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INTRODUCTION: The threshold of a Leg Length Discrepancy (LLD) by clinical examination on a sheet or centimeter paper (CP) is not known precisely whether or not it concerns limbs equipped with a hip prosthesis. We therefore conducted a prospective in silico study in order to: (1) determine the reproducibility and sensitivity of the clinical measurement of the LLD in different ideal and "degraded" clinical situations, (2) determine the threshold from which the human eye is capable of detecting a length inequality in clinic, (3) to determine whether the use of a graduated support (centimeter paper) improves the clinical measurement threshold. HYPOTHESIS: Our hypothesis was that clinical measurement on a centimeter support would improve clinical measurement accuracy. MATERIAL AND METHODS: This was an in silico study, the experiment was conducted on a mannequin. Different inequalities were created on a mannequin and photographed with a total of 30 inequalities from -22 to +22 mm on sheet or centimeter paper (CP). This was a multicenter study, with 40 different readers. We asked the readers to make a second measurement one month later. We evaluated the inter- and intra-observer reproducibility. The error rate at the threshold of 3 mm and 5 mm were calculated versus the gold standard. Finally, we determined at which thresholds respectively 75% and 95% of the measurements were correct. RESULTS: A total of 4140 measurements were performed and compared to the gold standard. With a threshold of 75% accurate measurement, the LLD detection threshold was 2.8 mm on centimeter paper and 4.5 mm on sheet. With a threshold of 95% accurate measurement, the LLD detection threshold was 3.4 mm on centimeter paper and 5.2 mm on sheet. Interobserver agreement (assessed overall on the 40 observers by Krippendorff's generalized Kappa) was 0.86 (95% confidence interval (CI95%) = 0.79 to 0.92) on CP and 0.71 (CI95% = 0.63 to 0.79) on sheet. Intra-observer agreement assessed by the intraclass correlation coefficient among observers who made 2 measurements had a median value (IQR) of 0.96 (0.94 to 0.99) on CP and 0.90 (0.83 to 0.94) on sheet. DISCUSSION: The clinical detection threshold on sheet at the patient's bed appears close to 5 mm. A more precise measurement is possible with graduated centimeter paper. A study in daily practice on patients in real situations would confirm our results. LEVEL OF EVIDENCE: III; prospective diagnostic comparative in Silico study.
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BACKGROUND: Prosthetic joint infection (PJI) caused by Candida spp is a severe complication of arthroplasty. We investigated the outcomes of Candida PJI. METHODS: This was a retrospective observational multinational study including patients diagnosed with Candida-related PJI between 2010 and 2021. Treatment outcome was assessed at 2-year follow-up. RESULTS: A total of 269 patients were analyzed. Median age was 73.0 (interquartile range [IQR], 64.0-79.0) years; 46.5% of patients were male and 10.8% were immunosuppressed. Main infection sites were hip (53.0%) and knee (43.1%), and 33.8% patients had fistulas. Surgical procedures included debridement, antibiotics, and implant retention (DAIR) (35.7%), 1-stage exchange (28.3%), and 2-stage exchange (29.0%). Candida spp identified were Candida albicans (55.8%), Candida parapsilosis (29.4%), Candida glabrata (7.8%), and Candida tropicalis (5.6%). Coinfection with bacteria was found in 51.3% of cases. The primary antifungal agents prescribed were azoles (75.8%) and echinocandins (30.9%), administered for a median of 92.0 (IQR, 54.5-181.3) days. Cure was observed in 156 of 269 (58.0%) cases. Treatment failure was associated with age >70 years (OR, 1.811 [95% confidence interval {CI}: 1.079-3.072]), and the use of DAIR (OR, 1.946 [95% CI: 1.157-3.285]). Candida parapsilosis infection was associated with better outcome (OR, 0.546 [95% CI: .305-.958]). Cure rates were significantly different between DAIR versus 1-stage exchange (46.9% vs 67.1%, P = .008) and DAIR versus 2-stage exchange (46.9% vs 69.2%, P = .003), but there was no difference comparing 1- to 2-stage exchanges (P = .777). CONCLUSIONS: Candida PJI prognosis seems poor, with high rate of failure, which does not appear to be linked to immunosuppression, use of azoles, or treatment duration.
