We consider that the observed uneven prevalence of SL users can reflect underlying hereditary forms of congenital HL accumulated in certain populations by specific genetic background and population structure. At least, the data from this study indicate that the highest proportions of SL users detected in some Siberian regions are consistent with the reported accumulation of specific hereditary HL forms in indigenous Yakut, Tuvinian and Altaian populations.The absence of comparable epidemiological data challenges the correct estimation of the prevalence of congenital hearing loss (HL) around the world. Sign language (SL) is known as the main type of communication of deaf people. We suggest that the distribution of SL can be interpreted as an indirect indicator of the prevalence of congenital HL. Since a significant part of congenital HL is due to genetic causes, an assessment of the distribution of SL users can reveal regions with an extensive accumulation of hereditary HL. For the first time, we analyzed the data on the distribution of SL users that became available for the total population of Russia by the 2010 census. Seventy-three out of 85 federal regions of Russia were ranked into three groups by the 25 Hearing loss (HL) is one of the most common sensory disorders that makes it a serious public health problem. According to the World Health Organization (WHO), the number of people with disabling HL is approximately 466 million worldwide, including 34 million children . The WHOUniversal newborn hearing screening (UNHS) programs are potentially the best source of data on the prevalence of congenital deafness –6. The rAt the same time, there are studies assessing HL prevalence by other approaches in particular countries. One of the most well known is the 1972 National Census of the Deaf Population (NCDP) in the USA, which gathered information on the size, geographic distribution, and characteristics of the deaf population in the USA . HoweverIn Italy, the data from the Italian National Institute of Social Insurance (INPS) were applied as a basis for estimating the prevalence of HL . As, in In this work, we propose to consider the data on the prevalence of the knowledge of sign language (SL) as an alternative indicator of the prevalence of congenital and prelingual HL. Congenital or early-onset HL is the most socially significant form of HL, greatly affecting the healthy development and socialization of deaf people, most of whom use SL as their only way of communication. Therefore, a high incidence of SL users in certain locations might indicate the accumulation of hereditary forms of deafness, as half of the congenital HL cases have a genetic etiology . For theThe prevalence of congenital HL in Russia was described previously only for certain populations either by broad surveys on inherited diseases –16 or byTo analyze the prevalence of SL users in Russia, we used the data of the last (2010) comprehensive nationwide population census conducted simultaneously according to a unified statistical methodology in all regions of Russia and published by the Federal State Statistics Service (FSSS) of the Russian Federation . The repth and 75th percentiles for available values (the number and proportion of SL users). We divided regional values for SL users into three groups by lower and upper quartiles. Values significantly lower than the 25th percentile were considered “low”, values significantly higher than the 25th percentile and lower than the 75th percentile were considered “average”, and values significantly higher than the 75th percentile were considered “high”.In 2010, the Russian Federation constitutionally consisted of 85 federal subjects, and the total population amounted to 142,946,788 people . A distiThis study was approved by the local Biomedical Ethics Committee of Federal State Budgetary Scientific Institution “Yakut Science Centre of Complex Medical Problems”, Yakutsk, Russia . The data were analyzed anonymously, and no informed consent forms were required.It should be noted that the number of SL users cannot be directly interpreted as the number of hard-of-hearing people since a certain part of hearing people also know and use SL . Nevertheless, we suggest that the proportion of hearing SL users can be neglected when analyzing the prevalence of SL across regions of Russia due to a relatively uniform distribution of educational institutes and accessibility of social services for deaf people in federal subjects of Russia.According to the final report of the 2010 census, 138,312,535 people answered the question about their language knowledge. Among them, 18,591,655 respondents indicated that they know and use another language besides Russian . In totaTo study the prevalence of SL users, we had two sets of relevant data: aggregated statistics and regional reports of local offices of the FSSS. According to the aggregated statistics from the census results, there were 120,528 SL users among 138,312,535 respondents, with a proportion of 0.087%. Information on SL users reported by local FSSS offices was available for 73 out of 85 federal subjects of Russia, and in this case, the total number of SL users was 107,064 out of 122,527,891 respondents, which corresponds to the same proportion of 0.087%. We assume that the “missing” 13,464 SL users are uniformly distributed among 12 regions with unavailable census data.We analyzed the regional distribution of the number and proportion of SL users across different regions of Russia. According to the number of SL users, the regions of Russia were divided (by lower and upper quartiles) into three groups: “low number” - 19 regions; “average number” - 36 regions; and “high number” (18 regions). There were some regions with the largest number of SL users: Moscow– 9,342; Moscow Oblast’– 4,162; the Republic of Bashkortostan– 4,059; Sverdlovsk Oblast’– 3,887; Chelyabinsk Oblast’– 3,732; Rostov Oblast’– 3,557; and Saint-Petersburg– 3,553 . The regThe proportion of SL users varied across federal regions of Russia, from 0.045% to 0.261%. We subdivided all regions into three categories according to the proportion of SL users: “low proportion”– 14 regions; “average proportion”– 48 regions; and “high proportion”– 11 regions . The higIn this study, by using census data, we determined the proportion and regional distribution of SL users in the total population (~ 140 million people) of Russia. Sign language, according to the total number of SL users, is in the thirty-second position in the list of 119 different native languages used in Russia . The totThe number of SL users varied from 29 in Chukotka Autonomous Okrug to 9,342 in Moscow, with a mean number of 1,466.63 per region, and corresponded to the number of respondents in the studied regions . The numth percentile. Some of these regions of Russia , the Republic of Adygeya, the Republic of Altai, the Republic of Khakasiya, and the Republic of Kalmykiya) are inhabited, in addition to Russians, by different small indigenous peoples . We suggest that the observed uneven distribution of SL users in these regions can represent the underlying hereditary forms of hearing impairment accumulated by a certain genetic background, a particular ethnic history and specific demographic factors in different ethnic groups living in Russia.To identify regions with values different from the general proportion of SL users in Russia (0.087%), we divided them into three groups by lower and upper quartiles: “low proportion” (14 regions), “average proportion” (48 regions), and “high proportion” (11 regions) . The higNumerous studies have shown that the frequencies of Mendelian diseases and different pathogenic variants can vary significantly among different regions of Russia –42. AlthThe obtained data on high proportions of SL users in some regions of Siberia , and the Republic of Altai) are consistent with available data on the high prevalence of unique pathogenic variants in the genes associated with hearing impairment among indigenous populations in these regions  , 30, 49.Therefore, while SL knowledge is not defined exclusively by hearing status, the prevalence of SL users might be used as an indirect indicator of the accumulation of congenital or early-onset hereditary deafness, which, in turn, would determine the direction for more detailed genetic and epidemiological studies.S1 Figth percentile) in red, average values (between the 25th and the 75th percentile) in yellow, and low values (under the 25th percentile) in green.A–Number of SL users across federal regions of Russia. The regions without available information are shown in gray. B–Comparison of the number of SL users in different regions of Russia. The regions are shown in three different colors according to the number of SL users: high values (above the 75(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


The family of all ω-open sets of X which is finer than τ and this topology is denoted by ω-open sets played a vital role in general topology research see, A in θ-closure and the A is θ-closed A is θ-open θ-closed  Since by f is weakly continuous. Since (b) Suppose to the contrary that Theorem 2.39Every faintly-continuous function is faintly continuous.ProofThe implication in Example 2.40f is faintly continuous but not faintly Consider the identity function We can summarize the results and examples above by means of the following diagram:S. Al Ghour, B. Irshidat: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare no conflict of interest.No additional information is available for this paper.


