Staphylococcus lugdunensis (SL) belongs to the group of coagulase negative Staphylococci (CoNS). SL infections may have a more virulent course compared to other CoNS, bearing resemblance to infections caused by S. aureus (SA). This study aims to investigate the clinical characteristics of SL infections at a tertiary care center in Lebanon.We enrolled all patients presenting from 2017 to 2021 with positive cultures for SL from four clinical sites . We then selected controls with SA matched 1:1 by site of infection. Since CoNS is not a well-described respiratory pathogen, CoNS were only matched to urine, wound, and blood. Patients who were deemed to have colonization were excluded. We determined the risk factors and outcomes associated with SL infections compared to SA and CoNS infections. We then identified predictors of mortality in the SL cohort.We identified 101 patients with SL infection, who were matched to 103 patients with SA and 97 patients with CoNS. Most SL isolates were obtained from wound samples (72%), followed by urine (16%), blood (8%) and sputum (4%). Compared to SA, SL patients were less likely to be admitted to the hospital for more than 6 days within the past 30 days . On the other hand, compared to CoNS, patients in the SL cohort were more likely to have a history of endocarditis . There were no other significant differences between baseline characteristics of SL, SA, and CoNS patients. As far as outcomes, compared to the SA group, patients with SL were more likely to develop renal insufficiency  and less likely to have persistent bacteremia . No difference in outcomes was observed between the SL and CoNS cohorts. The mortality rate among SL patients was 8%. The predictors of mortality in this cohort are found in the table.Bivariable and multivariable analysis of predictors of mortality among patients with SL infectionsSL appears to be more associated with patients in the community rather than the hospital setting and may be more likely to occur in patients with damaged heart valves. Chronic pulmonary disease and a blood source of infection are associated with a fatal outcome in SL patients.All Authors: No reported disclosures


Recently, silsesquioxanes (SSQ) and polyhedral oligomeric silsesquioxanes (POSS) have gained much interest in the area of biomaterials, mainly due to their intrinsic properties such as biocompatibility, complete non-toxicity, the ability to self-assemble and to form a porous structure, facilitating cell proliferation, creating a superhydrophobic surface, osteoinductivity, and ability to bind hydroxyapatite. All the above has resulted in new developments in medicine. However, the application of POSS-containing materials in dentistry is still at initial stage and deserves a systematic description to ensure future development. Significant problems, such as reduction of polymerization shrinkage, water absorption, hydrolysis rate, poor adhesion and strength, unsatisfactory biocompatibility, and corrosion resistance of dental alloys, can be addressed by the design of multifunctional POSS-containing materials. Because of the presence of silsesquioxanes, it is possible to obtain smart materials that allow the stimulation of phosphates deposition and repairing of micro-cracks in dental fillings. Hybrid composites result in materials exhibiting shape memory, as well as antibacterial, self-cleaning, and self-healing properties. Moreover, introducing POSS into polymer matrix allows for materials for bone reconstruction, and wound healing. This review covers the recent developments in the field of POSS application in dental materials and gives the future perspectives within a promising field of biomedical material science and chemical engineering. For over twenty years, dentistry has been using composite polymeric materials, mainly methacrylate resins, that have outperformed amalgams, ceramics, and metals . The priTo meet the requirements for hydrolytic stability, bioactive/biomimetic and antibacterial capabilities, abrasion resistance, long-term mechanical strength, and other properties, modification of polymeric matrices is usually needed, e.g., by introducing fillers to form composites . Thus, cPolyhedral oligomeric silsesquioxanes (POSS) are organosilicon hybrid materials with a three-dimensional, chemically substituted, and thermally stable inorganic silicon-oxygen core (cage) to which organic substituents are covalently bonded . The subPolyhedral oligomeric silsesquioxanes exhibit unique properties, such as biocompatibility, non-toxicity, stability, tastelessness, well-defined structure, and hydrolytic degradation to neutral orthosilicic acid ,13,14. IThe most significant advantage of POSS is the ability to tailor their properties by selecting proper substituents ,18. ChanIntroducing POSS particles into a polymer matrix may enhance the material’s toughness, strength, thermal stability, ultraviolet steadiness, abrasion resistance, hardness, and biocompatibility (improved cell attachment and cell proliferation) ,22. In aAll the above explains considerable interest that POSS have gained in terms of application in biomedical fields , e.g., PMuch of the attention devoted to POSS and POSS-based composites in biomedicine concerns the research in the field of dentistry. In this review we present and discuss the most recent advances in the area of POSS and POSS-based polymer composites as materials for dental applications. This work provides also an outlook for the future of POSS/polymer composites applications in dentistry and dental surgery.anti-shrinkage additive for dental materials since Culberston et al. introduced MA-POSS to dental composites in 2001 -pyrimidinone (UPy) groups and then crosslinked by thiol-click chemistry [Since then, numerous articles about POSS in bone regeneration have been published ,145,146.v/v) . Subsequn (TIPS) . The buln (TIPS) . In the n (TIPS) . By tranhosphate . POSS sehosphate . Comparehemistry . CompareCombining chitosan and POSS-1 appears to be beneficial. Celesti et al. tested different concentrations of POSS-1 in CS-POSS hybrid hydrogels that were doped with ketoprofen to test drug release . The posSimilarly, covalent attachment of POSS were used in another work  in whichFinally, a macroporous polyetheretherketone (PEEK) scaffold modified with methacrylated chitosan and MA-POSS was obtained . PEEK isIn recent years, many publications have been devoted to hydrogels as materials for wound dressing. The above originates from hydrogel properties, such as adjustable mechanical properties that mimic natural tissue, good water absorption and retention capacity, controlled oxygen and nutrient permeability, easy and inexpensive processing, good wear resistance, and comfort. Modification of hydrogels with POSS allowed for obtaining bio-materials with superior properties. One of the first hydrogel systems containing POSS was obtained in the year 2005  and thenSuch shrinking hydrogels were described by Bu et al. . They arPolyhedral oligomeric silsesquioxanes are promising fillers for dental polymer composites. POSS incorporation not only may enhance resin properties, but also give rise to new ones, e.g., osteoinductivity. The latest works showed that POSS may be successfully incorporated into a polymer matrix to promote hydroxyapatite formation, reduce shrinkage during curing, increase the mechanical performance of the fillings, or ensure antimicrobial properties. Simultaneously, silsesquioxanes may act as co-initiators, which creates additional opportunities to combine the role of filler and a crosslinking agent. Selecting the proper POSS type and its content allows for tailoring the properties of dental composites.Among all studied POSS for dental materials, POSS functionalized with methacrylate groups receive the most attention. Out of methacrylate silsesquioxanes, octamethacrylate POSS (MA-POSS) seems to be the most examined one. Its incorporation in dental resins reduces water sorption, enhances strength, and promotes osseointegration, reducing either the emerging cavities or microleakage, and may be used for bone reconstruction. Multiple reactive groups secure high homogeneity, and since the POSS nanocage is surrounded by long polymeric chains, the agglomeration effects are reduced. POSS concentration also plays an important role—most materials remained homogeneous or self-assembled into nanodomains when POSS concentration was less than 10 wt.%. Exceeding this content most often results in deterioration of mechanical properties such as flexural strength, hardness, Young’s modulus, and scratch resistance, which are a key properties in dental fillings. With these properties in mind, it would be good to use even 5 wt.% POSS. However, if controlling the shrinkage is the only issue to address, the amount of POSS may be increased up to 10 wt.%.POSS hydrophobicity allows for the reduction of hydrolysis in both methacrylate dental fillings and PEG hydrogels composites. The above results in delaying their bio-degradation, e.g., until the alveolar bone is recovered. Thus, POSS may also act as a factor regulating the in vivo lifespan of dental materials. Moreover, hydrophobicity plays an important role in the case of tissue adhesives. Silsequioxanes might therefore be used as adhesives, since the strong adhesive effect on living tissue occurs due to the presence of the hydrophobic POSS segments. The future perspectives of POSS-containing materials in dentistry include new developments in the design of materials exhibiting shape memory, self-cleaning, and bactericidal properties. Another class of promising hybrid materials with POSS are injectable or 3D-printed scaffolds in which silsesquioxanes may ensure controlled microporous morphology and facilitate cell proliferation. Since the research focused mostly on octa-functional MA-POSS, many possible architectures that may be interesting due to various substituents (such as double-decker or open-cage POSS) have not been tested. One POSS molecule may play two or more different roles in a composite if equipped with different substituents. Combining POSS with chitosan, or HAp seems to be also attractive for self-repairing fillings, where osteoinductivity plays an important role. POSS self-assembling abilities may be used for drug release systems or enhancing the bactericidal properties of composites. Such approaches seem to be more advanced in other fields of medicine; therefore, taking advantage of these achievements and introducing them in dentistry might prove beneficial.


