The Effect of Ficin Immobilized on Carboxymethyl Chitosan on Biofilms of Oral Pathogens.

In the last decade, Ficin, a proteolytic enzyme extracted from the latex sap of the wild fig tree, has been widely investigated as a promising tool for the treatment of microbial biofilms, wound healing, and oral care. Here we report the antibiofilm properties of the enzyme immobilized on soluble carboxymethyl chitosan (CMCh) and CMCh itself. Ficin was immobilized on CMCh with molecular weights of either 200, 350 or 600 kDa. Among them, the carrier with a molecular weight of 200 kDa bound the maximum amount of enzyme, binding up to 49% of the total protein compared to 19-32% of the total protein bound to other CMChs. Treatment with pure CMCh led to the destruction of biofilms formed by Streptococcus salivarius, Streptococcus gordonii, Streptococcus mutans, and Candida albicans, while no apparent effect on Staphylococcus aureus was observed. A soluble Ficin was less efficient in the destruction of the biofilms formed by Streptococcus sobrinus and S. gordonii. By contrast, treatment with CMCh200-immobilized Ficin led to a significant reduction of the biofilms of the primary colonizers S. gordonii and S. mutans. In model biofilms obtained by the inoculation of swabs from teeth of healthy volunteers, the destruction of the biofilm by both soluble and immobilized Ficin was observed, although the degree of the destruction varied between artificial plaque samples. Nevertheless, combined treatment of oral Streptococci biofilm by enzyme and chlorhexidine for 3 h led to a significant decrease in the viability of biofilm-embedded cells, compared to solely chlorhexidine application. This suggests that the use of either soluble or immobilized Ficin would allow decreasing the amount and/or concentration of the antiseptics required for oral care or improving the efficiency of oral cavity sanitization.

Complexation of Bromelain, Ficin, and Papain with the Graft Copolymer of Carboxymethyl Cellulose Sodium Salt and N-Vinylimidazole Enhances Enzyme Proteolytic Activity.

This study investigates the features of interactions between cysteine proteases (bromelain, ficin, and papain) and a graft copolymer of carboxymethyl cellulose sodium salt with N-vinylimidazole. The objective is to understand the influence of this interactions on the proteolytic activity and stability of the enzymes. The enzymes were immobilized through complexation with the carrier. The interaction mechanism was examined using Fourier-transform infrared spectroscopy and flexible molecular docking simulations. The findings reveal that the enzymes interact with the functional groups of the carrier via amino acid residues, resulting in the formation of secondary structure elements and enzyme's active sites. These interactions induce modulation of active site of the enzymes, leading to an enhancement in their proteolytic activity. Furthermore, the immobilized enzymes demonstrate superior stability compared to their native counterparts. Notably, during a 21-day incubation period, no protein release from the conjugates was observed. These results suggest that the complexation of the enzymes with the graft copolymer has the potential to improve their performance as biocatalysts, with applications in various fields such as biomedicine, pharmaceutics, and biotechnology.

Harmonic radiation contribution and X-ray transmission at the Small Quantum Systems instrument of European XFEL.

Transmission measurements of the soft X-ray beamline to the Small Quantum Systems (SQS) scientific instrument at the SASE3 undulator of European XFEL are presented. Measurements are reported for a wide range of photon energies (650 eV to 2400 eV), using X-ray gas monitors as well as a bolometric radiometer. The results are in good agreement with simulations for the beam transport and show a transmission of up to 80% over the whole photon energy range. The contribution of second- and third-harmonic radiation of the soft X-ray undulator is determined at selected photon energies by performing transmission measurements using a gas absorber to provide variable attenuation of the incoming photon flux. A comparison of the results with semi-analytic calculations for the generation of free-electron laser pulses in the SASE3 undulator reveals an influence of apertures along the beam transport on the exact harmonic content to be accounted for at the experiment. The second-harmonic content is measured to be in the range of 0.1% to 0.3%, while the third-harmonic contributed a few percent to the SASE3 emission. For experiments at the SQS instrument, these numbers can be reduced through specific selections of the mirror reflection angles.

The beam transport system for the Small Quantum Systems instrument at the European XFEL: optical layout and first commissioning results.

