Reactive Halogen Species: Role in Living Systems and Current Research Approaches.

Reactive halogen species (RHS) are highly reactive compounds that are normally required for regulation of immune response, inflammatory reactions, enzyme function, etc. At the same time, hyperproduction of highly reactive compounds leads to the development of various socially significant diseases - asthma, pulmonary hypertension, oncological and neurodegenerative diseases, retinopathy, and many others. The main sources of (pseudo)hypohalous acids are enzymes from the family of heme peroxidases - myeloperoxidase, lactoperoxidase, eosinophil peroxidase, and thyroid peroxidase. Main targets of these compounds are proteins and peptides, primarily methionine and cysteine residues. Due to the short lifetime, detection of RHS can be difficult. The most common approach is detection of myeloperoxidase, which is thought to reflect the amount of RHS produced, but these methods are indirect, and the results are often contradictory. The most promising approaches seem to be those that provide direct registration of highly reactive compounds themselves or products of their interaction with components of living cells, such as fluorescent dyes. However, even such methods have a number of limitations and can often be applied mainly for in vitro studies with cell culture. Detection of reactive halogen species in living organisms in real time is a particularly acute issue. The present review is devoted to RHS, their characteristics, chemical properties, peculiarities of interaction with components of living cells, and methods of their detection in living systems. Special attention is paid to the genetically encoded tools, which have been introduced recently and allow avoiding a number of difficulties when working with living systems.

The European Nucleotide Archive in 2021.

The European Nucleotide Archive (ENA, https://www.ebi.ac.uk/ena), maintained at the European Molecular Biology Laboratory's European Bioinformatics Institute (EMBL-EBI) provides freely accessible services, both for deposition of, and access to, open nucleotide sequencing data. Open scientific data are of paramount importance to the scientific community and contribute daily to the acceleration of scientific advance. Here, we outline the major updates to ENA's services and infrastructure that have been delivered over the past year.

Mechanical Tensions Regulate Gene Expression in the Xenopus laevis Axial Tissues.

During gastrulation and neurulation, the chordamesoderm and overlying neuroectoderm of vertebrate embryos converge under the control of a specific genetic programme to the dorsal midline, simultaneously extending along it. However, whether mechanical tensions resulting from these morphogenetic movements play a role in long-range feedback signaling that in turn regulates gene expression in the chordamesoderm and neuroectoderm is unclear. In the present work, by using a model of artificially stretched explants of Xenopus midgastrula embryos and full-transcriptome sequencing, we identified genes with altered expression in response to external mechanical stretching. Importantly, mechanically activated genes appeared to be expressed during normal development in the trunk, i.e., in the stretched region only. By contrast, genes inhibited by mechanical stretching were normally expressed in the anterior neuroectoderm, where mechanical stress is low. These results indicate that mechanical tensions may play the role of a long-range signaling factor that regulates patterning of the embryo, serving as a link coupling morphogenesis and cell differentiation.

Meta-analysis of the effects of anti-migraine therapeutics in the rat trigeminovascular nociception model as a tool for design optimization.

The main reasons for the low reliability of results from preclinical studies are the lack of prior sample size calculations and poor experimental design. Here, we demonstrate how the tools of meta-analysis can be implemented to tackle these issues. We conducted a systematic search to identify controlled studies testing established migraine treatments in the electrophysiological model of trigeminovascular nociception (EMTVN). Drug effects on the two outcomes, dural stimulation-evoked responses and ongoing neuronal activity were analysed separately using a three-level model with robust variance estimation. According to the meta-analysis, which included 21 experiments in rats reported in 13 studies, these drugs significantly reduced trigeminovascular nociceptive traffic, affecting both outcomes. Based on the estimated effect sizes and outcome variance, we provide guidance on sample sizes allowing to detect such effects with sufficient power in future experiments. Considering the revealed methodological features that potentially influence the results and the main source of statistical bias of the included studies, we discuss the translational potential of the EMTVN and the steps needed to improve it. We believe that the presented approach can be used for design optimization in research with other animal models and as such deserves further validation.

The European Nucleotide Archive in 2020.

The European Nucleotide Archive (ENA; https://www.ebi.ac.uk/ena), provided by the European Molecular Biology Laboratory's European Bioinformatics Institute (EMBL-EBI), has for almost forty years continued in its mission to freely archive and present the world's public sequencing data for the benefit of the entire scientific community and for the acceleration of the global research effort. Here we highlight the major developments to ENA services and content in 2020, focussing in particular on the recently released updated ENA browser, modernisation of our release process and our data coordination collaborations with specific research communities.