Cows were randomly assigned to 1 of 4 experimental treatments: (1) control; no supplemental Met or choline (CON), (2) 13 g/d of choline ion delivered via abomasal infusion (CHO), (3) 13 g/d of Met delivered via abomasal infusion (MET), and (4) 13 g/d of choline and 13 g/d of Met delivered via abomasal infusion (CHO + MET). Cows received the same basal diet throughout the experiment, which was formulated to be deficient in Met . Periods were 7 d in length with d 1 to 2 serving as a wash-out period and cows being infused on d 3 to 7. Milk samples were collected twice daily on d 5 to 7 and were analyzed for fat, true protein, lactose, and choline metabolites including betaine, phosphocholine, and free choline using hydrophilic interaction liquid chromatography-tandem mass spectrometry. Blood samples were collected via venipuncture of the coccygeal vein at 1100, 1300, and 1500 h on d 7 of each period and were analyzed for free AA as well as choline metabolites. Plasma Met increased in response to Met infusion and an interaction with choline and Met infusion was observed in the plasma concentration of branched-chain AA. Cows receiving choline exhibited the greatest Cho yield in milk. Milk phosphocholine yield tended to be highest when both choline and Met were infused.Four lactating, ruminally cannulated Jersey cows, (mean ± standard deviation) 264 ± 54.2 d in milk and 484 ± 24.1 kg of body weight, were arranged in a 4 × 4 Latin square design to measure the effects of abomasal infusion of choline chloride with or without  RPC) and Met (RPM), have been explored as production- and health-enhancing feed additives in dairy cattle nutrition, particularly in periparturient cattle in the case of RPC. Furthermore, because they both participate in one-carbon metabolism, their interplay has been examined when fed in combination. For example, in a meta-analysis summarizing the effects of RPC supplementation , supplementing choline ion at a rate of 12.9 g/d increased milk yield by an average of 2.0 kg/d; however, when METMP was high , the same amount of choline supplementation resulted in an increase of 1.02 kg/d of milk. A similar pattern was observed for ECM yield . There was also an interaction between the linear effects of choline and METMP on milk protein yield, where supplementing 12.9 g/d of choline ion when METMP was low resulted in an average increase in milk protein yield of 0.07 kg/d, but when METMP was high, increased 0.04 kg/d. In an experiment conducted by dl-Met prepartum and 13.5 g/d dl-Met postpartum), RPC and RPM (13.0 g/d choline ion and 9 g/d dl-Met prepartum and 13.5 g/d dl-Met postpartum), or control (no RPM or RPC) during the periparturient period. The Met concentrations were 1.77 and 1.82 Met, % of MP in pre- and postpartum diets, respectively. The researchers observed an 8.7 kg/d increase in milk yield for multiparous cows when RPC was fed without RPM, though the increase was not observed when RPC and RPM were fed in combination. Additionally, despite the notion that choline may have a sparing effect on Met and promote milk protein synthesis, RPC fed with RPM did not show any significant impact on milk protein concentration.Choline and Met are both methyl donors involved in one-carbon metabolism, a variety of pathways that work together to promote lipid, nucleotide, and protein synthesis, as well as an abundance of methylation reactions and regulators of the animals' oxidative status . Accordientation , positivWhile the interplay of supplemental choline and Met on production performance has been explored, the effects of supplementation in combination on the appearance of choline metabolites and plasma Met levels has not been fully established. The objective of this experiment was to investigate potential changes in the appearance of choline metabolites in milk and plasma when choline was abomasally infused with or without infusion of Met. Choline metabolites appearing in plasma and milk have been utilized previously as bioavailability markers for choline ; therefoBefore conducting the experiment, procedures using animals were approved by the University of Nebraska–Lincoln Institutional Animal Care and Use Committee. Four lactating, ruminally cannulated Jersey cows, averaging (±SD) 264 ± 54.2 DIM at the start of the experiment and averaging 484 ± 24.1 kg of BW, were used in this experiment. Cows were housed in a temperature-controlled barn at the Dairy Metabolism Facility in the Animal Science Complex of University of Nebraska–Lincoln  in individual tiestalls equipped with rubber mats, and were milked at 0700 and 1800 h. Cows were fed the same basal diet for 30 d before and throughout the experiment, which was formulated to meet expected dietary requirements determined using the CON), (2) 13 g/d of choline ion  delivered via abomasal infusion (CHO), (3) 13 g/d of Met  delivered via abomasal infusion (MET), and (4) 13 g/d of choline ion and 13 g/d of Met delivered via abomasal infusion (CHO + MET). The abomasal infusion treatments were continuously infused each of the last 5 d of each period at a rate of 4 L per d (approximately 167 mL/h) using a peristaltic pump . Days 1 to 2 served as a wash-out period; we anticipated that 2 d of wash-out and 5 d of treatments would be sufficient for absorption of choline and incorporation into milk as described by dl-Met 99%, so 13.1 g/d was infused in solution to deliver 13 g/d postruminally. The abomasal infusion line was constructed according to The experimental design was a 4 × 4 Latin square with 4 periods of 7 d. Cows received 1 of 4 experimental treatments that were designed to deliver choline ion, Met, or both via abomasal infusion. The experimental treatments were as follows: (1) control; no infusion of supplemental Met or choline (Cho), betaine (Bet), and phosphocholine (Pcho) were measured using hydrophilic interaction liquid chromatography-tandem mass spectrometry at an external laboratory . Additional samples were collected at the time of milking in 50-mL tubes containing 2-bromo-2-nitropropane-1,3 diol and shipped to DHIA  where they were analyzed for fat, true protein, and lactose  were measured using hydrophilic interaction liquid chromatography-tandem mass spectrometry at an external laboratory . An aliquot of 3 mL of plasma was deproteinized with 15% sulfosalicylic acid . Samples were placed in an ice bath for 10 min before centrifuging at 1,500 × g at 4°C for 20 min. The supernatant was collected, and 0.75-mL aliquots were placed into Nunc CryoTube vials  and stored at −80°C. Plasma samples were submitted to the University of Missouri–Columbia Agricultural Experiment Station Chemical Laboratory for free AA analysis (M) were adjusted for the use of 15% sulfosalicylic acid.Blood samples were collected via the coccygeal vein into Vacutainer tubes containing EDTA  at 1100, 1300, and 1500 h on d 7 of each period. The samples were immediately placed on ice and centrifuged within 45 min at 4°C at 1,500 × analysis . Plasma Milk + 0.032[Pcho]Milk. Data were analyzed as a 4 × 4 Latin square using the PROC MIXED procedure of SAS . The model fixed factors included the period, choline, methionine, and interaction between choline and methionine. Cow was considered the random effect. Treatment means were generated using the LSMEANS statement. Significance was declared at P < 0.05 and trends were defined as 0.05 < P ≤ 0.10.Net portal flux of choline was predicted using milk choline metabolites according to The primary purpose of this experiment was to explore the potential changes in the appearance of choline metabolites in milk and plasma when choline was abomasally infused with or without Met in cows consuming a Met-deficient diet. We wished to investigate the potential impact of a Met-deficient diet on plasma free AA and the appearance of choline metabolites used as biomarkers . AlthougP < 0.01) plasma Met, but no interaction (P = 0.68) between infused choline and Met was observed. Plasma Met concentration averaged 15.8, 15.3, 27.9, and 25.1 ± 3.20 μM for CON, CHO, MET, and CHO + MET treatments, respectively. The results indicate that Met was successfully delivered postruminally under the conditions of the abomasal infusion system. Additionally, plasma Cys concentration was increased by infusion of Met (P < 0.01) and no interaction (P = 0.36) between choline and Met was observed. These results were likely related to the increased concentration of plasma Met observed, as Cys may be synthesized metabolically from dietary Met. This process occurs endogenously by first demethylating Met to homocysteine (via intermediate forms of S-adenosylmethionine and S-adenosylhomocysteine) and subsequent combination with Ser to yield cystathionine, which is then cleaved by cystathionase to yield Cys and α-ketoglutarate (BCAA), namely, Val (P = 0.02) and Leu (P = 0.01). These were reduced when comparing the CHO to the CON treatment, but were not different from MET and CHO + MET treatments. To our knowledge, this effect has not been observed in dairy cows; however, M, decreased (P = 0.05) to 122 ± 10.2 μM when choline was infused, and returned to 149 ± 10.2 μM when Met was infused with choline (P = 0.01). The plasma concentration of Lys was increased in cows infused with MET (P = 0.01), but no interaction between choline and Met was observed (P = 0.17). An interaction of choline and Met infusion was observed on the plasma concentration of Trp, which was greatest in cows receiving CHO + MET (P = 0.01).Plasma AA concentrations are listed in lutarate . An inteP > 0.05) between treatments (P = 0.05) and Cho tended (P = 0.10) to increase with choline infusion. We did not observe an effect (P 0.11) on Pcho, which was expected as choline infusion typically results in increases in all 3 metabolites as observed by M) and yield (g/d) of Bet, Cho, and Pcho, and estimated net portal flux (g/d) of Cho. The concentrations of Bet in milk tended (P = 0.08) to increase with choline infusion, whereas Cho and Pcho were not affected (P ≥ 0.13) by treatments. Estimated net portal flux of choline tended (P = 0.08) to increase with choline infusion. The yield of milk Cho also increased (P = 0.04) in response to choline infusion, but not with Met infusion (P = 0.57); treatments were observed to be 0.09, 0.11, 0.08, and 0.15 ± 0.027 g/d for CON, CHO, MET, and CHO + MET, respectively. Several crossover designs have employed abomasal infusions to test the effects of choline or AA on plasma appearance (P = 0.12) between choline and Met infusion on milk Pcho yield. Using a post hoc LSMEANS test (superscripts not shown in tables), we tended to observe a difference (P = 0.06) between the CHO + MET versus CHO treatments (0.11 vs. 0.05 ± 0.02 g/d). This result is noteworthy in terms of our hypothesis in that when cows were fed a Met-deficient diet and infused with choline, the yield of Pcho in milk tended to be greater when choline was infused in combination with Met compared with choline alone. The response could provide preliminary evidence to the notion that choline may be diverted for use as a methyl donor to regenerate Met (S-methyltransferase (BHMT) and Overall DMI, milk yield, milk fat, and milk true protein measures were not different (eatments . Due to pearance  and the rate Met . This pr


