Concrete technology is becoming more and more sustainable and ecological following more extensive and focused research. The usage of industrial waste and by-products, such as steel ground granulated blast-furnace slag (GGBFS), mine tailing, fly ash, and recycled fibers, is a very important step toward a good transition of concrete into a “green” future and significant improvement in waste management in the world. However, there are also several known durability-related problems with some types of eco-concretes, including exposure to fire. The general mechanism occurring in fire and high-temperature scenarios is broadly known. There are many variables that weightily influence the performance of this material. This literature review has gathered information and results regarding more sustainable and fire-resistant binders, fire-resistant aggregates, and testing methods. Mixes that utilize industrial waste as a total or partial cement replacement have been consistently achieving favorable and frequently superior outcomes when compared to conventional ordinary Portland cement (OPC)-based mixes, especially at a temperature exposure up to 400 °C. However, the primary emphasis is placed on examining the impact of the matrix components, with less attention given to other factors such as sample treatment during and following exposure to high temperatures. Furthermore, there is a shortage of established standards that could be utilized in small-scale testing. Sufficient fire resistance is one of the properties that must be ensured. Concrete is a composite material composed of fine and coarse aggregates, water, active and inert fillers, and cementitious binder. Each of these components affect the ultimate performance, including the damage mechanism at high temperatures and when exposed to fire.There is a high global demand for sustainable and more environmentally friendly concretes characterized by a low CO3 [Common fire damage of concrete appears as discoloration, cracking, spalling, and decomposition of the binder matrix ,2,3. The3 . The mea3 . Najm et3  reported3 . Concret3 . Short e3  and theiThe main source of crazing and cracking, which leads to delamination and spalling and then, afterward, to the destruction of the inner structure, is the thermal incompatibility between components of the binder matrix . SEM imaThe amount of free and bound water has a significant impact on the dehydration. Thermogravimetric analysis (TGA) and magnetic resonance (NMR) of samples made of geopolymer showed that the majority of water is bound physically rather than chemically, as reported by Vickers et al. . When thSpalling can be defined as a gradual peeling and scaling off of the external layers of concrete exposed to high temperatures. The mechanism remains largely unknown, but the increased vapor pressure created at elevated temperatures from entrapped water has been indicated as the leading factor . The creRef.  the dura2 into calcium oxide (CaO). The absolute water evaporates. Other factors, such as the melting point of aggregates and the thermal expansion of concrete, also contribute to the degradation of OPC concrete at elevated temperatures.OPC (ordinary Portland cement)-based concrete faces chemical decomposition at elevated temperatures, as shown in There are several performance limitations of concrete based on OPC when subjected to high temperatures. This review paper will center on exploring alternatives to the usage of OPC and examine several environmentally friendly binding materials and their performance at high temperatures. It will also consider other critical factors such as an alkali-activated system, aggregates, and the significance of large-scale testing.2 are present in the concrete dehydrate. The GGBFS particles themselves can also undergo changes and transform into crystalline phases. Studies showed that reference OPC concrete lost 14% of its residual compressive strength and mixes containing OPC/GGBFS blends only 4–5% when exposed to 800 °C. Furthermore, an OPC specimen showed cracking after exposure to 400 °C, while the OPC/GGBFS had no signs of any disintegration [One of the commonly used construction materials in the building industry is steel . During egration . Samplesegration . GGBFS hegration ,49,50. Tegration . The preegration .Tests of concretes based on cement blends with 3 wt% and 40 wt% of BFS showed that the orientation of the crack has a major impact on the spalling scenario . InclinePoon et al.  compared2 [In general, GGBFS, but also concretes with fly ash (FA), seem to perform better in fire with less spalling and with narrower cracks. This trend was linked with the decreased amount of Ca(OH)2 . The repLow-calcium content fly ash is considered as a good pozzolanic material . The res2 present in the concrete dehydrates results in the formation of new phases such as gehlenite and anhydrite. Additionally, the fly ash particles themselves change and transform into crystalline phases such as mullite and hematite. These changes result in microstructural alterations and reduce the overall performance of the concrete in high temperature conditions.Fly ash-based concrete, under exposure to elevated temperatures, can undergo chemical decomposition. Between 200 and 400 °C, the new gel is formed, the density is increased, and the peak strength happens. The decomposition at higher temperatures results in the loss of strength and overall durability. The calcium silicate hydrates (C–S–H) and calcium hydroxide Ca(OH)2 emissions, is considered a suitable and ecological option for an OPC replacement [Calcium sulfoaluminate cement, due to lower COlacement . The comlacement . Monosullacement  occur asThis type of cement shows superior results in compressive strength tests, shrinkage behavior, gaining early strength, and resistance compared to OPC in ambient conditions . The useGood insulation properties are essential in such solutions, as they help to prevent the spread of fire by reducing the amount of heat transferred from the fire-exposed side to the unexposed side of the structure. This can significantly increase the time available for evacuation and intervention by the fire department, potentially saving lives and reducing damage to the building. Moreover, a passive fire-protection solution made of materials with good insulation properties can reduce the cost and complexity of active fire-protection measures, such as sprinkler systems and fire-retardant coatings. Therefore, further research and development in this area is highly desirable. The investigation of mechanical and chemical changes occurring in CSA mixes at elevated temperatures and during fire has not been adequately examined. Most of the existing literature on CSA mixes concentrates on aspects such as their performance in normal conditions, manufacturing difficulties, such as a slow hydration rate caused by the presence of belite after 28 days , and theAlkali-activated concrete, also commonly called geopolymer, was officially introduced in 1978 by French scientist Joseph Davidovits . HoweverThe binder plays the main role due to its high chemical reactivity. Many studies in this field have reported that polymerizing materials rich with alumina-silicates, such as GGBFS , fly ashElzaig ferrochrome slag (EFS)-based geopolymer concrete has shown a significantly improved fire resistance and less reduction in compressive strength and cracking or spalling than OPC concrete . The maiFly ash geopolymer concrete has recently received a lot of attention due to its performance at elevated temperatures. Initial research has reported a slight increase in strength during exposure to a moderately low temperature of ~200 °C and relatively low loss of strength when exposed to higher temperatures compared to concrete specimens based on OPC . Saker e4), albite (NaAlSi3O8), and tridymite (SiO2).The enhanced performance of these blends was linked to the improved development of alumina-silicate networks during the process of geopolymerization. The better formation of this network is essential for improving the fire resistance of the geopolymer mixes, as it increases the stability of the material at high temperatures . The micTherefore, it is important to carefully consider the geopolymerization process when designing and manufacturing geopolymer mixes with improved fire-resistance properties. Although finer aggregates could enhance the formation of microcracks, which leads to the presence of bigger cracks and spalling phenomena . Higher The Si/Al ratio tends to influence the microstructure and performance of geopolymer concrete. Geopolymers consisting of fly ash with high Si/Al (≥5) revealed an improved compressive strength and better volume stability at a temperature of 1000 °C compared to the geopolymer with the same binder but lower Si/Al ratio (<2) .Another approach to enhancing the fire and high temperature properties of geopolymer concrete is the usage of selected filler and aggregates, which are more thermally resistant and stable. The application of a fine ceramic filler in a calcined kaolin clay and potassium silica solution geopolymer mix following Fuller’s gradation design (1 F/250) stood out with an undisturbed mechanical strength after a 1000 °C temperature exposure . Thus, NThe fibers also minimized the mass loss and limited crack formation and spalling. Generally, there were no clear indications of enhanced mechanical properties. Geopolymer concrete, as a high-temperature and fire-resistant material with excellent thermal performance, chemical, and physical stability ,111, couRecent research and tests have proven the possible usage of a PFP system in the function of thermal resistance materials . The merThe increased density of geopolymer concrete prevent spalling and cracking