Mammalian Antigen Display for Pandemic Countermeasures.

Pandemic countermeasures require the rapid design of antigens for vaccines, profiling patient antibody responses, assessing antigen structure-function landscapes, and the surveillance of emerging viral lineages. Cell surface display of a viral antigen or its subdomains can facilitate these goals by coupling the phenotypes of protein variants to their DNA sequence. Screening surface-displayed proteins via flow cytometry also eliminates time-consuming protein purification steps. Prior approaches have primarily relied on yeast as a display chassis. However, yeast often cannot express large viral glycoproteins, requiring their truncation into subdomains. Here, we describe a method to design and express antigens on the surface of mammalian HEK293T cells. We discuss three use cases, including screening of stabilizing mutations, deep mutational scanning, and epitope mapping. The mammalian antigen display platform described herein will accelerate ongoing and future pandemic countermeasures.

Diagnostics of COVID-19 Based on CRISPR-Cas Coupled to Isothermal Amplification: A Comparative Analysis and Update.

The emergence of the COVID-19 pandemic prompted fast development of novel diagnostic methods of the etiologic virus SARS-CoV-2. Methods based on CRISPR-Cas systems have been particularly promising because they can achieve a similar sensitivity and specificity to the benchmark RT-qPCR, especially when coupled to an isothermal pre-amplification step. Furthermore, they have also solved inherent limitations of RT-qPCR that impede its decentralized use and deployment in the field, such as the need for expensive equipment, high cost per reaction, and delivery of results in hours, among others. In this review, we evaluate publicly available methods to detect SARS-CoV-2 that are based on CRISPR-Cas and isothermal amplification. We critically analyze the steps required to obtain a successful result from clinical samples and pinpoint key experimental conditions and parameters that could be optimized or modified to improve clinical and analytical outputs. The COVID outbreak has propelled intensive research in a short time, which is paving the way to develop effective and very promising CRISPR-Cas systems for the precise detection of SARS-CoV-2. This review could also serve as an introductory guide to new labs delving into this technology.

A dos metros de ti / Five feet apart (2019) de Justin Baldoni: una herramienta de aprendizaje sobre fibrosis quística / Five Feet Apart (2019) by Justin Baldoni: a tool to learn about cystic fibrosis

El cine es una efectiva herramienta de enseñanza - aprendizaje que permite ilustrar desde diversos enfoques los procesos complejos de la enfermedad. En este artículo hacemos hincapié sobre estas herramientas educativas que nos brinda la película A dos metros de ti / Five feet apart, sobre la fibrosis quística. En la que se retratan los aspectos diarios que sobrellevan los pacientes dentro de un hospital desde su alimentación, tratamiento e infecciones. A la vez, la necesidad del contacto físico que tienen con las personas que conviven; ya que estos pacientes deben mantenerse alejados entre sí, a dos metros de distancia. Además, trata de las infecciones causadas por bacterias multirresistentes como Burkholderia cepacia y métodos alternativos como el proceso de trasplante pulmonar que permite prolongar la vida en paciente con fibrosis quística. (AU)

Movies are a helpful tool to learn and teach which allow us to depict from different points of view the complexity of the illness. In this paper we focus on the educational tools we saw in the movie Five feet apart about cystic fibrosis, where we can see the daily aspects that patients have to endure in a hospital, from their feeding, treatment and infections, as well as the importance of physical contact with the people around because they must keep a distance of 2 m between them. It also depicts the infections caused by multi-resistant bacteria like Burkholderia cepacia and the alternative methods to treat it like lung transplant which allows patients to extend their life expectancy. (AU)