The Small Quantum Systems instrument is one of the six operating instruments of the European XFEL, dedicated to the atomic, molecular and cluster physics communities. The instrument started its user operation at the end of 2018 after a commissioning phase. The design and characterization of the beam transport system are described here. The X-ray optical components of the beamline are detailed, and the beamline performances, transmission and focusing capabilities are reported. It is shown that the X-ray beam can be effectively focused as predicted by ray-tracing simulations. The impact of non-ideal X-ray source conditions on the focusing performances is discussed.

Carboxymethyl Cellulose-Based Polymers as Promising Matrices for Ficin Immobilization.

The present work is devoted to research on the interaction between carboxymethyl cellulose sodium salt and its derivatives (graft copolymer of carboxymethyl cellulose sodium salt and N,N-dimethyl aminoethyl methacrylate) with cysteine protease (ficin). The interaction was studied by FTIR and by flexible molecular docking, which have shown the conjugates' formation with both matrices. The proteolytic activity assay performed with azocasein demonstrated that the specific activities of all immobilized ficin samples are higher in comparison with those of the native enzyme. This is due to the modulation of the conformation of ficin globule and of the enzyme active site by weak physical interactions involving catalytically valuable amino acids. The results obtained can extend the practical use of ficin in biomedicine and biotechnology.

Novel Biocatalysts Based on Bromelain Immobilized on Functionalized Chitosans and Research on Their Structural Features.

Enzyme immobilization on various carriers represents an effective approach to improve their stability, reusability, and even change their catalytic properties. Here, we show the mechanism of interaction of cysteine protease bromelain with the water-soluble derivatives of chitosan-carboxymethylchitosan, N-(2-hydroxypropyl)-3-trimethylammonium chitosan, chitosan sulfate, and chitosan acetate-during immobilization and characterize the structural features and catalytic properties of obtained complexes. Chitosan sulfate and carboxymethylchitosan form the highest number of hydrogen bonds with bromelain in comparison with chitosan acetate and N-(2-hydroxypropyl)-3-trimethylammonium chitosan, leading to a higher yield of protein immobilization on chitosan sulfate and carboxymethylchitosan (up to 58 and 65%, respectively). In addition, all derivatives of chitosan studied in this work form hydrogen bonds with His158 located in the active site of bromelain (except N-(2-hydroxypropyl)-3-trimethylammonium chitosan), apparently explaining a significant decrease in the activity of biocatalysts. The N-(2-hydroxypropyl)-3-trimethylammonium chitosan displays only physical interactions with His158, thus possibly modulating the structure of the bromelain active site and leading to the hyperactivation of the enzyme, up to 208% of the total activity and 158% of the specific activity. The FTIR analysis revealed that interaction between N-(2-hydroxypropyl)-3-trimethylammonium chitosan and bromelain did not significantly change the enzyme structure. Perhaps this is due to the slowing down of aggregation and the autolysis processes during the complex formation of bromelain with a carrier, with a minimal modification of enzyme structure and its active site orientation.

Novel Immobilized Biocatalysts Based on Cysteine Proteases Bound to 2-(4-Acetamido-2-sulfanilamide) Chitosan and Research on Their Structural Features.

Briefly, 2-(4-Acetamido-2-sulfanilamide) chitosan, which is a chitosan water-soluble derivative, with molecular weights of 200, 350, and 600 kDa, was successfully synthesized. The immobilization of ficin, papain, and bromelain was carried out by complexation with these polymers. The interaction mechanism of 2-(4-acetamido-2-sulfanilamide) chitosan with bromelain, ficin, and papain was studied using FTIR spectroscopy. It was found that the hydroxy, thionyl, and amino groups of 2-(4-acetamido-2-sulfanilamide) chitosan were involved in the complexation process. Molecular docking research showed that all amino acid residues of the active site of papain formed hydrogen bonds with the immobilization matrix, while only two catalytically valuable amino acid residues took part in the H-bond formation for bromelain and ficin. The spectral and in silico data were in good agreement with the catalytic activity evaluation data. Immobilized papain was more active compared to the other immobilized proteases. Moreover, the total and specific proteolytic activity of papain immobilized on the carrier with a molecular weight of 350 kDa were higher compared to the native one due to the hyperactivation. The optimal ratio of protein content (mg × g -1 of carrier), total activity (U × mL-1 of solution), and specific activity (U × mg-1 of protein) was determined for the enzymes immobilized on 2-(4-acetamido-2-sulfanilamide) chitosan with a molecular weight of 350 kDa.

