Targeted bacterial conjugation mediated by synthetic cell-to-cell adhesions.

Genetic interventions on microbiomes, for clinical or biotechnological purposes, remain challenging. Conjugation-based delivery of genetic cargo is still unspecific and limited by low conjugation rates. Here we report an approach to overcome these problems, based on a synthetic bacterial adhesion system. Mating assemblers consist on a synthetic adhesion formed by the expression on the surface of donor and target cells of specific nanobodies (Nb) and their cognate antigen (Ag). The Nb-Ag bridge increased 1-3 logs transfer of a variety of plasmids, especially in liquid media, confirming that cell-cell docking is a main determinant limiting mating efficiency. Synthetic cell-to-cell adhesion allows efficient conjugation to targeted recipients, enhancing delivery of desired genes to a predefined subset of prey species, or even specific pathogenic strains such as enterohemorrhagic Escherichia coli (EHEC), within a bacterial community. The synthetic conjugation enhancer presented here optimizes plasmid delivery by selecting the target hosts with high selectivity.

Decellularized tracheal prelamination implant: A proposed bilateral double organ technique.

In tracheal replacement transplantation, prelamination is a critical stage. Nowadays, the most widely used prelamination technique is the prethoracic fascia flap with lateral thoracic artery. We propose a flap based on the internal thoracic artery, which allows a relatively non-aggressive double organ implant, and we have tested its efficacy in decellularized tracheas. Tracheas of albino New Zealand rabbits were decellularized following a protocol that uses detergents and cryogenization, sterilized with 1kGy gamma radiation, and tutorized with a stent. Bilateral pedicled flaps made of pectoral fascia and a muscular component were harvested through a longitudinal 3-cm central thoracic incision, wrapping the tracheas with them in 16 rabbits, remaining them implanted for 2, 4, 8, and 12 weeks. The tracheas were then studied histologically using standard stainings plus immunohistochemistry (CD31). The models were adjusted with Bayesian statistics using ordinal regression; results as odds ratios and credibility intervals. All analysis were performed using R software. Acute inflammatory cell invasion was observed at 2 weeks, which almost disappeared at week 8 after implant. Only macrophages and giant cells increased between Weeks 8 and 12 (OR 10.487, CI [1.603-97.327]). The cartilage maintained its structure, with slight signs of ischemia in a few cases. New CD31-positive vessels were observed from Week 2 and increasing thereafter, reaching a maximum peak at Week 8. We propose a bilateral implant technique that is viable and effective as a prelamination option for two concurrent tracheas, achieving perfect vascularization and integration of the organ with hardly any inflammatory response in the medium or long term.

Negative feedback and transcriptional overshooting in a regulatory network for horizontal gene transfer.

Horizontal gene transfer (HGT) is a major force driving bacterial evolution. Because of their ability to cross inter-species barriers, bacterial plasmids are essential agents for HGT. This ability, however, poses specific requisites on plasmid physiology, in particular the need to overcome a multilevel selection process with opposing demands. We analyzed the transcriptional network of plasmid R388, one of the most promiscuous plasmids in Proteobacteria. Transcriptional analysis by fluorescence expression profiling and quantitative PCR revealed a regulatory network controlled by six transcriptional repressors. The regulatory network relied on strong promoters, which were tightly repressed in negative feedback loops. Computational simulations and theoretical analysis indicated that this architecture would show a transcriptional burst after plasmid conjugation, linking the magnitude of the feedback gain with the intensity of the transcriptional burst. Experimental analysis showed that transcriptional overshooting occurred when the plasmid invaded a new population of susceptible cells. We propose that transcriptional overshooting allows genome rebooting after horizontal gene transfer, and might have an adaptive role in overcoming the opposing demands of multilevel selection.

The stb operon balances the requirements for vegetative stability and conjugative transfer of plasmid R388.