Sacsin Deletion Induces Aggregation of Glial Intermediate Filaments.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurodegenerative disorder commonly diagnosed in infants and characterized by progressive cerebellar ataxia, spasticity, motor sensory neuropathy and axonal demyelination. ARSACS is caused by mutations in the SACS gene that lead to truncated or defective forms of the 520 kDa multidomain protein, sacsin. Sacsin function is exclusively studied on neuronal cells, where it regulates mitochondrial network organization and facilitates the normal polymerization of neuronal intermediate filaments (i.e., neurofilaments and vimentin). Here, we show that sacsin is also highly expressed in astrocytes, C6 rat glioma cells and N9 mouse microglia. Sacsin knockout in C6 cells (C6Sacs-/-) induced the accumulation of the glial intermediate filaments glial fibrillary acidic protein (GFAP), nestin and vimentin in the juxtanuclear area, and a concomitant depletion of mitochondria. C6Sacs-/- cells showed impaired responses to oxidative challenges (Rotenone) and inflammatory stimuli (Interleukin-6). GFAP aggregation is also associated with other neurodegenerative conditions diagnosed in infants, such as Alexander disease or Giant Axonal Neuropathy. Our results, and the similarities between these disorders, reinforce the possible connection between ARSACS and intermediate filament-associated diseases and point to a potential role of glia in ARSACS pathology.

Zero-mode waveguides visualize the first steps during gelsolin-mediated actin filament formation.

Actin filament dynamics underlie key cellular processes. Although the elongation of actin filaments has been extensively studied, the mechanism of nucleation remains unclear. The micromolar concentrations needed for filament formation have prevented direct observation of nucleation dynamics on the single molecule level. To overcome this limitation, we have used the attoliter excitation volume of zero-mode waveguides to directly monitor the early steps of filament assembly. Immobilizing single gelsolin molecules as a nucleator at the bottom of the zero-mode waveguide, we could visualize the actin filament nucleation process. The process is surprisingly dynamic, and two distinct populations during gelsolin-mediated nucleation are observed. The two populations are defined by the stability of the actin dimers and determine whether elongation occurs. Furthermore, by using an inhibitor to block flattening, a conformational change in actin associated with filament formation, elongation was prevented. These observations indicate that a conformational transition and pathway competition determine the nucleation of gelsolin-mediated actin filament formation.

Dynamic interactions and Ca2+-binding modulate the holdase-type chaperone activity of S100B preventing tau aggregation and seeding.

The microtubule-associated protein tau is implicated in the formation of oligomers and fibrillar aggregates that evade proteostasis control and spread from cell-to-cell. Tau pathology is accompanied by sustained neuroinflammation and, while the release of alarmin mediators aggravates disease at late stages, early inflammatory responses encompass protective functions. This is the case of the Ca2+-binding S100B protein, an astrocytic alarmin which is augmented in AD and which has been recently implicated as a proteostasis regulator, acting over amyloid ß aggregation. Here we report the activity of S100B as a suppressor of tau aggregation and seeding, operating at sub-stoichiometric conditions. We show that S100B interacts with tau in living cells even in microtubule-destabilizing conditions. Structural analysis revealed that tau undergoes dynamic interactions with S100B, in a Ca2+-dependent manner, notably with the aggregation prone repeat segments at the microtubule binding regions. This interaction involves contacts of tau with a cleft formed at the interface of the S100B dimer. Kinetic and mechanistic analysis revealed that S100B inhibits the aggregation of both full-length tau and of the microtubule binding domain, and that this proceeds through effects over primary and secondary nucleation, as confirmed by seeding assays and direct observation of S100B binding to tau oligomers and fibrils. In agreement with a role as an extracellular chaperone and its accumulation near tau positive inclusions, we show that S100B blocks proteopathic tau seeding. Together, our findings establish tau as a client of the S100B chaperone, providing evidence for neuro-protective functions of this inflammatory mediator across different tauopathies.

Unravelling the local crystallographic structure of ferromagnetic [Formula: see text]-[Formula: see text]N nanocrystals embedded in GaN.