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Labelled leucocyte scintigraphy (LS) is regarded as helpful when exploring bone and joint infections. The aim of this study was to evaluate the utility of LS for the diagnosis of chronic periprosthetic joint infections (PJIs) in patients exhibiting arthroplastic loosening. One hundred sixty-eight patients were referred to centres for treatment of complex PJI. One hundred fifty underwent LS using 99mTc-HMPAO (LLS); 18 also underwent anti-granulocyte scintigraphy (AGS) and 13 additional SPECT with tomodensitometry imaging (SPECT-CT). The LS results were compared with bone scan data. For all, the final diagnoses were determined microbiologically; perioperative samples were cultured. LS values were examined, as well as sensitivity by microorganism, anatomical sites, and injected activity. LS results were also evaluated according to the current use of antibiotics or not. The sensitivity, specificity, and positive predictive value of LLS were 72%, 60%, and 80%, respectively. LLS performed better than did AGS. SPECT-CT revealed the accurate locations of infections. The sensitivity of LS was not significantly affected by the causative pathogen or the injected activity. No correlation was evident between the current antibiotic treatment and the LS value. The test was more sensitive for knee (84%) than hip arthroplasty (57%) but was less specific for knee (52% vs. 75%). Sensitivity and specificity of LLS varied by the location of infection bone scan provide no additional value in PJI diagnosis. Current antibiotic treatment seems to have no influence on LS sensitivity as well as labelling leukocyte activity or pathogens responsible for chronic PJI.
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Articulaciones/microbiología , Leucocitos/inmunología , Infecciones Relacionadas con Prótesis/diagnóstico , Cintigrafía/normas , Anciano , Femenino , Prótesis de Cadera/microbiología , Humanos , Prótesis de la Rodilla/microbiología , Masculino , Valor Predictivo de las Pruebas , Infecciones Relacionadas con Prótesis/inmunología , Infecciones Relacionadas con Prótesis/microbiología , Cintigrafía/métodos , Estudios Retrospectivos , Sensibilidad y Especificidad , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón ÚnicoAsunto(s)
Profilaxis Antibiótica/métodos , Artroplastia/efectos adversos , Pruebas Diagnósticas de Rutina/métodos , Técnicas Microbiológicas/métodos , Infecciones Relacionadas con Prótesis/epidemiología , Infecciones Relacionadas con Prótesis/prevención & control , Humanos , Infecciones Relacionadas con Prótesis/diagnósticoRESUMEN
Anionic lipids play a variety of key roles in membrane function, including functional and structural effects on respiratory complexes. However, little is known about the molecular basis of these lipid-protein interactions. In this study, NarGHI, an anaerobic respiratory complex of Escherichia coli, has been used to investigate the relations in between membrane-bound proteins with phospholipids. Activity of the NarGHI complex is enhanced by anionic phospholipids both in vivo and in vitro. The anionic cardiolipin tightly associates with the NarGHI complex and is the most effective phospholipid to restore functionality of a nearly inactive detergent-solubilized enzyme complex. A specific cardiolipin-binding site is identified on the basis of the available X-ray diffraction data and of site-directed mutagenesis experiment. One acyl chain of cardiolipin is in close proximity to the heme b(D) center and is responsible for structural adjustments of b(D) and of the adjacent quinol substrate binding site. Finally, cardiolipin binding tunes the interaction with the quinol substrate. Together, our results provide a molecular basis for the activation of a bacterial respiratory complex by cardiolipin.
