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Iron storage proteins, e.g., vertebrate ferritin, and the ferritin-like bacterioferritin (Bfr) and bacterial ferritin (Ftn), are spherical, hollow proteins that catalyze the oxidation of Fe2+ at binuclear iron ferroxidase centers (FOC) and store the Fe3+ in their interior, thus protecting cells from unwanted Fe3+/Fe2+ redox cycling and storing iron at concentrations far above the solubility of Fe3+. Vertebrate ferritins are heteropolymers of H and L subunits with only the H subunits having FOC. Bfr and Ftn were thought to coexist in bacteria as homopolymers, but recent evidence indicates these molecules are heteropolymers assembled from Bfr and Ftn subunits. Despite the heteropolymeric nature of vertebrate and bacterial ferritins, structures have been determined only for recombinant proteins constituted by a single subunit type. Herein we report the structure of Acinetobacter baumannii bacterioferritin, the first structural example of a heteropolymeric ferritin or ferritin-like molecule, assembled from completely overlapping Ftn homodimers harboring FOC and Bfr homodimers devoid of FOC but binding heme. The Ftn homodimers function by catalyzing the oxidation of Fe2+ to Fe3+, while the Bfr homodimers bind a cognate ferredoxin (Bfd) which reduces the stored Fe3+ by transferring electrons via the heme, enabling Fe2+ mobilization to the cytosol for incorporation in metabolism.
Assuntos
Acinetobacter baumannii , Proteínas de Bactérias , Grupo dos Citocromos b , Ferritinas , Ferritinas/química , Ferritinas/metabolismo , Acinetobacter baumannii/metabolismo , Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Multimerização Proteica , Ferro/metabolismo , Ferro/química , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Oxirredução , Conformação ProteicaRESUMO
In diderm bacteria, the Lol pathway canonically mediates the periplasmic transport of lipoproteins from the inner membrane (IM) to the outer membrane (OM) and therefore plays an essential role in bacterial envelope homeostasis. After extrusion of modified lipoproteins from the IM via the LolCDE complex, the periplasmic chaperone LolA carries lipoproteins through the periplasm and transfers them to the OM lipoprotein insertase LolB, itself a lipoprotein with a LolA-like fold. Yet, LolB homologs appear restricted to ψ-proteobacteria and are missing from spirochetes like the tick-borne Lyme disease pathogen Borrelia burgdorferi , suggesting a different hand-off mechanism at the OM. Here, we solved the crystal structure of the B. burgdorferi LolA homolog BB0346 (LolA Bb ) at 1.9 Å resolution. We identified multiple structural deviations in comparative analyses to other solved LolA structures, particularly a unique LolB-like protruding loop domain. LolA Bb failed to complement an Escherichia coli lolA knockout, even after codon optimization, signal I peptide adaptation, and a C-terminal chimerization which had allowed for complementation with an α-proteobacterial LolA. Analysis of a conditional B. burgdorferi lolA knockout strain indicated that LolA Bb was essential for growth. Intriguingly, protein localization assays indicated that initial depletion of LolA Bb led to an emerging mislocalization of both IM and periplasmic OM lipoproteins, but not surface lipoproteins. Together, these findings further support the presence of two separate primary secretion pathways for periplasmic and surface OM lipoproteins in B. burgdorferi and suggest that the distinct structural features of LolA Bb allow it to function in a unique LolB-deficient lipoprotein sorting system. SIGNIFICANCE: Borrelia spirochetes causing Lyme disease and relapsing fever have unusual double-membrane envelopes that instead of lipopolysaccharide (LPS) display abundant surface lipoproteins. We recently showed that secretion of these surface lipoproteins in Borrelia burgdorferi depends on a distant homolog of the canonical LPS outer membrane translocase LptD. Here, we probed the role of the B. burgdorferi Lol pathway in lipoprotein sorting and secretion. We show that the periplasmic chaperone LolA is essential, functionally different from E. coli LolA, with structural features of a bifunctional lipoprotein carrier protein operating without a downstream LolB outer membrane lipoprotein insertase. Depletion of LolA did not impact surface lipoprotein localization but led to a marked mislocalization of inner membrane lipoproteins to the outer membrane. This further supports two parallel, yet potentially interacting Borrelia lipoprotein transport pathways that are responsible for either secreting surface lipoprotein virulence factors or maintaining proper distribution of lipoproteins within the periplasmic space.
