Atomic-Scale Variations of the Mechanical Response of 2D Materials Detected by Noncontact Atomic Force Microscopy.

We show that noncontact atomic force microscopy (AFM) is sensitive to the local stiffness in the atomic-scale limit on weakly coupled 2D materials, as graphene on metals. Our large amplitude AFM topography and dissipation images under ultrahigh vacuum and low temperature resolve the atomic and moiré patterns in graphene on Pt(111), despite its extremely low geometric corrugation. The imaging mechanisms are identified with a multiscale model based on density-functional theory calculations, where the energy cost of global and local deformations of graphene competes with short-range chemical and long-range van der Waals interactions. Atomic contrast is related with short-range tip-sample interactions, while the dissipation can be understood in terms of global deformations in the weakly coupled graphene layer. Remarkably, the observed moiré modulation is linked with the subtle variations of the local interplanar graphene-substrate interaction, opening a new route to explore the local mechanical properties of 2D materials at the atomic scale.

Nanoscale reduction of graphene oxide thin films and its characterization.

In this paper, we report on a method to reduce thin films of graphene oxide (GO) to a spatial resolution better than 100 nm over several tens of micrometers by means of an electrochemical scanning probe based lithography. In situ tip-current measurements show that an edged drop in electrical resistance characterizes the reduced areas, and that the reduction process is, to a good approximation, proportional to the applied bias between the onset voltage and the saturation thresholds. An atomic force microscope (AFM) quantifies the drop of the surface height for the reduced profile due to the loss of oxygen. Complementarily, lateral force microscopy reveals a homogeneous friction coefficient of the reduced regions that is remarkably lower than that of native graphene oxide, confirming a chemical change in the patterned region. Micro Raman spectroscopy, which provides access to insights into the chemical process, allows one to quantify the restoration and de-oxidation of the graphitic network driven by the electrochemical reduction and to determine characteristic length scales. It also confirms the homogeneity of the process over wide areas. The results shown were obtained from accurate analysis of the shift, intensity and width of Raman peaks for the main vibrational bands of GO and reduced graphene oxide (rGO) mapped over large areas. Concerning multilayered GO thin films obtained by drop-casting we have demonstrated an unprecedented lateral resolution in ambient conditions as well as an improved control, characterization and understanding of the reduction process occurring in GO randomly folded multilayers, useful for large-scale processing of graphene-based material.

Vipericidins: a novel family of cathelicidin-related peptides from the venom gland of South American pit vipers.

Cathelicidins are phylogenetically ancient, pleiotropic host defense peptides-also called antimicrobial peptides (AMPs)-expressed in numerous life forms for innate immunity. Since even the jawless hagfish expresses cathelicidins, these genetically encoded host defense peptides are at least 400 million years old. More recently, cathelicidins with varying antipathogenic activities and cytotoxicities were discovered in the venoms of poisonous snakes; for these creatures, cathelicidins may also serve as weapons against prey and predators, as well as for innate immunity. We report herein the expression of orthologous cathelicidin genes in the venoms of four different South American pit vipers (Bothrops atrox, Bothrops lutzi, Crotalus durissus terrificus, and Lachesis muta rhombeata)-distant relatives of Asian cobras and kraits, previously shown to express cathelicidins-and an elapid, Pseudonaja textilis. We identified six novel, genetically encoded peptides: four from pit vipers, collectively named vipericidins, and two from the elapid. These new venom-derived cathelicidins exhibited potent killing activity against a number of bacterial strains (S. pyogenes, A. baumannii, E. faecalis, S. aureus, E. coli, K. pneumoniae, and P. aeruginosa), mostly with relatively less potent hemolysis, indicating their possible usefulness as lead structures for the development of new anti-infective agents. It is worth noting that these South American snake venom peptides are comparable in cytotoxicity (e.g., hemolysis) to human cathelicidin LL-37, and much lower than other membrane-active peptides such as mastoparan 7 and melittin from bee venom. Overall, the excellent bactericidal profile of vipericidins suggests they are a promising template for the development of broad-spectrum peptide antibiotics.

Laser-induced disaggregation of TiO2 nanofillers for uniform nanocomposites.