The primary goal of the testing campaign was to study the shrinkage behaviour of large-scale specimens exposed to real environmental conditions. Large-scale prismatic concrete specimens were equipped with vibrating wire strain gauges to monitor the strain evolution inside the specimens. To analyse the shrinkage behaviour of the specimens, the thermal strain had to be deducted from the measured strain. To study the influence of seasonal environmental conditions, different specimen production dates (in summer and winter) were examined. The measured shrinkage strains of the large-scale specimens are compared with the results of shrinkage models developed by two engineering entities  and RILEM ). The comparison shows a poor agreement of the measurements with the models, even though the results from the model for small specimens tested in the laboratory under constant environmental condition agree well with the experimental results. This leads to the conclusion that the poor agreement between the measurements and the shrinkage models must be due to the seasonally changing environmental conditions. The comparison of the results from specimens with different production dates shows that different shrinkage behaviour occurs, especially in the first year of measurements.This article presents an experimental testing campaign on large-scale concrete specimens with cross-sectional areas of up to 1 m NU Database of Laboratory Creep and Shrinkage Data [Shrinkage of concrete is usually defined as the load-independent strain due to drying of the material. From an engineering point of view, shrinkage of concrete can be decomposed into drying shrinkage and basic shrinkage . Drying age Data . This daage Data ,10,11. Tage Data  and moreage Data : (i) Theage Data  had a duge times . To highge times  as a funIn addition to the fact that the size and service life of concrete structures exceed the size and test duration of the specimens of the shrinkage tests contained in the database, it has also been shown that the influence of seasonal changes in environmental conditions on the results is significant; see, e.g., ,16. The To carry out long-term tests on specimens exposed to real environmental conditions, a monitoring concept allowing for continuous measurements is required. Furthermore, to capture the influence of daily temperature cycles, sensors with high resolution and accuracy are needed. Ge et al.  tested fAs indicated by The goal of the long-term testing campaign was to carry out creep and shrinkage tests on large-scale test specimens subjected to real environmental conditions. Initially, four test series were produced, with six large-scale specimens per test series. Every test series consisted of three shrinkage and three creep specimens, resulting in a total number of 24 large-scale specimens. In this publication, the results of the shrinkage specimens of three of the test series  are presented.fib model code 2010 [u is the perimeter subjected to drying. The test specimens were prisms with a length of 3000 mm for the large and medium specimens and 2900 mm for the small specimens; see The large-scale specimens were designed to cover a broad range of notional member sizes ode 2010 , h0 is dThe first two test series, S1 and S2, were produced in July 2017 and series S4 was produced in February 2018. Series S1 and S4 have the same concrete composition which yields concrete strength class C30/37. A different concrete composition was chosen for test series S2, which yields concrete strength class C50/60.The large-scale specimens were produced to simulate the behaviour of real concrete structures. Therefore, all the production steps such as installing the formwork, casting the concrete, vibrating, curing, and formwork removal was carried out by a construction company; see The final storage location was the storage yard of a precast plant where the specimens were stored outdoors; see The concrete compositions were chosen to represent two concrete mixes commonly used in Austria. All the series were prepared from a single batch of ready-mixed concrete with compositions as specified in E, the compressive strength The basic mechanical properties at the age of 28 days are summarised in 12390-13 , EN-123912390-13 .The aggregate was the same for both concrete compositions; see To monitor the continuous evolution of the strain inside the large-scale specimens, vibrating wire strain gauges  were usThe VWSGs had an effective gauge length of 153 mm and a resolution of rding to . The senThe measurements of the VWSGs were recorded using a Campbell Scientific CR6 measurement and control data logger. Every large-scale specimen also had four extensometer measurement points on each side. The extensometer measurements were used to verify the VWSG measurements.Since concrete is a mixture of cement, aggregate, and water, the hardening of concrete is characterised by the transformation of the liquid phase to a hardened phase, which occurs due to the chemical reactions of the hydration process. The liquid phase of fresh concrete is represented by the cement paste. The hardening of the cement paste can be divided into three stages. The first stage is the dormant period which starts after water has been added to the cement. During the dormant period the cement paste is liquid. The setting period starts when the hydration products start to intersect with each other. During the setting period the cement paste starts to gain stiffness, and at the end of setting the cement paste solidifies. After the setting period finishes, the hardening period starts, during which the cement paste continues to harden and its mechanical strength increases.The time of setting is therefore important as the point in time at which the VWSGs and the surrounding concrete start to act compositely. Nam et al.  defined t, respectively, B is the batch calibration factor, and B and Since the strain readings obtained from the VWSGs are calculated from the changes in the natural frequency of the wire  and theTo separate the thermal strain from the measured strain, two different approaches are used to identify the linear coefficient of thermal expansion (CTE). The first approach is used to determine the CTE of the concrete up until the formwork removal. The second approach is used to calculate the CTE during the whole measurement period. For the first approach, the strain measurements of the first four days were used. To determine the CTE at the beginning of the measurements, the hydration heat, as shown in i-th day of the whole measurement period are calculated. For every daily mean temperature T. T in To determine the CTE for the entire measurement period, the following approach was used. The daily mean strains Equation :2)αc,i=αc,i=ε¯i In general, the time-dependent behaviour of concrete can be formulated mathematically within the framework of linear ageing viscoelasticity as follows :4)ε(t)=ε(t)=(4)εSince the large-scale specimens are unloaded and can freely contract or expand, the terms for the stress-induced strain and the strain due to cracking in Equation  are assut in Since VWSGs allow measurements to be carried out from the time the sensor and the surrounding concrete start acting compositely, measurements documenting the strain development due to hydration heat can be taken. For the separation of the thermal strain, mens see , and themens see  have to mens see  and are mens see , using tUtilising the calculated CTEs, the separation of the shrinkage strain Equation :5)εsh; and (ii) the International Union of Laboratories and Experts in Construction, Materials, Systems and Structures  the Fédération Internationale du Béton   fib MC 2The laboratory measurements, which were performed at a constant temperature and humidity, agree well with the results of both models, with In the large-scale specimens produced in the summer (S1 and S2), the influence of the specimen size on the measured shrinkage strain was relatively small, contrary to the estimates from the considered models.The varying environmental conditions over the first 100 days significantly influenced the rate of shrinkage (decreased shrinkage rate for the summer series (S1 and S2) and increased rate for the winter series S4). This is not reflected by either model.As can be seen in From the comparison of the models with the experimental measurements the following conclusions can be drawn:Although experimental proof of the influence of seasonal changes in environmental humidity and temperature on the shrinkage behaviour of concrete is scarce, some studies have dealt with this topic; see, e.g. ,50,51,52A testing campaign to investigate the influence of the production date on the time-dependent behaviour of concrete was performed by Vandewalle . The inforted in  were carorted in  observedThe difference between the findings of Vandewalle  and the yard see b, the avyard see c. It canThe production date has a significant influence on the evolution of the shrinkage strain. Specimens produced in summer exhibited less shrinkage than those produced in winter. Similar observations have previously been documented in ,18.Contrary to the observations of Vandewalle , the shrTo summarise the findings regarding the influence of different production dates on the shrinkage strain, the following observations can be made:VWSGs can be used for long-term measurements. In the presented study, VWSGs took strain measurements for over six years and the accuracy of the measurements is confirmed by additional measurements carried out with an extensometer.VWSGs allow for early-age concrete strain measurements as soon as the concrete and the sensor start acting compositely .The coefficient of thermal expansion (CTE) of concrete was back calculated from the measurements of the VWSGs. Two different calculation procedures were used to determine the CTE at an early age and during the whole measurement period.The CTE calculated for the whole measurement period shows a dependence on the measured concrete temperature. The calculated CTE values increase with increasing temperature.fib and RILEM) did not agree well with each other. Since the models do not capture the influence of changing environmental conditions, the measured shrinkage strains of the large-scale specimens were not predicted accurately by the models. Shrinkage strains from the tests carried out in the laboratory agreed well with the results from the models, since the shrinkage tests were performed under constant environmental conditions  embedded in the specimens. The large-scale specimens were unloaded and exposed to real environmental conditions. The analysis of the results of the long-term measurements has led to the following conclusions:


