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Background: The microbiome likely plays a role in tuberculosis (TB) pathogenesis. We evaluated the site-of-disease microbiome and predicted metagenome in people with presumptive tuberculous pericarditis, a major cause of mortality, and explored for the first time, the interaction between its association with C-reactive protein (CRP), a potential diagnostic biomarker and the site-of-disease microbiome in extrapulmonary TB. Methods: People with effusions requiring diagnostic pericardiocentesis (n=139) provided background sampling controls and pericardial fluid (PF) for 16S rRNA gene sequencing analysed using QIIME2 and PICRUSt2. Blood was collected to measure CRP. Results: PF from people with definite (dTB, n=91), probable (pTB, n=25), and non- (nTB, n=23) tuberculous pericarditis differed in ß-diversity. dTBs were, vs. nTBs, Mycobacterium-, Lacticigenium-, and Kocuria- enriched. Within dTBs, HIV-positives were Mycobacterium-, Bifidobacterium- , Methylobacterium- , and Leptothrix -enriched vs. HIV-negatives and HIV-positive dTBs on ART were Mycobacterium - and Bifidobacterium -depleted vs. those not on ART. Compared to nTBs, dTBs exhibited short-chain fatty acid (SCFA) and mycobacterial metabolism microbial pathway enrichment. People with additional non-pericardial involvement had differentially PF taxa (e.g., Mycobacterium -enrichment and Streptococcus -depletion associated with pulmonary infiltrates). Mycobacterium reads were in 34% (31/91), 8% (2/25) and 17% (4/23) of dTBs, pTBs, and nTBs, respectively. ß-diversity differed between patients with CRP above vs. below the median value ( Pseudomonas -depleted). There was no correlation between enriched taxa in dTBs and CRP. Conclusions: PF is compositionally distinct based on TB status, HIV (and ART) status and dTBs are enriched in SCFA-associated taxa. The clinical significance of these findings, including mycobacterial reads in nTBs and pTBs, requires evaluation.
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Rationale: Chronic obstructive pulmonary disease (COPD) is associated with high morbidity, mortality, and healthcare costs. Cigarette smoke is a causative factor; however, not all heavy smokers develop COPD. Microbial colonization and infections are contributing factors to disease progression in advanced stages. Objectives: We investigated whether lower airway dysbiosis occurs in mild-to-moderate COPD and analyzed possible mechanistic contributions to COPD pathogenesis. Methods: We recruited 57 patients with a >10 pack-year smoking history: 26 had physiological evidence of COPD, and 31 had normal lung function (smoker control subjects). Bronchoscopy sampled the upper airways, lower airways, and environmental background. Samples were analyzed by 16S rRNA gene sequencing, whole genome, RNA metatranscriptome, and host RNA transcriptome. A preclinical mouse model was used to evaluate the contributions of cigarette smoke and dysbiosis on lower airway inflammatory injury. Measurements and Main Results: Compared with smoker control subjects, microbiome analyses showed that the lower airways of subjects with COPD were enriched with common oral commensals. The lower airway host transcriptomics demonstrated differences in markers of inflammation and tumorigenesis, such as upregulation of IL-17, IL-6, ERK/MAPK, PI3K, MUC1, and MUC4 in mild-to-moderate COPD. Finally, in a preclinical murine model exposed to cigarette smoke, lower airway dysbiosis with common oral commensals augments the inflammatory injury, revealing transcriptomic signatures similar to those observed in human subjects with COPD. Conclusions: Lower airway dysbiosis in the setting of smoke exposure contributes to inflammatory injury early in COPD. Targeting the lower airway microbiome in combination with smoking cessation may be of potential therapeutic relevance.