The conjugative plasmid R388 and a number of other plasmids carry an operon, stbABC, adjacent to the origin of conjugative transfer. We investigated the role of the stbA, stbB, and stbC genes. Deletion of stbA affected both conjugation and stability. It led to a 50-fold increase in R388 transfer frequency, as well as to high plasmid loss. In contrast, deletion of stbB abolished conjugation but provoked no change in plasmid stability. Deletion of stbC showed no effect, neither in conjugation nor in stability. Deletion of the entire stb operon had no effect on conjugation, which remained as in the wild-type plasmid, but led to a plasmid loss phenotype similar to that of the R388ΔstbA mutant. We concluded that StbA is required for plasmid stability and that StbA and StbB control conjugation. We next observed the intracellular positioning of R388 DNA molecules and showed that they localize as discrete foci evenly distributed in live Escherichia coli cells. Plasmid instability of the R388ΔΔstbA mutant correlated with aberrant localization of the plasmid DNA molecules as clusters, either at one cell pole, at both poles, or at the cell center. In contrast, plasmid molecules in the R388ΔΔstbB mutant were mostly excluded from the cell poles. Thus, results indicate that defects in both plasmid maintenance and transfer are a consequence of variations in the intracellular positioning of plasmid DNA. We propose that StbA and StbB constitute an atypical plasmid stabilization system that reconciles two modes of plasmid R388 physiology: a maintenance mode (replication and segregation) and a propagation mode (conjugation). The consequences of this novel concept in plasmid physiology will be discussed.

Optical Modification of a Nanoporous Alumina Structure Associated with Surface Coverage by the Ionic Liquid AliquatCl.

This manuscript analyses changes in the optical parameters of a commercial alumina nanoporous structure (AnodiscTM or AND support) due to surface coverage by the ionic liquid (IL) AliquatCl (AlqCl). XPS measurements were performed for chemical characterization of the composite AND/AlqCl and the AND support, but XPS resolved angle analysis (from 15° to 75°) was carried out for the homogeneity estimation of the top surface of the ANDAlqCl sample. Optical characterization of both the composite AND/AlqCl and the AND support was performed by three non-destructive and non-invasive techniques: ellipsometry spectroscopy (SE), light transmittance/reflection, and photoluminescence. SE measurements (wavelength ranging from 250 nm to 1250 nm) allow for the determination of the refraction index of the AND/AlqCl sample, which hardly differs from that corresponding to the IL, confirming the XPS results. The presence of the IL significantly increases the light transmission of the alumina support in the visible region and reduces reflection, affecting also the maximum position of this latter curve, as well as the photoluminescence spectra. Due to these results, illuminated I-V curves for both the composite AND/AlqCl film and the AND support were also measured to estimate its possible application as a solar cell. The optical behaviour exhibited by the AND/AlqCl thin film in the visible region could be of interest for different applications.

Optical and Electrochemical Properties of a Nanostructured ZnO Thin Layer Deposited on a Nanoporous Alumina Structure via Atomic Layer Deposition.

This study explores the optical and electrochemical properties of a ZnO coating layer deposited on a nanoporous alumina structure (NPAS) for potential multifunctional applications. The NPAS, synthesized through an electrochemical anodization process, displays well-defined nanochannels with a high aspect ratio (~3000). The ZnO coating, achieved via atomic layer deposition, enables the tuning of the pore diameter and porosity of the NPAS, thereby influencing both the optical and electrochemical interfacial properties. A comprehensive characterization using photoluminescence, spectroscopy ellipsometry and impedance spectroscopy (with the sample in contact with NaCl solutions) provides insights into optical and electrochemical parameters, including the refractive index, absorption coefficient, and electrolyte-ZnO/NPAS interface processes. This research demonstrates potential for tailoring the optical and interfacial properties of nanoporous structures by selecting appropriate coating materials, thus opening avenues for their utilization in various technological applications.

Modification of the Physical Properties of a Nafion Film Due to Inclusion of n-Dodecyltriethylammonium Cation: Time Effect.

This study investigates the effects of modifying commercial Nafion-212 thin films with dodecyltriethylammonium cation (DTA+) on their electrical resistance, elastic modulus, light transmission/reflection and photoluminescence properties. The films were modified through a proton/cation exchange process for immersion periods ranging from 1 to 40 h. X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) were employed to analyze the crystal structure and surface composition of the modified films. The electrical resistance and the different resistive contributions were determined via impedance spectroscopy. Changes in the elastic modulus were evaluated using stress-strain curves. Additionally, optical characterization tests, including light/reflection (250-2000 nm) and photoluminescence spectra, were also performed on both unmodified and DTA+-modified Nafion films. The results reveal significant changes in the electrical, mechanical and optical properties of the films, depending on the exchange process time. In particular, the inclusion of the DTA+ into the Nafion structure improved the elastic behavior of the films by significantly decreasing the Young modulus. Furthermore, the photoluminescence of the Nafion films was also enhanced. These findings can be used to optimize the exchange process time to achieve specific desired properties.

