Enzymatic synthesis of mono- and trifluorinated alanine enantiomers expands the scope of fluorine biocatalysis.

Fluorinated amino acids serve as an entry point for establishing new-to-Nature chemistries in biological systems, and novel methods are needed for the selective synthesis of these building blocks. In this study, we focused on the enzymatic synthesis of fluorinated alanine enantiomers to expand fluorine biocatalysis. The alanine dehydrogenase from Vibrio proteolyticus and the diaminopimelate dehydrogenase from Symbiobacterium thermophilum were selected for in vitro production of (R)-3-fluoroalanine and (S)-3-fluoroalanine, respectively, using 3-fluoropyruvate as the substrate. Additionally, we discovered that an alanine racemase from Streptomyces lavendulae, originally selected for setting an alternative enzymatic cascade leading to the production of these non-canonical amino acids, had an unprecedented catalytic efficiency in ß-elimination of fluorine from the monosubstituted fluoroalanine. The in vitro enzymatic cascade based on the dehydrogenases of V. proteolyticus and S. thermophilum included a cofactor recycling system, whereby a formate dehydrogenase from Pseudomonas sp. 101 (either native or engineered) coupled formate oxidation to NAD(P)H formation. Under these conditions, the reaction yields for (R)-3-fluoroalanine and (S)-3-fluoroalanine reached >85% on the fluorinated substrate and proceeded with complete enantiomeric excess. The selected dehydrogenases also catalyzed the conversion of trifluoropyruvate into trifluorinated alanine as a first-case example of fluorine biocatalysis with amino acids carrying a trifluoromethyl group.

Synthesis of fluorinated amino acids by low-specificity, promiscuous aldolases coupled to in situ fluorodonor generation.

Fluorine (F) is an important element in the synthesis of molecules broadly used in medicine, agriculture, and materials. F addition to organic structures represents a unique strategy for tuning molecular properties, yet this atom is rarely found in Nature and approaches to produce fluorometabolites (such as fluorinated amino acids, key building blocks for synthesis) are relatively scarce. This chapter discusses the use of L-threonine aldolase enzymes (LTAs), a class of enzymes that catalyze reversible aldol addition to the α-carbon of glycine. The C-C bond formation ability of LTAs, together with their known substrate promiscuity, make them ideal for in vitro F biocatalysis. Here, we describe protocols to harness the activity of the low-specificity LTAs isolated from Escherichia coli and Pseudomonas putida on 2-fluoroacetaldehyde to efficiently synthesize 4-fluoro-L-threonine in vitro. This chapter also provides a comprehensive account of experimental protocols to implement these activities in vivo. These methods are illustrative and can be adapted to produce other fluorometabolites of interest.

An Approach to Develop Digital Twins in Industry.

The industry is currently undergoing a digital revolution driven by the integration of several enabling technologies. These include automation, robotics, cloud computing, industrial cybersecurity, systems integration, digital twins, etc. Of particular note is the increasing use of digital twins, which offer significant added value by providing realistic and fully functional process simulations. This paper proposes an approach for developing digital twins in industrial environments. The novelty lies in not only focusing on obtaining the model of the industrial system and integrating virtual reality and/or augmented reality but also in emphasizing the importance of incorporating other enabled technologies of Industry 4.0, such as system integration, connectivity with standard and specific industrial protocols, cloud services, or new industrial automation systems, to enhance the capabilities of the digital twin. Furthermore, a proposal of the software tools that can be used to achieve this incorporation is made. Unity is chosen as the real-time 3D development tool for its cross-platform capability and streamlined industrial system modeling. The integration of augmented reality is facilitated by the Vuforia SDK. Node-RED is selected as the system integration option, and communications are carried out with MQTT protocol. Finally, cloud-based services are recommended for effective data storage and processing. Furthermore, this approach has been used to develop a digital twin of a robotic electro-pneumatic cell.

The pAblo·pCasso self-curing vector toolset for unconstrained cytidine and adenine base-editing in Gram-negative bacteria.