Antibiotic use is associated with development of antimicrobial resistance and dysregulation of the microbiome . These changes have in turn been associated with downstream adverse health outcomes. This review analyses recent important publications in a rapidly evolving field, contextualizing the available evidence to assist clinicians weighing the potential risks of antibiotics on a patient's microbiome.Although the majority of microbiome research is observational, we highlight recent interventional studies probing the associations between antibiotic use, microbiome disruption, and ill-health. These studies include germ-free mouse models, antibiotic challenge in healthy human volunteers, and a phase III study of the world's first approved microbiome-based medicine.The growing body of relevant clinical and experimental evidence for antibiotic-mediated microbiome perturbation is concerning, although further causal evidence is required. Within the limits of this evidence, we propose the novel term ‘microbiotoxicity’ to describe the unintended harms of antibiotics on a patient's microbiome. We suggest a framework for prescribers to weigh microbiotoxic effects against the intended benefits of antibiotic use. Antibiotics, when appropriately prescribed, are lifesaving, indispensable weapons in our clinical armoury. However, decades of inappropriately broad, lengthy or even unnecessary antibiotics have led to the global emergence of antimicrobial resistance (AMR) ▪. The WHIn recent years, there has been exponential growth in research into and interest in the human microbiome. Such research has highlighted associations between antimicrobial use, microbiome perturbation, and adverse health outcomes. This review analyses the role of the microbiome as a complex immunological, endocrine and neurological organ system, and the potentially harmful effects of antibiotics on this microbial ecosystem. We propose the novel term ‘microbiotoxicity’ to encompass the unintended side effects of antibiotics on a patient's microbiome. In doing so, we urge clinicians to weigh the benefits of antibiotics in treating infection against these microbiotoxic effects when making individual prescribing decisions. The microbiome is the total community of living microorganisms colonizing all outer and inner body surfaces, along with their microbial metabolites, organic compounds and genetic material . There aGut microbes are involved in a wide range of physiological functions, including production of essential vitamins, bile salts and short-chain fatty acids (SCFAs) such as butyrate . These SLike any organ system, the microbiome demonstrates predictable developmental trajectory. Newborns are born virtually free of bacteria, becoming rapidly colonized with a diverse pioneer microbiome derived largely from their mothers’ vaginal, faecal, skin, mucosal and breastmilk flora . Within Some argue it is inappropriate to consider the microbiome an organ system, because of its mutability and inter-individual variability . HoweverIt might appear redundant to point out that antibiotics kill bacteria; and yet we see no redundancy in warning our patients of the side effects of antibiotics on their own microbiota. We already warn oncology patients of the cytotoxic effects of chemotherapy, and consider liver and renal function tests before prescribing hepatotoxic or nephrotoxic agents. So why not pay heed to our patients’ microbiomes when prescribing microbiotoxic agents Fig. ?Bifidobacterium, Lactobacillus and Bacteroides species . This may lead to infections with AMR pathogens, and onward transmission of bacteria carrying AMR genes.The association between antibiotic use and microbiome perturbation is becoming increasingly compelling, and has been most intensively studied for the gut microbiome –16. Immed Candida. There id Candida▪,19▪▪. TIn many cases, antibiotic-associated microbiome perturbations stabilize within a few weeks . HoweverClostridium difficile diarrhoea, a debilitating acute or chronic infection associated with significant morbidity and cost. Faecal microbiota transplants, which restore host microbiota, are curative in over 80% of treated patients with recurrent C. difficile infection, compared with less than one-third of patients treated with vancomycin alone . Significant microbiota changes are also seen in babies whose mothers received peri-partum antibiotics, even if the babies themselves were untreated [▪▪]. The impact of antibiotics on microbiota are also pronounced in the elderly, and in acute inflammation, such as intercurrent infection or comorbidities . The mechanisms underlying microbiotoxicity are becoming increasingly understood, including direct and indirect effects: bacteria targeted by antibiotics may have co-dependence with other resident bacteria, either producing metabolites required by their symbionts, or degrading waste products toxic to their symbionts . However, the real-word utility of such interventions remains unclear.As sequencing technologies and bioinformatic analyses continue to improve in efficiency and accessibility, more nuanced research quantifying microbiotoxicity is within reach, and even clinical diagnostic tests based on a patient's microbiome may be on the horizon . Lookingnfection . This confection ▪▪. HowevWe propose the term ‘microbiotoxicity’ when weighing antibiotic side effects on this multitudinous, complex and oft-neglected organ system. By acknowledging the indispensable role of the microbiome in human health, the duty of care of prescribers should be extended to include their patients’ microbiomes. In cases of severe infection, these unfortunate microbiotoxic effects may be entirely justified and unavoidable, and we do not suggest withholding antibiotics when clinically indicated. Rather, each antimicrobial prescription should involve careful weighing of the risk of infection against the risk of antibiotic-induced microbiotoxicity Fig. . CurrentThe drivers underlying AMR are multifactorial and deeply entrenched, including antibiotic overuse in animal agriculture, population pressure, sanitation and public health infrastructure . AlthougThe microbiome is a complex immune, metabolic, endocrine and neurological organ system, integral to the human-microbial superorganism. Antimicrobials are associated with harm to the microbiome and downstream ill-health, although these bystander effects vary with host and antibiotic factors. We hope that the concepts and framework presented here will help clinicians make more nuanced and individualized antimicrobial choices, and even empower them to challenge inappropriate prescribing practices around them. We, therefore, urgently invite our colleagues to add the term ‘microbiotoxicity’ to their clinical vocabulary as a call to arms: a reminder to first do no harm, microbes and all.Figures are original and were produced using Mindthegraph.com.AT is funded by a Medical Research Council Clinical Research Training Fellowship (MR/V002015/1).D.B. is supported by a CSO Senior Scottish Clinical Fellowship (SCAF/16/03).There are no conflicts of interest.


Limosilactobacillus reuteri TF-7, Enterococcus faecium TF-18, and Bifidobacterium animalis TA-1) in high-cholesterol diet (HCD)-induced hypercholesterolemic rats. The results showed that the administration of single probiotics contributed to a reduction in the body weight gain, visceral organ indexes, hyperlipidemia, and hepatic steatosis and also an improvement in the gastrointestinal microbiota. Besides the effect of single cholesterol-lowering probiotics, three probiotics strains could also synergize their hypocholesterolemic effect when administered simultaneously. These findings indicate that three cholesterol-lowering probiotic strains are suitable for development as probiotic supplements to reduce the risk of diseases caused by cholesterol and exert health benefits with synergistic effect when administered simultaneously.Hypercholesterolemia is a significant risk factor for cardiovascular disease and metabolic disorders. Probiotics are the essential constituents of the gastrointestinal microbiota that provide health-promoting effects. Cholesterol-lowering activity is a specific property of probiotics, improving the cholesterol metabolism without adverse effects. Thus, the purpose of this study was to investigate the hypocholesterolemic effect of single and mixed cholesterol-lowering probiotic strains (including  Hypercholesterolemia, which is a form of hyperlipidemia, is estimated to cause approximately 40% of deaths by 2030 ,2. HyperProbiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” . ProbiotLactobacillus, Enterococcus, Bifidobacterium, and Streptococcus, residng in the gastrointestinal microbiota strikingly exerts different functional characteristics [Limosilactobacillus reuteri TF-7 (formerly named as Lactobacillus reuteri TF-7), E. faecium TF-18, and B. animalis TA-1. They also possessed other probiotic characteristics. Thus, with their properties, these probiotics are suitable to be applied in this study [Probiotics can be obtained by food consumption. Consuming a variety of products with probiotics can promote diversity of gastrointestinal microbiota, which plays a significant role in health-promoting effects . Each preristics . Accordiis study . Therefog, 4 °C for 10 min and diluted in sterile phosphate-buffered saline  to a concentration of 109 colony forming unit (CFU)/mL.Three cholesterol-lowering probiotics, originally isolated from traditional Thai foods, were cultured in de Man, Rogosa, and Sharpe (MRS) agar  under anaerobic conditions at 37 °C for 48 h. A single colony of probiotics was picked up to culture in MRS broth and then incubated at the same conditions. After that, bacterial cells were harvested using centrifugation at 5000× w/w) carbohydrate, 24% (w/w) protein, 4.5% (w/w) fat, 5% (w/w) fiber, 0.9% (w/w) phosphorus, 1% (w/w) calcium, and 12.6% (w/w) moisture. All rats were then randomly assigned to seven groups with similar average body weight. All groups were fed with basal diet, had free water access (ad libitum), and received different supplements by daily oral gavage for eight weeks as follows:(Control), control group received 2 mL of PBS;9 CFU/mL mixed three probiotic strains and 1 mL of PBS;(Probiotics), probiotics control group received 1 mL of 1 × 10(HCD), HCD group received 1 mL of HCD and 1 mL of PBS;L. reuteri TF-7 group received 1 mL of HCD and 1 mL of 1 × 109 CFU/mL L. reuteri TF-7;(HCD-L), HCD supplemented with E. faecium TF-18 group received 1 mL of HCD and 1 mL of 1 × 109 CFU/mL E. faecium TF-18;(HCD-E), HCD supplemented with B. animalis TA-1 group received 1 mL of HCD and 1 mL of 1 × 109 CFU/mL B. animalis TA-1;(HCD-B), HCD supplemented with 9 CFU/mL mixed three probiotic strains.