In the Fe-doped GaN phase-separated magnetic semiconductor Ga[Formula: see text]FeN, the presence of embedded [Formula: see text]-[Formula: see text]N nanocrystals determines the magnetic properties of the system. Here, through a combination of anomalous X-ray diffraction and diffraction anomalous fine structure, the local structure of Ga in self-assembled face-centered cubic (fcc) [Formula: see text]-[Formula: see text]N nanocrystals embedded in wurtzite GaN thin layers is investigated in order to shed light onto the correlation between fabrication parameters, local structural arrangement and overall magnetic properties of the material system. It is found, that by adjusting the growth parameters and thus, the crystallographic surroundings, the Ga atoms can be induced to incorporate into 3c positions at the faces of the fcc crystal lattice, reaching a maximum occupancy of 30%. The magnetic response of the embedded nanocrystals is ferromagnetic with Curie temperature increasing from 450 to 500 K with the Ga occupation. These results demonstrate the outstanding potential of the employed experimental protocol for unravelling the local structure of magnetic multi-phase systems, even when embedded in a matrix containing the same element under investigation.

Standard deviation of microscopy images used as indicator for growth stages.

Photoelectron emission microscopy (PEEM) and low energy electron microscopy (LEEM) can easily distinguish between organic molecules adsorbed in crystallites or in the wetting layers as well as the bare metal substrate due to their different electronic properties. Already before (and during) the condensation of such solid phases (2D islands or 3D crystallites), there is a dilute 2D gas phase. Such a 2D gas phase consists of molecules, which are highly mobile and diffuse across the surface. The individual molecules are too small to be resolved in PEEM/LEEM images. Here, we discuss, how image features below and above the resolution limit of a PEEM/LEEM affect the mean electron yield and its (normalized) standard deviation. We support our findings with two experimental examples: the deposition of cobalt phthalocyanine (CoPc) on Ag(100) and of perfluoro-pentacene on Ag(110). Our results demonstrate, how a spatial and temporal analysis of image series can be used to obtain information about molecular phases, which cannot be directly resolved in microscopy images.

Out-of-Plane Magnetic Anisotropy in Ordered Ensembles of FeyN Nanocrystals Embedded in GaN.

Phase-separated semiconductors containing magnetic nanostructures are relevant systems for the realization of high-density recording media. Here, the controlled strain engineering of Ga δ FeN layers with Fe y N embedded nanocrystals (NCs) via Al x Ga 1 - x N buffers with different Al concentration 0 < x Al < 41 % is presented. Through the addition of Al to the buffer, the formation of predominantly prolate-shaped ε -Fe 3 N NCs takes place. Already at an Al concentration x Al ≈ 5% the structural properties-phase, shape, orientation-as well as the spatial distribution of the embedded NCs are modified in comparison to those grown on a GaN buffer. Although the magnetic easy axis of the cubic γ '-Ga y Fe 4 - y N nanocrystals in the layer on the x Al = 0 % buffer lies in-plane, the easy axis of the ε -Fe 3 N NCs in all samples with Al x Ga 1 - x N buffers coincides with the [ 0001 ] growth direction, leading to a sizeable out-of-plane magnetic anisotropy and opening wide perspectives for perpendicular recording based on nitride-based magnetic nanocrystals.

Allogeneic Cellular Therapy in a Mature Tooth with Apical Periodontitis and Accidental Root Perforation: A Case Report.

INTRODUCTION: Cell therapy in regenerative endodontics introduces an alternative option to classic treatment strategies for complex endodontic cases. The aim of this case report was to describe cell-based therapy using allogeneic umbilical cord mesenchymal stem cells (UC-MSCs) encapsulated in a bioscaffold for a complex case of a mature permanent tooth with apical periodontitis and accidental root perforation. METHODS: A healthy 19-year-old man undergoing orthodontic treatment was referred for endodontic treatment in tooth #7; he was diagnosed with apical periodontitis during a previously initiated treatment associated with accidental perforation of the radicular cervical third. The root perforation was sealed with glass ionomer and composite resin, and the root canal was instrumented, disinfected, and dressed with calcium hydroxide. After 3 weeks, allogeneic UC-MSCs were encapsulated in platelet-poor plasma and then implanted into the root canal, and Biodentine (Septodont, Saint-Maur-des-Fosses, France) was placed below the cementoenamel junction. Finally, the tooth was restored with composite resin. RESULTS: Follow-up examinations were performed 6 months and 1 year later. The examinations included periapical radiography, cone-beam computed tomographic imaging, and sensitivity and vitality tests. Radiographic and cone-beam computed tomographic images indicated remission of the apical lesion. Clinical evaluations revealed normal responses to percussion and palpation tests; the tooth was responsive to the electric pulp test, and the vitality test indicated low blood perfusion units. CONCLUSIONS: This case report reveals the potential use of allogeneic cellular therapy using encapsulated UC-MSCS in a platelet-poor plasma scaffold for a complex case of a permanent tooth with apical periodontitis and root perforation.

