The development of antibiotic resistance poses an increasing threat to global health. Understanding how resistance develops in bacteria is critical for the advancement of new strategies to combat antibiotic resistance. In the 1980s, it was discovered that certain antibiotics induce elevated rates of mutation in bacteria. From this, an “increased evolvability” hypothesis was proposed: antibiotic-induced mutagenesis increases the genetic diversity of bacterial populations, thereby increasing the rate at which bacteria develop antibiotic resistance. However, antibiotic-induced mutagenesis is one of multiple competing factors that act on bacterial populations exposed to antibiotics. Its relative importance in shaping evolutionary outcomes, including the development of antibiotic resistance, is likely to depend strongly on the conditions. Presently, there is no quantitative model that describes the relative contribution of antibiotic-induced mutagenesis to bacterial evolution. A far more complete understanding could be reached if we had access to technology that enabled us to study antibiotic-induced mutagenesis at the molecular-, cellular-, and population-levels simultaneously. Direct observations would, in principle, allow us to directly link molecular-level events with outcomes in individual cells and cell populations. In this review, we highlight microscopy studies which have allowed various aspects of antibiotic-induced mutagenesis to be directly visualized in individual cells for the first time. These studies have revealed new links between error-prone DNA polymerases and recombinational DNA repair, evidence of spatial regulation occurring during the SOS response, and enabled real-time readouts of mismatch and mutation rates. Further, we summarize the recent discovery of stochastic population fluctuations in cultures exposed to sub-inhibitory concentrations of bactericidal antibiotics and discuss the implications of this finding for the study of antibiotic-induced mutagenesis. The studies featured here demonstrate the potential of microscopy to provide direct observation of phenomena relevant to evolution under antibiotic-induced mutagenesis. Mycobacterium tuberculosis, appears to acquire antibiotic resistance exclusively through mutation, as opposed to lateral gene transfer , most of which have functions relevant to DNA damage . The evolutionary dynamics at play within this near-MIC regime are more complex than those that occur at lethal concentrations of antibiotic . At concWe propose that to better understand the role of antibiotic-induced mutagenesis on the evolution of antibiotic-resistance mutations, we need to be able to measure its effects while simultaneously monitoring cell and population level behaviors. This is not achievable with conventional microbiology techniques , which operate as end-point assays and have indirect read-outs.E. coli, due to its prevalence as a laboratory model organism, and with fluoroquinolones because these drugs are strongly mutagenic and are commonly utilized as model compounds in laboratory experiments. For a more comprehensive review of antibiotic-induced mutagenesis, which includes work in other systems, we suggest a recent review by In recent years, researchers have begun to explore microscopy as a means to obtain direct, real-time observations of individual cells, and even individual molecules within them. The purpose of the current review is to highlight studies in which microscopy has improved our understanding of either the molecular mechanisms underlying antibiotic-induced mutagenesis or cell‐ and population-level events that occur when bacteria are challenged with sub-inhibitory concentrations of antibiotics. Much of the microscopy work presented here has been conducted in E. coli cells treated with ciprofloxacin, a fluoroquinolone antibiotic that is a key model compound in the study of antibiotic-induced mutagenesis , pol II, pol IV, and pol V (The most widely studied form of antibiotic-induced mutagenesis is associated with the SOS response and involves increased production of error-prone DNA polymerases. nd pol V . Of thesnd pol V .E. coli cells (via homologous recombination (umuDC (2 (UmuD homodimer) and UmuC proteins are only expressed once RecA* accumulates in cells. At this stage, however, UmuD2 and UmuC do not form an active polymerase. Second, UmuD2 homodimers undergo autocatalytic cleavage in the presence of RecA*, in a mechanism analogous to that of LexA  or pol V Mut (UmuD′2C-RecA-ATP). It remains unknown whether pol V Mut formation occurs on the membrane or in the cytosol. In the microscopy images, pol V spatial regulation presents as a redistribution of fluorescently labeled UmuC from the membrane to the cytosol. Due to the fact that UmuC is expressed at very low levels in E. coli  break repair was originally discovered through genetics experiments  involved in double-strand break repair , double-strand break repair factors , the SOS response (mutagenesis was blocked in a lexA [Ind−] background), and the RpoS response  is induced under a variety of circumstances, including stationary growth, starvation, and oxidative damage (S) is an alternative σ-factor that governs the expression of hundreds of different genes (70 (or RpoD), for binding to RNA polymerase, and in doing so dramatically changes the gene expression profile of the cell . Expansion at 1x MIC strongly favored the gyrA (S83A) mutation, which appeared in 7/8 isolates. The via microscopy.What might evolution to antibiotic resistance look like under the microscope? A 2015 study by Antibiotics can induce mutagenesis, tolerance, and cell death. Evolution to antibiotic resistance is often limited by the mutation supply rate, which is a product of the cellular mutation rate and the size of the population . ElevateThe fact that population growth in this near-MIC regime is the product of both cell growth and cell death means that many more generations are required for the population to reach a certain size than would be required in the absence of drug. When rates of antibiotic-induced mutagenesis are measured using fluctuation analyses, the results are reported in units of mutations per genome per generation. Typically, the number of generations is calculated based on the population size after treatment, under the assumption that cells are not dying when the antibiotic concentration is below the MIC. In light of the stochastic population dynamics reported by the Kim group, it appears that many of the mutation rates reported in the literature may be overestimated because more generations occurred than were accounted for in the measurements .The second important ramification of stochastic population dynamics for the study of antibiotic-induced mutagenesis is that when the population size is small, as would be the case in a mother machine for example; many populations will become extinct when exposed to sub-MIC concentrations of bactericidal antibiotics. Evidence of this is visible in Uphoff’s work with sub-MIC norfloxacin , where tThe studies highlighted throughout this review demonstrate the potential of microscopy in helping to achieve a more thorough understanding of antibiotic-induced mutagenesis. There is enormous potential for other types of correlative measurements as well. Microscopy shows a great degree of promise as an important platform for uncovering functional linkages between the molecular-level actions of error-prone DNA repair processes, the cell-level behaviors of stress responses and growth dynamics, and population-level behaviors that determine mutation supply rates. The development of advanced techniques for microscopy and microfluidic technology is sure to continue, however, we feel it is important to point out that great strides can be taken with the technology that is already available. As has been noted previously , our undAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Staphylococus aureus (MRSA) to vancomycin-resistent Enterococcus faecium (VRE) and to multidrug-resistant gramnegative bacteria, while antibiotic consumption volumes, trends and patterns across countries do not show major and highly significant improvements. The way to go further is to recognize that better prescribing comes at a cost and requires investment in expert personnel, practice guideline drafting, and implementation aids, and, secondly, the setting of clear goals and quantitative targets for prescribing quality.It is more than two decades ago that a European Union conference on “The Microbial Threat” hosted by the Danish Government in Copenhagen in September 1998 issued recommendations to encourage good practice in the use of antimicrobial agents and reduce inappropriate prescribing. Essential components of those recommendations were antimicrobial teams in hospitals and the use of feedback to prescribers as well as educational activities. Two decades later, important surveillance systems on both antimicrobial resistance as well as on antibiotic consumption are functioning at the European level and in most European countries; European Committee on Antimicrobial Susceptibility Testing (EUCAST) has thoroughly re-evaluated, standardized and harmonized antibiotic susceptibility testing and breakpoints; there have been educational activities in many countries; and stewardship teams are now included in many guidelines and policy papers and recommendations. Yet, antimicrobial resistance problems in Europe have shifted from methicillin-resistant  Infections due to antibiotic-resistant microorganisms are increasing in prevalence and of growing global concern. They may be associated with prolonged in-hospital stay, increased morbidity and mortality, and heightened cost , 2. AntiEducational initiatives for both health professionals  and the general public are of major importance for improving the use of antimicrobial agents.Antimicrobials for therapeutic use should be prescription-only medicines and so should not be advertised to the public.Antimicrobial teams  should be introduced in every hospital. They should have the authority to modify antimicrobial prescriptions of individual clinicians in accordance with locally accepted guidelines, always taking account of the needs of the patient. Clinicians should be given an opportunity to approve the remit and recommendations of the teams. The teams should also cover the community, including nursing homes and other residential institutions, and the primary/secondary care interface. Feedback should be provided to clinicians.Guidelines for appropriate antimicrobial usage should be introduced in all aspects of both medical and veterinary practice.Treatment should be limited to bacterial infections, using antibiotics directed against the causative agent, given in optimal dosage, dosage intervals and length of treatment with steps taken to ensure maximum patient concordance with the treatment regimen, and only when the benefit of the treatment outweighs the individual and global risks.Steps must be taken to increase access to diagnostic testing for patients with infections, and the range of tests needs to be improved.Antibiotic stewardship in human medicine can be defined as ongoing efforts by a healthcare institution—whether local, regional/national or international—to optimize antimicrobial use among patients with the aim to improve patient outcomes, ensure cost-effective therapy and reduce adverse sequelae of antimicrobial use including antimicrobial resistance. The overall aim is to preserve the efficacy of antibiotics for use in the future. The term antibiotic stewardship was probably first coined in 1996 by John McGowan and Dale Gerding , mentionOnly a few years later, the European Commission issued the Council recommendation “2002/77/EC” on the prudent use of antimicrobial agents in human medicine . SurveysTo facilitate and further develop surveillance in the areas of antibiotic resistance and antibiotic consumption at the European level, the new European Centre for Disease Control and Prevention  was a major step. Two surveillance projects most important for antibiotic stewardship were transferred to the ECDC in the following years. One was the European Antimicrobial Resistance Surveillance System (EARSS), established in 1998 and initially funded by the European Commission’s Directorate General for Health and Consumer Affairs (DG SANCO) and the Dutch Ministry of Health, Welfare and Sports . The admThe second important surveillance programme, the European Surveillance of Antimicrobial Consumption (ESAC) project, also granted by DG SANCO, was launched in 2001. ESAC, at that time coordinated by the University of Antwerp, initiated an international network of surveillance systems, aiming to collect comparable and reliable data on antibiotic use in Europe. It was transferred to the ECDC in 2011 and renamed “ESAC-Net”. The ESAC investigators published a number of most critical papers on antibiotic use density in European countries at a population-based level –12. ESACwww.eucast.org), a committee that standardizes antimicrobial susceptibility testing and harmonizes breakpoints, organized by ESCMID, ECDC and national breakpoint committees, and the ESCMID study groups for Antimicrobial Stewardship (ESGAP), for Antimicrobial Resistance Surveillance (ESGARS) and for PK/PD of Anti-Infectives (EPASG).Other most relevant initiatives in Europe were projects associated with the European Society of Clinical Microbiology and Infectious Diseases (ESCMID), namely the European Committee on Antimicrobial Susceptibility Testing (EUCAST)  as well as in the rate of carbapenem resistance (<1% to >50%) [Data from surveillance and research projects are prerequisites for defining areas for potential improvement and further research need and investment. What do the data tell us after two decades of surveillance? For some it may be a surprise, for others it may be as expected that the published data on antibiotic consumption and resistance continue to show wide variations in Europe , 18, andisolates . Also, twww.strame.se) and the Dutch Working Party on Antibiotic Policy .It is important to note that the “makers” of resistance surveillance systems in Europe as well as within countries in Europe (and elsewhere) are neither politicians nor prescribers themselves, and all sides may have substantial conceptual misunderstandings of the bacterial resistance problem and its consequences and solutions . While sThere has been some work on defining and refining prescribing quality indicators, but very limited use of these –33. ThisAt the European and also at the level of many countries in Europe, quantitative targets—for antibiotic use, for resistance containment or reduction, as well as for structural requirements and investments—are too often missing. Only 8 out of 28 European countries have defined quantitative targets in their national plans according to a survey in 2017 . Among tThe setting of such targets in the field of antibiotic stewardship after thorough discussion and consensus between clinicians, public health officials and politicians is where Europe and its countries and regions need to improve which requires specific, measurable, achievable, relevant and timebound (SMART) objectives .


