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Purpose: To evaluate whether the clinical, biochemical and radiological features of patients with primary aldosteronism (PA) can predict both main subtypes of PA. Methods: A retrospective multicenter study of PA patients followed in 27 Spanish tertiary hospitals (SPAIN-ALDO Register). Only patients with confirmed unilateral or bilateral PA based on adrenal venous sampling (AVS) and/or postsurgical biochemical cure after adrenalectomy were included. Supervised regression techniques were used for model development. Results: 328 patients [270 unilateral PA (UPA), 58 bilateral PA (BPA)] were included. The area under the curve (AUC) for aldosterone/potassium ratio and aldosterone responses following saline infusion test were 0.602 [95%CI 0.520 to 0.684] and 0.574 [95% CI 0.446–0.701], respectively, to differentiate UPA from BPA. The AUC was 0.825 [95% 0.764–0.886] when the prediction model with seven parameters – comorbidities (dyslipidemia, cerebrovascular disease, sleep apnea syndrome [SAS]), systolic blood pressure (SBP), plasma aldosterone levels (PAC), hypokalemia and unilateral adrenal nodule >1 cm and normal contralateral adrenal gland on CT/MRI – was used. In patients without comorbidities, hypokalemia, SBP > 160 mmHg, PAC > 40 ng/dL, and unilateral adrenal lesions were associated with a likelihood of having a UPA of 98.5%. The chance of BPA was higher in individuals with comorbidities, SBP < 140 mmHg, normokalemia, low PAC levels, and no adrenal tumors on the CT/MRI (91.5%). Conclusion: A combination of high PAC, SBP > 160 mmHg, low serum potassium, a unilateral adrenal nodule>1 cm and no comorbidities could predict a UPA with a 98.5% accuracy.

Purpose: To evaluate the prevalence, risk factors and evolution of diabetes mellitus (DM) after targeted treatment in patients with primary aldosteronism (PA). Methods: A retrospective multicenter study of PA patients in follow-up at 27 Spanish tertiary hospitals (SPAIN-ALDO Register). Results: Overall, 646 patients with PA were included. At diagnosis, 21.2% (n = 137) had DM and 67% of them had HbA1c levels < 7%. In multivariate analysis, family history of DM (OR 4.00 [1.68–9.53]), the coexistence of dyslipidemia (OR 3.57 [1.51–8.43]) and advanced age (OR 1.04 per year of increase [1.00–1.09]) were identified as independent predictive factors of DM. Diabetic patients were on beta blockers (46.7% (n = 64) vs. 27.5% (n = 140), P < 0.001) and diuretics (51.1% (n = 70) vs. 33.2% (n = 169), p < 0.001) more frequently than non-diabetics. After a median follow-up of 22 months [IQR 7.5–63.0], 6.9% of patients developed DM, with no difference between those undergoing adrenalectomy and those treated medically (HR 1.07 [0.49–2.36], p = 0.866). There was also no significant difference in the evolution of glycemic control between DM patients who underwent surgery and those medically treated (p > 0.05). Conclusion: DM affects about one quarter of patients with PA and the risk factors for its development are common to those of the general population. Medical and surgical treatment provides similar benefit in glycemic control in patients with PA and DM.

We use the free radical polymerization initiator 4,4′-azobis(cyanovaleric acid) coordinated to the open metal sites of metal-organic frameworks (MOFs) to give rise to highly uniform MOF/polymer hybrids. We demonstrate this strategy on two robust zirconium MOFs (NU-1000 and MOF-808), which are the most effective catalysts for degradation of chemical warfare nerve agents. The resulting hybrid materials maintain their hydrolytic catalytic activity and have substantially improved adhesion to polypropylene and activated carbon textile fibers, yielding highly robust MOF/polymer/textile hybrid systems. These composites are suitable for the green production of active protective clothing and filters capable of detoxifying organophosphorus warfare agents.

BackgroundThere is limited literature regarding the re-fracture of a previously augmented vertebral compression fracture (VCF). These re-fractures may present as an asymptomatic remodeling of the vertebral body around the cement cast while in other cases they involve the middle column, at the transition zone between the cement-augmented and non-augmented vertebral body. In the latter, a posterior wall retropulsion is possible and, if left untreated, might progress to vertebral body splitting, central canal stenosis, and kyphotic deformity. There is no consensus regarding the best treatment for these re-fractures. There are cases in which a repeated augmentation relieves the pain, but this is considered an undertreatment in cases with middle column involvement, posterior wall retropulsion, and kyphosis.MethodsWe report four cases of re-fracture with middle column collapse of a previously augmented VCF, treated with the stent-screw assisted internal fixation (SAIF) technique. A modified more postero-medial deployment of the anterior metallic implants was applied, to target the middle column fracture. This modified SAIF allowed the reduction and stabilization of the middle column collapse as well as the partial correction of the posterior wall retropulsion and kyphosis.ResultsComplete relief of back pain with stable clinical and radiographic findings at follow-up was obtained in all cases.ConclusionsIn selected cases, the middle column SAIF technique is safe and effective for the treatment of the re-fracture with middle column collapse of a previously cement-augmented VCF. This technique requires precision in trocar placement and could represent a useful addition to the technical armamentarium for VCF treatment.

Video Abstract16VV1Marta Araujo-Castro

Objective: The aim of this study was to evaluate the rate of adrenal venous sampling (AVS) performance in patients with primary aldosteronism (PA), the main reasons for its non-performance, and the success and complications rate of this procedure in Spain. Moreover, the concordance between CT/MRI and AVS for PA subtyping was evaluated. Methods: A retrospective multicenter study of PA patient follow-up in 20 Spanish tertiary hospitals between 2018–2021 was performed (SPAIN-ALDO Register). Results: Of the 440 patients with PA included in the study, 153 underwent AVS (34.8%). The main reasons for not performing AVS were: patient rejection to the procedure, low catheterization rate in the center and unilateral disease based on CT/MRI. The overall success rate was 44.4% (the left adrenal vein was properly canulated in 77.8% and the right adrenal vein in 48.4%). Only 3 patients experienced minor complications. In the 45 patients with unilateral disease according to AVS, CT/MRI indicated bilateral disease or normal adrenal glands in 17. In the 23 patients with bilateral disease, CT/MRI indicated unilateral disease in 14. However, no significant differences were observed in biochemical response (P= 0.051) and hypertension resolution (P= 0.150) between patients who underwent surgery based on CT/MRI results and those who underwent surgery based on AVS results. Conclusion: In our setting, AVS is still an underused technique in patients with PA. The low experience and success rate in AVS partially justify these results. More training for providers and patients needs to be done to include appropriate well performed AVS in the diagnosis algorithm of PA.