events. According to the current state of the art, the data obtained are reliable enough to demonstrate significant similarities in the performance and properties of alkali-activated materials and ceramics, which indicates enhanced resistance to high temperatures and fire compared to OPC concrete.Large-scale fire tests are considered to be the most reliable method of determining the fire resistance of materials and structures, as they provide a realistic scenario of a fire situation. However, they come at a high cost and require significant resources, making them challenging to conduct on a regular basis. These tests involve subjecting a full-scale structural element or system to a fire scenario that closely resembles real-world conditions, including fire load, temperature, and duration. Due to the expense and complexity of large-scale tests, most of the tests are conducted in specialized laboratories or institutions with accreditation for such testing. There are few standards applied in some countries. A Euroclass system was introduced in all European Union countries by the European Committee for Standardization, CEN . Most scSmall-scale fire tests are commonly used by researchers to investigate the fire resistance of concrete materials due to their low cost and easy logistics. These tests involve subjecting small concrete specimens to elevated temperatures and measuring their response. While small-scale tests have limitations, such as the inability to replicate the complex behavior of large-scale structures, they provide a valuable starting point for exploring the fire resistance of concrete materials. One of the primary distinctions is that the specimens are subjected to elevated temperatures in an electric furnace, rather than actual fire or flames . Howeverinc [These types of tests are usually restricted to a single event, which helps to determine the degree of fire performance produced by a selected structural composition . There ainc , as showSuch factors as the maximum temperature, duration of fire exposure, time of decay, and heat release are crucial parameters to be considered in the fire testing of materials. The maximum temperature is the highest temperature reached during the test, and it is an important indicator of the material’s thermal resistance. The duration of fire exposure represents the time the material is exposed to fire, and it can affect the material’s structural integrity and mechanical properties. The time of decay is the time taken by the material to reach a certain temperature after the end of the fire exposure, and it can provide insight into the material’s cooling behavior. Lastly, the heat release is the total amount of heat released during the combustion process. It is an important factor in evaluating the fire hazard of a material. All these factors must be considered to accurately assess the fire performance of materials and ensure their safety in real-life fire situations. Nowadays, there are few developed custom-made investigations for specific types of concrete structures, especially underground objects where fire safety is a priority ,142,143.It is well known that aggregates expand when exposed to elevated temperatures . This evAggregates are categorized into two types based on their mineralogy, i.e., siliceous (S) and calcareous (C) . EurocodThe application of fibers in concrete can enhance its performance by an improvement in the durability and mechanical properties, lower shrinkage, or inducement of the ability to self-repair ,160,161.The cooling regime of concrete after exposure to high temperatures is a crucial factor ,176,177.There are two common methods currently used . In the Others studied the effects of the cooling methods on a concrete mix containing fly ash and siliceous aggregate. The results showed that instant cooling influenced the concrete properties only when the sample was exposed to <330 °C . Furnace2, and the level of disorientation of the concrete samples was similar between water cooling and exposure to ambient humidity for one week. This allowed for the identification of two ranges of disorientation of concrete, with and without dehydration/rehydration of Ca(OH)2. These tests were supplemented with SEM and IR studies. The cooling process is a critical stage in the postfire assessment of concrete structures, as it can significantly affect the residual properties of the material. However, there is a lack of comprehensive studies that investigate the effects of different cooling methods on the mechanical and physical properties of blended cement and ecological concrete. In order to develop more resilient and sustainable concrete structures, it is crucial to gain a deeper understanding of how cooling methods impact the behavior of these materials under fire exposure. This requires more research and experimentation to identify the optimal cooling techniques and parameters for different types of blended cement and ecological concrete mixtures.The experiments demonstrated that exposure to ambient moisture resulted in the rehydration of CaO to Ca(OH)The aspect of heat transfer and thermal conductivity of concrete are important factors to be considered when investigating fire damage mechanisms ,182,183.In addition, the transient plane method can be applied to evaluate the thermal performance of high-volume fly ash (HVFA) mortars and concretes at ambient temperatures. The Transient Plane Source (TPS) method is a technique used to measure the thermal conductivity and thermal diffusivity of different materials. It involves inserting a small, flat sensor between two specimens of the material being tested and applying a constant heat flux to one side. The sensor reads the temperature increase on the other side of the sample and calculates the thermal properties based on the rate of temperature alteration over time .HVFA concrete with reduced thermal dynamism is characterized by a lower cost of utility cooling and heating in commercial and residential structures. The determination of the moisture resistance factor and thermal conductivity decrease with rising temperatures was reported by Kizilkanat et al. . Li et aAs previously demonstrated, it was generally observed that mixes with a 70 weight percentage of ground granulated blast-furnace slag (GGBFS) had an improved performance in terms of fire resistance at high temperatures . The thiHigher temperatures influenced the acceleration of heat absorption. The thermal conductivity of concrete with a 30% fly ash replacement was found to be lower than that of ordinary concrete under identical conditions. One of the biggest problems is a lack of standardized methodology to define the thermal properties of concrete. One method which could be applied is the Transient Plane Source (TPS) technique. It allows for carrying out a test regarding the heat transfer in ambient and at elevated temperature conditions . Due to The mechanical properties and durability of eco-concretes are, in some cases, compromised when exposed to fire. Fire-related damage of concretes is usually assessed using small-scale indirect tests. These tests are not sufficient to provide the full set of data needed for modeling. The performance of eco-concrete is always reduced after exposure to elevated temperatures. Eco-concrete has different temperature thresholds depending on its composition. Color alteration, cracks, delamination, and spalling are very often the accompanying events which can be easily identified. Partial replacement of Portland cement by alternative binders and secondary cementitious materials also enables, in some cases, an enhancement in their performance when exposed to fire ,125,129.GGBFS and FA have gained popularity as research subjects for material behavior under elevated temperatures due to their wide availability and easy accessibility. They have shown promising results as binder replacements in cement blends and in alkali-activated systems, as evidenced in The performance of eco-concrete is influenced by a range of factors, and its behavior during a fire event is particularly difficult to predict due to the complex nature of the material. Therefore, it is essential to evaluate the various stages of the eco-concrete life cycle, including laboratory tests, casting, service life, fire events, and postfire scenarios, to ensure its optimal performance. All these stages play a critical role in the final performance of eco-concrete, and their thorough evaluation is necessary for a reliable and effective fire-performance assessment.It has been demonstrated that concrete can be made more environmentally friendly by improving its fire resistance and ability to withstand high temperatures ,202. ThiA sintering process refers to the bonding and densification of the particles at high temperatures, leading to the formation of a stronger and more cohesive structure. In many cases, the sintering event of the eco-concrete matrix resulted in an improved adhesion between the fibers and matrix. As a consequence, the overall mechanical properties were enhanced in comparison with OPC concrete.Ecological concrete generally showed reduced spalling and less cracking compared to OPC concrete when exposed to elevated temperatures. The visible color alteration after exposure to fire does not necessarily indicate a compromise in the structural integrity or performance of the material. It could be a good indicator for nondestructive methods.It was reported that some types of fibers did not show any noticeable signs of deterioration. The favorable effect of crack and spalling prevention was observed. It was assumed that fibers were able to withstand thermal stress and maintained their integrity. Some also indicated the induction of a self-healing process in fire-damaged