Specific and Selective Inhibitors of Proprotein Convertases Engineered by Transferring Serpin B8 Reactive-Site and Exosite Determinants of Reactivity to the Serpin α1PDX.

The molecular determinants of substrate specificity and selectivity in the proprotein convertase (PC) family of proteases are poorly understood. Here we demonstrate that the natural serpin family inhibitor, serpin B8, is a specific and selective inhibitor of furin relative to the other PCs of the constitutive protein secretion pathway, PC4, PC5, PACE4, and PC7 (PC4-PC7, respectively), and identify reactive-site (P6-P5' residues) and exosite elements of the serpin that contribute to this specificity and selectivity through studies of chimeras of serpin B8 and α1PDX, an engineered serpin inhibitor of furin. Kinetic studies revealed that the specificity and selectivity of the serpin chimeras for inhibiting PCs were determined by P6-P5 and P3-P2 residue-dependent recognition of the P4Arg-X-X-P1Arg PC consensus sequence and exosite-dependent recognition of the reactive loop P2' residue of the chimeras by the PCs. Both productive and nonproductive binding of the chimeras to PC4-PC7 but not to furin contributed to a decreased specificity for inhibiting PC4-PC7 and an increased selectivity for inhibiting furin. Molecular dynamics simulations suggested that nonproductive binding of the chimeras to the PCs was correlated with a greater conformational variability of the catalytic sites of PC4-PC7 relative to that of furin. Our findings suggest a new approach for designing selective inhibitors of PCs using α1PDX as a scaffold, as evidenced by our ability to engineer highly specific and selective inhibitors of furin and PC4-PC7.

A strategy based on thermal flexibility to design triosephosphate isomerase proteins with increased or decreased kinetic stability.

Kinetic stability of proteins determines their susceptibility to irreversibly unfold in a time-dependent process, and therefore its half-life. A residue displacement analysis of temperature-induced unfolding molecular dynamics simulations was recently employed to define the thermal flexibility of proteins. This property was found to be correlated with the activation energy barrier (Eact) separating the native from the transition state in the denaturation process. The Eact was determined from the application of a two-state irreversible model to temperature unfolding experiments using differential scanning calorimetry (DSC). The contribution of each residue to the thermal flexibility of proteins is used here to propose multiple mutations in triosephosphate isomerase (TIM) from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), two parasites closely related by evolution. These two enzymes, taken as model systems, have practically identical structure but large differences in their kinetic stability. We constructed two functional TIM variants with more than twice and less than half the activation energy of their respective wild-type reference structures. The results show that the proposed strategy is able to identify the crucial residues for the kinetic stability in these enzymes. As it occurs with other protein properties reflecting their complex behavior, kinetic stability appears to be the consequence of an extensive network of inter-residue interactions, acting in a concerted manner. The proposed strategy to design variants can be used with other proteins, to increase or decrease their functional half-life.

Molecular Reorientation during the Initial Growth of Perfluoropentacene on Ag(110).

Perfluoropentacene (PFP) is an organic material that has been widely studied over the last years and has already found applications in organic electronics. However, fundamental physical questions, such as the structural formation and the preferential orientation of the molecules during deposition on metal surfaces, are still not fully understood. In this work, we report on a unique in-plane molecular reorientation during the completion of the first monolayer of PFP on the Ag(110) surface. To characterize the molecular alignment, we have monitored the deposition process in real time using polarization-dependent differential reflectance spectroscopy and reflectance anisotropy spectroscopy. Abrupt changes in the optical signals reveal an intricate sequence of reorientation transitions of the PFP molecules upon monolayer completion and during the formation of the second monolayer, eventually leading to a full alignment of the long molecular axis along the [001] direction of the substrate and an enhanced structural ordering. Scanning tunneling microscopy and low-energy electron diffraction confirm the observed molecular reorientation upon monolayer compression and provide further details on the structural and orientational ordering of the PFP monolayer before and after compression.