A synthetic biology toolkit, exploiting clustered regularly interspaced short palindromic repeats (CRISPR) and modified CRISPR-associated protein (Cas) base-editors, was developed for genome engineering in Gram-negative bacteria. Both a cytidine base-editor (CBE) and an adenine base-editor (ABE) have been optimized for precise single-nucleotide modification of plasmid and genome targets. CBE comprises a cytidine deaminase conjugated to a Cas9 nickase from Streptococcus pyogenes (SpnCas9), resulting in C→T (or G→A) substitutions. Conversely, ABE consists of an adenine deaminase fused to SpnCas9 for A→G (or T→C) editing. Several nucleotide substitutions were achieved using these plasmid-borne base-editing systems and a novel protospacer adjacent motif (PAM)-relaxed SpnCas9 (SpRY) variant. Base-editing was validated in Pseudomonas putida and other Gram-negative bacteria by inserting premature STOP codons into target genes, thereby inactivating both fluorescent proteins and metabolic (antibiotic-resistance) functions. The functional knockouts obtained by engineering STOP codons via CBE were reverted to the wild-type genotype using ABE. Additionally, a series of induction-responsive vectors was developed to facilitate the curing of the base-editing platform in a single cultivation step, simplifying complex strain engineering programs without relying on homologous recombination and yielding plasmid-free, modified bacterial cells.

Self-regulating arousal via pupil-based biofeedback.

The brain's arousal state is controlled by several neuromodulatory nuclei known to substantially influence cognition and mental well-being. Here we investigate whether human participants can gain volitional control of their arousal state using a pupil-based biofeedback approach. Our approach inverts a mechanism suggested by previous literature that links activity of the locus coeruleus, one of the key regulators of central arousal and pupil dynamics. We show that pupil-based biofeedback enables participants to acquire volitional control of pupil size. Applying pupil self-regulation systematically modulates activity of the locus coeruleus and other brainstem structures involved in arousal control. Furthermore, it modulates cardiovascular measures such as heart rate, and behavioural and psychophysiological responses during an oddball task. We provide evidence that pupil-based biofeedback makes the brain's arousal system accessible to volitional control, a finding that has tremendous potential for translation to behavioural and clinical applications across various domains, including stress-related and anxiety disorders.

Structural Control of Plasmon Resonance in Molecularly Linked Metal Oxide Nanocrystal Gel Assemblies.

Nanocrystal gels exhibit collective optical phenomena based on interactions among their constituent building blocks. However, their inherently disordered structures have made it challenging to understand, predict, or design properties such as optical absorption spectra that are sensitive to the coupling between the plasmon resonances of the individual nanocrystals. Here, we bring indium tin oxide nanocrystal gels under chemical control and show that their infrared absorption can be predicted and systematically tuned by selecting the nanocrystal sizes and compositions and molecular structures of the link-mediating surface ligands. Thermoreversible assemblies with metal-terpyridine links form reproducible gel architectures, enabling us to derive a plasmon ruler that governs the spectral shifts upon gelation, predicated on the nanocrystal and ligand compositions. This empirical guide is validated using large-scale, many-bodied simulations to compute the optical spectra of gels with varied structural parameters. Based on the derived plasmon ruler, we design and demonstrate a nanocrystal mixture whose spectrum exhibits distinctive line narrowing upon assembly.

Expression and Characterization of Two α-l-Arabinofuranosidases from Talaromyces amestolkiae: Role of These Enzymes in Biomass Valorization.

