, and 20 years of virus discovery studies have uncovered thousands of new coronavirus sequences in nature. Most of the animal-derived sarbecoviruses have never been isolated in culture due to cell incompatibilities and a poor understanding of the in vitro requirements for their propagation. Here, we built on our growing body of work characterizing viral entry mechanisms of bat sarbecoviruses in human cells and have developed a virus isolation protocol that allows for the exploration of these understudied viruses. Our protocol is robust and practical, leading to successful isolation of more sarbecoviruses than previous approaches and from field samples that had been collected over a 10-year longitudinal study.
Assuntos
Enzima de Conversão de Angiotensina 2 , Betacoronavirus , Quirópteros , Receptores Virais , Animais , Humanos , Enzima de Conversão de Angiotensina 2/metabolismo , Quirópteros/virologia , População do Leste Asiático , Estudos Longitudinais , Receptores Virais/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Tripsina , Betacoronavirus/isolamento & purificação , ZoonosesRESUMO
It has been shown that prostaglandin (PG) E2 synthesized in the lateral parabrachial nucleus (LPBN) is involved in lipopolysaccharide-induced fever. But the neural mechanisms of how intra-LPBN PGE2 induces fever remain unclear. In this study, we investigated whether the LPBN-preoptic area (POA) pathway, the thermoafferent pathway for feed-forward thermoregulatory responses, mediates fever induced by intra-LPBN PGE2 in male rats. The core temperature (Tcore) was monitored using a temperature radiotelemetry transponder implanted in rat abdomen. We showed that microinjection of PGE2 (0.28 nmol) into the LPBN significantly enhanced the density of c-Fos-positive neurons in the median preoptic area (MnPO). The chemical lesioning of MnPO with ibotenate or selective genetic lesioning or inhibition of the LPBN-MnPO pathway significantly attenuated fever induced by intra-LPBN injection of PGE2. We demonstrated that EP3 receptor was a pivotal receptor for PGE2-induced fever, since microinjection of EP3 receptor agonist sulprostone (0.2 nmol) or EP3 receptor antagonist L-798106 (2 nmol) into the LPBN mimicked or weakened the pyrogenic action of LPBN PGE2, respectively, but this was not the case for EP4 and EP1 receptors. Whole-cell recording from acute LPBN slices revealed that the majority of MnPO-projecting neurons originating from the external lateral (el) and dorsal (d) LPBN were excited and inhibited, respectively, by PGE2 perfusion, initiating heat-gain and heat-loss mechanisms. The amplitude but not the frequency of spontaneous and miniature glutamatergic excitatory postsynaptic currents (sEPSCs and mEPSCs) in MnPO-projecting LPBel neurons increased after perfusion with PGE2; whereas the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) and the A-type potassium (IA) current density did not change. In MnPO-projecting LPBd neurons, neither sEPSCs nor sIPSCs responded to PGE2; however, the IA current density was significantly increased by PGE2 perfusion. These electrophysiological responses and the thermoeffector reactions to intra-LPBN PGE2 injection, including increased brown adipose tissue thermogenesis, shivering, and decreased heat dissipation, were all abolished by L-798106, and mimicked by sulprostone. These results suggest that the pyrogenic effects of intra-LPBN PGE2 are mediated by both the inhibition of the LPBd-POA pathway through the EP3 receptor-mediated activation of IA currents and the activation of the LPBel-POA pathway through the selective enhancement of glutamatergic synaptic transmission via EP3 receptors.
Assuntos
Regulação da Temperatura Corporal , Dinoprostona , Febre , Núcleos Parabraquiais , Área Pré-Óptica , Receptores de Prostaglandina E Subtipo EP3 , Animais , Masculino , Ratos , Regulação da Temperatura Corporal/efeitos dos fármacos , Dinoprostona/farmacologia , Febre/induzido quimicamente , Febre/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Núcleos Parabraquiais/efeitos dos fármacos , Núcleos Parabraquiais/fisiologia , Área Pré-Óptica/efeitos dos fármacos , Área Pré-Óptica/metabolismo , Ratos Sprague-Dawley , Receptores de Prostaglandina E Subtipo EP3/metabolismoRESUMO
The interest in cluster chemistry lies not only in the development of new types of geometric structures but also in the higher-level connectivity and assembly of clusters at the supramolecular level. Here, we report a novel windmill-like Al10 cluster and consider this geometrically unique cluster as an anionic node assembled together with different cationic guests such as imidazolium and guanidinium. These guests with different hydrogen-bond angles can help to obtain a series of diverse hydrogen-bonding networks and then manipulate the stacking mode of hosts and guests. Furthermore, we realized a supramolecular approach to fine-tune the optical limiting properties of the cluster. This work not only enriches the host-guest chemistry of ionic windmill-like clusters but also opens up more possibilities for aluminum oxo cluster-based hydrogen-bonded frameworks.
