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Shell-isolated nanoparticle-enhanced fluorescence (SHINEF) is a variation of surface-enhanced fluorescence (SEF) that involves the use of core–shell nanostructures to enhance fluorescence signals. This increase in signals is achieved due to the enhanced local electric field produced by light stimulation toward the plasmonic metal core of the core–shell nanostructure. In SHINEF, a thin insulating shell is introduced around a plasmonic metal core, creating a structure that enhances the fluorescence of nearby molecules while minimizing the unwanted interactions with the metal core, such as energy transfer events. In this study, we explore the fluorescence enhancement produced by silver nanoparticles (AgNPs) coated with SiO2for two triphenylamine–thiophene aggregation-induced emission (AIE) luminogens, 5-(4-(bis(4-methoxyphenyl)amino)phenyl)thiophene-2-carbaldehyde (TTY) and 5'-(4-(bis(4-methoxyphenyl)amino)phenyl)-[2,2'-bithiophene]-5-carbaldehyde (TTO), deposited on glass substrates. The results derived from emission and extinction spectra reveal that enhancement depends mainly on the concentrations of both molecules and AgNPs on the substrate as well as the selected excitation wavelength to carry out the measurement. A maximum experimental enhancement factor of 8.0 is achieved when the molecular concentration is at its lowest level, in combination with a higher AgNP concentration. These results are also rationalized in terms of computational simulation based on Mie theory. Simulated optical extinction spectra and the enhanced local external electric field around AgNPs have been carried out to compare with the obtained experimental results. This enhancement induces a significant decrease in lifetimes when the molecules are in the proximity of the nanostructured surface. These findings underscore the potential use of plasmonic nanoparticles as an effective alternative for enhancing the fluorescence of molecules with AIE characteristics.

The hydrogen evolution reaction (HER) is a crucial electrochemical process for the proposed hydrogen economy since it has the potential to provide pure hydrogen for fuel cells. Nowadays, hydrogen electroproduction is considerably expensive, so promoting the development of new non-noble catalysts for the cathode of alkaline electrolyzers appears as a suitable way to reduce the costs of this technology. In this sense, a series of tungsten-based carbide materials have been synthesized by the urea-glass route as candidates to improve the HER in alkaline media. Moreover, two different pyridinium-based ionic liquids were employed to modify the surface of the carbide grains and control the amount and nature of their surface species. The main results indicate that the catalyst surface composition is modified in the hybrid materials, which are then distinguished by the appearance of tungsten suboxide structures. This implies the action of ionic liquids as reducing agents. Consequently, differential electrochemical mass spectrometry (DEMS) is used to precisely determine the onset potentials and rate-determining steps (RDS) for the HER in alkaline media. Remarkably, the modified surfaces show high catalytic performance (overpotentials between 45 and 60 mV) and RDS changes from Heyrovsky–Volmer to Heyrovsky as the surface oxide structures get reduced. H2O molecule reduction is then faster at tungsten suboxide, which allows the formation of the adsorbed hydrogen at the surface, boosting the catalytic activity and the kinetics of the alkaline HER.

Despite recent advances in the development of scaffold-based three-dimensional (3D) cell models, challenges persist in imaging and monitoring cell behavior within these complex structures due to their heterogeneous cell distribution and geometries. Incorporating sensors into 3D scaffolds provides a potential solution for real-time, in situsensing and imaging of biological processes such as cell growth and disease development. We introduce a 3D printed hydrogel-based scaffold capable of supporting both surface-enhanced Raman scattering (SERS) biosensing and imaging of 3D breast cancer cell models. The scaffold incorporates plasmonic nanoparticles and SERS tags, for sensing and imaging, respectively. We demonstrate the scaffold’s adaptability and modularity in supporting breast cancer spheroids, thereby enabling spatial and temporal monitoring of tumor evolution.

Downshifters refer to compounds with the capacity to absorb UV photons and transform them into visible light. The integration of such downshifters has the potential to improve the efficiency of commercial photovoltaic modules. Initially, costly lanthanide derivatives and organic fluorescent dyes were introduced, resulting in a heightened module efficiency. In a novel research direction guided by the same physicochemical principles, the utilization of copper(I) coordination compounds is proposed. This choice is motivated by its simpler and more economical synthesis, primarily due to copper being a more abundant and less toxic element. Our proposal involves employing 1,2-bis(4-pyridyl) ethane (bpe), an economically viable commercial ligand, in conjunction with CuI to synthesize coordination polymers: [CuI(bpe)]n(1), [Cu3I3(bpe)3]n(2),and [CuI(bpe)0.5]n(3). These polymers exhibit the ability to absorb UV photons and emit light within the green and orange spectra. To conduct external quantum efficiency studies, the compounds are dispersed on glass and then encapsulated with ethylene vinyl acetate through heating to 150 °C. Interestingly, during these procedural steps, the solvents and temperatures employed induce a phase transformation, which has been thoroughly examined through both experimental analysis and theoretical calculations. The outcomes of these studies reveal an enhancement in external quantum efficiency with [Cu3I3(bpe)3]n(2), at a cost significantly lower (between 340 and 350 times) than that associated with lanthanide DS complexes.

