RESUMO
Biofilm is the primary cause of persistent infections caused by Streptococcus suis (S. suis). Metabolism and AI-2 quorum sensing are intricately linked to S. suis biofilm formation. Although the role of the AI-2 quorum sensing luxS gene in S. suis biofilm has been reported, its specific regulatory mechanism remains unclear. This study explored the differences in biofilm formation and monosaccharide metabolism among the wild type (WT), luxS mutant (ΔluxS) and complement strain (CΔluxS), and Galleria mellonella larvae were used to access the effect of luxS gene deletion on the virulence of S. suis in different monosaccharide medias. The results indicated that deletion of the luxS gene further compromised the monosaccharide metabolism of S. suis, impacting its growth in media with fructose, galactose, rhamnose, and mannose as the sole carbon sources. However, no significant impact was observed in media with glucose and N-acetylglucosamine. This deletion also weakened EPS synthesis, thereby diminishing the biofilm formation capacity of S. suis. Additionally, the downregulation of adhesion gene expression due to luxS gene deletion was found to be independent of the monosaccharide medias of S. suis.
Assuntos
Proteínas de Bactérias , Biofilmes , Liases de Carbono-Enxofre , Monossacarídeos , Percepção de Quorum , Streptococcus suis , Biofilmes/crescimento & desenvolvimento , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Streptococcus suis/genética , Streptococcus suis/metabolismo , Streptococcus suis/crescimento & desenvolvimento , Percepção de Quorum/genética , Monossacarídeos/metabolismo , Animais , Regulação Bacteriana da Expressão Gênica , Deleção de Genes , Virulência/genética , Lactonas/metabolismo , Larva/microbiologia , Homosserina/análogos & derivados , Homosserina/metabolismoRESUMO
Many cyanobacteria, both unicellular and filamentous, exhibit surface motility driven by type IV pili (T4P). While the component parts of the T4P machinery described in other prokaryotes are largely conserved in cyanobacteria, there are also several T4P proteins that appear to be unique to this phylum. One recently discovered component is EbsA, which has been characterized in two unicellular cyanobacteria. EbsA was found to form a complex with other T4P proteins and is essential for motility. Additionally, deletion of ebsA in one of these strains promoted the formation of biofilms. To expand the understanding of ebsA in cyanobacteria, its role in motility and biofilm formation were investigated in the model filamentous cyanobacterium Nostoc punctiforme. Expression of ebsA was strictly limited to hormogonia, the motile filaments of N. punctiforme. Deletion of ebsA did not affect hormogonium development but resulted in the loss of motility and the failure to accumulate surface pili or produce hormogonium polysaccharide (HPS), consistent with pervious observations in unicellular cyanobacteria. Protein-protein interaction studies indicated that EbsA directly interacts with PilB, and the localization of EbsA-GFP resembled that previously shown for both PilB and Hfq. Collectively, these results support the hypothesis that EbsA forms a complex along with PilB and Hfq that is essential for T4P extension. In contrast, rather than enhancing biofilm formation, deletion of both ebsA and pilB abolish biofilm formation in N. punctiforme, implying that distinct modalities for the relationship between motility, T4P function and biofilm formation may exist in different cyanobacteria.
Assuntos
Proteínas de Bactérias , Biofilmes , Fímbrias Bacterianas , Nostoc , Nostoc/genética , Nostoc/metabolismo , Nostoc/fisiologia , Nostoc/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Deleção de GenesRESUMO
African swine fever (ASF) is an acute infectious disease with a high mortality rate in both domestic and wild boars. Commercial vaccines or antiviral drugs for ASF were not available due to the complex diversity of the structure and genome of its pathogen African swine fever virus (ASFV). In recent years, there have been many reports on candidate strains of attenuated vaccines for ASFV. In this study, we obtained a recombinant virus named SY18ΔL60LΔCD2v by simultaneously deleting the L60L gene and CD2v gene from highly virulent strain SY18. In vitro, SY18ΔL60LΔCD2v displayed a decreased growth kinetic compared to that of parental SY18. In vivo, high doses (105 TCID50) of SY18ΔL60LΔCD2v can protect pigs (5/5) from attacks by the parental SY18 strain (102 TCID50). Low doses (102 TCID50) of SY18ΔL60LΔCD2v only protected 20% of pigs (1/5) from attacks by the parental SY18 strain (102 TCID50). The results indicated that the absence of these two genes in SY18 could induce protection against the homologous parental strain, and there were no obvious clinical symptoms or viremia. These results indicate that the SY18ΔL60LΔCD2v strain can serve as a new live attenuated vaccine candidate for the prevention and control of ASFV infection.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Deleção de Genes , Vacinas Atenuadas , Vacinas Virais , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/imunologia , Vírus da Febre Suína Africana/patogenicidade , Animais , Suínos , Febre Suína Africana/prevenção & controle , Febre Suína Africana/virologia , Febre Suína Africana/imunologia , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/administração & dosagem , Vacinas Virais/imunologia , Vacinas Virais/genética , Proteínas Virais/genética , Proteínas Virais/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Viremia/prevenção & controleRESUMO