We report the controlled synthesis of thin films of prototypical zirconium metal–organic frameworks [Zr6O4(OH)4(benzene-1,4-dicarboxylate-2-X)6] (X = H, UiO-66 and X = NH2, UiO-66-NH2) over the external surface of shaped carbonized substrates (spheres and textile fabrics) using a layer-by-layer method. The resulting composite materials contain metal–organic framework (MOF) crystals homogeneously distributed over the external surface of the porous shaped bodies, which are able to capture an organophosphate nerve agent simulant (diisopropylfluorophosphate, DIFP) in competition with moisture (very fast) and hydrolyze the P–F bond (slow). This behavior confers the composite material self-cleaning properties, which are useful for blocking secondary emission problems of classical protective equipment based on activated carbon.

The optimal planning of preoperative diagnosis, management and treatment of pituitary tumors (PT) candidates to pituitary surgery (PS) requires a multidisciplinary approach involving a team of endocrinologists, neurosurgeons, ENT, neuro-ophthalmologists and neuroradiologists with experience in pituitary diseases. Such teams improve surgical results, minimize complications and facilitate their correct treatment if occurring, and optimize the hormonal, ophthalmological and radiological preoperative and follow-up evaluation. We have developed a clinical practice protocol for patients with PT who are candidates to PS based on the most recent national and international guidelines and the relevant literature regarding PT published in the last years. The protocol has been elaborated by a multidisciplinary team of a Spanish Pituitary Tumor Center of Excellence (PTCE) that includes at least one neurosurgeon, ENT, neuroradiologist, neuro-ophthalmologist, endocrine pathologist and endocrinologist specialized in pituitary diseases. We elaborated this guideline with the aim of sharing our experience with other centers involved in the perioperative and surgical management of PT thereby facilitating the management of patients undergoing PS.

Carbon footprint and nonrenewability are among the motives to mitigate dependence on oil resources. On the other hand, palm trees exceeded 100 million around globe, which poses enormous amount of biowastes to get exploited per annum. Moreover, nanocellulose is emerging as efficient low cost material, which shows increasing versatility throughout time. In this investigation we will attempt to extract nanocellulose from palm wastes (fronds, leaves, and coir), and characterize them. Dry biomass was pulped by being subjected to 10% (wt/wt) NaOH alkaline treatment at 160 °C for 2 h. Pulped non-bleached biomass underwent acidic hydrolysis by mechanical stirring in 20% H2SO4(v/v) and heating to 120 °C for 30 min. After filtration, neutralization, and centrifugion, resultant particles were characterized to assess their morphology, size, particle charge, existing chemical groups, and crystallinity. For the three types of palm residues, our preparation technique was successful to isolate lignin containing nanocellulose particles. However, coir was more recalcitrant to acidic hydrolysis than fronds and leaves. Our palm residues yielded 42–82 nm spherical particles, and zeta potential ranging between −11 and −19 mV. Crystallinity was higher after pulping, and lower after hydrolysis, which suggests promotion of amorphous content of cellulose. Lignin-containing nanocellulose prepared in this study initiates promising horizon especially in heavy metal (cations) removal from water in environmental applications and sustained drug delivery for medical applications.

There is a lack of evidence on timing, frequency, and duration of postoperative endocrine, radiologic, and ophthalmologic assessments that should be performed after pituitary surgery (PS). However, it is known that careful optimization of treatment and follow-up strategies as well as a multidisciplinary approach may have a significant impact on long-term outcomes, improving surgical results, minimize complications and facilitate their correct treatment if occurring, and optimize the hormonal, ophthalmological, and radiological reassessment throughout the follow-up. Considering that there are no specific guidelines on the postoperative management of patients with pituitary tumors (PT), we present our protocol for the postoperative management of patients with PT. It has been elaborated by the multidisciplinary team of a Spanish Pituitary Tumor Center of Excellence (PTCE) that includes at least one neurosurgeon, ENT, neuroradiologist, neuro-ophthalmologist, endocrine pathologist and endocrinologist specialized in pituitary diseases. We elaborated this guideline with the aim of sharing our experience with other centers involved in the management of PT thereby facilitating the postoperative management of patients submitted to PS.

A series of Ce/Zr mixed-metal–organic frameworks with different topology/connectivity, namely, Ce/Zr-UiO-66 (U01, U02, and U03) (fcu (12-c)), Ce/Zr-DUT-67-PZDC (D01 and D02) (reo (8-c)), and Ce/Zr-MOF-808 (M01, M02, and M03) (spn (6-c)) were evaluated toward the detoxification of toxic nerve agent model diisopropylfluorophosphate (DIFP) at room temperature in unbuffered aqueous solution. Noteworthily, the catalytic rate for P–F bond cleavage increased with increasing Ce/Zr molar ratio. A further increase in catalytic activity can be achieved by Mg(OMe)2doping of the mixed-metal MOFs as exemplified with M01@Mg(OMe)2and M02@Mg(OMe)2systems. The results show that Mg(OMe)2incorporation into the mesoporous cavities of M01 and M02 give rise to P–F hydrolytic degradation half-lives of nearly 5 and 2 min with 100% degradation of DIFP after 55 and 65 min for M01@Mg(OMe)21:2 and M02@Mg(OMe)21:4, respectively.