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Lesão Pulmonar , Doença Pulmonar Obstrutiva Crônica , Humanos , Animais , Camundongos , Disbiose/complicações , RNA Ribossômico 16S , Doença Pulmonar Obstrutiva Crônica/genética , Inflamação/complicações , Lesão Pulmonar/complicações , Pulmão/patologiaRESUMO
Background: Isolated small airway abnormalities may be demonstrable at rest in patients with normal spirometry; however, the relationship of these abnormalities to exertional symptoms remains uncertain. This study uses an augmented cardiopulmonary exercise test (CPET) to include evaluation of small airway function during and following exercise to unmask abnormalities not evident with standard testing in individuals with dyspnoea and normal spirometry. Methods: Three groups of subjects were studied: 1) World Trade Center (WTC) dust exposure (n=20); 2) Clinical Referral (n=15); and Control (n=13). Baseline evaluation included respiratory oscillometry. Airway function during an incremental workload CPET was assessed by: 1) tidal flow versus volume curves during exercise to assess for dynamic hyperinflation and expiratory flow limitation; and 2) post-exercise spirometry and oscillometry to evaluate for airway hyperreactivity. Results: All subjects demonstrated normal baseline forced expiratory volume in 1â s (FEV1)/forced vital capacity (FVC). Dyspnoea was reproduced during CPET in WTC and Clinical Referral groups versus Control without abnormality in respiratory pattern and minute ventilation. Tidal flow-volume curves uncovered expiratory flow limitation and/or dynamic hyperinflation with increased prevalence in WTC and Clinical Referral versus Control (55%, 87% versus 15%; p<0.001). Post-exercise oscillometry uncovered small airway hyperreactivity with increased prevalence in WTC and Clinical Referral versus Control (40%, 47% versus 0%, p<0.05). Conclusions: We uncovered mechanisms for exertional dyspnoea in subject with normal spirometry that was attributable to either small airway dysfunction during exercise and/or small airway hyperreactivity following exercise. The similarity of findings in WTC environmentally exposed and clinically referred cohorts suggests broad relevance for these evaluations.
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Malignant pleural effusions (MPE) complicate malignancies and portend worse outcomes. MPE is comprised of various components, including immune cells, cancer cells, and cell-free DNA/RNA. There have been investigations into using these components to diagnose and prognosticate MPE. We hypothesize that the microbiome of MPE is unique and may be associated with diagnosis and prognosis. We compared the microbiota of MPE against microbiota of pleural effusions from non-malignant and paramalignant states. We collected a total of 165 pleural fluid samples from 165 subjects; Benign (n = 16), Paramalignant (n = 21), MPE-Lung (n = 57), MPE-Other (n = 22), and Mesothelioma (n = 49). We performed high throughput 16S rRNA gene sequencing on pleural fluid samples and controls. We showed that there are compositional differences among pleural effusions related to non-malignant, paramalignant, and malignant disease. Furthermore, we showed differential enrichment of bacterial taxa within MPE depending on the site of primary malignancy. Pleural fluid of MPE-Lung and Mesothelioma were associated with enrichment with oral and gut bacteria that are commonly thought to be commensals, including Rickettsiella, Ruminococcus, Enterococcus, and Lactobacillales. Mortality in MPE-Lung is associated with enrichment in Methylobacterium, Blattabacterium, and Deinococcus. These observations lay the groundwork for future studies that explore host-microbiome interactions and their influence on carcinogenesis.
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Neoplasias Pulmonares , Mesotelioma Maligno , Mesotelioma , Microbiota , Derrame Pleural Maligno , Derrame Pleural , Humanos , RNA Ribossômico 16S/genética , Derrame Pleural Maligno/diagnóstico , Mesotelioma/diagnóstico , Mesotelioma/patologia , Biomarcadores , Derrame Pleural/diagnóstico , Prognóstico , Microbiota/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/complicaçõesRESUMO
OBJECTIVE: Although systemic thrombolysis (ST) is the standard of care in the treatment of high-risk pulmonary embolism (PE), large variations in real-world usage exist, including its use to treat intermediate-risk PE. A paucity of data is available to define the outcomes and practice patterns of the ST dose, duration, and treatment of presumed and imaging-confirmed PE. METHODS: We performed a multicenter retrospective study to evaluate the real-world practice patterns of ST use in the setting of acute PE (presumed vs imaging-confirmed intermediate- and high-risk PE). Patients who had received tissue plasminogen activator for PE between 2017 and 2019 were included. We compared the baseline clinical characteristics, tissue plasminogen activator practice patterns, and outcomes for patients with confirmed vs presumed PE. RESULTS: A total of 104 patients had received ST for PE: 52 with confirmed PE and 52 with presumed PE. Significantly more patients who had been treated for presumed PE had experienced cardiac arrest (n = 47; 90%) compared with those with confirmed PE (n = 23; 44%; P < .01). Survival to hospital discharge was 65% for the patients with confirmed PE vs 6% for those with presumed PE (P < .01). The use of ST was contraindicated for 56% of the patients with confirmed PE, with major bleeding in 26% but no intracranial hemorrhage. CONCLUSIONS: The in-hospital mortality of patients with confirmed acute PE has remained high (35%) in contemporary practice for those treated with ST. A large proportion of these patients had had contraindications to ST, and the rates of major bleeding were significant. Those with confirmed PE had had a higher survival rate compared with those with presumed PE, including those with cardiac arrest. This observation suggests a limited role for empiric thrombolysis in cardiac arrest situations.
