Current cancer diagnosis techniques are often dependent on the collection of tumour tissue, involving invasive processes for the patient. Circulating Tumour DNA (ctDNA) emerges as an alternative resource for cancer detection and monitoring, that can be har vested from simple blood samples. Digital Polymerase Chain Reaction (dPCR) is a fast and sensitive technique for DNA amplification, suitable for low DNA concentrations such as ctDNA. Advances in microfluidics allow the partition of PCR samples into droplets based in water-in-oil emulsions, so that PCR amplification occurs within each droplet. In this way, the PCR reaction is a well controlled process with a low probability of contami nation and allowing a high throughput analysis. The aimed of this work was to develop droplet-based microfluidic device for application to dPCR technique coupled with real-time droplet monitoring. This work focused on the design and fabrication of a microfluidic device capable of producing a large number of uniform droplets with volumes in the nanoliter range and constant frequency. For this, a polydimethylsiloxane (PDMS) droplet generator device was developed, through photo and soft-lithography techniques, and tested with several oil/water flow rates ratios. Then, the droplets generated were characterized in terms of droplet size, velocity and frequency through the implementation of a powerful open-source software for real-time analysis. Several tests on different devices were carried out to evaluate the device reproducibility. Finally, the droplet generator was incorporated with a serpentine design, allowing the PCR cycles to occur in continuous flow. The results revealed that was possible to generate droplets with radius between 22-99 µm and a coefficient of variation bellow 10%. The correspondents volumes ranged between 90 pL-4.18 nL. Moreover, the velocities obtained situated between 0.05 mm/s-7.62 mm/s with droplet generating frequency of 2-50 Hz. Regarding to the droplet monitoring, the results of the workflows developed revealed similarity with the results obtained trough a widely used software for this purposes, with the advantage of allowing real-time analysis for a larger sample of results.

Advances in concrete technology during the last decades have resulted in the development of materials with enhanced mechanical properties, such as High Strength Concrete (HSC), Fibre Reinforced Concrete (FRC) and Ultra-High Performance Fibre Reinforced Concrete (UHPFRC). The application of these materials in flat slabs, which are a popular structural solution in Reinforced Concrete (RC) buildings worldwide, has the potential of significantly reducing raw material consumption by enabling the design of slenderer and therefore lighter structures. However, flat slabs are susceptible to punching shear failure, which is a complex phenomenon that remains challenging, even though significant efforts have been made to experimentally study it. For advanced concrete materials (HSC, FRC and UHPFRC), the challenge is further accentuated by the continuous and rapid development of these materials. With the purpose of identifying and highlighting gaps in the published literature, a review of tests with HSC, FRC and UHPFRC slab-column connections in non-seismic and seismic loading applications is presented in this paper. It is shown that future research directions in this field include, among others, testing thicker slabs, HSC slabs with higher concrete compressive strength, HSC combined with FRC and several more cases related to seismic loading conditions.

This study describes for the first time the iron- and copper-mediated gelation of FucoPol, fucose-rich bacterial polysaccharide. The ability of FucoPol to gel in the presence of metal cations, including iron(III) and copper(II), was used for the preparation of hydrogel beads. Iron mediated the formation of stable and not cytotoxic gel beads, while copper resulted in fragile and cytotoxic ones. Copper-mediated beads coated with an iron-mediated gel layer were more stable and had reduced cytotoxicity. The resulting polymeric structures had differing morphology, physical properties and cytotoxicity, which support their use in several applications, including biomedicine, agriculture and bioremediation.

Two piano-stool iron(II) complexes bearing N-heterocyclic carbene ligands outfitted with acetamide- and amine-pendant arms [Cp*Fe(NHCR)(CO)I] {Cp* = η5-tetramethylcyclopentadienyl; R = CH2CONEt2 (3), (CH2)2NEt2 (4)}, have been prepared and fully characterized. Their catalytic activity in transfer hydrogenation (TH) of ketones using iPrOH as a hydrogen source and catalytic amounts of base (LiOtBu) has been explored, along with that of previously reported [CpFe(NHCR)(CO)I] {R = nBu (5), (CH2)2OH (6), Et (7), and (CH2)3OH (8)} complexes containing hydroxyl and nonfunctionalized alkyl arms. Complex 3 displayed the highest catalytic activity of the whole series 3–8, reaching a TOF50 value of 533 h–1. NMR monitoring of the stoichiometric reaction of 3 with LiOtBu, allowed the identification of a new species 3' containing a deprotonated amidate moiety, which has been fully characterized by 1H, 13C, and 15N NMR. Finally, a green protocol for the reduction of ketones through TH using glycerol as a hydrogen source, under microwave irradiation in the presence of catalytic amounts of 3 and base has been developed.