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Zoonotic coronaviruses are known to produce severe infections in humans and have been the cause of significant morbidity and mortality worldwide. SARS-CoV-2 was the largest and latest contributor of fatal cases, even though MERS-CoV has the highest case-fatality ratio among zoonotic coronaviruses. These infections pose a high risk to public health worldwide warranting efforts for the expeditious discovery of antivirals. Hence, we hereby describe a novel series of inhibitors of coronavirus 3CLpro embodying an N-substituted 2-pyrrolidone scaffold envisaged to exploit favorable interactions with the S3-S4 subsites and connected to an invariant Leu-Gln P2-P1 recognition element. Several inhibitors showed nanomolar antiviral activity in enzyme and cell-based assays, with no significant cytotoxicity. High-resolution crystal structures of inhibitors bound to the 3CLpro were determined to probe and identify the molecular determinants associated with binding, to inform the structure-guided optimization of the inhibitors, and to confirm the mechanism of action of the inhibitors.
Assuntos
Antivirais , Proteases 3C de Coronavírus , Desenho de Fármacos , SARS-CoV-2 , Antivirais/farmacologia , Antivirais/química , Antivirais/síntese química , Humanos , SARS-CoV-2/efeitos dos fármacos , Cristalografia por Raios X , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/metabolismo , Pirrolidinonas/farmacologia , Pirrolidinonas/química , Relação Estrutura-Atividade , Animais , Modelos MolecularesRESUMO
In efforts towards eliminating malaria, a discovery program was initiated to identify a novel antimalarial using KAF156 as a starting point. Following the most recent TCP/TPP guidelines, we have identified mCMQ069 with a predicted single oral dose for treatment (â¼40-106 mg) and one-month chemoprevention (â¼96-216 mg). We have improved unbound MPC and predicted human clearance by 18-fold and 10-fold respectively when compared to KAF156.
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Lactate dehydrogenase-A (LDHA) is the major isoform of lactate dehydrogenases (LDH) that is overexpressed and linked to poor survival in pancreatic ductal adenocarcinoma (PDAC). Despite some progress, current LDH inhibitors have poor structural and physicochemical properties or exhibit unfavorable pharmacokinetics that have hampered their development. The present study reports the synthesis and biological evaluation of a novel class of LDHA inhibitors comprising a succinic acid monoamide motif. Compounds 6 and 21 are structurally related analogs that demonstrated potent inhibition of LDHA with IC50s of 46 nM and 72 nM, respectively. We solved cocrystal structures of compound 21-bound to LDHA that showed that the compound binds to a distinct allosteric site between the two subunits of the LDHA tetramer. Inhibition of LDHA correlated with reduced lactate production and reduction of glycolysis in MIA PaCa-2 pancreatic cancer cells. The lead compounds inhibit the proliferation of human pancreatic cancer cell lines and patient-derived 3D organoids and exhibit a synergistic cytotoxic effect with the OXPHOS inhibitor phenformin. Unlike current LDHA inhibitors, 6 and 21 have appropriate pharmacokinetics and ligand efficiency metrics, exhibit up to 73% oral bioavailability, and a cumulative half-life greater than 4 h in mice.
Assuntos
Antineoplásicos , Proliferação de Células , Inibidores Enzimáticos , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Animais , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Proliferação de Células/efeitos dos fármacos , Administração Oral , Camundongos , Relação Estrutura-Atividade , Estrutura Molecular , Ensaios de Seleção de Medicamentos Antitumorais , Disponibilidade Biológica , Relação Dose-Resposta a Droga , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/metabolismo , Linhagem Celular Tumoral , Modelos MolecularesRESUMO
Infected cell protein 0 (ICP0) is an immediate-early regulatory protein of herpes simplex virus 1 (HSV-1) that possesses E3 ubiquitin ligase activity. ICP0 transactivates viral genes, in part, through its C-terminal dimer domain (residues 555-767). Deletion of this dimer domain results in reduced viral gene expression, lytic infection, and reactivation from latency. Since ICP0's dimer domain is associated with its transactivation activity and efficient viral replication, we wanted to determine the structure of this specific domain. The C-terminus of ICP0 was purified from bacteria and analyzed by X-ray crystallography to solve its structure. Each subunit or monomer in the ICP0 dimer is composed of nine ß-strands and two α-helices. Interestingly, two adjacent ß-strands from one monomer "reach" into the adjacent subunit during dimer formation, generating two ß-barrel-like structures. Additionally, crystallographic analyses indicate a tetramer structure is formed from two ß-strands of each dimer, creating a "stacking" of the ß-barrels. The structural protein database searches indicate the fold or structure adopted by the ICP0 dimer is novel. The dimer is held together by an extensive network of hydrogen bonds. Computational analyses reveal that ICP0 can either form a dimer or bind to SUMO1 via its C-terminal SUMO-interacting motifs but not both. Understanding the structure of the dimer domain will provide insights into the activities of ICP0 and, ultimately, the HSV-1 life cycle.