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Parada Cardíaca , Embolia Pulmonar , Doença Aguda , Fibrinolíticos/efeitos adversos , Parada Cardíaca/tratamento farmacológico , Hemorragia/induzido quimicamente , Humanos , Embolia Pulmonar/diagnóstico por imagem , Embolia Pulmonar/tratamento farmacológico , Estudos Retrospectivos , Terapia Trombolítica/efeitos adversos , Ativador de Plasminogênio Tecidual/efeitos adversos , Resultado do TratamentoRESUMO
OBJECTIVES: Patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may develop end-stage lung disease requiring lung transplantation. We report the clinical course, pulmonary pathology with radiographic correlation, and outcomes after lung transplantation in three patients who developed chronic respiratory failure due to postacute sequelae of SARS-CoV-2 infection. METHODS: A retrospective histologic evaluation of explanted lungs due to coronavirus disease 2019 was performed. RESULTS: None of the patients had known prior pulmonary disease. The major pathologic findings in the lung explants were proliferative and fibrotic phases of diffuse alveolar damage, interstitial capillary neoangiogenesis, and mononuclear inflammation, specifically macrophages, with varying numbers of T and B lymphocytes. The fibrosis varied from early collagen deposition to more pronounced interstitial collagen deposition; however, pulmonary remodeling with honeycomb change was not present. Other findings included peribronchiolar metaplasia, microvascular thrombosis, recanalized thrombi in muscular arteries, and pleural adhesions. No patients had either recurrence of SARS-CoV-2 infection or allograft rejection following transplant at this time. CONCLUSIONS: The major pathologic findings in the lung explants of patients with SARS-CoV-2 infection suggest ongoing fibrosis, prominent macrophage infiltration, neoangiogenesis, and microvascular thrombosis. Characterization of pathologic findings could help develop novel management strategies.
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COVID-19 , Transplante de Pulmão , Trombose , COVID-19/complicações , Fibrose , Humanos , Pulmão/patologia , Transplante de Pulmão/efeitos adversos , Estudos Retrospectivos , SARS-CoV-2 , Trombose/patologiaRESUMO
Eukaryotic cells have evolved highly orchestrated protein catabolic machineries responsible for the timely and selective disposal of proteins and organelles, thereby ensuring amino acid recycling. However, how protein degradation is coordinated with amino acid supply and protein synthesis has remained largely elusive. Here we show that the mammalian proteasome undergoes liquid-liquid phase separation in the nucleus upon amino acid deprivation. We termed these proteasome condensates SIPAN (Starvation-Induced Proteasome Assemblies in the Nucleus) and show that these are a common response of mammalian cells to amino acid deprivation. SIPAN undergo fusion events, rapidly exchange proteasome particles with the surrounding milieu and quickly dissolve following amino acid replenishment. We further show that: (i) SIPAN contain K48-conjugated ubiquitin, (ii) proteasome inhibition accelerates SIPAN formation, (iii) deubiquitinase inhibition prevents SIPAN resolution and (iv) RAD23B proteasome shuttling factor is required for SIPAN formation. Finally, SIPAN formation is associated with decreased cell survival and p53-mediated apoptosis, which might contribute to tissue fitness in diverse pathophysiological conditions.