(HCD-mix), HCD supplemented with mixed three probiotic strains group received 1 mL of HCD and 1 mL of 1 × 10Thirty-five male Wistar rats (aged 8 weeks with 120–140 g weight) were purchased from the Nomura Siam International . All animal procedures were performed according to the guidelines of the Ethics and Research Standardization Section, Srinakharinwirot University . All rats were randomly divided into 7 groups of 5 individuals and were housed in plastic cages with wire mesh clover in room conditions at a temperature of 24 ± 1 °C, 12/12 h light-dark cycle, and 60 ± 5% relative humidity. The acclimatization was performed prior to experiments for a week on a basal diet  containing 52% (w/v) cholesterol, 0.5% (w/v) sodium cholate, 12.5% (w/v) sucrose, and 17.2% (w/v) fat as the major constituents [HCD was prepared based on Paigen atherogenic diet with minor modifications, i.e., 1.25% (tituents . Body wev/v) formaldehyde.Feces of rats were collected at the end of the experiment to analyze the fecal microbiota. At the end of the experiment, all rats were made to fast for 10 h prior to euthanasia. Then, they were sacrificed to collect blood using cardiac puncture. Abdominal fat and liver were harvested, rinsed, and weighed after blotting them dry. After that, the liver was fixed with 10% , and alanine transaminase (ALT), were examined in the plasma by the Professional Laboratory Management Corp, Co., Ltd. .Plasma was collected using centrifugation of heparinized blood at 1500× Right lobe of a rat’s liver was sectioned and embedded in paraffin. Liver tissue sections with a thickness of 5 μm were stained with hematoxylin and eosin (H&E) and Masson’s trichome. Immunohistochemical staining for F4/80 surface molecules of macrophages was performed.For Masson’s trichrome staining, liver tissue sections were deparaffinized and rehydrated using gradient alcohol. Then, re-fixed in Bouin’s solution for 1 h at 56 °C, rinsed with running tap water for 5 min, and stained in Weigert’s iron hematoxylin for 5 min. The sections were rinsed with running tap water again and stained in Biebrich’s scarlet-acid fuchsin solution for 20 min and washed in distilled water. After that, the sections were differentiated in phosphotungstic-phosphomolybdic acid for 10 min, transferred to aniline blue solution for 8 min, rinsed briefly in distilled water, and dipped in 1% acetic acid for 1 time and then washed again in distilled water and mounted on slides.For F4/80 immunohistochemical staining, the paraffin-embedded liver tissues were section at 5 μm, mounted on charged glass slides, deparaffinized in xylene, and stained with F4/80 antibody for 32 min. Then, the tissues were counterstained briefly using one drop of hematoxylin and one drop of bluing reagent. The histology of liver tissues was studied using light microscopy at an original magnification of 400× .Microbial DNA was extracted from 200 μg of feces using QIAamp DNA Stool Mini Kit  according to the manufacturer’s instruction. After extraction, DNA concentration was determined using NanoDrop One . Sequencing of 16 s rRNA of microbiota were performed using next-generation sequencing . The fecal microbiota changes from different groups were analyzed and expressed as a heatmap correlation .p value less than 0.05 was considered significant.All experiments were performed three times independently. The results were expressed as means ± standard deviation (SD). Statistically significant analysis of data was performed with ANOVA with Dunnett’s or Tukey’s multiple comparison test using the GraphPad Prism software . A L. reuteri TF-7, E. faecium TF-18, and B. animalis TA-1 were investigated in Wistar rat model. During the experimental period, body weight of rats in all groups increased with the highest body weight gain observed in the HCD group (p < 0.05). Interestingly, the body weight gains were significantly reduced in all treated groups after administration of probiotics for 8 weeks (p < 0.05) when compared with the HCD group as shown in Individual and synergistic cholesterol-lowering effects of CD group . AlthougConsidering the result in p < 0.05). Similar levels of TC, TG, and LDL-C among HCD-L, HCD-E, HCD-B, and HCD-mix groups were observed. These results were significantly lower when compared with HCD group (p < 0.05). Interestingly, administration of mixed cholesterol-lowering probiotics (HCD-mix group) reduced TC and LDL-C levels to levels similar to those in normal diet-fed rats in both control and probiotics groups. However, there was no statistical difference in HDL-C levels in all experimental groups.To evaluate whether single and mixed cholesterol-lowering probiotic administrations can reduce hyperlipidemia in HCD-fed rats, the levels of TC, TG, HDL-C, and LDL-C were measured. Based on the result in p < 0.05) with an increase in AST/ALT ratio, which was consistent with control and probiotics groups  reduced lipid profiles and liver injury by modulating the mevalonate pathway in hypercholesterolemic rats [As expected, our results showed significant elevation in body weight gain, abdominal fat and liver indexes , lipid lmic rats . Considemic rats ,22. Excemic rats . Thus, tL. plantarum DSM20174 reduced the progression of non-alcoholic fatty liver disease by gut microbiota modulation and improved lipid accumulation and adipose tissue inflammation [L. reuteri TF-7 exhibited the most effect in reducing abdominal fat liver weight, TC, and TG levels. These results were in agreement with our previous study that showed L. reuteri TF-7 (out of 17 probiotic strains) possessed the strongest BSH activity and the highest levels of cholesterol assimilation with statistical significance [Lactobacillus, Bifidobacterium, and Enterococcus species exhibited relatively high therapeutic effect in reducing hypercholesterolemic diet-induced cardiovascular disease by BSH activity and cholesterol assimilation [Excess cholesterol is also accumulated in hepatic cells, leading to non-alcoholic fatty liver disease (NAFLD) ,24. Therammation . Therefoificance . Moreoveificance . Thus, iificance . Overallmilation ,33,34.L. plantarum Lp3 that showed the ability to ameliorate dysbiosis of gastrointestinal microbiota [Lactobacillus and Bifidobacterium were negatively correlated with obesity [Akkermansia can inhibit atherosclerotic lesion formation and decrease circulating endotoxin levels through prevention of metabolic endotoxemia-induced inflammation in circulation and local vascular lesions [Bacteroides was found in atherosclerotic cardiovascular patients [Previous studies have revealed that oral administration of probiotics could improve the gastrointestinal microbiota in order to exert health-promoting effects . Thus, tcrobiota . Lactoba obesity . Akkerma lesions . In prevpatients  and postpatients . ImportaIn summary, this result indicated that the three cholesterol-lowering probiotic strains are good supplements to prevent metabolic disorders, liver injury, and cardiovascular disease. Furthermore, administration with mixed cholesterol-lowering probiotic strains causes a synergistic hypocholesterolemic effect. Overall, these findings suggest that these cholesterol-lowering probiotic strains can be developed as probiotic functional foods.


The optimal metric for outpatient antimicrobial stewardship has not been well defined. The number of antibiotic prescriptions per clinic visit does not account for the therapeutic duration. We found only moderate association between prescription-based metrics and days-supplied–based metrics. Outpatient antibiotic consumption metrics should incorporate the duration of therapy. However, defining appropriate metrics for outpatient antibiotic consumption is challenging. The Society of Infectious Diseases Pharmacists (SIDP) suggests tracking prescription records. The Centers for Disease Control and Prevention (CDC) Core Elements of Antimicrobial Stewardship guideline recommends that every outpatient setting track the percentage of all visits leading to antimicrobial prescriptions.Monitoring antibiotic usage is critical for promoting the appropriate use of antibiotics. Ambulatory antibiotic prescriptions reached 251.1 million in 2019. The information for medication dispensing at pharmacies may not be immediately transmitted to providers, whereas the number of prescriptions can be more easily obtained through electronic medical record (EMR) systems. In addition, outpatient antibiotic prescriptions are often given for an extended period at a single visit , and a single prescription may account for multiple dispensings from pharmacies if refills are included. Therefore, the number of antibiotic prescriptions may not accurately reflect the amount of antibiotics dispensed, especially for prescriptions with longer durations. These limitations can lead to inappropriate assessment of trends within the same system or for interfacility comparison.These recommendations are constrained by data availability. The duration of therapy is not a required component in prescriptions and often requires calculations from the total quantity and the direction of use. Also, the presence of a prescription does not necessarily mean the medication is dispensed from pharmacies.From pharmacies at the Veterans’ Health Administration (VHA), which operates both outpatient care deliveries and pharmacies, we compared 2 metrics for outpatient antibiotic consumption: the number of prescriptions in EMRs and the number of dispensed days, or days supplied. and only systemic oral agents prescribed on weekdays during business hours. Data for antibiotic prescriptions filled outside the VHA were not available for analysis. We also excluded outpatient prescriptions between 2 days before and 1 day after hospital discharge as discharge prescriptions to avoid interfacility bias between facilities with inpatient care services and those without.We conducted a retrospective study of all patients who received antibiotic prescriptions at outpatient clinics through in-person or video telehealth visits within 129 VHA healthcare systems filled at VHA pharmacies from January 2010 to December 2019. We obtained data from the VHA Corporate Data Warehouse, and we counted antibiotic prescriptions and days supplied (as specified by prescribers) from VHA pharmacies. We included antibiotics in the National Healthcare Safety Network (NHSN) protocolτB coefficient. We interpreted |τB| ≥ 0.7 as strong correlations, 0.5 ≤ |τB| < 0.7 as moderate correlations, and |τB| < 0.5 as weak correlations. All statistical analyses were performed using R version 4.1.2 software .To assess differences in longitudinal trends for the 2 metrics, we calculated the VHA system-wide number of prescriptions and days supplied per clinic visit for each year and plotted a line graph showing how the prescription number and days supplied changed from 2010. To assess the impact of differences on interfacility comparisons, we calculated the number of prescriptions and days supplied per clinic visit for each healthcare system and ranked facilities using each of the 2 metrics. We then compared rankings using the Kendall The Institutional Review Board of the University of Iowa and the Research and Development Committee at Iowa City Veterans’ Health Care System approved this study with a waiver of informed consent.1 (online). The mean days supplied per prescription remained largely unchanged during the study period.During the study period, 13,373,460 clinic visits (5.0%) of the 265,613,607 total clinic visits had an antibiotic prescription, and the total of days supplied was 182,793,572. Clinic visits, prescriptions, days supplied, and mean days supplied are shown in Supplementary Table Figure τB coefficient was 0.68 , indicating only moderate correlations between interfacility comparisons based on 2 metrics.For interfacility comparisons, we detected substantial differences in rankings of healthcare systems based on the 2 metrics . Lastly, the VHA provides services to more elderly populations with no pediatric care, which may limit the generalizability of our findings to non-VHA populations.We have demonstrated that the number of antibiotic prescriptions may be useful only when assessing the overall trend within a healthcare system. It may not provide accurate estimates when used for interfacility comparisons for antibiotic consumption or trends for the management of acute infections (short-course therapy) or long-term therapy. As the interoperability and data transmissibility of EMR systems improve, traditional but suboptimal metrics should be replaced by metric(s) that better reflect dispensed amounts, whenever feasible, to provide a higher content validity. Additionally, metrics that capture the duration of therapy may encourage prescriptions with shorter durations and lessen the ecological impact of outpatient antimicrobial use.