Optical Characterization of ALD-Coated Nanoporous Alumina Structures: Effect of Sample Geometry or Coated Layer Material.

Optical characterization of nanoporous alumina-based structures (NPA-bSs), obtained by ALD deposition of a thin conformal SiO2 layer on two alumina nanosupports with different geometrical parameters (pore size and interpore distance), was performed by two noninvasive and nondestructive techniques such as spectroscopic ellipsometry (SE) and photoluminescence (Ph) spectra. SE measurements allow us to estimate the refraction index and extinction coefficient for the studied samples and their dependence with wavelength for the 250-1700 nm interval, showing the effect of sample geometry and cover-layer material (SiO2, TiO2, or Fe2O3), which significantly affect the oscillatory character of both parameters, as well as changes associated with the light incidence angle, which are attributed to surface impurities and inhomogeneity. Photoluminescence curves exhibit a similar shape independently of sample pore-size/porosity, but they seem to affect intensity values. This analysis shows the potential application of these NPA-bSs platforms to nanophotonics, optical sensing, or biosensing.

Determination of conjugation rates on solid surfaces.

A cytometric method for the estimation of end-point conjugation rates is developed and adapted to surface conjugation. This method improves the through-put of conjugation assays based on replica-plating and results in less noisy experimental data. Although conjugation on solid surfaces deviates from ideal conditions in which cells are continuously mixed, results show that, within the limits of high initial population densities and short mating times, end-point estimates of the conjugation rates are robust measurements. They are independent of the donor/recipient ratios and, to some extent, of the sampling time. Remixing the mating population in the course of a conjugation experiment results in a boost in the frequency of transconjugants.

Effects of Tepary bean (Phaseolus acutifolius) protease inhibitor and semipure lectin fractions on cancer cells.

Some natural and synthetic protease inhibitors (PI), such as the Bowman-Birk PI from soybean, have anticancer effects. We previously purified and characterized a Bowman-Birk-type PI from Tepary bean (Phaseolus acutifolius) seeds (TBPI). A semipure protein fraction containing this inhibitor, when tested its in vitro effect on transformed cells, showed a differential cytotoxic effect, as well as an increase in cell attachment to culture dishes. In this article we report that lectins were responsible for the cytotoxic effect previously observed, exhibiting a differential, antiproliferative effect on nontransformed cells and on different lineages of cancer cells. Although the purified TBPI lacked cytotoxicity, it was found to be responsible for the increase in cell adhesion, decreasing culture dishes' extracellular matrix degradation, leading to a decrease of the in vitro cell invasion capacity. This effect coincided with the suppression of Matrix Metalloproteinase-9 activity. These results indicate that Tepary bean seeds contain at least 2 different groups of bioactive proteins with distinct effects on cancer cells.

Nanoporous Alumina Support Covered by Imidazole Moiety-Based Ionic Liquids: Optical Characterization and Application.

This work analyzes chemical surface and optical characteristics of a commercial nanoporous alumina structure (NPAS) as a result of surface coverage by different imidazolium-based ionic liquids (1-butyl-3-metylimidazolium hexafluorophosphate, 3-methyl-1-octylimidazolium hexafluorophosphate, or 1-ethyl-3-methylimidazolium tetrafluoroborate). Optical characteristics of the IL/NPAS samples were determined by photoluminescence (at different excitation wavelengths (from 300 nm to 400 nm), ellipsometry spectroscopy, and light transmittance/reflectance measurements for a range of wavelengths that provide information on modifications related to both visible and near-infrared regions. Chemical surface characterization of the three IL/NPAS samples was performed by X-ray photoelectron spectroscopy (XPS), which indicates almost total support coverage by the ILs. The IL/NPAS analyzed samples exhibit different photoluminescence behavior, high transparency (<85%), and a reflection maximum at wavelength ~380 nm, with slight differences depending on the IL, while the refractive index values are rather similar to those shown by the ILs. Moreover, the illuminated I−V curves (under standard conditions) of the IL/NPAS samples were also measured for determining the efficiency energy conversion to estimate their possible application as solar cells. On the other hand, a computational quantum mechanical modeling method (DFT) was used to establish the most stable bond between the ILs and the NPAS support.

