Development of Luminescent Biosensors for Calprotectin.

Calprotectin, a metal ion-binding protein complex, plays a crucial role in the innate immune system and has gained prominence as a biomarker for various intestinal and systemic inflammatory and infectious diseases, including inflammatory bowel disease (IBD) and tuberculosis (TB). Current clinical testing methods rely on enzyme-linked immunosorbent assays (ELISAs), limiting accessibility and convenience. In this study, we introduce the Fab-Enabled Split-luciferase Calprotectin Assay (FESCA), a novel quantitative method for calprotectin measurement. FESCA utilizes two new fragment antigen binding proteins (Fabs), CP16 and CP17, that bind to different epitopes of the calprotectin complex. These Fabs are fused with split NanoLuc luciferase fragments, enabling the reconstitution of active luciferase upon binding to calprotectin either in solution or in varied immobilized assay formats. FESCA's output luminescence can be measured with standard laboratory equipment as well as consumer-grade cell phone cameras. FESCA can detect physiologically relevant calprotectin levels across various sample types, including serum, plasma, and whole blood. Notably, FESCA can detect abnormally elevated native calprotectin from TB patients. In summary, FESCA presents a convenient, low-cost, and quantitative method for assessing calprotectin levels in various biological samples, with the potential to improve the diagnosis and monitoring of inflammatory diseases, especially in at-home or point-of-care settings.

Ultrafast Laser-Induced Dynamics of Non-Equilibrium Electron Spill-Out in Nanoplasmonic Bilayers.

Contemporary quantum plasmonics capture subtle corrections to the properties of plasmonic nano-objects in equilibrium. Here, we demonstrate non-equilibrium spill-out redistribution of the electronic density at the ultrafast time scale. As revealed by time-resolved 2D spectroscopy of nanoplasmonic Fe/Au bilayers, an injection of the laser-excited non-thermal electrons induces transient electron spill-out thus changing the plasma frequency. The response of the local electronic density switches the electronic density behavior from spill-in to strong (an order of magnitude larger) spill-out at the femtosecond time scale. The superdiffusive transport of hot electrons and the lack of a direct laser heating indicate significantly non-thermal origin of the underlying physics. Our results demonstrate an ultrafast and non-thermal way to control surface plasmon dispersion through transient variations of the spatial electron distribution at the nanoscale. These findings expand quantum plasmonics into previously unexplored directions by introducing ultrashort time scales in the non-equilibrium electronic systems.

Effect of Shot Peening on the Low-Cycle Fatigue Behavior of an AA2519-T62 Friction-Stir-Welded Butt Joint.

In this investigation, an AA2519-T62 FSW butt joint was subjected to shot peening with an air pressure of p = 0.6 MPa, a processing time of t = 10 min (per side), and a steel ball diameter of dk = 1.5 mm. In order to evaluate the impact of shot peening on the low-cycle behavior, the samples were tested with coefficient R = 0.1 at total strain amplitudes of 0.35%, 0.4%, and 0.5%. The shot-peened welds are characterized by a higher value of stress amplitude, a lower value of plastic strain amplitude, and their fatigue life increased slightly. The cyclic strength coefficient and the cyclic strain hardening exponent were reduced by 45% and 55%, respectively, as the result of the surface layer hardening. The shot peening process had no noticeable effect on the character of crack initiation and propagation. Almost in all cases, the cracking started in the area under the weld face, located close to the boundary between the thermo-mechanically affected zone and the stir zone at the advancing side. Only at the heaviest loadings (εac = 0.5%) were cracks initiated in the heat-affected zone at the retreating side. Despite the introduction of small cracks in the stir zone, their presence did not affect the decohesion character of the welded joint. Overall, it was observed that there is a minimal, positive impact of shot peening on the properties of the investigated joints.

A Comprehensive Study of a Novel Explosively Hardened Pure Titanium Alloy for Medical Applications.