Chitosan Graft Copolymers with N-Vinylimidazole as Promising Matrices for Immobilization of Bromelain, Ficin, and Papain.

This work aims to synthesize graft copolymers of chitosan and N-vinylimidazole (VI) with different compositions to be used as matrices for the immobilization of cysteine proteases-bromelain, ficin, and papain. The copolymers are synthesized by free radical solution copolymerization with a potassium persulfate-sodium metabisulfite blend initiator. The copolymers have a relatively high frequency of grafting and yields. All the synthesized graft copolymers are water-soluble, and their solutions are characterized by DLS and laser Doppler microelectrophoresis. The copolymers are self-assembled in aqueous solutions, and they have a cationic nature and pH-sensitivity correlating to the VI content. The FTIR data demonstrate that synthesized graft copolymers conjugate cysteine proteases. The synthesized copolymer adsorbs more enzyme macromolecules compared to non-modified chitosan with the same molecular weight. The proteolytic activity of the immobilized enzymes is increased up to 100% compared to native ones. The immobilized ficin retains up to 97% of the initial activity after a one-day incubation, the immobilized bromelain retains 69% of activity after a 3-day incubation, and the immobilized papain retains 57% of the initial activity after a 7-day incubation. Therefore, the synthesized copolymers can be used as matrices for the immobilization of bromelain, ficin, and papain.

p66Shc signaling does not contribute to tubular damage induced by renal ischemia-reperfusion injury in rat.

Renal ischemia-reperfusion (IR) injury is one of the major causes of acute kidney injury and represents a significant risk factor for renal transplantation. The level of renal damage is influenced by the ischemic duration and is caused by excessive amounts of produced reactive oxygen species (ROS). Adaptor protein p66Shc is known to regulate cellular and organ's sensitivity to oxidative stress and to contribute significantly to mitochondrial production of hydrogen peroxide in stress conditions. Studies carried out in cultured renal cells suggest that p66Shc-mediated mitochondrial dysfunction and ROS production are responsible for renal ischemic injury. We used our genetically modified rats, which either lack p66Shc expression, or express p66Shc variant, which constitutively generates increased quantities of hydrogen peroxide, to evaluate potential contribution of p66Shc signaling to renal damage in ischemia reperfusion rat model. Analysis of outer medulla tubule damage revealed the lack of contribution of either p66Shc expression or its constitutive signaling to IR injury in rat model.

Interaction of the nitrogen-containing carbon backbone polymers with essential α-amino acids.

The aim of this work is to research the interactions of water-soluble nitrogen-containing copolymers with essential amino acids in aqueous media. For this, poly(N-vinylformamide-co-N-vinylimidazole) and poly(N-vinylcaprolactam-co-N-vinylimidazole) random copolymers were synthesized by free radical polymerization. The products obtained are characterized by GPC, DLS, and FTIR. The copolymers have a narrow molecular weight distribution and low dispersity. The interactions of the obtained copolymers with histidine, proline, arginine, leucine, phenylalanine, and methionine were researched by UV spectroscopy, FTIR, and TEM. It was found that conjugation of the copolymers with amino acids correlates with the copolymer composition and hydrodynamic radius Rh, and depends on the pH of the medium and amino acid structures. It is shown that chloride anion presence in the polymer-amino acid-water systems affects the mechanism of their interactions. The research shows that the synthesized copolymers can be used for the creation of effective eco-friendly amino acid extraction systems or matrices for enzyme immobilization.

Microwave Simulation Experiments on Regolith (Lunar Dust) Deposition on Stainless Steel.