RESUMO
Xanthomonas fragariae usually causes angular leaf spot (ALS) of strawberry, a serious bacterial disease in many strawberry-producing regions worldwide. Recently, a new strain of X. fragariae (YL19) was isolated from strawberry in China and has been shown to cause dry cavity rot in strawberry crown. In this study, we constructed a green fluorescent protein (GFP)-labeled Xf YL19 (YL19-GFP) to visualize the infection process and pathogen colonization in strawberries. Foliar inoculation of YL19-GFP resulted in the pathogen migrating from the leaves to the crown, whereas dip inoculation of wounded crowns or roots resulted in the migration of bacteria from the crowns or roots to the leaves. These two invasion types both resulted in the systematic spread of YL19-GFP, but inoculation of a wounded crown was more harmful to the strawberry plant than foliar inoculation. Results increased our understanding of the systemic invasion of X. fragariae, and the resultant crown cavity caused by Xf YL19.
Assuntos
Fragaria , Xanthomonas , Fragaria/microbiologia , ChinaRESUMO
The coronavirus disease 2019 (COVID-19) has been a global epidemic for more than three years, causing more than 6.9 million deaths. COVID-19 has the clinical characteristics of strong infectivity and long incubation period, and can cause multi-system damage, mainly lung damage, clinical symptoms of acute respiratory distress syndrome (ARDS) and systemic multiple organ damage. The SARS-CoV-2 virus is still constantly mutating. At present, there is no global consensus on the pathological changes of COVID-19 associated deaths and even no consensus on the criteria for determining the cause of death. The investigation of the basic pathological changes and progression of the disease is helpful to guide the clinical treatment and the development of therapeutic drugs. This paper reviews the autopsy reports and related literature published worldwide from February 2020 to June 2023, with a clear number of autopsy cases and corresponding pathological changes of vital organs as the inclusion criteria. A total of 1 111 autopsy cases from 65 papers in 18 countries are included. Pathological manifestations and causes of death are classified and statistically analyzed, common pathological changes of COVID-19 are summarized, and analytical conclusions are drawn, suggesting that COVID-19 infection can cause life-threatening pathological changes in vital organs. On the basis of different health levels of infected groups, the direct cause of death is mainly severe lung damage and secondary systemic multiple organ failure.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/patologia , Causas de Morte , Pulmão/patologia , AutopsiaRESUMO
Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the most fatal diseases in the world. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the production of 5-methyltetrahydrofolate (5-CH3-THF), which is required for the de novo biosynthesis of methionine in bacteria. Here, we identified Rv2172c as an MTHFR in M. tuberculosis through in vitro and in vivo analyses and determined that the protein is essential for the in vitro growth of the bacterium. Subsequently, we constructed rv2172c R159N and L214A mutants in M. tuberculosis and found that these mutants were more sensitive to the antifolates para-aminosalicylic acid (PAS) and sulfamethoxazole (SMX). Combining biochemical and genetic methods, we found that rv2172c R159N or L214A mutation impaired methionine production, leading to increased susceptibility of M. tuberculosis to PAS, which was largely restored by adding exogenous methionine. Moreover, overexpression of rv2172c in M. tuberculosis could increase methionine production and lead to PAS resistance. This research is the first to identify an MTHFR in M. tuberculosis and reveals that the activity of this enzyme is associated with susceptibility to antifolates. These findings have particular value for antitubercular drug design for the treatment of drug-resistant TB.