Bovine herpesvirus type 1 (BoHV-1) establishes lifelong latency in trigeminal ganglionic (TG) neurons following intranasal and ocular infection in cattle. Periodically, the latent virus reactivates in the TG due to stress and is transported anterogradely to nerve endings in the nasal epithelium, where the virus replicates and sheds. Consequently, BoHV-1 is transmitted to susceptible animals and maintained in the cattle population. Modified live BoHV-1 vaccine strains (BoHV-1 MLV) also have a similar latency reactivation. Therefore, they circulate and are maintained in cattle herds. Additionally, they can regain virulence and cause vaccine outbreaks because they mutate and recombine with other circulating field wild-type (wt) strains. Recently, we constructed a BoHV-1 quadruple mutant virus (BoHV-1qmv) that lacks immune evasive properties due to UL49.5 and glycoprotein G (gG) deletions. In addition, it also lacks the gE cytoplasmic tail (gE CT) and Us9 gene sequences designed to make it safe, increase its vaccine efficacy against BoHV-1, and restrict its anterograde neuronal transport noted above. Further, we engineered the BoHV-1qmv-vector to serve as a subunit vaccine against the Rift Valley fever virus (BoHV-1qmv Sub-RVFV) (doi: 10.3390/v15112183). In this study, we determined the latency reactivation and nasal virus shedding properties of BoHV-1qmv (vector) and BoHV-1qmv-vectored subunit RVFV (BoHV-1qmv sub-RVFV) vaccine virus in calves in comparison to the BoHV-1 wild-type (wt) following intranasal inoculation. The real-time PCR results showed that BoHV-1 wt- but not the BoHV-1qmv vector- and BoHV-1qmv Sub-RVFV-inoculated calves shed virus in the nose following dexamethasone-induced latency reactivation; however, like the BoHV-1 wt, both the BoHV-1qmv vector and BoHV-1qmv Sub-RVFV viruses established latency, were reactivated, and replicated in the TG neurons. These results are consistent with the anterograde neurotransport function of the gE CT and Us9 sequences, which are deleted in the BoHV-1qmv and BoHV-1qmv Sub-RVFV.
Assuntos
Herpesvirus Bovino 1 , Mucosa Nasal , Neurônios , Gânglio Trigeminal , Ativação Viral , Latência Viral , Eliminação de Partículas Virais , Animais , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 1/fisiologia , Herpesvirus Bovino 1/imunologia , Bovinos , Mucosa Nasal/virologia , Gânglio Trigeminal/virologia , Neurônios/virologia , Deleção de Genes , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/administração & dosagem , Replicação Viral , Doenças dos Bovinos/virologia , Doenças dos Bovinos/prevenção & controle , Doenças dos Bovinos/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/genética , Infecções por Herpesviridae/veterinária , Infecções por Herpesviridae/virologia , Infecções por Herpesviridae/prevenção & controle , Infecções por Herpesviridae/imunologia , Vacinas Virais/imunologia , Vacinas Virais/genética , Vetores Genéticos/genética , Rinotraqueíte Infecciosa Bovina/virologia , Rinotraqueíte Infecciosa Bovina/prevenção & controle , Rinotraqueíte Infecciosa Bovina/imunologia , Vacinas contra Herpesvirus/genética , Vacinas contra Herpesvirus/imunologiaRESUMO
BACKGROUND: False negative rapid diagnostic tests (RDTs) accruing to the non-detection of Plasmodium falciparum histidine-rich protein 2/3 (Pfhrp2/3) is threatening the diagnosis and management of malaria. Although regular monitoring is necessary to gauge the level of efficacy of the tool, studies in Cameroon remain limited. This study assessed Plasmodium spp. prevalence and Pfhrp2/3 gene deletions across ecological and transmission zones in Cameroon. METHODS: This is a cross-sectional, multi-site, community- and hospital- based study, in 21 health facilities and 14 communities covering all five ecological settings in low seasonal (LS) and intense perennial (IPT) malaria transmission zones between 2019 and 2021. Participants were screened for malaria parasite using Pfhrp2 RDT and light microscopic examination of thick peripheral blood smears. DNA was extracted from dried blood spot using chelex®-100 and P. falciparum confirmed using varATS real-time quantitative Polymerase Chain Reaction (qPCR), P. malariae and P. ovale by real-time qPCR of Plasmepsin gene, and P. vivax using a commercial kit. Isolates with amplified Pfcsp and Pfama-1 genes were assayed for Pfhrp 2/3 gene deletions by conventional PCR. RESULTS: A total of 3,373 participants enrolled, 1,786 Plasmodium spp. infected, with 77.4% P. falciparum. Discordant RDT and qPCR results (False negatives) were reported in 191 (15.7%) P. falciparum mono-infected samples from LS (29%, 42) and IPT (13.9%, 149). The Pfhrp2+/Pfhrp3 + genotype was most frequent, similar between LS (5.5%, 8/145) and IPT (6.0%, 65/1,076). Single Pfhrp2 and Pfhrp3 gene deletions occurred in LS (0.7%, 1/145 each) and IPT (3.6%, 39/1,076 vs. 2.9%, 31/1,076), respectively. Whilst a single sample harboured Pfhrp2-/Pfhrp3- genotype in LS, 2.4% (26/1,076) were double deleted at IPT. Pfhrp2+/Pfhrp3- (0.3%, 3/1,076) and Pfhrp2-/Pfhrp3+ (1.2%, 13/1,076) genotypes were only observed in IPT. Pfhrp2, Pfhrp3 deletions and Pfhrp2-/Pfhrp3- genotype accounted for 78.8% (26), 69.7% (23) and 63.6% (21) RDT false negatives, respectively. CONCLUSION: Plasmodium falciparum remains the most dominant and widely distributed Plasmodium species across transmission and ecological zones in Cameroon. Although the low prevalence of Pfhrp2/3 gene deletions supports the continued use of HRP2-based RDTs for routine malaria diagnosis, the high proportion of false-negatives due to gene deleted parasites necessitates continued surveillance to inform control and elimination efforts.