Assuntos
Herpesvirus Humano 1 , Proteínas Imediatamente Precoces , Multimerização Proteica , Ubiquitina-Proteína Ligases , Proteínas Imediatamente Precoces/química , Proteínas Imediatamente Precoces/metabolismo , Proteínas Imediatamente Precoces/genética , Cristalografia por Raios X , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Modelos Moleculares , Humanos , Domínios Proteicos , Dobramento de Proteína , Sequência de Aminoácidos , Conformação Proteica em Folha betaRESUMO
Infected cell protein 0 (ICP0) is an immediate-early regulatory protein of herpes simplex virus 1 (HSV-1) that possesses E3 ubiquitin ligase activity. ICP0 transactivates viral genes, in part, through its C-terminal dimer domain (residues 555-767). Deletion of this dimer domain results in reduced viral gene expression, lytic infection, and reactivation from latency. Since ICP0's dimer domain is associated with its transactivation activity and efficient viral replication, we wanted to determine the structure of this specific domain. The C-terminus of ICP0 was purified from bacteria and analyzed by X-ray crystallography to solve its structure. Each subunit or monomer in the ICP0 dimer is composed of nine ß-strands and two α-helices. Interestingly, two adjacent ß-strands from one monomer "reach" into the adjacent subunit during dimer formation, generating two ß-barrel-like structures. Additionally, crystallographic analyses indicate a tetramer structure is formed from two ß-strands of each dimer, creating a "stacking" of the ß-barrels. The structural protein database searches indicate the fold or structure adopted by the ICP0 dimer is novel. The dimer is held together by an extensive network of hydrogen bonds. Computational analyses reveal that ICP0 can either form a dimer or bind to SUMO1 via its C-terminal SUMO-interacting motifs but not both. Understanding the structure of the dimer domain will provide insights into the activities of ICP0 and, ultimately, the HSV-1 life cycle.
RESUMO
NADH cytochrome b5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b5 (b5 ) and cytochrome b5 reductase (b5 R) domains separated by a CHORD-Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full-length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high-resolution atomic structures of the b5 domain and a construct containing the CS and b5 R domains (CS/b5 R). Ncb5or also contains an N-terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L34 MDWIRL40 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to a b5 domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N-terminal region naturally fused to the b5 domain, we were able to obtain a high-resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25-129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp120 (corresponding to invariant Trp37 in Ncb5or) to be part of an 11-residue α-helix (S116 QMDWLKLTRT126 ) packing against two of the four helices in the b5 domain that surround heme (α2 and α5). The Trp120 side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr85 and Tyr88 (α2), Cys124 (α5), and Leu47 in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp37 in nucleating the formation of the N-terminal helix, whose location in the N/b5 module suggests a role in regulating the function of this multi-domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b5 module as reflected by its existence in a class of cytochrome b5 proteins from three kingdoms among eukaryotes.