α-l-arabinofuranosidases are glycosyl hydrolases that catalyze the break between α-l-arabinofuranosyl substituents or between α-l-arabinofuranosides and xylose from xylan or xylooligosaccharide backbones. While they belong to several glycosyl hydrolase (GH) families, there are only 24 characterized GH62 arabinofuranosidases, making them a small and underrepresented group, with many of their features remaining unknown. Aside from their applications in the food industry, arabinofuranosidases can also aid in the processing of complex lignocellulosic materials, where cellulose, hemicelluloses, and lignin are closely linked. These materials can be fully converted into sugar monomers to produce secondary products like second-generation bioethanol. Alternatively, they can be partially hydrolyzed to release xylooligosaccharides, which have prebiotic properties. While endoxylanases and ß-xylosidases are also necessary to fully break down the xylose backbone from xylan, these enzymes are limited when it comes to branched polysaccharides. In this article, two new GH62 α-l-arabinofuranosidases from Talaromyces amestolkiae (named ARA1 and ARA-2) have been heterologously expressed and characterized. ARA-1 is more sensitive to changes in pH and temperature, whereas ARA-2 is a robust enzyme with wide pH and temperature tolerance. Both enzymes preferentially act on arabinoxylan over arabinan, although ARA-1 has twice the catalytic efficiency of ARA-2 on this substrate. The production of xylooligosaccharides from arabinoxylan catalyzed by a T. amestolkiae endoxylanase was significantly increased upon pretreatment of the polysaccharide with ARA-1 or ARA-2, with the highest synergism values reported to date. Finally, both enzymes (ARA-1 or ARA-2 and endoxylanase) were successfully applied to enhance saccharification by combining them with a ß-xylosidase already characterized from the same fungus.

Osteoblastic and Bacterial Response of Hybrid Dental Implants.

Bacterial infections in dental implants generate peri-implantitis disease that causes bone loss and the mobility of the dental implant. It is well known that specific ranges of roughness favor the proliferation of bacteria, and it is for this reason that new dental implants called hybrids have appeared. These implants have a smooth area in the coronal part and a rough surface in the apical part. The objective of this research is the physico-chemical characterization of the surface and the osteoblastic and microbiological behavior. One-hundred and eighty discs of titanium grade 3 with three different surfaces (smooth, smooth-rough, and completely rough) were studied. The roughness was determined by white light interferometry, and the wettability and surface energy by the sessile drop technique and the application of Owens and Wendt equations. Human osteoblast SaOS-2 was cultured to determine cell adhesion, proliferation, and differentiation. Microbiological studies were performed with two common bacterial strains in oral infection, E. faecalis and S. gordonii, at different times of culture. The roughness obtained for the smooth surface was Sa = 0.23 and for the rough surface it was 1.98 µm. The contact angles were more hydrophilic for the smooth surface (61.2°) than for the rough surface (76.1°). However, the surface energy was lower for the rough surface (22.70 mJ/m2) in both its dispersive and polar components than the smooth surface (41.77 mJ/m2). Cellular activity in adhesion, proliferation, and differentiation was much higher on rough surfaces than on smooth surfaces. After 6 h of incubation, the osteoblast number in rough surfaces was more than 32% higher in relation to the smooth surface. The cell area in smooth surfaces was higher than rough surfaces. The proliferation increased and the alkaline phosphatase presented a maximum after 14 days, with the mineral content of the cells being higher in rough surfaces. In addition, the rough surfaces showed greater bacterial proliferation at the times studied and in the two strains used. Hybrid implants sacrifice the good osteoblast behavior of the coronal part of the implant in order to obstruct bacterial adhesion. The following fact should be considered by clinicians: there is a possible loss of bone fixation when preventing peri-implantitis.

The Use of Human Sterilized Crushed Tooth Particles Compared with BTCP Biomaterial and Empty Defects in Bone Formation inside Critical Rabbit Calvaria Sites.

This study aimed to assess the bone regeneration of critical-size defects in rabbit calvaria filled with freshly crushed extracted teeth, comparing them with BTCP biomaterial and empty sites. Materials and methods: Twenty-one female New Zealand rabbits were used in this study. Two critical-size defects 6 mm in size were created in the skull bone, each with a 3 mm separation between them. Three experimental groups were evaluated: Group A (human sterilized crushed teeth granules alone), Group B (Bioner Bone, Bioner Sitemas Implantológicos), and Group C (unfilled defects). The animals were sacrificed at 4 and 8 weeks. Evaluation of the samples involved histological and histomorphometric analyses with radiographic evaluation. The histological evaluation showed a higher volume reduction in Group A compared with Group B (p < 0.05) and Control. Group A showed the highest values for cortical closure and bone formation around the particles, followed by Group B and Group C (p < 0.05). Within the limitations of this animal study, we can conclude that the use of human tooth particles leads to increased bone formation and reduced connective tissue in critical-size defects in rabbit calvaria when compared to BTCP biomaterial. The calvarial model is a robust base for the evaluation of different biomaterials.