Assuntos
Antígenos de Protozoários , Testes Diagnósticos de Rotina , Deleção de Genes , Malária Falciparum , Plasmodium falciparum , Proteínas de Protozoários , Estudos Transversais , Camarões/epidemiologia , Proteínas de Protozoários/genética , Humanos , Antígenos de Protozoários/genética , Plasmodium falciparum/genética , Adulto , Adolescente , Masculino , Malária Falciparum/epidemiologia , Malária Falciparum/transmissão , Malária Falciparum/parasitologia , Feminino , Criança , Adulto Jovem , Pré-Escolar , Pessoa de Meia-Idade , Reações Falso-Negativas , Lactente , Prevalência , Estações do Ano , IdosoRESUMO
BACKGROUND: Gliomas are the most common central nervous system (CNS) tumors in infant but with incidence rate only 1.38 per 100,000. Due to distinctive clinical, histologic, and molecular features, the current World Health Organization (WHO) CNS5 separate gliomas in children from adult as pediatric-type diffuse high-grade and low-grade gliomas. Infant hemispheric gliomas constitute a biologically and clinically distinct subgroup of pediatric-type diffuse high-grade. In this case we present clinical, radiographic, intraoperative, and methylation profiling of the first infant-type hemispheric glioma diagnosed in Indonesia. CASE DESCRIPTION: This is a case report of infant operated at Dr. Cipto Mangunkusumo National General Hospital, Jakarta, Indonesia in February 2024. A 6-month-old male infant brought to regional hospital due to head enlargement compared to infant of the same age, head circumference was 50 cm [>2 standard deviation (SD)] with frontal bossing. Brain MRI showed large multi-loculated cystic lesion at left parietooccipital region, which appeared hypointense on T1-weithgted (T1W), hyperintense on T2-weighted (T2W) and fluid-attenuated inversion recovery (FLAIR), with irregular contrast enhancing border. There was isointense lesion on T1W with inhomogeneous contrast enhancement. The largest volume of cystic lesion was 216 cm. Intraoperatively, parietal bone was thinner than usual. The brain was tense, purplish, and non-pulsating, giving the impression of a tumor with indistinct borders with the normal cortex. Dark clear yellowish fluid was spurt after the cortex was incised. Histopathological findings revealed moderate to high cellularity tumor tissue with mitosis, microvascular proliferation, palisading necrosis. In collaboration with German Cancer Research Center (DKFZ), DNA methylation array analysis showed the tumor to match the Infant-type Hemispheric Glioma methylation class (calibrated score 0.94) with deletion of cyclin dependent kinase inhibitor 2A/B (CDKN2A/B). CONCLUSIONS: Methylation class (MC) infant-type hemispheric glioma may present with macrocephaly. On magnetic resonance imaging (MRI) it may appear as large multi-loculated cystic lesion and irregular contrast enhancing border. The key diagnostic criteria for infant-type hemispheric glioma involve combination of clinical, pathological, and molecular feature.