Interplay between Protein Thermal Flexibility and Kinetic Stability.

Kinetic stability is a key parameter to comprehend protein behavior and it plays a central role to understand how evolution has reached the balance between function and stability in cell-relevant timescales. Using an approach that includes simulations, protein engineering, and calorimetry, we show that there is a clear correlation between kinetic stability determined by differential scanning calorimetry and protein thermal flexibility obtained from a novel method based on temperature-induced unfolding molecular dynamics simulations. Thermal flexibility quantitatively measures the increment of the conformational space available to the protein when energy in provided. The (ß/α)8 barrel fold of two closely related by evolution triosephosphate isomerases from two trypanosomes are used as model systems. The kinetic stability-thermal flexibility correlation has predictive power for the studied proteins, suggesting that the strategy and methodology discussed here might be applied to other proteins in biotechnological developments, evolutionary studies, and the design of protein based therapeutics.

The effect of specific proline residues on the kinetic stability of the triosephosphate isomerases of two trypanosomes.

The effect of specific residues on the kinetic stability of two closely related triosephosphate isomerases (from Trypanosoma cruzi, TcTIM and Trypanosoma brucei, TbTIM) has been studied. Based on a comparison of their ß-turn occurrence, we engineered two chimerical enzymes where their super secondary ß-loop-α motifs 2 ((ßα)2 ) were swapped. Differential scanning calorimetry (DSC) experiments showed that the (ßα)2 motif of TcTIM inserted into TbTIM (2Tc) increases the kinetic stability. On the other hand, the presence of the (ßα)2 motif of TbTIM inserted into TcTIM (2Tb) gave a chimerical protein difficult to purify in soluble form and with a significantly reduced kinetic stability. The comparison of the contact maps of the (ßα)2 of TbTIM and TcTIM showed differences in the contact pattern of residues 43 and 49. In TcTIM these residues are prolines, located at the N-terminal of loop-2 and the C-terminal of α-helix-2. Twelve mutants were engineered involving residues 43 and 49 to study the effect over the unfolding activation energy barrier (EA ). A systematic analysis of DSC data showed a large decrease on the EA of TcTIM (ΔEA ranging from 468 to 678 kJ/mol) when the single and double proline mutations are present. The relevance of Pro43 to the kinetic stability is also revealed by mutation S43P, which increased the free energy of the transition state of TbTIM by 17.7 kJ/mol. Overall, the results indicate that protein kinetic stability can be severely affected by punctual mutations, disturbing the complex network of interactions that, in concerted action, determine protein stability. Proteins 2017; 85:571-579. © 2016 Wiley Periodicals, Inc.

Manipulating Mn-Mgk cation complexes to control the charge- and spin-state of Mn in GaN.

Owing to the variety of possible charge and spin states and to the different ways of coupling to the environment, paramagnetic centres in wide band-gap semiconductors and insulators exhibit a strikingly rich spectrum of properties and functionalities, exploited in commercial light emitters and proposed for applications in quantum information. Here we demonstrate, by combining synchrotron techniques with magnetic, optical and ab initio studies, that the codoping of GaN:Mn with Mg allows to control the Mn(n+) charge and spin state in the range 3≤n≤5 and 2≥S≥1. According to our results, this outstanding degree of tunability arises from the formation of hitherto concealed cation complexes Mn-Mg(k), where the number of ligands k is pre-defined by fabrication conditions. The properties of these complexes allow to extend towards the infrared the already remarkable optical capabilities of nitrides, open to solotronics functionalities, and generally represent a fresh perspective for magnetic semiconductors.