ABSTRACT This paper describes the inspection for cavity detection in an urban area in Torrente (Valencia, Spain). A shallow cave was found during the excavation work for a new sewerage [...]

Energy transfer and exciplex emission are not only crucial photophysical processes in many living organisms but also important for the development of smart photonic materials. We report, herein, the rationally designed synthesis and characterization of two highly charged bischromophoric homo[2]catenanes and one cyclophane incorporating a combination of polycyclic aromatic hydrocarbons, i.e., anthracene, pyrene, and perylene, which are intrinsically capable of supporting energy transfer and exciplex formation. The possible coconformations of the homo[2]catenanes, on account of their dynamic behavior, have been probed by Density Functional Theory calculations. The unique photophysical properties of these exotic molecules have been explored by steady-state and time-resolved absorption and fluorescence spectroscopies. The tetracationic pyrene–perylene cyclophane system exhibits emission emanating from a highly efficient Förster resonance energy transfer (FRET) mechanism which occurs in 48 ps, while the octacationic homo[2]catenane displays a weak exciplex photoluminescence following extremely fast (<0.3 ps) exciplex formation. The in-depth fundamental understanding of these photophysical processes involved in the fluorescence of bischromophoric cyclophanes and homo[2]catenanes paves the way for their use in future bioapplications and photonic devices.

Five judges, most award-winners themselves, share the insights and tips that may get one an award. Michael Good feels that technical proficiency is important, both commercially and aesthetically and so flawed execution will greatly diminish a visually attractive design.

To determine the prevalence of burnout syndrome and the associated variables in medical specialists in Mexico.

Donor–acceptor (DA) π-interactions are weak attractive forces that are exploited widely in molecular and supramolecular chemistry. They have been characterized extensively by ensemble techniques, providing values for their energies that are useful for the design of soft materials. For implementation of motions or operations based on these DA π-interactions in wholly synthetic molecular machines, the mechanical strength and force associated with their out-of-equilibrium performance are the key parameters, in addition to their energies obtained at thermodynamic equilibrium. In this context, we have used single-molecule force spectroscopy as a nonequilibrium technique to determine the mechanical strength of individual DA π-interactions in solution. We designed and synthesized a molecular tweezer that is able to encapsulate π-donors and also demonstrated a precise opening extension. The mechanical breaking of the noncovalent interactions between viologen units—π-acceptors commonly employed in mechanically interlocked molecules—and several π-donors afforded a characteristic force–distance signature, revealing the opening of individual viologen tweezers with an unambiguous extension. Single-tweezer host-exchange experiments performed in situdemonstrated the sensitivity of the technique. This simple design could be exploited in quantifying the force of a large range of weak noncovalent bonding interactions as well as the potential work that molecular machines can generate at the single-molecule level.

The phenomenon of photon upconversion, in which a system absorbs two or more photons of lower energy and emits a photon of higher energy, has been used in numerous applications, including non-destructive bioimaging, deep-penetrating photodynamic therapy, catalysis, and photovoltaic devices. To date, photon upconversion has been observed typically in inorganic nanocrystals, nanoparticles, metal–organic frameworks, supramolecular assemblies, and organic dyads. Herein, we demonstrate a new strategy for harnessing photon upconversion—supramolecular upconversion—based on host–guest chemistry. We have identified a box-like fluorescent tetracationic host incorporating a thiazolothiazole emitter, which can accommodate a guest-sensitizer, 5,15-diphenylporphyrin, inside its cavity, and demonstrated that the host–guest inclusion complex displays triplet-fusion upconversion when the guest is excited with low-energy light. The strategy of supramolecular upconversion has been employed successfully in two other host–guest complexes—with hosts comprised of anthracene emitters and a 5,15-diphenylporphyrin guest—corroborating the fact that this strategy is a general one and can be applied to the design of a new family of host–guest complexes for photon upconversion. More importantly, supramolecular upconversion is accessible in solution under dilute conditions (μM) compared to most of the existing approaches that require significantly higher concentrations (mM) of emitters and/or sensitizers. Transient absorption spectroscopy and density functional theory have been employed in order to confirm a triplet-fusion upconversion mechanism. Host–guest complexation-mediated supramolecular photon upconversion eliminates multiple issues in the existing systems related to high working concentrations, high incident laser power, and low optical penetration depths.