Optical and Physicochemical Characterizations of a Cellulosic/CdSe-QDs@S-DAB5 Film.

CdSe quantum dots nanoparticles were coated with the thiolated (DiAminoButane based dendrimer) DAB dendrimer of fifth generation (S-DAB5) and embedded in a highly hydrophilic regenerated cellulose (RC) film by simple dip-coating method (immersion in QD-dendrimer aqueous solution) as a way to get a flexible nano-engineered film (RC-4/CdSe-QDs@S-DAB5) with high transparency and photoluminescence properties for different applications. Optical changes in the RC film associated with QDs inclusion were determined by spectroscopic ellipsometry (SE) measurements, which provide information on changes caused in the refraction index and the extinction coefficients of the film, as well as by light transmittance/reflectance curves and photoluminescence (PL) spectra. Impedance spectroscopy (IS) and other typical physicochemical techniques for material characterization (TEM, SEM and XPS) have also been used in order to have more complete information on film characteristics. A comparison of RC-4/CdSe-QDs@S-DAB5 film optical characteristics with those exhibited by other RC-modified films depending on the type of dendrimer was also carried out.

Characterization of the DNA Binding Domain of StbA, A Key Protein of A New Type of DNA Segregation System.

Low-copy-number plasmids require sophisticated genetic devices to achieve efficient segregation of plasmid copies during cell division. Plasmid R388 uses a unique segregation mechanism, based on StbA, a small multifunctional protein. StbA is the key protein in a segregation system not involving a plasmid-encoded NTPase partner, it regulates the expression of several plasmid operons, and it is the main regulator of plasmid conjugation. The mechanisms by which StbA, together with the centromere-like sequence stbS, achieves segregation, is largely uncharacterized. To better understand the molecular basis of R388 segregation, we determined the crystal structure of the conserved N-terminal domain of StbA to 1.9 Å resolution. It folds into an HTH DNA-binding domain, structurally related to that of the PadR subfamily II of transcriptional regulators. StbA is organized in two domains. Its N-terminal domain carries the specific stbS DNA binding activity. A truncated version of StbA, deleted of its C-terminal domain, displays only partial activities in vivo, indicating that the non-conserved C-terminal domain is required for efficient segregation and subcellular plasmid positioning. The structure of StbA DNA-binding domain also provides some insight into how StbA monomers cooperate to repress transcription by binding to the stbDR and to form the segregation complex with stbS.

Depression and Suicide Risk in Mild Cognitive Impairment: The Role of Alzheimer's Disease Biomarkers.

BACKGROUND: Patients with depression and mild cognitive impairment (MCI) are at greater risk of developing dementia. Depression involves a higher risk of suicidal ideation (SI) and suicide attempts (SA). Biomarkers of Alzheimer's Disease (AD) could help to clarify the role of depression and SI in AD. METHOD: Fifty-nine participants aged > 50 with criteria of MCI positive (MCI-AD) (n=22) and negative (MCI-Non AD) (n=24) AD and healthy controls (HC) (n=13) were evaluated. We used the Geriatric Depression Scale (GDS-30) and the GDS-SI factor to measure depression and indirect risk for suicide, respectively. Additionally, AD biomarkers such as amyloid-ß (Aß), hyperphosphorilated tau (P-tau), and total tau (T-tau) in cerebrospinal fluid (CSF) were analyzed. RESULTS: No significant differences between the groups were found in depression. However, in the MCI-AD group, lower P-tau and T-tau levels were related to higher GDS-SI scores, suggesting that MCI-AD patients with lower AD pathology are at a higher risk of suicide. CONCLUSIONS: The result highlights the importance of considering SI in the initial phases of AD, and the potential role of AD biomarkers in early detection of symptoms.

Does a 3-month multidisciplinary intervention improve pain, body composition and physical fitness in women with fibromyalgia?