Exploiting the intrinsic photosensitivity of TiO2 nanoparticles, we demonstrated how ultraviolet (UV) pulsed laser irradiation of acrylate polymer nanocomposite solutions can separate the initial clusters of these colloidal semiconductor nanorods into clearly distinct units. From the irradiated solutions, optically clear nanocomposite films are obtained which exhibit enhanced optical properties with respect to the nanocomposites obtained without previous UV treatment.

Influence of conjugation chemistry and B epitope orientation on the immune response of branched peptide antigens.

Multimeric presentation, a well-proven way of enhancing peptide immunogenicity, has found substantial application in synthetic vaccine design. We have reported that a combination of four copies of a B-cell epitope with one of a T-cell epitope in a single branched construct results in a peptide vaccine conferring total protection against foot-and-mouth disease virus in swine, a natural host (Cubillos et al. (2008) J. Virol. 82, 7223-7230). More recently, a downsized version of this prototype with only two copies of the B epitope has proven as effective as the tetravalent one in mice. Here we evaluate three approaches to bivalent platforms of this latter type, involving different chemistries for the conjugation of two B epitope peptides to a branching T epitope. Comparison of classical thioether, "reverse" thioether (Monsó et al. (2012) Org. Biomol. Chem. 10, 3116-3121) and thiol-ene conjugation chemistries in terms of synthetic efficiency clearly singles out the latter, maleimide-based strategy as most advantageous. We also examine how minor structural differences among the conjugates--including the N- or C-terminal attachment of the B epitope to the branching T epitope--bear on the immunogenicity of these vaccine candidates, with the maleimide-based conjugate again emerging as the most successful.

Disclosing and overcoming the trade-off between noise and scanning speed in atomic force microscopy.

Traditional dynamic modalities for atomic force microscopy imaging suffer from a stringent trade-off between fast scanning speed, weak interaction forces and accurate topography reconstruction. Finding an effective compromise between these aspects is often challenging, especially for soft biological samples for which stringent requirements hold when imaged in vivo. In this paper the main causes of this undesired trade-off in standard systems are analyzed and the exploitation of the intrinsic dynamics of the cantilever through a nonlinear control strategy is proposed as a method to overcome this limitation. A direct application to imaging of biological samples is reported to validate the results and show the effectiveness of the proposed technique.

Visualization of π-hole in molecules by means of Kelvin probe force microscopy.

Submolecular charge distribution significantly affects the physical-chemical properties of molecules and their mutual interaction. One example is the presence of a π-electron-deficient cavity in halogen-substituted polyaromatic hydrocarbon compounds, the so-called π-holes, the existence of which was predicted theoretically, but the direct experimental observation is still missing. Here we present the resolution of the π-hole on a single molecule using the Kelvin probe force microscopy, which supports the theoretical prediction of its existence. In addition, experimental measurements supported by theoretical calculations show the importance of π-holes in the process of adsorption of molecules on solid-state surfaces. This study expands our understanding of the π-hole systems and, at the same time, opens up possibilities for studying the influence of submolecular charge distribution on the chemical properties of molecules and their mutual interaction.

Incidence and risk factors of bacterial sepsis and invasive fungal infection in neonates and infants requiring major surgery: an Italian multicentre prospective study.

BACKGROUND: Limited data are currently available on the incidence rates and risk factors for bacterial sepsis and invasive fungal infections (IFIs) among neonates and infants undergoing major surgery. AIM: To assess the incidence of bacterial sepsis and IFI, fungal colonization, risk factors for sepsis, and mortality in neonates and infants aged <3 months undergoing major surgery. METHODS: A multicentre prospective study was conducted involving 13 level-3 neonatal intensive care units in Italy, enrolling all infants aged ≤3 months undergoing major surgery. FINDINGS: From 2018 to 2021, 541 patients were enrolled. During hospitalization, 248 patients had a bacterial infection, and 23 patients had a fungal infection. Eighty-four patients were colonized by fungal strains. Overall, in-hospital mortality was 2.8%, but this was higher in infected than in uninfected infants (P = 0.034). In multivariate analysis, antibiotic exposure before surgery, ultrasound-guided or surgical placement of vascular catheters, vascular catheterization duration, and gestational age ≤28 weeks were all associated with bacterial sepsis. The risk of IFI was markedly higher in colonized infants (odds ratio (OR): 8.20; P < 0.001) and was linearly associated with the duration of vascular catheterization. Fungal colonization in infants with abdominal surgery increased the probability of IFI 11-fold (OR: 11.1; P < 0.001). CONCLUSION: Preventive strategies such as early removal of vascular catheters and the fluconazole prophylaxis should be considered to prevent bacterial and fungal sepsis in infants undergoing abdominal surgery, and even more so in those with fungal colonization.