A fundamental challenge in developing treatments for autism spectrum disorders is the heterogeneity of the condition. More than one hundred genetic mutations confer high risk for autism, with each individual mutation accounting for only a small fraction of cases1–3. Subsets of risk genes can be grouped into functionally related pathways, most prominently those involving synaptic proteins, translational regulation, and chromatin modifications. To attempt to minimize this genetic complexity, recent therapeutic strategies have focused on the neuropeptides oxytocin and vasopressin4–6, which regulate aspects of social behaviour in mammals7. However, it is unclear whether genetic risk factors predispose individuals to autism as a result of modifications to oxytocinergic signalling. Here we report that an autism-associated mutation in the synaptic adhesion molecule Nlgn3results in impaired oxytocin signalling in dopaminergic neurons and in altered behavioural responses to social novelty tests in mice. Notably, loss of Nlgn3is accompanied by a disruption of translation homeostasis in the ventral tegmental area. Treatment of Nlgn3-knockout mice with a new, highly specific, brain-penetrant inhibitor of MAP kinase-interacting kinases resets the translation of mRNA and restores oxytocin signalling and social novelty responses. Thus, this work identifies a convergence between the genetic autism risk factor Nlgn3, regulation of translation, and oxytocinergic signalling. Focusing on such common core plasticity elements might provide a pragmatic approach to overcoming the heterogeneity of autism. Ultimately, this would enable mechanism-based stratification of patient populations to increase the success of therapeutic interventions.

Collisional intermolecular interactions between excited states form short-lived dimers and complexes that lead to the emergence of excimer/exciplex emission of lower energy, a phenomenon which must be differentiated from the photoluminescence (PL) arising from the monomeric molecules. Although the utilization of noncovalent bonding interactions, leading to the generation of excimer/exciplex PL, has been investigated extensively, precise control of the aggregates and their persistence at very low concentrations remains a rare phenomenon. In the search for a fresh approach, we sought to obtain exciplex PL from permanent structures by incorporating anthracene moieties into pyridinium-containing mechanically interlocked molecules. Beyond the optical properties of the anthracene moieties, their π-extended nature enforces [π···π] stacking that can overcome the Coulombic repulsion between the pyridinium units, affording an efficient synthesis of an octacationic homo[2]catenane. Notably, upon increasing the ionic strength by adding tetrabutylammonium hexafluorophosphate, the catenane yield increases significantly as a result of the decrease in Coulombic repulsions between the pyridinium units. Although the ground-state photophysical properties of the free cyclophane and the catenane are similar and show a charge-transfer band at ∼455 nm, their PL characters are distinct, denoting different excited states. The cyclophane emits at ∼562 nm (quantum yield ϕF= 3.6%, emission lifetime τs= 3 ns in MeCN), which is characteristic of a disubstituted anthracene–pyridinium linker. By contrast, the catenane displays an exciplex PL at low concentration (10–8M) with an emission band centered on 650 nm (ϕF= 0.5%, τs= 14 ns) in MeCN and at 675 nm in aqueous solution. Live-cell imaging performed in MIAPaCa-2 prostate cancer cells confirmed that the catenane exciplex emission can be detected at micromolar concentrations.

•We meta-analyzed 42 studies representing 35 cohorts and 3.8 million adults to compare the associations of objectively measured exercise-estimated, and non-exercise-estimated cardiorespiratory fitness (CRF) with all-cause and cardiovascular disease (CVD) mortality in adults.•We found 14% and 16% reductions in all-cause and CVD mortality risk per higher metabolic equivalent of task ((MET) i.e., 3.5 mL/kg/min), respectively, with no differences in risk reduction between objectively measured, exercise-estimated, and non-exercise-estimated CRF.•Exercise and non-exercise estimated CRF provide practical and robust alternatives to the more costly and time-consuming objectively measured CRF to enhance patient risk stratification in clinical settings.

We present a rapid and efficient method to generate a family of platinum supramolecular square complexes, including previously inaccessible targets, through the use of ball milling mechanochemistry. This one-pot, two-step process occurs in minutes and enables the synthesis of the squares [Pt4(en)4(N∩N)4][CF3SO3]8(en= ethylenediamine, N∩N = 4,4′-bipyridine derivatives) from commercially available precursor K2PtCl4in good to excellent yields. In contrast, solution-based assembly requires heating the reagents for weeks and gives lower yields. Mechanistic investigations into this remarkable rate acceleration revealed that solution-based assembly (refluxing for days) results in the formation of large oligomeric side-products that are difficult to break down into the desired squares. On the other hand, ball milling in the solid state is rapid and appears to involve smaller intermediates. We examined the binding of the new supramolecular squares to guanine quadruplexes, including oncogene and telomere-associated DNA and RNA sequences. Sub-micromolar binding affinities were obtained by fluorescence displacement assays (FID) and isothermal titration calorimetry (ITC), with binding preference to telomere RNA (TERRA) sequences. ITC showed a 1:1 binding stoichiometry of the metallosquare to TERRA, while the stoichiometry was more complex for telomeric quadruplex DNA and a double-stranded DNA control.