In this article, results are presented of experiments on depositing charged particles, which imitate the levitating dust on the Moon, on stainless steel. Ensembles of particles are created above the surface of laboratory regolith whose composition and particle size distribution imitate the dust that covers the Moon's surface. Under the action of the gyrotron radiation on regolith, non-linear physical-chemical processes develop (breakdown, chain plasmachemical reactions, and particle scattering by the Coulomb mechanism), which lead to the appearance of a levitating cloud of particles. The simulation experiment is based on the similarity between the processes that develop in the laboratory experiments with regolith and the processes that occur on the Moon during its bombardment by micrometeorites. The effect of the levitating cloud on stainless steel plates is studied and it is shown that regolith particles in the shape of spheroids of different sizes are deposited on the surface of the plates. The dimensions of the deposited particles and the density of their placement depend on the quality of treatment of the plate surface. It is shown that the laboratory-produced dusty plasma can be used in simulation experiments to study the modification of surfaces of different materials for space technology.

Modulation of p66Shc impairs cerebrovascular myogenic tone in low renin but not low nitric oxide models of systemic hypertension.

Cerebral blood flow and perfusion are tightly maintained through autoregulation despite changes in transmural pressure. Oxidative stress impairs cerebral blood flow, precipitating cerebrovascular events. Phosphorylation of the adaptor protein p66Shc increases mitochondrial-derived oxidative stress. The effect of p66Shc gain or loss of function in nonhypertensive rats is unclear. We hypothesized that p66Shc gain of function would impair autoregulation of cerebral microcirculation under physiological and pathological conditions. Three previously established transgenic [salt-sensitive (SS) background] p66Shc rats were used, p66-Del/SS (express p66Shc with a nine-amino acid deletion), p66Shc-knockout (KO)/SS (frameshift premature termination codon), and p66Shc signaling and knock-in substitution of Ser36Ala (p66Shc-S36A)/SS (substitution of Ser36Ala). The p66Shc-Del were also bred on Sprague-Dawley (SD) backgrounds (p66-Del/SD), and a subset was exposed to a hypertensive stimulus [NG-nitro-l-arginine methyl ester (l-NAME)] for 4 wk. Active and passive diameters to increasing transmural pressure were measured and myogenic tone was calculated in all groups (SS and SD). Myogenic responses to increasing pressure were impaired in p66Shc-Del/SS rats relative to wild-type (WT)/SS and knock-in substitution of Ser36Ala (S36A; P < 0.05). p66-Del/SD rats did not demonstrate changes in active/passive diameters or myogenic tone relative to WT/SD but did demonstrate attenuated passive diameter responses to higher transmural pressure relative to p66-Del/SS. Four weeks of a hypertensive stimulus (l-NAME) did not alter active or passive diameter responses to increasing transmural pressure (P = 0.86-0.99), but increased myogenic responses relative to p66-Del/SD (P < 0.05). Collectively, we demonstrate the functional impact of p66Shc within the cerebral circulation and demonstrate that the genetic background of p66Shc rats largely drives changes in cerebrovascular function.NEW & NOTEWORTHY We demonstrate that the modulation of p66Shc signaling impairs cerebral artery myogenic tone in a low renin model of hypertension. This impairment is dependent upon the genetic background, as modulated p66Shc signaling in Sprague-Dawley rats does not impair cerebral artery myogenic tone.

Double p52Shc/p46Shc Rat Knockout Demonstrates Severe Gait Abnormalities Accompanied by Dilated Cardiomyopathy.

The ubiquitously expressed adaptor protein Shc exists in three isoforms p46Shc, p52Shc, and p66Shc, which execute distinctly different actions in cells. The role of p46Shc is insufficiently studied, and the purpose of this study was to further investigate its functional significance. We developed unique rat mutants lacking p52Shc and p46Shc isoforms (p52Shc/46Shc-KO) and carried out histological analysis of skeletal and cardiac muscle of parental and genetically modified rats with impaired gait. p52Shc/46Shc-KO rats demonstrate severe functional abnormalities associated with impaired gait. Our analysis of p52Shc/46Shc-KO rat axons and myelin sheets in cross-sections of the sciatic nerve revealed the presence of significant anomalies. Based on the lack of skeletal muscle fiber atrophy and the presence of sciatic nerve abnormalities, we suggest that the impaired gait in p52Shc/46Shc-KO rats might be due to the sensory feedback from active muscle to the brain locomotor centers. The lack of dystrophin in some heart muscle fibers reflects damage due to dilated cardiomyopathy. Since rats with only p52Shc knockout do not display the phenotype of p52Shc/p46Shc-KO, abnormal locomotion is likely to be caused by p46Shc deletion. Our data suggest a previously unknown role of 46Shc actions and signaling in regulation of gait.

p66Shc-mediated hydrogen peroxide production impairs nephrogenesis causing reduction of number of glomeruli.