The waste corresponds to soft and hard plastic, including household polymers vastly disposed of in landfills, although technically recyclable. Mechanical and physical properties, cracking, mass loss, and the occurrence of spalling were assessed in high strength concrete samples produced with either plastic waste or polypropylene fibers after 2-h exposure to 600 °C. The analysis was supported by Scanning Electron Microscopy and X-Ray Computed Tomography images. The plastic waste is composed of different polymers with a thermal degradation between 250 to 500 °C. Polypropylene (PP) fibers and plastic waste dispersed in concrete have proved to play an essential role in mitigating heat-induced concrete spalling, contributing to the release of internal pressure after the polymer melting. The different morphology of plastic waste and polypropylene fibers leads to distinct mechanisms of action. While the vapor pressure dissipation network originated by polypropylene fibers is related to the formation of continuous channels, the plastic waste seems to cause discontinuous reservoirs and fewer damages into the concrete matrix. The incorporation of plastic waste improved heat-induced concrete spalling performance. While 6 kg/m The growing public concern on excessive plastic waste generation and poor final disposal results in global commitments to the production of recyclable, reusable, or compostable industrial products . On the The annual production of plastics increased about 200-fold from 1950 (2 million tons) to 2015  and from the all plastics ever produced, only about 9% have been recycled, 12% incinerated, and 79% accumulated in landfills or the natural environment . Mismana2 emissions and waste generation [The construction sector is one of the major contributors to COneration ,11 but sneration . In thisneration ,14,15, lneration ,17, selfneration ,19,20, aneration ,22,23 coValue-added applications of plastic waste in concrete buildings and paving blocks , fiber-rRegarding the use of HSC in reinforced concrete structures, the main concern is the increased tendency for heat-induced concrete spalling ,31,32 whSeveral technological solutions have been adopted to ensure safety the of reinforced concrete structures designed with HSC: limiting the use of HSC to low fire exposure risk structural elements, considering the use of external fire protection systems, or even reducing thermal stresses on the structural elements through design options . On the 3 [The main mechanisms behind fiber effectiveness to mitigate the heat-induced spalling are related to the formation of discontinuous reservoirs, continuous channels, and vacated channels in the concrete upon reaching the fiber melting point ,35,44. S3 .Thus, this paper investigates a potential application of plastic waste as a polymeric addition in HSC, with a focus on the potential to mitigate heat-induced concrete spalling and the consequent effects on the mechanical properties. The plastic waste corresponds to the polymeric fraction of the discarded recyclable waste gathered in a waste collectors’ cooperative in Southern Brazil. Physical and mechanical properties, crack width, mass loss, and the evidence of heat-induced concrete spalling were monitored in concrete samples produced with plastic waste (PW) exposed to high temperatures. The results are compared to the ones of concrete samples produced with commercial polypropylene (PP) fibers. Scanning Electron Microscopy (SEM) and X-Ray Computed Tomography (CT) images supported the analysis of experimental data, endorsed by two-way ANOVA  and Student t-test with α 0.05.The discarded plastic waste used in this experimental program was collected in a waste collectors’ cooperative of a medium-sized municipality with 496.8 km² and 1.5 million residents in Southern Brazil.The assessment of a representative sample of about 4 tons of solid waste sorted by the same collector’s cooperative over one year  quantifiComminution processes were applied to reduce the plastic waste size . The polThe identification of plastic waste constituents was carried out based on usual appearance, density, and behavior on heating including, transparency, consistency, flammability, the appearance of flame, and odor of vapors, as proposed by Braun .m) and thermal degradation temperatures of the plastic waste using a Shimadzu DTG-60 equipment  under Argon, following the ASTM 3850-19 and ASTM 3418-15 [Thermogravimetric analysis and Differential Scanning Calorimetry were performed to characterize the melting point , crushed gravel , sand , polycarboxylate superplasticizer, and 2% entrapped air. Commercial PP fibers  and plastic waste (PW) were added to concrete mixes with dosages of 3 kg/m3 (0.125% by volume) and 6 kg/m3 (0.250% by volume).Concrete mixes  were desConcrete mixes were produced in a planetary mixer with materials added in the following order: cement, rice husk ash, sand, 50% of water, gravel, 50% of water, superplasticizer, PP or PW. The mixing process lasted approximately 5 min. Forty cylindrical ∅ 100 × 200 mm specimens were cast  for eachAn electric muffle furnace was used to heat 20 saturated specimens of each mixture . HeatingHeat-induced concrete spalling was assessed by cracking visual inspection and mass loss of specimens submitted to 600 °C for two hours. Cracking visual inspection was carried out in 10 randomly selected specimens of each concrete mix. The number of cracks with openings between 0.2 mm and 0.5 mm was quantified in 3 chosen demarcated areas of each specimen , according to the pattern presented in 2, 2240 × 2368 pixel2, pixel size 50 µm, Hamamatsu, Japan) images were performed to support the assessment of concrete behavior at high temperature. Porosity was quantified in reconstructed volumes using the volume analysis tool of Octopus V8.6 software , based on a back-projection algorithm with convolution and correction for cone-beam. Young’s modulus [Water absorption by capillarity test , Scannin modulus , compres modulus , and ten modulus  tests weThe identification process revealed that the plastic waste sample is composed by Polycarbonate (PC), High-Density Polyethylene (HDPE), Low-Density Polyethylene (LDPE), Polyvinyl Chloride (PVC), Polypropylene (PP), Acrylonitrile Butadiene Styrene (ABS), Polystyrene (EPS), and a low concentration of other plastics, according to The thermal degradation of plastic waste samples extends to near 500 °C with a high mass loss . These r3 of plastic waste, which is statistically comparable to 3 kg/m3 and 6 kg/m3 of PP fibers, with a confidence level of 95%. Considering that the volume change is negligible after exposure to elevated temperatures [eratures , the masThe SEM 3D anaglyph images in A rough estimate of porosity performed based on CT images  of concrThe results of absorption by capillarity bring some insights on the porosity and permeability characteristics of concrete samples before and after the heating process, which is important since the heat-induced concrete spalling phenomenon is extremely related to the capillary pore structure . The absThe results show no statistically significant difference for absorption by capillarity of samples before heating. After heating, the absorption by capillarity of concrete samples with PP fibers is statistically higher than CREF, which is related to the presence of larger and interconnected pores and channels , as showCPW samples showed lower absorption by capillarity when compared to CREF after heating, which could be related to the surface morphology of the plastic waste and potentially less internal damage caused by the exposure to high temperature ,68. Ozawck of 60 MPa .As addressed in Even though the compressive strength of CPW3 is 10.20% lower than CREF at ambient temperature, after exposure to a high temperature, both compressive strengths are statistically equivalent, evidencing the positive effects of the plastic waste addition. For CPW6, the contribution of plastic waste is even more evident, since the compressive strength is 7.8% lower than CREF at ambient temperature and 49.51% higher after exposition to high temperature. Additionally, the compressive strength of CPW6 and CPP6 exposed to high temperatures are statistically equivalent.3 of PP fibers and plastic waste did not show any significant difference to CREF, the incorporation of 6 kg/m3 increased the tensile strength by 100.98% (CPP6) and 29.41% (CPW6) compared to CREF. The most prominent result is the tensile strength of CPW6, 49.51% higher than CREF after exposure to high temperature, while other concrete mixtures do not show any significant difference when compared to CREF.While 3 kg/mLower Young’s modulus resulted in higher residual modulus, which corroborates to the findings of Xiong and Liew  and Ali These results demonstrate the potential of plastic waste, especially considering the mechanical properties after heating, which corroborates to the findings of mitigation of heat-induced spalling phenomenon presented in 3 of polymeric addition. Georgali and Tsakiridis [A post-fire compressive strength factor of 0.45 is recommended in the general rules for the structural fire design of Eurocode 2  for normakiridis  reportedAs PP fibers and plastic waste melt at temperatures of about 200 °C, a reduction in the tensile strength of CPP and CPW samples to the same level of CREF would be expected after exposure to 600 °C temperatures . The resThe results of The overall results of residual mechanical properties demonstrate the similar performance of CPW3 and CREF, although the tensile strength of CPW3 is not as severely affected by high temperature as it is for CREF. It is noteworthy that CPW6 showed the lowest losses in mechanical properties after exposure to high temperature.This paper investigated the effects of plastic waste on the heat-induced spalling performance and mechanical properties of high strength concrete. The main findings are summarized below.Plastic waste dispersed in concrete helped mitigate heat-induced concrete spalling by releasing internal pressure after polymer melting.The vapor pressure dissipation network caused by PP fibers are mainly related to the formation of continuous channels, while plastic waste caused discontinuous reservoirs that lead to less damage in the concrete matrix.3 of plastic waste is comparable to the reference concrete, with the advantage of heat-induced concrete spalling mitigation.The residual mechanical performance of concrete with 3 kg/m3 not only improved the heat-induced concrete spalling, but also the mechanical performance after exposure to high temperature.The incorporation of 6 kg/mThese results are limited to this case study since the heat-induced concrete spalling is highly dependent on experimental and mix design parameters. Workability, dispersion, and durability issues related to the use of plastic waste in HSC should be further investigated to confirm the trends reported in this paper.