Assuntos
Citocromos b , NAD , Animais , Humanos , Citocromo-B(5) Redutase/química , Oxirredutases , Heme/químicaRESUMO
The high morbidity and mortality associated with SARS-CoV-2 infection, the etiological agent of COVID-19, has had a major impact on global public health. Significant progress has been made in the development of an array of vaccines and biologics, however, the emergence of SARS-CoV-2 variants and breakthrough infections are an ongoing major concern. Furthermore, there is an existing paucity of small-molecule host and virus-directed therapeutics and prophylactics that can be used to counter the spread of SARS-CoV-2, and any emerging and re-emerging coronaviruses. We describe herein our efforts to address this urgent need by focusing on the structure-guided design of potent broad-spectrum inhibitors of SARS-CoV-2 3C-like protease (3CLpro or Main protease), an enzyme essential for viral replication. The inhibitors exploit the directional effects associated with the presence of a gem-dimethyl group that allow the inhibitors to optimally interact with the S4 subsite of the enzyme. Several compounds were found to potently inhibit SARS-CoV-2 and MERS-CoV 3CL proteases in biochemical and cell-based assays. Specifically, the EC50 values of aldehyde 1c and its corresponding bisulfite adduct 1d against SARS-CoV-2 were found to be 12 and 10 nM, respectively, and their CC50 values were >50 µM. Furthermore, deuteration of these compounds yielded compounds 2c/2d with EC50 values 11 and 12 nM, respectively. Replacement of the aldehyde warhead with a nitrile (CN) or an α-ketoamide warhead or its corresponding bisulfite adduct yielded compounds 1g, 1eand1f with EC50 values 60, 50 and 70 nM, respectively. High-resolution cocrystal structures have identified the structural determinants associated with the binding of the inhibitors to the active site of the enzyme and, furthermore, have illuminated the mechanism of action of the inhibitors. Overall, the high Safety Index (SI) (SI=CC50/EC50) displayed by these compounds suggests that they are well-suited to conducting further preclinical studies.
Assuntos
COVID-19 , Hepatite C Crônica , Coronavírus da Síndrome Respiratória do Oriente Médio , Humanos , SARS-CoV-2/metabolismo , Antivirais/farmacologia , Antivirais/química , Peptídeo Hidrolases , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Cisteína Endopeptidases/metabolismoRESUMO
Somatic activating mutations of PIK3CA are associated with development of vascular malformations (VMs). Here, we describe a microfluidic model of PIK3CA-driven VMs consisting of human umbilical vein endothelial cells expressing PIK3CA activating mutations embedded in three-dimensional hydrogels. We observed enlarged, irregular vessel phenotypes and the formation of cyst-like structures consistent with clinical signatures and not previously observed in cell culture models. Pathologic morphologies occurred concomitant with up-regulation of Rac1/p21-activated kinase (PAK), mitogen-activated protein kinase cascades (MEK/ERK), and mammalian target of rapamycin (mTORC1/2) signaling networks. We observed differential effects between alpelisib, a PIK3CA inhibitor, and rapamycin, an mTORC1 inhibitor, in mitigating matrix degradation and network topology. While both were effective in preventing vessel enlargement, rapamycin failed to reduce MEK/ERK and mTORC2 activity and resulted in hyperbranching, while inhibiting PAK, MEK1/2, and mTORC1/2 mitigates abnormal growth and vascular dilation. Collectively, these findings demonstrate an in vitro platform for VMs and establish a role of dysregulated Rac1/PAK and mTORC1/2 signaling in PIK3CA-driven VMs.
Assuntos
Serina-Treonina Quinases TOR , Malformações Vasculares , Humanos , Serina-Treonina Quinases TOR/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Sirolimo/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Malformações Vasculares/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
IMPORTANCE: Indwelling catheters are a known source of dissatisfaction for postoperative patients. There is a paucity of data describing patient-perceived outcomes associated with the alternative of intermittent self-catheterization (ISC). OBJECTIVES: The aim of this study was to describe patient satisfaction and outcomes associated with ISC after outpatient female pelvic reconstructive surgery. STUDY DESIGN: This was a secondary analysis of a prospective cohort study at an academic tertiary referral center from September 2018 to June 2021. Participants completed preoperative ISC instruction that included an instructional video, 1:1 demonstration with a health care provider, and provision of ISC supplies. Participants were instructed to perform ISC postoperatively until they had 2 consecutive outpatient PVRs less than one-half the voided volume. Participant satisfaction was assessed 2 weeks postprocedure, with adverse events evaluated at 6 weeks. RESULTS: One hundred sixty participants completed preoperative ISC instruction and were included in this analysis. Mean age was 52.1 (SD +/- 11.4) years, mean body mass index was 28.9 (SD +/- 5.8), and mean time from ISC instruction to surgery was 16.4 (SD +/- 15.7) days. Most participants reported no difficulty with ISC (124/160 [78%]) and had high levels of satisfaction (148/151 [98%]). Difficulty performing ISC was not associated with time since ISC instruction ( P = 0.32), difficulty noted at ISC instruction by the health care provider ( P = 0.24), or the duration of ISC instruction ( P = 0.16). On multiple logistic regression, age, body mass index, and prolapse beyond the hymen did not predict difficulty learning or performing ISC. At 6 weeks postprocedure, 22 of 155 participants (14%) endorsed symptoms of a urinary tract infection, and 15 of 160 (9%) had a culture-proven urinary tract infection. CONCLUSIONS: Women undergoing outpatient pelvic reconstructive surgery report ease and satisfaction with ISC.