Owing to their extraordinary magnetic properties and low-cost production, iron oxide nanoparticles (IONs) are in the focus of research. In order to better understand interactions of IONs with biomolecules, a tool for the prediction of the propensity of different peptides to interact with IONs is of great value. We present an effective implicit surface model (EISM), which includes several interaction models. Electrostatic interactions, van der Waals interactions, and entropic effects are considered for the theoretical calculations. However, the most important parameter, a surface accessible area force field contribution term, derives directly from experimental results on the interactions of IONs and peptides. Data from binding experiments of ION agglomerates to different peptides immobilized on cellulose membranes have been used to parameterize the model. The work was carried out under defined environmental conditions; hence, effects because of changes, for example structure or solubility by changing the surroundings, are not included. EISM enables researchers to predict the binding of peptides to IONs, which we then verify with further peptide array experiments in an iterative optimization process also presented here. Negatively charged peptides were identified as best binders for IONs in Tris buffer. Furthermore, we investigated the constitution of peptides and how the amount and position of several amino acid side chains affect peptide-binding. The incorporation of glycine leads to higher binding scores compared to the incorporation of cysteine in negatively charged peptides.

Introduction: Neuromyelitis optica (NMO) and Multiple Sclerosis (MS) have been reported in different populations. NMO is more frequent in non-Caucasians, and clinical phenotype differences between populations are likely influenced by genetic susceptibility. In Brazil, it has been reported that NMO patients have a mainly European ancestry background. Like other autoimmune diseases, NMO has a multifactorial origin (i.e., genetics, environmental and infectious factors). Regarding the genetics of NMO, epidemiological findings suggest that a polygenic background has an important role in the development of this type of disease. In this context, various genes have been studied, such as those involved in the synthesis of the T cell beta chain receptor, the VH2-5 gene, myelin basic protein, the CTLA-4 gene, and the interleukin-1 gene. Materials and Methods: We performed a study with the main objective of identifying candidate genes involved in the susceptibility to develop NMO in a Mexican population. Result: We included 35 patients with an NMO diagnosis according to the Wingerchuk 2006 criteria. The mean age of the patients was 43.3 years old (20-67), and 80 percent of the patients were women. The presence of HLA-DRB1*03 and HLA-DRB1*10 alleles were more frequent in NMO patients than in controls (n=198; p=0.03 and 0.005, respectively). Conclusion: There were no differences in the other alleles that have been described in MS subjects, such as HLA-DRB1*04, HLA-DRB1*08 and HLA-DRB1*13. These findings may support the hypothesis that implicated immune-genetics as a key factor in development of this type of disease.

Making use of the inherent surface anisotropy of different high index surface planes vicinal to the low index Au(111) orientation, one-dimensional polymers have been synthesized following established procedures from two different precursor molecules. The successful polymerization of both 4,4?-dibromo-p-terphenyl and 5,5'-dibromo-salophenato-Co(II) precursors into poly(p-phenylene) and poly[salophenato-Co(II)], respectively, has been confirmed by scanning tunneling microscopy and low energy electron diffraction. Angle-resolved photoemission spectroscopy data reveal a highly dispersive band in the case of poly(p-phenylene) while no significant dispersion is resolved for poly[salophenato-Co(II)]. On the basis of density functional theory calculations, we explain this observation as a result of a high conjugation along the aromatic phenyl groups in poly(p-phenylene) that is absent in the case of poly[salophenato-Co(II)], where intramolecular conjugation is interrupted in the salophenato-Co(II) unit. Furthermore, we make use of multicenter and delocalization indexes to characterize the electron mobility (corresponding to a high band dispersion) along different paths associated with individual molecular orbitals.

A new kind of low molecular-mass organic gelator (LMOG) p-electron-deficient N,N-octyl-7,7'-diazaisoindigo (1) with aggregation-induced enhanced emission (AIEE) phenomenon is described. This organogel is capable of self-assembling through intermolecular H-bonding and p–p interactions between diazaisoindigo molecules. Its rheological properties, X-ray diffraction pattern, optical properties and theoretical calculations were investigated. The AIEE effect is exhibited in fluorescence during the formation of the supramolecular organogel, which persisted in the xerogel state, and the spectral red-shifts suggest the formation of J-type aggregates during the gelation process via p–p interactions in microbelts or 3D networks. Fluorescence lifetime and quantum yield significantly increase from dilute solution to the aggregate state. From a theoretical perspective, the effect of the aggregation of 1on the photophysical properties was also studied by means of the density functional theory (DFT). In this sense, the lowest energy electronic transitions were calculated for both the single molecule and different size aggregates in order to predict spectral shifts. In addition, the geometry and molecular properties of the excited state were analyzed in different material states.