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Aminoácidos/metabolismo , Apoptose/fisiologia , Núcleo Celular/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inanição , Animais , Autoantígenos , Linhagem Celular Tumoral , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Eucarióticas , Exercício Físico , Fibroblastos , Humanos , Camundongos , Nutrientes , Biossíntese de Proteínas , Proteólise , Estresse Fisiológico , UbiquitinaRESUMO
KIF14 is a mitotic kinesin whose malfunction is associated with cerebral and renal developmental defects and several cancers. Like other kinesins, KIF14 couples ATP hydrolysis and microtubule binding to the generation of mechanical work, but the coupling mechanism between these processes is still not fully clear. Here we report 20 high-resolution (2.7-3.9 Å) cryo-electron microscopy KIF14-microtubule structures with complementary functional assays. Analysis procedures were implemented to separate coexisting conformations of microtubule-bound monomeric and dimeric KIF14 constructs. The data provide a comprehensive view of the microtubule and nucleotide induced KIF14 conformational changes. It shows that: 1) microtubule binding, the nucleotide species, and the neck-linker domain govern the transition between three major conformations of the motor domain; 2) an undocked neck-linker prevents the nucleotide-binding pocket to fully close and dampens ATP hydrolysis; 3) 13 neck-linker residues are required to assume a stable docked conformation; 4) the neck-linker position controls the hydrolysis rather than the nucleotide binding step; 5) the two motor domains of KIF14 dimers adopt distinct conformations when bound to the microtubule; and 6) the formation of the two-heads-bound-state introduces structural changes in both motor domains of KIF14 dimers. These observations provide the structural basis for a coordinated chemo-mechanical kinesin translocation model.
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Cinesinas/química , Cinesinas/metabolismo , Proteínas Oncogênicas/química , Proteínas Oncogênicas/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Cinesinas/genética , Ligantes , Camundongos , Microtúbulos/química , Microtúbulos/genética , Microtúbulos/metabolismo , Simulação de Acoplamento Molecular , Proteínas Oncogênicas/genética , Ligação Proteica , Conformação Proteica , Domínios ProteicosRESUMO
Coronavirus disease 2019 (COVID-19) is associated with increased rates of deep vein thrombosis (DVT) and pulmonary embolism (PE). Pulmonary Embolism Response Teams (PERT) have previously been associated with improved outcomes. We aimed to investigate whether PERT utilization, recommendations, and outcomes for patients diagnosed with acute PE changed during the COVID-19 pandemic. This is a retrospective cohort study of all adult patients with acute PE who received care at an academic hospital system in New York City between March 1st and April 30th, 2020. These patients were compared against historic controls between March 1st and April 30th, 2019. PE severity, PERT utilization, initial management, PERT recommendations, and outcomes were compared. There were more cases of PE during the pandemic (82 vs. 59), but less PERT activations (26.8% vs. 64.4%, p < 0.001) despite similar markers of PE severity. PERT recommendations were similar before and during the pandemic; anticoagulation was most recommended (89.5% vs. 86.4%, p = 0.70). During the pandemic, those with PERT activations were more likely to be female (63.6% vs. 31.7%, p = 0.01), have a history of DVT/PE (22.7% vs. 1.7%, p = 0.01), and to be SARS-CoV-2 PCR negative (68.2% vs. 38.3% p = 0.02). PERT activation during the pandemic is associated with decreased length of stay (7.7 ± 7.7 vs. 13.2 ± 12.7 days, p = 0.02). PERT utilization decreased during the COVID-19 pandemic and its activation was associated with different biases. PERT recommendations and outcomes were similar before and during the pandemic, and led to decreased length of stay during the pandemic.