This paper aims to assess the influence of clogging on paving material (pervious concrete) drainage characteristics as well as the influence of the properties of an unbound base layer on drainage characteristics of the whole paving system. The clogging influence has been studied measuring the drainage characteristics on pervious concrete flags before and after their clogging, according to ASTM C1701-09. Additionally, the drainage characteristics of uncontaminated pervious concrete as a paving material was assessed using the falling head method. To assess the influence of properties of an unbound base course (UBC) on drainage characteristics of the whole paving system, the unbound base layer was compacted in two different levels of compaction and the drainage characteristics were measured (according to ASTM C1701-09). It is concluded that pervious concrete prepared with a smaller aggregate fraction is more prone to clogging. Regarding the influence of UBC, it is important to find a balance between pervious concrete infiltration and UBC exfiltration rate, particularly in a case of pervious concrete flags made of coarse aggregate. Good drainage presents a necessity for quality road projects. This is a particularly important issue within highly populated areas or urban areas where good surface drainage presents an imperative for quality daily life. Past years of investigations have provided some new materials and technologies for effective drainage, and pervious pavements were emphasized as a green, sustainable paving solution. Such a pavement is typically considered as the surface course comprised of permeable block pavers, porous asphalt or pervious concrete, generally with the underlying bedding layer separated from the coarse aggregates beneath by geotextiles. As a dominant building material for many years, concrete is used for various applications which utilize its specific characteristics. Green paving with pervious concrete is accentuated by its ability to reduce runoff water, improve runoff water quality, reduce on-site noise level as well as relieve the heat island effect ,2,3,4. EPermeability, as one of the main features of pervious concrete, is assessed with different laboratory and field methods. The most used ones are constant and falling head methods and the method described in standard ASTM C1701-09 , for in-Pervious concrete has certain limiting usage factors, of which the most important ones are durability issues related to abrasion and freeze-thaw cycles resistance. Researchers have shown that increasing mechanical properties by different additives  drainage characteristics as well as the influence of the properties of the unbound base layer on drainage characteristics of the whole paving system. The clogging influence has been studied measuring the drainage characteristics on pervious concrete flags before and after their clogging, according to ASTM C1701-09. The clogging was simulated using a mixture of clay and sand as suggested in . Additio3, respectively, according to EN 1097-6 [Pervious concrete mixtures with three different types of aggregates and different proportions of aggregate fractions were used within this study, accounting for 11 different pervious concrete mixtures in total . CrushedN 1097-6 .w/c) was 0.33, with tap water. The cement was CEM II/A-M(S-V) 42.5 N, per EN 197-1 [3 per EN 196-6 [3 for all mixtures. Specimens of all concrete mixtures were cast with a compacting rod, by rodding 25 times. All specimens were extracted from moulds 24 h after casting and placed in a water tank for 27 days, at a temperature of 20 ± 5 °C, per EN 12390 2 [For all mixtures, the water to cement ratio  method was used on small cylindrical samples 5 cm diameter and 10 cm in height on uncontaminated pervious concrete samples, and the standardized test method per ASTM C1701-09  was usedTo simulate and consider realistic parameters for drainage characteristics valorisation of laboratory-made pervious concrete samples, a new test setup is devised. As the unbound base course (UBC), natural gravel is used with grain size distribution presented in Maximum dry density and water content were determined by vibrating hammer according to standard EN 13286-4 . The matFor simulating clogging, a combination of silt (0–80 µm) and sand (0–2 mm) suspended in water were used in a ratio of 75–25%, respectively . The graPresented results are the average value of three samples per mixture with the variation of the results within 10%.Results of the hardened concrete mechanical characteristics investigation are presented in As presented in Porosity is usually discussed in a view of infiltration rate so it will be discussed within Results of infiltration rate obtained on small cylindrical samples of uncontaminated pervious concrete by the falling head method (FHM), on pervious concrete flags before and after clogging, as well as on paving systems tested following ASTM C1701 are presented in The influence of clogging in pervious concrete flags expressed as a reduction in infiltration rate due to clogging , is calcWhen the results obtained by two utilized methods, the falling head method (FHM) and method for inbuilding pervious concrete per ASTM C1701-09, are analysed it can be seen that the FHM results are higher . On averThe pervious concrete mixtures with coarse aggregate have better permeability characteristics, regardless of aggregate type . This isDifferent coarse to small fraction ratios were used to analyse in more detail how the variation of aggregate size influences pervious concrete characteristics, i.e., does it enhance strength characteristics while preserving porosity and permeability. For dolomite aggregate, as presented in As presented in Small-size aggregate mixtures have a higher infiltration rate reduction , primari3 lower density, with the difference being 1% to 30%. The highest drops were observed for coarse aggregate mixtures, with up to 38% for M8, which indicates that the influence of UBC density is higher for coarse aggregate mixtures. For mixtures with higher porosity and large pores at the surface, water velocity is high and UBC exfiltration is insufficient, causing the creation of a water film at the contact of the pervious concrete slab and UBC Mechanical characteristics of pervious concrete can be significantly influenced by an educated selection of the type of fine fraction. Using 10% sand instead of 10% diabase of the fraction 0–2 mm results in a significant increase in compressive and flexural strength with negligible reduction in porosity.(2)Pervious concrete mixtures with coarse aggregate show beneficial permeability characteristics, regardless of aggregate type.(3)Optimal porosity and infiltration rate with dolomite aggregate can be achieved with a mixture having 30% of 4–8 mm aggregate fraction.(4)Design of pervious concrete mixture composition should consider the type of used aggregate with its surface characteristics.(5)Small-size aggregate mixtures of pervious concrete have higher infiltration rate reduction due to clogging. However, due to a small difference in infiltration rate drop for small and coarse aggregate mixtures and favourable influence on mechanical characteristics, using a portion of small aggregate is desirable, particularly in wearing (surface) course.(6)A significant infiltration rate drop, particularly within coarse aggregate mixtures, occurs when a real pavement system is simulated by the unbound base course beneath the pervious concrete slab.(7)Mixtures with a combination of small and coarse dolomite aggregate exhibit less than 10% infiltration rate drop under the influence of clogging or UBC. Generally, it is important to find a balance between pervious concrete infiltration and UBC exfiltration rate. An excessive pervious concrete infiltration rate could adversely influence the pavement system due to UBC inability of sufficient exfiltration, which could lead to pavement damage. Pervious concrete mixtures with a combination of small and coarse aggregate should be considered.Drainage characteristics of 11 pervious concrete mixtures were investigated within this research to define a suitable mixture for low-traffic pavement surfaces. The aim was to define the influence of clogging and the impact of the unbound base layer on pervious concrete infiltration ratio reduction of a pavement surface course. Based on the results, the following conclusions can be drawn:The conclusions presented here are drawn from the research conducted on a limited number of samples and, to reach general conclusions, more tests are to be conducted.