samples.The postfire scenario and cooling process has a significant impact on the properties and behavior of ecological concrete. Rapid cooling can cause high thermal stress and potential cracking due to the differential volume change in its components.Nanoscale research to fully understand the mechanism of influence of elevated temperatures on an eco-concrete element;An investigation about heating and the cooling rate and its influence on ecological and sustainable concrete performance. In addition, the postfire behavior of eco-concrete must be studied more profoundly;A proper approach and methodology to formulate a user-friendly concrete mix design with a minimized presence of hazardous materials and alkali solutions which can withstand high temperatures;Evidence that admixtures do not change the concrete properties under elevated temperatures;The bonding properties of different materials, such as reinforcement, in ecological concrete;A standardized method and methodology to carry out small-scale testing of specimens;Large-scale fire tests on ecological concrete. It remains unknown how the extremal factors such as the shape of the structure, wind, snow, applied loads, additional elements located in concrete structures, and fire-extinguishing systems (passive and active) could disturb the properties and overall performance of concrete elements;A developed and classified database of sustainable concrete components and possible configurations;A database, building codes, fire regulations, design and material approach, and user standards in a variety of scenarios, situations, and timelines;A life-cycle assessment analysis and a determination of the potential reusage of recycled concrete to minimalize the volume of waste;Digitalization, collaborations, and open access combined with artificial intelligences.Summarizing, there is still a lack of reliable and replicable data related to the performance of eco-concretes in fire. The following research needs could be indicated:


Antibiotic resistance remains a global public health concern with significant patient morbidity and tremendous associated health care costs. Drivers of antibiotic resistance are multifaceted and differ between developing and developed countries. Under evolutionary pressure, microbes acquire antibiotic tolerance through a variety of mechanisms at the cellular level. Patients after orthopaedic trauma are vulnerable to drug-resistant pathogens, particularly after open fractures. Traumatologists practicing appropriate antibiotic prophylaxis and treatment regimens mitigate infection and propagation of antibiotic resistance. In 2019, ABR was listed by the World Health Organization (WHO) as the fifth most significant threat to global health. The conservative estimate for annual deaths worldwide due to ABR is currently around 700,000.Salmonella typhi appeared.Staphylococcus aureus (MRSA) infection.7Antibacterial resistance emerged as a concern in the 1970s, when the first drug-resistant strains of Unfortunately, the development of new classes of antibiotics has lagged behind the emergence of resistance. Meanwhile, antibiotic access and utilization have increased. While high-income countries have maintained a steady rate of consumption, lower and middle-income countries (LMICs) are increasing antibiotic utilization. This is influenced by lack of surveillance, low barriers for use and associated misuse, decreased costs, and high availability.2Microbes inherently possess genetic variability, with mechanisms that confer resistance to different classes of antibiotics. The 3 main contributing mechanisms include intrinsic resistance, genetic mutations, and horizontal gene transfer (including plasmid-mediated transfer). 1113141516131415 Notably, practitioners in private practice tend to overprescribe antibiotics compared with those working in community hospitals.18 Nosocomial infections represent a large number of drug-resistant infections, with transmission between patients and healthcare workers, who may practice infrequent handwashing and sanitation procedures. The routine prophylactic administration of antibiotics for invasive medical or surgical procedures can also contribute to ABR.Societal influences contribute to ABR in high-income countries and LMICs Fig. . Noncomp202120 Along with the disproportionate increase in ABR prevalence, LMICs can lack the infrastructure necessary to effectively track multidrug-resistant infections or enforce protocolized antibiotic administration. A report from the WHO in 2014 found great variation in level and quality of antibiotic surveillance programs in LMICs, and subsequently founded the Global Antimicrobial Resistance Surveillance System to address this critical gap.Resistance rates in LMICs rise at a 4–7× faster rate compared with high-income countries, secondary to increases in antibiotic utilization.24 Antibiotic-resistant bacteria of animal origin are then transmitted to human hosts through meat consumption or contaminated water supply.There are some unique drivers of ABR in developed countries. The proliferation of widespread antibiotic use with minimal regulation in the agricultural industry is a huge contributor to AMR.3MRSA present.Drug-resistant organisms are particularly prevalent in deep infections after fracture fixation. A study of 214 postoperative infections associated with fracture surgery demonstrated that 32% of all infections had Acinetobacter baumannii and Pseudomonas aeruginosa were the most common GNRs isolated in open and closed fractures, respectively, both of which exhibited low rates of susceptibility to carbapenems. A retrospective cohort study of 202 various open fractures demonstrated a 10% incidence of infection. A total of 25% of these infections were MRSA. The overall incidence of MRSA infection after open fractures was 2.5% in their population. Amongst those patients with infection, 55% grew at least 1 GNR.Open fractures are associated with increased risk of infection. In a Brazilian study of 11,030 patients who underwent internal fixation for open (17.1%) and closed fractures, the overall incidence of infection was 6%, which disproportionally occurred more in open fractures (14.7%).Drug-resistant infections have been extensively profiled in patients with hip fractures. In a study of 3686 patients at a British teaching hospital, 48% of patients with infection were attributed to MRSA . An MRSA infection after hip fracture was associated with increased costs, length of stay, and mortality compared with non-MRSA infection. In a multicenter study of 22 hospitals in France, the most common infectious isolate after femoral neck fracture was MRSA (32%).Another study looked into variations in bacterial pathogens causing infection after fracture treatment.435 In a recent survey of OTA members, 96% of surgeons recommended cefazolin as first-line prophylaxis in closed fractures. Most surgeons (58%) administered antibiotics within 1 hour of incision with redosing for 24 hours postoperatively, whereas 39% used a single dose and the remainder varied depending upon the clinical scenario.38 There are a few meta-analyses that suggest single dosing has similar outcomes as multiple doses, but these conclusions are based upon low-quality data.4041424041 Therefore, specific recommendations as to single versus 24-hour dosing for closed fractures remain unclear based upon existing evidence.Although routine antibiotic prophylaxis to prevent infection after fracture surgery has been widely accepted as standard of care for decades, some questions remain as to specific regimens in the era of evolving antibiotic resistance.Clostridium species for those wounds with fecal or soil contamination.Intravenous antibiotic administration is critical in preventing infection in open fractures, but the optimal duration remains controversial. Currently, coverage for gram-positive organisms is recommended with the addition of gram-negative coverage for type III open fractures and anaerobic coverage against Improved antibiotic stewardship does not translate to increased infection risk. A level-one academic trauma center implemented a protocol to guide antibiotic prophylaxis in open fractures based upon Gustilo-Anderson type.P = 0.07). This reduced infection risk was attributed to effective prophylaxis against gram-positive organisms, as the rate of gram-negative infections was unchanged. The long-term ramifications of routine adjunctive local delivery of antibiotics in open fractures with respect to ABR remain unclear.Appropriate indications for local delivery of antibiotics are evolving. A meta-analysis of open fractures reported a risk reduction of 11.9% in fracture-related infection if additional local antibiotics were given prophylactically.49 Advanced diagnostic techniques, such as bacterial or metagenomic sequencing, allow for efficient identification of causative organisms and targeted antibiotic administration. Also, bacteriophages and other nonantibiotic therapies the activate the host's innate immune system may translate into mainstream treatments but are currently developmental. The ideal duration of antibiotics for both prophylaxis and proven infection is still unclear and also warrants future investigation.Novel strategies to improve antibiotic stewardship are being developed.5Antibiotic resistance remains a significant global health issue. Bacteria inherently evolve resistance to antimicrobials over time which is accelerated through selective pressures generated by antibiotic exposure. Orthopaedic trauma surgeons should be aware of evidence-based prophylaxis and treatment strategies to effectively prevent and manage postoperative infection without contributing to the development of antibiotic resistance through indiscriminate prescribing.


