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Infrastruktura transportu kolejowego zapewnia codzienny przewóz dużej liczby osób i ładunków. Znaczenie kolei pod względem zapewnienia sprawności obsługi na danym obszarze czyni z niej infrastrukturę krytyczną. Obserwuje się istotny rozwój wykorzystania technologii informatycznych na kolei, podobnie jak we wszystkich innych branżach. Z tego względu zarządzanie koleją, jako systemem fizycznym, należy zastąpić zarządzaniem koleją, jako systemem cyberfizycznym. Infrastruktura kolejowa jest narażona na znaczące ataki zarówno w przestrzeni fizycznej, jak i w cyberprzestrzeni. Artykuł jest poświęcony zarządzaniu sieciami komunikacyjnymi, służącymi do transmisji danych i partycjami, rozumianymi jako logicznie wydzielone zasoby informatyczne (służące do przetwarzania danych), wykorzystywanymi na potrzeby trans-portu kolejowego. Systemowo, wielopoziomowe, bezpieczne i niezależne (ang. System Multiple Independent Levels of Security – MILS) przetwarzanie danych spełnia wysokie wymagania dotyczące bezpieczeństwa systemu. MILS jest niezawodną architekturą bezpieczeństwa opartą na koncepcji separacji i kontrolowanego przepływu danych. W artykule opisano możliwości wykorzystania platformy MILS w systemie teleinformatycznym i systemie sterowania ruchem kolejowym. Słowa kluczowe: Systemy cyberfizyczne, infrastruktura krytyczna, systemowo wielopoziomowe, bezpieczne i niezależne przetwarzanie danych (MILS)

The railway transport infrastructure ensures the transfer of large numbers of people and cargo every day. The importance of the railway in terms of ensuring the serviceability of the territory makes it a critical infrastructure. We can observe the development of the use of IT technologies on railway, as in all areas of the human system. The management of the railway as a physical system needs to be superseded by management of the railway as a cyberphysical system. The railway infra-structure has a large area of attack in both, physical space and cyber space. Multiple Independent Levels of Security (MILS) can meet the high system security requirements. The MILS is a high-assurance security architecture based on the concepts of separation and controlled information flow. The article discusses the possibilities of using the MILS platform in the data communication system and the control system of the railway. Keywords: Cyber Physical Systems, Critical Infrastructures, Multiple Independent Levels of Security

The article shows the results of research directed to detection of technical facilities accidents and failures sources at their operation. The research aim is to create the effective tools for management of risks so the coexistence of technical facilities with their vicinity would be ensured throughout their life cycles. The problems solution way is based on the simultaneously preferred concept, in which the safety is preferred over the reliability. Respecting the present knowledge on technical facilities´ safety and the lessons learned from the past technical facilities accidents and failures, the causes of which were connected with their operation, two tools are developed: Decision Support System and Risk Management Plan that were reviewed by experts and tested in practice.

MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer's DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicer•–miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways.

Mammalian Dicer is the gatekeeper into the essential gene-regulating miRNA pathway. What is committing mammalian Dicer to the miRNA pathway remains unknown. We report that Dicer’s highly conserved DExD/H helicase domain is the key structural element supporting accurate miRNA biogenesis. While ATPase activity of the domain is non-essential, its loss is lethal in mice. It is required during canonical miRNA biogenesis for efficient recognition, high-fidelity cleavage, and strand selection. Structure of Dicer-miRNA precursor complexes showed that the DExD/H domain acquired helicase-unrelated function defining Dicer conformations, which affect substrate loading and facilitate pre-selection of miRNA precursors. Dicer lacking the DExD/H domain favors conformations enabling reduced substrate selectivity and supporting RNA interference, a different small RNA pathway. Therefore, Dicer’s DExD/H domain ensures indispensable high-fidelity precursor processing of mammalian miRNAs, which constitutes a structural “mold” for adaptive miRNA evolution.