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Anticoagulantes/administração & dosagem , Tratamento Farmacológico da COVID-19 , COVID-19 , Hospitais Universitários , Pandemias , Embolia Pulmonar , SARS-CoV-2/metabolismo , Idoso , Idoso de 80 Anos ou mais , COVID-19/sangue , COVID-19/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Cidade de Nova Iorque/epidemiologia , Guias de Prática Clínica como Assunto , Embolia Pulmonar/sangue , Embolia Pulmonar/tratamento farmacológico , Embolia Pulmonar/epidemiologia , Estudos Retrospectivos , Índice de Gravidade de DoençaRESUMO
Amputation of a salamander limb triggers a regeneration process that is perfect. A limited number of genes have been studied in this context and even fewer have been analyzed functionally. In this work, we use the BMP signaling inhibitor LDN193189 on Ambystoma mexicanum to explore the role of BMPs in regeneration. We find that BMP signaling is required for proper expression of various patterning genes and that its inhibition causes major defects in the regenerated limbs. Fgf8 is downregulated when BMP signaling is blocked, but ectopic injection of either human or axolotl protein did not rescue the defects. By administering LDN193189 treatments at different time points during regeneration, we show clearly that limb regeneration progresses in a proximal to distal fashion. This demonstrates that BMPs play a major role in patterning of regenerated limbs and that regeneration is a progressive process like development.
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Ambystoma mexicanum/metabolismo , Proteínas de Anfíbios/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Extremidades/fisiologia , Regeneração/fisiologia , Transdução de Sinais , Ambystoma mexicanum/crescimento & desenvolvimento , Proteínas de Anfíbios/genética , Animais , Proteínas Morfogenéticas Ósseas/genética , Proliferação de Células/efeitos dos fármacos , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Fator de Transcrição MSX1/genética , Fator de Transcrição MSX1/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Regeneração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismoRESUMO
By using the Cre-mediated genetic switch technology, we were able to successfully generate a conditional knock-in mouse, bearing the KIF2A p.His321Asp missense point variant, identified in a subject with malformations of cortical development. These mice present with neuroanatomical anomalies and microcephaly associated with behavioral deficiencies and susceptibility to epilepsy, correlating with the described human phenotype. Using the flexibility of this model, we investigated RosaCre-, NestinCre- and NexCre-driven expression of the mutation to dissect the pathophysiological mechanisms underlying neurodevelopmental cortical abnormalities. We show that the expression of the p.His321Asp pathogenic variant increases apoptosis and causes abnormal multipolar to bipolar transition in newborn neurons, providing therefore insights to better understand cortical organization and brain growth defects that characterize KIF2A-related human disorders. We further demonstrate that the observed cellular phenotypes are likely to be linked to deficiency in the microtubule depolymerizing function of KIF2A.
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Comportamento Animal , Cinesinas/fisiologia , Malformações do Desenvolvimento Cortical/patologia , Mutação , Neurônios/patologia , Proteínas Repressoras/fisiologia , Animais , Masculino , Malformações do Desenvolvimento Cortical/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismoRESUMO
DNA double strand breaks (DSBs) have detrimental effects on cell survival and genomic stability, and are related to cancer and other human diseases. In this study, we identified microtubule-depolymerizing kinesin Kif2C as a protein associated with DSB-mimicking DNA templates and known DSB repair proteins in Xenopus egg extracts and mammalian cells. The recruitment of Kif2C to DNA damage sites was dependent on both PARP and ATM activities. Kif2C knockdown or knockout led to accumulation of endogenous DNA damage, DNA damage hypersensitivity, and reduced DSB repair via both NHEJ and HR. Interestingly, Kif2C depletion, or inhibition of its microtubule depolymerase activity, reduced the mobility of DSBs, impaired the formation of DNA damage foci, and decreased the occurrence of foci fusion and resolution. Taken together, our study established Kif2C as a new player of the DNA damage response, and presented a new mechanism that governs DSB dynamics and repair.