A colorimetric assay of DNA cleavage by bleomycin (BLM) derivatives was developed utilizing high colloidal stability on double‐stranded (ds) DNA‐modified gold nanoparticles (dsDNA‐AuNPs) possessing a cleavage site. The assay was performed using dsDNA‐AuNPs treated with inactive BLM or activated BLM (Fe(II)⋅BLM). A 10‐min exposure in dsDNA‐AuNPs with inactive BLM treatment resulted in a rapid color change from red to purple because of salt‐induced non‐crosslinking aggregation of dsDNA‐AuNPs. In contrast, the addition of active Fe(II)⋅BLM retained the red color, probably because of the formation of protruding structures at the outermost phase of dsDNA‐AuNPs caused by BLM‐mediated DNA cleavage. Furthermore, the results of our model experiments indicate that oxidative base release and DNA‐cleavage pathways could be visually distinguished with color change. The present methodology was also applicable to model screening assays using several drugs with different mechanisms related to antitumor activity. These results strongly suggest that this assay with a rapid color change could lead to simple and efficient screening of potent antitumor agents. The colorimetric detection of bleomycin (BLM)‐mediated DNA cleavage with sequence selectivity is successfully demonstrated using dsDNA‐AuNPs with high colloidal stability. Notably, the cleavage pathways between oxidative base release and DNA cleavage can be identified according to a change in color using a colloidal stability test applied to dsDNA‐AuNPs with a blunt end and dangling end. This includes the availability of the BLM gene cluster for biosynthesiseled DNA,14, 15et al. reported that gold nanoparticles functionalized with single‐stranded (ss) DNA (ssDNA‐AuNP), through S‐Au interactions show unique colloidal properties not expressed in free‐form DNA.et al. also reported a non‐crosslinking aggregation occurrence that depends on the terminal base pairing of double‐stranded DNA (dsDNA) under high ionic conditions.2C19 gene SNP genotyping, which plays a role in the metabolism of pharmaceutical agents.Mirkin In this study, we developed a colorimetric method for detecting BLM‐mediated DNA cleavage to facilitate the selection of potent antitumor BLM analogs. The method is based on the high colloidal stability of the dsDNA‐AuNPs containing the main cleavage site for BLM. By using this method, we also identified the cleavage pathway via C4′‐hydroxy and C4′‐hydroperoxy intermediates. Furthermore, model colorimetric screening assays using several DNA‐associated drugs with different mechanisms were investigated.8 L ⋅ mol−1 ⋅ cm−1 at 520 nm).2 and B2) and BLM A5 were purchased from AdooQ Bioscience . The BLM concentrations were calculated using a molar extinction coefficient of 14,500 L ⋅ mol−1 ⋅ cm−1 at 292 nm in H2O.A colloidal dispersion of AuNPs with a nominal diameter of 15 nm was purchased from BBI Solutions . The concentration of AuNPs was determined using the molar extinction coefficient  solution (0.5 mol ⋅ L−1) and NaCl (2.5 mol ⋅ L−1) to obtain a final concentration of 10 mmol ⋅ L−1 PB solution containing 100 mmol ⋅ L−1 NaCl. Next, the buffer solution was incubated at 50 °C for 2 d and purified via centrifugation at 18,700×g at 10 °C for 25 min to obtain ssDNA‐AuNP. The ssDNA‐AuNP concentration was estimated using a molar extinction coefficient (4.10×108 L ⋅ mol−1 ⋅ cm−1 at 525 nm). The final dsDNA‐AuNP concentration for the colorimetric assay was set at 5 nmol ⋅ L−1. PB solution  containing 100 mmol ⋅ L−1 NaCl was used as the dispersion medium.ssDNA‐AuNPs were prepared according to the literature.43, 56−1 ssDNA‐AuNP was treated with 60 μL of 1 mol ⋅ L−1 DTT in an aqueous solution to release the ssDNA‐SH on the AuNP surface. The mixture was incubated at 20–25 °C overnight. Next, after removing the AuNPs via centrifugation at 18,700×g for 25 min, the released probe in the supernatant was quantified using the OliGreen ssDNA Quantitation Kit . The experiment was performed in triplicate (n=3).A stock solution of 60 μL of 10 nmol ⋅ L−1 ssDNA‐AuNP in 10 mmol ⋅ L−1 PB (pH 7.0) containing 100 mmol ⋅ L−1 NaCl was added to a solution containing 1 μL of 1 % Tween 20 and 2.5 μL of 4 μmol ⋅ L−1 complementary ssDNA to form dsDNA‐AuNP. Next, 2.5 μL of 80 μmol ⋅ L−1 BLM was added to this mixture, followed by incubation at 20–25 °C for 10 min. Next, 5 μL of 40 μmol ⋅ L−1 Fe(NH4)2(SO4)4 was added to the solution. Afterwards, 5 μL of water was added to the control sample without Fe2+, and the mixture was incubated at 20–25 °C for 20 min. Finally, 4 μL of 2.5 mol ⋅ L−1 NaCl was added to induce non‐crosslinking dsDNA‐AuNP aggregation . The final concentrations of dsDNA‐AuNP, NaCl, and Fe(II)⋅BLM were 5, 0.5, and 10 μmol ⋅ L−1, respectively. These procedures were repeated for the colorimetric assay to determine Fe(II)⋅BLM concentration dependency ranging between 0–10 μmol ⋅ L−1 and for screening the BLM model with several DNA‐associated drugs .A stock solution of 5 μL of 20 nmol ⋅ L−1 and 0.5 mol ⋅ L−1, respectively.To examine the colorimetric cleavage pathway, a 20 % feed ratio was provided by mixing complementary 16‐nt and 18‐nt DNA, followed by the addition of ssDNA‐AuNP to form a 20 % dangling end on the outermost surface of dsDNA‐AuNP. Finally, NaCl was added to induce non‐crosslinking dsDNA‐AuNP aggregation . The final concentrations of dsDNA‐AuNP and NaCl were 5 nmol ⋅ L−1) and activated Fe(II)⋅BLM  was incubated for 30 min at 20–25 °C, followed by the addition of NaCl  to evaluate the time‐dependent colloidal stability of dsDNA‐AuNPs. The changes in the UV‐vis absorption spectra were evaluated using the ratio of the absorbance at 530 nm and 700 nm (A530/A700).dsDNA‐AuNP aggregation via BLM‐mediated DNA cleavage was evaluated using a total volume of 100 μL and a procedure similar to that described above. A solution containing dsDNA‐AuNPs  in 2.5 mmol ⋅ L−1 PB solution with 0.5 mol ⋅ L−1 NaCl was dropped onto an elastic‐carbon‐coated copper grid  followed by removal of the excess solution by blotting with a filter paper. The measurements were performed using TEM  operated at an accelerating voltage of 80 kV.For sample preparation, 1 μL of dsDNA‐AuNPs  for evaluation of BLM analog activities.15 (not at cytidine3), allowing us to design the base sequence of dsDNA substrate containing a major cleavage site (5′‐GC‐3′) on the polyA strand. Consequently, 5′‐CGC‐3′ was chosen for 18‐nt 3′‐thiolated ssDNA with TA base pairs; it was attached to AuNPs with a 15‐nm diameter and mixed with 18‐nt complementary ssDNA to form dsDNA on AuNP (dsDNA‐AuNP) ⋅BLM containing a mixture of BLM A2 and BLM B2 , followed by the addition of NaCl to induce non‐crosslinking aggregation.2O, Fe2+, or BLM, were adapted using the same procedure as those for Fe(II)⋅BLM.To achieve a colorimetric method to detect BLM‐mediated DNA cleavage, we focused on DNA substrates for BLM‐mediated DNA cleavage. Hecht ) Figure a. This d2+ or BLM showed rapid aggregation, resulting in a drastic color change from red to purple owed to a shift in SPR 2(SO4)2 (Fe(II)⋅BLM) retained the red color. In addition, treatment of dsDNA‐AuNPs with Fe(II)⋅BLM resulted in change in the optical properties of the dsDNA‐AuNP dispersion (λmax=525 nm), as revealed via UV‐vis absorption spectroscopy. Treatment of dsDNA‐AuNP with several controls  exhibited a red shift in the characteristic SPR (λmax=548 nm) and an increase in the intensity at approximately 700 nm related to aggregation state ⋅BLM also exhibited apparent colorimetric and spectroscopic changes between BLM and Fe(II)⋅BLM .The dsDNA‐AuNP solution treated with FeR Figure a. The tee Figure b. These −1 ). On the other hand, both Fe(II)⋅BLM and BLM at 10 μmol ⋅ L−1 in AT‐dsDNA‐AuNP did not produce a color change at the salt concentration ranging from 0.4 to 0.7 mol ⋅ L−1 ). Additionally, a higher salt concentration of 0.7 mol ⋅ L−1 in