Furthermore, the c-Met expression frequency was decreased by AFP knockdown and increased by AFP overexpression (p < 0.01). The cell counting kit-8 cell proliferation assay, cell invasion, and migration assays confirmed that the AFP could affect the malignant biological behavior of AFP-GC. These findings suggest that AFP contributes to the malignant biological properties of AFP-GC and the high expression of c-Met in AFP-GC.This study aimed to investigate whether α-fetoprotein (AFP) could affect the malignant behavior of AFP-producing gastric cancer (AFP-GC) and to explore the relationship between AFP and mesenchymal–epithelial transition factor (c-Met) in AFP-GC. In this study, 23 patients with AFP-GC (AFP[+]) and 18 patients with common gastric cancer (AFP[−]) were evaluated for the c-Met expression using immunohistochemical analysis. The AFP-GC cell line, GCIY, was used. The AFP endoribonuclease-prepared small interfering RNA (siRNA) and eukaryotic AFP overexpression vector were used to increase/knockdown the expression of AFP. Afterward, the c-Met expression was evaluated by polymerase chain reaction and western blot. The proliferation, migration, and invasion of GCIY cells were estimated before and after the AFP overexpression/knockdown. The c-Met expression in both groups was the same ( AFP was synthesized in the fetus’s liver by the sixth week of conception . AFP-proGenerally, AFP-positive GC had more aggressive behavior than the AFP-negative GC . Even thHepatocyte growth factor (HGF) is a pleiotropic cytokine composed of an α-chain and a β-chain . HGF and22.1A total of 248 patients with GC were admitted for surgery at the Department of General Surgery, Huashan Hospital, Fudan University, China. A total of 28 patients had elevated preoperative serum AFP levels (AFP > 10 ng/mL). AFP was detected in GC cells by immunohistochemical staining in 23 of these 28 patients composed of the AFP-GC group (AFP[+]). Other 23 patients with GC and normal serum AFP levels were selected at random for comparison, and samples of the correspondent GC were tested for AFP immunoreactivity after surgical removal. AFP-negative was confirmed in 18 of these patients, composed of the AFP-negative GC group (AFP[−]). The essential characteristics of these AFP[+] and AFP[−] patients are shown in Informed consent: Informed consent has been obtained from all individuals included in this study.Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies, and in accordance with the tenets of the Helsinki Declaration, and has been approved by the Medical Ethics Committee of Huashan Hospital, Fudan University .2.2Subject inclusion criteria were as follows: (i) age from 18 to 80 years; (ii) the preoperative clinical diagnosis was a malignant gastric tumor, (iii) the tumor could be resected locally by preoperative assessment, (iv) the postoperative pathological specimen was more than 1 cm × 1 cm, (v) the patient chooses to undergo surgery first, and (vi) AFP-positive blood test and immunohistochemical staining of pathological specimens were included in the AFP[+] group; otherwise, they were included in the AFP[−] group.Subject exclusion criteria were as follows: (i) postoperative pathological non-adenocarcinoma, (ii) the patient was suffering from other malignant tumors at the same time, (iii) pregnant patients, and (iv) patients already participated in other research.2.3®, Rabbit mAb). The following day, the sections were washed in phosphate buffered saline (PBS) and incubated at 20–25°C with biotinylated secondary antibodies. The sections were incubated with diaminobenzidine to visualize the antigens and afterward counterstained with hematoxylin, dehydrated, and mounted. Negative control sections were treated with PBS instead of the primary antibodies, and AFP- and Met-positive liver cancer sections were used as a positive control. All sections were classified according to the grade of immunostaining in the carcinoma cells: negative (−), no carcinoma cells were stained; moderate positive (+), less than two-thirds of the cells were stained; and strong positive (++), more than two-thirds of the cells were stained  with 10% FBS.The AFP-GC cell line GCIY purchased from RIKEN BioResource Center, Japan, was cultured in a minimum essential medium containing 15% fetal bovine serum (FBS) at 37°C in a 5% COCells were divided into the following groups: blank control group, GV230 group (vector GV230 with no AFP gene was transfected into the cells), GV230-AFP group (vector GV230-AFP was transfected into the cells), Met esiRNA group (Met esiRNA was transfected into the cells), GV230-AFP + Met esiRNA group (both GV230-AFP and Met esiRNA were transfected into the cells), NC esiRNA group (negative control esiRNA was transfected into the cells), and AFP esiRNA group (AFP esiRNA was transfected into the cells).2.5in vitro  group. The frequency of the c-Met expression was increased by overexpression, whereas it was decreased by knockdown of AFP. In addition, our study also revealed that the AFP could affect the malignant biological properties of AFP-GC.AFP-GC was first reported by Bourreille et al. in 1973. However, AFP-GC with hepatoid differentiation was described for the first time by Ooi et al. in 1985 and named “hepatoid gastric cancer (hepatoid GC)” . TherefoCorrespondingly, the high frequency of liver metastasis in AFP-GC may be linked with overexpression of c-Met, a receptor of HGF and encoded by the c-Met proto-oncogene. Gardner et al., in their study, have shown that the expression of c-Met can enhance the ability of metastatic liver melanomas . SubsequNevertheless, the mechanism linking the c-Met expression and AFP remains uncertain. Our research revealed that there is no significant difference between the incidence of the c-Met expression in AFP-positive and AFP-negative GC. However, the strong positive incidence of the c-Met expression was much higher in AFP-positive GC than in AFP-negative GC, reinforcing the previous studies.While the Met pathway plays a critical role in cancer cells’ invasion and metastasis abilities, the Met proto-oncogene encodes a transmembrane receptor–protein tyrosine kinase. Overexpression of this transmembrane receptor is associated with poor prognosis in various cancers . On the Furthermore, the results of this study may indicate that AFP regulates the expression of c-Met, and the effect might be direct. It would require the translocation of AFP to the nucleus or indirectly through the action of AFP on one or more transcription factors that regulate c-Met directly. However, more studies are needed for further exploration of these assumptions. For instance, recent reports have demonstrated that AFP may function as a regulator of the phosphatidylinositol 3-kinase/Akt pathway hepatocellular carcinoma cells in humans ,27. TheyTaken together, a better understanding of the biological activities of AFP as a growth regulatory cell-signaling factor has emerged . This stThis study also has several limitations. (i) This study does not provide the detailed mechanisms by which AFP affects the c-Met expression. At this stage, some hypotheses can only be made and require further exploration through studies. (ii) Some of the SDs of this study seem high. (iii) Furthermore, some figures do not show all data points and proper error bars. However, additional experiments/results could not be provided due to the ongoing pandemic situation in China and will consider in future studies.In conclusion, based on these studies, we suggest that AFP might regulate the expression of c-Met through the activation of the Akt pathway. We plan to investigate this hypothesis in the future.


MET gene is crucial for cell growth and has shown promise as a cancer treatment target. However, distinguishing between focal amplification and polysomy, different types of gene multiplication, is challenging. Accurate differentiation requires techniques such as in situ hybridization (ISH) or next generation sequencing (NGS). As the effectiveness of MET inhibitors can vary, careful patient selection and defining the perfect amplification threshold are critical. Future studies should focus on determining optimal therapy combinations and innovating new treatments targeting MET amplification.The MET gene plays a vital role in cellular proliferation, earning it recognition as a principal oncogene. Therapies that target MET amplification have demonstrated promising results both in preclinical models and in specific clinical cases. A significant obstacle to these therapies is the ability to distinguish between focal amplification and polysomy, a task for which simple MET copy number measurement proves insufficient. To effectively differentiate between the two, it is crucial to utilize comparative measures, including in situ hybridization (ISH) with the centromere or next generation sequencing (NGS) with adjacent genes. Despite the promising potential of MET amplification treatment, the judicious selection of patients is paramount to maximize therapeutic efficacy. The effectiveness of MET inhibitors can fluctuate depending on the extent of MET amplification. Future research must seek to establish the ideal threshold value for MET amplification, identify the most efficacious combination therapies, and innovate new targeted treatments for patients exhibiting MET amplification.The  MET gene, also referred to as c-MET, encodes a receptor tyrosine kinase (RTK) known as MET. This gene has garnered significant attention in the oncology field due to its pivotal role in tumorigenesis and metastasis [The tastasis . The abetastasis , gastrictastasis ,4, colortastasis , papillatastasis , hepatoctastasis , and bretastasis ,9.MET have been identified as oncogenic drivers in cancer. One such alteration is exon 14 skipping, which results in an aberrant MET protein lacking a critical regulatory domain [MET exon 14 skipping, highlighting its clinical relevance [Multiple genetic alterations within y domain . The evey domain . Notablyelevance .MET amplification is characterized by an increased copy number of the MET gene, leading to elevated MET protein levels and hyperactivation of downstream signaling cascades, including the RAS-ERK/MAPK, PI3K-AKT-mTOR, or PLCgamma-PKC pathways [In addition to exon 14 skipping, gene amplification and single nucleotide variants have been implicated in MET-driven oncogenesis. pathways ,13,14,15pathways ,17.MET alterations has paved the way for targeted therapies in cancer treatment. For instance, in the case of NSCLC patients with MET exon 14 skipping, the development of MET tyrosine kinase inhibitors (TKIs), such as capmatinib and tepotinib, has shown remarkable efficacy in clinical trials [The identification of specific l trials ,19,20. TMET amplification has emerged as an intriguing therapeutic target. Targeted therapies designed to counteract MET amplification have shown promise in preclinical studies and a subset of clinical studies [MET amplification [Furthermore,  studies ,21. Howefication ,24,25,26MET alterations in cancer, this review aims to comprehensively summarize the measurement, frequency, and therapeutic implications of MET amplification. We seek to elucidate the underlying mechanisms driving MET amplification and explore its potential as a therapeutic target. Additionally, we will discuss the challenges and prospects associated with targeting MET amplification, including the emergence of resistance mechanisms.In light of the significance of HER2 (ERBB2) gene on chromosome 17 and the overexpression of HER2 protein play a crucial role in the pathogenesis of HER2-positive breast cancer [ERRB2 gene amplification, while HER2 immunochemistry (IHC) identifies overexpression of HER2 protein. These two assays are commonly used and exhibit a strong correlation, serving as biomarkers for anti-HER2 therapy [Gene amplification is a prevalent genetic alteration observed in cancer ,28,29. It cancer . Fluores therapy ,32. In t therapy .MET amplification is controversial, MET amplification has been reported in a number of cancer types. It occurs 1% to 6% in NSCLC patients [However, the situation regarding MET differs significantly from HER2. Although the method of measurement and threshold for patients ,35,36,37patients ,38,39, 1patients ,41, 3% tpatients , and 8% patients .MET amplification [MET amplification techniques such as FISH and next generation sequencing (NGS) may differ substantially from those identified using IHC for treatment selection. The underlying reasons for the discordance between MET protein expression and gene amplification are not yet fully understood, but intra-tumor heterogeneity has been suggested as a contributing factor [It has been observed that MET protein expression assessed by IHC does not always align with fication ,45,46. Tg factor . Previoug factor ,49,50. CERRB2 to centromere enumerator probe (CEP)17. However, it is crucial to consider the presence of polysomy when interpreting FISH results [Gene copy number gain in cancer can manifest as either focal gene amplification or polysomy (or aneuploidy of chromosome) . Focal a results . Polysom results ,54. Cons results .MET gene entails a specific gain of gene copies, while polysomy involves an increase in chromosome 7 copy number, often accompanied by co-amplification of adjacent genes such as CDK6 and BRAF [MET amplification as a therapeutic target [Similarly, in the context of MET, focal amplification of the and BRAF  MET copy number ≥ 2.2.