The COVID-19 Data Portal: accelerating SARS-CoV-2 and COVID-19 research through rapid open access data sharing.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic will be remembered as one of the defining events of the 21st century. The rapid global outbreak has had significant impacts on human society and is already responsible for millions of deaths. Understanding and tackling the impact of the virus has required a worldwide mobilisation and coordination of scientific research. The COVID-19 Data Portal (https://www.covid19dataportal.org/) was first released as part of the European COVID-19 Data Platform, on April 20th 2020 to facilitate rapid and open data sharing and analysis, to accelerate global SARS-CoV-2 and COVID-19 research. The COVID-19 Data Portal has fortnightly feature releases to continue to add new data types, search options, visualisations and improvements based on user feedback and research. The open datasets and intuitive suite of search, identification and download services, represent a truly FAIR (Findable, Accessible, Interoperable and Reusable) resource that enables researchers to easily identify and quickly obtain the key datasets needed for their COVID-19 research.

Ex vivo observation of granulocyte activity during thrombus formation.

BACKGROUND: The process of thrombus formation is thought to involve interactions between platelets and leukocytes. Leukocyte incorporation into growing thrombi has been well established in vivo, and a number of properties of platelet-leukocyte interactions critical for thrombus formation have been characterized in vitro in thromboinflammatory settings and have clinical relevance. Leukocyte activity can be impaired in distinct hereditary and acquired disorders of immunological nature, among which is Wiskott-Aldrich Syndrome (WAS). However, a more quantitative characterization of leukocyte behavior in thromboinflammatory conditions has been hampered by lack of approaches for its study ex vivo. Here, we aimed to develop an ex vivo model of thromboinflammation, and compared granulocyte behavior of WAS patients and healthy donors. RESULTS: Thrombus formation in anticoagulated whole blood from healthy volunteers and patients was visualized by fluorescent microscopy in parallel-plate flow chambers with fibrillar collagen type I coverslips. Moving granulocytes were observed in hirudinated or sodium citrate-recalcified blood under low wall shear rate conditions (100 s-1). These cells crawled around thrombi in a step-wise manner with an average velocity of 90-120 nm/s. Pre-incubation of blood with granulocyte priming agents lead to a significant decrease in mean-velocity of the cells and increase in the number of adherent cells. The leukocytes from patients with WAS demonstrated a 1.5-fold lower mean velocity, in line with their impaired actin polymerization. It is noteworthy that in an experimental setting where patients' platelets were replaced with healthy donor's platelets the granulocytes' crawling velocity did not change, thus proving that WASP (WAS protein) deficiency causes disruption of granulocytes' behavior. Thereby, the observed features of granulocytes crawling are consistent with the neutrophil chemotaxis phenomenon. As most of the crawling granulocytes carried procoagulant platelets teared from thrombi, we propose that the role of granulocytes in thrombus formation is that of platelet scavengers. CONCLUSIONS: We have developed an ex vivo experimental model applicable for observation of granulocyte activity in thrombus formation. Using the proposed setting, we observed a reduction of motility of granulocytes of patients with WAS. We suggest that our ex vivo approach should be useful both for basic and for clinical research.

Ceruloplasmin Reduces the Lactoferrin/Oleic Acid Antitumor Complex-Mediated Release of Heme-Containing Proteins from Blood Cells.

Our previous study showed that not only bovine lactoferrin (LF), the protein of milk and neutrophils, but also the human species forms complexes with oleic acid (OA) that inhibit tumor growth. Repeated injections of human LF in complex with OA (LF/8OA) to hepatoma-carrying mice decelerated tumor growth and increased animals' longevity. However, whether the effect of the LF/8OA complex is directed exclusively against malignant cells was not studied. Hence, its effect on normal blood cells was assayed, along with its possible modulation of ceruloplasmin (CP), the preferred partner of LF among plasma proteins. The complex LF/8OA (6 µM) caused hemolysis, unlike LF alone or BSA/8OA (250 µM). The activation of neutrophils with exocytosis of myeloperoxidase (MPO), a potent oxidant, was induced by 1 µM LF/8OA, whereas BSA/8OA had a similar effect at a concentration increased by an order. The egress of heme-containing proteins, i.e., MPO and hemoglobin, from blood cells affected by LF/8OA was followed by a pronounced oxidative/halogenating stress. CP, which is the natural inhibitor of MPO, added at a concentration of 2 mol per 1 mol of LF/8OA abrogated its cytotoxic effect. It seems likely that CP can be used effectively in regulating the LF/8OA complex's antitumor activity.

Incorporation of Pectin into Vaterite Microparticles Prevented Effects of Adsorbed Mucin on Neutrophil Activation.