IgG4-related disease (IgG4-RD) is a multi-organ autoimmune disease characterized by elevated serum IgG4 concentration. Although serum IgG4 concentration is widely used as a biomarker for IgG4-RD and type 1 autoimmune pancreatitis (AIP), a pancreatic manifestation of IgG4-RD, a significant number of patients have normal serum IgG4 levels, even in the active phase of the disease. Recently, we reported that the development of experimental AIP and human type 1 AIP is associated with increased expression of IFN-α and IL-33 in the pancreas. In this study, we assessed the utility of serum IFN-α and IL-33 levels as biomarkers for type 1 AIP and IgG4-RD. Serum IFN-α and IL-33 concentrations in patients who met the diagnostic criteria for definite type 1 AIP and/or IgG4-RD were significantly higher than in those with chronic pancreatitis or in healthy controls. Strong correlations between serum IFN-α, IL-33, and IgG4 concentrations were observed. Diagnostic performance of serum IFN-α and IL-33 concentrations as markers of type 1 AIP and/or IgG4-RD was comparable to that of serum IgG4 concentration, as calculated by the receiver operating characteristic curve analysis. Induction of remission by prednisolone treatment markedly decreased the serum concentration of these cytokines. We conclude that serum IFN-α and IL-33 concentrations can be useful as biomarkers for type 1 AIP and IgG4-RD. This autoimmune disorder is also characterized by multiple organ involvement3. The clinicopathological features of IgG4-RD were first identified in patients with autoimmune pancreatitis (AIP) and then, various kinds of autoimmune diseases formerly classified as AIP, autoimmune sialadenitis, autoimmune sclerosing cholangitis, and retroperitoneal fibrosis, have become recognized as organ-specific manifestations of systemic IgG4-RD4. Now AIP is classified into type 1 and type 2 AIP, the former being the pancreatic manifestation of IgG4-RD5.IgG4-related disease (IgG4-RD) is a recently discovered autoimmune disorder characterized by elevated IgG4 serum levels and by the accumulation of IgG4-expressing plasmacytes in the affected organs7. Moreover, serum IgG4 concentration over 280 mg/dL is associated with multiple organ involvement and the risk of relapse9. Thus, the measurement of serum IgG4 concentration is useful not only for the diagnosis but also for the evaluation of disease severity in IgG4-RD. It should be noted, however, that the levels of this particular IgG subtype are not always useful for the differential diagnosis or evaluation of disease severity in IgG4-RD and type 1 AIP. About 20% of patients with type 1 AIP have normal levels of IgG4 in the serum10. Furthermore, serum IgG4 concentrations are sometimes elevated in patients with pancreatic cancer11. In addition, disease flare-ups are seen in about 10% of patients with type 1 AIP even in the presence of normal IgG4 levels12. Therefore, it is necessary to develop novel biomarkers that could be useful for the diagnosis and disease progression monitoring of type 1 AIP and IgG4-RD.Elevated IgG4 serum levels are a diagnostic marker for type 1 AIP and IgG4-RD14. Recently, we reported that the production of interferon (IFN)-α and interleukin (IL)-33 by plasmacytoid dendritic cells (pDCs) mediates chronic fibroinflammatory responses in experimental AIP and human type 1 AIP19. Our previous studies provided evidence that pancreatic accumulation of pDCs, which may produce large amounts of IFN-α and IL-33, underlies the immunopathogenesis of type 1 AIP and IgG4-RD19. Furthermore, we had a case of type 1 AIP and IgG4-RD in which the serum concentrations of IFN-α and IL-33, rather than the serum concentration of IgG4, were highly associated with disease activity20. Based on our previous data, in the present study, we tested the hypothesis that serum concentrations of IFN-α and IL-33 could be useful biomarkers for type 1 AIP and IgG4-RD.Cytokines and chemokines serum levels may also serve as biomarkers in several autoimmune diseases. For example, serum concentrations of IL-6 and thymus and activation-regulated chemokine are well-established biomarkers of multicentric Castleman’s disease (MCD) and atopic dermatitis, respectively7 and/or IgG4-RD6.Twelve patients met the diagnostic criteria for both IgG4-RD and type 1 AIP. Two patients met the criteria for IgG4-RD alone whereas seven patients met that for type 1 AIP alone. The 21 patients that fulfilled the diagnostic criteria for definite type 1 AIP and/or IgG4-RD are thereafter called type 1 AIP/IgG4-RD patients in this manuscript. Histological specimens (n = 18) were obtained by endoscopic ultrasound-guided fine-needle aspiration in 16 patients, endoscopic retrograde cholangiography-guided biopsy in one patient, and surgical resection in one patient, and analyzed by a pathologist. Histologic analysis was not performed for the three patients that were diagnosed with type 1 AIP based on typical radiographic findings and serum IgG4 concentrations7. Positron emission tomography/computed tomography (PET/CT) using 18F-2-fluoro-2-deoxy-D-glucose was performed in 19 patients (90.5%) for the diagnosis and evaluation of disease activity21. The number of affected organs was determined by PET/CT in 19 patients and contrast-enhanced whole-body CT in two patients. IgG4-RD responder index (IgG4-RD RI) was calculated as previously described22.To validate the utility of serum IFN-α and IL-33 concentrations as biomarkers of type 1 AIP and IgG4-RD, we enrolled 21 consecutive, newly diagnosed patients who visited the Kindai University Hospital between June 2018 and December 2019 and met the diagnostic criteria for definite type 1 AIP23. We selected typical CP patients exhibiting pancreatic stones and main pancreatic duct dilatation, as per CT examinations.Twelve chronic pancreatitis (CP) patients who met diagnostic criteria for definite alcoholic CP were also enrolled as the disease control groupSerum samples were obtained at the diagnosis of type 1 AIP/IgG4-RD or CP. In addition, serum samples were also obtained from eight healthy control (HC) cases. The study protocol conformed to the ethical guidelines for human clinical research established by the Japanese Ministry of Health, Labor and Welfare and was approved by the ethics committee of the Kindai University, Faculty of Medicine. Written informed consent was obtained from all patients and healthy controls at enrollment.Twelve patients with type 1 AIP/IgG4-RD received oral administration of prednisolone (PSL) at the initial dose of 0.6–0.7 mg/kg for the first two weeks followed by the scheduled tapering regimen (5 mg every two weeks). In all of these 12 patients, PET-CT confirmed improved disease activity at the time of serum sampling after PSL administration . However, serum samples were not collected from the remaining nine patients (of the 21 type 1 AIP/IgG4-RD patients), since six months had not passed after the initiation of PSL treatment.24. Briefly, IgG and IgM serum concentrations were measured using a turbidimetric immunoassay and that of IgE were measured using a fluorescence enzyme immunoassay. Serum levels of IgG4  were measured using a latex immunoturbidimetric method. Serum concentrations of IgG1, IgG2, and IgG3 were determined using enzyme-linked immunosorbent assay (ELISA) commercial kits, as described previously 25. Serum IgG subtypes’ concentrations in the HCs analyzed in this study ranged as follows: IgG1 (245.5–1129.1 mg/dL), IgG2 (141.4–948.6 mg/dL), and IgG3 (17.1–150.9 mg/dL).Serum concentrations of total IgG, IgG4, IgA, and IgE were measured as described previously24. Serum concentrations of IL-1β, IL-6, and TNF-α were determined using other commercial ELISA kits, as previously described (Thermo Fisher Scientific)26.Serum concentrations of IFN-α and IL-33 were measured using commercial ELISA kits from R&D systems 2 + (1 − specificity)2.Diagnostic performance of serum markers  was assessed by receiver operating characteristic (ROC) curve analysis; the area under the curve (AUC) values for each biomarker were calculated. The optimal cut-off points of serum markers were determined based on the minimum values of (1 − sensitivity)P-value was derived by multiplying a P-value derived by the number of tests. The Wilcoxon signed rank test, which is a nonparametric version of paired t test, was used to compare serum markers before PSL treatment to those at maintenance treatment. The Spearman correlation coefficient was used to evaluate the association between the different serum markers evaluated in this study. All statistical analyses were performed using the GraphPad Prism 6.0 software . P-values < 0.05 indicated statistical significance.Baseline characteristics are indicated as percentages for categorical variables or means ± standard deviation for continuous variables. The Mann–Whitney U test, which is a nonparametric version of unpaired t test, was used to evaluate the differences between two groups for baseline characteristics and serum markers. The Kruskal–Wallis test, which is a nonparametric version of one-way ANOVA, was used to evaluate the differences between more than two groups for serum markers. For post hoc analysis , the Bonferroni corrected Mann–Whitney U test was performed for comparison between two groups, where each 7 and/or IgG4-RD6 were enrolled in this study. Twelve CP patients met the diagnostic criteria for definite CP23. The characteristics of patients with type 1 AIP/IgG4-RD and CP are shown in Table A total of 21 type 1 AIP/IgG4-RD patients that fulfilled the diagnostic criteria for definite type 1 AIP27, serum levels of total IgG were significantly higher in patients with type 1 AIP/IgG4-RD than in CP patients or HCs  of patients with type 1 AIP/IgG4-RD, as compared to its upper limits. In addition to IgG4, patients with type 1 AIP/IgG4-RD exhibited elevated serum IgG1 levels as compared to those in CP patients and HCs. In contrast, serum concentrations of IgG3 were comparable between patients with type 1 AIP/IgG4-RD, CP patients and HCs. Last but not least, patients with type 1 AIP/IgG4-RD exhibited lower serum IgG2 levels than those from CP patients. Thus, these data suggest that patients with type 1 AIP and IgG4-RD are characterized by elevated concentrations of serum total IgG, IgG1, and IgG4.We then turned our attention to the serum concentrations of IgG subtypes in patients with type 1 AIP/IgG4-RD. In line with the previous reports25. Indeed, peripheral blood pDCs from type 1 AIP/IgG4-RD patients enhanced IgG4 production by healthy B cells in a T cell-independent and IFN-α-dependent manner15. More importantly, increased production of IFN-α and IL-33 by pDCs mediated chronic