Assuntos
Glioma , Humanos , Masculino , Glioma/genética , Lactente , Inibidor p16 de Quinase Dependente de Ciclina/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Inibidor de Quinase Dependente de Ciclina p15/genética , Deleção de GenesRESUMO
BACKGROUND: We are primarily investigating the prognostic role of cell-cycle-dependent kinase inhibitor (CDKN)-2A homozygous deletion in central nervous system (CNS) World Health Organization (WHO) grade 4 gliomas. Additionally, traditional prognostic factors for grade 4 gliomas will be examined, and our results will be validated. METHODS: We conducted a retrospective analysis of glioma cohorts in our institute. Medical records were reviewed for 142 glioblastoma patients for 15 years, and pathological slides were examined again for the updated diagnosis according to the 2021 WHO classification of CNS tumors. The isocitrate dehydrase (IDH) mutation and CDKN2A deletion were examined by next generation sequencing (NGS) analysis using ONCO accuPanel®. Traditional prognostic factors including age, WHO performance status, extent of resection, and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation were examined. RESULTS: After the exclusion of 6 patients with poor status of pathologic samples, 136 glioblastoma that were diagnosed by previous WHO criteria were changed into 29 (21.3%) astrocytoma, IDH-mutant, CNS WHO grade 4 and 107 (78.7%) glioblastoma, IDH-wildtype, CNS WHO grade 4. Among them, 61 patients (56.0%) had CDKN2A deletion. Group A with IDH-wildtype and CDKN2A deletion had a mean overall survival (OS) of 15.70 months [95% confident interval (CI): 13.86-17.54], group B with IDH-mutant and CDKN2A deletion had a mean OS of 19.37 months (95% CI: 13.43-25.30), group C with IDH-wildtype and intact CDKN2A had a mean OS of 22.63 months (95% CI: 20.10-25.17), and group D with IDH-mutant and intact CDKN2A had a mean OS of 33.38 months (95% CI: 29.35-37.40). Multifactor analysis showed following factors were independently associated with OS: age [≥50 vs. <50 years; hazard ratio (HR) 4.642], extent of resection (gross total resection vs. others; HR 5.523), WHO performance (0, 1 vs. 2; HR 5.007), MGMT promoter methylation, (methylated vs. unmethylated; HR 5.075), IDH mutation (mutant vs. wildtype; HR 6.358), and CDKN2A deletion (absence vs. presence; HR 13.452). CONCLUSIONS: The presenting study suggests that CDKN2A deletion should play a powerful prognostic role in CNS WHO grade 4 gliomas as well as low-grade glioma. Even if CNS WHO grade 4 gliomas had mutant IDH, they can have poor clinical outcomes due to CDKN2A deletion.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina , Glioma , Isocitrato Desidrogenase , Mutação , Humanos , Glioma/genética , Glioma/patologia , Masculino , Feminino , Pessoa de Meia-Idade , Isocitrato Desidrogenase/genética , Estudos Retrospectivos , Inibidor p16 de Quinase Dependente de Ciclina/genética , Adulto , Organização Mundial da Saúde , Gradação de Tumores , Idoso , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Homozigoto , Deleção de Genes , Adulto JovemRESUMO
Aeromonas veronii is an opportunistic pathogen that poses great threat to aquaculture and human health, so there is an urgent need for green and efficient methods to deal with its infection. In this study, single and double gene deletion strains (AV-ΔaroA, AV-Δppk1 and AV-ΔaroA/ppk1) that can be stably inherited were constructed. Pathogenicity test showed that the toxicity of AV-ΔaroA and AV-ΔaroA/ppk1 was significantly lower compared to wild-type A. veronii. Biological characterization analysis revealed that the decrease in pathogenicity might be due to the declined growth, motility, biofilm formation abilities and the expression of virulence-related genes in mutants. Subsequently, we evaluated the efficacy of AV-ΔaroA/ppk1 as a live attenuated vaccine (LAV). Safety assessment experiments showed that AV-ΔaroA/ppk1 injected at a concentration of 3 × 107 CFU/mL was safe for C. carassius. The relative percentage survival of AV-ΔaroA/ppk1 was 67.85 %, significantly higher than that of the inactivated A. veronii, which had an RPS of 54.84 %. This improved protective effect was mainly attributed to the increased levels of A. veronii specific IgM antibody, enhanced alkaline phosphatase, lysozyme and superoxide dismutase activities, as well as higher expression levels of several immune related genes. Together, these findings deepen our understanding of the functional roles of aroA and ppk1 in A. veronii pathogenicity, provide a good candidate of LAV for A. veronii.
