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Histidine kinases (HKs) are a central part of bacterial environmental-sensing two-component systems. They provide their hosts with the ability to respond to a wide range of physical and chemical signals. HKs are multidomain proteins consisting of at least a sensor domain, dimerization and phosphorylation domain (DHp), and a catalytic domain. They work as homodimers and the existence of two different autophosphorylation mechanisms (cis and trans) has been proposed as relevant for pathway specificity. Although several HKs have been intensively studied, a precise sequence-to-structure explanation of why and how either cis or trans phosphorylation occurs is still unavailable nor is there any evolutionary analysis on the subject. In this work, we show that AlphaFold can accurately determine whether an HK dimerizes in a cis or trans structure. By modeling multiple HKs we show that both cis- and trans-acting HKs are common in nature and the switch between mechanisms has happened multiple times in the evolutionary history of the family. We then use AlphaFold modeling to explore the molecular determinants of the phosphorylation mechanism. We conclude that it is the difference in lengths of the helices surrounding the DHp loop that determines the mechanism. We also show that very small changes in these helices can cause a mechanism switch. Despite this, previous evidence shows that for a particular HK the phosphorylation mechanism is conserved. This suggests that the phosphorylation mechanism participates in system specificity and mechanism switching provides these systems with a way to diverge.
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Electroactive coatings for smart wearable textiles based on a furan bio-epoxy monomer (BOMF) crosslinked with isophorone diamine (IPD) and additivated with carbon nanotubes (CNTs) are reported herein. The effect of BOMF/IPD molar ratio on the curing reaction, as well as on the properties of the crosslinked resins was first assessed, and it was found that 1.5:1 BOMF/IPD molar ratio provided higher heat of reaction, glass transition temperature, and mechanical performance. The resin was then modified with CNT to prepare electrically conductive nanocomposite films, which exhibited conductivity values increased by eight orders of magnitude upon addition of 5 phr of CNTs. The epoxy/CNT nanocomposites were finally applied as coatings onto a cotton fabric to develop electrically conductive, hydrophobic and breathable textiles. Notably, the integration of CNTs imparted efficient and reversible electrothermal behavior to the cotton fabric, showcasing its potential application in smart and comfortable wearable electronic devices.
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Smart protective coatings and devices are currently of great interest. In particular, they can absorb or reflect harmful waves of electromagnetic interference (EMI). In this work, novel binary and ternary composites with highly amorphous poly(vinyl alcohol) (HAVOH) as a matrix and single-walled carbon nanotubes (SWCNTs) and MXenes as nanofillers were prepared. HAVOH is a recently patented kind of poly(vinyl alcohol) (PVOH) that was modified with diol monomers. MXenes are a new type of inorganic two-dimensional (2D) nanoparticle consisting of carbides, nitrides and carbonitrides. Three series of composites, HAVOH/SWCNTs, HAVOH/MXenes and HAVOH/SWCNTs/MXenes, were prepared using the solvent casting method. Samples were tested with various methods to study their structure, electrical properties, thermal behavior and EMI-shielding properties. HAVOH/3.0 wt.% SWCNTs/3.0 wt.% MXene specimens revealed a shielding effectiveness of 55 dB, which is 122 times better than that of the neat matrix. These results are promising for the fabrication of films with protective effects against EMI.
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This work proposes a biorefinery approach for utilizing tomato pomace (TP) through a top-down deconstructing strategy, combining mild chemical hydrolysis with high-pressure homogenization (HPH). The objective of the study is to isolate cellulose pulp using different combinations of chemical and physical processes: (i) direct HPH treatment of the raw material, (ii) HPH treatment following acid hydrolysis, and (iii) HPH treatment following alkaline hydrolysis. The results demonstrate that these isolation routes enable the production of cellulose with tailored morphological properties from TP with higher yields (up to +21% when HPH was applied before hydrolysis and approximately +6% when applied after acid or after alkaline hydrolysis). Additionally, the side streams generated by this cascade process show a four-fold increase in phenolic compounds when HPH is integrated after acid hydrolysis compared to untreated sample, and they also contain nanoparticles composed of hemicellulose and lignin, as shown by FT-IR and SEM. Notably, the further application of HPH treatment enables the production of nanostructured cellulose from cellulose pulp derived from TP, offering tunable properties. This approach presents a sustainable pathway for the extraction of cellulose and nanocellulose, as well as the valorization of value-added compounds found in residual biomass in the form of side streams.