fibroinflammatory responses in both experimental AIP and human type 1 AIP19. Thus, T cell-independent cytokines produced by antigen-presenting cells, including pDCs, may be involved in the elevation of serum IgG1 and IgG4 concentrations in patients with type 1 AIP/IgG4-RD. Based on these findings from our previous reports, we evaluated the profiles of cytokines involved in innate rather than adaptive responses in this study. As shown in Fig. 19.We have previously reported that peripheral blood monocytes and pDCs isolated from patients with type 1 AIP/IgG4-RD promoted the production of IgG1 and IgG4 by B cells from HCs in the absence of T cellsA strong positive correlation between the serum concentrations of IFN-α and IL-33 was seen in patients with type 1 AIP/IgG4-RD  is essential, and will be the focus of our next study.The measurement of IgG4 serum levels is useful for the differential diagnosis between type 1 AIP and pancreatic cancer. It should be noted, however, that a significant fraction of patients with type 1 AIP and pancreatic cancer exhibit normal and elevated levels of this IgG subtype, respectively. Thus, the differential diagnosis between type 1 AIP and pancreatic cancer cannot be exclusively based on the presence or absence of elevated serum IgG4 levels19. Importantly, the development of experimental AIP depends on the activation of signaling pathways triggered by pDC-derived IFN-α and IL-33, since the depletion of pDCs or inhibition of IFN-α or IL-33-mediated signaling pathways effectively prevented the development of experimental AIP16. In this study, we found that type 1 AIP/IgG4-RD patients exhibited higher serum concentrations of IFN-α and IL-33 than patients with CP or HCs. Thus, patients with type 1 AIP/IgG4-RD are characterized by enhanced IFN-α and IL-33 responses not only in the pancreas but also in the serum. Therefore, it is likely that systemic as well as in situ production of IFN-α and IL-33 might underlie the immunopathogenesis of type 1 AIP and IgG4-RD. In addition, the positive correlations between serum concentrations of these cytokines and IgG4 serum levels reported in this study are fully consistent with the results of our in vitro studies, in which peripheral blood pDCs isolated from type 1 AIP/IgG4-RD patients promoted IgG4 production by HC B cells in an IFN-α-dependent manner15. In this study, we have extended these previous data with regard to a possible clinical application by providing evidence that the AUC values of serum IFN-α or IL-33 levels are comparable to those of serum IgG4 AUC values in the ROC curve analysis.We have previously demonstrated that murine experimental AIP and human type 1 AIP/IgG4-RD are characterized by pancreatic accumulation of pDCs, which produce large amounts of IFN-α and IL-3332. In contrast, elevated serum concentrations of IL-33 seen in type 1 AIP/IgG4-RD have not been reported in SLE33. IL-33 is a profibrogenic cytokine that induces fibrogenic cytokines such as IL-5 and IL-1334. Activation of the IFN-α-IL-33 axis may contribute to the development of a specific type of tissue fibrosis, called storiform fibrosis, in type 1 AIP/IgG4-RD3 since the inhibition of this axis markedly decreased, not only chronic inflammation, but also pancreatic fibrosis in experimental AIP18. Therefore, the activation of signaling pathways triggered by IL-33 and IFN-α may be involved in the clinical manifestations specific to type 1 AIP and IgG4-RD, but not to SLE. In the future, it will be interesting to determine whether serum concentrations of these two cytokines correlate with the degree of tissue fibrosis in type 1 AIP/IgG4-RD.Type 1 AIP/IgG4-RD and systemic lupus erythematosus (SLE) are characterized by elevated serum concentrations of IFN-α37. Moreover, clinical cases with overlapping IgG4-RD and MCD were also described39. As for the clinical manifestations, Tsukuda et al. reported that elevated serum levels of IL-6 are associated with the involvement of the bile duct, spleen, and liver in IgG4-RD patients40. Although we could not confirm the association between serum IL-6 concentrations and specific clinical manifestations, probably due to a relatively small number of patients in this study, further studies are required to sub-classify the MCD-like subtype within type 1 AIP/IgG4-RD patients based on the evaluation of serum IL-6 levels.Patients with CP and type 1 AIP/IgG4-RD exhibited comparable serum concentrations of the prototypical innate immunity cytokines such as IL-1β, IL-6, and TNF-α. It should be noted, however, that a significant fraction  of patients with type 1 AIP/IgG4-RD displayed elevated serum concentrations of IL-6 in this study. One concern regarding type 1 AIP/IgG4-RD patients exhibiting elevated IL-6 levels might be the simultaneous occurrence of type 1 AIP/IgG4-RD and MCD. Indeed, it has been reported that differentiation between MCD and IgG4-RD may be difficult in some cases3. IgG1, which activates the complement system and Fcγ receptors play pathogenic roles in various autoimmune and infectious diseases42. In contrast, IgG4 induces anti- rather than pro-inflammatory responses as this IgG subtype lacks the ability to activate the complement and Fcγ receptors42. Therefore, it is likely that an enhanced IgG4 response is an epiphenomenon associated with chronic inflammation in type 1 AIP/IgG4-RD. This idea is supported by translational studies in which IgG1 isolated from the serum of type 1 AIP patients was found to cause more severe pancreatic injury than IgG4 upon intraperitoneal injection into neonatal mice43. Moreover, the injection of IgG4 attenuated pancreatic injury induced by the injection of IgG143. In line with these findings, two recent studies that identified candidate autoantigens in type 1 AIP and IgG4-RD provided evidence that the IgG4 subtype inhibits the binding of the IgG1 subtype to the autoantigen epitopes45. These reports suggested pathogenic roles for the IgG1 rather than the IgG4 subtype. Given the fact that relapse sometimes occurs even in type 1 AIP/IgG4-RD patients with normal IgG4 concentrations12, monitoring serum concentrations of both IgG1 and IgG4 might be necessary in this autoimmune disorder.Our serum IgG subtype analysis revealed higher serum concentrations of IgG1 and IgG4 in type 1 AIP/IgG4-RD patients than in CP patients. Importantly, serum levels of both IgG subtypes were considerably reduced by PSL treatment. Thus, our results suggest that both IgG1 and IgG4 faithfully reflect disease progression. It should be noted, however, that the subtype of IgG causing chronic fibroinflammatory responses has not been identified in type 1 AIP/IgG4-RD46. This discrepancy can be partially explained by the difference in the distribution of affected organs in IgG4-RD. Chan’s study examined serum IgG2 concentrations in 43 patients with orbital IgG4-RD, including two type 1 AIP cases46. Our study examined serum IgG subtypes in 21 type 1 AIP/IgG4-RD patients that did not present eye involvement. Whether IgG subtype responses depend on the distribution of affected organs requires further investigation.In contrast to the determined IgG1 and IgG4 levels, we did not detect any significant elevation of serum IgG2 or IgG3 concentrations in patients with type 1 AIP/IgG4-RD. Recently, Chan et al. reported significantly elevated serum IgG2 levels in patients with IgG4-RD3.Although the data presented in this study support the utility of serum IFN-α and IL-33 as biomarkers of type 1 AIP/IgG4-RD, our study had several limitations. First, this retrospective study was conducted with patient samples collected in a single center. Future multicenter studies addressing the utility of these cytokine markers in a prospective manner are required to confirm our data. Secondly, most patients enrolled in this study were type 1 AIP patients, and thus, abnormal innate immune responses in the pancreas might have some influence on their serum cytokine responses. Given the fact that IgG4-RD affects almost all the organs in the body, it is possible that serum cytokine responses may differ depending on the predominantly affected ones. Therefore, serum cytokine responses need to be evaluated in various types of IgG4-RD, such as IgG4-related sialadenitis, IgG4-related lung disease, and IgG4-related kidney disease, in addition to type 1 AIPIn conclusion, we found that the serum of patients with type 1 AIP/IgG4-RD showed high concentrations of IFN-α and IL-33, which, in turn, positively correlated with the serum IgG4 levels. Furthermore, their levels were markedly decreased following PSL treatment. Therefore, we conclude that the serum concentrations of IFN-α and IL-33 may serve as biomarkers for the diagnosis of type 1 AIP/IgG4-RD, as well as for monitoring purposes.Supplementary Information.


Objective: This study aimed to investigate the effect of environmental conditions including nutrient content, acetic acid concentration, salt concentration, and temperature on the formation of viable but nonculturable (VBNC) state of Pediococcus acidilactici, as well as its control and detection in food system.Methods: Representing various environmental conditions in different food systems, 16 induction groups were designed for the formation of VBNC state of P. acidilactici. Traditional plate counting was applied to measure the culturable cell numbers, and Live/Dead Bacterial Viability Kit combined with fluorescent microscopy was used to identify viable cells numbers. The inhibition of bacterial growth and VBNC state formation by adjusting the environmental conditions were investigated, and the clearance effect of VBNC cells in crystal cake system was studied. In addition, a propidium monoazide-polymerase chain reaction (PMA-PCR) assay was applied to detect the VBNC P. acidilactici cells in crystal cake food system.Results: Among the environmental conditions included in this study, acetic acid concentration had the greatest effect on the formation of VBNC state of P. acidilactici, followed by nutritional conditions and salt concentration. Reducing nutrients in the environment and treating with 1.0% acetic acid can inhibit P. acidilactici from entering the VBNC state. In the crystal cake system, the growth of P. acidilactici and the formation of VBNC state can be inhibited by adding 1.0% acetic acid and storing at −20°C. In crystal cake system, the PMA-PCR assay can be used to detect VBNC P. acidilactici cells at a concentration higher than 104 cells/ml.Conclusion: The VBNC state of P. acidilactici can be influenced by the changing of environmental conditions, and PMA-PCR assay can be applied in food system for the detection of VBNC P. acidilactici cells. P. acidilactici, followed