Assuntos
Ácido Aminossalicílico , Mycobacterium tuberculosis , Ácido Aminossalicílico/metabolismo , Ácido Aminossalicílico/farmacologia , Antituberculosos/metabolismo , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/metabolismoRESUMO
Due to the potential hazard of perfluorooctanoic acid (PFOA), hexafluoropropylene oxide dimer acid (HFPO-DA, GenX) has become a typical alternative since 2009. However, GenX has recently been reported to have equal or even greater toxicity and bioaccumulation than PFOA. Considering the suitability of alternatives, it is quite essential to study and compare the degradation degree between PFOA and GenX in water. Therefore, in the present study, a comprehensive degradation comparison between them via electrooxidation with a titanium suboxide membrane anode was conducted. The degradation rate decreased throughout for PFOA, while it first increased and then decreased for GenX when the permeate flux increased from 17.3 L to 100.3 L m-2·h-1. The different responses of PFOA and GenX to flux might be attributed to their different solubilities. In addition, the higher kobs of PFOA demonstrated that it had a better degradability than GenX by 2.4-fold in a mixed solution. The fluorinated byproduct perfluoropropanoic acid (PFPrA) was detected as a GenX intermediate, suggesting that ether bridge splitting was needed for GenX electrooxidation. This study provides a reference for assessing the degradability of GenX and PFOA and indicates that it is worth reconsidering whether GenX is a suitable alternative for PFOA from the point of view of environmental protection.
Assuntos
Fluorocarbonos , Poluentes Químicos da Água , Bioacumulação , Caprilatos , Fluorocarbonos/análise , Poluentes Químicos da Água/análiseRESUMO
BACKGROUND: Lung ultrasound (LUS) is a useful tool for assessing the severity of lung disease, without radiation exposure. However, there is little data on the practicality of LUS in assessing the severity of bronchopulmonary dysplasia (BPD) and evaluating short-term clinical outcomes. We adapted a LUS score to evaluate BPD severity and assess the reliability of mLUS score correlated with short-term clinical outcomes. METHODS: Prospective diagnostic accuracy study was designed to enroll preterm infants with gestational age < 34 weeks. Lung ultrasonography was performed at 36 weeks postmenstrual age. The diagnostic and predictive values of new modified lung ultrasound (mLUS) scores based on eight standard sections were compared with classic lung ultrasound (cLUS) scores. RESULTS: A total of 128 infants were enrolled in this cohort, including 30 without BPD; 31 with mild BPD; 23 with moderate BPD and 44 with severe BPD. The mLUS score was significantly correlated with the short-term clinical outcomes, superior to cLUS score. The mLUS score well correlated with moderate and severe BPD (AUC = 0.813, 95% CI 0.739-0.888) and severe BPD (AUC = 0.801, 95% CI 0.728-0.875), which were superior to cLUS score. The ROC analysis of mLUS score to evaluate the other short-term outcomes also showed significant superiority to cLUS score. The optimal cutoff points for mLUS score were 14 for moderate and severe BPD and 16 for severe BPD. CONCLUSIONS: The mLUS score correlates significantly with short-term clinical outcomes and well evaluates these outcomes in preterm infants.
Assuntos
Displasia Broncopulmonar , Displasia Broncopulmonar/diagnóstico por imagem , Humanos , Lactente , Recém-Nascido , Recém-Nascido Prematuro , Pulmão/diagnóstico por imagem , Estudos Prospectivos , Reprodutibilidade dos Testes , UltrassonografiaRESUMO
Dehydrocostus lactone (DHL), a natural sesquiterpene lactone isolated from the traditional Chinese herbs Saussurea lappa and Inula helenium L., has important anti-inflammatory properties used for treating colitis, fibrosis, and Gram-negative bacteria-induced acute lung injury (ALI). However, the effects of DHL on Gram-positive bacteria-induced macrophage activation and ALI remains unclear. In this study, we found that DHL inhibited the phosphorylation of p38 MAPK, the degradation of IκBα, and the activation and nuclear translocation of NF-κB p65, but enhanced the phosphorylation of AMP-activated protein kinase (AMPK) and the expression of Nrf2 and HO-1 in lipoteichoic acid (LTA)-stimulated RAW264.7 cells and primary bone-marrow-derived macrophages (BMDMs). Given the critical role of the p38 MAPK/NF-κB and AMPK/Nrf2 signaling pathways in the balance of M1/M2 macrophage polarization and inflammation, we speculated that DHL would also have an effect on macrophage polarization. Further studies verified that DHL promoted M2 macrophage polarization and reduced M1 polarization, then resulted in a decreased inflammatory response. An in vivo study also revealed that DHL exhibited anti-inflammatory effects and ameliorated methicillin-resistant Staphylococcus aureus (MRSA)-induced ALI. In addition, DHL treatment significantly inhibited the p38 MAPK/NF-κB pathway and activated AMPK/Nrf2 signaling, leading to accelerated switching of macrophages from M1 to M2 in the MRSA-induced murine ALI model. Collectively, these data demonstrated that DHL can promote macrophage polarization to an anti-inflammatory M2 phenotype via interfering in p38 MAPK/NF-κB signaling, as well as activating the AMPK/Nrf2 pathway in vitro and in vivo. Our results suggested that DHL might be a novel candidate for treating inflammatory diseases caused by Gram-positive bacteria.