Assuntos
Aeromonas veronii , Vacinas Bacterianas , Doenças dos Peixes , Infecções por Bactérias Gram-Negativas , Vacinas Atenuadas , Aeromonas veronii/patogenicidade , Aeromonas veronii/fisiologia , Aeromonas veronii/imunologia , Vacinas Atenuadas/imunologia , Vacinas Bacterianas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/prevenção & controle , Doenças dos Peixes/prevenção & controle , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Animais , Virulência , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Carpas/imunologia , Deleção de GenesRESUMO
Outbreaks of the foot-and-mouth disease (FMD) have major economic impact on the global livestock industry by affecting the animal health and product safety. L-protease, a non-structural protein of FMDV, is a papain-like cysteine proteinase involved in viral protein processing as well as cleavage of host proteins for promoting the virus growth. FMDV synthesizes two forms of leader proteinase, Lpro (Labpro and Lbpro), where the deletion of Labpro is lethal and Lbpro deletion is reported to be attenuated. Defective replicons have been used by trans-complementing the deleted gene to produce one time replicating virus; thus, the bio-safety procedure can be compromised in the production units. Attempts are made to rescue of ΔLbproFMDV Asia1 virus by co-expressing the Lbpro protein carried in pcDNA plasmid. Mutant FMDV cDNA, pAsia-ΔLbpro, was constructed by PCR mediated mutagenesis using inverse primers. Transfection of BHK-21 cells with in-vitro transcribed RNA from the constructs failed to produce an infective mutant FMDV. Genetic trans-complementation of the Lbpro, which was done by co-transfecting the pcDNALbpro plasmid DNA along with the pAsia-ΔLbpro RNA in BHK-21 cells also failed to produce viable virus. Expression experiments of reporter genes and indirect immune-fluorescence confirmed the production of the viral proteins in wild type FMDV pAsiaWT; however, it was absent in the pAsia-ΔLbpro indicating that the leaderless virus was unable to produce infectious progeny and infect the cells. Failure to produce virus either by Lbpro deleted mutant clone or by genetic complementation suggests little chance of reversion of the disabled virus with large deletions of FMDV genome.
Assuntos
Vírus da Febre Aftosa , Vírus da Febre Aftosa/genética , Animais , Linhagem Celular , Genoma Viral/genética , Replicação Viral , Febre Aftosa/virologia , Cricetinae , Plasmídeos/genética , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Deleção de Genes , EndopeptidasesRESUMO
INTRODUCTION: Caused by mutation or deletion of the CHM gene, choroideremia is a rare X-linked recessive chorioretinal dystrophy characterized by progressive degeneration of the retinal pigment epithelium, photoreceptors, and the choriocapillaris. There are few published reports of choroideremia associated with complex syndromic phenotypes due to large or contiguous gene deletions. METHODS: Case report and review of literature. RESULTS: We present a case of a 46-year-old male with a prior clinical diagnosis of syndromic retinitis pigmentosa, who was found to have syndromic choroideremia associated with a novel multi-gene deletion of 13.5 megabase pairs. This deletion encompassing 18 genes is one of the largest deletions reported in the literature. A total of 18 male cases of choroideremia associated with confirmed large or contiguous gene deletions have been published to date. Previously reported deletions range in size from 4 to 15 megabase pairs, and observed phenotypes include cleft lip and palate, ptosis, obesity, metabolic diseases, developmental delay, and hearing loss. DISCUSSION: The contribution of our case aims to expand our understanding of Xq21 deletions and prompts further investigation of genes found in this locus. Furthermore, it highlights the importance of including syndromic choroideremia on the differential diagnosis in the workup of other syndromic retinopathies, particularly those that feature obesity, hearing loss, or intellectual disability.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Coroideremia , Deleção de Genes , Humanos , Coroideremia/genética , Coroideremia/diagnóstico , Masculino , Pessoa de Meia-Idade , Proteínas Adaptadoras de Transdução de Sinal/genética , Síndrome , FenótipoRESUMO
Hyperfunction of the dopamine system has been implicated in manic episodes in bipolar disorders. How dopaminergic neuronal function is regulated in the pathogenesis of mania remains unclear. Histaminergic neurons project dense efferents into the midbrain dopaminergic nuclei. Here, we present mice lacking dopaminergic histamine H2 receptor (H2R) in the ventral tegmental area (VTA) that exhibit a behavioral phenotype mirroring some of the symptoms of mania, including increased locomotor activity and reduced anxiety- and depression-like behavior. These behavioral deficits can be reversed by the mood stabilizers lithium and valproate. H2R deletion in dopaminergic neurons significantly enhances neuronal activity, concurrent with a decrease in the γ-aminobutyric acid (GABA) type A receptor (GABAAR) membrane presence and inhibitory transmission. Conversely, either overexpression of H2R in VTA dopaminergic neurons or treatment of H2R agonist amthamine within the VTA counteracts amphetamine-induced hyperactivity. Together, our results demonstrate the engagement of H2R in reducing VTA dopaminergic activity, shedding light on the role of H2R as a potential target for mania therapy.