UiO-66, MOF-808and NU-1000metal–organic frameworks exhibit a differentiated reactivity toward [Mg(OMe)2(MeOH)2]4related to their pore accessibility. Microporous UiO-66remains unchanged while mesoporous MOF-808and hierarchical micro/mesoporous NU-1000materials yield doped systems containing exposed MgZr5O2(OH)6clusters in the mesoporous cavities. This modification is responsible for a remarkable enhancement of the catalytic activity toward the hydrolytic degradation of P–F and P–S bonds of toxic nerve agents, at room temperature, in unbuffered aqueous solutions.

Horizontal gene transfer (HGT) mediated by the spread of plasmids fuels evolution in prokaryotes. Although plasmids provide bacteria with new adaptive genes, they also produce physiological alterations that often translate into a reduction in bacterial fitness. The fitness costs associated with plasmids represent an important limit to plasmid maintenance in bacterial communities, but their molecular origins remain largely unknown. In this work, we combine phenomics, transcriptomics and metabolomics to study the fitness effects produced by a collection of diverse plasmids in the opportunistic pathogen Pseudomonas aeruginosaPAO1. Using this approach, we scan the physiological changes imposed by plasmids and test the generality of some main mechanisms that have been proposed to explain the cost of HGT, including increased biosynthetic burden, reduced translational efficiency, and impaired chromosomal replication. Our results suggest that the fitness effects of plasmids have a complex origin, since none of these mechanisms could individually provide a general explanation for the cost of plasmid carriage. Interestingly, our results also showed that plasmids alter the expression of a common set of metabolic genes in PAO1, and produce convergent changes in host cell metabolism. These surprising results suggest that there is a common metabolic response to plasmids in P. aeruginosaPAO1.

In this paper, the damage behaviour of aerospace composite structures has been studied. The analysed structure is an all-composite wing with inter-laminar manufacturing induced damage. The manufacturing induced delaminations are located at the leading edge on the extrados and in the area near the fuselage. Different analyses have been performed to correctly predict the pre-existent damages evolution under service loading conditions. Preliminary linear buckling analyses have been performed to check for instability of the delaminations under service loads; then the risk of delaminations’ propagation has been evaluated by means of a Virtual Crack Closure Technique based approach.

The use of donation after circulatory death (DCD) has increased significantly during the past decade. However, warm ischemia results in a greater risk for transplantation. Indeed, controlled DCD(cDCD) was associated with inferior outcomes compared with donation after brain death. The use of abdominal normothermic regional perfusion (nRP) to restore blood flow before organ recovery in cDCDhas been proposed as better than rapid recovery to reverse the effect of ischemia and improve recipients’ outcome. Here, the first Spanish series using abdominal nRPas an in situconditioning method is reported. A specific methodology to avoid restoring circulation to the brain after death determination is described. Twenty‐seven cDCDdonors underwent abdominal nRPduring at least 60 min. Thirty‐seven kidneys, 11 livers, six bilateral lungs, and one pancreas were transplanted. The 1‐year death‐censored kidney survival was 91%, and delayed graft function rate was 27%. The 1‐year liver survival rate was 90.1% with no cases of ischemic cholangiopathy. Transplanted lungs and pancreas exhibited primary function. The use of nRPmay represent an advance to increase the number and quality of grafts in cDCD. Poor results in cDCDlivers could be reversed with nRP. Concerns about restoring brain circulation after death are easily solved. Abdominal normothermic regional perfusion with premortem interventions represents an advance to increase the number and quality of grafts recovered in controlled donation after circulatory death.

The development of protective self-detoxifying materials is an important societal challenge to counteract risk of attacks employing highly toxic chemical warfare agents (CWAs). In this work, we have developed bifunctional zirconium metal–organic frameworks (MOFs) incorporating variable amounts of nucleophilic amino residues by means of formation of the mixed ligand [Zr6O4(OH)4(bdc)6(1–x)(bdc-NH2)6x] (UiO-66-xNH2) and [Zr6O4(OH)4(bpdc)6(1–x)(bpdc-(NH2)2)6x] (UiO-67-x(NH2)2) systems where bdc = benzene-1,4-dicarboxylate; bdc-NH2= benzene-2-amino-1,4-dicarboxylate; bpdc = 4,4′-biphenyldicarboxylate; bpdc-(NH2)2= 2,2′-diamino-4,4′-biphenyldicarboxylate and x= 0, 0.25, 0.5, 0.75, 1. In a second step, the UiO-66-xNH2and UiO-67-x(NH2)2systems have been postsynthetically modified by introduction of highly basic lithium tert-butoxide (LiOtBu) on the oxohydroxometallic clusters of the mixed ligand MOFs to yield UiO-66-xNH2@LiOtBuand UiO-67-x(NH2)2@LiOtBumaterials. The results show that the combination of pre and postsynthetic modifications on these MOF series gives rise to fine-tuning of the catalytic activity toward the hydrolytic degradation of both simulants and real CWAs in unbuffered aqueous solutions. Indeed, UiO-66-0.25NH2@LiOtBuis able to hydrolyze both CWAs simulants (diisopropylfluorophosphate (DIFP), 2-chloroethylethylsulfide (CEES), and real CWAs (soman (GD), sulfur mustard (HD)) quickly in aqueous solution. These results are related to a suitable combination of robustness, nucleophilicity, basicity, and accessibility to the porous framework.

This paper proposes a Fractional-order modification of the traditional Integral Resonant Controller named as FIRC. The fractional integral action utilised in the proposed FIRC is a simple, robust, and well-performing technique for vibration control in smart structures with collocated sensor-actuator pairs such as nanopositioning stages. The proposed control scheme is robust in the sense of being insensitive to spillover dynamics and maintaining closed-loop stability even in the presence of model inaccuracies or time-delays in the system. The experimental and simulated results have showed that the proposed FIRC can provide a closed-loop bandwidth which spans up to a 95.2% of the first resonant mode of the experimental system, thus improving the bandwidth achieved by classical integer-order IRC implementations.

This work studies wear kinetics of index able inserts (II) made of mixed ceramics based on Russian-manufactured in case of fine milling of special hardened cast iron with globular graphite. In a wide range of cutting parameters typical for fine milling, an impact of tool insert wearing parameters on qualitative and quantitative characteristics of the cutting process have been studied. Morphological traits and nature of wear of work surfaces has been studied using optical and scanning electron microscopy. Criteria of II states, as well as specifics of contact work surface transformation, correlating with force parameters of the face milling process, have been determined.