OBJECTIVE: To determine the effects of a 3-month multidisciplinary intervention on pain (primary outcome), body composition and physical fitness (secondary outcomes) in women with fibromyalgia (FM). METHODS: 75 women with FM were allocated to a low-moderate intensity 3-month (three times/week) multidisciplinary (pool, land-based and psychological sessions) programme (n=33) or to a usual care group (n=32). The outcome variables were pain threshold, body composition (body mass index and estimated body fat percentage) and physical fitness (30 s chair stand, handgrip strength, chair sit and reach, back scratch, blind flamingo, 8 feet up and go and 6 min walk test). RESULTS: The authors observed a significant interaction effect (group*time) for the left (L) and right (R) side of the anterior cervical (p<0.001) and the lateral epicondyle R (p=0.001) tender point. Post hoc analysis revealed that pain threshold increased in the intervention group (positive) in the anterior cervical R (p<0.001) and L (p=0.012), and in the lateral epicondyle R (p=0.010), whereas it decreased (negative) in the anterior cervical R (p<0.001) and L (p=0.002) in the usual care group. There was also a significant interaction effect for chair sit and reach. Post hoc analysis revealed improvement in the intervention group (p=0.002). No significant improvement attributed to the training was observed in the rest of physical fitness or body composition variables. CONCLUSIONS: A 3-month multidisciplinary intervention three times/week had a positive effect on pain threshold in several tender points in women with FM. Though no overall improvements were observed in physical fitness or body composition, the intervention had positive effects on lower-body flexibility.

Obstructive sleep apnea and Alzheimer's disease-related cerebrospinal fluid biomarkers in mild cognitive impairment.

Previous studies have demonstrated that sleep-breathing disorders, and especially obstructive sleep apnea (OSA), can be observed in patients with a higher risk of progression to Alzheimer's disease (AD). Recent evidence indicates that cerebrospinal fluid (CSF) AD-biomarkers are associated with OSA. In this study, we investigated these associations in a sample of patients with mild cognitive impairment (MCI), a condition that is considered the first clinical phase of AD, when patients showed biomarkers consistent with AD pathology. A total of 57 patients (mean age = 66.19; SD = 7.13) with MCI were included in the study. An overnight polysomnography recording was used to assess objective sleep parameters (i.e. apnea/hypopnea index [AHI], total sleep time, sleep efficiency, sleep latency, arousal index, awakening, stage 1, 2, and slow-wave sleep and rapid eye movement sleep, periodic limb movement index, O2 saturation during sleep, and percentage of time O2 saturation <90%). Phosphorylated-tau (P-tau), total-tau (T-tau), and amyloid-beta 42 (Aß42) were measured in CSF. Unadjusted correlation analyses showed that a higher AHI (reflecting higher OSA severity) was related to higher P-tau and T-tau (both results remained significant after Bonferroni correction, p = 0.001). Importantly, these associations were observed even after adjusting for potential confounders (i.e. age, sex, body mass index, sleep medication, smoking, hypertension, and heart disease). Although more research is needed to establish a causal link, our findings provide evidence that OSA could be related to the pathophysiological mechanisms involved in neurodegeneration in MCI patients.

Surface Modification of Nanoporous Anodic Alumina during Self-Catalytic Atomic Layer Deposition of Silicon Dioxide from (3-Aminopropyl)Triethoxysilane.

Changes associated to atomic layer deposition (ALD) of SiO2 from 3-aminopropyl triethoxysilane (APTES) and O3, on a nanoporous alumina structure, obtained by two-step electrochemical anodization in oxalic acid electrolyte (Ox sample) are analysed. A reduction of 16% in pore size for the Ox sample, used as support, was determined by SEM analysis after its coverage by a SiO2 layer (Ox+SiO2 sample), independently of APTES or O3 modification (Ox+SiO2/APTES and Ox+SiO2/APTES/O3 samples). Chemical surface modification was determined by X-ray photoelectron spectroscopy (XPS) technique during the different stages of the ALD process, and differences induced at the surface level on the Ox nanoporous alumina substrate seem to affect interfacial effects of both samples when they are in contact with an electrolyte solution according to electrochemical impedance spectroscopy (EIS) measurements, or their refraction index as determined by spectroscopic ellipsometry (SE) technique. However, no substantial differences in properties related to the nanoporous structure of anodic alumina (photoluminescent (PL) character or geometrical parameters) were observed between Ox+SiO2/APTES and Ox+SiO2/APTES/O3 samples.