non‐crosslinking AT‐dsDNA‐AuNP aggregation by BLM than that of the G‐C base pairs in dsDNA‐AuNP was required, presumably because of differences in number of hydrogen bond between the A‐T and G‐C base pairs.Furthermore, dsDNA with 18 A‐T base pairs (without main cleavage site) was attached to AuNPs (AT‐dsDNA‐AuNPs) using the same procedure for dsDNA‐AuNP with a main cleavage site. The colloidal stability of AT‐dsDNA‐AuNP thus obtained was evaluated by salt concentration dependency of color changes in comparison to dsDNA‐AuNP. In the case of dsDNA‐AuNPs, Fe(II)⋅BLM and BLM treatment gave clear color changes in the salt concentration range of 0.4 to 0.7 mol ⋅ L−1) in dsDNA‐AuNP solutions. Solutions with Fe(II)⋅BLM concentrations higher than 8 μmol ⋅ L−1 retained the red color, in contrast to those of lower concentrations, ranging from 2–6 μmol ⋅ L−1 ⋅BLM was maintained because of the appearance of a protruding structure of at least 10 % on the particles. Furthermore, the results of Fe(II)⋅BLM, regarding the aggregated state at 2 μmol ⋅ L−1 (left panel) and dispersed state at 10 μmol ⋅ L−1 (right panel), were supported by TEM images ⋅BLM  was adapted.530/A700 value of all samples, at all concentration ranges was 20, which is consistent with results of a previous study.530/A700 value of the solution treated with 8–10 μmol ⋅ L−1 of Fe(II)⋅BLM was maintained at approximately 70 % (A530/A700=14) after 15 min and retained a red color ⋅BLM decreased to 40 % (A530/A700=5.0) in 5 min  was separately treated with NaCl  and incubated at room temperature for 10 min. The sample of dsDNA‐AuNPs having inactive and oxidative base release showed spontaneous aggregation ,5 and clinically used BLM (Blenoxane), via the same mechanism of DNA cleavage, maintained the red color derived from the dispersed state. Therefore, we demonstrated the colorimetric screening of the activity of Fe(II)⋅BLM from sequence‐specific DNA cleavage of Fe(II)⋅BLM.After confirming that colorimetric BLM‐mediated DNA cleavage and the colorimetric cleavage pathway can work as expected under Fe(II)⋅BLM concentration dependency, we performed model colorimetric screening using several DNA‐associated agents with different medicinal mechanisms, as summarized in Table S1. CPAThese results validate our dsDNA‐AuNP design. Notably, the cleavage sites in dsDNA introduced at the outermost surface of the particles could be essential for controlling colloidal stability with specificity against Fe(II)⋅BLM. If the strand with a 5′‐GC‐3′ site in the middle is adapted to dsDNA‐AuNPs, then (i) the presence of a dense DNA layer may introduce steric hindrance to the interaction between BLM and DNA, resulting in a loss of sequence properties; (ii) the duplex structure will be maintained with a nick even if BLM cleavage occurs. This may be attributed to the attractive blunt‐end stacking at the dsDNA terminal, resulting in the spontaneous non‐crosslinking aggregation of dsDNA‐AuNPs; and (iii) the translation of small structural changes corresponding to the cleavage pathway into the control of dsDNA‐AuNP dispersibility is difficult, as shown in our model experiment ⋅BLM, and the reaction was concentration‐dependent. This concentration‐dependent behavior led to colorimetric changes, with 6 μmol ⋅ L‐1 being the detection limit for the BLM concentration. Furthermore, AT‐dsDNA‐AuNPs treated with BLM and Fe(II)⋅BLM exhibited the same color change tendencies resulting from spontaneous salt‐induced aggregation at the same salt concentration, suggesting sequence‐specific cleavage by Fe(II)⋅BLM could be detected colorimetrically. Consequently, we demonstrated the colorimetric detection of BLM‐mediated DNA cleavage using dsDNA‐AuNPs. More importantly, we demonstrated that the cleavage pathways between oxidative base release and DNA cleavage could be identified based on the change in color using a colloidal stability test in dsDNA‐AuNPs with abasic sites and protrusion structures of two bases. Experiments regarding the cleavage pathway for the characterization of decomposed fragments released from BLM‐mediated dsDNA‐AuNP cleavage according to mass analysis and the detailed kinetics will be published elsewhere. The methodology implemented could provide a practical non‐fluorescence‐based analysis method in which potent DNA‐damaging agents are colorimetrically screened, and sequence‐specific cleavage is more accurate based on combinatorial solid‐phase libraries.The authors declare no conflict of interest.4As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


However, the most commonly used Equations for such estimations do not take into account the influence of concrete moisture. The aim of this paper was to establish this influence for two series of structural lightweight aggregate concrete (LWAC) varying in their strength (40.2 and 54.3 MPa) and density (1690 and 1780 kg/m3). The effect of LWAC moisture content turned out to be much more pronounced in the case of dynamic modulus measurements than for static ones. The achieved results indicate that the moisture content of the concrete should be taken into consideration in modulus measurements as well as in Equations estimating c,sE on the basis of dE specified by the ultrasonic pulse velocity method. The static modulus of LWACs was lower on average by 11 and 24% in relation to dynamic modulus, respectively when measured in air-dried and water-saturated conditions. The influence of LWAC moisture content on the relationship between specified static and dynamic moduli was not affected by the type of tested lightweight concrete.The dynamic modulus of elasticity ( The modulus of elasticity is one of the most important mechanical properties of structural concrete in the designing process for concrete construction. The knowledge of a value of this characteristic is necessary to determine the deflection of a structural member, stress–strain relation, losses of prestress force, crack width as well as stresses induced by strains associated with environmental effects. The values of modulus of elasticity given in the European Standard for designing concrete structures EN-1992-1-1 [A specified value of modulus of elasticity is affected by numerous material and technological parameters such as type and content of concrete constituent materials, concrete condition and method of testing. Nevertheless, it is the type of aggregate which determines the modulus especially strongly ,3,4. ThaA parameter that can significantly affect the value of the modulus of elasticity, but which is rarely taken into account, is concrete moisture content. However, unlike many studies on the effect of concrete moisture content on its compressive strength e.g., ,10,11), ,11, 10,10E [tangent initial modulus specified at stress equaled to 0 (E0 ),c,0E [secant initial modulus determined at the first cycle ;c,sE [secant stabilized modulus determined after the third cycle .dE) is also specified for concrete. It is carried out in non-destructive tests causing vibrations in concrete, which result in stresses being vanishingly small. For this reason, the value of the dynamic and static initial moduli are often equated [Besides the static moduli, mentioned above, a dynamic modulus  and a constant depending on the unit system were taken into consideration (k). Nevertheless, parameter k was specified for units in psi (0.23), while in SI is unidentified [Equations 2) and (3) describing relationships between  and (3) entified .