Near-infrared (NIR) light is known to have outstanding optical penetration in biological tissues and to be non-invasive to cells compared with visible light. These characteristics make NIR-specific light optimal for numerous biological applications, such as the sensing of biomolecules or in theranostics. Over the years, significant progress has been achieved in the synthesis of fluorescent cyclophanes for sensing, bioimaging, and making optoelectronic materials. The preparation of NIR-emissive porphyrin-free cyclophanes is, however, still challenging. In an attempt for fluorescence emissions to reach into the NIR spectral region, employing organic tetracationic cyclophanes, we have inserted two 9,10-divinylanthracene units between two of the pyridinium units in cyclobis(paraquat-p-phenylene). Steady-state absorption, fluorescence, and transient-absorption spectroscopies reveal the deep-red and NIR photoluminescence of this cyclophane. This tetracationic cyclophane is highly soluble in water and has been employed successfully as a probe for live-cell imaging in a breast cancer cell line (MCF-7).

ABSTRACTA 1-year prospective multicenter study was performed to explore the significance of the presence of enterococci in cultures of peritoneal fluid from patients with secondary bacterial peritonitis in seven Spanish hospitals. The clinical records of patients with positive peritoneal fluid cultures were reviewed and distributed into cases (patients with cultures yielding enterococci) and controls (patients with cultures not yielding enterococci). Of a total of 158 records, 38 (24.1%) were cases and 120 (75.9%) were controls. The percentages or the scores (cases versus controls) for the variables included in the multivariate analysis were as follows: age of >50 years, 89.5% versus 68.3%; malignancy, 39.5% versus 18.3%; chronic obstructive pulmonary disease (COPD), 15.8% versus 4.2%; postoperative peritonitis, 55.3% versus 30.1%; nosocomial onset, 57.9% versus 34.2%; a higher Charlson comorbidity index, 3.29 ± 3.38 versus 1.84 ± 2.31; APACHE II score, 10.71 ± 4.37 versus 8.76 ± 5.49; ultimately or rapidly fatal disease, 63.2% versus 34.8%; need for surgical reintervention, 36.1% versus 15.1%; and admission to an intensive care unit, 45.9% versus 30.8%. In the multivariate analysis, enterococci were associated only with postoperative peritonitis (P= 0.009; odds ratio [OR] = 5.0; 95% confidence interval [CI] = 1.49 to 16.80), a higher Charlson comorbidity index (P= 0.002; OR = 1.30; 95% CI = 1.11 to 1.54), and COPD (P= 0.046; OR = 6.50; 95% CI = 1.04 to 40.73). The results of this study showed that enterococci were associated with comorbidity. An association with mortality could not be demonstrated.

ABSTRACTWe used confocal microscopy to evaluate nuclear dynamics in mature, growing hyphae of Neurospora crassawhose nuclei expressed histone H1-tagged green fluorescent protein (GFP). In addition to the H1-GFP wild-type (WT) strain, we examined nuclear displacement (passive transport) in four mutants deficient in microtubule-related motor proteins (ro-1, ro-3, kin-1, and a ro-1 kin-1double mutant). We also treated the WT strain with benomyl and cytochalasin A to disrupt microtubules and actin microfilaments, respectively. We found that the degree of nuclear displacement in the subapical regions of all strains correlated with hyphal elongation rate. The WT strain and that the ro-1 kin-1double mutant showed the highest correlation between nuclear movement and hyphal elongation. Although most nuclei seemed to move forward passively, presumably carried by the cytoplasmic bulk flow, a small proportion of the movement detected was either retrograde or accelerated anterograde. The absence of a specific microtubule motor in the mutants ro-1, ro-3, or kin-1did not prevent the anterograde and retrograde migration of nuclei; however, in the ro-1 kin-1double mutant retrograde migration was absent. In the WT strain, almost all nuclei were elongated, whereas in all other strains a majority of nuclei were nearly spherical. With only one exception, a sizable exclusion zone was maintained between the apex and the leading nucleus. The ro-1mutant showed the largest nucleus exclusion zone; only the treatment with cytochalasin A abolished the exclusion zone. In conclusion, the movement and distribution of nuclei in mature hyphae appear to be determined by a combination of forces, with cytoplasmic bulk flow being a major determinant. Motor proteins probably play an active role in powering the retrograde or accelerated anterograde migrations of nuclei and may also contribute to passive anterograde displacement by binding nuclei to microtubules.