Assuntos
Anti-Inflamatórios/farmacologia , Lactonas/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Pneumonia Estafilocócica/etiologia , Sesquiterpenos/farmacologia , Doença Aguda , Animais , Plasticidade Celular/efeitos dos fármacos , Plasticidade Celular/imunologia , Modelos Animais de Doenças , Ativação de Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Modelos Biológicos , NF-kappa B/metabolismo , Fosforilação , Pneumonia Estafilocócica/tratamento farmacológico , Pneumonia Estafilocócica/metabolismo , Pneumonia Estafilocócica/patologia , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVES: To study the transcriptomic changes of astrocytes in the brain of rats exposed to methamphetamine (METH) and its possible mechanism in neurotoxicity. METHODS: The rats were intraperitoneally injected with METH (15 mg/kg) every 12 h for 8 times in total to establish the subacute rat model of METH. After the model was successfully established, the striatum was extracted, and astrocytes were separated by the magnetic bead method. Transcriptome sequencing was performed on selected astrocytes, and the differentially expressed genes were analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. RESULTS: A total of 876 differentially expressed genes were obtained by transcriptome sequencing, including 321 up-regulated genes and 555 down-regulated genes. GO analysis revealed that differentially expressed genes were mainly concentrated in cell structure, biological process regulation, extracellular matrix and organelle functions. KEGG pathway enrichment analysis showed that steroids biosynthesis, fatty acid biosynthesis, peroxisome proliferators-activated receptor (PPAR), adenosine 5'-monophosphate-activated protein kinase (AMPK) and other signaling pathways were significantly changed. CONCLUSIONS: METH can cause structural changes of astrocytes through multiple targets, among which cellular structure, steroids biosynthesis and fatty acid biosynthesis may play an important role in nerve injury, providing a new idea for forensic identification of METH related death.
Assuntos
Metanfetamina , Transcriptoma , Animais , Astrócitos , Encéfalo , Perfilação da Expressão Gênica , Metanfetamina/farmacologia , Ratos , Transdução de SinaisRESUMO
An efficient solvent-free protocol for the Buchwald-Hartwig cross-coupling reaction of aryl and heteroaryl chlorides with primary and secondary amines using the Pd(dba)2/ligand 1 catalytic system has been developed. Notably, the catalytic system also efficiently catalyzed the reaction under aqueous conditions.
RESUMO
An easily available Pd(OAc)2/(2-(anthracen-9-yl)-1H-inden-3-yl) dicyclohexylphosphine/toluene/iPrOH/water catalytic system was developed, which shows high catalytic activity in the Suzuki-Miyaura cross-coupling reactions of a diverse array of aryl and heteroaryl chlorides with Pd loadings down to 0.01 mol%.
RESUMO
Deaths involved with environmental hazards and intoxication might present with minimal or nonspecific morphological features, which are insufficient to establish a diagnosis. The present study investigated the postmortem brain mRNA and immunohistochemical expressions of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), inducible nitric oxide synthase (iNOS) and nuclear factor erythroid-2-related factor-2 (Nrf2) in forensic cases. Relative mRNA quantification using Taqman real-time PCR assay demonstrated higher expression of IL-1ß, TNF-α and iNOS, and lower expression of Nrf2 in methamphetamine intoxication and hyperthermia cases, higher expression of iNOS in phenobarbital intoxication cases, and higher expression of Nrf2 in phenobarbital intoxication and hypothermia cases. Immunostaining results showed substantial inter-individual variations in each group, showing no evident differences in distribution or intensity. These findings suggest that different inflammatory and antioxidant responses were involved in deaths from different etiologies, and these markers may be useful for evaluating brain damage and responses.