Bleomycin (BLM) is a medication introduced used to treat various types of cancer, including testicular cancer, ovarian cancer, and Hodgkin’s disease. Its most serious side effect is pulmonary fibrosis and impaired lung function. Using A549 human lung cells it is shown that, in parallel to an increased cell toxicity and DNA damage, BLM causes a marked enlargement of the cell nucleus. This effect is abolished by inorganic polyphosphate (polyP), if this physiological polymer is administered together with BLM. The detoxification of BLM is–most likely–caused by the upregulation of the gene encoding the BLM hydrolase which inactivates BLM in vitro and in vivo. This study contributes also to a rational application in COVID-19 patients since polyP prevents binding of SARS-CoV-2 to host cells.−1 causes a decrease in cell viability (by ~70%), accompanied by an increased DNA damage and chromatin expansion (by amazingly 6-fold). Importantly, the BLM-caused effects on cell growth and DNA integrity are substantially suppressed by polyP. In parallel, the enlargement of the nuclei/chromatin in BLM-treated cells  is normalized to ~12 µm after co-incubation of the cells with BLM and polyP. A sequential application of the drugs  does not cause this normalization. During co-incubation of BLM with polyP the gene for the BLM hydrolase is upregulated. It is concluded that by upregulating this enzyme polyP prevents the toxic side effects of BLM. These data might also contribute to an application of BLM in COVID-19 patients, since polyP inhibits binding of SARS-CoV-2 to cellular ACE2.The anti-cancer antitumor antibiotic bleomycin(s) (BLM) induces athyminic sites in DNA after its activation, a process that results in strand splitting. Here, using A549 human lung cells or BEAS-2B cells lunc cells, we show that the cell toxicity of BLM can be suppressed by addition of inorganic polyphosphate (polyP), a physiological polymer that accumulates and is released from platelets. BLM at a concentration of 20 µg ml The bleomycins (BLMs) are glycopeptides size of ~1415 g/mol) with antineoplastic activity, which bind to DNA in a particular manner and cause strand-scissions in a reaction in which Fe(II) and oxygen are required 415 g/mol who firs5 and A2 , is forming with O2 a complex. The formed BLM-Fe(II)-OO accepts an electron and H+, a process during which the complex is converted to BLM-Fe(III)-OOH  were stained with DRAQ5  ,52. AlteThe images were taken with a fluorescence microscope  using a wavelength of 646 nm/662 nm (DRAQ5) and 495 nm . Where indicated Nomarski optics was used to contrast the light microscopical images. The nuclear sizes were obtained by application of ImageJ .The cells either A549 adenocarcinomic human alveolar basal epithelial cells, BEAS-2B cells which are immortalized but non-tumorigenic human cell, or human osteogenic sarcoma cells   were uset test was applied. The average values came from ten independent experiments. Values of p < 0.05 were considered statistically significant (*). The calculations were performed with the GraphPad Prism 7.0 software .For the statistical analyses the Student’s The Fast Micromethod was applied to determine DNA damage in A549 cells as described . This sy−2 in 8-well plates. After an incubation period of 3 days the cells were collected and used for the break determinations. Incubations with BLM, polyP or combinations are specified with the respective experiments. Five parallel assays were performed. The significance was calculated by ANOVA (by Bonferroni adjustment) program [A549 cells were seeded at a density of 40,000 cells cm program .−2) and incubated for 1 or 3 days. Then RNA was extracted, reverse-transcribed into cDNA using reverse transcriptase  and an oligo(dT)16 primer (Promega). The amplified PCR products were obtained after 28–32 cycles . For the amplification of the BLM hydrolase [GAPDH  with the primer pair Fwd, 5′-ACTTTGTGAAGCTCATTTCC-3′ and Rev, 5′-TTGCTGGGGCTGGTGGTCCA-3′. The amplifications were performed in triplicate in an iCycler , and the mean Ct values and the efficiencies were calculated applying the iCycler software (Bio-Rad) [A detailed description of the qRT-PCR (reverse transcriptase-PCR) procedure applied has been given before . The A54ydrolase ,61,62, tBio-Rad) ; the estp < 0.05 were considered statistically significant (*). The calculations were performed with the GraphPad Prism 7.0 software.Student t test was applied to perform comparisons between two groups. Values of −1 a maximum is achieved with about 60% reduction of the proliferation rate. Based on this finding a BLM concentration of  20 µg mL−1 was applied for the A549 cell studies presented here. In the latter cell system (A549 cell) an ~70% reduction of the viability was obtained by using the MTT assay .It is surprising that BLM does not cause complete inhibition of cell proliferation in vitro not only for tumor cells  but alsoTT assay A. Lower −1 was applied since this concentration has earlier been detected to cause a morphogenetic effect on cells in vitro, for example to induce mineralization in osteoblast-like SaOS-2 cells [−1) caused a 66% increase in A549 cell growth (−1) a gradual decrease of BLM-caused cell toxicity is measured. At the level of 1 µg mL−1 of co-addition the reduction of BLM-caused toxicity  comes to ~60%, a value that further increases close to a value seen in the polyP assays alone.It was the aim of the present study to elucidate if the physiological, morphogenetically acting polymer polyP interferes with the growth of the cells. The soluble Na-polyP was used for the study, here a concentration of also 20 µg mL-2 cells . This col growth A. If inc−1 to 70.7%, at 3 µg L−1 to 43.9%, and at 10 µg L−1 to 26.8%, respectively. In contrast, polyP increase cell viability at concentrations >3 µg mL−1 to 126% (3 µg mL−1) or to 163% (10 µg mL−1). In combination, addition of BLM together with polyP to the BEAS-2B cells: If polyP is added at a concentration of 10 µg L−1 to the cultures, exposed to 3 µg L−1 BLM, the degree of viability increased to 134%. Likewise, cultures incubated with 10 µg L−1 BLM, together with 10 µg L−1 of polyP, showed still a higher viability with 111%; the controls (without BLM or polyP) were set to 100%  value of 0.61. In contrast, cells incubated with 20 µg mL−1 of polyP alone, or in combination with 20 µg mL−1 of BLM reached again control values, of around 0.05.The A549 cells were–after seeding–incubated for 3 days in the presence of those drug concentrations that have also been used for the co-incubation experiments in the MTT study. In the controls, only a low level of DNA breaks is detected with an SSF × (−1) value of 0.04 B. This l−1 of polyP the morphology does not change and also the number of nuclear division figures remain the same between the controls and the polyP treated samples  treated assays is similarly dense (−1) show a much larger cell nucleus, with even ~25 µm in diameter after the 3 days incubation period (−1) together with polyP (20 µg mL−1). Under those conditions, the morphology of the cells and the nuclei in them show normal sizes -caused increase in cell volume is reversed by a secondary application of polyP (20 µg mL−1) to the cells (−1) for a period of 3 days the size of the nuclei decreases slightly to 23.3 ± 8.4 µm  to ~5000 µm3 (BLM-treated cultures for 3 days) during the 3 days incubation period. However, if in a pre-incubation period the drug BLM exposure was shortened to 12 h or 24 h followed by the addition of polyP (in the absence of BLM) the size of the nuclei increased only slightly to to ~800 µm3 (12 h BLM) or ~1500 µm3 (24 h BLM). These data show that the aberrant effect of BLM is much reduced during a shorter incubation period.A quantitative assessment of the rescue effect of polyP was performed both with A549 cells and with BEAS-2B cells . The siz−1) or polyP (20 µg mL−1) alone or in combination and the steady-state expression of the gene encoding the BLM hydrolase was measured by qRT-PCR. The exposure of the cells to the single components does not cause a significant difference in the expression level within one group. After 1 day of incubation, an approximately 70% increase of the expression levels is seen  drug complex together with H2O2 in one step. The oxidation state of Fe in BLM is reversible [2, the DNA is attacked via a 4′-radical intermediate a process which causes the releases of thymine from the DNA und the formation of an apyrimidinic site. From there DNA damage starts and allows the 3′-phosphoglycolate and the 5′-phosphate termini to form [BLM is a member of a first class of DNA splitting drugs that remove preferentially thymine from double- and single-stranded DNA . After tversible   or with Calcein-AM (whole cell morphology) or by light microscopy using Nomarski optics. Again the finding that the size of the cells in BLM-treated cultures dramatically increased from a volume seen in the controls, with ~900 µmeviously . Alreadyeviously . It remaeviously ,77. Howeeviously . A furtheviously , or durieviously  is  A549 cells can be abolished by co-incubation with polyP. This finding will contribute to a further rational application of this drug during the initiation of lung toxicity, fibrosis, also with respect to a use of BLM in the treatment schedule in COVID-19 patients. In one in vivo study, using the dextran sodium sulfate (DSS)-induced chronic colitis model in mice, it had been disclosed that polyP ameliorate fibrosis in vivo in the mouse model . The gatWith respect to a potential therapeutic intervention controlling COVID-19 disorders a modulation with the transforming growth factor β had been discussed . This co


Among hundreds of thousands of signal receptors contributing to oncogenic activation, tumorigenesis, and metastasis, the hepatocyte growth factor (HGF) receptor – also called tyrosine kinase MET – is a promising target in cancer therapy as its axis is involved in several different cancer types. It is also associated with poor outcomes and is involved in the development of therapeutic resistance. Several HGF/MET-neutralizing antibodies and MET kinase-specific small molecule inhibitors have been developed, resulting in some context-dependent progress in multiple cancer treatments. Nevertheless, the concomitant therapeutic resistance largely inhibits the translation of such targeted drug candidates into clinical application. Until now, numerous studies have been performed to understand the molecular, cellular, and upstream mechanisms that regulate HGF/MET-targeted drug resistance, further explore novel strategies to reduce the occurrence of resistance, and improve therapeutic efficacy after resistance. Intriguingly, emerging evidence has revealed that, in addition to its conventional function as an oncogene, the HGF/MET axis stands at the crossroads of tumor autophagy, immunity, and microenvironment. Based on current progress, this review summarizes the current challenges and simultaneously proposes future opportunities for HGF/MET targeting for therapeutic cancer interventions. MET is a tyrosine kinase receptor with one well-established ligand, hepatocyte growth factor (HGF) . MET is Since the HGF/MET axis plays an important role in cancer, various approaches have been explored to inhibit it, including the use of MET-neutralizing antibodies, HGF antagonists, and tyrosine kinase activity-targeted inhibitors (TKIs). MET-specific small-molecule TKIs are divided into two functionally distinct classes: type I  and type II  inhibitors, which preferentially bind to the active and inactive conformations of MET, respectively . PreclinIt is of the utmost importance to understand the molecular mechanisms regulating resistance to specific therapies to facilitate the application of alternative agents and improve clinical efficacy through the early identification of resistance and its relevant mechanisms. Therefore, an in-depth investigation of drug resistance is critical to improving the efficacy of MET-targeted drugs. Recently, as genotype-targeted therapies have been adopted for the treatment of tumors, several new mechanisms that explain the resistance to targeted therapies have been discovered. Additionally, the resistance to MET-targeting TKIs has been partially analyzed and is mainly attributed to the mutation, fusion, and focal amplification of MET and/or other genes involved in survival. Additionally, the coordination of tumor metabolism and autophagy are critical for the resistance to HGF/MET-targeted therapy . Interesp-21-activated kinase 1 (PAK1) amplification may be the cause of resistance to onartuzumab. Moreover, onartuzumab and emibetuzumab did not achieve satisfactory clinical results in clinical trials  is required to acquire resistance to MET inhibitors in the MET-dependent tumor model. PIM 1/3 upregulation is associated with acquired resistance to MET inhibitors. PIM kinases mediate resistance to MET inhibitors through the control of cap-independent Bcl-2 translation . Indeed, in vivo .A number of