Corrosion Resistance and Titanium Ion Release of Hybrid Dental Implants.

One of the strategies for the fight against peri-implantitis is the fabrication of titanium dental implants with the part close to the neck without roughness. It is well known that roughness favors osseointegration but hinders the formation of biofilm. Implants with this type of structure are called hybrid dental implants, which sacrifice better coronal osseointegration for a smooth surface that hinders bacterial colonization. In this contribution, we have studied the corrosion resistance and the release of titanium ions to the medium of smooth (L), hybrid (H), and rough (R) dental implants. All implants were identical in design. Roughness was determined with an optical interferometer and residual stresses were determined for each surface by X-ray diffraction using the Bragg-Bentano technique. Corrosion studies were carried out with a Voltalab PGZ301 potentiostat, using Hank's solution as an electrolyte at a temperature of 37 °C. Open-circuit potentials (Eocp), corrosion potential (Ecorr), and current density (icorr) were determined. Implant surfaces were observed by JEOL 5410 scanning electron microscopy. Finally, for each of the different dental implants, the release of ions into Hank's solution at 37 °C at 1, 7, 14, and 30 days of immersion was determined by ICP-MS. The results, as expected, show a higher roughness of R with respect to L and compressive residual stresses of -201.2 MPa and -20.2 MPa, respectively. These differences in residual stresses create a potential difference in the H implant corresponding to Eocp of -186.4 mV higher than for the L and R of -200.9 and -192.2 mV, respectively. The corrosion potentials and current intensity are also higher for the H implants (-223 mV and 0.069 µA/mm2) with respect to the L (-280 mV and 0.014 µA/mm2 and R (-273 mV and 0.019 µA/mm2). Scanning electron microscopy revealed pitting in the interface zone of the H implants and no pitting in the L and R dental implants. The titanium ion release values to the medium are higher in the R implants due to their higher specific surface area compared to the H and L implants. The maximum values obtained are low, not exceeding 6 ppb in 30 days.

Formation of PLGA-PEDOT: PSS Conductive Scaffolds by Supercritical Foaming.

The usage of conjugated materials for the fabrication of foams intended to be used as therapeutic scaffolds is gaining relevance these days, as they hold certain properties that are not exhibited by other polymer types that have been regularly used until the present. Hence, this work aims to design a specific supercritical CO2 foaming process that would allow the production of porous polymeric devices with improved conductive properties, which would better simulate matrix extracellular conditions when used as therapeutic scaffolds (PLGA-PEDOT:PSS) systems. The effects of pressure, temperature, and contact time on the expansion factor, porosity, mechanical properties, and conductivity of the foam have been evaluated. The foams have been characterized by scanning electron and atomic force microscopies, liquid displacement, PBS degradation test, compression, and resistance to conductivity techniques. Values close to 40% porosity were obtained, with a uniform distribution of polymers on the surface and in the interior, expansion factors of up to 10 orders, and a wide range of conductivity values (2.2 × 10-7 to 1.0 × 10-5 S/cm) and mechanical properties (0.8 to 13.6 MPa Young's modulus in compression test). The conductive and porous scaffolds that have been produced by supercritical CO2 in this study show an interesting potential for tissue engineering and for neural or cardiac tissue regeneration purposes due to the fact that electrical conductivity is a crucial factor for proper cell function and tissue development.

Plasmonic Response of Complex Nanoparticle Assemblies.