The Cellulosome is an intricate macromolecular protein complex that centralizes the cellulolytic efforts of many anaerobic microorganisms through the promotion of enzyme synergy and protein stability. The assembly of numerous carbohydrate processing enzymes into a macromolecular multiprotein structure results from the interaction of enzyme-borne dockerin modules with repeated cohesin modules present in noncatalytic scaffold proteins, termed scaffoldins. Cohesin–dockerin (Coh-Doc) modules are typically classified into different types, depending on structural conformation and cellulosome role. Thus, type I Coh-Doc complexes are usually responsible for enzyme integration into the cellulosome, while type II Coh-Doc complexes tether the cellulosome to the bacterial wall. In contrast to other known cellulosomes, cohesin types from Bacteroides cellulosolvens, a cellulosome-producing bacterium capable of utilizing cellulose and cellobiose as carbon sources, are reversed for all scaffoldins, i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. It has been previously shown that type I B. cellulosolvens interactions possess a dual-binding mode that adds flexibility to scaffoldin assembly. Herein, we report the structural mechanism of enzyme recruitment into B. cellulosolvens cellulosome and the identification of the molecular determinants of its type II cohesin–dockerin interactions. The results indicate that, unlike other type II complexes, these possess a dual-binding mode of interaction, akin to type I complexes. Therefore, the plasticity of dual-binding mode interactions seems to play a pivotal role in the assembly of B. cellulosolvens cellulosome, which is consistent with its unmatched complexity and size.

Background: The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive. Methods: The fraction capacity of Butyl- and Octyl-Sepharose matrices on LNCaP lysates was evaluated by manipulating the ionic strength of binding and elution phases, followed by a Co-Immunoprecipitation (Co-IP) polishing. Several potential stabilizing additives were assessed, and the melting temperature (Tm) values ranked the best/worst compounds. The secondary structure of STEAP1 was identified by circular dichroism. Results: The STEAP1 was not fully captured with 1.375 M (Butyl), in contrast with interfering heterologous proteins, which were strongly retained and mostly eluted with water. This single step demonstrated higher selectivity of Butyl-Sepharose for host impurities removal from injected crude samples. Co-IP allowed recovering a purified fraction of STEAP1 and contributed to unveil potential physiologically interacting counterparts with the target. A Tm of  55 °C was determined, confirming STEAP1 stability in the purification buffer. A predominant α-helical structure was identified, ensuring the protein’s structural stability. Conclusions: A method for successfully isolating human STEAP1 from LNCaP cells was provided, avoiding the use of detergents to achieve stability, even outside a membrane-mimicking environment.

Aiming to rationalize the release profile of an incorporated pharmaceutical drug in terms of its mobility, driven by guest-host interactions, the poorly water-soluble ibuprofen drug was loaded in a mesoporous inorganic silica matrix with unmodified (MCM-41) and modified surface (MCM-41sil) by post-synthesis silylation, both having pore sizes   3 nm. The single calorimetric detection of a broad glass transition step for both ibuprofen composites indicates full drug amorphization, confirmed by the only appearance of an amorphous halo in the powder XRD patterns. Moreover, a gradient profile is disclosed by the heat flux derivative plot in the glass transition, in coherence with the thermogravimetric profile that shows a multi-step decomposition trace for confined ibuprofen in these matrixes. While identical guest dynamics, as probed by dielectric relaxation spectroscopy, were found in both dehydrated composites, a significant molecular population with faster relaxation exists in the hydrated state for the drug inside the unmodified matrix. This was rationalized as the concurrence of true confinement effects, which manifest under nanometer dimensions, and greater water affinity of the unmodified matrix, forcing the drug molecules to be placed mostly in the pore core. Finite size effects are also felt in both dehydrated composites, however guest-host interactions give origin to a dominant population with slowed down mobility that governs the overall guest dynamics. In spite of an inferior number of active sites for drug adsorption in the silylated matrix, a faster ibuprofen delivery in phosphate buffer (pH = 6.8) was observed when the drug is released from unmodified MCM-41 in the hydrated state. Therefore, our results suggest that a relevant role is played by water molecules, which impair a strong guest adsorption in the host surface more efficiently than the limited surface modification, influence the higher ratio of a faster population in the pore core and facilitate the diffusion of the aqueous releasing media inside pores.