publications have reported that several micro-RNAs (miRNAs) inhibit tumor progression by targeting MET . FurtherOverall, acquired resistance to HGF/MET-targeted therapy can occur due to a variety of mechanisms: (i) second-site mutations in the MET kinase domain or abnormal activation of the HGF/MET signaling pathway, (ii) oncogene overexpression or the induction of bypass signaling pathways, (iii) copy number changes, and (iv) upregulation of downstream signaling molecules or formation of fusion proteins . TherefoAutophagy, one of several cellular adaptive responses to therapeutic stresses caused by anti-cancer agents, is an evolutionarily conserved proteolytic process in which damaged cellular components are incorporated into lysosomes for degradation and material recycling . Thus, aHelicobacter pylori CagA protein negatively regulates autophagy via the MET-PI3K/AKT-mTOR signaling pathway . Moreover, exposure to pro-inflammatory cytokines can lead to the induction or upregulation of MET expression in various cell types. In addition, HGF/MET signaling affects numerous immune cells, including mast cells , DCs Ba, LangerhListeria monocytogenes internalin B to MET provides the functionality required for peritoneal mast cell activation, and crosstalk between MET and α2β1 integrin may contribute to mast-cell activation in autoimmune conditions , immune and other cells, and molecules. It has recently been recognized as an important factor in sustained resistance to targeted therapies. For example, the production of ligands in a paracrine manner can activate signals that are able to compensate for drug-inhibited pathways in tumor cells . Tumor-aIn addition, hypoxia is a characteristic of the tumor microenvironment and is closely related to resistance to MET-targeted inhibitors. Recent studies have shown that under hypoxic conditions, tumor cells developed resistance to MET inhibitors (PHA-665752 and SU11274) and, interestingly, could recover their sensitivity to MET-TKI when normoxia was restored. Hypoxia significantly reduces the phosphorylation of critical MET residues, whereas MET downstream signaling, AKT, and ERK are not affected. After restoration from hypoxia, MET phosphorylation and activity can be quickly recovered . These pAlthough many inhibitors and antibodies against HGF/MET are available, most of them are designed to block HGF-mediated MET activation or directly inhibit the kinase activity of MET. However, due to the aforementioned reasons, the targeting mechanisms of MET inhibitors may potentially cause drug resistance and poor clinical efficacy. Therefore, it is important to improve the clinical efficacy of MET inhibitors to reduce the occurrence of drug resistance. According to these needs, we have constructed an anti-MET single-domain nanoantibody (VHH) pool to improve the efficacy of MET inhibitors. The anti-MET VHH pool against the whole MET ectodomain can effectively decrease MET phosphorylation and protein expression, and inhibit tumor cell proliferation, invasion, and tumor growth . HoweverAs we have previously explained, the HGF/MET signaling pathway is closely related to tumor immunity and, thus, researchers reconsidered an HGF/MET-targeted strategy for immunological stimulation and activation to obtain better tumor-killing effects. A bispecific monoclonal antibody targeting MET and PD-1 has been designed, developed, and tested for multiple cancer models by Acquired resistance to targeted therapy can occur through numerous mechanisms, including MET point mutations, increasing copy number, and bypassing signaling pathway activation. Some studies suggested that switching from type I to type II inhibitors, or vice versa, results in an acquired resistance mutation to each type . For insCancer promotion and survival depend on the complex signaling network between tumor and stromal cells in the surrounding microenvironment. Furthermore, HGF is secreted by both cancer and stromal cells. As The HGF/MET pathway is considered a promising target in multiple cancer types. Resistance is the result of the complex interactions among various receptor tyrosine kinases and other proliferative signals. Thus, the mere inhibition of MET phosphorylation with a single drug treatment may be insufficient to suppress the HGF/MET pathway . EncouraTaken together, the role of HGF/MET in cancer seems to be quite complicated. HGF/MET has oncogenic and pro-metastatic effects, but also has anti-cancer functions. Accumulating evidence suggests a close relationship between the HGF/MET signaling pathway and induction of the immune response. Thus, it remains uncertain whether it acts as an immunosuppressive or an immune-positive stimulus. Therefore, more in-depth investigations of these controversies should be performed to provide a better understanding of the mechanisms of resistance and accelerate the development of next-generation MET-TKI-targeting drugs.XH, XB, and TL designed this project and supervised the work. XH, EL, and HS performed the reference collection, analysis, and interpretation, wrote the manuscript, and contributed equally to the drafting process. XW, TT, XZ, JX, ZT, and CG discussed and commented on the arrangement of the manuscript. All authors were involved in the revision of the manuscript, read and approved the final manuscript version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A slump flow test, a viscosity test, and setting time measurement were conducted to identify the impacts of NS and MgO on the rheological properties of UWC. The pH and turbidity were measured to investigate the anti-washout performance of UWC mixes. To analyze the hydration characteristics and mechanical properties, hydration heat analysis, a compressive strength test, and thermogravimetric analyses were conducted. The experimental results showed that the fine particles of NS and MgO reduced slump flow, increased viscosity, and enhanced the anti-washout resistance of UWC. In addition, both NS and MgO shortened the initial and final setting times, and the replacement of MgO specimens slightly prolonged the setting time. NS accelerated the peak time and increased the peak temperature, and MgO delayed the hydration process and reduced the temperature due to the formation of brucite. The compressive results showed that NS improved the compressive strength of the UWC, and MgO slightly decreased the strength. The addition of NS also resulted in the formation of extra C–S–H, and the replacement of MgO caused the generation of a hydrotalcite phase.In this paper, the effect of nano-SiO As bridges continue to age, experts have been increasingly concerned about their structural safety, and suggest strengthening procedures. However, structures located underwater are more difficult to strengthen than superstructures. The existing strengthening methods ,3,4,5,6 UWC is generally produced using viscosity-modified admixtures (VMAs) and anti-washout admixtures (AWAs) to fabricate viscous concrete mixes . It is dSeveral attempts have been made to control the rheological properties and washout resistance of UWC. Park et al.  studied 2 (NS) is the common name for nano-sized silica particles with a diameter between 5 nm and 100 nm. NS particles are about 1000 times smaller than OPC particles [Nano-SiOarticles . Previouarticles ,22 foundarticles ,26,27,28articles  of NS haMagnesium oxide (MgO) is widely used in concrete manufacturing. Previous studies have demonstrated that the hydration and expansion characteristics of MgO positively affected concrete by reducing the hydration heat, decreasing the shrinkage, and changing the porosity and pore size distribution ,30,31,32In summary, this study investigated the effects of NS and MgO as mineral admixtures on the workability, washout properties, strength development, and hydration process of UWC. A slump flow test and a viscosity test were conducted to analyze these effects on the workability of UWC; the pH and turbidity measurement tested the anti-washout resistance of UWC; the setting time and isothermal conduction calorimetry were used to determine the hydration characteristics of UWC; and a compressive strength test measured the mechanical property of UWC.2 (NS) powder with a purity of 99.5% and average particle sizes of approximately 20–40 nm was used as an additional binder material.Ordinary Portland Cement (OPC) obtained from A company  that complies with ASTM C-150  was usedFive mix proportions of non-dispersible UWC and control concrete mixtures were prepared. The concrete mixes were prepared with a laboratory-scale concrete mixer . The fresh properties of the UWC mixes were determined by testing their workability. The slump test was conducted according to ASTM C 143 . The visTo measure the anti-washout resistance of the UWC mixes, the pH and turbidity measurements preceded via a method used in a previous study . First, To evaluate the mechanical properties of UWC concrete mixes, the compressive strength of the concrete cylinders with dimensions of Φ 100 mm × 200 mm at 7 and 28 days of curing was measured using a universal testing machine  and following ASTM C39 . Three rIsothermal calorimetry is a convenient means of examining the hydration characteristic of cementitious materials at early ages. The heat evolution was investigated using a semi-adiabatic calorimeter, as in a previous study . The spe2 environment at a heating rate of 10 K/min within a temperature range of 2 to 1000 °C.The hydration product of the UWC concrete specimens at later ages was measured via thermogravimetric analysis (TGA). The powder samples were obtained from pieces of the concrete specimens used for the compressive strength test. A thermogravimetric analyzer  was used for TGA analysis in an NTo evaluate the effects of NS and MgO on the workability of UWC concrete mixes, a slump flow test was conducted, as shown in −1, the shear stress was significantly reduced, probably because of destruction of the flocculated structures due to rotor rotation [rotation . The sherotation . As NS h2 formation during MgO hydration. Polat et al. [2 formation slowed the hydration process and delayed the setting time by encircling the cement particles.The initial setting time, the final setting time, and the setting time that is the duration between the initial and final setting times of the UWC pastes with different NS and MgO contents, are illustrated in t et al.  found thTo evaluate the anti-washout resistance of the UWC concrete mixes, pH measurements and a turbidity test were conducted. 2 and water during the hydration process [To investigate the effects of NS and MgO on the mechanical properties of the UWC concrete, a compressive strength test was conducted after 7 days and 28 days of curing. The results are shown in  process ,57. Unlu process  reportedThe isothermal calorimetry method is used to investigate the effects of temperature on the reaction kinetics of cementitious materials . Figure The first sharp peak was produced from the dissolution of the dry mixtures, and occurred immediately after the mixtures were mixed . The sec3. However, this carbonate region can be generated through powder manufacturing [acturing . The resThis study investigated the effects of NS and MgO on the hydration characteristics and anti-washout resistance of non-dispersible UWC concrete. The following conclusions were reached.The results of the rheological experiments revealed that the fine particles and large specific areas of NS and MgO reduced the flowability and increased the viscosity of UWC. However, the impact of the smaller NS on the rheological properties was greater than that of MgO;Both NS and MgO shortened the initial and final setting times compared to the control specimens due to their fine particle sizes. However, MgO prolonged the setting time  due to the formation of an insoluble hydration product. The hydration heat analysis showed that NS accelerated the hydration process and increased the hydration heat, whereas MgO delayed the peak time and reduced the temperature;The anti-washout performance results showed similar pH and turbidity tendencies in all the mixes. Both the addition of NS and replacement OPC with MgO reduced the pH and turbidity values, demonstrating that both NS and MgO increased the cohesion of the UWC mixes due to their finer particle size compared to that of OPC, which enhanced the anti-washout resistance of the UWC;The addition of NS slightly increased the compressive strength of the UWC because the high pozzolanic activity of NS generated extra nucleation sites and accelerated the hydration process. However, MgO decreased the compressive strength of M5 and M10 by 7.13% and 10.48%, respectively, compared to that of the control specimen due to the formation of brucite;The TGA results showed that NS promoted the generation of C–S–H gel due to its pozzolanic activity and nucleation effect. In addition, the specimens that contained MgO showed enhanced mass loss fractions at 25 to 400 °C, which generated larger amounts of hydrotalcite.