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Smart polymer coatings embedding stimuli-responsive corrosion inhibitor nanocarriers are commonly exploited, in the literature, for the development of high-performance active coatings. In this work, high-surface-area amino-functionalized mesoporous silica nanoparticles (MSN-NH2) were developed with a one-step synthesis process and then functionalized with benzoyl chloride (MSN-BC) through a reaction with amino groups. MSN-BC are able to release benzoic acid (BA) in acid and alkaline conditions as a result of the hydrolysis of the pH-sensitive amide bond. MSN-BC were embedded in polymer coatings to exploit the pH-dependent release of corrosion-inhibiting BA. After an in-depth characterization of the developed functional nanoparticles and of their pH-dependent release kinetics of BA, MSN-BC were embedded in an acrylic matrix, realizing coatings for the corrosion protection of aluminum AA2024 alloys. Results demonstrate the effectiveness of the nanoparticles' porous structure for a high loading of the anticorrosive active agent BA and the long-lasting efficiency of the coating for the corrosion protection of aluminum alloys, as validated by morphological and electrochemical impedance spectroscopy (EIS) measurements. EIS experiments were carried out with up to 21 days of exposure to a corrosive environment, revealing the potentialities of the acrylic coatings embedding MSN-BC for the protection of aluminum alloys.
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A hybrid montmorillonite (MMT)/reduced graphene oxide (rGO) film was realised and used as a non-invasive sensor for the monitoring of water absorption and desorption in pristine and consolidated tuff stones. This film was obtained by casting from a water dispersion containing graphene oxide (GO), montmorillonite and ascorbic acid; then the GO component was thermo-chemically reduced and the ascorbic acid phase was removed by washing. The hybrid film showed electrical surface conductivity that varied linearly with the relative humidity, ranging from 2.3 × 10-3 S in dry conditions to 5.0 × 10-3 S at 100% RH. The sensor was applied onto tuff stone samples through the use of a high amorphous polyvinyl alcohol layer (HAVOH) adhesive, which guaranteed good water diffusion from the stone to the film and was tested during water capillary absorption and drying tests. Results show that the sensor is able to monitor water content changes in the stone, being potentially useful to evaluate the water absorption and desorption behaviour of porous samples both in laboratory environments and in situ.
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Phosphorylation carries chemical information in biological systems. In two-component systems (TCSs), the sensor histidine kinase and the response regulator are connected through phosphoryl transfer reactions that may be uni- or bidirectional. Directionality enables the construction of complex regulatory networks that optimize signal propagation and ensure the forward flow of information. We combined x-ray crystallography, hybrid quantum mechanics/molecular mechanics (QM/MM) simulations, and systems-integrative kinetic modeling approaches to study phosphoryl flow through the Bacillus subtilis thermosensing TCS DesK-DesR. The allosteric regulation of the histidine kinase DesK was critical to avoid back transfer of phosphoryl groups and futile phosphorylation-dephosphorylation cycles by isolating phosphatase, autokinase, and phosphotransferase activities. Interactions between the kinase's ATP-binding domain and the regulator's receiver domain placed the regulator in two distinct positions in the phosphotransferase and phosphatase complexes, thereby determining whether a key glutamine residue in DesK was properly situated to assist in the dephosphorylation reaction. Moreover, an energetically unfavorable phosphotransferase conformation when DesK was not phosphorylated minimized reverse phosphoryl transfer. DesR dimerization and a dissociative phosphoryl transfer reaction also enforced the direction of phosphoryl flow. Shorter or longer distances between the phosphoryl acceptor and donor residues shifted the phosphoryl transfer equilibrium by modulating the stabilizing effect of the Mg2+ cofactor. These mechanisms control the directionality of signal transmission and show how structure-encoded allostery stores and transmits information in signaling systems.
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Bacillus subtilis , Transducción de Señal , Histidina Quinasa/metabolismo , Bacillus subtilis/genética , Fosforilación , Monoéster Fosfórico Hidrolasas , Proteínas Bacterianas/metabolismoRESUMEN
In this work, engineered stimuli-responsive mesoporous silica nanoparticles (MSNs) were developed and exploited in polymer coatings as multifunctional carriers of a typical corrosion inhibitor, benzotriazole (BTA). In detail, a new capping system based on a BTA-silver coordination complex, able to dissolve in acid and alkaline conditions and to simultaneously tailor the BTA release and the capture of chloride ions, was properly designed and realized. Acrylic coatings embedding the engineered MSNs were deposited onto iron rebar samples and tested for their protective capability in acid and alkaline environments. Results highlighted the high potential of the proposed system for the protection of metals, due to the synergistic effect of the mesoporous structure and the capping system, which guaranteed both the sequestration of chloride ions and the on-demand release of the effective amount of anticorrosive agents able to ensure the enhanced protection of the substrate.