The application of vaterite microparticles for mucosal delivery depends on their interaction with mucin and immune cells. As we have shown previously, the binding of mucin onto particles enhances the generation of reactive oxygen species by neutrophils. The attenuation of the pro-oxidant effect of the bound mucin through the modification of vaterite could improve its biocompatibility. Hybrid microparticles composed of vaterite and pectin (CCP) were prepared using co-precipitation. In comparison with vaterite (CC), they had a smaller diameter and pores, a greater surface area, and a negative zeta-potential. We aimed to study the cytotoxicity and mucin-dependent neutrophil-activating effect of CCP microparticles. The incorporated pectin did not influence the neutrophil damage according to a lactate dehydrogenase test. The difference in the CC- and CCP-elicited luminol or lucigenin chemiluminescence of neutrophils was insignificant, with no direct pro- or antioxidant effects from the incorporated pectin. Unlike soluble pectin, the CCP particles were ineffective at scavenging radicals in an ABAP-luminol test. The fluorescence of SYTOX Green demonstrated a CCP-stimulated formation of neutrophil extracellular traps (NETs). The pre-treatment of CC and CCP with mucin resulted in a 2.5-times-higher CL response of neutrophils to the CC-mucin than to the CCP-mucin. Thus, the incorporation of pectin into vaterite microspheres enabled an antioxidant effect to be reached when the neutrophils were activated by mucin-treated microparticles, presumably via exposed ligands.

Caught red handed: modeling and confirmation of the myeloperoxidase ceruloplasmin alpha-thrombin complex.

The work is devoted to the study of the structural characteristics of the myeloperoxidase-ceruloplasmin-thrombin complex using small-angle neutron scattering methods in combination with computer modeling, as well as surface plasmon resonance and solid-phase enzyme assay. We have previously shown that the functioning of active myeloperoxidase during inflammation, despite the presence in the blood of an excess of ceruloplasmin which inhibits its activity, is possible due to the partial proteolysis of ceruloplasmin by thrombin. In this study, the myeloperoxidase-ceruloplasmin-thrombin heterohexamer was obtained in vitro. The building of a heterohexamer full-atomic model in silico, considering the glycosylation of the constituent proteins, confirmed the absence of steric barriers for the formation of protein-protein contacts. It was shown that the partial proteolysis of ceruloplasmin does not affect its ability to bind to myeloperoxidase, and a structural model of the heterohexamer was obtained using the small-angle neutron scattering method.

The implementation of transcranial Doppler ultrasonography for preclinical study of migraine.

Transcranial Doppler ultrasonography is used to study intracranial blood flow changes associated with migraine in humans, but whether this method is helpful in preclinical settings is yet unknown. To identify changes in rat intracranial blood flow specific to trigeminovascular activation-a key process in migraine pathophysiology-we measured Doppler indices in the middle cerebral artery and basilar artery before, during, and after dural or somatosensory electrical stimulation. Hemodynamic changes specific to dural stimulation were tested further in separate experiments. After baseline recordings, the animals received cumulative infusions of valproate (100 mg/kg, trice), sumatriptan (0.3, 1, and 3 mg/kg), or saline, and dural stimulation with measurement of Doppler indices was repeated every 10 min for 1 h. Several parameters of blood flow in the rat middle cerebral artery underwent alterations specific to trigeminovascular activation. These changes, however, were insensitive to valproate and sumatriptan and diminished over time. These findings question the reliability of blood flow velocity variations in large intracranial vessels as biological markers of migraine-related processes and do not support the idea of using transcranial Doppler ultrasonography for preclinical screening of antimigraine treatments, at least in the model of acute trigeminovascular activation in rats.

The European Nucleotide Archive in 2019.

The European Nucleotide Archive (ENA, https://www.ebi.ac.uk/ena) at the European Molecular Biology Laboratory's European Bioinformatics Institute provides open and freely available data deposition and access services across the spectrum of nucleotide sequence data types. Making the world's public sequencing datasets available to the scientific community, the ENA represents a globally comprehensive nucleotide sequence resource. Here, we outline ENA services and content in 2019 and provide an insight into selected key areas of development in this period.

Insulin Receptor-Related Receptor Regulates the Rate of Early Development in Xenopus laevis.