Clostridioides difficile disease and attenuated emergence of antibiotic resistant organisms. SYN-007 is a delayed release formulation of ribaxamase intended for use with oral beta-lactams. In dogs treated with oral amoxicillin, SYN-007 diminished antibiotic-mediated microbiome disruption and reduced the emergence of antibiotic resistance without altering amoxicillin systemic absorption. Here, SYN-007 function in the presence of clavulanate, a beta-lactamase inhibitor, was investigated. Dogs received amoxicillin , three times a day (TID)) or the combined antibiotic/beta-lactamase inhibitor, amoxicillin/clavulanate  +/™ SYN-007  for five days. Serum amoxicillin levels were not significantly different +/™ SYN-007 compared to amoxicillin alone or amoxicillin/clavulanate alone as controls for both first and last doses, indicating SYN-007 did not interfere with systemic absorption of the antibiotic. Whole genome shotgun metagenomics analyses of the fecal microbiomes demonstrated both amoxicillin and amoxicillin/clavulanate significantly reduced diversity and increased the frequency of antibiotic resistance genes. Microbiome damage appeared more severe with amoxicillin/clavulanate. In contrast, with SYN-007, microbiome diversity was not significantly altered, and frequency of antibiotic resistance genes did not increase. Importantly, SYN-007 functioned in the presence of clavulanate to protect the gut microbiome indicating that SYN-007 activity was not inhibited by clavulanate in the dog gastrointestinal tract. SYN-007 has the potential to expand microbiome protection to beta-lactam/beta-lactamase inhibitor combinations delivered orally or systemically.Beta-lactamases, enzymes produced by bacteria to degrade beta-lactam antibiotics, have been harnessed as therapeutics to protect the gut microbiome from damage caused by antibiotics. Proof-of-concept of this approach using SYN-004 (ribaxamase), a beta-lactamase formulated for oral delivery with intravenous (IV) penicillins and cephalosporins, was demonstrated with animal models and in humans. Ribaxamase degraded ceftriaxone in the gastrointestinal tract, protected the gut microbiome, significantly reduced the incidence of  Clostridioides difficile infection (CDI) [The gut microbiome, composed of the commensal microbiota and their genetic material, represents a complex ecosystem important to human health. Antibiotics disrupt this bionetwork, resulting in alterations of the normal microbial balance that can weaken colonization resistance and result in pathobiont overgrowth. Broad-spectrum antimicrobials, of which the beta-lactams are the most commonly used, are especially harmful to the beneficial anaerobes inhabiting the gut ,2,3, andon (CDI) ,5. In adon (CDI) . Selection (CDI) ,7. ThereBeta-lactamases are enzymes naturally produced by bacteria that specifically inactivate beta-lactam antibiotics via hydrolysis. SYN-004 (ribaxamase) is a beta-lactamase enzyme formulated for oral administration and intended for use with intravenous (IV) beta-lactams to degrade antibiotics excreted through bile into the GI tract to protect the intestinal microbiota. Ribaxamase is formulated with a pH-sensitive enteric coating that protects the beta-lactamase from stomach acid and proteases and releases the enzyme at pH 5.5 or greater, the pH of the upper small intestine, proximal to the site of bile release . In animHowever, the majority of beta-lactams are delivered orally, not systemically , and theThe efficacy of SYN-007 in protecting the gut microbiome was evaluated in dogs . AmoxiciBroadening microbiome protection to include oral beta-lactam antibiotics greatly expands the utility of oral beta-lactamase therapy. Oral amoxicillin is the most commonly used antimicrobial, and alone or in combination with the beta-lactamase inhibitor, clavulanate, accounted for over 70 million prescriptions in the U.S. in 2016 . AmoxiciHere, SYN-007 efficacy was evaluated in dogs that received oral amoxicillin or oral amoxicillin/clavulanate with or without SYN-007 for five days. After the first and last doses, serum antibiotic levels were measured to evaluate whether SYN-007 interfered with amoxicillin systemic absorption. Metagenomic and resistome analyses of fecal DNA that was collected before and after antibiotic exposure was performed to assess changes to the gut microbiome and gut resistome.® L30-D55  [® FS30D (Evonik) by spray coating as previously described [SYN-007 comprises of sugar pellets coated with ribaxamase and then covered with EudragitGermany) . The preescribed . A 10 mgescribed . SYN-007escribed .n = 5 each); Amoxicillin alone, Amoxicillin + SYN-007, Amoxicillin/Clavulanate alone, and Amoxicillin/Clavulanate + SYN-007.Female Beagle dogs, 7 to 8 months old and 6.5 to 7.7 kg, were purchased from Covance Research Products . Animals that were never exposed to antibiotics were ordered specifically for this study. Animals were received at the test site, Calvert Laboratories, Inc. , 30 days before study initiation and first sample collection to allow acclimation to their new environment. Animal health was evaluated daily. Individual animal housing was in compliance with United States Department of Agriculture Guidelines and animals were allowed to comingle prior to study initiation. Animals received antibiotic-free PMI Canine Diet. Animals were randomly divided into 4 cohorts  after each antibiotic +/™ SYN-007 dose. On study day 6, animals were fed 1.5 h after the last dose of antibiotic +/™ SYN-007. Body weights, recorded daily, were used to calculate the dose of amoxicillin or amoxicillin/clavulanate received on study days 1–6 to achieve 40 mg/kg per dose. Dogs were allowed free access to water.Amoxicillin was supplied as a powder  and resuspended in 100% Mott’s Apple Juice (pH 3.0) instead of water, per package instructions, at 400 mg/5 mL as previously described [The animal study protocol, 0832DS123.002, was approved on 8 February 2018 by Calvert Laboratories, Inc., Institutional Animal Care and Use Committee (IACUC). All animal procedures were performed in accordance with the Animal Welfare Act at Calvert Laboratories, Inc.  and in agreement with the guidelines established by the Calvert Institutional IACUC. Calvert Laboratories, Inc. is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AALAC).Liquid chromatography turbo ion spray tandem mass spectrometry (LC/MS/MS) was used for amoxicillin serum measurements as previously described . An amoxhttps://jgi.doe.gov/data-and-tools/) was used to evaluate read quality prior to metagenomics analyses. Samples had an average read quality of Q20 demonstrating 99% sequencing accuracy . Reads per sample were 60,000,000 ± 9,000,000 (mean and standard deviation), demonstrating similar read depth.Total DNA was isolated from fecal specimens, and libraries were constructed, quantified, and sequenced using a single Illumina HiSeq v3 flowcell aiming for 25 million 100 bp reads per sample as prevoiusly described . Open soCosmosID, Inc.  bioinformatics software was used to analyze unassembled whole genome metagenomic sequencing, as previously described , for bacBacterial metagenomics data were analyzed for alpha diversity using the Shannon Index , and bethttps://submit.ncbi.nlm.nih.gov/subs/sra/), Accession SRP093227.Fecal DNA metagenomics sequencing data are available in Sequence Read Archive (SRA) (p > 0.99). Similarly, by day 6, after 16 doses of antibiotic + SYN-007, there was no significant difference in amoxicillin serum level AUC for either group . Notably, amoxicillin blood levels appeared to be lower at the 3, 4, and 6 h time points in the amoxicillin + SYN-007 cohort, although this difference was not significant  comparing pretreatment microbiome composition to post-treatment was performed with a greater distance between points designating a more pronounced difference in microbiome composition . For easEscherichia coli was observed following amoxicillin/clavulanate alone exposure in two dogs resulting in E. coli monodomination, defined as >30% of microbiome composed of one species  [Megamonas hypermegale  or Bacteroides vulgantus . Species monodomination was not observed in the amoxicillin alone, or either SYN-007 cohorts. However, increased relative abundance of E. coli and/or M. hypermegale was observed in all amoxicillin alone treated dogs while increased abundance of Fusobacterium mortiferum was observed in two amoxicillin alone treated dogs and three amoxicillin/clavulanate + SYN-007-treated dogs. These data demonstrate that amoxicillin/clavulanate altered gut microbiome composition more drastically than amoxicillin alone, and that antibiotic-mediated changes to the gut microbiome were attenuated with SYN-007.Stacked bar graphs, constructed based on the relative abundance of each bacterial species in each sample, were used to visualize specific changes in microbiota composition of the fecal microbiomes before and after antibiotic exposure . Compare15, 56%) . LikewisFecal resistomes were analyzed to evaluate if SYN-007 affected antibiotic resistance gene emergence and propagation. Heatmaps of antibiotic resistance genes in the fecal microbiome of each animal before and after antibiotic treatment were generated. Emergence of beta-lactamase genes, which confer resistance specifically to beta-lactam antibiotics, was observed following antibiotic exposure . The amoIn addition to beta-lactamases, additional resistance genes were affected by antibiotic exposure . FollowiResults of the microbiome and resistome analyses demonstrate that oral administration of amoxicillin and amoxicillin/clavulanate caused alterations to gut microbiomes and resistomes. Amoxicillin/clavulanate exposure resulted in more dramatic shifts in gut microbiome and resistome composition compared to amoxicillin alone. Co-administration of SYN-007 with amoxicillin or amoxicillin/clavulanate attenuated gut microbiome and resistome changes.Antibiotic inactivation within the GI tract represents a novel strategy to preserve the gut microbiome from antibiotic-mediated damage. SYN-007, an oral delayed-release formulation of the beta-lactamase enzyme ribaxamase , was shoNotably, ribaxamase, derived from a class A serine beta-lactamase, is sensitive to beta-lactamase inhibitors in vitro , but wasWhile SYN-007 did not significantly alter amoxicillin systemic absorption when delivered with oral amoxicillin or amoxicillin/clavulanate, it is noteworthy that amoxicillin serum pharmacokinetic (PK) curves were indistinguishable with amoxicillin/clavulanate +/™ SYN-007 at day 6 D. In conE. coli, M. hypermegale, or B. vulgantus was observed after amoxicillin/clavulanate exposure in four of the five treated animals. The increased abundance of these bacterial species was detected after amoxicillin exposure, but levels approaching monodomination were not reached.Despite the potential advantage of clavulanate in augmenting SYN-007 GI tract utility, amoxicillin/clavulanate was more disruptive to the gut microbiome than amoxicillin alone. While both amoxicillin and amoxicillin/clavulanate exposure resulted in changes in the gut microbiome, including significant reduction alpha diversity, principal coordinate analysis revealed that gut microbiome beta-diversity was altered more severely with amoxicillin/clavulanate A. This cEscherichia species [Saccharomyces boulardii, to healthy subjects with amoxicillin/clavulanate limited microbiome damage and reduced AAD rates [These data may have