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Cateteres de Demora , Satisfação do Paciente , Infecções Urinárias , Feminino , Humanos , Pessoa de Meia-Idade , Cateterismo , Estudos Prospectivos , Adulto , IdosoRESUMO
In heart failure with preserved ejection fraction (HFpEF), the occurrence of myocardial fibrosis is associated with adverse outcome. Whether pirfenidone, an oral antifibrotic agent without hemodynamic effect, is efficacious and safe for the treatment of HFpEF is unknown. In this double-blind, phase 2 trial ( NCT02932566 ), we enrolled patients with heart failure, an ejection fraction of 45% or higher and elevated levels of natriuretic peptides. Eligible patients underwent cardiovascular magnetic resonance and those with evidence of myocardial fibrosis, defined as a myocardial extracellular volume of 27% or greater, were randomly assigned to receive pirfenidone or placebo for 52 weeks. Forty-seven patients were randomized to each of the pirfenidone and placebo groups. The primary outcome was change in myocardial extracellular volume, from baseline to 52 weeks. In comparison to placebo, pirfenidone reduced myocardial extracellular volume (between-group difference, -1.21%; 95% confidence interval, -2.12 to -0.31; P = 0.009), meeting the predefined primary outcome. Twelve patients (26%) in the pirfenidone group and 14 patients (30%) in the placebo group experienced one or more serious adverse events. The most common adverse events in the pirfenidone group were nausea, insomnia and rash. In conclusion, among patients with HFpEF and myocardial fibrosis, administration of pirfenidone for 52 weeks reduced myocardial fibrosis. The favorable effects of pirfenidone in patients with HFpEF will need to be confirmed in future trials.
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Insuficiência Cardíaca/tratamento farmacológico , Piridonas/uso terapêutico , Idoso , Feminino , Humanos , Masculino , Piridonas/efeitos adversos , Fatores de Tempo , Resultado do TratamentoRESUMO
The metabotropic glutamate receptor 5 (mGlu5) is a recognized central nervous system therapeutic target for which several negative allosteric modulator (NAM) drug candidates have or are continuing to be investigated for various disease indications in clinical development. Direct measurement of target receptor occupancy (RO) is extremely useful to help design and interpret efficacy and safety in nonclinical and clinical studies. In the mGlu5 field, this has been successfully achieved by monitoring displacement of radiolabeled ligands, specifically binding to the mGlu5 receptor, in the presence of an mGlu5 NAM using in vivo and ex vivo binding in rodents and positron emission tomography imaging in cynomolgus monkeys and humans. The aim of this study was to measure the RO of the mGlu5 NAM HTL0014242 in rodents and cynomolgus monkeys and to compare its plasma and brain exposure-RO relationships with those of clinically tested mGlu5 NAMs dipraglurant, mavoglurant, and basimglurant. Potential sources of variability that may contribute to these relationships were explored. Distinct plasma exposure-response relationships were found for each mGlu5 NAM, with >100-fold difference in plasma exposure for a given level of RO. However, a unified exposure-response relationship was observed when both unbound brain concentration and mGlu5 affinity were considered. This relationship showed <10-fold overall difference, was fitted with a Hill slope that was not significantly different from 1, and appeared consistent with a simple Emax model. This is the first time this type of comparison has been conducted, demonstrating a unified brain exposure-RO relationship across several species and mGlu5 NAMs with diverse properties. SIGNIFICANCE STATEMENT: Despite the long history of mGlu5 as a therapeutic target and progression of multiple compounds to the clinic, no formal comparison of exposure-receptor occupancy relationships has been conducted. The data from this study indicate for the first time that a consistent, unified relationship can be observed between exposure and mGlu5 receptor occupancy when unbound brain concentration and receptor affinity are taken into account across a range of species for a diverse set of mGlu5 negative allosteric modulators, including a new drug candidate, HTL0014242.