by nutritional conditions and salt concentration.Acetic acid concentration had the greatest effect on the formation of VBNC state of P. acidilactici from entering into the VBNC state.Reducing nutrients and treating with 1.0% acetic acid are able to inhibit P. acidilactici and the formation of VBNC state can be inhibited by adding 1.0% acetic acid and storing at −20°C.In the crystal cake food system, the growth of P. acidilactici cells at a concentration higher than 104 cells/ml.In the crystal cake food system, the PMA-PCR assay can be used to detect VBNC Under stress conditions, various species of bacteria can enter a physiologically viable but nonculturable (VBNC) state. Bacteria entering into the VBNC state are a survival strategy to cope with adverse conditions. However, food-borne spoilage and pathogenic bacteria pose a threat to food safety and even public health after entering the VBNC state . The preLactobacillus plantarum  glycerol. It was streaked on MRS agar plate for 48 h at 37°C. Subsequently, a single colony was inoculated into a culture tube containing 2 ml of MRS broth and cultured at 37°C for 24 h. P. acidilactici was grown overnight in MRS broth at 37°C for 12 h. The bacterial suspension was diluted 1:100 in fresh MRS medium. Subsequently, the cultivation continued to the log phase.P. acidilactici, and in accordance with the principle of reverse adjustment, conditions which are unfavorable to bacterial growth were selected as candidate factors for inducing VBNC state. Three factors, including nutrition, salt concentration, and acid, were taken as a single variable. Three factors and four levels of orthogonal experiments were designed to reach 16 experimental groups  was used. The fluorescent probes SYTO 9 and PI were used with fluorescence microscope to distinguish intact from membrane-permeabilized cells. For these assays, 500 μl sample was obtained and centrifuged at 5,000 rpm for 15 min. Subsequently, the cells of P. acidilactici were washed twice with physiological saline (PBS). The supernatant was removed, and the pellet was resuspended in 500 μl of PBS. The cell suspensions were incubated with 1.5 μl of a dye mixture containing SYTO 9 and PI for 30 min at room temperature in the dark. Five microliter of cells incubated with the dye mixture was trapped between a slide and a square coverslip. The slide was placed under a fluorescent microscope for further observation. Live cells were observed under red excitation light with a wavelength of A480 nm/450 nm, and dead cells were observed under blue excitation light with a carrier length of A490 nm/635 nm. The results were then superimposed using Image J software. Under fluorescence microscope, the viable cells showed green, whereas dead cells exhibited red.The induction culture was removed from the refrigerator and placed at room temperature. When the induction culture completely melted, it was serially diluted in 0.9% NaCl, spread on MRS agar plates, and incubated at 37°C for 24 h. When the culturable cell number of NC state . In ordeP. acidilactici VBNC state, the effect of acid (0.7 and 1.0%) and nutrition (0 and 25%) on the formation of P. acidilactici VBNC state were studied  were added to a 1.5 ml centrifuge tube, and they were thoroughly mixed and left at room temperature for 10 min. Subsequently, the centrifuge tube was placed on a crushed ice box, and light treatment was performed for 15 min at a distance of 15 cm from a 650 W halogen lamp to complete the binding of PMA and DNA. The PMA molecules remaining after the treatment are passivated. The PMA-treated bacteria suspension was centrifuged at 10,000 rpm/min for 5 min, and the supernatant was discarded. DNA was then extracted using a bacterial group DNA extraction kit . The extracted DNA was detected by PCR. PCR assay was performed in a 25-μl volume and with 0.6 μM primers . The thermal profile for PCR mixtures were 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 60 s and a final extension cycle at 72°C for 5 min. The amplified products (5 μl/well) were analyzed by gel electrophoresis in 2% agarose gels and stained with ethidium bromide for 10 min. A negative control was performed using sterile water instead of culture or DNA template, and then visualized by UV transilluminator.According to the National Food Safety standards (GB4789.3–2016) in China, 25 g of ground crystal cake was added to 225 ml of PBS and sterilized. Subsequently, P. acidilactici were reduced to zero (4 and −20°C) within 3 days . In the induction culture with an acetic acid concentration of 1% . However, when the culture medium concentration was 100% and P. acidilactici culture was stored at 4 and −20°C, it could survive for 37 and 45 days, respectively . This shows that the concentration of nutrients has a certain effect on the survival of P. acidilactici, and sufficient nutrients can enhance the resistance of P. acidilactici to external acidic substances. When the acetic acid concentration was 0.1 and 0%, the culturable cell number of experimental group 6 reduced to zero in 3 days, the culturable cell number of experimental group 2 reduced to 0 in 34 and 44 days, and the culturable cell number of experimental group 1 reduced to 0 in 31 days at 4°C. P. acidilactici in the experimental groups 5, 9, 13, 10, and 14 could survive for more than 45 days. This shows that the acetic acid concentrations used in this study have the most significant effect on the survival of P. acidilactici.Under the conditions in which the acetic acid concentration is 2%, the medium concentration ranges from 0 to 100%, and the salt concentration ranges from 0.9 to 15%, the culturable and viable cell numbers of on of 1% , P. acidP. acidilactici cells could survive for 45 days, and the culturable cell number of P. acidilactici was not significantly different. The salt concentration used in this experiment had no significant effect on the survival of P. acidilactici. Therefore, among the environmental conditions discussed in this study, acid has the greatest impact on the survival of P. acidilactici, followed by nutrition, and salt concentration has the least effect on the survival of P. acidilactici.In the experimental groups 5, 9, 10, 13 and 14, with the increase of the salt concentration, the P. acidilactici VBNC. Among the 16 induction cultures, only in three induction cultures successfully induced P. acidilactici entering into the VBNC state within 45 days. (1) The medium concentration is 0, the sodium chloride mass fraction is 0.9%, and the acetic acid volume fraction is 0. (2) The concentration of the medium is 25%, the mass fraction of sodium chloride is 0.9%, and the volume fraction of acetic acid is 0.1%. (3) Medium concentration is 100%, sodium chloride mass fraction is 5%, and acetic acid volume fraction is 1%. This shows that when the concentration of the medium is less than 25%, the concentration of sodium chloride is 0.9%, and the concentration of acetic acid is less than 0.1%, it is beneficial to the formation of VBNC state of Escherichia coli. That is, nutritional deficiencies and relatively low concentrations of sodium chloride and acetic acid are benificial to VBNC state formation. Under this environmental pressure, P. acidilactici does not die directly, but enters the VBNC state. In addition, when the concentration of the culture medium is 100%, the concentration of sodium chloride is 5%, and the volume fraction of acetic acid is 1%, P. acidilactici can enter the VBNC state. This shows that under the condition of sufficient nutrition, P. acidilactici can resist higher sodium chloride concentration and acid . However, when the nutrient concentration was 0 and 25%, P. acidilactici was not culturable and viable , by adding a certain amount of acetic acid (acetic acid volume fraction reaches 0.7 or 1.0%), the culturability of r 3 days . And bacP. acidilactici under food processing and storage conditions. P. acidilactici was inoculated in food system with 25, 50, and 100% nutrient content and cultured at 4 and −20°C, respectively. After 3 days of culture, the culturability and activity of P. acidilactici were measured. Only when the nutrient content is 100% and stored at 4°C, P. acidilactici is not culturable and viable, but can survive in other experimental groups. Therefore, adding 1% acetic acid to rice flour products and storing them at −20°C can prevent P. acidilactici in rice flour products from entering the VBNC state and avoid potential threats caused by the presence of P. acidilactici in the VBNC state.Crystal cake is a traditional Chinese snack, which has the characteristics of rich nutrition. The rich nutrients and sufficient water in crystal cake make it a natural medium for the growth of various pathogenic and spoilage bacteria. In this study, crystal cake was applied as a representative food system to study the inhibition and detection of the VBNC state of P. acidilactici in the VBNC state in the crystal cake food system is higher than 104 cells/ml, the P. acidilactici in the VBNC state can be successfully detected using the PMA-PCR technology (Propidium monoazide is a DNA intercalating molecule that can differentiate between live and dead or membrane-damaged bacteria. It can selectively penetrate the damaged cells and form a stable covalent high affinity bonds with DNA, following photo-activation exposure to strong visible light . The DNAchnology .P. acidilactici, followed by nutrition, and the effect of salt concentration on the survival of P. acidilactici was the least significant. In addition to the influence of the composition of the induction culture, 4°C is more favorable for P. acidilactici to enter into the VBNC state. By reducing the amount of nutrients in the environment and treating with 1.0% acetic acid, the formation of VBNC state can be suppressed. The addition of 1% acetic acid in rice flour products and storage at −20°C can inhibit P. acidilactici in rice flour products from entering into the VBNC state and avoid potential threats caused by the presence of P. acidilactici in the VBNC state. Also, PMA-PCR method can be applied to detect VBNC P. acidilactici cells with concentration higher than 104 cells/ml.Among the environmental conditions discussed in this study, acid had the greatest effect on the formation of VBNC state of All datasets presented in this study are included in the article.JL and KW conceived of the study and participated in its design and coordination. T-YH, YM, YC, FS, RP, and JC performed the experimental work. CB and LC analyzed the data. JL prepared and revised this manuscript. All authors reviewed and approved the final manuscript.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.