(2)Ec,sc,sE and dE may be significantly different from estimations given by the proposed relationships [c,sE was higher by up 50% and the difference in overestimation of the static modulus was up to 15 GPa. In turn, the analysis of results for structural lightweight concretes, presented in [c,sE. by up to 4.5 GPa (on average by 12%) and 3.0 GPa (on average by 8%), respectively when applied Equations (1) and (2). It should be noted that the difference between measured and estimated values of static modulus of elasticity was higher when the initial moisture content of the lightweight aggregate was bigger. Such an observation may be explained by the different microstructure of LWACs with aggregates of different initial moisture content. As it was proved in [The above Equations, especially (1), are often used to estimate the static secant modulus of concrete built into a construction instead of its direct testing on specimens cored from the structure. Meanwhile, some scientific papers, in which the results of both types of moduli were tested, indicate that the relationship between ionships ,24,25,26ionships ,23 proveented in ,23,26 : 0.37 and 0.55. To ensure similar workability of both fresh concretes, a superplasticizer was applied for LWAC with lower w/c. However, both concretes were made of Portland cement CEM I 42.5 R, potable water, natural sand 0/2 mm as fine aggregate and sintered fly ash aggregate 4/8 mm (Lytag) as coarse aggregate. Both series were characterized by the same volume proportions between cement matrix (mortar) and lightweight aggregate (55%:45%). The tests were carried out on standard cylindrical specimens cast of two series of structural lightweight aggregate concretes characterized by different strengths and densities. The various properties of these two LWACs were achieved by different nominal water and cement ratios of cement matrix  were molded and compacted on a vibration table. All 42 specimens were demolded after 24 h and cured in a climatic chamber  according to EN 12390-2  for 28 dThe concretes were tested at two ages: 28 days and 3 years. At the first age, the concrete density and strength were determined to classify both concretes. The actual tests of dynamic and static moduli of elasticity were carried out at the age of 3 years. This late age of the research ensured that the time of testing had no effect on the specified values of the modulus of elasticity. The tests carried out, the LWACs specimens’ number and age at testing, as well as the standard test procedures for each concrete series are given in The tests of compressive strength and modulus of elasticity at 28 days were carried out on specimens in saturated conditions as it is required by the European Standards EN 12390-3 [c,sE) was tested according to Method B of the standard EN 12390-13 [dE) was specified using ultrasonic pulse velocity method as described in EN 12504-4 [cm, cylf) was calculated. Finally, the nominal lower stress (pσ) and the nominal upper stress (aσ) were assumed as 0.5 MPa and cm, cylf/3, respectively. The standard scheme of loading and unloading cycles, as well as an example of cycles registered at testing, are presented in pσ up to the upper stress a.σ The stabilized secant modulus of elasticity was determined according to Equation (4), where amσ and bmσ are stress values measured at the third cycle, while a,3ε and p,2ε are corresponding strains.At a given moisture condition, the static secant modulus of elasticity  adopted from ASTM C 215 , where VSince EN 12504-4  assumed cm, cylf/3) that is significantly lower than the fatigue strength. As was stated in [To eliminate the influence of concrete heterogeneity on the result of static and dynamic modulus measurements, both tests were carried out in sequences on the same set of specimens including both moisture conditions. Such an approach was assumed reliable owing to the relatively low upper stress considered in multiple cyclic loading  at the age of three years was 1710 and 1810 kg/mThe water absorption, calculated on the basis of concrete density in water-saturated and oven-dried conditions, was 10.1 and 7.9%, respectively for LC1 and LC2. The moisture content in concrete to be tested in air-dried conditions at the age of three years was 1.2 and 1.7%, respectively for LC1 and LC2.The failure and fracture form of cylindrical specimens at compressive strength tests showed good structural homogeneity of both tested concretes . There wAccording to achieved results of compressive strength see , the uppThe specified values of secant modulus of elasticity tested in two conditions: air dried and water saturated are presented in The values of ultrasonic pulse velocity, measured in two conditions: air dried and water saturated, are presented in The values of dynamic modulus of elasticity, calculated on the basis of measured ultrasonic pulse velocity using Equation (5), for both conditions: air dried and water saturated are presented in The relationship between measured values of ultrasonic pulse velocity and values of dynamic modulus of elasticity, specified on these measurements, are shown in As it was assumed, various water-cement ratios ensured significantly different levels of compressive strength, modulus of elasticity and density for both concrete series. As a result, in accordance with the standard EN-206 , concretAt the age of three years, both series revealed pronounced strength development. The mean value of compressive strength at this age time was higher by 20% in relation to the strength determined at the standard age of 28 days. Such an increment in strength should be assessed as relatively high as for structural concretes ,42,43. IThe comparison of specified values of static secant and dynamic moduli tested in both considered moisture conditions for series LC1 and LC2 are presented in The carried out tests proved considerably higher values of dynamic modulus of elasticity in relation to static secant values, regardless of concrete series and the moisture content of specimens . c,sE and dE determined for individual specimens tested in air-dried conditions ranged from 0.85 to 0.93, while specified in water-saturated conditions ranged from 0.71 to 0.80. The effect of the concrete series was not observed. As a result, the average ratio c,sE / dE was assessed as 0.89 and 0.76 for air-dried conditions and water-saturated conditions, respectively. In both cases, the standard deviation of the ratio was 0.03. The obtained ratio values for tested lightweight concretes were slightly lower than those calculated for LWACs reported in [c,sE / dE turned out to be insignificantly higher than those calculated on the basis of data given in [c,sE / dE determined in air-dried conditions was considerably higher than for water-saturated concretes. The above comparison of ratios achieved and calculated on the basis of reported data indicates that c,sE / dE is affected by both moisture content and the homogeneity of the concrete composite structure. The less homogeneous structure of a composite , the lower the ratio.The ratio of orted in ,25. On tgiven in  for normgiven in ,23. As igiven in , in thisThe obtained results showed that the application of Equation (1) or (2) to determine the secant modulus of elasticity of tested lightweight concretes in standard water-saturated conditions would lead to overestimation on average by 9 and 16%, respectively. For lightweight concretes tested in standard water-saturated conditions, Equation (6) should be proposed as more reliable:c,sE, on average by 6 and 3%, respectively. In turn, for LWACs in air-dried conditions, both Equations (1) and (2) slightly underestimate the value of If the values of the secant modulus of elasticity of concretes in a saturated condition, as meeting the requirements of the standard EN 12390-13 [It should be noted that since there were no visible differences in the relationship between the specified moduli for both tested concretes, Equation (3) taking into consideration concrete density, would not be appropriate in this case.c,sE determined in water-saturated conditions were higher than the values tested in the air-dried conditions by only 2% on average. Meanwhile, for the dynamic modulus, such a mean increment was as high as 19%. No visible influence of the concrete