(b)MET is amplified without co-amplification of CDK6 and BRAF. Co-amplification status was defined as “increased together” when the copy number of the other gene (CDK6 or BRAF) ≥ 2.2, and the difference with MET amplification is within +/−0.5.(c)MET amplification that satisfies both (a) and (b) is defined as focal.Several examples were given to illustrate this: a sample with only MET alterations related to abnormal signaling in approximately 10.7% (110 patients) of the entire patient population across nine different cancer types, most notably non-small cell and small cell lung cancers, gastroesophageal cancer, and prostate adenocarcinoma. MET alterations were found in 37 out of 291 patients. Among these, 24 had amplifications, 5 had exon 14 skipping, and 13 had single nucleotide variants (SNVs). Co-alterations, such as amplification, SNVs, were found in four samples.Results of analyzing patient cohort consisting with the testing cohort (291 patients), and validation cohort (734 patients) showed MET amplifications, about 30% (7/24) were classified as focal amplification. The MET copy number was significantly higher with focal amplification compared to non-focal amplification (polysomy). In a validation cohort, focal MET amplification was detected in 4.2% of patients. Overall, the rate of focal amplification was 3.7% (=38/1025) across all patients.Of the 24 MET amplification using comprehensive genomic profiling with NGS in patients with advanced cancer. The study also suggests that only 30% of all MET amplifications detected by the NGS are focal, and these are associated with a higher plasma MET copy number.This study showed that this approach can distinguish focal from non-focal MET, EGFR, BRAF, and CDK6. Inclusion of a broader array of genes or single nucleotide polymorphisms (SNPs) has the potential to substantially enhance the precision and fidelity of detecting focal amplification events. Furthermore, the absence of a well-defined correlation between EGFR and MET necessitates a more comprehensive exposition.Nevertheless, it is pertinent to acknowledge a limitation inherent in this study, namely the selective focus on a restricted gene set localized within chromosome 7, encompassing MET amplification analysis due to its widespread use in clinical practice and the potential to simultaneously examine other driver genes, such as EGFR. The integration of NGS with established methods such as FISH has been proposed in HER2-positive cases to capture those missed by the current IHC and FISH-based approaches [MET amplification identification, leading to improved patient stratification and personalized treatment decisions.Although clinical investigations are warranted, NGS holds promise as a valuable tool for proaches . By leveMET amplification assessment, further research is needed to establish standardized guidelines for interpreting NGS results, particularly in distinguishing between polysomy and focal amplification. Additionally, the development of bioinformatics algorithms and computational tools specific to MET amplification analysis will aid in the accurate classification of NGS data. Efforts to validate the clinical utility of NGS-based detection of focal amplification and its correlation with treatment response are crucial steps toward integrating NGS into routine clinical practice for precision cancer care.To fully exploit the potential of NGS in MET amplification has emerged as a significant mechanism of acquired resistance in various targeted therapies, particularly in EGFR-mutant NSCLCs. MET amplification is known to stimulate signaling pathways such as RAS-ERK/MAPK, STAT, and PI3K/AKT downstream of EGFR, leading to resistance against EGFR TKIs [GFR TKIs ,71. ThisGFR TKIs ,73,74.MET amplification as a resistance mechanism in NSCLC. Coleman et al. conducted a comprehensive analysis of key clinical trials in NSCLC and reported that MET amplification was identified as a mechanism of resistance in 7–15% of patients who experienced treatment failure with first-line osimertinib and in 10–22% following second-line osimertinib [MET amplification in acquired resistance scenarios.Clinical studies have shed light on the prevalence of mertinib . These fMET amplification may contribute to treatment resistance not only in EGFR-positive NSCLCs but also in other oncogenic gene-positive NSCLCs, such as those with ALK fusion [RET-fusion [ROS1-fusion [NTRK-fusion [KRAS [EGFR, KRAS, ALK fusion, and RET fusion alterations exhibit MET amplification [MET amplification as a potential resistance mechanism in the management of various oncogenic driver alterations.Moreover, it has been recognized that K fusion , RET-fusT-fusion ,77, ROS11-fusion , NTRK-fuK-fusion , or KRASon [KRAS . Collectfication . This unMET amplification in acquired resistance scenarios is crucial for optimizing treatment strategies. Efforts are underway to develop effective therapeutic approaches targeting MET amplification to overcome acquired resistance. These include the investigation of combination therapies involving MET inhibitors with other targeted agents or immunotherapies to enhance treatment efficacy and prevent the emergence of resistance. Additionally, ongoing research aims to elucidate the underlying mechanisms and molecular interactions associated with MET amplification-mediated resistance, which will inform the development of novel therapeutic strategies for patients with MET-amplified NSCLC.Understanding the prevalence and clinical implications of MET amplification (MET/CEP ≥ 2.0) [As previously discussed, initial studies have indicated limited effectiveness of MET inhibitors in patients with P ≥ 2.0) . HoweverP ≥ 2.0) ,86,87,88MET amplification. For instance, the PROFILE 1001 study examined the activity of crizotinib, an ALK/ROS1/MET tyrosine kinase inhibitor (TKI), in NSCLC patients categorized into high (MET/CEP ≥ 4), medium (4 > MET/CEP > 2.2), or low (2.2 ≥ MET/CEP ≥ 1.8) amplification groups [n groups . The higMET amplification, revealing ORR rates of 29% (GCN ≥ 10), 12% (GCN 6 to 9), 9% (GCN 4 or 5), and 7% (GCN < 4), indicating an increased therapeutic effect with higher degrees of MET amplification  [MET amplification, consistently showing improved outcomes in the higher amplification groups  . Severaln groups .However, it is worth noting that the cutoff criteria for defining amplification varied across trials, and establishing standardized criteria for identifying optimal treatment targets remains a future challenge.MET amplification. In recent years, significant advancements have been made in targeting both MET and EGFR, leading to the initiation of several clinical trials investigating the combination of MET inhibitors and EGFR inhibitors for acquired resistance in NSCLC. MET amplification in EGFR-mutant NSCLC.Combination therapy involving primary oncogene TKIs and MET TKIs has emerged as a rational strategy for addressing acquired resistance resulting from MET exon14 skipping [EGFR mutations and MET amplification [MET amplification (GCN ≥ 4 by FISH) or MET overexpression (IHC 3+) showed improved activity, particularly in the high MET amplification group  [MET amplification (GCN ≥ 5 or MET/CEP ≥ 2.0 by FISH), or MET overexpression (IHC 2+/3+). The ORR in this study was 67%, with a median PFS of 16.6 months compared to 4.2 months with chemotherapy . Additionally, the overall survival was 37.3 months vs. 13.1 months with chemotherapy  [Capmatinib and tepotinib, both FDA-approved for NSCLC with skipping ,19, havefication ,90. In a months) . AnotherEGFR mutation-positive lung cancers with MET amplification [MET amplification (GCN ≥ 5 or MET/CEP ≥ 2.0 by FISH or GCN ≥ 5 by NGS), the ORR ranged from 64% to 67%. For patients with resistance after third-generation EGFR-TKIs and MET amplification, the objective response rate was 30%. A phase I study examining the combination of savolitinib and gefitinib in patients with acquired resistance to EGFR-TKI and MET amplification (GCN ≥ 5 or MET/CEP ≥ 2.0 by FISH) reported an objective response rate of 52% [MET amplification varied across these studies. Furthermore, resistance-related amplification is often considered a subclonal population, which may warrant a lower threshold for defining the degree of amplification compared to de novo amplification [Savolitinib, a potent and selective MET TKI, in combination with osimertinib, was evaluated in the TATTON study for its efficacy in pretreated fication . In patie of 52% . It is ification ,58. Nevefication .MET amplification out of a total of 28 patients), ORR was 32%, and mPFS was 5.9 months [Ongoing investigations are exploring the use of antibody-based therapies targeting MET. Telisotuzumab vedotin, an antibody-drug conjugate targeting MET, exhibited promising antitumor activity and acceptable toxicity in NSCLC cases with acquired resistance to EGFR-TKI and MET overexpression (H-score ≥ 150 by IHC). In a study involving patients with MET overexpression . This trial seeks to assess the potential benefits of combining a MET inhibitor (savolitinib) with a third-generation EGFR inhibitor (osimertinib) in patients who have developed resistance to previous treatments.Several ongoing clinical trials are currently investigating treatment strategies targeting ClinicalTrials.gov Identifier: NCT03778229). This study aims to provide further insights into the potential synergistic effects of these targeted therapies in overcoming resistance mechanisms, specifically in patients with MET amplification.In addition, a phase II trial is underway to investigate the efficacy of savolitinib in combination with osimertinib for acquired resistance in NSCLC . This trial explores the potential of combining MET inhibition with immune checkpoint blockade to enhance therapeutic outcomes in this patient population.Furthermore, a phase II trial is evaluating the combination of savolitinib and durvalumab, an immune checkpoint inhibitor, for advanced gastric cancer . This study aims to assess the safety and efficacy of this novel antibody therapy in patients with advanced NSCLC harboring EGFR mutations and MET amplification.Lastly, a phase I trial is investigating amivantamab, a human bispecific antibody targeting both EGFR and MET, in advanced NSCLC (MET amplification as a therapeutic target and may contribute to the development of more effective treatment strategies for patients with acquired resistance in various cancer types. The results from these studies will provide valuable insights into the clinical utility of targeting MET amplification and may guide future treatment decisions in precision oncology.These ongoing trials represent important steps in advancing our understanding of MET amplification represents a promising avenue, particularly in combination with EGFR-TKI therapy for pretreated NSCLC patients. However, the appropriate selection of patients is crucial for maximizing treatment efficacy. The underlying mechanisms contribute to the varying effectiveness of MET inhibitors based on the degree of MET amplification. Nevertheless, there remains a lack of consensus regarding the specific threshold or cut-off value for MET amplification across different studies.The treatment of MET amplification, to identify the best combination therapies, and to develop new targeted therapies for patients with MET amplification.Further research is needed to determine the optimal cutoff value for