Escherichia coli resistance rates, evaluating all antibiotic use and patients with positive cultures hospitalized between 2011 and 2018. Data on antibiotics were collected quarterly as the defined daily dose (DDD)/100 days hospitalization. In 2014, an intervention was introduced, targeting the reduction of overall antibiotic use as well as specifically targeting quinolones and other broad-spectrum antibiotics. Using interrupted time series analysis (ITS), we compared the rates and trends of antibiotic use and resistant E. coli. We included 6001 patients, 3182 pre-ASP and 2819 post-ASP. We observed significant changes in absolute numbers as well as in trends for use of DDD/100 days of all antibiotics by 31% from 76 to 52, and by 52% from 10.4 to 4.9 for quinolones. ITS demonstrated that before the ASP intervention, there was a slope pattern for increased E. coli resistance to antibiotics. This slope was reversed following the intervention for quinolones −1.52, aminoglycosides −2.04, and amoxicillin clavulanate (amox/clav) −1.76; the effect of the intervention was observed as early as three months after the intervention and continued to decrease over time until the end of the study, at 48 months. We conclude that the ASP can positively impact the resistance rate of Gram-negative infections over time, regardless of the targeted combination of antibiotics, if the overall use is reduced.Infections caused by multidrug-resistant Gram-negative bacteria increase hospitalizations and mortality rates; antibiotic pressure increases resistance rates. We evaluated the impact of the antibiotics stewardship program (ASP) on  In recent years, bacterial resistance to antibiotics has increased globally. Multidrug-resistant (MDR) Gram-negative bacteria have become one of the greatest risks to public health ,2, causiOne of the major contributors to the increasing resistance rates is the unnecessary use of antibiotics, particularly broad-spectrum antibiotics, with reports of over 50% of antibiotics provided to patients without good cause . Use of The main goals of ASPs are to reduce the incidence of resistant infections ,13 and tE. coli in urine cultures following an educational community-based ASP intervention [p = 0.03) in hospital-acquired antibiotic-resistant organisms and a 13%  reduction in hospital-acquired MDR organisms [Although successful ASPs have reduced costs and improved resource utilization, there are limited data on their success in reducing the rate of resistant pathogens ,16. A mervention , and PerE. coli bacteria grown in urine and blood cultures.Soroka University Medical Center (SUMC) is a 1100-bed tertiary/referral hospital, which serves as the only major medical provider for a population of approximately 900,000 people in the southern district of Israel. In recent years, we have observed a steady increase in Gram-negative-resistant infections in the southern district; in particular, we have seen a high prevalence of ESBL (~25% of urine pathogens) and MDR infections, which prompted an ASP intervention . In thisE. coli bacterial cultures were identified: 5488 urine cultures and 505 blood cultures. Duplicates (n = 3394) were excluded. Baseline characteristics between the pre-ASP and post-ASP periods are compared in p < 0.001). The highest percentage of cultures was drawn in the emergency department  and in the internal medicine department . A significantly shorter length of hospital stay, from 4 to 3 days, was also noted between the pre-ASP and post-ASP periods (p < 0.001).We collected medical records of 6001 cases: 3182 from the pre-ASP and 2819 from the post-ASP period. A total of 9387 positive Following implementation of the ASP, overall use of antibiotics in the hospital significantly decreased by 31% from 76 defined daily doses (DDD)/100 bed days in the pre-ASP period to 51 DDD/100 bed days in the post-ASP period. Quinolone use decreased by 52% (from 10.4 to 4.9 DDD/bed 100 days), aminoglycoside use decreased by 32% (from 2.2 to 1.5 DDD/100 bed days), and amoxicillin clavulanate (amox/clav) use decreased by 58% (from 18.7 to 7.8 DDD/100 bed days). Antibiotic consumption by quarter is shown in p = 0.003; ITS analysis of antibiotic use showed that the ASP’s intervention significantly reduced the overall antibiotic use at SUMC from a slight negative slope of −0.26 to a strong negative slope of −1.87 (Aminoglycoside use decreased prior to the ASP intervention (a slope of −0.036) and maintained a negative slope throughout the study period, without any significant changes. A decreasing slope for use of amox/clav was noted before the intervention, with a negative slope of −1.67. This pattern continued, albeit mostly at a non-significantly slower rate.p < 0.001; 29.6% to 32.6%, p = 0.015, respectively). In contrast, gentamicin and ciprofloxacin resistance significantly decreased in urine cultures between the pre-ASP and post-ASP periods . A non-statistically significant decrease in resistance was also noted for ampicillin and trimethoprim/sulfamethoxazole in urine cultures.As shown in p = 0.012) for aminoglycosides and −4.41 (p = 0.01) for quinolones , a black box warning was added in 2016 , which mIn the fourth quarter of 2014, we formed an ASP team comprising an infectious disease physician and two clinical pharmacists, and a hospital-wide intervention was implemented in 2015. The intervention included educational lectures on the ASP to medical staff, including nurses, residents, and attending physicians in each ward. In addition, a computerized authorization form for restricted antibiotics, which had to be renewed every three days (automatic stop order), was introduced. Restricted antibiotics included all antibiotics except for penicillin, metronidazole, 1st and 2nd generation cephalosporins, and 3rd generation ceftriaxone. All restricted antibiotics prescribed were authorized by an infectious disease specialist and then reviewed by a dedicated clinical pharmacist for adjustment to renal function, interactions with other medications, and appropriate dosing for the intended use. Each department head in the hospital received daily feedback by electronic notification of all patients under their care that had been treated with antibiotics for more than 5 days. All department heads were required to include, in their yearly reports to management, the rates of antimicrobial consumption using defined daily doses (DDD)/100 bed days compared to that of previous years. The program did not change any existing treatment protocols or practices of restricting antibiotics; however, it aimed at overall reduction of antibiotics use, particularly targeting a reduction in the use of quinolones and amoxicillin/clavulanate (amox/clav).E. coli cultures in blood or urine were included in the analysis.The study was approved by SUMC’s ethics committee, with ID Sor-19-008. The requirement for a patient’s informed consent was waived. The study was designed and reported utilizing STROBE-AMS methodology . ClinicaPatient data were collected retrospectively from SUMC’s electronic medical records.® 2 microbial identification system and antibiotic susceptibility testing . Interpretation of test results was performed according to the guidelines proposed by the Clinical Laboratory Standards Institute (CLSI), which are updated yearly [All urine and blood cultures were performed by SUMC’s Microbiology Laboratory Service according to standard techniques. Antimicrobial susceptibility tests were performed using the Kirby–Bauer disk diffusion method  or the Vd yearly .For the purpose of analysis, culture results that included a sensitivity panel and were defined as intermediate or resistant according to CLSI standards were classified as resistant. Since the SUMC lab only began to electronically mark cultures as ESBL-positive in 2016, we were unable to collect this information for the whole study period. To prevent duplication, the first positive culture was chosen for each admission.Data on antibiotic use were collected from SUMC’s bioinformatics systems, which collects information from patients’ electronic medical records as well as from logistic and supply sources. Data on antibiotic consumption are presented per annual quarter as DDD per 100 days of admission.The percentage of resistant strains in urine and blood cultures was compared for each antibiotic between the pre-ASP—from the beginning of Q1 2013 (January 2013) to the end of Q4 2014 (December 2014)—and post-ASP—from the beginning of Q1 2015 (January 2015) to the end of Q4 2018 (December 2018)—periods. All quarterly antibiotic use per DDD/100 bed days beginning in the first quarter of 2013 (data were not available prior this date) and up to the fourth quarter of 2018 was analyzed.t test for normally distributed data, or the Mann–Whitney U test for data without a normal distribution. The prevalence of resistant E. coli was examined for each antibiotic, by the percentage of total positive E. coli found in the same period. To examine the effect of the intervention on DDD and antibiotic resistance over time, interrupted time series analysis (ITS) was performed. ITS analysis is a quasi-experimental design, which uses Autoregressive Integrated Moving Average models. This statistical method investigates time trends before and after an intervention. The ITS analysis was based on the quarterly DDD value for each antibiotic that was consumed in SUMC, in addition to the quarterly percentage of antibiotic resistance, calculated for each antibiotic. We examined the trend of the slope before and after the intervention; furthermore, we examined the level effect—specifically, the change in level for every period comprised with the preintervention predicted value. Statistical calculations were performed using IBM SPSS Statistics for Windows, Version 25.0 . A p-value less than 0.05 was considered statistically significant.Categorical variables were summarized as frequencies and percentages, and continuous variables were summarized as mean and standard deviation (SD) or median with interquartile range as appropriate. Chi-squared test or Fisher’s exact test were employed for comparing categorical nominal variables. Continuous values were compared using the unpaired Student E. coli. This is an important affirmation that investing in ASP will have a clinical impact in decreasing antibiotic-resistant bacteria.In conclusion, an ASP that targets antibiotic selection pressure is an effective way to impact the resistance patterns of Gram-negative infections, specifically


Acoustic Emission (AE) nondestructive tests have attracted great interest for their use in the determination of structural properties and behavior of reinforced concrete (RC) elements. One of the applications this method can contribute to is in high-strength concrete (HSC) columns. These elements have a great advantage in the lower stories of high-rise buildings. However, the premature failure of the concrete cover and the brittleness nature of the failure is of a concern for engineers. This paper presents a study on the AE monitoring of HSC columns subjected to compressive axial loading. The study consists of four large-scale reinforced HSC columns with different confinement reinforcement and height. It is shown that the AE distributions in the columns are categorized by three stages. Moreover, the levels of loads reached at the first AE macro event are similar to the lower range levels of the nominal axial compressive strengths of the tested specimens, while the majority of macro AE events are located at the concrete cover. Based on the results of this study, AE monitoring can provide indications for the damage and load levels attained by reinforced high-strength concrete columns subjected to compressive axial loading. The use of high-strength concrete (HSC) for the lower story columns in high-rise buildings is very attractive for architects and engineers . One of The Acoustic Emission (AE) nondestructive test method was used by several research studies to examine the failure mode and the development of cracks in structural RC elements ,14. StudS for L for An experimental program was designed and performed to investigate the effectiveness of the AE technique for monitoring the cracking development and the damage levels of HSC columns. The experimental program included four circular reinforced HSC column specimens of The target concrete unconfined strength of the designed specimens was set to 78 MPa. The concrete mixture of the specimens was prepared in the laboratory. The concrete mixture properties, which had a 0.27 water–cement ratio, are given in me), fc′ .The longitudinal steel reinforcement consisted of six strength .The RC columns and the concrete cylinders were cured for 7 days and after that were laid in the laboratory ambient conditions to the day of testing. Specimen axial displacement was recorded using four Linear Variable Differential Transformers (LVDTs). Two LVDTs (with a gauge length of l plates . A load l plates . The spel plates .E) and specifically, by calculation of AE cumulative absolute energy, E) is an AE hit parameter derived by a squared sum of AE signal’s voltage along the signal’s duration divided on input impedance  Initiation of AE activity, (B) peak AE absolute energy rate distribution (C) beginning of steady damage accumulation, and (D) the first AE macro event and initiation of macro-damage development. Reference points A, B, and D were obtained from AE absolute energy rate, while event C was obtained from the cumulative AE absolute energy distribution at the moment, when a principle drop in the distribution’s slope was detected. Thus, point C was determined at the intersection of two tangential lines defined along the cumulative absolute energy curve (between points B and D). Moreover, point D was determined as the first AE event with discrete burst energy that is significantly higher than the preceding stage (which is characterized by concrete micro-cracking). The recorded loads at these events are given in e column . It shoue column  for NSC The leading RC design standards ,24,25,27given as ,24,25,278 to 1.0 , is a co8 to 1.0 . One of It is interesting to note  that thetandards ,24,25) atandards ,30,31,32AE data was also analyzed to study 3D source location of the AE events detected during loading tests. Location calculations were performed using Vallen AE software with 3D solid algorithm  and usinPD see b. This rThis paper presents a study on the acoustic emission (AE) monitoring of high-strength concrete columns subjected to compressive axial loading. The study consists of four large-scale reinforced high-strength concrete columns with different confinement reinforcement amounts and heights. The results of the study show the following.The AE distributions in the columns are categorized by three stages defined by the initiation of AE activity, the peak AE absolute energy rate distribution, the beginning of steady damage accumulation, and the first AE macro event and initiation of macro-damage development.The levels of loads reached at the peak AE energy distribution are similar to the levels of the assumed serviceability axial compressive strengths of the tested specimens.The levels of loads reached at the beginning of steady damage accumulation are similar to the levels of the design axial compressive strengths of the tested specimens.The levels of loads reached at the first AE macro event are similar to the lower range levels of the nominal axial compressive strengths of the tested specimens.The majority of AE events are located at the concrete cover and especially those associated with the macro-cracking.It is believed that the first AE macro event indicates the beginning of the premature failure of the concrete cover. Thus, this phenomenon should be taken into account in the determination of the nominal axial load compressive strength as prescribed in part of the leading standards. Moreover, based on the results of this study, AE monitoring can provide correlation between the damage and load levels  attained by reinforced high-strength concrete columns subjected to compressive axial loading and the AE results. Future studies can include additional loading schedules like loading/unloading to provide more information regarding stages of damage/cracks development in the columns and specifically regarding the load levels at which irreversible damage is initiated.