Long-range and long-term interferometric tracking by static and dynamic force-clamp optical tweezers.

Optical tweezers are recognized single-molecule technique to resolve forces and motion on the molecular scale. Complex biological phenomena, such as cell differentiation and locomotion, require long range tracking capabilities with nanometer resolution over an extended period, to resolve molecular processes on the cellular scale. Here we introduce a real-time control of the microscope stage position to perform long-term tracking, with sub-millisecond resolution, of a bead attached to a neuron, preserving sub-nanometer sensitivity on a spatial range of centimeters, seven orders of magnitude larger. Moreover, the suitability of the system is tested by time- modulating the force-clamp condition to study the role of statically and dynamically applied forces in neuronal differentiation.

Efficacy of cecropin A-melittin peptides on a sepsis model of infection by pan-resistant Acinetobacter baumannii.

Pan-resistant Acinetobacter baumannii have prompted the search for therapeutic alternatives. We evaluate the efficacy of four cecropin A-melittin hybrid peptides (CA-M) in vivo. Toxicity was determined in mouse erythrocytes and in mice (lethal dose parameters were LD(0), LD(50), LD(100)). Protective dose 50 (PD(50)) was determined by inoculating groups of ten mice with the minimal lethal dose of A. baumannii (BMLD) and treating with doses of each CA-M from 0.5 mg/kg to LD(0). The activity of CA-Ms against A. baumannii was assessed in a peritoneal sepsis model. Mice were sacrificed at 0 and 1, 3, 5, and 7-h post-treatment. Spleen and peritoneal fluid bacterial concentrations were measured. CA(1-8)M(1-18) was the less haemolytic on mouse erythrocytes. LD(0) (mg/kg) was 32 for CA(1-8)M(1-18), CA(1-7)M(2-9), and Oct-CA(1-7)M(2-9), and 16 for CA(1-7)M(5-9). PD(50) was not achieved with non-toxic doses (≤ LD(0)). In the sepsis model, all CA-Ms were bacteriostatic in spleen, and decreased bacterial concentration (p < 0.05) in peritoneal fluid, at 1-h post-treatment; at later times, bacterial regrowth was observed in peritoneal fluid. CA-Ms showed local short-term efficacy in the peritoneal sepsis model caused by pan-resistant Acinetobacter baumannii.

Real-space imaging of anisotropic charge of σ-hole by means of Kelvin probe force microscopy.

An anisotropic charge distribution on individual atoms, such as σ-holes, may strongly affect the material and structural properties of systems. However, the spatial resolution of such anisotropic charge distributions on an atom represents a long-standing experimental challenge. In particular, the existence of the σ-hole on halogen atoms has been demonstrated only indirectly through the determination of the crystal structures of organic molecules containing halogens or with theoretical calculations, consequently calling for its direct experimental visualization. We show that Kelvin probe force microscopy with a properly functionalized probe can image the anisotropic charge of the σ-hole and the quadrupolar charge of a carbon monoxide molecule. This opens a new way to characterize biological and chemical systems in which anisotropic atomic charges play a decisive role.

[Prevalence of cardiovascular disease in uraemia and relevance of cardiovascular risk factors]. / Prevalencia de enfermedad cardiovascular en la uremia y relevancia de los factores de riesgo cardiovascular.