DNA can be damaged in many ways, and a double strand break is one of the most dangerous. This occurs when both strands of the double helix snap at the same time, leaving two broken ends. When cells detect this kind of damage, they race to get it fixed as quickly as possible. Fixing these double strand breaks is thought to involve the broken ends being moved to 'repair centers' in the nucleus of the cell, but it was unclear how the broken ends were moved. One possibility was that the cells transport the broken ends along protein filaments called microtubules. Cells can assemble these track-like filaments on-demand to carry cargo attached to molecular motors called kinesins. However, this type of transport happens outside of the cell's nucleus, and while there are different kinesin proteins localized inside the nucleus, their roles are largely unknown. In an effort to understand how broken DNA ends are repaired, Zhu, Paydar et al. conducted experiments that simulated double strand breaks and examined the proteins that responded. The first set of experiments involved mixing cut pieces of DNA with extracts taken from frog eggs or human cells. Zhu, Paydar et al. found that one kinesin called Kif2C stuck to the DNA fragments, and attached to many proteins known to play a role in DNA damage repair. Kif2C had previously been shown to help separate the chromosomes during cell division. To find out more about its potential role in DNA repair, Zhu, Paydar et al. then used a laser to create breaks in the DNA of living human cells and tracked Kif2C movement. The kinesin arrived within 60 seconds of the DNA damage and appeared to transport the cut DNA ends to 'repair centers'. Getting rid of Kif2C, or blocking its activity, had dire effects on the cells' abilities to mobilize and repair breaks to its DNA. Without the molecular motor, fewer double strand breaks were repaired, and so DNA damage started to build up. Defects in double strand break repair happen in many human diseases, including cancer. Many cancer treatments damage the DNA of cancer cells, sometimes in combination with drugs that stop cells from building and using their microtubule transport systems. Understanding the new role of Kif2C in DNA damage repair could therefore help optimize these treatment combinations.
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Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Recombinação Homóloga , Cinesinas/fisiologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microtúbulos/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ligação Proteica , XenopusRESUMO
BACKGROUND: Postoperative ileus (POI) is a temporary delay of coordinated intestinal peristalsis. Alvimopan, an oral peripherally acting mu-opioid receptor antagonist approved for accelerating gastrointestinal recovery, has never been studied specifically in patients with inflammatory bowel disease (IBD). AIM: To investigate the efficacy of alvimopan in preventing POI among IBD patients. METHODS: A retrospective chart review was conducted on 246 IBD patients undergoing bowel surgery between 2012 and 2017. Data collected included demographics, IBD subtype, length of stay (LOS), postoperative gastrointestinal symptoms, and administration of alvimopan. The primary outcome was POI; secondary gastrointestinal recovery outcomes were: time to first flatus, time to first bowel movement, time to tolerating a liquid diet, time to tolerating solid food, and LOS. RESULTS: When compared with the control group, patients in the alvimopan group had shorter times to tolerating liquids and solids, first flatus, and first bowel movements (p < 0.01). LOS was shorter in the alvimopan group when compared with controls (p < 0.01). The overall incidence of POI was higher in controls than in the alvimopan group (p = 0.07). For laparoscopic surgeries, the incidence of POI was also higher in controls than in the alvimopan group (p < 0.01). On multivariable analysis, alvimopan significantly decreased time to all gastrointestinal recovery endpoints when compared to controls (p < 0.01). CONCLUSIONS: Alvimopan is effective in accelerating time to gastrointestinal recovery and reducing POI in IBD patients. While the benefits of alvimopan have been demonstrated previously, this is the first study of the efficacy of alvimopan in IBD patients.
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Fármacos Gastrointestinais/uso terapêutico , Íleus/prevenção & controle , Doenças Inflamatórias Intestinais/cirurgia , Piperidinas/uso terapêutico , Complicações Pós-Operatórias/prevenção & controle , Adulto , Feminino , Humanos , Íleus/diagnóstico , Íleus/etiologia , Doenças Inflamatórias Intestinais/diagnóstico , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/etiologia , Estudos RetrospectivosRESUMO
Viruses have transformed our understanding of mammalian RNA processing, including facilitating the discovery of the methyl-7-guanosine (m7G) cap on the 5' end of RNAs. The m7G cap is required for RNAs to bind the eukaryotic translation initiation factor eIF4E and associate with the translation machinery across plant and animal kingdoms. The potyvirus-derived viral genome-linked protein (VPg) is covalently bound to the 5' end of viral genomic RNA (gRNA) and associates with host eIF4E for successful infection. Divergent models to explain these observations proposed either an unknown mode of eIF4E engagement or a competition of VPg for the m7G cap-binding site. To dissect these possibilities, we resolved the structure of VPg, revealing a previously unknown 3-dimensional (3D) fold, and characterized the VPg-eIF4E complex using NMR and biophysical techniques. VPg directly bound the cap-binding site of eIF4E and competed for m7G cap analog binding. In human cells, VPg inhibited eIF4E-dependent RNA export, translation, and oncogenic transformation. Moreover, VPg formed trimeric complexes with eIF4E-eIF4G, eIF4E bound VPg-luciferase RNA conjugates, and these VPg-RNA conjugates were templates for translation. Informatic analyses revealed structural similarities between VPg and the human kinesin EG5. Consistently, EG5 directly bound eIF4E in a similar manner to VPg, demonstrating that this form of engagement is relevant beyond potyviruses. In all, we revealed an unprecedented modality for control and engagement of eIF4E and show that VPg-RNA conjugates functionally engage eIF4E. As such, potyvirus VPg provides a unique model system to interrogate eIF4E.