To evaluate general shortcomings and faculty-specific pitfalls as well as to improve antibiotic prescription quality (ABQ) in non-ICU wards, we performed a prospective cluster trial.  An infectious-disease (ID) consulting service performed a prospective investigation consisting of three 12-week phases with point prevalence evaluation conducted once per week  at seven non-ICU wards, followed by assessment of sustainability (weeks 37–48). Baseline evaluation (phase 1) defined multifaceted interventions by identifying the main shortcomings. Then, to distinguish intervention from time effects, the interventions were performed in four wards, and the 3 remaining wards served as controls; after assessing effects (phase 2), the same interventions were performed in the remaining wards to test the generalizability of the interventions (phase 3). The prolonged responses after all interventions were then analyzed in phase 4. ABQ was evaluated by at least two ID specialists who assessed the indication for therapy, the adherence to the hospital guidelines for empirical therapy, and the overall antibiotic prescription quality.  In phase 1, 406 of 659 (62%) patients cases were adequately treated with antibiotics; the main reason for inappropriate prescription was the lack of an indication . The antibiotic prescription quality (ABQ) significantly increased, reaching 86% in all wards after the focused interventions . In phase 2 the effect was only seen in wards that already participated in interventions . No improvement was seen in wards that received interventions only after phase 2 . A given indication significantly increased from about 80% to more than 90% (p<.0001). No carryover effects were observed. ABQ can be improved significantly by intervention bundles with apparent sustainable effects. Clostridiodis difficile infections, is a major concern , , , , 6], , . HoweverTo this end, we conducted a prospective cluster trial using a protocol with three periods in seven non-ICU wards at a university hospital in Germany. This study aimed to  evaluateSince data on antibiotic usage from non-ICU wards are scarce, the present study included seven non-ICU wards, defining the clusters in the statistical analysis. The wards belonged to the following faculties: gastroenterology, nephrology/immunology, visceral/transplant surgery, orthopedics, cardiology/pneumology, traumatology, and cardiothoracic surgery. Patients were included if they were present on that ward on the day of collection and were receiving antibiotics assumed to be for therapy of infectious diseases. Patients were excluded if they were not receiving antibiotic therapy on the day of enrollment or were receiving antibiotics for non-infectious reasons .Three 12-week phases during which point prevalence evaluation were conducted once per week and were interrupted by three 6- to 10-week phases, which were occasionally used for interventions. These interventions were performed directly after baseline observation in four wards: cardiology/pneumology, nephrology/immunology, visceral/transplant surgery, and orthopedics. The remaining three wards served as controls to distinguish true intervention effects from time trends and the Hawthorne effect during phase 2. To prove the universality of the approach, the same interventions were performed on these three wards  after phase 2. Thus, the efficacy of the interventions and the prolonged effect of the interventions were assessed during phase 3. For a detailed timeline, see Figure 1 All patients in any of the wards receiving antibiotics were enrolled on a weekly level, and antibiotic prescription practice was evaluated by a board of three infectious disease (ID) specialists. As before the study, the ID consultant service could be requested on demand for ID rounds and/or single ID case visits throughout the trial as necessary. The purpose of this prospective cluster trial was to improve antibiotic prescription quality. To this end, we addressed two key questions: How often is the antibiotic therapy appropriate and what are the main shortcomings and faculty-specific pitfalls at baseline? What is the influence of the interventions on the rate of appropriateness? Thus, the endpoint was to assess whether the antibiotic therapy administered was appropriate or not depending on general and ward-specific interventions.Since appropriateness quality is hard to define and because it considers many aspects and issues of antibiotic therapy , all of adherence to the hospital guidelines in the empirical setting the pathogen-directed situation adequate dosing including adaptation given organ insufficiencies and allergiesusing the appropriate application form streamlining the therapeutic approach in accordance with the microbiological results It should be mentioned that at the University Hospital Aachen, local guidelines for the empirical antibiotic therapy of the main infectious disease entities were already issued several years ago.The intervention strategies were communicated to all staff members and are based on the results obtained during the baseline phases and their interpretation. All interventions were targeted at the identified shortcomings and faculty-specific pitfalls.On the individual ward, strategies were developed in interdisciplinary teams consisting of members of the ID consultant service and faculty/ward-specific physicians. The interventions comprised:teaching the principles of adequate antibiotic prescriptiondistribution of already existing pocket cards explaining the empiric antibiotic therapy regimen based on local resistance patternscompilation and distribution of pocket cards for the best pathogen-oriented therapy regimenflow charts to facilitate better diagnoses (and thus improved prescription of an appropriate antibiotic regimen) for urinary tract infectionsAll wards received one instruction session in which all the results were presented along with interpretation of the same, aiming at improving antibiotic prescription quality. The other intervention tools were included upon the decision of ward- and faculty-specific physicians. One topic of special interest which represents a major issue antibiotic prescription quality is the indication for therapy in general. Thus, we also specifically analyzed this endpoint. The indication for therapy in general means that a given indication for antibiotic prescription was based on a highly suspected infection according to national and international criteria for diagnosing pneumonia, sepsis, urinary tract infections, bone and joint infections, endocarditis, surgical site infections, etc. th phase was conducted according to the same rules as described for phases 1, 2 and 3, in order to define the sustainability of the achieved improvements.After all interventions were performed, a 4The study was approved by the local ethics committee .For data handling, a data management file using the Access program (Microsoft Office) was compiled and used. ® version 9.4 [TS1M1] under Windows X64 7 Pro was used for computations. Effects were considered significant at p<0.05. We described our results as odds ratios (OR) with corresponding 95% confidence intervals.We fitted generalized mixed-effects random-intercept models with logit links to the data of the response rates for “Indication for AB”; “Appropriate AB”; “Appropriate AB empirical setting”; “Appropriate AB pathogen-oriented setting”, taking into account the fixed effects intervention, in addition to phase as well as first and second order carryover effects. We assumed unstructed covariance within the clusters. Random intercept was applied to account for ward heterogeneity. PROC GLIMMIX  were receiving antibiotics Figure 2 . During the first 12 weeks , 406/659 (62%) patient cases received appropriate antibiotic therapy. The appropriateness quality did not depend on the hospital setting in which antibiotics were used, i.e., the empirical setting versus pathogen-oriented setting , phase 2  and phase 3 . No significant carryover effects were observed  meaning that the treatment difference was not affected by the phases.We observed infection rates of 359/476 (75%) in phase 1; 328/457 (72%) in phase 2, and 354/416 (85%) in phase 3. Compared to the primary endpoints in the 4After the first intervention had taken place on the four intervention wards, the rate of appropriateness increased on each of these wards. In contrast, no changes were observed on any of the control wards  as well as for the pathogen-oriented therapies   we conducted the trials in a nonrandomized manner. Further, we were more interested on description of the effects and intended to provide some planning suggestions for a cluster-randomized trial.At baseline, about 40% of antibiotic therapies were inappropriate. The major reasons for this were lack of indication, the therapy regimen lasting too long or being too broad, as well as an empirically false regimen on organisms that were not susceptible in vitro. After the interventions, these general shortcomings significantly decreased to less than 10% patients taking antibiotics without a true indication, and more than 85% patients were receiving an appropriate antibiotic regimen. Importantly, due to the design of this cluster trial, it seems that there will be positive lasting effects in terms of improved antibiotic prescription quality. For example, without any new intervention, the ABQ was observed to steadily increase. Even more importantly, only three wards asked for ID visits in phase four, indicating that a behavioral change had taken place and that physicians felt more competent when prescribing an appropriate antibiotic regime. For example, two of the surgery wards began analyzing all microbiological results on a daily basis. Note that at the start of the trial, microbiological results were only analyzed on a daily basis for patients who did not show adequate signs of recovery. Such an analysis is a basic requirement for the adaptation of antibiotic therapy in general and for streamlining in particular.Clostridium difficile infections and antibiotic prescription quality . This can be explained by the steady antibiotic usage rate per patient during the study. Antibiotic resistance, particularly in gram-negative rods, is rapidly increasing. Unfortunately, only a few new antibiotics are expected to be developed in the near future. Therefore, medical interventions aiming at improving appropriate antibiotic prescription practices are urgently needed. Using a portfolio of only a few basic tools and strategies, we succeeded not only in significantly increasing antibiotic prescription quality for more than two years, but also found indications that our trial will have long-lasting positive effects on antibiotic therapy.Finally, we attempted to correlate the improvements in overall antibiotic prescription quality with factors of collateral damage caused by the antibiotic administered. However, in contrast to other studies, we found no correlation between the rate of nosocomial The authors declare that they have no competing interests.Ralf-Dieter Hilgers appreciates the discussions about statistical analysis of the presented data with PD. Dr. Nicole Heussen and Prof. Dr. Maria Kateri, who were funded by the DAAD for the project “Poisson Response Models: Bayesian Analysis and Model Selection”. This study was partly funded by the EU Aspire Award 2013 given to Simone Scheithauer  by Pfizer.The study was approved by the local ethics committee .Nothing to declare.