implications regarding the clinical observation that amoxicillin/clavulanate therapy results in significantly increased incidence of antibiotic-associated diarrhea (AAD) compared to treatment with amoxicillin alone . Clavula species , and co-AD rates . To dateE. coli, M. hypermegale, and B. vulgantus , enzymes capable of inactivating a broad range of beta-lactam antibiotics including penicillins and cephalosporins . Perhapshe ESBLs . Infectihe ESBLs . Here, ghe ESBLs , were obsistance , to antiOther resistance genes were also affected by antibiotic exposure. Amoxicillin/clavulanate administration resulted in more pronounced changes to fecal resistomes than amoxicillin alone. An increased frequency of genes encoding multidrug efflux transporter system components that confer resistance to many antibiotic classes was observed with amoxicillin/clavulanate. Genes that displayed a decreased frequency with antibiotic exposure included genes conferring resistance to tetracycline and aminoglycosides. Co-administration of SYN-007 with amoxicillin and amoxicillin/clavulanate attenuated gut resistome changes. These observations suggest that alterations in the resistome were influenced by both direct selection of species resistant to beta-lactams, and by fluctuations in gut microbiota composition following antibiotic exposure. Changes in the frequency of a diverse set of antibiotic resistance genes following antibiotic exposure in dogs and pigs was reported previously ,30,32,33Protection of the gut microbiome from damage caused by antibiotics is critical for maintenance of health, preservation of colonization resistance, and mitigation of antibiotic resistance emergence. Broad-spectrum antibiotics such as the beta-lactams are especially harmful. Here, the beta-lactam/beta-lactamase inhibitor combination amoxicillin/clavulanate was demonstrated to cause more damage to the gut microbiome than amoxicillin alone and resulted in the emergence of genes encoding ESBL and IRT beta-lactamases. SYN-007, a beta-lactamase formulated for delayed release following oral delivery with oral beta-lactam antibiotics, protected the gut microbiome of dogs from amoxicillin/clavulanate and reduced emergence of antibiotic resistance. The use of beta-lactamase enzymes to degrade antibiotics within the GI tract may represent a viable strategy for the protection of the gut microbiome and offers a pharmacologic stewardship approach to combat the emergence of antibiotic resistance.


Our meta-analysis concluded that METs gave rise to pregnancies but were associated with a reduced ongoing/live birth rate and a higher miscarriage rate. All 37 MET live births were confirmed viable, among which 8 completed prenatal genetic testing with normal results. Longitudinal investigation on one MET pregnancy evidenced the aneuploidy depletion hypothesis. This is the first multi-center prospective study reporting a full MET pregnancy outcome with complementary information from prenatal genetic testing as compared to euploid and non-PGT cohorts.Chromosomal mosaicism is at high occurrence in early developmental-stage embryos, but much lower in those at prenatal stage. Recent studies provided evidence on the viability of mosaic embryos by reporting pregnancy outcomes. Expanded research is warranted to evaluate its clinical significance. This is a multi-center prospective cohort study on 137 mosaic, 476 euploid and 835 non-preimplantation genetic testing (non-PGT) embryos from three in vitro fertilization (IVF) providers of three countries in Asia, applying the same preimplantation genetic testing for aneuploidies (PGT-A) reporting criteria. Mosaic embryo transfers (METs) resulted in a significantly lower clinical pregnancy rate (40.1% versus 59.0% versus 48.4%), lower ongoing/live birth rate (27.1% versus 47.0% versus 35.1%) and higher miscarriage rate (33.3% versus 20.5% versus 27.4%) than euploid and non-PGT transfers, respectively. Pregnancy losses after METs were different between embryos carrying numerical and segmental chromosomal abnormalities ( Mosaicism has been reported in cleavage and blastocyst stage embryo biopsies. Chromosomal mis-segregation during either meiosis or mitosis results in aneuploidy. Age has been well characterized as the cause of aberrant chromosomal separation in gametogenesis, which leads to constitutional aneuploidy. Mitotic errors in early embryonic development have been characterized as the mechanism of chromosomal mosaicism, producing two or more cell lines within one embryo or organism. Current preimplantation genetic testing for aneuploidies (PGT-A) has a common resolution at 5 Mb to 10 Mb by sequencing and a reproducible detection limit of the mosaic level at 20% or 30% . On the Greco et al.  first reSince several studies have confirmed the lowered implantation potential of mosaic embryos, the major concern of METs remains to be the factor(s) that may affect live birth rate, as well as the antenatal and postnatal health of babies derived from mosaic embryos. Therefore, we conducted this multi-center prospective cohort study with the aim of characterizing factors that affect live birth rate and obtaining additional information from prenatal and/or postnatal genetic follow-ups of MET pregnancies.This study was approved by the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee (CREC-2010.432 and CREC-2007.059). Patient gave their written informed consent before inclusion.This study was conducted at three in vitro fertilization- preimplantation genetic testing (IVF-PGT) centers in Hong Kong , Malaysia  and Thailand (Safe Fertility Center). The study period spanned from 2016 to 2018 and recruited all IVF and Intracytoplasmic sperm injection (ICSI) cycles with embryo transfers. The viability of mosaic embryos was studied by reviewing the pregnancy outcome and making comparisons with an age-matched control group. A total of 137 mosaic embryos transferred in 133 frozen-embryo transfer (FET) cycles (including 129 single- and 4 double-embryo transfers) and 476 euploid embryo transferred in 447 FET cycles (including 418 single- and 29 double-embryo transfers) from 3 centers were included in the MET group and euploid control group, respectively. In addition, 835 embryos from 777 non-PGT cycles were included as a non-PGT control group .Prior to the detection and reporting of mosaicism, cell line mixes by gradual proportions of mosaicism  were tested for validation of both chromosomal microarray analysis  and next-generation sequencing (NGS) by VeriSeq  platforms. This study was designed in three parts. The first part was a multicenter prospective cohort study of pregnancy outcome of METs. Two centers used VeriSeq platform while one center used chromosomal microarray analysis (CMA) for PGT-A. With the multi-center data, a meta-analysis was performed to summarize the overall risk ratio of pregnancy outcome by the MET approach. Thirdly, one MET patient was longitudinally followed up from preimplantation to the prenatal and postnatal stages. Biopsies from multiple placental sites were tested by CMA.Two centers cultured embryos in G-TL medium  and one in G1/G2 sequential media  supplemented with 10% synthetic serum substitute . Assisted hatching was performed using non-contact laser . Laser-assisted isolation of trophectoderm (TE) was performed on day 5 or day 6 of embryo development if a blastocyst had discernable inner cell mass and TE. Morphological grading of blastocysts was assessed according to Schoolcraft and Gardner . BlastocChromosomal microarray analysis by Agilent 8 × 60 K microarray chip and the NGS by VeriSeq PGS kit were conducted according to the manufacturer’s protocols. Data analysis was performed with Cytogenomics 2.7.8.0 software (Agilent) for CMA and BlueFuse Multi for NGS . Both pipelines used human genome build 19 (hg19/GRCh37) as a reference genome.The NGS-based PGT-A method is characterized by having high detection resolution for segmental aneuploidies ,20. BaseThe option of mosaic embryo transfer would only be provided to patients when they had no euploid embryo. Genetic counseling was provided by clinical geneticists for the reasons, options, potential outcomes and prenatal management of transferring mosaic embryos ,23,24.Patients were highly recommended to perform prenatal diagnosis during genetic counselling. Prenatal genetic testing results were traced on pregnancies that were sustained beyond the first trimester. Non-invasive prenatal testing (NIPT) as the prenatal screening approach was traced and recorded by all three centers in this study. Genetic testing on amniotic fluid or chorionic villi by either karyotyping or CMA was performed in the case of prenatal genetic diagnosis. Karyotyping or CMA was performed on the product of gestation (POG) on patients’ request.Pregnancy outcome was traced and calculated at four stages according to nomenclatures ,26: (i) One MET pregnancy had full-term delivery with the whole placenta donated. Placenta biopsy was performed on 16 sites: 8/16 from the maternal side and 8/16 from the fetal side. Biopsy sites were randomly selected from different cotyledons that were naked-eye identifiable. Chromosomal microarray analysis  was performed on all 16 sites. As the sex of the fetus is known to be a male, male control was selected as a reference for CMA.The present meta-analysis was performed following the preferred reporting item for systematic reviews and meta-analysis (PRISMA) statement . The elePearson chi-square test was performed to test the outcome difference between MET and control groups. Fisher’s exact test was used for sample size smaller than 30. Two-sided significance of <0.05 was regarded as statistically significant compared to the null hypothesis. Statistics for this study was performed by SPSS .p-value of the heterogeneity test >0.1, the studies had sufficient similarities to be pooled together for analysis and a fixed model was performed. When the heterogeneity test had p < 0.1, a random effect model was applied with the outlier study subgrouped for comparison. Moreover, I2 > 50% was considered as moderate to high heterogeneity between studies and therefore was not eligible for inclusion of meta-analysis. Mantel–Haenszel (M–H) pooled risk ratios (RR) with a 95% confidence interval of implantation, miscarriage and ongoing pregnancy/live births were undertaken.Meta-analysis was performed by Review Manager version 5.4 . Publicap < 0.001) when compared with the euploid control , but not when compared with the non-PGT control . Moreover, embryos with mosaicism had a statistically lower chance of reaching clinical pregnancy in the MET group  than in the euploid control , but not in the non-PGT control . Thirty-six pregnancies (37 embryos) in the MET group led to ongoing pregnancies and live births, with an OPR of 27.1% (36/133), which was significantly lower (p < 0.001) than the OPR of 47.0% (210/447) in the euploid control. The OPR was also lower in the MET group than the non-PGT control , but with no statistical significance (p = 0.468). The miscarriages in the MET group occurred ranging from week 6 to week 17 of gestation, with the majority during the first trimester. The miscarriage rate was higher in the MET group  than in the euploid control , but there was no statistical difference between the MET group and the non-PGT control   with statistical difference (= 0.652) . In geneEmbryos in the MET cohort had an average mosaic level of 37% ± 11% with a range of 