Optical properties of nanoparticle assemblies reflect distinctive characteristics of their building blocks and spatial organization, giving rise to emergent phenomena. Integrated experimental and computational studies have established design principles connecting the structure to properties for assembled clusters and superlattices. However, conventional electromagnetic simulations are too computationally expensive to treat more complex assemblies. Here we establish a fast, materials agnostic method to simulate the optical response of large nanoparticle assemblies incorporating both structural and compositional complexity. This many-bodied, mutual polarization method resolves limitations of established approaches, achieving rapid, accurate convergence for configurations including thousands of nanoparticles, with some overlapping. We demonstrate these capabilities by reproducing experimental trends and uncovering far- and near-field mechanisms governing the optical response of plasmonic semiconductor nanocrystal assemblies including structurally complex gel networks and compositionally complex mixed binary superlattices. This broadly applicable framework will facilitate the design of complex, hierarchically structured, and dynamic assemblies for desired optical characteristics.

Complex regulation in a Comamonas platform for diverse aromatic carbon metabolism.

Critical to a sustainable energy future are microbial platforms that can process aromatic carbons from the largely untapped reservoir of lignin and plastic feedstocks. Comamonas species present promising bacterial candidates for such platforms because they can use a range of natural and xenobiotic aromatic compounds and often possess innate genetic constraints that avoid competition with sugars. However, the metabolic reactions of these species are underexplored, and the regulatory mechanisms are unknown. Here we identify multilevel regulation in the conversion of lignin-related natural aromatic compounds, 4-hydroxybenzoate and vanillate, and the plastics-related xenobiotic aromatic compound, terephthalate, in Comamonas testosteroni KF-1. Transcription-level regulation controls initial catabolism and cleavage, but metabolite-level thermodynamic regulation governs fluxes in central carbon metabolism. Quantitative 13C mapping of tricarboxylic acid cycle and cataplerotic reactions elucidates key carbon routing not evident from enzyme abundance changes. This scheme of transcriptional activation coupled with metabolic fine-tuning challenges outcome predictions during metabolic manipulations.

Physiotherapy in palliative medicine: patient and caregiver wellness.

OBJECTIVES: Despite the demonstrated efficacy of physiotherapy in palliative care programmes, there are scarce data of its real-life impact on patients' and caregivers' wellness and stress. Our aim was to assess effectiveness of a 30-day physiotherapy programme in psychological wellness and health-related quality of life (HRQoL) of patients with advanced chronic diseases or cancer and in their caregivers' stress. METHODS: Quasiexperimental before-after study applying personalised kinesitherapy, exercise with curative effects, respiratory physiotherapy, therapeutic massages and ergotherapy. Psychological wellness, HRQoL and caregiver's strain outcomes were measured. RESULTS: 207 patients (60% men, with a mean age of 73.6±12 years) were included; 129 (62.3%) with advanced cancer, and the remaining 78 with advanced chronic diseases. Psychological wellness (Emotional Stress Detection Tool decreased from 12.4±3 to 11±3; p<0.0001), caregiver's strain (Caregiver Strain Index decreased from 8.5±3.2 to 7.9±3.5; p<0.0001) and HRQoL (WHO-BREF physical health domain increased from 8.3±2.6 to 9.4±2.9; p<0.0001) showed a significant improvement after the physiotherapy programme. Global satisfaction with the physiotherapy intervention was also high (Client Satisfaction Questionnaire-8 of 28.3±3.3 points). CONCLUSIONS: A personalised physiotherapy programme incorporated to integral palliative care improved psychological wellness, HRQoL and caregivers' strain of patients with advanced chronic diseases and cancer.

The HA4M dataset: Multi-Modal Monitoring of an assembly task for Human Action recognition in Manufacturing.

This paper introduces the Human Action Multi-Modal Monitoring in Manufacturing (HA4M) dataset, a collection of multi-modal data relative to actions performed by different subjects building an Epicyclic Gear Train (EGT). In particular, 41 subjects executed several trials of the assembly task, which consists of 12 actions. Data were collected in a laboratory scenario using a Microsoft® Azure Kinect which integrates a depth camera, an RGB camera, and InfraRed (IR) emitters. To the best of authors' knowledge, the HA4M dataset is the first multi-modal dataset about an assembly task containing six types of data: RGB images, Depth maps, IR images, RGB-to-Depth-Aligned images, Point Clouds and Skeleton data. These data represent a good foundation to develop and test advanced action recognition systems in several fields, including Computer Vision and Machine Learning, and application domains such as smart manufacturing and human-robot collaboration.