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Cardiolipinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Complejos Multienzimáticos/metabolismo , Nitrato-Reductasa/metabolismo , Sitios de Unión , Cardiolipinas/química , Espectroscopía de Resonancia por Spin del Electrón , Proteínas del Complejo de Cadena de Transporte de Electrón/química , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Hemo/química , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Modelos Moleculares , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Nitrato-Reductasa/química , Nitrato-Reductasa/genética , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Fosfolípidos/química , Fosfolípidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteolípidos/metabolismo , Electricidad EstáticaRESUMEN
We have developed a method for rapid quantification of fish spoilage bacteria based on quantitative PCR with degenerated oligonucleotides that hybridize on the torA gene coding for trimethylamine N-oxide reductase, one of the major bacterial enzymes in fish spoilage. To show the utility of this gene, we used a regular PCR with DNA extracts from whiting (Merlangius merlangus) and plaice (Pleuronectes platessa) stored in ice. Quantitative PCR showed that the number of copies of the torA gene, i.e., the number of spoilage bacteria, increases with length of storage. This approach can therefore be used to evaluate freshness for the two fish species studied (whiting and plaice).
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Lenguado/metabolismo , Gadiformes/metabolismo , Oxidorreductasas N-Desmetilantes/genética , Alimentos Marinos/análisis , Alimentos Marinos/normas , Animales , Comportamiento del Consumidor , Seguridad de Productos para el Consumidor , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , ADN/análisis , Manipulación de Alimentos/métodos , Humanos , Oxidorreductasas N-Desmetilantes/metabolismo , Reacción en Cadena de la Polimerasa , Especificidad de la EspecieRESUMEN
Intraosseous leiomyoma is an exceedingly rare tumor, with about 15 cases in the literature. Gradually worsening nonspecific pain is the main manifestation. A unilocular or multilocular radiolucency with a rim of sclerosis is seen on standard radiographs. The findings may simulate nonossifying fibroma of a metaphysis or histiocytofibroma of the pelvis. Neither computed tomography nor magnetic resonance imaging can establish the definitive diagnosis, which requires histological examination with immunohistochemistry stains. Intraosseous leiomyosarcoma is the main differential diagnosis. The best discriminating criterion is the mitotic index, which is less than 1 to 4 mitoses per 50 high-power fields in leiomyomas. En bloc excision with wide margins is the treatment of choice for avoiding local recurrences, which are extremely rare.
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Neoplasias Óseas/diagnóstico , Neoplasias Óseas/cirugía , Leiomioma/diagnóstico , Leiomioma/cirugía , Adulto , Articulación del Tobillo , Biopsia con Aguja , Trasplante Óseo/métodos , Femenino , Estudios de Seguimiento , Articulación de la Cadera , Humanos , Inmunohistoquímica , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Procedimientos Ortopédicos/métodos , Enfermedades Raras , Medición de Riesgo , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
We carried out a prospective study to assess the value of mini-invasive osteosynthesis with the PerCutaneous Compression Plate (PCCP) in the treatment of trochanteric hip fractures in elderly patients. Sixty five consecutive patients were included, with an average age at surgery of 84.2 years (range : 63 to 99), with more than 93% ASA 2, 3 or 4 patients. No perioperative complications were noted. All but two patients were ambulatory with weight bearing two days after operation. The postoperative blood transfusion rate was only 25%. No patient was lost to follow-up. We noted 11 deaths (17%) at the end of follow-up (5 during hospitalisation and 6 during the first 9 months). The re-operation rate was 4.5%. Osteosynthesis with the PCCP allows for decreased intraoperative bleeding and minimises injury to muscle and tendon structures. The stability and quality of the construct enable early recovery of patients while limiting morbidity for this type of fracture.
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Placas Óseas , Fijación Interna de Fracturas/métodos , Fracturas de Cadera/cirugía , Anciano , Anciano de 80 o más Años , Transfusión Sanguínea , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Resultado del TratamientoRESUMEN
Understanding when and how metal cofactor insertion occurs into a multisubunit metalloenzyme is of fundamental importance. Molybdenum cofactor insertion is a tightly controlled process that involves specific interactions between the proteins that promote cofactor delivery, enzyme-specific chaperones, and the apoenzyme. In the assembly pathway of the multisubunit molybdoenzyme, membrane-bound nitrate reductase A from Escherichia coli, a NarJ-assisted molybdenum cofactor (Moco) insertion step, must precede membrane anchoring of the apoenzyme. Here, we have shown that the NarJ chaperone interacts at two distinct binding sites of the apoenzyme, one interfering with its membrane anchoring and another one being involved in molybdenum cofactor insertion. The presence of the two NarJ-binding sites within NarG is required to ensure productive formation of active nitrate reductase. Our findings supported the view that enzyme-specific chaperones play a central role in the biogenesis of multisubunit molybdoenzymes by coordinating subunits assembly and molybdenum cofactor insertion.