Assuntos
Neurônios Dopaminérgicos , Mania , Receptores Histamínicos H2 , Área Tegmentar Ventral , Animais , Área Tegmentar Ventral/metabolismo , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Camundongos , Receptores Histamínicos H2/metabolismo , Receptores Histamínicos H2/genética , Mania/metabolismo , Comportamento Animal , Masculino , Camundongos Knockout , Camundongos Endogâmicos C57BL , Receptores de GABA-A/metabolismo , Deleção de Genes , Transtorno Bipolar/metabolismo , Transtorno Bipolar/patologia , Transtorno Bipolar/genéticaRESUMO
In the healing process of myocardial infarction, cardiac fibroblasts are activated to produce collagen, leading to adverse remodeling and heart failure. Our previous study showed that ASPP1 promotes cardiomyocyte apoptosis by enhancing the nuclear trafficking of p53. We thus explored the influence of ASPP1 on myocardial fibrosis and the underlying mechanisms. Here, we observed that ASPP1 was increased after 4 weeks of MI. Both global and myofibroblast knockout of ASPP1 in mice mitigated cardiac dysfunction and fibrosis after MI. Strikingly, ASPP1 produced the opposite influence on p53 level and cell fate in cardiac fibroblasts and cardiomyocytes. Knockdown of ASPP1 increased p53 levels and inhibited the activity of cardiac fibroblasts. ASPP1 accumulated in the cytoplasm of fibroblasts while the level of p53 was reduced following TGF-ß1 stimulation; however, inhibition of ASPP1 increased the p53 level and promoted p53 nuclear translocation. Mechanistically, ASPP1 is directly bound to deubiquitinase OTUB1, thereby promoting the ubiquitination and degradation of p53, attenuating myofibroblast activity and cardiac fibrosis, and improving heart function after MI.
Assuntos
Fibrose , Infarto do Miocárdio , Miocárdio , Miofibroblastos , Proteína Supressora de Tumor p53 , Animais , Humanos , Masculino , Camundongos , Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Deleção de Genes , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/genética , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Proteólise , Fator de Crescimento Transformador beta1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , UbiquitinaçãoRESUMO
Biofilms in nature often exist as communities. In this study, an experimental mixed-species community consisting of Pseudomonas aeruginosa, Pseudomonas protegens and Klebsiella pneumoniae was used to investigate how AI-2 transporters affect interspecies interactions and composition. The K. pneumoniae lsrB/lsrD deletion mutants had a 10-25-fold higher concentration of extracellular AI-2 compared to the wild-type. Although these deletion mutants produced monospecies biofilms of similar biomass, the substitution of these mutants for the parental strain significantly altered composition. Dual-species biofilm assays demonstrated that the changes in composition were due to the cumulative effect of pairwise interactions. It was further revealed that K. pneumoniae being present physically in the consortium was important in AI-2 mediating composition in the consortium, and that AI-2 transporters were crucial in achieving maximum biomass in the community. In conclusion, these findings demonstrate that AI-2 transporters mediate interspecies interactions and is important in maintaining the compositional equilibrium of the community.
Assuntos
Proteínas de Bactérias , Biofilmes , Klebsiella pneumoniae , Pseudomonas aeruginosa , Biofilmes/crescimento & desenvolvimento , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Interações Microbianas , Homosserina/análogos & derivados , Homosserina/metabolismo , Pseudomonas/genética , Pseudomonas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Deleção de Genes , Biomassa , LactonasRESUMO
Trichothecenes are toxins produced by certain species from several fungal genera, including Aspergillus, Fusarium, Isaria, Paramyrothecium, Stachybotrys, Trichoderma, and Trichothecium. These toxins are of interest because they contribute to the toxigenicity, plant pathogenicity, and/or biological control activities of some fungi. All trichothecenes have the same core (12,13-epoxytrichothec-9-ene or EPT) structure but can differ from one another by the presence or absence of a macrocyclic ring formed from polyketide and isoprenoid substituents esterified to carbon atoms 4 and 15 of EPT, respectively. Genes required for formation and some modifications of EPT have been elucidated, but almost nothing is known about genes specific to the formation of the macrocyclic ring. Therefore, we used genomic, transcriptomic, metabolomic, and gene deletion analyses to identify genes that are required specifically for the formation of the macrocyclic ring. These analyses identified one gene, TRI24, that is predicted to encode an acyltransferase and that is required for macrocyclic ring formation during biosynthesis of macrocyclic trichothecenes by the fungus Paramyrothecium roridum. In addition, a TRI24 deletion mutant of P. roridum caused less severe disease symptoms on common bean and had less antifungal activity than its wild-type progenitor strain. We propose that the reduced aggressiveness and antifungal activity of the mutant resulted from its inability to produce trichothecenes with a macrocyclic ring. To our knowledge, this is the first report of a gene required specifically for the formation of the macrocyclic ring of trichothecenes and that loss of the macrocyclic ring of trichothecenes can alter the biological activities of a fungus. KEY POINTS: ⢠TRI24 gene is found in all known macrocyclic trichothecene-producing fungi. ⢠A tri24-deletion mutant exhibits a reduction in antifungal and plant disease activities. ⢠TRI24 is the first described gene specific to macrocyclic trichothecene biosynthesis.