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Fator de Iniciação 4E em Eucariotos/química , Potyvirus/genética , Biossíntese de Proteínas/fisiologia , RNA/química , Ribonucleoproteínas/química , Proteínas Virais/química , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Capuzes de RNA/química , Processamento Pós-Transcricional do RNA , Ribonucleoproteínas/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais/fisiologiaRESUMO
Mutations in KIF14 have previously been associated with either severe, isolated or syndromic microcephaly with renal hypodysplasia (RHD). Syndromic microcephaly-RHD was strongly reminiscent of clinical ciliopathies, relating to defects of the primary cilium, a signalling organelle present on the surface of many quiescent cells. KIF14 encodes a mitotic kinesin, which plays a key role at the midbody during cytokinesis and has not previously been shown to be involved in cilia-related functions. Here, we analysed four families with fetuses presenting with the syndromic form and harbouring biallelic variants in KIF14. Our functional analyses showed that the identified variants severely impact the activity of KIF14 and likely correspond to loss-of-function mutations. Analysis in human fetal tissues further revealed the accumulation of KIF14-positive midbody remnants in the lumen of ureteric bud tips indicating a shared function of KIF14 during brain and kidney development. Subsequently, analysis of a kif14 mutant zebrafish line showed a conserved role for this mitotic kinesin. Interestingly, ciliopathy-associated phenotypes were also present in mutant embryos, supporting a potential direct or indirect role for KIF14 at cilia. However, our in vitro and in vivo analyses did not provide evidence of a direct role for KIF14 in ciliogenesis and suggested that loss of kif14 causes ciliopathy-like phenotypes through an accumulation of mitotic cells in ciliated tissues. Altogether, our results demonstrate that KIF14 mutations result in a severe syndrome associating microcephaly and RHD through its conserved function in cytokinesis during kidney and brain development.
Assuntos
Anormalidades Congênitas/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Nefropatias/congênito , Rim/anormalidades , Cinesinas/genética , Mutação com Perda de Função , Microcefalia/genética , Proteínas Oncogênicas/genética , Animais , Anormalidades Congênitas/metabolismo , Citocinese/genética , Modelos Animais de Doenças , Feminino , Imunofluorescência , Genes Letais , Estudos de Associação Genética/métodos , Loci Gênicos , Humanos , Rim/metabolismo , Nefropatias/genética , Nefropatias/metabolismo , Cinesinas/química , Cinesinas/metabolismo , Masculino , Microcefalia/metabolismo , Microcefalia/patologia , Proteínas Oncogênicas/química , Proteínas Oncogênicas/metabolismo , Linhagem , Fenótipo , Relação Estrutura-Atividade , Peixe-ZebraRESUMO
KEY POINTS: Studies of urothelial cells, bladder sheets or lumens of filled bladders have suggested that mediators released from urothelium into suburothelium (SubU)/lamina propria (LP) activate mechanisms controlling detrusor excitability. None of these approaches, however, has enabled direct assessment of availability of mediators at SubU/LP during filling. We developed an ex vivo mouse bladder preparation with intact urothelium and SubU/LP but no detrusor, which allows direct access to the SubU/LP surface of urothelium during filling. Pressure-volume measurements during filling demonstrated that bladder compliance is governed primarily by the urothelium. Measurements of purine mediators in this preparation demonstrated asymmetrical availability of purines in lumen and SubU/LP, suggesting that interpretations based solely on intraluminal measurements of mediators may be inaccurate. The preparations are suitable for assessments of release, degradation and transport of mediators in SubU/LP during bladder filling, and are superior to experimental approaches previously used for urothelium research. ABSTRACT: The purpose of this study was to develop a decentralized (ex vivo) detrusor smooth muscle (DSM)-denuded mouse bladder preparation, a novel model that enables