This paper investigates and analyses use of numerical modeling by finite element method (FEM) at studying of consolidation processes of materials from powder by spark plasma sintering (SPS). Tasks of SPS process optimization is discussed in detail. Examples of numeric analysis of SPS of current conducting and non-conducting materials are given. Numeric modeling of sample sintering with hybrid method when SPS process is combined with hot pressing (HP) process is studied. Also paper presents development prospects of principles of SPS process numeric modeling.

ABSTRACTPlasmids play a key role in the horizontal spread of antibiotic resistance determinants among bacterial pathogens. When an antibiotic resistance plasmid arrives in a new bacterial host, it produces a fitness cost, causing a competitive disadvantage for the plasmid-bearing bacterium in the absence of antibiotics. On the other hand, in the presence of antibiotics, the plasmid promotes the survival of the clone. The adaptations experienced by plasmid and bacterium in the presence of antibiotics during the first generations of coexistence will be crucial for the progress of the infection and the maintenance of plasmid-mediated resistance once the treatment is over. Here we developed a model system using the human pathogen Haemophilus influenzaecarrying the small plasmid pB1000 conferring resistance to β-lactam antibiotics to investigate host and plasmid adaptations in the course of a simulated ampicillin therapy. Our results proved that plasmid-bearing clones compensated for the fitness disadvantage during the first 100 generations of plasmid-host adaptation. In addition, ampicillin treatment was associated with an increase in pB1000 copy number. The augmentation in both bacterial fitness and plasmid copy number gave rise to H. influenzaepopulations with higher ampicillin resistance levels. In conclusion, we show here that the modulations in bacterial fitness and plasmid copy number help a plasmid-bearing bacterium to adapt during antibiotic therapy, promoting both the survival of the host and the spread of the plasmid.

ZrO2 and Al2O3 are monolithic ceramics used today in a wide variety of structural components. However, both materials present important drawbacks for some specific applications. In the case of Al2O3, its moderate strength (500 MPa) and toughness (4 MPa.√m) makes it unsuitable for high loading conditions. On the other hand, ZrO2 presents higher strength and toughness values (900 MPa and 6 MPa.√m) than Al2O3 but it is a material limited in its long-term behaviour due to its bad response to hydrothermal ageing and a pronounced tendency for subcritical crack growth. Due to this fact, ceramic nanocomposites made of Al2O3 and ZrO2 (ATZ and ZTA) have been developed in the last years in order to overcome the main drawbacks of the monolithic materials as they can combine the properties of both, strong and tough materials, simultaneously, with null ageing and even higher biocompatibility. In this work, several amounts of Al2O3 disperse phase (15, 35 and 50 vol %) were added to one ZrO2 matrix (CeO2 - 10 mol %) in order to see their effect on the mechanical properties, subcritical crack propagation and long-term reliability.

Microwave sintering has emerged in recent years as a new, fast, cheap and green technology for sintering a variety of materials. The main advantages of microwave heating can be summarized as follow: reduced processing times, energy costs and environmental benefits. Nevertheless, understanding how this specific heating drives to obtain ceramic materials with a combination of unique, structural and functional properties is the big challenge. The present work shows the different and improved properties achieved with β-eucryptite nanocomposite ceramic materials by microwave heating compared with the conventional method. Microcracking evolution in addition to the microstructure of the sintered materials along the several thermal cycles has been studied. Mechanical properties changes observed can be related to this process. Thus, the microwave technique is a promising tool for sintering new materials by controlling the composition of the phases, chemical reactivity and nanostructure, using up to 70% less energy in the whole sintering process than conventional heating. This technique becomes part of the new and innovative technologies "eco-green".

This paper presents a method for the on-line identification of the payload carried by a single-link flexible manipulator whose movement is constrained to the vertical plane. The vertical movement of the robot makes the effect of gravity not negligible, and determines therefore, the non-linear behaviour of the analysed system. The proposed estimator is based on the algebraic derivative approach in the frequency domain. The short period of time it takes for the algorithm to make an estimate, and its low computational burden make it suitable for real-time and adaptive control applications. The proposed estimator is experimentally tested in several situations carefully chosen to reproduce routine operations of the flexible manipulator.

ABSTRACTStreptococcus suisis an emerging zoonotic agent responsible for high-mortality outbreaks among the human population in China. In this species, the ABC transporter SatAB mediates fluoroquinolone resistance when overexpressed. Here, we describe and characterize satR, an open reading frame (ORF) encoding a MarR superfamily regulator that acts as a repressor of satAB. satRis cotranscribed with satAB, and its interruption entails the overexpression of the pump, leading to a clinically relevant increase in resistance to fluoroquinolones.

ABSTRACTArm/Rmt methyltransferases have emerged recently in pathogenic bacteria as enzymes that confer high-level resistance to 4,6-disubstituted aminoglycosides through methylation of the G1405 residue in the 16S rRNA (like ArmA and RmtA to -E). In prokaryotes, nucleotide methylations are the most common type of rRNA modification, and they are introduced posttranscriptionally by a variety of site-specific housekeeping enzymes to optimize ribosomal function. Here we show that while the aminoglycoside resistance methyltransferase RmtC methylates G1405, it impedes methylation of the housekeeping methyltransferase RsmF at position C1407, a nucleotide that, like G1405, forms part of the aminoglycoside binding pocket of the 16S rRNA. To understand the origin and consequences of this phenomenon, we constructed a series of in-frame knockout and knock-in mutants of Escherichia coli, corresponding to the genotypes rsmF+, ΔrsmF, rsmF+rmtC+, and ΔrsmF rmtC+. When analyzed for the antimicrobial resistance pattern, the ΔrsmFbacteria had a decreased susceptibility to aminoglycosides, including 4,6- and 4,5-deoxystreptamine aminoglycosides, showing that the housekeeping methylation at C1407 is involved in intrinsic aminoglycoside susceptibility in E. coli. Competition experiments between the isogenic E. colistrains showed that, contrary to expectation, acquisition of rmtCdoes not entail a fitness cost for the bacterium. Finally, matrix-assisted laser desorption ionization (MALDI) mass spectrometry allowed us to determine that RmtC methylates the G1405 residue not only in presence but also in the absence of aminoglycoside antibiotics. Thus, the coupling between housekeeping and acquired methyltransferases subverts the methylation architecture of the 16S rRNA but elicits Arm/Rmt methyltransferases to be selected and retained, posing an important threat to the usefulness of aminoglycosides worldwide.