AIM: To evaluate the prevalence of cardiovascular disease (CVD) and its association with cardiovascular risk factors, as well as their control in end-stage renal disease (ESRD) patients under maintenance hemodialysis (HD). PATIENTS AND METHODS: A total of 265 patients with ESRD on maintenance HD from a University Hospital and 4 dialysis units were included in this multicenter and cross-sectional study that analyzed the prevalence of CVD and the possible association with classic and new cardiovascular risk factors. Usual biochemical and haemathological parameters were analyzed, as well as plasma levels of homocysteine, troponin-I, BNP, lipoprotein(a), C reactive protein, IL-6, fibrinogen, asymmetrical dimethylarginine (ADMA), advanced oxidation protein products (AOPP), malondialdehyde, adiponectin, osteoprotegerin, and fetuin. In a subset of patients an echocardiography and carotid artery Doppler echography were also performed. RESULTS: The prevalence of CVD was 52.8%. Factors positively associated with prevalent CVD were age, BMI, left ventricular hypertrophy, hypertension, dyslipidemia and diabetes mellitus, dialysis vintage, Charlson s comorbility index, levels of fibrinogen, osteoprotegerin, BNP and CRP, as well as carotid intima-media thickness, left ventricular mass and pulse pressure. Factors negatively associated with prevalent CVD were: previous renal transplant, ejection fraction or levels of LDL-c and phosphorous. In the multivariate analysis dyslipidemia, left ventricular hypertrophy, age and LDL-c (negatively) were associated with CVD. CONCLUSIONS: In HD patients the prevalence of CVD is high and is associated with the presence of cardiovascular risk factors and subclinical CVD.

Experimental study of the application of a new bone cement loaded with broad spectrum antibiotics for the treatment of bone infection. / Estudio experimental de la aplicación de un nuevo cemento óseo cargado con antibióticos de amplio espectro para el tratamiento de la infección ósea.

OBJECTIVES: To evaluate the in vivo behaviour of a new bone cement loaded with antibiotics, in a rabbit bone infection model. MATERIAL AND METHODS: Sixteen New Zealand rabbits divided into 4 groups were used, depending on the cement (commercial or experimental) and the antibiotic (vancomycin or linezolid) used to control a bone infection caused by Staphylococcus aureus. The commercial cement is Palacos® R and the experimental cement has been achieved by adding PLGA to the solid phase of Palacos® R cement. A novel histological staging method based on bone histoarchitecture has been used. This staging allows us a global vision of bone repair capacity, in the presence of modified cement, and also allows us to correlate the damage generated with the functionality of the tissue. RESULTS: The degree of bone destructuration found depended on the type of cement and antibiotic, and was higher in the groups with commercial cement than in the experimental group (P<.01) and in the groups with linezolid with respect to vancomycin (P=.04) The percentage of macrophages varied exclusively depending on the antibiotic used, and was higher in the vancomycin groups (P=.04). DISCUSSION: The development of new formulations of bone cement that release more, and more prolonged, new generation antibiotics such as linezolid, present an in vivo behaviour superior to commercial cement, respecting the bone structure. This behaviour would have a clinical implication in fighting infections by increasingly resistant germs in the treatment of prosthetic infection.

Physicochemical characterization of several types of naturally colored cotton fibers from Peru.

Elemental composition, physical dimensions (length and apparent diameter), and crystallinity of different types of naturally colored cotton (NCCs) fibers from Peru were investigated using a CHNS organic elemental analyzer, optical microscopy and X-Ray Diffraction (XRD). Spectroscopic studies involving Fourier Transform Infrared Spectroscopy and X-Ray photoelectron spectroscopy (XPS) were conducted; and the thermal stability of cotton samples were also investigated. Results from organic elemental analyzer and XPS showed that cotton samples contain mainly carbon, oxygen and hydrogen, but darker color samples also presented nitrogen. It was also found that the white cotton sample exhibited the longest fibers whereas the darker color samples showed the shortest values in length. Interestingly, the crystallinity seems also decrease with color intensity of NCCs. Finally, the thermal stability of white cotton fibers was similar to those obtained for the NCCs.

Ground laser remote sensing measurements of a Saharan dust outbreak in Central Spain. Influence on PM10 concentrations in the lower and upper Spanish plateaus.