Simple Summary The formation of metastasis is not only intricately orchestrated by cancer cells but is also affected by the surrounding extracellular matrix (ECM). The barrier function of the ECM represents an obstacle that cancer cells have to overcome to disseminate from the primary tumor to form metastasis in distant organs. Here, we demonstrate an approach to studying the remodeling of a collagen-rich ECM by colorectal tumor cells using multiphoton microscopy (MPM). This approach allows the analysis of the invasion front of tumors grown on the CAM in 3D. MPM is superior to conventional histology, which is limited to 2D analysis and needs extensive tissue preparation. Abstract Cancer cells facilitate tumor growth by creating favorable tumor micro-environments (TME), altering homeostasis and immune response in the extracellular matrix (ECM) of surrounding tissue. A potential factor that contributes to TME generation and ECM remodeling is the cytoskeleton-associated human death-associated protein kinase 1 (DAPK1). Increased tumor cell motility and de-adhesion (thus, promoting metastasis), as well as upregulated plasminogen-signaling, are shown when functionally analyzing the DAPK1 ko-related proteome. However, the systematic investigation of how tumor cells actively modulate the ECM at the tissue level is experimentally challenging since animal models do not allow direct experimental access while artificial in vitro scaffolds cannot simulate the entire complexity of tissue systems. Here, we used the chorioallantoic membrane (CAM) assay as a natural, collagen-rich tissue model in combination with all-optical experimental access by multiphoton microscopy (MPM) to study the ECM remodeling potential of colorectal tumor cells with and without DAPK1 in situ and even in vivo. This approach demonstrates the suitability of the CAM assay in combination with multiphoton microscopy for studying collagen remodeling during tumor growth. Our results indicate the high ECM remodeling potential of DAPK1 ko tumor cells at the tissue level and support our findings from proteomics.

The choroid plexus (ChP) produces cerebrospinal fluid and forms an essential brain barrier. ChP tissues form in each brain ventricle, each one adopting a distinct shape, but remarkably little is known about the mechanisms underlying ChP development. Here, we show that epithelial WNT5A is crucial for determining fourth ventricle (4V) ChP morphogenesis and size in mouse. Systemic Wnt5a knockout, or forced Wnt5a overexpression beginning at embryonic day 10.5, profoundly reduced ChP size and development. However, Wnt5a expression was enriched in Foxj1-positive epithelial cells of 4V ChP plexus, and its conditional deletion in these cells affected the branched, villous morphology of the 4V ChP. We found that WNT5A was enriched in epithelial cells localized to the distal tips of 4V ChP villi, where WNT5A acted locally to activate non-canonical WNT signaling via ROR1 and ROR2 receptors. During 4V ChP development, MEIS1 bound to the proximal Wnt5a promoter, and gain- and loss-of-function approaches demonstrated that MEIS1 regulated Wnt5a expression. Collectively, our findings demonstrate a dual function of WNT5A in ChP development and identify MEIS transcription factors as upstream regulators of Wnt5a in the 4V ChP epithelium.

Enamel is the hardest tissue in mammalian organisms and is the layer covering the tooth. It consists of hydroxyapatite (HAP) crystallites, which mineralize on a protein scaffold known as the enamel matrix. Enamel matrix assembly is a very complex process mediated by enamel matrix proteins (EMPs). Altered HAP deposition or disintegration of the protein scaffold can cause enamel defects. Various methods have been established for enamel phenotyping, including MicroCT scanning with various resolutions from 9 µm for in vivo imaging to 1.5 µm for ex vivo imaging. With increasing resolution, we can see not only the enamel layer itself but also a detailed map of mineralization. To study enamel microstructure, we combine the MicroCT analysis with scanning electron microscopy (SEM), which enables us to perform element analyses such as calcium-carbon ratio. However, the methods mentioned above only show the result-already formed enamel. Stimulated emission depletion (STED) microscopy provides extra information about protein structure in the form of EMP localization and position before enamel mineralization. A combination of all these methods allows analyzing the same sample on multiple levels-starting with the live animal being scanned harmlessly and quickly, followed by sacrifice and high-resolution MicroCT scans requiring no special sample preparation. The biggest advantage is that samples remain in perfect condition for SEM or STED microscopic analysis. © 2022 Wiley Periodicals LLC. Basic Protocol 1: In vivo MicroCT scanning of mouse Basic Protocol 2: Ex vivo HR-MicroCT of the teeth Basic Protocol 3: SEM for teeth microstructure Basic Protocol 4: Stimulated emission depletion (STED) microscopy.

1AbstractCold and nutrient activated brown adipose tissue (BAT) is capable of increasing systemic energy expenditure via uncoupled respiration and secretion of endocrine factors thereby protecting mice against diet-induced obesity and improving insulin response and glucose tolerance in men. Long non-coding RNAs (lncRNAs) have recently been identified as fine tuning regulators of cellular function. While certain lncRNAs have been functionally characterised in adipose tissue, their overall contribution in the activation of BAT remains elusive. We identified lncRNAs correlating to inter-scapular brown adipose tissue (iBAT) function in high fat diet (HFD) and cold stressed mice. We focused on Gm15551 which has an adipose tissue specific expression profile, is highly upregulated during adipogenesis and downregulated by β-adrenergic activation in mature adipocytes. Albeit we performed comprehensive transcriptional and adipocyte physiology profiling in vitro and in vivo, we could not detect an effect of gain or loss of function of Gm15551.