Synthetic metabolism for in vitro acetone biosynthesis driven by ATP regeneration.

In vitro ketone production continues to be a challenge due to the biochemical features of the enzymes involved-even when some of them have been extensively characterized (e.g. thiolase from Clostridium acetobutylicum), the assembly of synthetic enzyme cascades still face significant limitations (including issues with protein aggregation and multimerization). Here, we designed and assembled a self-sustaining enzyme cascade with acetone yields close to the theoretical maximum using acetate as the only carbon input. The efficiency of this system was further boosted by coupling the enzymatic sequence to a two-step ATP-regeneration system that enables continuous, cost-effective acetone biosynthesis. Furthermore, simple methods were implemented for purifying the enzymes necessary for this synthetic metabolism, including a first-case example on the isolation of a heterotetrameric acetate:coenzyme A transferase by affinity chromatography.

Fe3O4-TiO2 Thin Films in Solar Photocatalytic Processes.

The optical properties of 5wt% Fe3O4-TiO2 thin films were evaluated in detail with the aim of proposing a mechanism for solar photocatalytic processes and highlighting the advantages over the use of bare TiO2. The results showed that the incorporation of 5wt% Fe3O4 enhanced the optical properties by a redshift to a wavelength in the visible range, reducing the anatase/rutile band gap energy from 3.2 eV to 2.8 eV. Photoluminescence studies reveal a superior separation efficiency of photoexcited electron-hole pairs when Fe3O4 nanoparticles (NPs) are present in the photocatalyst. X-ray photoelectron spectroscopy spectra confirm the presence of Fe3O4 and existence of a chemical bonding between TiO2 and Fe3O4 NPs. Moreover, in this study, a mechanism of solar photocatalytic processes involving Fe3O4-TiO2 thin films is proposed and it is supported by experimental results. Finally, solar photocatalytic experiments were carried out, indicating that the effectiveness for the removal of the selected pharmaceutical is considerably improved when the composite material is used as catalyst. Furthermore, it was demonstrated that the photocatalytic activity of the prepared Fe3O4-TiO2 thin films depends on their thickness, achieving the highest pharmaceutical removal yields using the 2 µm thick sample. The stability and reusability of the catalyst was confirmed studying the photocatalytic activity over three cycles.

Effects of auditory sleep modulation approaches on brain oscillatory and cardiovascular dynamics.

Slow waves, the hallmark feature of deep nonrapid eye movement sleep, do potentially drive restorative effects of sleep on brain and body functions. Sleep modulation techniques to elucidate the functional role of slow waves thus have gained large interest. Auditory slow wave stimulation is a promising tool; however, directly comparing auditory stimulation approaches within a night and analyzing induced dynamic brain and cardiovascular effects are yet missing. Here, we tested various auditory stimulation approaches in a windowed, 10 s ON (stimulations) followed by 10 s OFF (no stimulations), within-night stimulation design and compared them to a SHAM control condition. We report the results of three studies and a total of 51 included nights and found a large and global increase in slow-wave activity (SWA) in the stimulation window compared to SHAM. Furthermore, slow-wave dynamics were most pronouncedly increased at the start of the stimulation and declined across the stimulation window. Beyond the changes in brain oscillations, we observed, for some conditions, a significant increase in the mean interval between two heartbeats within a stimulation window, indicating a slowing of the heart rate, and increased heart rate variability derived parasympathetic activity. Those cardiovascular changes were positively correlated with the change in SWA, and thus, our findings provide insight into the potential of auditory slow wave enhancement to modulate cardiovascular restorative conditions during sleep. However, future studies need to investigate whether the potentially increased restorative capacity through slow-wave enhancements translates into a more rested cardiovascular system on a subsequent day.