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Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Chaperonas Moleculares/química , Molibdeno/química , Nitrato-Reductasa/química , Nitratos/química , Sitios de Unión , Técnicas Biosensibles , Escherichia coli/metabolismo , Eliminación de Gen , Modelos Biológicos , Modelos Moleculares , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Fracciones Subcelulares , Thermus/enzimología , Técnicas del Sistema de Dos HíbridosRESUMEN
The maturation of Escherichia coli nitrate reductase A requires the incorporation of the Mo-(bis-MGD) cofactor to the apoprotein. For this process, the NarJ chaperone is strictly required. We report the first description of protein interactions between molybdenum cofactor biosynthetic proteins (MogA, MoeA, MobA, and MobB) and the aponitrate reductase (NarG) using a bacterial two-hybrid approach. Two conditions have to be satisfied to allow the visualization of the interactions, (i) the presence of an active and mature molybdenum cofactor and (ii) the presence of the NarJ chaperone and of the NarG structural partner subunit, NarH. Formation of tungsten-substituted cofactor prevents the interaction between NarG and the four biosynthetic proteins. Our results suggested that the final stages of molybdenum cofactor biosynthesis occur on a complex made up by MogA, MoeA, MobA, and MobB, which is also in charge with the delivery of the mature cofactor onto the aponitrate reductase A in a NarJ-assisted process.
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Proteínas de Escherichia coli/biosíntesis , Molibdeno , Nitrato Reductasas/biosíntesis , Apoproteínas/metabolismo , Proteínas de Escherichia coli/metabolismo , Sustancias Macromoleculares , Chaperonas Moleculares/metabolismo , Nitrato-Reductasa , Subunidades de Proteína/metabolismo , Sulfurtransferasas/metabolismo , Transactivadores/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Molybdopterin guanine dinucleotide (MGD) is the form of the molybdenum cofactor that is required for the activity of most bacterial molybdoenzymes. MGD is synthesized from molybdopterin (MPT) and GTP in a reaction catalyzed by the MobA protein. Here we report that wild type MobA can be copurified along with bound MPT and MGD, demonstrating a tight binding of both its substrate and product. To study structure-function relationships, we have constructed a number of site-specific mutations of the most highly conserved amino acid residues of the MobA protein family. Variant MobA proteins were characterized for their ability to support the synthesis of active molybdenum enzymes, to bind MPT and MGD, to interact with the molybdenum cofactor biosynthesis proteins MobB and MoeA. They were also characterized by x-ray structural analysis. Our results suggest an essential role for glycine 15 of MobA, either for GTP binding and/or catalysis, and an involvement of glycine 82 in the stabilization of the product-bound form of the enzyme. Surprisingly, the individual and double substitution of asparagines 180 and 182 to aspartate did not affect MPT binding, catalysis, and product stabilization.
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Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/fisiología , Secuencia de Aminoácidos , Asparagina/química , Ácido Aspártico/química , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Escherichia coli/enzimología , Prueba de Complementación Genética , Glicina/química , Modelos Moleculares , Datos de Secuencia Molecular , Molibdeno/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Nitrato-Reductasa , Nitrato Reductasas/metabolismo , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Sulfurtransferasas/química , Transactivadores/química , Técnicas del Sistema de Dos HíbridosRESUMEN
The final stages of bacterial molybdenum cofactor (Moco) biosynthesis correspond to molybdenum chelation and nucleotide attachment onto an unique and ubiquitous structure, the molybdopterin. Using a bacterial two-hybrid approach, here we report on the in vivo interactions between MogA, MoeA, MobA, and MobB implicated in several distinct although linked steps in Escherichia coli. Numerous interactions among these proteins have been identified. Somewhat surprisingly, MobB, a GTPase with a yet unclear function, interacts with MogA, MoeA, and MobA. Probing the effects of various mo. mutations on the interaction map allowed us (i) to distinguish Moco-sensitive interactants from insensitive ones involving MobB and (ii) to demonstrate that molybdopterin is a key molecule triggering or facilitating MogA-MoeA and MoeA-MobA interactions. These results suggest that, in vivo, molybdenum cofactor biosynthesis occurs on protein complexes rather than by the separate action of molybdenum cofactor biosynthetic proteins.