The purpose of the study is to demonstrate coherent optical tomography and electroretinography techniques adopted from the human clinical practice to assess the morphology and function of the mouse retina in a high-throughput phenotyping environment. We present the normal range of wild-type C57Bl/6NCrl retinal parameters in six age groups between 10 and 100 weeks as well as examples of mild and severe pathologies resulting from knocking out a single protein-coding gene. We also show example data obtained by more detailed analysis or additional methods useful in eye research, for example, the angiography of a superficial and deep vascular complex. We discuss the feasibility of these techniques in conditions demanding a high-throughput approach such as the systemic phenotyping carried out by the International Mouse Phenotyping Consortium.

We describe compositional architectures and certifications in the research project certMILS. Compositional architectures enable re-use of certified COTS (commercial off-the-shelf) components with a well-defined delegation of responsibilities between component developers and system integrators during cyber physical system design and certification. We show how we used a Common Criteria certified MILS (Multiple Independent Levels of Safety / Security) platform for compositional designs and IEC 62443-4-1/62443-4-2 security evaluations and certifications for composed systems from the domains of smart grid, railway, and subway, that are safety- and security-critical.

The paper is focused on analysing the competition system of the highest Polish football league, in particular the championship part after the main competition, introduced in the season 2013/2014 and modified in the season 2017/2018. It is a study of the changes in the rankings after the regular part of the season, both in the group playing for the title and in the relegation group. It simulates a hypothetical situation as if the system had not been changed for the 2017/2018 and 2018/2019 seasons and the situation as if the current system had been played since the 2013/2014 season. In both cases, the results do not show huge variations, which so far challenges the needs for the 2017 modifications. In the long term, we can see a trend of higher variation of standings in the championship part after the main competition rather than in the relegation group. However, there are no major variations regarding the last two positions meaning relegation. In the six analysed seasons, the standings varied only in the seasons 2015/2016 and 2016/2017 – when only one team moved regarding the relegation positions in both cases. The standing of the championship group teams varies much less; although only in two out of six analysed seasons, the regular season winning team have kept their positions and became the final champions.

In contrast to the continuous increase in survival rates for many cancer entities, colorectal cancer (CRC) and pancreatic cancer are predicted to be ranked among the top 3 cancer-related deaths in the European Union by 2025. Especially, fighting metastasis still constitutes an obstacle to be overcome in CRC and pancreatic cancer. As described by Fearon and Vogelstein, the development of CRC is based on sequential mutations leading to the activation of proto-oncogenes and the inactivation of tumour suppressor genes. In pancreatic cancer, genetic alterations also attribute to tumour development and progression. Recent findings have identified new potentially important transcription factors in CRC, among those the activating transcription factor 2 (ATF2). ATF2 is a basic leucine zipper protein and is involved in physiological and developmental processes, as well as in tumorigenesis. The mutation burden of ATF2 in CRC and pancreatic cancer is rather negligible; however, previous studies in other tumours indicated that ATF2 expression level and subcellular localisation impact tumour progression and patient prognosis. In a tissue- and stimulus-dependent manner, ATF2 is activated by upstream kinases, dimerises and induces target gene expression. Dependent on its dimerisation partner, ATF2 homodimers or heterodimers bind to cAMP-response elements or activator protein 1 consensus motifs. Pioneering work has been performed in melanoma in which the dual role of ATF2 is best understood. Even though there is increasing interest in ATF2 recently, only little is known about its involvement in CRC and pancreatic cancer. In this review, we summarise the current understanding of the underestimated ‘cancer gene chameleon’ ATF2 in apoptosis, epithelial-to-mesenchymal transition and microRNA regulation and highlight its functions in CRC and pancreatic cancer. We further provide a novel ATF2 3D structure with key phosphorylation sites and an updated overview of all so-far available mouse models to study ATF2 in vivo.