20% to 69%. On the other hand, pregnancy outcomes were globally decreased in the <50% level of mosaicism when compared to euploid transfers. The number of embryos with a mosaic level of <40% that reached clinical pregnancy and ongoing/live birth  was significantly less than euploid ETs. No significant difference was found within the MET group when 40% and 50% were set as the cut-off level of mosaicism .p = 0.04). However, no difference was observed in clinical pregnancies  and ongoing pregnancies/live births   and segmental (8 embryos) chromosomal abnormalities  .p = 0.021). Embryos with “fair” morphology consistently showed poorer outcome in the MET group  had a statistically lower chance of reaching clinical pregnancy than those of the control group . None of the nine studies opposed this conclusion (There were 421 mosaic (928 embryo population) and 3052 euploid (4585 embryo population) embryos transferred that resulted in implantation among the 9 studies that were included for meta-analysis of implantation rate. There was no statistical difference in overall heterogeneity. By a fixed model, the implantation rate was significantly lower in the MET group   .p = 0.17, this comparison was analyzed by a fixed model. The probability of miscarriage in MET is significantly higher than the control group   and 360 euploid (2819 embryo population) embryos were reported as miscarriage. Given that the overall heterogeneity showed no difference with  = 0.17) .p = 0.325) (Twenty-five out of 36 live births (69.4%) among MET and 64 out of 109 live births (58.7%) in the control had birth weight records. No difference was found in regard to birth weight between babies from MET (3180 ± 505 g) and those in the control group  . Among tThe MET case with negative amniocentesis had a full-term delivery and donated the placenta for research. The patient transferred a male embryo with 45% mosaic loss at [GRCh37] 8q21.13q24.3 (81900001_146300000), 64.4 Mb in size. By CMA on all 16 biopsies A,B, it wOur current multi-center study demonstrated the decreased viability of mosaic embryos and concluded with the same findings as all MET clinical studies by meta-analysis. With the previous proof-of-principle study  and mousDifferent to the previous study using 50% as cut-off , we testThe effectiveness of MET studies has been debatable on the detection accuracy of mosaicism and the aneuploid calling consistency between TE and inner cell mass (ICM). Capalbo et al. discussed the possibility of false positives in mosaic callings and suggested additional biopsies from the embryo reported, as mosaic embryos should be tested to confirm their true chromosomal constitution . Victor Although mouse model  and clinThis multicenter prospective study in Asia has fully followed up the pregnancy outcomes after mosaic embryo transfer to the perinatal stage. Meta-analysis confirmed the consistently lower pregnancy outcome of MET compared to euploid transfers. Nevertheless, the viable and karyotypically normal live births by MET suggest it as an alternative option for patients with no euploid embryo acquired from PGT-A cycles. However, this option could only be provided to patients under the condition of comprehensive genetic counselling and recommendation to follow-up with prenatal diagnosis. A larger-scale randomized controlled trial is necessary to further evaluate specific numerical and segmental chromosomal effects of mosaic embryo transfer. Even though there is no evidence on confined placental mosaicism due to MET, more data on multiple placental biopsies from MET pregnancies are warranted for further investigation of mosaic embryo development.


The paper presents experimental studies on axially compressed columns made of concrete-filled glass fiber reinforced polymer (GFRP) tubes. The infill concrete was C30/37 according to Eurocode 2. The investigated composite pipes were characterized by different angles of fiber winding in relation to the longitudinal axis of the element: 20, 55 and 85 degrees. Columns of two lengths, 0.4 m and 2.0 m, were studied. The internal diameter and wall thickness of all the pipes were identical and amounted to 200 mm and 6 mm, respectively. The mean values of two mechanical properties, elasticity modulus and compression strength, were determined. These properties were determined for longitudinal compression and for circumferential tension. The graphs of longitudinal and peripheral deformations of polymer shells as a function of load level are presented both for empty tubes and for concrete-filled ones. The results of long-term investigations of three identically made 0.4 m high concrete-filled GFRP tubes are also presented. CFFT columns are a modification of CFST (concrete-filled steel tubes) columns commonly used in construction ,2. They The use of FRP (fiber reinforced polymer) pipes for making columns is an interesting field of application of composites in concrete structures. Originally, the application of composites in concrete columns involved reinforcing them by sticking composite fabric bands in the overload zones. This well-known and widely used technology ,10 has tOne of the main disadvantages of steel is that it is subject to corrosion, and therefore it is necessary to incur additional costs related to maintaining load-bearing structures in good technical condition during the operation of the structure. In case of structures using corrosion-resistant composite reinforcement, significant maintenance costs are eliminated. Additionally, the concrete inside CFFT columns is fully protected against environmental impacts. Unfortunately, each material has its own disadvantages, and the main problem with composites is their low fire resistance.CFFT columns were used for a road bridge along road no. 40 over the Nottoway River in Virginia, USA ,14. One A more typical area of application for CFFT piles is the shielding structures of sea piers, in which traditionally used reinforced concrete or CFST piles are subject to accelerated corrosion due to the salinity of the water. The corrosion is particularly evident in the zone of the variable water table. One of many examples is the construction of a munitions dock pier in the military port of Hadlock on Indian Island in Washington, USA . The pilThe authors carried out research on six concrete-filled poles made of composite polymer pipes reinforced with glass fiber, produced by the Polish company Vitreo. Half of the pipes were 400 mm long and the other half were 2000 mm long. The internal diameter of the ordered pipes was 200 mm. The wall thickness of the pipes was 6 mm. The pipes were characterized by three different angles 20°, 55° and 85° of glass fiber cross winding in relation to the longitudinal axis of the pipe.The list of all the tested elements is presented in Six strain gauges were placed on the outside of the polymer pipes halfway along their length—three in a vertical position and three in a horizontal position, at equal spacing of one third of the circumference of the element .Unlike other researchers ,4,5,7, tThe glass fibers used for the production of the pipes were in the form of continuous rovings with a linear density of 2400 g/m (2400 tex). They were produced from E-type glass fibers in Slovakia by the American company Johns Manville. Polyester resin, with the trade name Crystic 2-420 PA, was produced in Croatia by the British company Scott Bader. The manufacturer declared the tensile strength of the hardened resin between 44 and 46 MPa, and the tensile modulus between 3.03 and 3.68 GPa. The resin hardener, called Butanox M-50, was produced by the Dutch company AkzoNobel. The pipes’ internal diameter declared by the manufacturer was 200 mm, and the wall thickness was to be between 5.5 and 6.0 mm. The former corresponded exactly to reality, while the wall thickness measured by the authors ranged from 4.9 to 8.8 mm. The measurement was made on ring samples cut from pipes . The aveThe glass fiber content of the composite, as stated by the manufacturer, was 60%. This content was verified on the basis of the standard method  used in The mechanical properties, compressive strength and modulus of elasticity of the pipe material were determined through the testing of 400-mm-long pipe sections . Prior tfFRP,c was carried out with the use of a hydraulic press produced by the Swiss company Walter and Bai AG, model 102/5000-HK4, with a load capacity of 5000 kN and piston extension from 0 to 100 mm. The load was controlled by the displacement, i.e., by the constant extension of the press piston. The press tooling made it possible to digitally record the measurement results. The measurement of the piston advancement and force values was carried out discretely at a time interval of several seconds. The duration of a single experiment was several dozen minutes.Experimental research of the pipes’ composite strength to longitudinal compression The mode of destroying the pipes is shown in The highest load capacity in the group of empty short pipes was obtained by a sample with fiber winding orientation closest to the longitudinal axis of the pipe. The load-bearing capacity of a pipe with a 20° winding angle had about 100% advantage over the other two tubes. It was probably obtained thanks to the compression strength of the glass fibers themselves. For pipes with fiber orientation more deviated from the vertical, the decisive factor was the compression strength of the resin itself, many times lower than the strength of reinforcing fibers  graph was taken into account, omitting only the falling part of the curve. It was assumed that the approximation line had to start at the beginning of the coordinate system. The resulting elasticity modulus values of three types of FRP pipes are given in These three groups were installed at a mutual distance of 120° around the circumference of the pipe. One strain gauge measuring the longitudinal deformations of the pipe and one strain gauge measuring the circumferential deformations were glued side by side. The strains of pipes were measured by the gauges discretely, every 0.5 mm of the press piston advancement. In this way, nine readings were obtained for each of the strain gauges in the case of the pipe with the winding angle θ = 20°, sixteen readings for θ = 55° and twenty readings for θ = 85°. The graphs presented in fFRP,c obtained in the tests described in the previous subchapter. It is worth noting that the value of elasticity modulus for glass fibers is about 75 GPa according to [The samples with the smallest winding angle in relation to the longitudinal axis (angle θ = 20°) reached the highest value of elasticity modulus in the longitudinal direction. Both remaining samples (winding angles θ = 55° and θ = 85°) were characterized by several-times-lower elasticity modulus, while the values of elasticity modulus of these samples were similar. In this respect, the results turned out to be similar to the results of composite longitudinal compression strength rding to , for polrding to  and betwfFRP,circ circumferential strength of the pipe and EFRP,circ elasticity modulus were performed with the standard method which consisted in tearing the ring cut out from the pipe. This method, called the split disk method, is used in Polish [The tests of n Polish , European Polish  standardn Polish , the rinAccording to the strain gauges manufacturer, the engineering tolerance of their gauge factors is 0.5%. A measuring uncertainty test was conducted for strains taking into account the tolerance of strain gauge factors along with other uncertainty factors: change in gauge factor and in amplifier characteristics due to change in ambient temperature, tolerance of measuring equipment including electric cables, imprecise gauge installing and a degradation in the measuring equipment sensitivity since the last calibration. The resulting total measuring uncertainty was equal to 1.4%.