series was observed.The carried out tests proved the effect of concrete moisture content on the specified values of both tested moduli . The valc,Es of normal-weight concrete tested in water saturated condition ranged from 12 to 32% in relation to the dry condition. It should be noted that although the referred concretes had similar strength to those tested in this research, the porosity structure of composites was different. To achieve a similar strength level of concrete made of a more porous aggregate, it is necessary to use cement paste tighter than in the case of normal-weight concrete. Therefore, in saturated condition there is less water in the cement matrix of LWAC than of NWAC. Moreover, the bond between aggregate and cement paste in LWAC is usually better. However, when comparing the results achieved in this research to those referred to in [c,sE determined in water-saturated conditions was bigger by 4 up to 14% in relation to results measured in air-dried condition. In this case, the explanation of a more pronounced moisture influence on the modulus in [The influence of the moisture content of the tested concretes on the values of the static secant modulus of elasticity turned out to be less significant than in the case of the studies discussed in ,13,14,15ed to in  for lighed to in , Ec,s dedulus in  is the mdulus in , air-driThe tests and the analyses of obtained results proved that lightweight concrete tested in standard water-saturated conditions revealed higher values of both specified moduli, static and dynamic, in relation to measurements achieved in air-dried conditions. However, the influence of the LWAC moisture content turned out to be much more pronounced in the case of dynamic modulus measurements. Such an observation indicates that the moisture content of the concrete should be taken into consideration in modulus tests as well as in Equations estimating the static secant modulus of elasticity on the basis of ultrasonic pulse velocity measurements.For tested concretes, the static modulus of elasticity was lower on average by 11 and 24% in relation to the dynamic modulus, respectively for lightweight concretes tested in air-dried and water-saturated conditions. Therefore, in the case of tested LWACs, the commonly used relationships between static and dynamic moduli ((1) and (2)) turned out to be more accurate than for some normal-weight concretes. Nevertheless, even for the tested lightweight concretes the Equations also required correction. c,sE and dE is influenced by both moisture content and the homogeneity of concrete composite structure. As a result, the tested lightweight concrete, despite their higher water absorption in comparison to NWACs, did not show a bigger effect of their moisture content on the specified values of static modulus of elasticity, probably due to better LWACs’ structural homogeneity and its more tight cement matrix. Nevertheless, it should be stated that the influence of the moisture content of concrete on its modulus of elasticity is not as unequivocal as in the case of compressive strength tests.The influence of LWAC moisture content on the specified static and dynamic module was not affected by the type of tested lightweight concrete, determined by its various strength and density level. However, the comparison of results achieved in this research and calculated on the basis of reported data indicates that the relationship between In order to establish a reliable and versatile relationship between the static and dynamic moduli of elasticity for structural concretes, further tests should be carried out on different types of cement composites in a wider range of their moisture content.


BackgroundUpper respiratory tract infections (URTIs) represent the most common diagnosis in ambulatory care settings. Some of these infections are properly treated with antibiotics, but evidence points to an inappropriate overuse of antibiotics in URTI management. This overuse is linked to antibiotic resistance, drug-related adverse effects, and increased costs.ObjectiveThis study evaluated the prevalence and predictors of antibiotic prescription for patients with URTI symptoms at the primary healthcare centers (PHCCs) and pediatric emergency department (ED) of the Maternity and Children Hospital (MCH) in Dammam, Saudi Arabia.MethodsA prospective study was conducted in the PHCCs and pediatric ED of MCH. Trained physicians collected data on patients with URTI symptoms aged three years and older. Scores based on modified Centor criteria were calculated, and rapid antigen detection tests (RADTs) were conducted for all study participants.ResultsOut of 469 patients with a URTI, 141 (30.1%) received a prescription for an antibiotic, with a smaller proportion in the PHCCs  than in the pediatric ED . The main significant predictors of antibiotic prescription in terms of odds ratio (OR) and 95% confidence interval (95%CI) were a positive RADT result , the presence of tonsillar exudate , tender and/or swollen anterior cervical lymph nodes , and fever . A higher Centor score was also a predictor (2 to 5 vs. −1 to 1) . The absence of a cough was not a significant predictor .ConclusionsAlthough a positive RADT increased the likelihood that a patient would be prescribed an antibiotic at the time of assessment, most antibiotic prescriptions were not justified. To control expenses, prevent adverse effects, and limit the spread of antibiotic resistance, efforts should be made to reduce unnecessarily high antibiotic usage. The inappropriate use of antimicrobial agents has been linked to antibiotic resistance, which is a global public health concern . Upper rThe Kingdom of Saudi Arabia (KSA) has a high rate of antibiotic use, ranging from 41% to 92% of total use, and the misuse is especially prevalent among children . This poThe aim of the current study was to discover the determinants of antibiotic prescription for URTIs in primary healthcare centers (PHCCs) and a pediatric emergency department (ED) affiliated with a hospital in the KSA. Most of the published literature regarding antibiotic prescription is retrospective or derived from records. Furthermore, these studies do not include Centor criteria or rapid antigen detection test (RADT) results. A prospective study to determine the factors influencing physicians’ practices in prescribing antibiotics for a disease mostly caused by viral infection could provide insight into the factors underlying the problem of antibiotic misuse. Therefore, the objective of this study was to explore the patterns and factors influencing antibiotic prescription for patients with URTIs.This study is expected to increase awareness about the overuse of antibiotics among general practitioners and pediatric physicians covering EDs and PHCCs. The results of this study could be used in planning and assessing the need for training and education in the judicious use of antibiotics and the proper treatment of URTIs.A sequential sample was collected prospectively from the ED of Maternity and Children Hospital (MCH), which is a secondary care hospital in Dammam, KSA, and from three PHCCs in the same city. Visitors aged three years and older with respiratory tract symptoms, such as sore throat, blocked or runny nose, cough, or any sign of pharyngitis were included.Data collection toolParticipants or their guardians were asked by well-trained physicians about their willingness to participate in the study. Those willing to participate were interviewed by their physician, who used a questionnaire created for this study. The questionnaire was previously validated by four expert consultants in infectious disease, community medicine, public health, and family medicine. After the questionnaire was completed, a standard throat swab was collected using polyester (Dacron)-tipped swabs. A rapid test for group A streptococcus infection