A number of human autoinflammatory diseases manifest with severe inflammatory bone destruction. Mouse models of these diseases represent valuable tools that help us to understand molecular mechanisms triggering this bone autoinflammation. The Pstpip2cmo mouse strain is among the best characterized of these; it harbors a mutation resulting in the loss of adaptor protein PSTPIP2 and development of autoinflammatory osteomyelitis. In Pstpip2cmo mice, overproduction of interleukin-1β (IL-1β) and reactive oxygen species by neutrophil granulocytes leads to spontaneous inflammation of the bones and surrounding soft tissues. However, the upstream signaling events leading to this overproduction are poorly characterized. Here, we show that Pstpip2cmo mice deficient in major regulator of Src-family kinases (SFKs) receptor-type protein tyrosine phosphatase CD45 display delayed onset and lower severity of the disease, while the development of autoinflammation is not affected by deficiencies in Toll-like receptor signaling. Our data also show deregulation of pro-IL-1β production by Pstpip2cmo neutrophils that are attenuated by CD45 deficiency. These data suggest a role for SFKs in autoinflammation. Together with previously published work on the involvement of protein tyrosine kinase spleen tyrosine kinase, they point to the role of receptors containing immunoreceptor tyrosine-based activation motifs, which after phosphorylation by SFKs recruit spleen tyrosine kinase for further signal propagation. We propose that this class of receptors triggers the events resulting in increased pro-IL-1β synthesis and disease initiation and/or progression.

Tooth formation requires complex signaling interactions both within the oral epithelium and between the epithelium and the underlying mesenchyme. Previous studies of the Wnt/β-catenin pathway have shown that tooth formation is partly inhibited in loss-of-function mutants, and gain-of-function mutants have perturbed tooth morphology. However, the stage at which Wnt signaling is first important in tooth formation remains unclear. Here, using an Fgf8-promoter-driven, and therefore early, deletion of β-catenin in mouse molar epithelium, we found that loss of Wnt/β-catenin signaling completely deletes the molar tooth, demonstrating that this pathway is central to the earliest stages of tooth formation. Early expression of a dominant-active β-catenin protein also perturbs tooth formation, producing a large domed evagination at early stages and supernumerary teeth later on. The early evaginations are associated with premature mesenchymal condensation marker, and are reduced by inhibition of condensation-associated collagen synthesis. We propose that invagination versus evagination morphogenesis is regulated by the relative timing of epithelial versus mesenchymal cell convergence regulated by canonical Wnt signaling. Together, these studies reveal new aspects of Wnt/β-catenin signaling in tooth formation and in epithelial morphogenesis more broadly.

Complex metabolic conditions such as type 2 diabetes and obesity result from the interaction of numerous genetic and environmental factors. While the family of Nme proteins has been connected so far mostly to development, proliferation, or ciliary functions, several lines of evidence from human and experimental studies point to the potential involvement of one of its members, NME7 (non-metastatic cells 7, nucleoside diphosphate kinase 7) in carbohydrate and lipid metabolism. As a complete lack of Nme7 is semilethal in rats, we compared morphometric, metabolic, and transcriptomic profiles of standard diet-fed heterozygous Nme7+/− on male rats vs. their wild-type Nme7+/+ controls. Nme7+/− animals showed increased body weight, adiposity, higher insulin levels together with decreased glucose tolerance. Moreover, they displayed pancreatic islet fibrosis and kidney tubular damage. Despite no signs of overt liver steatosis or dyslipidemia, we found significant changes in the hepatic transcriptome of Nme7+/− male rats with a concerted increase of expression of lipogenic enzymes including Scd1, Fads1, Dhcr7 and a decrease of Cyp7b1 and Nme7. Network analyses suggested possible links between Nme7 and the activation of Srebf1 and Srebf2 upstream regulators. These results further support the implication of NME7 in the pathogenesis of glucose intolerance and adiposity.

Maintenance of genome stability is essential for every living cell as genetic information is repeatedly challenged during DNA replication in each cell division event. Errors, defects, delays, and mistakes that arise during mitosis or meiosis lead to an activation of DNA repair processes and in case of their failure, programmed cell death, i.e. apoptosis, could be initiated. Fam208a is a protein whose importance in heterochromatin maintenance has been described recently. In this work, we describe the crucial role of Fam208a in sustaining genome stability during cellular division. The targeted depletion of Fam208a in mice using CRISPR/Cas9 led to embryonic lethality before E12.5. We also used the siRNA approach to downregulate Fam208a in zygotes to avoid the influence of maternal RNA in the early stages of development. This early downregulation increased arresting of the embryonal development at the two-cell stage and the occurrence of multipolar spindles formation. To investigate this further, we used the yeast two-hybrid (Y2H) system and identified new putative interaction partners Gpsm2, Svil, and Itgb3bp. Their co-expression with Fam208a was assessed by RT-qPCR profiling and in situ hybridization [1] in multiple murine tissues. Based on these results we proposed that Fam208a functions within the HUSH complex by interaction with Mphosph8 as these proteins are not only able to physically interact but also co-localise. We are bringing new evidence that Fam208a is a multi-interacting protein affecting genome stability on the cell division level at the earliest stages of development and by interaction with methylation complex in adult tissues. In addition to its epigenetic functions, Fam208a appears to have an important role in the zygotic division, possibly via interaction with newly identified putative partners Gpsm2, Svil, and Itgb3bp.