AIMS: Adaptor protein p66Shc, encoded by Shc1 gene, contributes to the pathogenesis of oxidative stress-related diseases. p66Shc ability to promote oxidative stress-related diseases requires phosphorylation of serine 36 residue (Ser36) and depends on translocation of p66Shc to the mitochondria. We tested the hypothesis that abnormal p66Shc-mediated reactive oxygen species (ROS) production could be critically involved in nephrons development during nephrogenesis. MAIN METHODS: We have generated unique mutant rats (termed p66Shc-Del), which express endogenous p66Shc with a 9-amino acid deletion, and lack regulatory Ser36. H2O2 renal production was measured by enzymatic microelectrode biosensors. Nephron numbers in 3-5 weeks old p66Shc-Del rats were quantified using the acid maceration method. KEY FINDINGS: p66Shc-Del rats, as wild type salt sensitive rats, display increased mean arterial blood pressure following chronic exposure to a high salt diet. In contrast to wild type rats, p66Shc-Del rats display increased H2O2 renal production and are characterized by a reduction in renal function. The number of glomeruli is significantly reduced in adult p66Shc-Del rats. SIGNIFICANCE: Since low nephron number is an established risk factor for kidney disease and hypertension in humans and rodents, our data suggest that H2O2 renal production, caused by irregular signaling of p66Shc, could be critical in regulating nephrogenesis and that abnormal p66Shc signaling negatively impacts kidney development and renal function by increasing susceptibility to diabetic nephropathy and hypertension-induced nephropathy.

Transmission measurement at the Bernina branch of the Aramis Beamline of SwissFEL.

The transmission of the optical components of the Bernina branch of the Aramis beamline at SwissFEL has been measured with an X-ray gas monitor from DESY and compared with a PSI gas detector upstream of the optical components. The transmission efficiencies of the Mo, Si and SiC mirror coatings of the Aramis beamline and the various other in-beam components were evaluated and compared with theoretical calculations, showing an agreement of 6% or better in all cases. The experiment has also shown the efficacy of the high-harmonic rejection mirrors at the Bernina branch of the Aramis beamline at SwissFEL, and characterized the transmission efficiency of the on-line spectrometer in the Aramis beamline. The theoretical transmission of the mirror coatings match the experimental data to within 7%. The accuracy of these measurements was checked against a radiative bolometer from a Japanese collaboration and found to agree to a level of 4% or better. Further comparisons with a diamond detector from a US-based inter-institute collaboration demonstrated a good agreement for the attenuator settings of the beamline.

X-ray photon diagnostics at the European XFEL.

The European X-ray Free-Electron Laser (European XFEL) (Altarelli et al., 2006; Tschentscher et al., 2017), the world's largest and brightest X-ray free-electron laser (Saldin et al., 1999; Pellegrini et al., 2016), went into operation in 2017. This article describes the as-built realization of photon diagnostics for this facility, the diagnostics commissioning and their application for commissioning of the facility, and results from the first year of operation, focusing on the SASE1 beamline, which was the first to be commissioned. The commissioning consisted of pre-beam checkout, first light from the bending magnets, X-rays from single undulator segments, SASE tuning with many undulator segments, first lasing, optics alignment for FEL beam transport through the tunnel up to the experiment hutches, and finally beam delivery to first users. The beam properties assessed by photon diagnostics throughout these phases included per-pulse intensity, beam position, shape, lateral dimensions and spectral properties. During this time period, the machine provided users with up to 14 keV photon energy, 1.5 mJ pulse energy, 300 FEL pulses per train and 4.5 MHz intra-bunch train repetition rate at a 10 Hz train repetition rate. Finally, an outlook is given into the diagnostic prospects for the future.

Operation of X-ray gas monitors at the European XFEL.