Pure titanium is gaining increasing interest due to its potential use in dental and orthopedic applications. Due to its relatively weak mechanical parameters, a limited number of components manufactured from pure titanium are available on the market. In order to improve the mechanical parameters of pure titanium, manufacturers use alloys containing cytotoxic vanadium and aluminum. This paper presents unique explosive hardening technology that can be used to strengthen pure titanium parameters. The analysis confirms that explosive induced α-ω martensitic transformation and crystallographic anisotropy occurred due to the explosive pressure. The mechanical properties related to residual stresses are very nonuniform. The corrosion properties of the explosive hardened pure titanium test do not change significantly compared to nonhardened titanium. The biocompatibility of all the analyzed samples was confirmed in several tests. The morphology of bone cells does not depend on the titanium surface phase composition and crystallographic orientation.

Mechanical Properties Analysis of Explosive Welded Sheet of AA2519-Ti6Al4V with Interlayer of AA1050 Subjected to Heat-Treatment.

The paper presents results of investigations of welding sheets of AA2519-Ti6Al4V, a difficult-to-joint components materials, produced by explosive welding with a thin technological interlayer of AA1050. The joining process leads to the formation of intermetalics in the vicinity of joint and generates significant residual stresses. In the next step the laminate was subjected to a heat treatment process in order to improve the mechanical properties by precipitation hardening. This treatment should not be carried out before welding because of negative influence on a ductility of the aluminum alloy. Material in this state was subjected to the tests of chemical composition, microstructure, and microhardness. A tensile test was carried out with accompanying strain analysis by the digital image correlation (DIC) method. Moreover, the residual stresses were determined which were measured by using two methods, the X-ray diffraction and the hole drilling. This approach made it possible to measure the residual stresses both in the plane parallel to the surface and in the cross section of the laminate.

Engineering of a synthetic antibody fragment for structural and functional studies of K+ channels.

Engineered antibody fragments (Fabs) have made major impacts on structural biology research, particularly to aid structural determination of membrane proteins. Nonetheless, Fabs generated by traditional monoclonal technology suffer from challenges of routine production and storage. Starting from the known IgG paratopes of an antibody that binds to the "turret loop" of the KcsA K+ channel, we engineered a synthetic Fab (sFab) based upon the highly stable Herceptin Fab scaffold, which can be recombinantly expressed in Escherichia coli and purified with single-step affinity chromatography. This synthetic Fab was used as a crystallization chaperone to obtain crystals of the KcsA channel that diffracted to a resolution comparable to that from the parent Fab. Furthermore, we show that the turret loop can be grafted into the unrelated voltage-gated Kv1.2-Kv2.1 channel and still strongly bind the engineered sFab, in support of the loop grafting strategy. Macroscopic electrophysiology recordings show that the sFab affects the activation and conductance of the chimeric voltage-gated channel. These results suggest that straightforward engineering of antibodies using recombinant formats can facilitate the rapid and scalable production of Fabs as structural biology tools and functional probes. The impact of this approach is expanded significantly based on the potential portability of the turret loop to a myriad of other K+ channels.

Engineered Ultra-High Affinity Synthetic Antibodies for SARS-CoV-2 Neutralization and Detection.

The Covid-19 pandemic is a centenarial global catastrophe. Similar events are likely to be recurring with more frequency in the future. The inability to control the virus' impact is caused by many factors, but the lack of a technology infrastructure to detect and impede the virus at an early stage are principal shortcomings. Using phage display mutagenesis, we have generated a cohort of high performance antibody fragments (Fabs) that can be used in a sensitive point of care (POC) assay and are potent inhibitors (IC50-0.5 nM) to viral entry into cells. The POC assay is based on a split-enzyme (ß-lactamase) complementation strategy that detects virus particles at low nM levels. We have shown that this assay is equally effective for detecting other viruses like Ebola and Zika. Importantly, its components can be freeze dried and stored, but becomes fully active when rehydrated.

Research on the Properties and Low Cycle Fatigue of Sc-Modified AA2519-T62 FSW Joint.