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Coenzimas , Escherichia coli/metabolismo , Metaloproteínas/biosíntesis , Escherichia coli/genética , Genes Bacterianos , Metaloproteínas/genética , Sondas Moleculares , Cofactores de Molibdeno , Mutación , PteridinasRESUMEN
The effect of the addition of trimethylamine N-oxide (TMAO) in the growth medium on Escherichia coli anaerobic fermentative and respiratory pathways was examined. Formate dehydrogenase H (FDH-H) activity was totally repressed by the addition of 40 mM TMAO, whereas the overall hydrogenase (HYD) activity was reduced by 25%. Accordingly, expression of lacZ operon fusions with the fdhF and hycB structural genes specifying FDH-H and HYD3 was reduced sevenfold and eightfold, respectively, leading to suppression of an active formate hydrogenlyase system. In contrast, global respiratory formate-dependent phenazine methosulphate reductase (FDH-PMS) activity, which consists of both the major anaerobic FDH-N enzyme and the aerobic FDH-Z isoenzyme, was increased approximately twofold. This was corroborated by a 2.5-fold stimulation of the sole fdoG-uidA transcriptional fusion which reflects the synthesis of the respiratory aerobic FDH-Z enzyme. In fdhD, fdhE or torA mutants lacking either FDH-PMS activity or TMAO reductase (TOR) activity, the formate hydrogenlyase pathway was no longer inhibited by TMAO. In addition, introduction of 30 mM formate in the growth medium was found to relieve the repressive effect of TMAO in the wild-type strain. When TMAO was added as terminal electron acceptor a significant enhancement of anaerobic growth was observed with the wild-type strain and the fdoG mutant. It was associated with the concomitant suppression of the formate hydrogenlyase enzymes. This was in contrast to the fdnG and torA mutants whose growth pattern and fermentative enzymes remained unaffected. Taken together, these results strongly suggest that formate-dependent reduction of TMAO via FDH-N and TOR reduces the amount of formate available for induction of the formate hydrogenlyase pathway.
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Escherichia coli/genética , Formiato Deshidrogenasas/genética , Formiatos/metabolismo , Regulación Bacteriana de la Expresión Génica , Hidrogenasas/genética , Metilaminas/farmacología , Complejos Multienzimáticos/genética , Oxidantes/farmacología , Anaerobiosis , Transporte de Electrón , Represión Enzimática , Escherichia coli/efectos de los fármacos , Formiato Deshidrogenasas/metabolismo , Genes Bacterianos , Metosulfato de Metilfenazonio/metabolismo , Mutación , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/metabolismoRESUMEN
Tellurite and selenate reductase activities were identified in extracts of Escherichia coli. These activities were detected on non-denaturing polyacrylamide gels using an in situ methyl viologen activity-staining technique. The activity bands produced from membrane-protein extracts had the same RF values as those of nitrate reductases (NRs) A and Z. Tellurite and selenate reductase activities were absent from membranes obtained from mutants deleted in NRs A and Z. Further evidence of the tellurite and selenate reductase activities of NR was demonstrated using rocket immunoelectrophoresis analysis, where the tellurite and selenate reductase activities corresponded to the precipitation arc of NR. Additionally, hypersensitivity to potassium tellurite was observed under aerobic growth conditions in nar mutants. The tac promoter expression of NR A resulted in elevated tellurite resistance. The data obtained also imply that a minimal threshold level of NR A is required to increase resistance. Under anaerobic growth conditions additional tellurite reductase activity was identified in the soluble fraction on non-denaturing gels. Nitrate reductase mutants were not hypersensitive under anaerobic conditions, possibly due to the presence of this additional reductase activity.