X-ray gas monitors (XGMs) are operated at the European XFEL for non-invasive single-shot pulse energy measurements and average beam position monitoring. They are used for tuning and maintaining the self-amplified spontaneous emission (SASE) operation and for sorting single-shot experimental data according to the pulse-resolved energy monitor data. The XGMs were developed at DESY based on the specific requirements for the European XFEL. In total, six XGM units are continuously in operation. Here, the main principle and experimental setup of an XGM are summarized, and the locations of the six XGMs at the facility are shown. Pulse energy measurements at 0.134 nm wavelength are presented, exceeding 1 mJ obtained with an absolute measurement uncertainty of 7-10%; correlations between different XGMs are shown, from which a SASE1 beamline transmission of 97% is deduced. Additionally, simultaneous position measurements close to the undulator and at the end of the tunnel are shown, along with the correlation of beam position data simultaneously acquired by an XGM and an imager.

An X-ray gas monitor for free-electron lasers.

A novel X-ray gas monitor (XGM) has been developed which allows the measurement of absolute photon pulse energy and photon beam position at all existing and upcoming free-electron lasers (FELs) over a broad spectral range covering vacuum ultraviolet (VUV), extreme ultraviolet (EUV) and soft and hard X-rays. The XGM covers a wide dynamic range from spontaneous undulator radiation to FEL radiation and provides a temporal resolution of better than 200 ns. The XGM consists of two X-ray gas-monitor detectors (XGMDs) and two huge-aperture open electron multipliers (HAMPs). The HAMP enhances the detection efficiency of the XGM for low-intensity radiation down to 105 photons per pulse and for FEL radiation in the hard X-ray spectral range, while the XGMD operates in higher-intensity regimes. The relative standard uncertainty for measurements of the absolute photon pulse energy is well below 10%, and down to 1% for measurements of relative pulse-to-pulse intensity on pulses with more than 1010 photons per pulse. The accuracy of beam-position monitoring in the vertical and horizontal directions is of the order of 10 µm.

The p52 isoform of SHC1 is a key driver of breast cancer initiation.

BACKGROUND: SHC1 proteins (also called SHCA) exist in three functionally distinct isoforms (p46SHC, p52SHC, and p66SHC) that serve as intracellular adaptors for several key signaling pathways in breast cancer. Despite the broad evidence implicating SHC1 gene products as a central mediator of breast cancer, testing the isoform-specific roles of SHC1 proteins have been inaccessible due to the lack of isoform-specific inhibitors or gene knockout models. METHODS: Here, we addressed this issue by generating the first isoform-specific gene knockout models for p52SHC and p66SHC, using germline gene editing in the salt-sensitive rat strain. Compared with the wild-type (WT) rats, we found that genetic ablation of the p52SHC isoform significantly attenuated mammary tumor formation, whereas the p66SHC knockout had no effect. Rats were dosed with 7,12-dimethylbenz(a)anthracene (DMBA) by oral gavage to induce mammary tumors, and progression of tumor development was followed for 15 weeks. At 15 weeks, tumors were excised and analyzed by RNA-seq to determine differences between tumors lacking p66SHC or p52SHC. RESULTS: Compared with the wild-type (WT) rats, we found that genetic ablation of the p52SHC isoform significantly attenuated mammary tumor formation, whereas the p66SHC knockout had no effect. These data, combined with p52SHC being the predominant isoform that is upregulated in human and rat tumors, provide the first evidence that p52SHC is the oncogenic isoform of Shc1 gene products in breast cancer. Compared with WT tumors, 893 differentially expressed (DE; FDR < 0.05) genes were detected in p52SHC KO tumors compared with only 18 DE genes in the p66SHC KO tumors, further highlighting that p52SHC is the relevant SHC1 isoform in breast cancer. Finally, gene network analysis revealed that p52SHC KO disrupted multiple key pathways that have been previously implicated in breast cancer initiation and progression, including ESR1 and mTORC2/RICTOR. CONCLUSION: Collectively, these data demonstrate the p52SHC isoform is the key driver of DMBA-induced breast cancer while the expression of p66SHC and p46SHC are not enough to compensate.

SwissFEL Aramis beamline photon diagnostics. Erratum.

The list of authors in the paper by Juranic et al. (2018) [J. Synchrotron Rad. 25, 1238-1248] is corrected.