σ-ε was determined for each of the fifteen tested rings (σ-ε graphs for each of the composites tested is negative strain (shortening) in the initial phase of the experiment. This was induced by local bending of the ring at the point where two semi-disks meet. In this place, the ring was locally straightened, which caused compression of its outer fibers  and tension in the internal fibers. After reaching a certain level of tensile force, the local bending effect passes and the further development of the σ-ε relationship is almost linear.Having the relationship between the strength measured by the testing machine and the composite strain at the notch point of the ring, as well as the pipe wall width and thickness measured at the notch before the test, the relationship of ed rings . A charaσ-ε graphs were considered only within their parts of tensile strains. The parts of compressive strains of the outer ring fibers were completely removed from the charts  columns were made simultaneously from a single shipment of concrete. The ordered concrete was class C30/37 according to Eurocode 2 , with a Concrete compression strength and modulus of elasticity in compression tests were carried out on standard concrete cylinders with a diameter of 150 mm and a height of 300 mm. Concrete samples were stored from the moment they were made to be tested in the same thermal-humidity conditions as the columns, i.e., at a temperature of about 20 °C, with relative air humidity of about 50%. Both tests of concrete samples were carried out with the use of a German Heckert strength press, model DP1600, with an operating range from 0 to 1600 kN. Elasticity modulus of concrete was tested using a compressometer-extensometer according to standard .Concrete used for the construction of axially compressed columns was tested in parallel with the experimental tests of the load capacity of these columns. They were carried out after a period of eighty to ninety-two days from the moment of filling them with concrete. Because almost three months passed between preparing the columns and testing them, it was decided to assign the same strength characteristics of concrete to all six columns and to disregard small differences resulting from the concrete’s varied age. The strength of concrete obtained this way in the uniaxial compression state was 38.1 MPa after 28 d and 42.1 MPa after 86 d. Elasticity modulus of concrete was 31.9 GPa and 33.0 GPa respectively. Concrete density was not investigated.Short columns  resulted from a more effective reduction in the column deformation in the circumferential direction. The shells of these columns protected the concrete core from damage, probably even after the limit elongations were exceeded. This was clearly demonstrated by the fact that the samples were barrel-shaped after destruction.While discussing the “force—vertical displacement of the piston” graphs , it is iLong poles  = 38.1 MPa was the basis for calculating the level of long-term load in the creep tests. After axial alignment, the specimens were left unloaded in the creep testing machine for one hour. After that time the initial reading was taken and the whole cross-section was loaded. The test was carried out continuously until the stresses σ = 0.46 fc(28) were reached , taking readings at levels 0.36·σ, 0.73·σ and σ. The total load was obtained within ten minutes. The creep tests were carried out with the use of a German Walter Bai strength press, model HKB1000, with the accuracy class 1 and an operating range from 0 to 1000 kN. The measuring uncertainty of the machine indicated in its calibration certificate was in range from 0.25% up to 0.63%, depending on the measured force magnitude.After concreting, the specimens were placed in an air-conditioned chamber in which the relative humidity was 50% and the temperature 20 °C; they were stored there until the test. An extensometer with a resolution of 1/100 mm and a measuring base of 250 mm was used to read the deformations. Frames were mounted along three lines evenly distributed on the side of the cylinder (every 120°). The tests commenced after sixty-three days, counting from the moment the samples were concreted. The determined average compression strength of concrete after twenty-eight days fThe first measurement was made after five minutes and the result was considered to be a so-called “temporary deformation”. Then the readings were taken for the period of 392 d. In the first week, readings were made daily, during the next three months, once a week, and after that time, once a month. Based on the obtained results, a curve of rheological deformations was determined taking into account the creep of the concrete and composite FRP as well as concrete shrinkage .c(28), the largest longitudinal deformations were also observed in samples with a winding angle of 55°. The pipes with winding angle θ = 55° seem to be insufficiently stiff in the longitudinal direction and at the same time insufficiently stiff in the circumferential direction. As a result, the stub CFFT columns made of these pipes deform most severely in the longitudinal direction.The smallest deformations were obtained for the sample with the 20° winding angle and the largest for the sample with the 55° winding angle. This refers to both immediate and long-term deformations resulting from creep and shrinkage of concrete. It is a consequence of the influence of the angle of glass fiber winding on the deformability of the composite. For pipes with fiber orientation more deviated from the vertical, the deformability of the resin itself is decisive see . It is wcolumns made of FRP pipes filled with concrete ) behave in a way that depends on the direction of fiber winding in the pipe;the load-bearing capacity of the CFFT columns is highest when the fibers are wound in a circumferential direction;the difference in load-bearing capacity caused by the direction of the fiber winding can be very large for stub columns and reach more than 150%;for slender columns used in the construction industry, however, the difference will be much lower—in the conducted studies the difference amounted to 33% at the most;brittle failure mode and low post-critical load capacity eliminate columns with a fiber winding angle close to the longitudinal one (in the study in question the winding angle was 20°) from applications in the construction industry;pipes with a winding angle close to the longitudinal one are characterized by increased resistance to longitudinal compression (in the studies described herein by as much as 100%) and higher modulus of elasticity, but these advantages are secondary to the disadvantages described above;in long-term tests, similar to short-term tests, the specimen with a fiber winding angle close to the longitudinal one showed the least strains;the biggest strains in long-term tests were obtained for the specimen with a fiber winding angle of 55°.The conducted experimental research allows the formulation of the following conclusions:


Mikulicz's disease is a unique condition involving the enlargement of the lacrimal and salivary glands, similar to that observed in Sjogren's syndrome; however, Mikulicz's disease is clinically characterized by infrequent autoimmune reactions and responsiveness to glucocorticoid treatment. The ultrasound features of the lacrimal and salivary glands in patients with IgG4-Mikulicz's disease were characterized by multiple hypoechoic areas of varying sizes within the enlarged glands. IgG4 serum level was also elevated, in contrast to the detection of normal levels in Sjogren's syndrome. In this article, we intended to illustrate a case of Mikulicz's disease with clinical and imaging features. IgG4-related disease (IGG4-RD) is a chronic inflammatory disease, characterized by swollen and thickened lesions of the affected organs, such as lacrimal glands, salivary glands, or pancreas, with high serum concentrations of IgG4, and marked IgG4-positive plasma cell infiltrations of the affected tissues. Inflammation can extend to multiple organs, including the biliary tree and kidney, during long-term follow-up. Therefore, IgG4-RD is generally regarded as a systemic disease. Diseases involving lacrimal and major salivary glands have often been referred to as “Mikulicz´s disease” or “Mikulicz´s syndrome” in the past . Since tA 32-year-old female patient was admitted to hospital with bilateral, symmetrical, painless swelling of the lacrimal, submandibular glands, with no history of dry eyes or mouth. The symptoms appeared since the first pregnancy and lasted for a period of longer than 3 years. Although she attended medical check-ups several times, she did not receive any definitive diagnosis or treatment.Imaging findings: on ultrasound examination of the patient´s orbit and neck, the diffuse enlargement of the lacrimal and submandibular glands was observed, in addition to hypervascularity and multiple hypoechoic areas (ic areas . Facial ic areas .Pathology: the patient underwent a biopsy of the lacrimal gland. The microscopic images showed lymphoplasmacytic infiltration and fibrotic areas. The immunohistochemical findings revealed CD138-positive cells (ve cells  and the ve cells .Serum IgG4: serum IgG4 levels were as high as 5,012 mg/L before the administration of steroid therapy, compared with the normal range of less than 1,350 mg/L [350 mg/L .Treatment: the patient was treated with 40 mg/day prednisolone for 4 weeks, after which the dose decreased by 5 mg/week until it reached 5 mg/day. After 9 months of treatment with corticoids, the serum IgG4 level was markedly decreased, returning to within the normal range, at 798 mg/L, accompanied by decreased gland sizes.During the past 2 decades, significant advancements have been made toward understanding MD. The association between the enlargement of lacrimal and salivary glands and elevated IgG4 serum levels was first reported in 2004  and was The 2019 American College of Rheumatology/ European League Against Rheumatism classification criteria for IgG4-RD is recommended for diagnosis. If the inclusion criteria are met, no exclusion criteria are present, and the total score is ≥20, then a diagnosis of IgG4-RD could be established . AlternaSystemic corticosteroid is the first-line treatment for MD, in most of the cases. The majority of patients respond well to glucocorticoid therapy within weeks, typically with reductions in mass sizes and improved symptoms, often associated with a significant drop in serum IgG4 levels . IntraorMD is a benign disease of unknown etiology, characterized by the bilateral enlargement of the lacrimal and salivary glands. The symptoms could be expected to improve following steroid therapy. We presented a case report of MD with a focus on the imaging. In our article, the association between lacrimal biopsy tissues, IgG4 serum levels, and treatment response were consistent with IgG4-RD. Ultrasound imaging may be a useful procedure for monitoring the efficacy of corticosteroid therapy in these patients.