IgG4-related kidney disease (IgG4-RKD) can affect multiple organs, which was first reported as a complication or extra-organ manifestation of autoimmune pancreatitis in 2004. It is characterized by abundant IgG4-positive plasma cells infiltration in tissues involved.A 69-year-old man presented with cough and renal dysfunction with medical history of hypertension and diabetes. Pathological findings revealed interstitial nephritis and he was initially diagnosed with IgG4-RKD. Prednisone helped the patient to get a remission of cough and an obvious decrease of IgG4 level. However, he developed invasive pulmonary fungal infection while steroid theatment. Anti-fungal therapy was initiated after lung puncture (around cavitary lung lesion). Hemodialysis had been conducted because of renal failure and he got rid of it 2 months later. Methylprednisolone was decreased to 8 mg/day for maintenance therapy. Anti-fungal infection continued for 4 months after discharge home. On the 4th month of follow-up, Chest CT revealed no progression of lung lesions.The corticosteroids are the first-line therapy of IgG4-RD and a rapid response helps to confirm the diagnosis. This case should inspire clinicians to identify IgG4-related lung disease and secondary pulmonary infection, pay attention to the complications during immunosuppressive therapy for primary disease control. IgG4-related disease (IgG4-RD) is an inflammatory and fibrotic disease which was first described in the pancreas and was called autoimmune pancreatitis (AIP) in 2001 . The preIgG4-related tubulointerstitial nephritis (IgG4-TIN), is one of the frequent pathological changes of IgG4-related kidney disease (IgG4-RKD), accounting for about 15–25% of all IgG-RD . IgG4-rePatients with IgG4-RKD have an increased risk of infection than general population. IgG4-TIN can be accompanied by eosinophilic lung disease  and pneuth because of gradually aggravated edema and cough. His medical history included hypertension, arrhythmia and diabetes. On 2019 May 8th, he had experienced cough and phlegm with temperature around 38 ~ 39 °C. Laboratory tests were presented in Table 18F-FDG-PET/CT showed interstitial pneumonia in both lungs. There was also elevated uptake abnormality in the upper kidney observed. Cefperazone-Sulbactam, doxycycline hydrochloride, imipenem, and linezolid were given. Because of no improvement, he took oral prednisone 24 mg per day. The body temperature recovered to normal and lower limb edema was alleviated after one week. On 2019 June 13th ,serum creatinine 157.7 umol/L , × 400). Electron microscopy demonstrated that there were no electron-dense deposits in the glomeruli  and cyclophosphamide  were prescribed by intravenous infusion. The patient had been followed up every month after the treatment Table . He pres+/CD8+ lymphocyte: 1.9. The patient had been followed up for 4 months. In the most recent follow-up examination, the serum creatinine level decreased to 101 umol/L and he got rid of hemodialysis  indicated the diagnosis of IgG4-RKD.This patient is characterized by serum IgG4 elevation, positive MPO-ANCA, which suggested the possibility of co-occurrence/concurrence of AAV and IgG4‐RD on his first visit from his serological presentation. Diagnosis of IgG4-RD requires particular pathological, serological and clinical features , 14, 15 Given the elevated MPO-ANCA and CRP, AAV related nephritis was a possible differential diagnosis (Proposed diagnostic criteria for kidney involvement in AAV listed in Table Since kidney involvement was firstly reported in a patient with IgG4-RD in 2004 , many siHowever, he developed intermittent fever, acute onset of left pleuritic chest pain and an emerging lung lesion after steroid use for one month. IgG4-RLD has been classified into four categories based on CT. Our case was the GGO type. This also was the primary feature of IPFI  of whichSystemic glucocorticoids are recommended as the first-line approach of renal injury in untreated IgG4-RD . A moderSince not every patient can be recover from renal disfunction, maintenance hemodialysis become necessary in patients with irreversible renal failure due to IgG4-RKD . And in Taken together, IgG4-RKD is an immune-mediated condition that can affect not only kidney but also several other organs, leading to a dense lymphoplasmacytic infiltration dominant in IgG4-positive plasma cells with fibrosis. This case should inspire clinicians to identify IgG4-related lung disease and secondary pulmonary infection, pay attention to the complications during immunosuppressive therapy for primary disease control Fig. .Fig. 6F


Nowadays, prestressed concrete sleepers are the most common type of sleepers. In different countries all over the world prestressed concrete sleepers are designed with different shapes, with different types of prestressed reinforcement and arrangements in cross-section. Additionally, different manufacturing methods and techniques are used for prestressed concrete sleepers. These railway members are stiff, durable and can withstand heavy loads. Despite that, damaged or deteriorated prestressed concrete sleepers appear on railway tracks even before the end of their service life. Therefore, there is a need for better understanding of the behavior of different types of prestressed concrete sleepers to optimize their design. The rail seat section of the sleeper is the most affected part of sleeper. Therefore, analysis of experimental results of bearing capacity, cracking, and deformation of the rail seat section of sleeper under static and dynamic loads are provided in this article. Furthermore, different types, diameters, and anchoring methods of prestressed reinforcement were analyzed in this article. Additionally, comparison of experimental results of different types of prestressed concrete sleepers is discussed. The railway track has the purpose to transfer adequately the trainload to the structure of railway track. During this transfer, it has to be ensured that the track components are not subjected to loads beyond their bearing capacity. Railway track consists of two main parts: superstructure and substructure. The superstructure consists of rails, rail pads, sleepers, and fastening systems while the substructure consists of ballast, sub-ballast, and subgrade. Each layer of the railway structure reduces the effect of the load on the underlying layer and thus distributes the load evenly between the layers. Furthermore, connecting the superstructure and substructure of the track, sleepers are the most affected part of the railway structure. They withstand static, cyclic, and impact loads of different types, directions and sizes caused by trains, and are affected by support reactions of the ballast ,2,3,4. ABallast in railway track performs an important function to reduce the stress to a level which the structure of the railway track can safely withstand without any unacceptable settlement. Additionally, it provides a suitable foundation for the sleepers and also holds the sleepers in their correct level and position, preventing lateral or longitudinal displacement. The performance of a sleeper to withstand lateral and longitudinal load is dependent upon the sleeper’s size, shape, surface geometry, weight, and spacing.The rail pad is one of the less visible parts of the fastening system. However, it has an essential function in damping the effect of vertical loading (particularly impact loading). This has two aspects: by providing an adjusting layer between the rail and the sleeper, the pad ensures even pressure on the rail seat area and by acting as a spring the pad reduces the transmission of vibration and impact from the rail into the sleeper.Historically, timber, steel, and concrete sleepers were introduced in different countries taking into account climatic conditions and accessibility of local materials. However, nowadays new types of material are under consideration of using it in railway sleepers for a specific application or to obtain desired properties eliminating disadvantages of conventionality used timber, steel, or concrete sleepers. Composite sleepers are introduced to withstand harsh environmental impacts and eliminate damage of reinforcement due to corrosion of steel . High stThe comparison of the behavior of timber, steel, and concrete sleepers showed that prestressed concrete sleepers have higher bearing capacity than timber and steel sleepers. Additionally, the deflection and ductility of prestressed concrete sleepers are lower and it provides more uniform load distribution under the sleeper compared to timber and steel sleepers . TherefoMany different types of sleepers have been designed and manufactured in recent decades. The quality of prestressed concrete sleepers is very important for the safety of the railway. Firstly, the quality of concrete sleepers is very dependent on the quality of manufacturing process. Two main manufacturing methods are currently used in the world that is a long line method and a single mold method ,10. In lSingle mold method uses one sleeper long molds with up to four parallel sleepers. As in the case of longline method, this method is based on pretensioning steel bars to molds before concrete is poured. Additionally, pretensioned bars are attached to the steel bearing plates by cold-formed buttons at their tips at each end of the mold. Therefore, the prestressing force is introduced to concrete through the steel bearing plates at the ends of the sleeper and bond between reinforcement and concrete. Usually, plain bars are used in this type of manufacturing method. Therefore, the bond portion is small. However, the bond can be increased by introducing indented bars.The behavior and stiffness of the sleeper are very dependent on