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Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacocinética , Receptor de Glutamato Metabotrópico 5/metabolismo , Administração Oral , Regulação Alostérica , Sítio Alostérico , Animais , Encéfalo/metabolismo , Estudos Clínicos como Assunto , Relação Dose-Resposta a Droga , Fármacos Atuantes sobre Aminoácidos Excitatórios/administração & dosagem , Fármacos Atuantes sobre Aminoácidos Excitatórios/sangue , Imidazóis/administração & dosagem , Imidazóis/sangue , Imidazóis/farmacocinética , Indóis/administração & dosagem , Indóis/sangue , Indóis/farmacocinética , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Piridinas/administração & dosagem , Piridinas/sangue , Piridinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Receptor de Glutamato Metabotrópico 5/químicaRESUMO
Postpartum urinary retention (PUR) is a common and potentially morbid condition if not recognised and managed promptly. We surveyed obstetrics and gynaecology (OBGYN) residents to determine residents' knowledge of the management of PUR. A total of 168 OBGYN residents in ACGME accredited programmes in the United States completed the survey. A percentage of 30.3 reported having a PUR prevention protocol at their institution, 43.3% reported not having a protocol and 26.7% did not know whether a protocol existed. About 89.3% of participants reported having previously taken care of a patient with PUR and 17.1% reported prior formal teaching on the management of PUR. Those who reported having a protocol were more likely to report feeling comfortable managing PUR. Overall, knowledge was low for management of PUR. Given the potential morbidity associated with inadequate management of PUR, formal education and standardisation through national guidelines may help improve care of patients with PUR.Impact statementWhat is already known on this subject? PUR is a common condition and if left untreated may lead to long-term impacts on patients' health. Early recognition of the condition and appropriate management can prevent these complications. Protocols have been shown to improve patient outcomes. Thus, it has been postulated that the implementation of protocols could improve recognition of the condition.What do the results of this study add? No previous studies have looked at the impact of PUR management protocols on physicians in training. Because physicians in training are often the first-in-line to manage patients at academic institutions, we sought to determine the proportion of obstetrics and gynaecology residents in the United States who report having a PUR management protocol at their institution and how this impacts their reported comfort at caring for patients with PUR, knowledge on PUR risk factors and recognition of scenarios concerning for PUR. While awareness of a PUR protocol did not lead to increased knowledge of risk factors or increased recognition of scenarios concerning for PUR, it did increase resident comfort with managing PUR patients. We also found that overall PUR knowledge was low.What are the implications of these findings for clinical practice and/or further research? Based on our findings, OBGYN residents would benefit from having protocols at their institutions since it increased their comfort at managing patients with PUR. Further, formal education on PUR is likely needed to improve knowledge of risk factors and recognition of scenarios concerning for PUR.
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Competência Clínica/estatística & dados numéricos , Internato e Residência/estatística & dados numéricos , Obstetrícia/educação , Transtornos Puerperais , Retenção Urinária , Adulto , Gerenciamento Clínico , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Masculino , Gravidez , Inquéritos e Questionários , Estados UnidosRESUMO
Ncb5or (NADH-cytochrome b5 oxidoreductase), a cytosolic ferric reductase implicated in diabetes and neurological diseases, comprises three distinct domains, cytochrome b5 (b5) and cytochrome b5 reductase (b5R) domains separated by a CHORD-Sgt1 (CS) domain, and a novel 50-residue N-terminal region. Understanding how interdomain interactions in Ncb5or facilitate the shuttling of electrons from NAD(P)H to heme, and how the process compares with the microsomal b5 (Cyb5A) and b5R (Cyb5R3) system, is of interest. A high-resolution structure of the b5 domain (PDB entry 3lf5) has previously been reported, which exhibits substantial differences in comparison to Cyb5A. The structural characterization of a construct comprising the naturally fused CS and b5R domains with bound FAD and NAD+ (PDB entry 6mv1) or NADP+ (PDB entry 6mv2) is now reported. The structures reveal that the linker between the CS and b5R cores is more ordered than predicted, with much of it extending the ß-sandwich motif of the CS domain. This limits the flexibility between the two domains, which recognize one another via a short ß-sheet motif and a network of conserved side-chain hydrogen bonds, salt bridges and cation-π interactions. Notable differences in FAD-protein interactions in Ncb5or and Cyb5R3 provide insight into the selectivity for docking of their respective b5 redox partners. The structures also afford a structural explanation for the unusual ability of Ncb5or to utilize both NADH and NADPH, and represent the first examples of native, fully oxidized b5R family members in which the nicotinamide ring of NAD(P)+ resides in the active site. Finally, the structures, together with sequence alignments, show that the b5R domain is more closely related to single-domain Cyb5R proteins from plants, fungi and some protists than to Cyb5R3 from animals.