ABSTRACTThe use of molecular tools for genotyping Mycobacterium tuberculosisisolates in epidemiological surveys in order to identify clustered and orphan strains requires faster response times than those offered by the reference method, IS6110restriction fragment length polymorphism (RFLP) genotyping. A method based on PCR, the mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping technique, is an option for fast fingerprinting of M. tuberculosis, although precise evaluations of correlation between MIRU-VNTR and RFLP findings in population-based studies in different contexts are required before the methods are switched. In this study, we evaluated MIRU-VNTR genotyping (with a set of 15 loci [MIRU-15]) in parallel to RFLP genotyping in a 39-month universal population-based study in a challenging setting with a high proportion of immigrants. For 81.9% (281/343) of the M. tuberculosisisolates, both RFLP and MIRU-VNTR types were obtained. The percentages of clustered cases were 39.9% (112/281) and 43.1% (121/281) for RFLP and MIRU-15 analyses, and the numbers of clusters identified were 42 and 45, respectively. For 85.4% of the cases, the RFLP and MIRU-15 results were concordant, identifying the same cases as clustered and orphan (kappa, 0.7). However, for the remaining 14.6% of the cases, discrepancies were observed: 16 of the cases clustered by RFLP analysis were identified as orphan by MIRU-15 analysis, and 25 cases identified as orphan by RFLP analysis were clustered by MIRU-15 analysis. When discrepant cases showing subtle genotypic differences were tolerated, the discrepancies fell from 14.6% to 8.6%. Epidemiological links were found for 83.8% of the cases clustered by both RFLP and MIRU-15 analyses, whereas for the cases clustered by RFLP or MIRU-VNTR analysis alone, links were identified for only 30.8% or 38.9% of the cases, respectively. The latter group of cases mainly comprised isolates that could also have been clustered, if subtle genotypic differences had been tolerated. MIRU-15 genotyping seems to be a good alternative to RFLP genotyping for real-time interventional schemes. The correlation between MIRU-15 and IS6110RFLP findings was reasonable, although some uncertainties as to the assignation of clusters by MIRU-15 analysis were identified.

ABSTRACTA new, efficient molecular method for detection of Bartonella, based on the 16S-23S rRNA intergenic spacer and 16S rRNA amplification by multiplex PCR combined with reverse line blotting, was designed. This assay could simultaneously detect 20 different known species and other Bartonellaspecies not described previously.

ABSTRACTVisceral leishmaniasis (VL) caused by Leishmania infantumis a common disease in human immunodeficiency virus (HIV)-infected people in the Mediterranean basin. However, most such cases are asymptomatic, and little information about the prevalence of these infections in HIV-infected individuals is available. The aim of this study was to assess the prevalence of subclinical infection and the relationship between several Leishmaniainfection markers by noninvasive methods in asymptomatic HIV-infected patients from Southern Spain. Ninety-two HIV-infected patients, who were consecutively attended at the participant hospitals in 2004, were invited to participate in this study. These patients were asymptomatic and without any history of cutaneous or visceral leishmaniasis. Leishmaniakinetoplast DNA (kDNA) was amplified from peripheral blood samples from 28 (30.4%) of these HIV-infected subjects. Sera from three (3.5%) patients tested positive for Leishmaniaby an enzyme-linked immunoassay. Two patients (2.4%) showed a specific 16-kDa band by Western blotting. In contrast, none of the patients showed a positive agglutination of urine. The leishmanin skin test was positive for four (4.3%) patients. None of the patients with a PCR-positive result showed a positive reaction by enzyme-linked immunoassay or by specific bands in Western blotting or had a positive leishmanin skin test. In conclusion, L. infantumkDNA was detected in a large proportion of asymptomatic HIV-infected patients, although a demonstrable cellular or humoral immune response to this parasite was not shown. Conversely, Leishmaniaantigen in urine was not detected in these patients.