This paper presents the hourly evolution of a severe Saharan dust outbreak, (SDO), affecting Central Spain over July 23-24, 2004 measured with a laser remote sensing device at a location close to the Guadarrama mountain range foothills and its impact on PM10 levels (particles with an aerodynamic diameter below 10 microm) recorded at four contrasting monitoring stations located in the upper and lower Spanish plateau, some 170km apart. During the period of study the Saharan dust layer, (SDL), presented significant hourly variability in height (3600-1500m), depth (1500-700m) and aerosol dust loading (extinction coefficient, EC, 0.22-1.28km(-1)). Overnight layering was generally observed whereas a well mixed layer prevailed in the afternoon. The (SDO) impact on the lower levels took place approximately 12h after the (SDL) was initially observed and triggered by a descending dust enriched, evidencing the important role of subsidence over the presence of dust in lower altitudes. During the event, PM10 levels at all the stations exceeded EU air quality daily mean standards, 50 microgm(-3), on 2-4days. The maximum values ranged from 185 to 245 microgm(-3) depending on the monitoring station. The impact on PM10 spread from 2days in the upper plateau to 3-4 in the lower plateau, in agreement with the geographical location of the monitoring stations with respect to the southwest origin of the intrusion. The impact was even more dramatic on PM10 hourly concentrations, leading to maximum hourly peaks ranging from 322 to 598 microgm(-3) again depending on the monitoring station. Correlations between EC vertical profiles and PM10 hourly concentrations at the monitoring stations showed the great influence of the (SDO) on surface concentrations. The best linear fits corresponded to the extinction coefficients in the lower altitudes (close to the lower range of the device 500-650m), EC2, yielding satisfactory correlation coefficients ranging from 0.68 to 0.71. The low variability of the slope of each individual linear fit, 19.2%, shows the similar impact of (SDO) on the PM10 hourly concentrations recorded in the area under study.

Imaging and structural studies of DNA-protein complexes and membrane ion channels.

In bio-imaging by electron microscopy, damage of the sample and limited contrast are the two main hurdles for reaching high image quality. We extend a new preparation method based on nanofabrication and super-hydrophobicity to the imaging and structural studies of nucleic acids, nucleic acid-protein complexes (DNA/Rad51 repair protein complex) and neuronal ion channels (gap-junction, K+ and GABAA channels) as paradigms of biological significance and increasing complexity. The preparation method is based on the liquid phase and is compatible with physiological conditions. Only in the very last stage, samples are dried for TEM analysis. Conventional TEM and high-resolution TEM (HRTEM) were used to achieve a resolution of 3.3 and 1.5 Å, respectively. The EM dataset quality allows the determination of relevant structural and metrological information on the DNA structure, DNA-protein interactions and ion channels, allowing the identification of specific macromolecules and their structure.

Development of advanced biantibiotic loaded bone cement spacers for arthroplasty associated infections.

The incidence increase of infections in patients with hip or knee implants with resistant pathogens (mainly some S. coagulase-negative and gram positive bacteria) demands advanced antibiotic loaded formulations. In this paper, we report the design of new biantibiotic acrylic bone cements for in situ delivery. They include a last generation antibiotic (daptomycin or linezolid) in combination with vancomycin and are performed based on a novel modification of the Palacos R® acrylic bone cement, which is based on two components, a liquid (methyl methacrylate) and a solid (polymeric phase). Hence, the solid component of the experimental formulations include 45wt% of microparticles of poly(D,L-lactic-co-glycolic) acid, 55wt% of poly(methyl methacrylate) beads and supplements (10wt-% each) of antibiotics. These formulations provide a selective and excellent control of the local release of antibiotics during a long time period (up to 2 months), avoiding systemic dissemination. The antimicrobial activity of the advanced spacers tested against S. aureus shows that single doses would be enough for the control of the infection. In vitro biocompatibility of cements on human osteoblasts is ensured. This paper is mainly focused on the preparation and characterization of cements and the studies of elution kinetics and bactericidal effects. Developed formulations are proposed as spacers for the treatment of infected arthroplasties, but also, they could be applied in other antibiotic devices to treat relevant bone-related infection diseases.

Naltrexone, an opioid antagonist, facilitates reepithelialization of the cornea in diabetic rat.