Assuntos
Citocromo-B(5) Redutase/química , Citocromos b5/química , NADP/química , Proteínas de Transporte/química , Domínio Catalítico , Cristalização , Heme/química , Humanos , Ligação de Hidrogênio , Cinética , Proteínas de Membrana/química , Modelos Moleculares , Complexos Multiproteicos , NAD/química , Oxirredução , Proteínas de Ligação a Fosfato , Conformação Proteica em Folha beta , Domínios Proteicos , Proteínas Recombinantes/químicaRESUMO
BACKGROUND: The PIROUETTE (PIRfenidOne in patients with heart failUre and preserved lEfT venTricular Ejection fraction) trial is designed to evaluate the efficacy and safety of the anti-fibrotic pirfenidone in patients with chronic heart failure and preserved ejection fraction (HFpEF) and myocardial fibrosis. HFpEF is a diverse syndrome associated with substantial morbidity and mortality. Myocardial fibrosis is a key pathophysiological mechanism of HFpEF and myocardial fibrotic burden is strongly and independently associated with adverse outcome. Pirfenidone is an oral anti-fibrotic agent, without haemodynamic effect, that leads to regression of myocardial fibrosis in preclinical models. It has proven clinical effectiveness in pulmonary fibrosis. METHODS: The PIROUETTE trial is a randomised, double-blind, placebo-controlled phase II trial evaluating the efficacy and safety of 52 weeks of treatment with pirfenidone in patients with chronic HFpEF (symptoms and signs of heart failure, left ventricular ejection fraction ≥ 45%, elevated natriuretic peptides [BNP ≥ 100 pg/ml or NT-proBNP ≥ 300 pg/ml; or BNP ≥ 300 pg/ml or NT-proBNP ≥ 900 pg/ml if in atrial fibrillation]) and myocardial fibrosis (extracellular matrix (ECM) volume ≥ 27% measured using cardiovascular magnetic resonance). The primary outcome measure is change in myocardial ECM volume. A sub-study will investigate the relationship between myocardial fibrosis and myocardial energetics, and the impact of pirfenidone, using 31phosphorus magnetic resonance spectroscopy. DISCUSSION: PIROUETTE will determine whether pirfenidone is superior to placebo in relation to regression of myocardial fibrosis and improvement in myocardial energetics in patients with HFpEF and myocardial fibrosis (NCT02932566). CLINICAL TRIAL REGISTRATION: clinicaltrials.gov (NCT02932566) https://clinicaltrials.gov/ct2/show/NCT02932566.
Assuntos
Insuficiência Cardíaca/tratamento farmacológico , Piridonas/uso terapêutico , Adulto , Idoso , Método Duplo-Cego , Feminino , Fibrose , Insuficiência Cardíaca/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeo Natriurético Encefálico , Fragmentos de Peptídeos , Volume Sistólico , Função Ventricular Esquerda/fisiologiaRESUMO
OBJECTIVE: Placenta accreta is a feared pathology, in part, because prenatal diagnosis is imperfect. It is not known whether clinical risk factors or sonographic features equally predict the entire graded pathological spectrum of placental overinvasion disease nor whether clinical outcomes differ along the spectrum. STUDY DESIGN: We conducted a mixed methods retrospective study of a cohort of women screened sonographically for placenta accreta, cross-referenced against cases identified by pathological diagnosis (N = 416). Demographic, diagnostic, and outcome information were compared across the spectrum of invasive placentation: percreta, increta, accreta, and focal accreta not requiring hysterectomy. The t-test, chi-square, Mann-Whitney, and Kruskal-Wallis tests were used for statistical analysis across groups. RESULTS: As the depth of invasion decreased, risk factors for placental overinvasion were less common, especially placenta previa and previous cesarean. There was also reduced anticipation by sonographic examination of the placenta. Rates of adverse outcomes were lower among women with focal accreta compared with those with deeper invasion. CONCLUSION: As the depth of invasion decreases, clinical risk factors and sonographic evaluation are less reliable in the antenatal prediction of placenta accreta. The potential for unanticipated morbidity underscores the need for improved diagnostic tools for placenta accreta spectrum.