Assuntos
Encéfalo/metabolismo , Interleucina-1beta/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Asfixia/metabolismo , Biomarcadores/metabolismo , Encéfalo/patologia , Causas de Morte , Feminino , Febre/metabolismo , Patologia Legal , Humanos , Hipotermia/metabolismo , Imuno-Histoquímica , Interleucina-1beta/genética , Masculino , Pessoa de Meia-Idade , Fator 2 Relacionado a NF-E2/genética , Óxido Nítrico Sintase Tipo II/genética , Intoxicação/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fator de Necrose Tumoral alfa/genética , Ferimentos e Lesões/metabolismo , Adulto JovemRESUMO
Sudden cardiac death (SCD) is the most frequent cause of sudden unexplained death in forensic practice. The most common cause of SCD is coronary artery disease related to coronary atherosclerosis. Previous study suggested the possible application of connexin 43 (Cx43) and zonula occludens-1 (ZO1) immunostaining in the early diagnosis of myocardial ischemia. However, there appears to be insufficient data with regard to their mRNA levels. The present study investigated the cardiac mRNA levels of Cx43 and ZO1, using forensic autopsy materials consisting of 41 control cases without any disease or structural abnormality of the heart (group 1), 32 deaths due to acute ischemic heart disease related to coronary atherosclerosis without apparent myocardial necrosis (group 2), and 29 traumatic deaths with coronary atherosclerosis (group 3). Ten candidate reference genes were evaluated in the left ventricles of 10 forensic autopsy cases. EEF1A1, PPIA, TPT1, and RPL13A were identified as the most stable reference genes. Using these validated reference genes, mRNA levels of Cx43 and ZO1 were examined in the bilateral ventricles and atria of the heart. Relative mRNA quantification demonstrated decreased calibrated normalized relative quantity (CNRQ) values of Cx43 and ZO1 in bilateral ventricles of group 2. When using one conventional reference gene (GAPDH or ACTB) for normalization, nearly no difference was detected among the three groups. These findings indicate that ventricular gap junction remodeling may be a key contributor to rhythm disturbances. Analysis of cardiac Cx43 and ZO1 using real-time PCR is useful in diagnosis of SCD, and validation of reference genes is crucial.
Assuntos
Conexina 43/genética , Doença da Artéria Coronariana/genética , Morte Súbita Cardíaca/patologia , RNA Mensageiro/metabolismo , Proteína da Zônula de Oclusão-1/genética , Estudos de Casos e Controles , Doença da Artéria Coronariana/metabolismo , Feminino , Genética Forense , Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Humanos , Masculino , Miocárdio/metabolismo , Projetos Piloto , Reação em Cadeia da Polimerase em Tempo Real , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
This study provides the candidate sequences in the identification of Radix et Rhizoma Clematidis and its adulterants using DNA barcoding. We amplified and sequenced the region psbA-trnH, with the data of 284 sequences from GenBank, the differential intra- and inter-specific divergences, genetic distance, barcoding gap were used to evaluate five barcodes, and the identification efficiency was assessed using BLAST1 and Nearest Distance methods. The results showed that psbA-trnH barcodes performed high identification efficiency and inter-specific divergences among the five different DNA barcodes. Analysis of the barcoding gap and NJ tree showed psbA-trnH was superior to other barcodes. Based on the identification and PCR amplification efficiency, psbA-trnH can be the ideal barcode to identify Radix et Rhizoma Clematidis and its adulterants accurately.
Assuntos
Código de Barras de DNA Taxonômico/métodos , DNA de Plantas/genética , Plantas Medicinais/genética , Ranunculaceae/genética , Contaminação de Medicamentos , Técnicas de Amplificação de Ácido Nucleico/métodos , Raízes de Plantas/genética , Plantas Medicinais/classificação , Ranunculaceae/classificação , Rizoma/genética , Especificidade da EspécieRESUMO
To establish a new method for identifying genus of Lilium by DNA barcoding technology, ITS, ITS2, psbA-trnH, matK and rbcL sequences were analyzed in term of variation of inter- and intra-species, barcoding gap, neighbor-joining tree to distinguish genus of Lilium based on 978 sequences from experimental and GenBank database, and identification efficiency was evaluated by Nearest distance and BLAST1 methods. The results showed that DNA barcoding could identify different species in genus of Lilium. ITS sequence performed higher identification efficiency, and had significant difference between intra- and inter-species. And NJ tree could also divide species into different clades. Results indicate that DNA barcoding can identify genus of Lilium accurately. ITS sequence can be the optimal barcode to identify species of Lilium.