ABSTRACTA total of 151 Escherichia colistrains resistant to cefotaxime and ceftazidime were isolated during a prospective surveillance study. These strains were characterized by clinical, microbiological, and molecular analyses and were distributed into four clusters of 103, 11, 6, and 5 isolates, along with 25 unrelated strains. The principal cluster was isolated from urine, wound, blood, and other samples in three hospitals, eight nursing homes, and a community healthcare center. This cluster was associated with both nosocomial (65%) and community-acquired (35%) infections. Most strains were resistant to ciprofloxacin, gentamicin, tobramycin, cefepime, amoxicillin-clavulanic acid, and trimethoprim-sulfamethoxazole but were susceptible to imipenem. All isolates from the four clusters expressed the extended-spectrum ß-lactamase (ESBL) CTX-M-15. This enzyme was also present in 8 (30.8%) of the 26 unrelated isolates. The other ESBLs, CTX-M-14 and CTX-M-32, were detected in five and seven cases, respectively, but they were detected in individual E. coliisolates only. In three clusters, blaCTX-M-15alleles were linked to an ISEcp1-like element, while in eight strains of cluster II an IS26element preceded the blaCTX-M-15allele. An additional pool of resistance genes included tetA, drfA14or dfrA17, sul1or sul2, aac(6')Ib, and aac(3)IIb. All except one of the 27 isolates tested for genetic virulence markers harbored the same three virulence genes: iutAand fyuA(siderophores), and traT(serum survival factor). Epidemic or occasional isolates of cefotaxime- and ceftazidime-resistant E. colican spread between distinct health facilities including hospitals, community health centers, and long-term care centers.

ABSTRACTThe antibody response to capsular polysaccharides of pneumococcal serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F elicited either naturally or after vaccination with Prevenar was investigated in a cohort of children (n= 163) with underlying chronic or recurrent lung diseases at risk of developing pneumococcal pneumonia and ultimately invasive disease. Serum concentrations of serotype-specific antibodies, as measured by enzyme-linked immunosorbent assay, in unvaccinated children (n= 88) were higher in nasopharyngeal carriers (n= 10) than in noncarriers (n= 78) both at baseline and during follow-up. However, the antibody levels depended on the serotype and age of the children. During the study period, 35% of unvaccinated noncarriers and 60% of unvaccinated carriers displayed serum antibodies to all serotypes above the reported WHO working group putative protective serum concentration against invasive disease (0.2 µg/ml). Overall, children vaccinated with Prevenar before enrollment (n= 61), irrespective of their carrier status, displayed significantly higher serum levels of antibodies to all serotypes than unvaccinated children. More than 85% of the vaccinated children had protective serum antibody concentrations at baseline; although antibody titers tended to decrease over time, the above-mentioned figure remained without change at the end of follow-up. The vaccine Prevenar elicited a significant rise in serum antibody concentrations against all serotypes in 14 children vaccinated at entry. All of these children acquired and maintained serum antibody levels of >0.2 µg/ml throughout the study (a mean of 13 months of follow-up). These data support the systematic use of the vaccine Prevenar in children with underlying chronic or recurrent lung diseases and stress the fact that a percentage of vaccinated children may need to be revaccinated in order to achieve protection against pneumococcal disease.

ABSTRACTThe aim of this study is to determine oncogenic human papillomavirus (HPV) types and HPV type 16 (HPV16) variant distribution in two Spanish population groups, commercial sex workers and imprisoned women (CSW/IPW) and the general population. A multicenter cross-sectional study of 1,889 women from five clinical settings in two Spanish cities was conducted from May to November 2004. Oncogenic HPV infection was tested by an Hybrid Capture II (HC2) test, and positive samples were genotyped by direct sequencing using three different primer sets in L1 (MY09/11 and GP5+/GP6+) and E6/E7. HPV16 variants were identified by sequencing the E6, E2, and L1 regions. Four hundred twenty-five samples were positive for the HC2 test, 31.5% from CSW/IPW and 10.7% from the general population. HPV16 was the most frequent type. Distinct profiles of oncogenic HPV type prevalence were observed across the two populations. In order of decreasing frequency, HPV types 16, 31, 58, 66, 56, and 18 were most frequent in CSW/IPW women, and types 16, 31, 52, 68, 51, and 53 were most frequent in the general population. We analyzed HPV16 intratype variants, and a large majority (78.7%) belonged to the European lineage. AA variants were detected in 16.0% of cases. African variants belonging to classes Af1 (4.0%) and Af2 (1.3%) were detected. Different HPV types and HPV16 intratype variants are involved in oncogenic HPV infections in our population. These results suggest that HPV type distribution differs in CSW/IPW women and in the general population, although further analysis is necessary.

ABSTRACTWe conducted a nationwide surveillance of the variable 5' emm-like (M-like protein gene) sequences from 214 pharyngeal group C and group G streptococci. Almost 75% of the isolates exhibited emmor emm-like sequences previously described. We identified six new 5' emm-like regions, and almost 23% of the isolates were nontypeable. Five emm-like sequences accounted for more than 50% of the isolates in group C and group G, suggesting horizontal gene transfer between strains of different species.