the ability to transfer prestressing force to concrete. Depending on the method of manufacturing of the sleepers the transfer of prestressing force to concrete can be ensured only through bond between reinforcement and concrete ,12  of monoblock prestressed concrete sleepers were tested. Type I sleepers were reinforced with four prestressed indented steel bars a. Type IUsually, sleepers are affected by positive bending moment at the rail seat section and can be affected by negative bending moment in the middle of the sleeper . Therefo28 monoblock prestressed concrete sleepers were tested during experimental research . Static Main geometrical properties of each type of sleeper are provided in 0/fpk = 0.79) and Type III sleepers (σ0/fpk = 0.78). Contrary, the lowest utilization of reinforcement strength is in Type I sleepers reinforced with Ø10.5 mm indented bars (σ0/fpk = 0.66)  .c), reinforcement strength (fpk), and reinforcement modulus of elasticity (Ep) are provided in The design concrete class of all tested sleepers was C50/60. Additionally, concrete cubes (100 × 100 × 100 mm) were cut from Type I, Type II, and Type III sleepers and standard concrete cubic (150 × 150 × 150 mm) strength was determined for each type of sleepers. Type I sleepers were reinforced with Ø9.6 mm or Ø10.5 mm prestressed indented steel bars. Type II and Type III sleepers were reinforced with Ø6.8 mm prestressed indented three-wire steel strands and with Ø7.0 mm prestressed plain steel bars respectively. The results of concrete compressive strength . Furthermore, the load was increased in steps every 10 kN until cracking load (Fcr.s). After the first crack appeared sleeper was unloaded at a constant speed of 2 kN/s. After that static load test was performed in steps. After every load step sleeper was unloaded. In each load step, static load was increased by 10 kN . This load is an upper value (Fmax.c) of the first stage of dynamic load. When Fmax.c is reached sleeper is unloaded at a constant speed of 2 kN/s until the lower value (Fmin.c) of dynamic load. Then the dynamic load test begins. In every next load stage, the dynamic load is increased in steps every 20 kN. After every dynamic load stage, the sleeper is unloaded. During the test, lower value of dynamic load was kept constant in every stage and was equal to Fmin.c = 50 kN. In the beginning and at the end of every load step crack widths and strains through the height of the cross-section were measured.The dynamic load test of the sleeper is divided into stages b. In eveLoading and supporting conditions were taken according to  and are Two techniques of measuring strains on the concrete surface through the height of the rail seat section during static and dynamic load tests were applied. On one side of sleeper concrete strains were measured with LVDT with an accuracy of 0.001 mm. Strains were measured at the level of mostly tensioned reinforcement, near the compressive zone and one measurement was performed at an intermediate level a. On theType I and Type III sleepers need to resist higher design cracking moment compared to Type II sleepers . Type IIAverage cracking moment of Type I sleepers prestressed with Ø9.6 mm indented bars was the lowest compared to other sleepers . This isThe stiffness of Type II sleepers was higher due to 5% higher precompression force in rail seat section and 5% lower negative eccentricity of prestressed reinforcement compared to Type I sleepers. However, the reinforcement ratio of sleepers prestressed with Ø10.5 mm indented bars was by 28% higher and concrete compressive strength was by 26% higher compared to Type II sleepers. Therefore, average cracking moment of Type II sleepers was slightly (2%) higher compared to sleepers reinforced with Ø10.5 mm indented bars. Additionally, the highest average cracking moment was determined for Type III sleepers. This was influenced by the highest precompression force in rail seat section and positive eccentricity of prestressed reinforcement. Therefore, Type III sleepers have the highest resistance to cracking.The experimental and design bearing capacities of all types of sleepers are provided in From The reinforcement ratio and concrete compressive strength in Type II sleepers were by 6% and 26% lower respectively compared to sleepers reinforced with Ø9.6 mm bars. However, the strength of reinforcement in Type II sleepers was by 18% higher. Therefore, in total bearing capacity of Type II sleepers and sleepers reinforced with Ø9.6 mm bars was approximately equal .cr) was used for the comparison. It was done to evaluate differences in geometrical parameters, initial prestress force  was in case of Type III sleepers and Type I sleepers reinforced with Ø9.6 mm bars. It was 25–31% higher compared to other types of sleepers. Therefore, cracking development stage was longer in Type III and Type I sleepers reinforced with Ø9.6 mm bars. Up to bending moment equal to 67% and 77% of bearing capacity of Type III sleepers and Type I sleepers reinforced with Ø9.6 mm bars respectively the difference between average residual crack widths was 0–20%. However, under the influence of higher bending moments, the residual crack widths of Type I sleepers reinforced with Ø9.6 mm bars were propagating much faster and the difference increased rapidly. Additionally, the stiffness of these sleepers also decreased much faster. It is influenced by a higher reserve of bearing capacity of Type III sleepers which in turn depend on the higher height and area of the rail seat section.The highest difference between experimental bearing capacity and experimental cracking moment  compared to sleepers reinforced with Ø9.6 mm bars. Types of failure for these sleepers were the same as in the case of static load . TherefoBearing capacity of Type II sleepers was by 2–6% higher compared to Type I sleepers after the influence of dynamic load. The highest bearing capacity was of Type III sleepers and it was by 4–10% higher compared to other types of sleepers.In all types of sleepers, dynamic load had negative impact on bearing capacity. Therefore, bearing capacity of sleepers reinforced with Ø9.6 mm indented bars, Ø10.5 mm indented bars, Ø6.8 mm three-wire strands and Ø7.0 mm plain bars decreased by 15%, 9%, 10%, and 21% respectively compared to static bearing capacity. The experimental results showed that the highest negative impact of dynamic load was on bearing capacity of Type III sleepers. Therefore, the difference in bearing capacity between Type II and Type III sleepers under dynamic load was smaller compare to the static test results .cr). The crack widths are provided at different stages of dynamic load test: crack widths before each dynamic load step and after each dynamic load step  was selected incorrectly and anchorage length of prestressed bars was insufficient. Conversely, in Type II sleepers reinforced with three-wire strands, the bond between reinforcement and concrete was adequate and strands did not slip during the test. Additionally, the anchorage and consequently bond of plain bars were ensured by additional anchor plates at the ends of Type III sleepers.The research results showed that experimental cracking and failure moments of Type I sleepers were lower than design moments. This means that the structural solution of these type of sleepers was not suitable, because their behavior did not correspond to design evaluation of sleepers. The research has shown that failure occurred due to insufficient anchorage of reinforcement in concrete. In the case of Type II and Type III sleepers, the experimental values of cracking and failure moments were higher than the design ones, because anchorage of reinforcement in concrete was sufficient.The research showed that reinforcement and rail seat section of Type III sleepers are chosen irrationally. During static and dynamic load tests, the sleepers failed in the concrete compression zone. This indicates that sleeper reinforcement does not match the selected cross-section of sleeper.The use of higher diameter (Ø10.5 mm) prestressed bars improved the Type I sleeper’s resistance to cracking at rail seat section. Therefore, propagation of residual crack width was slower compared to sleepers reinforced with lower diameter (Ø9.6 mm) prestressed bars. However, a higher reinforcement ratio negatively influenced anchorage zone of sleeper and sleepers failed due to slip of reinforcement accompanied by longitudinal splitting of concrete. Therefore, confining reinforcement or anchors could be introduced at the ends of the sleeper to minimize the concrete splitting effect.The distribution of crack width between each type of sleepers was more even under the influence of dynamic load compared to static load. Therefore, in contrast to static load test, crack widths of Type I sleepers reinforced with Ø10.5 mm bars were similar to crack widths of other types of sleepers under the influence of dynamic load. It means that Type I sleepers reinforced with Ø10.5 mm bars are more susceptible to dynamic load effect. Therefore, dynamic load reduces stiffness of this type of sleepers faster than for other types of sleepers.It was found that the bearing capacity of Type I sleepers reinforced with Ø9.6 mm and Ø10.5 mm indented bars, Type II and Type III sleepers after the influence of dynamic load decreased by 15%, 9%, 10%, and 21% respectively compared with static test results. The greater degradation of prestressed concrete sleepers under the influence of dynamic load is related to higher deformation of reinforcement in the crack plane and greater damage on bond between reinforcement and concrete near the crack plane. Additionally, the character of failure of each type  of the sleeper was the same after static and dynamic load tests.