The orphan insulin receptor-related receptor (IRR) encoded by insrr gene is the third member of the insulin receptor family, also including the insulin receptor (IR) and the insulin-like growth factor receptor (IGF-1R). IRR is the extracellular alkaline medium sensor. In mice, insrr is expressed only in small populations of cells in specific tissues, which contain extracorporeal liquids of extreme pH. In particular, IRR regulates the metabolic bicarbonate excess in the kidney. In contrast, the role of IRR during Xenopus laevis embryogenesis is unknown, although insrr is highly expressed in frog embryos. Here, we examined the insrr function during the Xenopus laevis early development by the morpholino-induced knockdown. We demonstrated that insrr downregulation leads to development retardation, which can be restored by the incubation of embryos in an alkaline medium. Using bulk RNA-seq of embryos at the middle neurula stage, we showed that insrr downregulation elicited a general shift of expression towards genes specifically expressed before and at the onset of gastrulation. At the same time, alkali treatment partially restored the expression of the neurula-specific genes. Thus, our results demonstrate the critical role of insrr in the regulation of the early development rate in Xenopus laevis.

The Mechanisms of L-Arginine Metabolism Disorder in Endothelial Cells.

L-arginine is a key metabolite for nitric oxide production by endothelial cells, as well as signaling molecule of the mTOR signaling pathway. mTOR supports endothelial cells homeostasis and regulates activity of L-arginine-metabolizing enzymes, endothelial nitric oxide synthase, and arginase II. Disruption of the L-arginine metabolism in endothelial cells leads to the development of endothelial dysfunction. Conflicting results of the use of L-arginine supplement to improve endothelial function reveals a controversial role of the amino acid in the endothelial cell biology. The review is aimed at analysis of the current data on the role of L-arginine metabolism in the development of endothelial dysfunction.

Advantages of imaging photoplethysmography for migraine modeling: new optical markers of trigemino-vascular activation in rats.

BACKGROUND: Existent animal models of migraine are not without drawbacks and limitations. The aim of our study was to evaluate imaging photoplethysmography (PPG) as a method of assessing intracranial blood flow in rats and its changes in response to electrical stimulation of dural trigeminal afferents. METHODS: Experiments were carried out with 32 anesthetized adult male Wistar rats. Trigeminovascular system (TVS) was activated by means of electrical stimulation of dural afferents through a closed cranial window (CCW). Parameters of meningeal blood flow were monitored using a PPG imaging system under green illumination with synchronous recording of an electrocardiogram (ECG) and systemic arterial blood pressure (ABP). Two indicators related to blood-flow parameters were assessed: intrinsic optical signals (OIS) and the amplitude of pulsatile component (APC) of the PPG waveform. Moreover, we carried out pharmacological validation of these indicators by determining their sensitivity to anti-migraine drugs: valproic acid and sumatriptan. For statistical analysis the non-parametric tests with post-hoc Bonferroni correction was used. RESULTS: Significant increase of both APC and OIS was observed due to CCW electrical stimulation. Compared to saline (n = 11), intravenous administration of both the sumatriptan (n = 11) and valproate (n = 10) by using a cumulative infusion regimen (three steps performed 30 min apart) lead to significant inhibitory effect on the APC response to the stimulation. In contrast, intravenous infusion of any substance or saline did not affect the OIS response to the stimulation. It was found that infusion of either sumatriptan or valproate did not affect the response of ABP or heart rate to the stimulation. CONCLUSIONS: Imaging PPG can be used in an animal migraine model as a method for contactless assessment of intracranial blood flow. We have identified two new markers of TVS activation, one of which (APC) was pharmacologically confirmed to be associated with migraine. Monitoring of changes in APC caused by CCW electrical stimulation (controlling efficiency of stimulation by OIS) can be considered as a new way to assess the peripheral mechanism of action of anti-migraine interventions.

Epitope specificity of two anti-morphine monoclonal antibodies: In vitro and in silico studies.

Monoclonal antibodies (mAbs) against morphine are important in the development of immunotherapeutic and diagnostic methods for the treatment and prevention of drug addiction. By the surface plasmon resonance (SPR) and enzyme immunoassay techniques, we characterized two previously obtained mAbs 3K11 and 6G1 and showed their ability to recognize free morphine and morphine-containing antigens in different ways because of the epitope specificity thereof. Using the defined amino acid sequences, we obtained three-dimensional models of the variable regions of Fab fragments of these antibodies and compared them with the known sequence and spatial structure of the anti-morphine antibody 9B1. Docking simulations are performed to obtain models of the antibodies complexes with morphine. Differences in the models of 3K11 and 6G1 complexes with morphine correlate with their experimentally detected epitope specificity. The results, in particular, can be used for the structure-based design of the corresponding humanized antibodies. According to our modeling and docking results, the very different modes of morphine binding to mAbs 3K11 and 6G1 are qualitatively similar to those previously reported for cocaine and two anti-cocaine antibodies. Thus, the obtained structural information brings more insight into the hapten recognition diversity.

The effect of myeloperoxidase isoforms on biophysical properties of red blood cells.