ABSTRACTStreptococcus suisis an emerging zoonotic pathogen. With the lack of an effective vaccine, antibiotics remain the main tool to fight infections caused by this pathogen. We have previously observed a reserpine-sensitive fluoroquinolone (FQ) efflux phenotype in this species. Here, SatAB and SmrA, two pumps belonging to the ATP binding cassette (ABC) and the major facilitator superfamily (MFS), respectively, have been analyzed in the fluoroquinolone-resistant clinical isolate BB1013. Genes encoding these pumps were overexpressed either constitutively or in the presence of ciprofloxacin in this strain. These genes could not be cloned in plasmids in Escherichia colidespite strong expression repression. Finally, site-directed insertion of smrAand satABin the amylocus of the Bacillus subtilischromosome using ligated PCR amplicons allowed for the functional expression and study of both pumps. Results showed that SatAB is a narrow-spectrum fluoroquinolone exporter (norfloxacin and ciprofloxacin), susceptible to reserpine, whereas SmrA was not involved in fluoroquinolone resistance. Chromosomal integration in Bacillusis a novel method for studying efflux pumps from Gram-positive bacteria, which enabled us to demonstrate the possible role of SatAB, and not SmrA, in fluoroquinolone efflux in S. suis.

Streptococcus suis is an emerging zoonotic pathogen. With the lack of an effective vaccine, antibiotics remain the main tool to fight infections caused by this pathogen. We have previously observed a reserpine-sensitive fluoroquinolone (FQ) efflux phenotype in this species. Here, SatAB and SmrA, two pumps belonging to the ATP binding cassette (ABC) and the major facilitator superfamily (MFS), respectively, have been analyzed in the fluoroquinolone-resistant clinical isolate BB1013. Genes encoding these pumps were overexpressed either constitutively or in the presence of ciprofloxacin in this strain. These genes could not be cloned in plasmids in Escherichia coli despite strong expression repression. Finally, site-directed insertion of smrA and satAB in the amy locus of the Bacillus subtilis chromosome using ligated PCR amplicons allowed for the functional expression and study of both pumps. Results showed that SatAB is a narrow-spectrum fluoroquinolone exporter (norfloxacin and ciprofloxacin), susceptible to reserpine, whereas SmrA was not involved in fluoroquinolone resistance. Chromosomal integration in Bacillus is a novel method for studying efflux pumps from Gram-positive bacteria, which enabled us to demonstrate the possible role of SatAB, and not SmrA, in fluoroquinolone efflux in S. suis.

ABSTRACTThe 16S rRNA methyltransferase ArmA is a worldwide emerging determinant that confers high-level resistance to most clinically relevant aminoglycosides. We report here the identification and characterization of a multidrug-resistant Salmonella entericasubspecies I.4,12:i:− isolate recovered from chicken meat sampled in a supermarket on February 2009 in La Reunion, a French island in the Indian Ocean. Susceptibility testing showed an unusually high-level resistance to gentamicin, as well as to ampicillin, expanded-spectrum cephalosporins and amoxicillin-clavulanate. Molecular analysis of the 16S rRNA methyltransferases revealed presence of the armAgene, together with blaTEM-1, blaCMY-2, and blaCTX-M-3. All of these genes could be transferred en blocthrough conjugation into Escherichia coliat a frequency of 10−5CFU/donor. Replicon typing and S1 pulsed-field gel electrophoresis revealed that the armAgene was borne on an ∼150-kb broad-host-range IncP plasmid, pB1010. To elucidate how armAhad integrated in pB1010, a PCR mapping strategy was developed for Tn1548, the genetic platform for armA.The gene was embedded in a Tn1548-like structure, albeit with a deletion of the macrolide resistance genes, and an IS26was inserted within the melgene. To our knowledge, this is the first report of ArmA methyltransferase in food, showing a novel route of transmission for this resistance determinant. Further surveillance in food-borne bacteria will be crucial to determine the role of food in the spread of 16S rRNA methyltransferase genes worldwide.

Plasmid pB1000 is a mobilizable replicon bearing the blaROB-1β-lactamase gene that we have recently described in Haemophilus parasuis and Pasteurella multocida animal isolates. Here we report the presence of pB1000 and a derivative plasmid, pB1000', in four Haemophilus influenzae clinical isolates of human origin. Pulsed-field gel electrophoresis showed unrelated patterns in all strains, indicating that the existence of pB1000 in H. influenzae isolates is not the consequence of clonal dissemination. The replicon can be transferred both by transformation and by conjugation into H. influenzae, giving rise to recipients resistant to ampicillin and cefaclor (MICs, ≥64 µg/ml). Stability experiments showed that pB1000 is stable in H. influenzae without antimicrobial pressure for at least 60 generations. Competition experiments between isogenic H. influenzae strains with and without pB1000 revealed a competitive disadvantage of 9% per 10 generations for the transformant versus the recipient. The complete nucleotide sequences of nine pB1000 plasmids from human and animal isolates, as well as the epidemiological data, suggest that animal isolates belonging to the Pasteurellaceae act as an antimicrobial resistance reservoir for H. influenzae. Further, since P. multocida is the only member of this family that can colonize both humans and animals, we propose that P. multocida is the vehicle for the transport of pB1000 between animal- and human-adapted members of the PASTEURELLACEAE:

ABSTRACTPlasmid pB1000 is a mobilizable replicon bearing the blaROB-1β-lactamase gene that we have recently described in Haemophilus parasuisand Pasteurella multocidaanimal isolates. Here we report the presence of pB1000 and a derivative plasmid, pB1000′, in four Haemophilus influenzaeclinical isolates of human origin. Pulsed-field gel electrophoresis showed unrelated patterns in all strains, indicating that the existence of pB1000 in H. influenzaeisolates is not the consequence of clonal dissemination. The replicon can be transferred both by transformation and by conjugation into H. influenzae, giving rise to recipients resistant to ampicillin and cefaclor (MICs, ≥64 μg/ml). Stability experiments showed that pB1000 is stable in H. influenzaewithout antimicrobial pressure for at least 60 generations. Competition experiments between isogenic H. influenzaestrains with and without pB1000 revealed a competitive disadvantage of 9% per 10 generations for the transformant versus the recipient. The complete nucleotide sequences of nine pB1000 plasmids from human and animal isolates, as well as the epidemiological data, suggest that animal isolates belonging to the Pasteurellaceaeact as an antimicrobial resistance reservoir for H. influenzae. Further, since P. multocidais the only member of this family that can colonize both humans and animals, we propose that P. multocidais the vehicle for the transport of pB1000 between animal- and human-adapted members of the Pasteurellaceae.

ABSTRACTIn most gram-negative bacteria, acquired multiresistance is conferred by large plasmids compiling numerous antimicrobial resistance genes. Here, we show an evolutionary alternative strategy used by Pasteurella multocidato become resistant to multiple clinically relevant antibiotics. Thirteen β-lactam-resistant clinical isolates, concomitantly resistant to tetracyclines and/or streptomycin as well as to sulfonamides, were studied. Pulsed-field gel electrophoresis analysis revealed different profiles among the isolates, showing that clonal dissemination was not the sole event responsible for the spread of multiresistance. Each P. multocidastrain carried two or three small plasmids between 4 and 6 kb in size. A direct association between resistance profile and plasmid content was found. Complete nucleotide sequencing of all plasmids revealed seven different replicons, six of them belonging to the ColE1 superfamily. All plasmids carried one, or a maximum of two, antimicrobial resistance determinants. Plasmids pB1000 and pB1002 bore blaROB-1, pB1001 carried tet(B), pB1003 and pB1005 carried sul2and strA, pB1006 harbored tet(O), and p9956 bore the tet(H) gene. All plasmids except pB1002 and pB1006 were successfully transformed into Escherichia coli. pB1000, also involved in β-lactam resistance in Haemophilus parasuis(A. San Millan et al., Antimicrob. Agents Chemother. 51:2260-2264, 2007), was mobilized in E. coliusing the conjugation machinery of an IncP plasmid. Stability experiments proved that pB1000 was stable in P. multocidabut highly unstable in E. coli. In conclusion, blaROB-1is responsible for β-lactam resistance in P. multocidain Spain. Coexistence and the spread of small plasmids are used by P. multocidato become multiresistant.

To compare the presentation and evolution of primary aldosteronism (PA) in the elderly (≥65 years) and young patients (<65 years).A retrospective multicenter study was performed in 20 Spanish hospitals of PA patients in follow-up between 2018 and 2021.Three hundred fifty-two patients with PA <65 years and 88 patients ≥65 years were included. Older PA patients had a two-fold higher prevalence of type 2 diabetes, dyslipidemia, and cerebrovascular disease, but these differences disappeared after adjusting for hypertension duration. At diagnosis, diastolic blood pressure was lower than in young patients (83.3 ± 11.54 vs 91.6 ± 14.46 mmHg, P< 0.0001). No differences in the rate of overall correct cannulation (56.5% vs 42.3%, P= 0.206) or the diagnosis of unilaterality (76.9% vs 62.5%, P= 0.325) in the adrenal venous sampling (AVS) was observed between the elderly and young groups. However, there was a lower proportion of PA patients who underwent adrenalectomy in the elderly group than in the younger group (22.7% (n= 20) vs 37.5% (n= 132), P= 0.009). Nevertheless, no differences in the rate of postsurgical biochemical (100% (n= 14) vs 92.8% (n= 90), P= 0.299) and hypertension cure (38.6% (n= 51) vs 25.0% (n= 5), P= 0.239) were observed between both groups.Older patients with PA have a worse cardiometabolic profile than young patients with PA that it is related to a longer duration of hypertension. However, the results of the AVS, and adrenalectomy are similar in both groups. Therefore, the management of elderly patients with PA should be based not only on age, but rather on the overall medical, physical, social, and mental characteristics of the patients.

Three Enterococcus faecium strains isolated successively from the same patient, vancomycin-resistant strain BM4659, vancomycin-dependent strain BM4660, and vancomycin-revertant strain BM4661, were indistinguishable by pulsed-field gel electrophoresis and harbored plasmid pIP846, which confers VanB-type resistance. The vancomycin dependence of strain BM4660 was due to mutation P175L, which suppressed the activity of the host Ddl D-Ala:D-Ala ligase. Reversion to resistance in strain BM4661 was due to a G-to-C transversion in the transcription terminator of the vanRSBoperon that lowered the free energy of pairing from –13.08 to –6.65 kcal/mol, leading to low-level constitutive expression of the resistance genes from the PRBpromoter, as indicated by analysis of peptidoglycan precursors and of VanXBD,D-dipeptidase activity. Transcription of the resistance genes, studied by Northern hybridization and reverse transcription, initiated from the PYBresistance promoter, was inducible in strains BM4659 and BM4660, whereas it started from the PRBregulatory promoter in strain BM4661, where it was superinducible. Strain BM4661 provides the first example of reversion to vancomycin resistance of a VanB-type dependent strain not due to a compensatory mutation in the ddl or vanSBgene. Instead, a mutation in the transcription terminator of the regulatory genes resulted in transcriptional readthrough of the resistance genes from the PRBpromoter in the absence of vancomycin.