The aim of this research was to examine the mechanical and fatigue properties of friction stir welded Sc-modified 5 mm thick AA2519-T62 extrusion. The joint was obtained using the following parameters: 800 rpm tool rotation speed, 100 mm/min tool traverse speed, 17 kN axial, and MX Triflute as a tool. The investigation has involved microstructure observations, microhardness distribution analysis, tensile test with digital image correlation technique, observations of the fracture surface, measurements of residual stresses, low cycle fatigue testing, and fractography. It was stated that the obtained weld is defect-free and has joint efficiency of 83%. The failure in the tensile test occurred at the boundary of the thermo-mechanically affected zone and stir zone on the advancing side of the weld. The residual stress measurements have revealed that the highest values of longitudinal stress are localized at the distance of 10 mm from the joint line with their values of 124 MPa (the retreating side) and 159 MPa (the advancing side). The results of low cycle fatigue testing have allowed establishing of the values of the cyclic strength coefficient (k' = 504.37 MPa) and cyclic strain hardening exponent (n' = 0.0068) as well as the factors of the Manson-Coffin-Basquin equation: the fatigue strength coefficient σ'f = 462.4 MPa, the fatigue strength exponent b = -0.066, the fatigue ductility coefficient ε'f = 0.4212, and the fatigue ductility exponent c = -0.911.

Mechanical Properties Analysis of the AA2519-AA1050-Ti6Al4V Explosive Welded Laminate.

Explosively welded layered materials made of (a) an AA2519 aluminum alloy (AlCuMgMn + ZrSc), (b) titanium alloy Ti6Al4V and (c) an intermediate layer composed of a thin aluminum alloyed AA1050 layer are considered herein. This study presents test results connected to measurement science including microstructural observations of the material combined with the explosive method, and a basic analysis of the strength properties based on microhardness and tensile tests. Owing to the joint's special manufacturing conditions, the laminate was subjected to deformation measurements with the digital image correlation (DIC) method. The research was supplemented by the residual stress measurements with the sin2ψ X-ray method based on the diffraction-reflection analysis that was verified by the bore trepanation method.

An engineered ultra-high affinity Fab-Protein G pair enables a modular antibody platform with multifunctional capability.

Engineered recombinant antibody-based reagents are rapidly supplanting traditionally derived antibodies in many cell biological applications. A particularly powerful aspect of these engineered reagents is that other modules having myriad functions can be attached to them either chemically or through molecular fusions. However, these processes can be cumbersome and do not lend themselves to high throughput applications. Consequently, we have endeavored to develop a platform that can introduce multiple functionalities into a class of Fab-based affinity reagents in a "plug and play" fashion. This platform exploits the ultra-tight binding interaction between affinity matured variants of a Fab scaffold (FabS ) and a domain of an immunoglobulin binding protein, protein G (GA1). GA1 is easily genetically manipulatable facilitating the ability to link these modules together like beads on a string with adjustable spacing to produce multivalent and bi-specific entities. GA1 can also be fused to other proteins or be chemically modified to engage other types of functional components. To demonstrate the utility for the Fab-GA1 platform, we applied it to a detection proximity assay based on the ß-lactamase (BL) split enzyme system. We also show the bi-specific capabilities of the module by using it in context of a Bi-specific T-cell engager (BiTE), which is a therapeutic assemblage that induces cell killing by crosslinking T-cells to cancer cells. We show that GA1-Fab modules are easily engineered into potent cell-killing BiTE-like assemblages and have the advantage of interchanging Fabs directed against different cell surface cancer-related targets in a plug and play fashion.

GnRH antagonist treatment of malignant adrenocortical tumors.