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Mesoporous silica nanoparticles (MSN) have attracted increasing interest for their applicability as smart nanocarriers of corrosion inhibitors, due to their porous structure, resistance to main corrosive environments and good compatibility with polymer coatings. In this review, the main synthetic routes to obtain MSN with tailored textural properties, the design of different loading and stimuli-induced release strategies, the development of advanced organic nanocomposite coatings with MSN and the validation of their anticorrosive performances are reviewed and compared. Through a critical analysis of the literature, the most promising research trends and perspectives to exploit the highly interesting properties of MSN in advanced organic coatings are proposed.
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PURPOSE: Congenital hypopituitarism (CH) can present in isolation or with other birth defects. Mutations in multiple genes can cause CH, and the use of a genetic screening panel could establish the prevalence of mutations in known and candidate genes for this disorder. It could also increase the proportion of patients that receive a genetic diagnosis. METHODS: We conducted target panel genetic screening using single-molecule molecular inversion probes sequencing to assess the frequency of mutations in known hypopituitarism genes and new candidates in Argentina. We captured genomic deoxyribonucleic acid from 170 pediatric patients with CH, either alone or with other abnormalities. We performed promoter activation assays to test the functional effects of patient variants in LHX3 and LHX4. RESULTS: We found variants classified as pathogenic, likely pathogenic, or with uncertain significance in 15.3% of cases. These variants were identified in known CH causative genes (LHX3, LHX4, GLI2, OTX2, HESX1), in less frequently reported genes (FOXA2, BMP4, FGFR1, PROKR2, PNPLA6) and in new candidate genes (BMP2, HMGA2, HNF1A, NKX2-1). CONCLUSION: In this work, we report the prevalence of mutations in known CH genes in Argentina and provide evidence for new candidate genes. We show that CH is a genetically heterogeneous disease with high phenotypic variation and incomplete penetrance, and our results support the need for further gene discovery for CH. Identifying population-specific pathogenic variants will improve the capacity of genetic data to predict eventual clinical outcomes.
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Enfermedades del Sistema Endocrino/genética , Pruebas Genéticas/estadística & datos numéricos , Hipopituitarismo/genética , Mutación/genética , Adolescente , Adulto , Argentina , Niño , Preescolar , Femenino , Heterogeneidad Genética , Humanos , Lactante , Proteínas con Homeodominio LIM/genética , Masculino , Fenotipo , Polimorfismo de Nucleótido Simple , Factores de Transcripción/genética , Adulto JovenRESUMEN
Interferon-stimulated gene 15 (ISG15) was the first ubiquitin-like modifier protein identified that acts by protein conjugation (ISGylation) and is thought to modulate IFN-induced inflammation. Here, we report a new patient from a non-consanguineous Argentinian family, who was followed for recurrent ulcerative skin lesions, cerebral calcifications and lung disease. Whole Exome Sequencing (WES) revealed two novel compound heterozygous variants (c.285del and c.299_312del, NM_005101.4 GRCh37(hg19), both classified as pathogenic according to ACMG criteria) in the ISG15 gene, resulting in a complete deficiency due to disruption of the second ubiquitin domain of the corresponding protein. The clinical phenotype of this patient is unique given the presence of recurrent pulmonary manifestations and the absence of mycobacterial infections, thus resulting in a phenotype distinct from that previously described in patients with biallelic loss-of-function (LOF) ISG15 variants. This case highlights the role of ISG15 as an immunomodulating factor whose LOF variants result in heterogeneous clinical presentations.