Assuntos
Tricotecenos , Tricotecenos/metabolismo , Tricotecenos/química , Doenças das Plantas/microbiologia , Deleção de Genes , Compostos Macrocíclicos/farmacologia , Compostos Macrocíclicos/química , Compostos Macrocíclicos/metabolismo , Metabolômica , Genes Fúngicos , Perfilação da Expressão GênicaRESUMO
African swine fever virus (ASFV) is the causative agent of an often lethal disease in domestic pigs, African swine fever (ASF). ASF is currently a pandemic disease challenging pig production in Eurasia. While the ASFV genome encodes for over 160 proteins, the function of most of them are still not characterized. Among those ASF genes with unknown functions is the E111R gene. It has been recently reported that the deletion of the E111R gene from the genome of the virulent Chinese field isolate SY18 strain produced a reduction of virus virulence when pigs were inoculated at relatively low doses. Conversely, we report here that deletion of the ASFV gene E111R in the Georgia 2010 isolate does not alter the virulence of the parental virus in experimentally inoculated pigs. A recombinant virus lacking the E111R gene, ASFV-G-∆E111R was intramuscularly (IM) inoculated in domestic pigs at a dose of 102 HAD50 of ASFV-G-∆E111R and compared with animals that received a similar dose of virulent ASFV-G. Both, animals inoculated with either the recombinant ASFV-G-∆E111R or the parental virus developed a fatal form of the disease and were euthanized around the 6th-7th day post-inoculation (dpi).
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Deleção de Genes , Replicação Viral , Animais , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/patogenicidade , Vírus da Febre Suína Africana/isolamento & purificação , Febre Suína Africana/virologia , Virulência/genética , Suínos , Proteínas Virais/genética , Genoma Viral , Sus scrofa/virologia , República da GeórgiaRESUMO
Yeast cell wall chitin has been shown to bind grape pathogenesis-related chitinases that are the primary cause of protein haze in wines, suggesting that yeast cell walls may be applied for haze protection. Here, we present a high-throughput screen to identify yeast strains with high cell wall chitin using a reiterative enrichment strategy and fluorescence-activated cell sorting of cells labelled with either GFP-tagged chitinase or Calcofluor white. To assess the validity of the strategy, we first used a pooled deletion strain library of Saccharomyces cerevisiae. The strategy enriched for deletion mutants with genes that had previously been described as having an impact on chitin levels. Genes that had not previously been linked to chitin biosynthesis or deposition were also identified. These genes are involved in cell wall maintenance and/or membrane trafficking functions. The strategy was then applied to a mutagenized population of a commercial wine yeast strain, S. cerevisiae EC1118. Enriched mutant strains showed significantly higher cell wall chitin than the wild type and significantly reduced the activity of chitinases in synthetic model wine, suggesting that these strains may be able to reduce haze formation in wine.
Assuntos
Parede Celular , Quitina , Quitinases , Citometria de Fluxo , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Parede Celular/metabolismo , Quitina/metabolismo , Quitinases/genética , Quitinases/metabolismo , Deleção de Genes , Vinho/microbiologia , Ensaios de Triagem em Larga Escala , BenzenossulfonatosRESUMO
The demand for sustainably produced bulk chemicals is constantly rising. Succinate serves as a fundamental component in various food, chemical, and pharmaceutical products. Succinate can be produced from sustainable raw materials using microbial fermentation and enzyme-based technologies. Bacteroides and Phocaeicola species, widely distributed and prevalent gut commensals, possess enzyme sets for the metabolization of complex plant polysaccharides and synthesize succinate as a fermentative end product. This study employed novel molecular techniques to enhance succinate yields in the natural succinate producer Phocaeicola vulgatus by directing the metabolic carbon flow toward succinate formation. The deletion of the gene encoding the methylmalonyl-CoA mutase (Δmcm, bvu_0309-0310) resulted in a 95% increase in succinate production, as metabolization to propionate was effectively blocked. Furthermore, deletion of genes encoding the lactate dehydrogenase (Δldh, bvu_2499) and the pyruvate:formate lyase (Δpfl, bvu_2880) eliminated the formation of fermentative end products lactate and formate. By overproducing the transketolase (TKT, BVU_2318) in the triple deletion mutant, succinate production increased from 3.9 mmol/g dry weight in the wild type to 10.9 mmol/g dry weight. Overall, succinate yield increased by 180% in the new mutant strain P. vulgatus Δmcm Δldh Δpfl pG106_tkt relative to the parent strain. This approach is a proof of concept, verifying the genetic accessibility of P. vulgatus, and forms the basis for targeted genetic optimization. The increase of efficiency highlights the huge potential of P. vulgatus as a succinate producer with applications in sustainable bioproduction processes. KEY POINTS: ⢠Deleting methylmalonyl-CoA mutase gene in P. vulgatus doubled succinate production ⢠Triple deletion mutant with transketolase overexpression increased succinate yield by 180% ⢠P. vulgatus shows high potential for sustainable bulk chemical production via genetic optimization.