Assuntos
Placenta Acreta/diagnóstico por imagem , Ultrassonografia Pré-Natal , Adulto , Cesárea , Feminino , Humanos , Histerectomia , Idade Materna , Gravidade do Paciente , Placenta/diagnóstico por imagem , Placenta/patologia , Placenta Acreta/patologia , Placenta Acreta/cirurgia , Gravidez , Resultado da Gravidez , Nascimento Prematuro/epidemiologia , Estudos Retrospectivos , Fatores de RiscoRESUMO
OBJECTIVE: To develop a novel method for capturing the discrepancy between objective tests and subjective dryness symptoms (a sensitivity scale) and to explore predictors of dryness sensitivity. METHODS: Archive data from the UK Primary Sjögren's Syndrome Registry (n = 688) were used. Patients were classified on a scale from -5 (stoical) to +5 (sensitive) depending on the degree of discrepancy between their objective and subjective symptoms classes. Sensitivity scores were correlated with demographic variables, disease-related factors, and symptoms of pain, fatigue, anxiety, and depression. RESULTS: Patients were on average relatively stoical for both types of dryness symptoms (mean ± SD ocular dryness -0.42 ± 2.2 and -1.24 ± 1.6 oral dryness). Twenty-seven percent of patients were classified as sensitive to ocular dryness and 9% to oral dryness. Hierarchical regression analyses identified the strongest predictor of ocular dryness sensitivity to be self-reported pain and that of oral dryness sensitivity to be self-reported fatigue. CONCLUSION: Ocular and oral dryness sensitivity can be classified on a continuous scale. The 2 symptom types are predicted by different variables. A large number of factors remain to be explored that may impact symptom sensitivity in primary Sjögren's syndrome, and the proposed method could be used to identify relatively sensitive and stoical patients for future studies.
Assuntos
Autoavaliação Diagnóstica , Síndrome de Sjogren/diagnóstico , Xeroftalmia/diagnóstico , Xerostomia/diagnóstico , Idoso , Fadiga/diagnóstico , Fadiga/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dor/diagnóstico , Dor/epidemiologia , Sistema de Registros , Síndrome de Sjogren/epidemiologia , Reino Unido/epidemiologia , Xeroftalmia/epidemiologia , Xerostomia/epidemiologiaRESUMO
In this chapter we demonstrate a method to produce virus-like particles (VLPs) from Escherichia coli. Standard bacterial protocols are used for the cloning, transformation, and expression of the protein subunits. A two-step protein purification method is highlighted: one step based on separating soluble proteins with ion-exchange affinity chromatography and a second polishing step using size-exclusion columns to isolate VLP species. The ensuing VLPs can be characterized with a variety of biophysical techniques including ultraviolet (UV)-visible spectroscopy for protein quantification, dynamic light scattering for size distribution determination, and transmission electron microscopy to ascertain size and morphology.
Assuntos
Escherichia coli/genética , Engenharia Genética/métodos , Vacinas de Partículas Semelhantes a Vírus/genética , Proteínas do Capsídeo/genética , Clonagem Molecular , Avaliação Pré-Clínica de Medicamentos , Difusão Dinâmica da Luz , Microscopia Eletrônica de Transmissão , Espectrofotometria Ultravioleta , Transformação Genética , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas de Partículas Semelhantes a Vírus/química , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificaçãoRESUMO
Nursing has given me the most amazing opportunities, from bedside nursing, where I have cared for some incredible people, to leading in national arenas.