WIZ (Widely Interspaced Zinc Finger) is associated with the G9a-GLP protein complex, a key H3K9 methyltransferase suggesting a role in transcriptional repression. However, its role in embryonic development is poorly described. In order to assess the loss of function of WIZ, we generated CRISPR/Cas9 WIZ knockout mouse model with 32 nucleotide deletion. Observing the lethality status, we identified the WIZ knockouts to be subviable during embryonic development and non-viable after birth. Morphology of developing embryo was analyzed at E14.5 and E18.5 and our findings were supported by microCT scans. Wiz KO showed improper development in multiple aspects, specifically in the craniofacial area. In particular, shorter snout, cleft palate, and cleft eyelids were present in mutant embryos. Palatal shelves were hypomorphic and though elevated to a horizontal position on top of the tongue, they failed to make contact and fuse. By comparison of proliferation pattern and histone methylation in developing palatal shelves we brought new evidence of importance WIZ dependent G9a-GLP methylation complex in craniofacial development, especially in palate shelf fusion.

The choroid plexus (ChP) produces cerebrospinal fluid and forms an essential brain barrier. ChP tissues form in each brain ventricle, each one adopting a distinct shape, but remarkably little is known about the mechanisms underlying ChP development. Here, we show that epithelial WNT5A is crucial for determining fourth ventricle (4V) ChP morphogenesis and size in mouse. Systemic Wnt5a knockout, or forced Wnt5a overexpression beginning at embryonic day 10.5, profoundly reduced ChP size and development. However, Wnt5a expression was enriched in Foxj1-positive epithelial cells of 4V ChP plexus, and its conditional deletion in these cells affected the branched, villous morphology of the 4V ChP. We found that WNT5A was enriched in epithelial cells localized to the distal tips of 4V ChP villi, where WNT5A acted locally to activate non-canonical WNT signaling via ROR1 and ROR2 receptors. During 4V ChP development, MEIS1 bound to the proximal Wnt5a promoter, and gain- and loss-of-function approaches demonstrated that MEIS1 regulated Wnt5a expression. Collectively, our findings demonstrate a dual function of WNT5A in ChP development and identify MEIS transcription factors as upstream regulators of Wnt5a in the 4V ChP epithelium.

Security concerns become increasingly important in safety-critical industrial cyberphysical systems. Different options for security certification exist. We describe a Common Criteria certification for a MILS separation kernel, and IEC 62443 analysis and certifications for the smart grid, railway and subway pilots using the MILS approach in the research project certMILS.

Neocortex expansion during human evolution provides a basis for our enhanced cognitive abilities. Yet, which genes implicated in neocortex expansion are actually responsible for higher cognitive abilities is unknown. The expression of human‐specific ARHGAP11B in embryonic/foetal mouse, ferret and marmoset neocortex was previously found to promote basal progenitor proliferation, upper‐layer neuron generation and neocortex expansion during development, features commonly thought to contribute to increased cognitive abilities. However, a key question is whether this phenotype persists into adulthood and if so, whether cognitive abilities are indeed increased. Here, we generated a transgenic mouse line with physiological ARHGAP11B expression that exhibits increased neocortical size and upper‐layer neuron numbers persisting into adulthood. Adult ARHGAP11B‐transgenic mice showed altered neurobehaviour, notably increased memory flexibility and a reduced anxiety level. Our data are consistent with the notion that neocortex expansion by ARHGAP11B, a gene implicated in human evolution, underlies some of the altered neurobehavioural features observed in the transgenic mice, such as the increased memory flexibility, a neocortex‐associated trait, with implications for the increase in cognitive abilities during human evolution., Effects of human‐specific ARHGAP11B, linked to higher cognitive abilities, on basal progenitor proliferation and upper‐layer neuron generation in rodent embryos persist into adulthood, correlating with altered neurobehaviour including reduced anxiety levels.