Assuntos
Fermentação , Ácido Succínico , Ácido Succínico/metabolismo , Humanos , Engenharia Metabólica/métodos , Deleção de Genes , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/metabolismo , Microbioma Gastrointestinal , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
Synechococcus sp. PCC7002 has been considered as a photosynthetic chassis for the conversion of CO2 into biochemicals through genetic modification. However, conventional genetic manipulation techniques prove inadequate for comprehensive genetic modifications in this strain. Here, we present the development of a genome editing tool tailored for S. PCC7002, leveraging its endogenous type I-D CRISPR-Cas system. Utilizing this novel tool, we successfully deleted the glgA1 gene and iteratively edited the genome to obtain a double mutant of glgA1 and glgA2 genes. Additionally, large DNA fragments encompassing the entire type I-A (â¼14â¯kb) or III-B CRISPR-Cas (â¼21â¯kb) systems were completely knocked-out in S. PCC7002 using our tool. Furthermore, the endogenous pAQ5 plasmid, approximately 38â¯kb in length, was successfully cured from S. PCC7002. Our work demonstrates the feasibility of harnessing the endogenous CRISPR-Cas system for genome editing in S. PCC7002, thereby enriching the genetic toolkit for this species and providing a foundation for future enhancements in its biosynthetic efficiency.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Genoma Bacteriano , Plasmídeos , Synechococcus , Edição de Genes/métodos , Synechococcus/genética , Plasmídeos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Deleção de GenesRESUMO
Cellular Communication Network Factor 2 (CCN2) is a matricellular protein implicated in cell communication and microenvironmental signaling. Overexpression of CCN2 has been documented in various cardiovascular pathologies, wherein it may exert either deleterious or protective effects depending on the pathological context, thereby suggesting that its role in the cardiovascular system is not yet fully elucidated. In this study, we aimed to investigate the effects of Ccn2 gene deletion on the progression of acute cardiac injury induced by doxorubicin (DOX), a widely utilized chemotherapeutic agent. To this end, we employed conditional knockout (KO) mice for the Ccn2 gene (CCN2-KO), which were administered DOX and compared to DOX-treated wild-type (WT) control mice. Our findings demonstrated that the ablation of CCN2 ameliorated DOX-induced cardiac dysfunction, as evidenced by improvements in ejection fraction (EF) and fractional shortening (FS) of the left ventricle. Furthermore, DOX-treated CCN2-KO mice exhibited a significant reduction in the gene expression and activation of oxidative stress markers (Hmox1 and Nfe2l2/NRF2) relative to DOX-treated WT controls. Additionally, the deletion of Ccn2 markedly attenuated DOX-induced cardiac fibrosis. Collectively, these results suggest that CCN2 plays a pivotal role in the pathogenesis of DOX-mediated cardiotoxicity by modulating oxidative stress and fibrotic pathways. These findings provide a novel avenue for future investigations to explore the therapeutic potential of targeting CCN2 in the prevention of DOX-induced cardiac dysfunction.
Assuntos
Fator de Crescimento do Tecido Conjuntivo , Doxorrubicina , Fibrose , Camundongos Knockout , Estresse Oxidativo , Animais , Doxorrubicina/efeitos adversos , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Deleção de Genes , Masculino , Miocárdio/metabolismo , Miocárdio/patologia , Camundongos Endogâmicos C57BL , Cardiotoxicidade/genética , Cardiotoxicidade/metabolismoRESUMO
Cellular NAD+ is continuously degraded and synthesized under resting conditions. In mammals, NAD+ synthesis is primarily initiated from nicotinamide (Nam) by Nam phosphoribosyltransferase, whereas poly(ADP-ribose) polymerase 1 (PARP1) and 2 (PARP2), sirtuin1 (SIRT1), CD38, and sterile alpha and TIR motif containing 1 (SARM1) are involved in NAD+ breakdown. Using flux analysis with 2H-labeled Nam, we found that when mammalian cells were cultured in the absence of Nam, cellular NAD+ levels were maintained and NAD+ breakdown was completely suppressed. In the presence of Nam, the rate of NAD+ breakdown (RB) did not significantly change upon PARP1, PARP2, SIRT1, or SARM1 deletion, whereas stable expression of CD38 did not increase RB. However, RB in PARP1-deleted cells was much higher compared with that in wild-type cells, in which PARP1 activity was blocked with a selective inhibitor. In contrast, RB in CD38-overexpressing cells in the presence of a specific CD38 inhibitor was much lower compared with that in control cells. The results indicate that PARP1 deletion upregulates the activity of other NADases, whereas CD38 expression downregulates the activity of endogenous NADases, including PARP1 and PARP2. The rate of cellular NAD+ breakdown and the resulting NAD+ concentration may be maintained at a constant level, despite changes in the NAD+-degrading enzyme expression, through the compensatory regulation of NADase activity.