Aberrantly expressed G protein-coupled receptors in tumors are considered as potential therapeutic targets. We analyzed the expressions of receptors of gonadotropin-releasing hormone (GNRHR), luteinizing hormone/chorionic gonadotropin (LHCGR) and follicle-stimulating hormone (FSHR) in human adrenocortical carcinomas and assessed their response to GnRH antagonist therapy. We further studied the effects of the GnRH antagonist cetrorelix acetate (CTX) on cultured adrenocortical tumor (ACT) cells (mouse Cα1 and Y-1, and human H295R), and in vivo in transgenic mice (SV40 T-antigen expression under inhibin α promoter) bearing Lhcgr and Gnrhr in ACT. Both models were treated with control (CT), CTX, human chorionic gonadotropin (hCG) or CTX+hCG, and their growth and transcriptional changes were analyzed. In situ hybridization and qPCR analysis of human adrenocortical carcinomas (n = 11-13) showed expression of GNRHR in 54/73%, LHCGR in 77/100% and FSHR in 0%, respectively. CTX treatment in vitro decreased cell viability and proliferation, and increased caspase 3/7 activity in all treated cells. In vivo, CTX and CTX+hCG (but not hCG alone) decreased ACT weights and serum LH and progesterone concentrations. CTX treatment downregulated the tumor markers Lhcgr and Gata4. Upregulated genes included Grb10, Rerg, Nfatc and Gnas, all recently found to be abundantly expressed in healthy adrenal vs ACT. Our data suggest that CTX treatment may improve the therapy of human adrenocortical carcinomas by direct action on GNRHR-positive cancer cells inducing apoptosis and/or reducing gonadotropin release, directing tumor cells towards a healthy adrenal gene expression profile.

Bovine ovarian follicular growth and development correlate with lysophosphatidic acid expression.

The basis of successful reproduction is proper ovarian follicular growth and development. In addition to prostaglandins and vascular endothelial growth factor, a number of novel factors are suggested as important regulators of follicular growth and development: PGES, TFG, CD36, RABGAP1, DBI and BTC. This study focuses on examining the expression of these factors in granulosa and thecal cells that originate from different ovarian follicle types and their link with the expression of lysophosphatidic acid (LPA), known local regulator of reproductive functions in the cow. Ovarian follicles were divided into healthy, transitional, and atretic categories. The mRNA expression levels for PGES, TFG, CD36, RABGAP1, DBI and BTC in granulosa and thecal cells in different follicle types were measured by real-time PCR. The correlations among expression of enzymes synthesizing LPA (autotaxin, phospholipase A2), receptors for LPA and examined factors were measured. Immunolocalization of PGES, TFG, CD36, RABGAP1, DBI and BTC was examined by immunohistochemistry. We investigated follicle-type dependent mRNA expression of factors potentially involved in ovarian follicular growth and development, both in granulosa and thecal cells of bovine ovarian follicles. Strong correlations among receptors for LPA, enzymes synthesizing LPA, and the examined factors in healthy and transitional follicles were observed, with its strongest interconnection with TFG, DBI and RABGAP1 in granulosa cells, and TFG in thecal cells; whereas no correlations in atretic follicles were detected. A greater number of correlations were found in thecal cells than in granulosa cells as well as in healthy follicles than in transitional follicles. These data indicate the role of LPA in the growth, development and physiology of the bovine ovarian follicle.

Luteinizing Hormone and GATA4 Action in the Adrenocortical Tumorigenesis of Gonadectomized Female Mice.

BACKGROUND/AIMS: Physiological role of luteinizing hormone (LH) and its receptor (LHCGR) in adrenal remains unknown. In inhibin-α/Simian Virus 40 T antigen (SV40Tag) (inhα/Tag) mice, gonadectomy-induced (OVX) elevated LH triggers the growth of transcription factor GATA4 (GATA4)-positive adrenocortical tumors in a hyperplasia-adenoma-adenocarcinoma sequence. METHODS: We investigated the role of LHCGR in tumor induction, by crossbreeding inhα/Tag with Lhcgr knockout (LuRKO) mice. By knocking out Lhcgr and Gata4 in Cα1 adrenocortical cells (Lhcgr-ko, Gata4-ko) we tested their role in tumor progression. RESULTS: Adrenal tumors of OVX inhα/Tag mice develop from the hyperplastic cells localized in the topmost layer of zona fasciculata. OVX inhα/Tag/LuRKO only developed SV40Tag positive hyperplastic cells that were GATA4 negative, cleaved caspase-3 positive and did not progress into adenoma. In contrast to Lhcgr-ko, Gata4-ko Cα1 cells presented decreased proliferation, increased apoptosis, decreased expression of Inha, SV40Tag and Lhcgr tumor markers, as well as up-regulated adrenal- and down-regulated sex steroid gene expression. Both Gata4-ko and Lhcgr-ko Cα1 cells had decreased expression of steroidogenic genes resulting in decreased basal progesterone production. CONCLUSION: Our data indicate that LH/LHCGR signaling is critical for the adrenal cell reprogramming by GATA4 induction prompting adenoma formation and gonadal-like phenotype of the adrenocortical tumors in inhα/Tag mice.