NME7 (non-metastatic cells 7, nucleoside diphosphate kinase 7) is a member of a gene family with a profound effect on health/disease status. NME7 is an established member of the ciliome and contributes to the regulation of the microtubule-organizing center. We aimed to create a rat model to further investigate the phenotypic consequences of Nme7 gene deletion. The CRISPR/Cas9 nuclease system was used for the generation of Sprague Dawley Nme7 knock-out rats targeting the exon 4 of the Nme7 gene. We found the homozygous Nme7 gene deletion to be semi-lethal, as the majority of SDNme7−/− pups died prior to weaning. The most prominent phenotypes in surviving SDNme7−/− animals were hydrocephalus, situs inversus totalis, postnatal growth retardation, and sterility of both sexes. Thinning of the neocortex was histologically evident at 13.5 day of gestation, dilation of all ventricles was detected at birth, and an external sign of hydrocephalus, i.e., doming of the skull, was usually apparent at 2 weeks of age. Heterozygous SDNme7+/− rats developed normally, we did not detect any symptoms of primary ciliary dyskinesia. The transcriptomic profile of liver and lungs corroborated the histological findings, revealing defects in cell function and viability. In summary, the knock-out of the rat Nme7 gene resulted in a range of conditions consistent with the presentation of primary ciliary dyskinesia, supporting the previously implicated role of the centrosomally located Nme7 gene in ciliogenesis and control of ciliary transport.

Summary: Osteoblasts orchestrate bone formation through the secretion of type I collagen and other constituents of the matrix on which hydroxyapatite crystals mineralize. Here, we show that TENT5A, whose mutations were found in congenital bone disease osteogenesis imperfecta patients, is a cytoplasmic poly(A) polymerase playing a crucial role in regulating bone mineralization. Direct RNA sequencing revealed that TENT5A is induced during osteoblast differentiation and polyadenylates mRNAs encoding Col1α1, Col1α2, and other secreted proteins involved in osteogenesis, increasing their expression. We postulate that TENT5A, possibly together with its paralog TENT5C, is responsible for the wave of cytoplasmic polyadenylation of mRNAs encoding secreted proteins occurring during bone mineralization. Importantly, the Tent5a knockout (KO) mouse line displays bone fragility and skeletal hypomineralization phenotype resulting from quantitative and qualitative collagen defects. Thus, we report a biologically relevant posttranscriptional regulator of collagen production and, more generally, bone formation.

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease., Author summary Patients affected by osteoporosis frequently present with decreased BMD and increased fracture risk. Genes are known to control the onset and progression of bone diseases such as osteoporosis. Therefore, we aimed to identify osteoporosis-related genes using BMD measures obtained from a large pool of mutant mice genetically modified for deletion of individual genes (knockout mice). In a collaborative endeavor involving several research sites world-wide, we generated and phenotyped 3,823 knockout mice and identified 200 genes which regulated BMD. Of the 200 BMD genes, 141 genes were previously not known to affect BMD. The discovery and study of novel BMD genes will help to better understand the causes and therapeutic options for patients with low BMD. In the long run, this will improve the clinical management of osteoporosis.

Bardet-Biedl Syndrome (BBS) is a pleiotropic genetic disease caused by the dysfunction of primary cilia. The immune system of patients with ciliopathies has not been investigated. However, there are multiple indications that the impairment of the processes typically associated with cilia may have influence on the hematopoietic compartment and immunity. In this study, we analyze clinical data of BBS patients and corresponding mouse models carrying mutations in Bbs4 or Bbs18. We find that BBS patients have a higher prevalence of certain autoimmune diseases. Both BBS patients and animal models have altered red blood cell and platelet compartments, as well as elevated white blood cell levels. Some of the hematopoietic system alterations are associated with BBS-induced obesity. Moreover, we observe that the development and homeostasis of B cells in mice is regulated by the transport complex BBSome, whose dysfunction is a common cause of BBS. The BBSome limits canonical WNT signaling and increases CXCL12 levels in bone marrow stromal cells. Taken together, our study reveals a connection between a ciliopathy and dysregulated immune and hematopoietic systems.

Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-191,2. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-193. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches. The bispecific IgG1-like CoV-X2 prevents SARS-CoV-2 spike binding to ACE2, neutralizes SARS-CoV-2 and its variants of concern, protects against disease in a mouse model, whereas the parental monoclonal antibodies generate viral escape.

The publication "Management of risks of processes, fittings and safety of complex technical facilities" is devoted to risks and their management for benefit of safety. It contains three parts. The introductory part solves problem of how to link existing norms and standards that contain the knowledge of entire generations of experts with the knowledge gained from the processing of original data using the engineering methods for working with risks. The next section shows results of comprehensive research on selected areas and contains 6 works. The last part contains 15 communications containing the expert results of partial problems related to risks and their management for safety. The publication contains results from a wide range of disciplines that contribute to the management and settlement of risks to safety. The articles contain: theoretical studies processing large data files; experimental works; and summarizing practical experience.