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Proteasas ATP-Dependientes/genética , ATPasas Asociadas con Actividades Celulares Diversas/genética , Discapacidad Intelectual/diagnóstico por imagen , Discapacidad Intelectual/genética , Espasticidad Muscular/diagnóstico por imagen , Espasticidad Muscular/genética , Atrofia Óptica/diagnóstico por imagen , Atrofia Óptica/genética , Ataxias Espinocerebelosas/diagnóstico por imagen , Ataxias Espinocerebelosas/genética , Adulto , Humanos , Imagen por Resonancia Magnética , Masculino , Metaloendopeptidasas/genética , Linaje , Hermanos , Secuenciación del Exoma , Adulto JovenRESUMEN
Histidine kinases (HK) of bacterial two-component systems represent a hallmark of allosterism in proteins, being able to detect a signal through the sensor domain and transmit this information through the protein matrix to the kinase domain which, once active, autophosphorylates a specific histidine residue. Inactive-to-active transition results in a large conformational change that moves the kinase on top of the histidine. In the present work, we use several molecular simulation techniques (Molecular Dynamics, Hybrid QM/MM, and constant pH molecular dynamics) to study the activation and autophosphorylation reactions in L. plantarum WalK, a cis-acting HK. In agreement with previous results, we show that the chemical step requires tight coupling with the conformational step in order to maintain the histidine phosphoacceptor in the correct tautomeric state, with a reactive δ-nitrogen. During the conformational transition, the kinase domain is never released and walks along the HK helix axis, breaking and forming several conserved residue-based contacts. The phosphate transfer reaction is concerted in the transition state region and is catalyzed through the stabilization of the negative developing charge of transferring phosphate along the reaction.
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Histidina Quinasa/química , Histidina Quinasa/metabolismo , Simulación de Dinámica Molecular , Teoría Cuántica , Concentración de Iones de Hidrógeno , Lactobacillus plantarum/enzimología , Fosforilación , Conformación Proteica , TermodinámicaRESUMEN
Electrophoresis (EP) and dielectrophoresis (DEP) are the two well-established methodologies to manipulate nanoparticles (NPs). Recently, DEP by a virtual electrode platform was demonstrated on ferroelectric substrates, where the driving force is due to the strong electric field generated by the pyroelectric effect, thus opening new scenarios for manipulating the matter. Such an innovative approach named pyroelectric-DEP has several advantages over traditional EP and DEP. However, a detailed study on this novel approach is required for understanding the complex pathways traced by NPs under the action of the pyroelectric-driven forces and thus for explaining the final patterns. Here, we investigate experimentally the dynamic behavior of CdSe NPs through time-lapse fluorescence microscopy imaging. Complete visualization and measurement of the directed-assembling process of NPs immersed in polydimethylsiloxane fluid is reported, which shows some unpredicted results with respect to the previous works, thus opening the route for designing in principle a reversible and switchable device allowing two different and reversible final NP-patterned states. The observed phenomena are fully analyzed by experimental and simulated analysis, and the movements of NPs is performed to elucidate in depth the involved processes. The investigation furnishes an interesting result that the complex behavior of the NPs can be fully comprehended and explained by considering the superposition of both EP and DEP forces.
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In this study, we report a direct writing method for the fabrication of microfluidic footpaths by pyro-electrohydrodynamic (EHD) jet printing. Here, we propose the use of a nozzle-free three-dimensional printing technique for the fabrication of printed structures that can be embedded in a variety of soft, transparent, flexible, and biocompatible polymers and thus easily integrated into lab-on-chip devices. We prove the advantage of the high resolution and flexibility of pyro-EHD printing for the realization of microfluidic channels well below the standard limit in dimension of conventional ink-jet printing technique and simply adaptable to the end-user desires in terms of geometry and materials. Starting from the description of the innovative approach proposed for the channel fabrication, we demonstrate the design, fabrication, and proof of a microfluidic matrix of interconnected channels. The method described here could be a breakthrough technology for the fabrication of in situ implantable, stretchable, and biocompatible devices, opening new routes in the field of biomedical engineering and wearable electronics.
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Lab-on-a-Chip (LoC) devices are extremely promising in that they enable diagnostic functions at the point-of-care. Within this scope, an important goal is to design imaging schemes that can be used out of the laboratory. In this paper, we introduce and test a pocket holographic slide that allows digital holography microscopy to be performed without an interferometer setup. Instead, a commercial off-the-shelf plastic chip is engineered and functionalized with this aim. The microfluidic chip is endowed with micro-optics, that is, a diffraction grating and polymeric lenses, to build an interferometer directly on the chip, avoiding the need for a reference arm and external bulky optical components. Thanks to the single-beam scheme, the system is completely integrated and robust against vibrations, sharing the useful features of any common path interferometer. Hence, it becomes possible to bring holographic functionalities out of the lab, moving complexity from the external optical apparatus to the chip itself. Label-free imaging and quantitative phase contrast mapping of live samples are demonstrated, along with flexible refocusing capabilities. Thus, a liquid volume can be analyzed in one single shot with no need for mechanical scanning systems.