Insights into the formation of an emissive CdTe-quantum-dots/cellulose hybrid film.

Optical and electrical modifications of a highly hydrophilic cellulose regenerated (RC) thin film as result of the inclusion in the film structure of cadmium-tellurium covered with mercaptosuccinic acid quantum dot nanoparticles (CdTe-MSA QDs) are analysed. CdTe-MSA QDs nanoparticles inclusion was performed by dip-coating of the RC film in an aqueous nano QDs solution, and two different immersion periods (2 h and 24 h) were considered. Impedance spectroscopy measurements, which can give information of changes in both film surface and bulk phase were performed for CdTe-MSA QDs inclusion confirmation as well as the estimation of electrical changes associated to QDs modification. Optical characterization of the original support and the CdTe-MSA(2 h) and CdTe-MSA(24 h) modified films was carried out by light transmittance and ellipsometry spectroscopy (at three different take-off angles). According to these results, the inclusion of CdTe-MSA QDs practically does not reduce the high transparency of the film support, but it increases the reflexion index and extinction coefficient of both modified films. Moreover, fluorescence and XPS measurements were also performed for more complete information on the effect caused by the presence of CdTe-MSA QDs in the cellulosic film. A comparison of changes in physicochemical properties of the cellulosic/CdTe-MSA QDs films and those modified with other nanoparticle quantum dots is also performed.

Clinical Utility of Plasma Lipid Peroxidation Biomarkers in Alzheimer's Disease Differential Diagnosis.

BACKGROUND: Differential diagnosis of Alzheimer's disease (AD) is a complex task due to the clinical similarity among neurodegenerative diseases. Previous studies showed the role of lipid peroxidation in early AD development. However, the clinical validation of potential specific biomarkers in minimally invasive samples constitutes a great challenge in early AD diagnosis. METHODS: Plasma samples from participants classified into AD (n = 138), non-AD (including MCI and other dementias not due to AD) (n = 70) and healthy (n = 50) were analysed. Lipid peroxidation compounds (isoprostanes, isofurans, neuroprostanes, neurofurans) were determined by ultra-performance liquid chromatography coupled with tandem mass spectrometry. Statistical analysis for biomarkers' clinical validation was based on Elastic Net. RESULTS: A two-step diagnosis model was developed from plasma lipid peroxidation products to diagnose early AD specifically, and a bootstrap validated AUC of 0.74 was obtained. CONCLUSION: A promising AD differential diagnosis model was developed. It was clinically validated as a screening test. However, further external validation is required before clinical application.

Lipid peroxidation biomarkers correlation with medial temporal atrophy in early Alzheimer Disease.

Alzheimer Disease (AD) is a pathology that causes millions of deaths every year and it also generates severe economic consequences for families and public health systems. Oxidative stress is related to neurodegenerative diseases damage. In fact, brain lipid oxidation could produce brain atrophy. The main objective of this study is the evaluation of atrophy and lipid peroxidation damage in AD patients. We studied medial temporal brain atrophy by magnetic resonance imaging (MRI) and a set of lipid peroxidation biomarkers from plasma samples, respectively. The participants were AD patients in early stages (n = 80) and healthy controls (n = 32). Some lipid peroxidation compounds (neuroprostanes, isoprostanes, neurofurans, isofurans, 17-epi-17-F2t-dihomo-IsoP, PGF2α) in plasma showed statistically significant correlation with medial temporal atrophy. So, they were selected to generate an AD diagnosis model, showing an AUC-ROC of 0.900, close to accuracy achieved by the model based on neuroimaging analysis (AUC-ROC 0.929). In addition, the new model showed suitable specificity, so it could be used as screening test. The developed model based on plasma biomarkers could reflect white and grey matter lipid peroxidation, which occurs in medial temporal lobe in early AD patients. Nevertheless, more studies are needed in this field in order to evaluate specificity against other dementias or neurodegenerative diseases.