SUMMARYDDI2 is an aspartic protease that cleaves polyubiquitinated substrates. Upon proteotoxic stress, DDI2 activates the ER-bound transcription factor TCF11/NRF1 (NFE2L1), a master regulator of proteostasis maintenance in mammalian cells, and ensures the expression of rescue factors including proteasome subunits. Here we describe the consequences of DDI2 ablation both in vivo and in cells. Knock-out of DDI2 in mice resulted in embryonic lethality at E12.5 with severe developmental failure. Molecular characterization of the embryos and surrogate DDI2 knock-out cell lines showed insufficient proteasome expression with proteotoxic stress, accumulation of high molecular weight ubiquitin conjugates, and induction of the unfolded protein and integrated stress responses. We also show that DDI2 KO-induced proteotoxic stress causes the cell-autonomous innate immune system to induce a type I interferon signature. These results indicate an important role for DDI2 in the proteostasis network of cells and tissues and in the maintenance of a balanced immune response.HighlightsDDI2-deficiency in mice causes severe developmental failure and embryonic lethality at mid-late gestationDDI2-deficiency causes severe proteotoxic stress and proteasome impairmentDDI2-deficiency induces the UPR and ISR signaling pathwaysDDI2-deficient cells survive via STAT3-dependent interferon signaling

Type 2 diabetes mellitus represents a major health problem with increasing prevalence worldwide. Limited efficacy of current therapies has prompted a search for novel therapeutic options. Here we show that treatment of pre-diabetic mice with mitochondrially targeted tamoxifen, a potential anti-cancer agent with senolytic activity, improves glucose tolerance and reduces body weight with most pronounced reduction of visceral adipose tissue due to reduced food intake, suppressed adipogenesis and elimination of senescent cells. Glucose-lowering effect of mitochondrially targeted tamoxifen is linked to improvement of type 2 diabetes mellitus-related hormones profile and is accompanied by reduced lipid accumulation in liver. Lower senescent cell burden in various tissues, as well as its inhibitory effect on pre-adipocyte differentiation, results in lower level of circulating inflammatory mediators that typically enhance metabolic dysfunction. Targeting senescence with mitochodrially targeted tamoxifen thus represents an approach to the treatment of type 2 diabetes mellitus and its related comorbidities, promising a complex impact on senescence-related pathologies in aging population of patients with type 2 diabetes mellitus with potential translation into the clinic.

Osteoblasts orchestrate bone formation by secreting dense, highly cross-linked type I collagen and other proteins involved in osteogenesis. Mutations in Col1α1, Col1α2, or collagen biogenesis factors lead to the human genetic disease, osteogenesis imperfecta (OI). Herein, we show that the TENT5A gene, whose mutation is responsible for poorly characterized type XVIII OI, encodes an active cytoplasmic poly(A) polymerase regulating osteogenesis. TENT5A is induced during osteoblast differentiation and TENT5A KO osteoblasts are defective in mineralization. The TENT5A KO mouse recapitulates OI disease symptoms such as bone fragility and hypomineralization. Direct RNA sequencing revealed that TENT5A polyadenylates and increases expression of Col1α1 and Col1α2 RNAs, as well as those of other genes mutated in OI, resulting in lower production and improper folding of collagen chains. Thus, we have identified the specific pathomechanism of XVIII OI and report for the first time a biologically relevant post-transcriptional regulator of collagen production. We further postulate that TENT5A, possibly together with its paralogue TENT5C, is responsible for the wave of cytoplasmic polyadenylation of mRNAs encoding secreted proteins occurring during bone mineralization.

The choroid plexus (ChP) produces cerebrospinal fluid and forms a critical barrier between the brain and the circulation. While the ChP forms in each brain ventricle, it adopts a different shape in each one and remarkably little is known about the mechanisms underlying its development. Here, we show that epithelial WNT5A is critical for determining fourth ventricle (4V) ChP morphogenesis and size. SystemicWnt5aknockout, or forced WNT5A overexpression beginning at E10.5, profoundly reduced the size and development of ChP in all ventricles. However, conditional deletion ofWnt5aexpression inFoxj1-expressing epithelial cells affected only the branched, villous morphology of the 4V ChP. We found that WNT5A was enriched in epithelial cells localized to the distal tips of 4V ChP villi, where WNT5A acted locally to activate non-canonical Wnt signaling via Ror1/Ror2 receptors. During 4V ChP development, MEIS1 bound to the proximalWnt5apromoter, and gain- and loss-of-function approaches demonstrated that MEIS1 regulatedWnt5aexpression. Collectively, our findings demonstrate a dual function of WNT5A in ChP development and identify MEIS1 and MEIS2 as upstream regulators ofWnt5ain the 4V ChP epithelium.