ABSTRACTThree Enterococcus faeciumstrains isolated successively from the same patient, vancomycin-resistant strain BM4659, vancomycin-dependent strain BM4660, and vancomycin-revertant strain BM4661, were indistinguishable by pulsed-field gel electrophoresis and harbored plasmid pIP846, which confers VanB-type resistance. The vancomycin dependence of strain BM4660 was due to mutation P175L, which suppressed the activity of the host Ddl d-Ala:d-Ala ligase. Reversion to resistance in strain BM4661 was due to a G-to-C transversion in the transcription terminator of the vanRSBoperon that lowered the free energy of pairing from −13.08 to −6.65 kcal/mol, leading to low-level constitutive expression of the resistance genes from the PRBpromoter, as indicated by analysis of peptidoglycan precursors and of VanXBd,d-dipeptidase activity. Transcription of the resistance genes, studied by Northern hybridization and reverse transcription, initiated from the PYBresistance promoter, was inducible in strains BM4659 and BM4660, whereas it started from the PRBregulatory promoter in strain BM4661, where it was superinducible. Strain BM4661 provides the first example of reversion to vancomycin resistance of a VanB-type dependent strain not due to a compensatory mutation in the ddlor vanSBgene. Instead, a mutation in the transcription terminator of the regulatory genes resulted in transcriptional readthrough of the resistance genes from the PRBpromoter in the absence of vancomycin.

ABSTRACTβ-Lactam resistance in Haemophilus parasuisis an emerging phenomenon that has not yet been characterized from a molecular perspective. Clinical high-level β-lactam-resistant isolates from Spain bore a novel plasmid, pB1000, expressing a functionally active ROB-1 β-lactamase. Pulsed-field gel electrophoresis was applied for the first time to H. parasuisand showed that β-lactam resistance is due to clonal spread of a resistant strain, BB1018, bearing pB1000.

ABSTRACTWe have identified and sequenced the genes encoding the quinolone-resistance determining region (QRDR) of ParC and GyrA in fluoroquinolone-susceptible and -resistant Streptococcus suisclinical isolates. Resistance is the consequence of single point mutations in the QRDRs of ParC and GyrA and is not due to clonal spread of resistant strains or horizontal gene transfer with other bacteria.

ZrO2 and Al2O3 are monolithic ceramics used today in a wide variety of structural components. However, both materials present important drawbacks for some specific applications. In the case of Al2O3, its moderate strength (500 MPa) and toughness (4 MPa.√m) makes it unsuitable for high loading conditions. On the other hand, ZrO2 presents higher strength and toughness values (900 MPa and 6 MPa.√m) than Al2O3 but it is a material limited in its long-term behaviour due to its bad response to hydrothermal ageing and a pronounced tendency for subcritical crack growth. Due to this fact, ceramic nanocomposites made of Al2O3 and ZrO2 (ATZ and ZTA) have been developed in the last years in order to overcome the main drawbacks of the monolithic materials as they can combine the properties of both, strong and tough materials, simultaneously, with null ageing and even higher biocompatibility. In this work, several amounts of Al2O3 disperse phase (15, 35 and 50 vol %) were added to one ZrO2 matrix (CeO2 - 10 mol %) in order to see their effect on the mechanical properties, subcritical crack propagation and long-term reliability.

Microwave sintering has emerged in recent years as a new, fast, cheap and green technology for sintering a variety of materials. The main advantages of microwave heating can be summarized as follow: reduced processing times, energy costs and environmental benefits. Nevertheless, understanding how this specific heating drives to obtain ceramic materials with a combination of unique, structural and functional properties is the big challenge. The present work shows the different and improved properties achieved with β-eucryptite nanocomposite ceramic materials by microwave heating compared with the conventional method. Microcracking evolution in addition to the microstructure of the sintered materials along the several thermal cycles has been studied. Mechanical properties changes observed can be related to this process. Thus, the microwave technique is a promising tool for sintering new materials by controlling the composition of the phases, chemical reactivity and nanostructure, using up to 70% less energy in the whole sintering process than conventional heating. This technique becomes part of the new and innovative technologies "eco-green".

OBJECTIVE: We have used receiver operating characteristic (ROC) analysis to determine the diagnostic performance of several serum parameters, in order to evaluate their potential usefulness in establishing the diagnosis of polycystic ovary syndrome (PCOS) in epidemiological studies. DESIGN: Prospective study. METHODS: One hundred and fourteen women reporting spontaneously for blood donation were included in the study. Menopausal and oral contraceptive-treated women were excluded. Serum samples were obtained at the moment of donation, independently of fasting, time of day or day of menstrual cycle. Measurements included total testosterone, sex hormone-binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), LH, FSH and estradiol. The free testosterone (FT) concentration and the free androgen index (FAI) were calculated from testosterone and SHBG levels. ROC curves were calculated for all these serum determinations. RESULTS: Eight patients were diagnosed with PCOS, according to the presence of oligomenorrhea, hirsutism, acne and/or hyperandrogenemia, and exclusion of non-classic congenital adrenal hyperplasia, hypothyroidism and hyperprolactinemia. Of the parameters studied SHBG, FAI, FT and DHEAS were considered adequate measures for the diagnosis of PCOS. For example, serum SHBG levels showed an area under the ROC curve of 0.875+/-(S.E.(w))0.045 (95% confidence interval 0.800-0.929). A SHBG decision threshold <37 nmol/l had a sensitivity of 87.5%, a specificity of 86.8%, a positive likelihood ratio of 6.63, and a negative likelihood ratio of 0.14, for the diagnosis of PCOS. CONCLUSIONS: Our present results strongly suggest that decreased SHBG levels, and increased FAI, free testosterone concentration and DHEAS concentrations, are highly effective as single analytical procedures in epidemiological studies for the detection of PCOS in women of reproductive age.

We evaluate the perianeurysmal environment and study parameters potentially influencing rupture of cerebral aneurysms. 101 consecutive aneurysm cases were retrospectively evaluated using radiological observation including imaging documents such as MR, CT and DSA studies. Aneurysm contact with perianeurysmal environment was classified and correlated with aneurysm shape, size, location and likely rupture point. Topographic relation of the aneurysm to the cisternal compartment was studied. Presence of contact with the surrounding structures was evaluated for bone, dura, brain, cranial nerves, arteries, and veins.The aneurysm shape and likely rupture point was found to be significantly influenced by the aneurysm environment. Depending on aneurysm type, location and size, the growth pattern also exhibited signs of interaction with the environment. Overall, there was no significant difference in the largest average diameter of the dome of ruptured (7.8 mm) and unruptured (6.7 mm) aneurysms. When compared to unruptured aneurysms (6/42), the presence of a bleb was more frequent in ruptured aneurysm (41/59).The perianeurysmal environment was found to have a significant influence on aneurysmal rupture pattern, whenever direct contact between the aneurysm and the anatomical structures in the perianeurysmal environment was visualized. This influence was independent of aneurysm size. Aneurysm size seems to be a poor indicator for the risk of rupture when compared to shape of the aneurysm and the degree of direct contact with the perianeurysmal environment.