Expression of factors involved in apoptosis and cell survival is correlated with enzymes synthesizing lysophosphatidic acid and its receptors in granulosa cells originating from different types of bovine ovarian follicles.

BACKGROUND: Lysophosphatidic acid (LPA) regulates reproductive processes in the cow. Ovarian granulosa cells play a pivotal role in follicle growth and development. Nevertheless, the role of LPA in the local regulation of granulosa cell function in different follicle categories in the bovine ovary has not been investigated. METHODS: Ovarian follicles were divided into healthy, transitional and atretic categories. The expression levels of AX, PLA2, LPARs and factors involved in apoptosis and cell survival processes in granulosa cells in different types of follicles were measured by real-time PCR. The correlations between the expression levels of AX, PLA2, LPARs and the examined factors were measured. The immunolocalization of AX, PLA2 and LPARs in different ovarian follicles was examined by immunohistochemistry. Statistical analyses were conducted in GraphPad using a one-way ANOVA followed by the Kruskal-Wallis multiple comparison test or a correlation analysis followed by Pearson's test. RESULTS: The expression levels of AX, PLA2 and LPARs, with the major role of LPAR2 and PLA2, were found in the granulosa cells originating from different follicle types. The expression levels of the factors involved in cell apoptosis (TNFα and its receptors, FAS, FASL, CASP3, CASP8, ß-glycan, and DRAK2) were significantly higher in the granulosa cells of the atretic follicles compared to the healthy follicles. A number of correlations between LPARs, AX, PLA2 and factors associated with apoptosis were observed in the atretic but not in the healthy follicles. A greater expression of the factors involved in differentiation and proliferation in the granulosa cells (DICE1 and SOX2) was found in the healthy follicles in comparison with the atretic. A number of correlations between LPARs, AX, PLA2 and the factors associated with cell survival were observed in the healthy but not in the atretic follicles. CONCLUSIONS: Granulosa cells are the target of LPA action and the source of LPA synthesis in the bovine ovarian follicle. We suggest that the participation of LPA in apoptosis in the atretic follicles mainly occurs through the regulation of TNF-α-dependent and caspase-induced pathways. In the transitional follicles, LPA might influence the inhibins to shift the balance between the number of healthy and atretic follicles. In the healthy follicle type, LPA, acting via LPAR1, might regulate MCL1 and estradiol-stimulating ERß mRNA expression, leading to the stimulation of anti-apoptotic processes in the granulosa cells and their differentiation and proliferation.

An ultrahigh vacuum system for in situ studies of thin films and nanostructures by nuclear resonance scattering of synchrotron radiation.

A multifunctional ultrahigh vacuum (UHV) system has been set up at the nuclear resonance beamline ID18 of the European Synchrotron Radiation Facility (ESRF). Thin and ultrathin films, nanoislands and -wires, multilayers, and stoichiometric oxides can be prepared by molecular beam epitaxy and characterized by low-energy electron diffraction, Auger electron spectroscopy, and reflection high-energy electron diffraction. Upon characterization the sample is transferred under UHV conditions to the chamber for experiments with the synchrotron beam. Electronic and magnetic properties, vibrational dynamics, and diffusion phenomena can be investigated by several synchrotron radiation based techniques, such as nuclear forward scattering, nuclear inelastic and quasielastic scattering, synchrotron radiation based perturbed angular correlations, and nuclear and electronic reflectivity. In addition, two portable UHV chambers serve to transfer the sample to other beamlines profiting from the available experimental techniques at the ESRF.