Ulcers and erosions of the corneal epithelium, as well as delays in resurfacing of the cornea after wounding, are major causes of ocular morbidity and visual loss in diabetes. To study whether intervention by the opioid antagonist naltrexone (NTX; 30 mg/kg, twice daily) can restore reepithelialization in diabetic cornea, we induced diabetes in rats by intravenous injection of 65 mg/kg streptozotocin. After confirmation of diabetes, 5-mm-diameter epithelial defects that did not include the limbus were created by mechanical scraping of the cornea. At 4 and 8 weeks, corneal reepithelialization was markedly subnormal, with delays ranging from 11% to 17-fold in the diabetic animals compared with control counterparts. Rats that were diabetic for 8 weeks also had a significant decrease in the incidence of complete wound closure. At 4 and 8 weeks, diabetic animals that were receiving NTX had an acceleration in reepithelialization compared with diabetic animals that were receiving vehicle and even surpassed controls. DNA synthesis in the corneal epithelium of diabetic rats was decreased up to 90% of control levels, and NTX exposure of diabetic subjects elevated the labeling index by up to eightfold from diabetic animals that were receiving vehicle. Opioid growth factor and opioid growth factor receptor distribution were comparable in diabetic and control animals. These results indicate a delay in reepithelialization that is dependent on the duration of diabetes and that intervention of endogenous opioid-receptor interfacing with an opioid antagonist can facilitate the process of wound healing.

Nucleocytoplasmic distribution of opioid growth factor and its receptor in tongue epithelium.

The subcellular distributions of the opioid growth factor (OGF), [Met(5)]-enkephalin, and opioid growth factor receptor (OGFr) in the epithelium of the rat tongue were determined in order to reveal structure-function relationships. Laser scanning confocal microscopic analysis showed that both OGF and OGFr were colocalized in the paranuclear cytoplasm and in the nuclei of keratinocytes in the stratum basale. Using immunoelectron microscopy and postembedding techniques, double labeling experiments disclosed that complexes of OGF-OGFr were colocalized on the outer nuclear envelope, in the paranuclear cytoplasm, perpendicular to the nuclear envelope in a putative nuclear pore complex, and in the nucleus adjacent to heterochromatin. Anti-OGF IgG alone was detected in the cytoplasm, and anti-OGFr IgG alone was associated with the outer nuclear envelope. Study of chronic treatment with the opioid antagonist, naltrexone (NTX), which blocks opioid-receptor binding, revealed the presence of OGFr immunoreactivity alone in the cytoplasm and the nucleus; some OGF-OGFr complexes were also observed. Colocalization of OGFr and karyopherin (importin) beta was recorded in the cytoplasm and nucleus. These results in tongue epithelium are the first to suggest that OGFr resides on the outer nuclear envelope, where OGF interacts with OGFr; that the OGF-OGFr complex translocates between cytoplasm and nucleus at the nuclear pore; and that the nuclear localization signal of OGFr interacts with karyopherin beta for nuclear transport. These novel data also indicate that signal transduction for cell proliferation appears to involve an OGF-OGFr complex that interfaces with chromatin in the nucleus. Moreover, the unique finding that OGFr was found in the cytoplasm and nucleus in NTX-treated specimens may suggest that NTX-OGFr complexes have the same pathway as OGF-OGFr.

Ground level ozone concentrations at a rural location in northern Spain.

A systematic analysis of surface ozone observations at a rural area of the upper Spanish plateau is presented. A near four-year study of ozone concentrations was carried out between February 2000 and October 2003. Diurnal and monthly variations of ozone are studied. The peak concentration levels are obtained between 13:00 and 15:00 GMT and the lowest levels at 4:00 and 5:00 GMT. The highest values together with a slight increase in data variability are found from April to August, when spring and summer maxima are appreciated. The relationship between synoptic-scale atmospheric transport patterns and the maximum ozone concentrations was also examined. Analysis of the 500-hPa synoptic weather patterns revealed that the highest values occur during continental and Atlantic ridges in summer and, to a lesser extent, during troughs in the east-northeast of the Iberian Peninsula in spring. Our approach entailed calculating 3-day isobaric backward air mass trajectories at the observational site sorted on the following main origins: European continent, African continent, Atlantic Ocean and local. The assessment of the results showed the influence of local conditions on the daily maximum ozone concentrations. Moreover, continental air masses provide certain evidence of long-range transport added to the local or regional contribution of the main cities affecting the sampling site. Conversely, Atlantic trajectories show the least contribution to ozone levels.