studies on availability of urothelium-derived mediators at the luminal and anti-luminal aspects of the urothelium during filling. Urinary bladders were excised from C57BL6/J mice and the DSM was removed by fine-scissor dissection without touching the mucosa. Morphology and cell composition of the preparation wall, pressure-volume relationships during filling, and fluorescent dye permeability of control, protamine sulfate- and lipopolysaccharide-treated denuded bladders were characterized. The preparation wall contained intact urothelium and suburothelium (SubU)/lamina propria (LP) and lacked the DSM and the serosa. The utility of the model for physiological research was validated by measuring release, metabolism and transport of purine mediators at SubU/LP and in bladder lumen during filling. We determined asymmetrical availability of purines (e.g. ATP, ADP, AMP and adenosine) in lumen and at SubU/LP during filling, suggesting differential mechanisms of release, degradation and bilateral transurothelial transport of purines during filling. Some observations were validated in DSM-denuded bladder of the cynomolgus monkey (Macaca fascicularis). The novel model was superior to current models utilized to study properties of the urothelium (e.g. cultured urothelial cells, bladder mucosa sheets mounted in Ussing chambers or isolated bladder strips in organ baths) in that it enabled direct access to the vicinity of SubU/LP during authentic bladder filling. The model is particularly suitable for understanding local mechanisms of urothelium-DSM connectivity and for broad understanding of the role of urothelium in regulating continence and voiding.
Assuntos
Músculo Liso/fisiologia , Bexiga Urinária/fisiologia , Urotélio/fisiologia , Animais , Feminino , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Músculo Liso/citologia , Músculo Liso/metabolismo , Técnicas de Cultura de Órgãos/métodos , Purinas/metabolismo , Bexiga Urinária/citologia , Bexiga Urinária/metabolismo , Urotélio/citologia , Urotélio/metabolismoRESUMO
Kinesin-13 proteins are major microtubule (MT) regulatory factors that catalyze removal of tubulin subunits from MT ends. The class-specific "neck" and loop 2 regions of these motors are required for MT depolymerization, but their contributing roles are still unresolved because their interactions with MT ends have not been observed directly. Here we report the crystal structure of a catalytically active kinesin-13 monomer (Kif2A) in complex with two bent αß-tubulin heterodimers in a head-to-tail array, providing a view of these interactions. The neck of Kif2A binds to one tubulin dimer and the motor core to the other, guiding insertion of the KVD motif of loop 2 in between them. AMPPNP-bound Kif2A can form stable complexes with tubulin in solution and trigger MT depolymerization. We also demonstrate the importance of the neck in modulating ATP turnover and catalytic depolymerization of MTs. These results provide mechanistic insights into the catalytic cycles of kinesin-13.
Assuntos
Cinesinas/metabolismo , Microtúbulos/metabolismo , Polimerização , Multimerização Proteica , Tubulina (Proteína)/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Humanos , Cinesinas/química , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Neospora caninum is a tissue cyst-forming coccidium capable of causing spinal cord or skeletal muscle disease in dogs. Infected bitches can transmit the parasite to their pups in utero. Seroprevalence of N. caninum was studied in naturally-infected, privately owned Bernese Mountain dogs, using antibody detection via an indirect fluorescent antibody test (IFAT) to identify infected individuals. A retrospective study was undertaken on available dogs from 14 litters. Five of eight dams tested seropositive. The index case was a bitch with a titre of 1:3200 by IFAT. Only one offspring from her first litter was seropositive. The frequency of putative congenital transmission in the breeding kennel was variable. Our results reiterate the importance of serological testing of all dams and littermates in a breeding kennel when clinical neosporosis is suspected in neonatal puppies.