Myeloperoxidase (MPO), an oxidant-producing enzyme, stored in azurophilic granules of neutrophils has been recently shown to influence red blood cell (RBC) deformability leading to abnormalities in blood microcirculation. Native MPO is a homodimer, consisting of two identical protomers (monomeric MPO) connected by a single disulfide bond but in inflammatory foci as a result of disulfide cleavage monomeric MPO (hemi-MPO) can also be produced. This study investigated if two MPO isoforms have distinct effects on biophysical properties of RBCs. We have found that hemi-MPO, as well as the dimeric form, bind to the glycophorins A/B and band 3 protein on RBC's plasma membrane, that lead to reduced cell resistance to osmotic and acidic hemolysis, reduction in cell elasticity, significant changes in cell volume, morphology, and the conductance of RBC plasma membrane ion channels. Furthermore, we have shown for the first time that both dimeric and hemi-MPO lead to phosphatidylserine (PS) exposure on the outer leaflet of RBC membrane. However, the effects of hemi-MPO on the structural and functional properties of RBCs were lower compared to those of dimeric MPO. These findings suggest that the ability of MPO protein to influence RBC's biophysical properties depends on its conformation (dimeric or monomeric isoform). It is intriguing to speculate that hemi-MPO appearance in blood during inflammation can serve as a regulatory mechanism addressed to reduce abnormalities on RBC response, induced by dimeric MPO.

Contribution of the Multiplicity Fluctuation in the Temperature Dependence of Phonon Spectra of Rare-Earth Cobaltites.

We have studied, both experimentally and theoretically, the unusual temperature dependence of the phonon spectra in NdCoO3, SmCoO3 and GdCoO3, where the Co3+ ion is in the low-spin (LS) ground state, and at the finite temperature, the high-spin (HS) term has a nonzero concentration nHS due to multiplicity fluctuations. We measured the absorption spectra in polycrystalline and nanostructured samples in the temperature range 3-550 K and found a quite strong breathing mode softening that cannot be explained by standard lattice anharmonicity. We showed that the anharmonicity in the electron-phonon interaction is responsible for this red shift proportional to the nHS concentration.

Effect of alpha-lactalbumin and lactoferrin oleic acid complexes on chromatin structural organization.

This work focuses on the study of multimeric alpha-lactalbumin oleic acid and lactoferrin oleic acid complexes. The purpose of the research is to study possible mechanisms involved in their pro-apoptotic activities, as seen in some tumor cell cultures. Complexes featuring oleic acid (OA) with human alpha-lactalbumin (hAl) or with bovine alpha-lactalbumin (bAl), and human lactoferrin (hLf) were investigated using small-angle neutron scattering (SANS). It was shown that while alpha-lactalbumin protein complexes were formed on the surface of polydisperse OA micelles, the lactoferrin complexes comprised a monodisperse system of nanoscale particles. Both hAl and hLf complexes appeared to interact with the chromatin of isolated nuclei affecting chromatin structural organization. The possible roles of these processes in the specific anti-tumor activity of these complexes are discussed.

Role of arginine deiminase in thymic atrophy during experimental Streptococcus pyogenes infection.

Expression of gene of arginine deiminase (AD) allows adaptation of Streptococcus pyogenes to adverse environmental conditions. AD activity can lead to L-arginine deficiency in the host cells' microenvironment. Bioavailability of L-arginine is an important factor regulating the functions of the immune cells in mammals. By introducing a mutation into S pyogenes M46-16, we obtained a strain with inactivated arcA/sagp gene (M49-16 delArcA), deficient in AD. This allowed elucidating the function of AD in pathogenesis of streptococcal infection. The virulence of the parental and mutant strains was examined in a murine model of subcutaneous streptococcal infection. L-arginine concentration in the plasma of mice infected with S pyogenes M49-16 delArcA remained unchanged in course of the entire experiment. At the same time mice infected with S pyogenes M49-16 demonstrated gradual diminution of L-arginine concentration in the blood plasma, which might be due to the activity of streptococcal AD. Mice infected with S pyogenes M49-16 delArcA demonstrated less intensive bacterial growth in the primary foci and less pronounced bacterial dissemination as compared with animals infected with the parental strain S pyogenes M46-16. Similarly, thymus involution, alterations in apoptosis, thymocyte subsets and Treg cells differentiation were less pronounced in mice infected with S pyogenes M49-16 delArcA than in those infected with the parental strain. The results obtained showed that S pyogenes M49-16 delArcA, unable to produce AD, had reduced virulence in comparison with the parental S pyogenes M49-16 strain. AD is an important factor for the realization of the pathogenic potential of streptococci.