Cre-loxP recombination system is a powerful tool for genome engineering. One of its applications is found in genetic mouse models that often require to induce Cre recombination in preimplantation embryos. Here, we describe a technically simple, affordable and highly efficient protocol for Cre protein delivery into mouse zygotes by electroporation. We show that electroporation based delivery of Cre has no negative impact on embryo survival and the method can be easily combined with in vitro fertilization resulting in a significantly faster generation of desired models. Lastly, we demonstrate that Cre protein electroporation is suitable for allelic conversion in primary cells derived from conditional mouse models.

Commercial LiNi1/3Mn1/3Co1/3O2 material is treated in appropriate way to provide cathode material with reasonable charge capacity and high cycling stability upon different charging/discharging rates. Electrochemical performance of the tested materials is studied by cyclic voltammetry of Li insertion and galvanostatic chronopotentiometry. An influence of calcination, surface area and particle size uniformity on the charge capacity and cycling stability is evaluated and the parameters of the optimized samples providing charge capacity of 141 mAh/g (cyclic voltammetry) and 144 and 135 mAh/g (galvanostatic chronopotentiometry) at 1 and 10C, respectively, represent a platform for scale up during the next period of the research.

The aim of all people is human security and development. Therefore, they need technological facilities, which ensure energy, services and products for life. Present cognition shows that safety of technological facilities is based on copping with risks. Human cognition contains a lot of knowledge and experiences with risks, but in reality only part of them are used in practice. The aim of research, the results of which are presented, was the judgment, how in practice connected with complex technological facility safety it is used the present human cognition on work with risks. With regard to world dynamic development it is necessary the priority risks to monitor and to cope with them during time, and also to measure the respective safety. At measure of rate for safety level we use the known experience that the better coping with relevant risks, the higher facility safety level is. The analysis of based publications and data from real practice shows that seven domains at work with risks are important. The paper shows the results of critical judgment of individual techniques that are used at work with risks in technological facilities in practice.

The Critical Infrastructure Protection (CIP) is a classic example of system of systems (SoS) management. We pay special attention to interdependencies among systems within SoS. The article deals with the requirements for the cyber gateway at the train transportation management system. More and more telemetry and remote control through communication systems are used on the track because of the technological development. The train at cyberspace acts as an end network that is connected to the operating center network via an open public network. The gateway must then meet the requirements of the railway infrastructure as well as the cyber infrastructure. We deal with the integration of various requirements for the train gateway into one verification and certification cycle in this paper. The requirement integration is important because, in the first it is necessary to ensure coherence and consistency between different requirements. The second reason is to speed up possible re-certification. A recertification is necessary because gateway adaptation or installation in a new environment. The European project certMILS deals with the issue of re-certification.

Předložený sborník odborných článků dokládá výsledky celoroční odborné a organizační práce v předmětné oblasti. Obsahuje výsledky z širokého spektra oborů, které přispívají k řízení a vypořádání rizik ve prospěch bezpečnosti. Články obsahují výsledky: - teoretických studií, - zpracování rozsáhlých datových souborů, - experimentálních prací, - zkušenosti z praxe. Sdělení ve sborníku jsou uspořádána alfabeticky dle příjmení prvního autora s přihlédnutím k počtu autorů. Řada článků obsahuje výsledky národních projektů. Články v předložené publikaci byly recenzovány a upraveny podle připomínek recenzentů. Výsledky uvedené ve sděleních ukazují, že i navzdory velkému množství poznatků o technických dílech, jejich zařízeních, ́ strukturách, vzájemných propojení, rizicích a bezpečnosti, se havárie a selhání technických zařízení a technických děl stále vyskytují. Příčin je několik: - dynamická proměnlivost světa, - nedostatečné lidské znalosti a schopnosti, - pomalé používání poznatků a zkušeností získaných v praxi, - neuspokojivé povědomí o rizicích a jejich důsledcích pro technická díla a veřejný zájem. Studie havárií a selhání technických zařízení a děl ukazují, že důležitým faktorem pro zajištění bezpečnosti je jak kvalitní technická obsluha, tak správná realizace odpovědnosti na různých úrovních řízení. Obsluha musí být založena na kvalitních datech z měření a na použití správných technických postupů. Lidský faktor se projevuje jako základním činitel při řízení rizik ve prospěch bezpečnosti, a to na všech úrovních řízení a provádění činností. Shromážděné poznatky také ukazují, že při prevenci havárií a selhání je třeba se vyvarovat, jak velkých chyb v prevenci rizik, tak výskytu drobných chyb, jejichž realizace v krátkém časovém intervalu je nebezpečná. Proto u řízení rizik ve prospěch bezpečnosti složitých technických děl je důležitá aplikace systémového pojetí a zvážení proměnnosti limitů a podmínek v prostoru a čase.