ABSTRACTWe assessed the performance of a rapid, single-well, real-time PCR assay for the detection of rifampin-resistant Mycobacterium tuberculosisby using clinical isolates from north India and Mexico, regions with a high incidence of tuberculosis. The assay uses five differently colored molecular beacons to determine if a short region of the M. tuberculosis rpoBgene contains mutations that predict rifampin resistance in most isolates. Until now, the assay had not been sufficiently tested on samples from countries with a high incidence of tuberculosis. In the present study, the assay detected mutations in 16 out of 16 rifampin-resistant isolates from north India (100%) and in 55 of 64 rifampin-resistant isolates from Mexico (86%) compared to results with standard susceptibility testing. The assay did not detect mutations (a finding predictive of rifampin susceptibility) in 37 out of 37 rifampin-susceptible isolates from India (100%) and 125 out of 126 rifampin-susceptible isolates from Mexico (99%). DNA sequencing revealed that none of the nine rifampin-resistant isolates from Mexico, which were misidentified as rifampin susceptible by the molecular beacon assay, contained a mutation in the region targeted by the molecular beacons. The one rifampin-susceptible isolate from Mexico that appeared to be rifampin resistant by the molecular beacon assay contained an S531W mutation, which is usually associated with rifampin resistance. Of the rifampin-resistant isolates that were correctly identified in the molecular beacon assay, one contained a novel L530A mutation and another contained a novel deletion between codons 511 and 514. Overall, the molecular beacon assay appears to have sufficient sensitivity (89%) and specificity (99%) for use in countries with a high prevalence of tuberculosis.

ABSTRACTWe conducted a nationwide survey of the variable 5' emm(M protein gene) sequences from 614 pharyngeal Streptococcus pyogenesisolates susceptible (299 isolates) and resistant (315 isolates) to erythromycin that were isolated in Spain from 1996 to 1999. Almost 98% of these isolates had emmsequences in agreement with previously recorded M antigen association. We only identified a new 5' emmsequence in 17 isolates. Nine different emmtypes accounted for 85% of the S. pyogenesisolates susceptible to erythromycin. By contrast, only 3 emmtypes accounted for 70% of the erythromycin-resistant isolates. Further characterization of these isolates by ribotyping and pulsed-field gel electrophoresis indicated that high frequency of erythromycin resistance in Spain is due to few clones.

GABABreceptors, the most abundant inhibitory G protein–coupled receptors in the mammalian brain, display pronounced diversity in functional properties, cellular signaling and subcellular distribution. We used high-resolution functional proteomics to identify the building blocks of these receptors in the rodent brain. Our analyses revealed that native GABABreceptors are macromolecular complexes with defined architecture, but marked diversity in subunit composition: the receptor core is assembled from GABAB1a/b, GABAB2, four KCTD proteins and a distinct set of G-protein subunits, whereas the receptor's periphery is mostly formed by transmembrane proteins of different classes. In particular, the periphery-forming constituents include signaling effectors, such as Cav2 and HCN channels, and the proteins AJAP1 and amyloid-β A4, both of which tightly associate with the sushi domains of GABAB1a. Our results unravel the molecular diversity of GABABreceptors and their postnatal assembly dynamics and provide a roadmap for studying the cellular signaling of this inhibitory neurotransmitter receptor.

ABSTRACTThe autolytic LytA amidase from 12 bile (deoxycholate)-insoluble streptococcal isolates (formerly classified as atypical Streptococcus pneumoniae) showing different antibiotic resistance patterns was studied. These atypical strains, which autolyze at the end of the stationary phase of growth, contain highly divergent lytAalleles (pairwise evolutionary distances of about 20%) compared to the lytAalleles of typical pneumococci. The atypical LytA amidases exhibit a peculiar deletion of two amino acids responsible for cell wall anchoring in the carboxy-terminal domain and have a reduced specific activity. These enzymes were inhibited by 1% deoxycholate but were activated by 1% Triton X-100, a detergent that could be used as an alternative diagnostic test for this kind of strain. Preparation of functional chimeric enzymes, PCR mutagenesis, and gene replacements demonstrated that the characteristic bile insolubility of these atypical strains was due to their peculiar carboxy-terminal domain and that the 2-amino-acid deletion was responsible for the inhibitory effect of deoxycholate. However, the deletion alone did not affect the specific activity of LytA. A detailed characterization of the genes encoding the 16S rRNA and SodA together with multilocus sequence typing indicated that the strains studied here are not a single clone and, although they cannot be strictly classified as typical pneumococci, they represent a quite diverse pool of organisms closely related to S. pneumoniae. The clinical importance of these findings is underlined by the role of the lytAgene in shaping the course of pneumococcal diseases. This study can also contribute to solving diagnostic problems and to understanding the evolution and pathogenic potential of species of the Streptococcus mitisgroup.