We report on linkage analysis and haplotype characterization in 12 Cuban families with autosomal dominant polycystic kidney disease (ADPK) using PKD1-linked markers. They included both standard restriction fragment length polymorphisms (26.6., BLu24, and pGGGl) as well as microsatellite polymorphisms (CW2, 16AC2.5, and SM6). All of the examined families were fully informative for genetic diagnosis and no evidence of unlinked families was found. Analysis of two recombination events places PKD1 distal to the marker BLu24 and reduces the size of the region likely to contain the disease gene by approximately 300 kb. The allele frequencies of each marker were similar in the ADPKD and normal populations.

Paget's disease of bone is a common condition characterized by bone pain, deformity, pathological fracture, and an increased incidence of osteosarcoma. Genetic factors play a role in the pathogenesis of Paget's disease but the molecular basis of the disease remains unclear. Previous genetic linkage studies have mapped the rare Paget's disease‐like bone dysplasia familial expansile osteolysis (FEO) to chromosome 18q21–22, and recent work has shown evidence of linkage between this locus and Paget's disease in one family. Here we studied the relationship between the 18q21–22 locus and Paget's disease in eight large multiplex families from diverse ethnic backgrounds with inherited Paget's disease. Paget's disease was inherited as an autosomal dominant trait in all families, with high penetrance by the sixth decade. Analysis of seven highly polymorphic markers from chromosome 18q21–22 showed positive summated two‐point log10odds ratio (lodscores) of +2.97 with the marker D18S42 at a recombination fraction (θ) = 0.05, and of +2.95 with the marker D18S60 at θ = 0.00, values which are close to the cut‐off of +3.0, which is generally accepted as evidence of linkage. Segregation analysis of the haplotypes and formal statistical analysis using the HOMOG program provided evidence for genetic heterogeneity, however, with evidence for linkage in five families and against linkage in the remaining three families (chi square 8.82; df = 2; p< 0.025). Multipoint linkage analysis in the five linked families showed lodscores of above +3.5 across the whole susceptibility region and a maximum summated lodscore of 3.89 at the marker D18S465. In the three nonlinked families, negative multipoint results were obtained for the whole region, with lodscores below –2.0 in one family, excluding this as a candidate locus for the disease. Our studies demonstrate the importance of hereditary factors in the pathogenesis of Paget's disease and confirm evidence of linkage between Paget's disease and chromosome 18q21–22 in some families. This raises the possibility that Paget's disease and FEO may share a common molecular basis, perhaps due to different mutations in the same gene or family of genes. Data from three families did not support evidence of linkage to 18q21–22 however, indicating that Paget's disease is genetically heterogeneous and suggests the presence of at least one additional locus which remains to be discovered.

Laryngeal tuberculosis, although the most common granulomatous disease of the larynx, is a rare form of extrapulmonary tuberculosis, never reported in immunosuppressed allograft recipients. We present two cases of laryngeal tuberculosis in renal transplant patients and a review of the literature. Two women, a 29-year-old and a 60-year-old, each more than 9 years after their cadaveric renal allograft, presented with a 2- week febrile illness with hoarseness and dysphagia, and both were found to have laryngeal tuberculosis by direct laryngoscopy. Although both radiographs were unremarkable, both patients had sputum positive for acid-fast bacilli that subsequently grew Mycobacterium tuberculosis. Clinical response promptly followed institution of isoniazid, rifampicin, and pyrazinamide in each case, although both required threefold increases in daily cyclosporin A dosage to maintain therapeutic levels. (Am J Kidney Dis 1998 Apr;31(4):701-5)

A topology of the Escherichia coli leader peptidase has been previously proposed on the basis of proteolytic studies. Here, a collection of alkaline phosphatase fusions to leader peptidase is described. Fusions to the periplasmic domain of this protein exhibit high alkaline phosphatase activity, while fusions to the cytoplasmic domain exhibit low activity. Elements within the cytoplasmic domain are necessary to stably anchor alkaline phosphatase in the cytoplasm. The amino-terminal hydrophobic segment of leader peptidase acts as a weak export signal for alkaline phosphatase. However, when this segment is preceded by four lysines, it acts as a highly efficient export signal. The coherence of in vitro studies with alkaline phosphatase fusion analysis of the topology of leader peptidase further indicates the utility of this genetic approach to membrane protein structure and insertion.

Plasmid pMccB17 (70 kilobases [kb]) codes for the production of microcin B17, a peptide that inhibits DNA synthesis, and for microcin B17 immunity. A BamHI-EcoRI fragment of 5.1 kb from pMccB17 was cloned into pBR322 in two steps. The resulting plasmid (pMM102) overproduced microcin B17 and expressed immunity against microcin. Mcc- and Mcc- Imm- mutants were isolated on plasmids pMccB17 and pMM102 by deleting various DNA fragments and by inserting different translocatable elements. Physical and phenotypic characterization of these mutants showed that a DNA region of 3.0 to 3.5 kb is required to produce microcin B17, whereas an adjacent region of about 1.0 kb is required to express microcin B17 immunity.

ABSTRACTTEM-1 is the dominant β-lactamase of Haemophilus influenzaeand can be located on small plasmids. Three distinct plasmids with sizes from 4,304 to 5,646 nucleotides (nt) were characterized: pA1606, pA1209, and pPN223. In addition to TEM-1 and a replication enzyme of the Rep 3 superfamily, pA1606 carries a Tn3resolvase gene and pA1606 and pA1209 carry an open reading frame (ORF) similar to a plasmid recombination enzyme gene described in Gram-positive bacteria. The plasmids transformed strain Rd to the ampicillin-resistant phenotype.