Nucleic acid amplification technologies (NAATs) have become fundamental tools in molecular diagnostics, due to their ability to detect small amounts of target molecules. Since its development, Polymerase Chain Reaction (PCR) has been the most exploited method, being stablished as the “gold standard” technique for DNA amplification. However, the requirement for different working temperatures leads to the need of a thermocycler machine or complex thermal apparatus, which have been preventing its application in novel integrated devices for single workflow and high throughput analysis. Conversely, isothermal amplification methods have been gaining attention, especially for point-of-care diagnosis and applications. These non-PCR based methods have been developed by mimicking the in vivo amplification mechanisms, while performing the amplification with high sensitivity, selectivity and allowing for high-throughput analysis. These favorable capabilities have pushed forward the implementation and commercialization of several platforms that exploit isothermal amplification methods, mostly against virus, bacteria and other pathogens in water, food, environmental and clinical samples. Nevertheless, the future of isothermal amplification methods is still dependent on achieving technical maturity and broader commercialization of enzymes and reagents.

After more than one hundred and thirty thousand protein structures determined by X-ray crystallography{,} the challenge of protein crystallization for 3D structure determination remains. In the quest for additives for efficient protein crystallization{,} inorganic materials emerge as an alternative. Magnetic particles (MPs) are versatile inorganic materials{,} easy to use{,} modify and manipulate in a wide range of biological assays. The potential of using functionalised MPs as crystallization chaperones for protein crystallization was shown in this work. MPs with distinct coatings were rationally designed to promote protein crystallization by affinity-triggered heterogeneous nucleation. Hen egg white lysozyme (HEWL) and trypsin{,} were crystallized in the presence of MPs either bare or coated with a polysaccharide (chitin) or a protein (casein){,} respectively. The addition of MPs was characterized in terms of bound protein to the MPs{,} crystal morphology{,} time-lapse of crystal emergence{,} crystallization yield fold change and crystal diffraction quality for structure determination. The MPs additives have shown to bind to the respective target protein{,} and to promote nucleation and crystal growth without compromising crystal morphology. On the other hand{,} MPs addition led to faster detectable crystal emergence and up to 13 times higher crystallization yield{,} addressing some the challenges in protein crystallization{,} the main bottleneck of macromolecular crystallography. Structure determination of the protein crystallized in the presence of MPs revealed that the structural characteristics of the protein remained unchanged{,} as shown by the superposition with PDB annotated proteins. Moreover{,} and unlike most reported cases{,} it was possible to exclude the inhibitor benzamidine during trypsin crystallisation{,} which is a remarkable result opening new prospects in enzyme engineering and drug design. Our results show that MPs coated with affinity ligands to target proteins can be used as controlled and tailor-made crystallization inducers.

Environmental changes trigger the continuous adaptation of bacteria to ensure their survival. This is possible through a variety of signal transduction pathways involving chemoreceptors known as methyl-accepting chemotaxis proteins (MCP) that allow the microorganisms to redirect their mobility towards favorable environments. MCP are two-component regulatory (or signal transduction) systems (TCS) formed by a sensor and a response regulator domain. These domains synchronize transient protein phosphorylation and dephosphorylation events to convert the stimuli into an appropriate cellular response. In this review, the variability of TCS domains and the most common signaling mechanisms are highlighted. This is followed by the description of the overall cellular topology, classification and mechanisms of MCP. Finally, the structural and functional properties of a new family of MCP found in Geobacter sulfurreducens are revisited. This bacterium has a diverse repertoire of chemosensory systems, which represents a striking example of a survival mechanism in challenging environments. Two G. sulfurreducens MCP—GSU0582 and GSU0935—are members of a new family of chemotaxis sensor proteins containing a periplasmic PAS-like sensor domain with a c-type heme. Interestingly, the cellular location of this domain opens new routes to the understanding of the redox potential sensing signaling transduction pathways.