was conducted using Alere TestPack Plus Strep A with On Board Controls, which is a rapid immunoassay for the qualitative detection of the group A streptococcal (group A strep) antigen from throat swab specimens for the diagnosis of group A strep pharyngitis.Modified Centor scores were computed based on the criteria presented in Table Ethical considerationsThe questionnaire and protocol were approved by the Dammam Medical Complex, KSA, the Institutional Review Board , and by the relevant authorities before conducting the study. The aim of the study was explained to the selected population. Prior to their participation in the study, consent was obtained from the patients or, in the case of children, from their parents or guardians. If children were able to communicate, their approval for participation was obtained. All names and data were handled with respect for confidentiality.Data processing and analysisData for all variables were checked for accuracy and completeness and then coded. The data were then entered into a personal computer using Statistical Package for the Social Sciences (SPSS) version 26  for cleaning and analysis. The chi-square test or Fisher’s exact test was used for the bivariate analysis of categorical variables. Binary logistic regression was performed for multivariate analysis, where a p-value of <0.05 was considered statistically significant.From March 13, 2022, to September 30, 2022, data were collected from 469 patients with respiratory symptoms at the PHCCs and ED of MCH. As shown in Table Regarding the Centor criteria, the most commonly reported symptoms were fever and cough in 190 (40.5%) and 318 (67.8%) participants, respectively. RADT was positive in a small proportion of participants .Thirty-seven (7.9%) of the patients included in the study had received antibiotics within the previous 28 days, and 141 (30.1%) received antibiotics by the end of the current visit, comprising 85 (24.4%) from the PHCCs and 56 (46.3%) from the ED.A comparison between the PHCCs and ED patients revealed a significant difference (p<0.05) across almost all relevant factors. ED patients were more likely to be younger and have more symptoms (both p<0.05). Moreover, ED patients were significantly more likely to have symptoms suggestive of strep A infection, positive RADT results, and a history of receiving antibiotics in the past 28 days and to be prescribed antibiotics by the current visit . The PHCC patients were more likely to be healthcare providers, have chronic illnesses, and be smokers .Unadjusted bivariate analysis showed that younger patients, those with chronic illnesses, and those with positive RADT results were more likely to be prescribed antibiotics during the current visit Table . Tender In a primary multivariate model including setting, age group, chronic illnesses, RADT result, and symptoms, the significant predictors of receiving antibiotics were visiting the ED , not being healthcare personnel , having a positive RADT result , and having suggestive symptoms . Absence of cough was not associated with higher odds of receiving antibiotics in this model , as seen in Table In a second multivariate model that replaced age groups and symptoms with the Centor score as a binary predictor , a high Centor score of 2 or more doubled the odds of being prescribed antibiotics . Except for setting , all other variables, including being healthcare personnel , having a chronic illness , and the strongest predictor, having a positive RADT result , were significant in this model.A third model was run after restricting age to 3-14 years and excluding job category as a predictor. Model 3 confirmed the findings of model 2, as a positive RADT result and a Centor score of 2 or more were significantly associated with an antibiotic prescription, whereas setting and comorbidities were not.The current study found that 30.1% of patients with URTI received antibiotics, which is relatively lower than percentages reported previously in the KSA -11 and mThe percentage of antibiotic prescriptions among visitors with URTI symptoms varies among healthcare facilities with different scopes of services, with lower rates in PHCCs (24%) than in the pediatric ED (46.3%). The intensity of symptoms and signs among ED visitors compared with PHCC visitors can explain the higher rate of antibiotic prescription in the ED, as supported by the results of this study. Statistically, setting was a significant factor when adjusted for age groups but not for Centor score, indicating that significance was related to symptoms and signs rather than setting. Furthermore, parents who bring their children to the ED are typically very worried about their health and may place greater pressure on physicians to prescribe antibiotics. In a 2018 KSA survey, the majority of physicians said that they feel pressured when patients ask for an antibiotic prescription . MoreoveOther than being a visitor to the ED, having a chronic illness was the other predictor of receiving antibiotics, which might reflect a belief that this group is vulnerable to bacterial infections that must be bolstered by antibiotics. Another predictor of prescribing antibiotics was the presence of fever, although this symptom does not differentiate between bacterial and viral infections . HoweverGuidelines for the proper management of URTIs do not appear to be adequate to dramatically reduce the unnecessary prescription of antibiotics. This problem could be the result of the difficulty in the implementation of the guidelines in daily clinical practice because of other factors that enhance antibiotic prescription, including patient behavior and lack of information or understanding ,21. For A systemic review of the effectiveness of physician-targeted interventions to improve appropriate antibiotic use for URTI revealed that, compared with written patient information and audit/feedback, educational meetings are more labor intensive but also appear to be more successful . FurtherMultiple interventions to increase appropriate antibiotic prescription in the KSA  have been associated with significant improvements . By makiLimitationsThe main limitation of this study was the inability to mask the RADT results from the physicians, which may have affected their decision to prescribe antibiotics. In addition, it was not possible to implement a systematic random selection of patients given the context of a busy clinic.To the best of our knowledge, this study is the first prospective investigation combining Centor criteria and RADT results to assess the practice of antibiotic prescription. The positive impact of restricting over-the-counter sale of antimicrobial drugs could be strengthened by addressing the root causes of antibiotic misuse, promoting physicians’ rational use of antibiotics through education and training on best evidence-based practices, and auditing their performance in a way that does not interfere with patient care and individualized treatment. Furthermore, supporting physicians’ decision-making with improved availability of RADT assessment when Centor criteria require confirmation can help minimize the prescription of unnecessary antibiotics.


Following a global shortage of piperacillin/tazobactam in 2017, a formulary decision was taken at a large District General Hospital in the East of England to partly replace piperacillin/tazobactam with either temocillin as monotherapy or as part of a combination regimen. A retrospective audit was then conducted to assess the clinical effectiveness of temocillin therapy.Data from patients admitted to Watford General Hospital between May and August 2017 and treated with temocillin were reviewed retrospectively. Clinical characteristics of patients, data related to the episode of infection, clinical success, tolerance and mortality were analysed.n = 46), the lung (n = 40) and the urinary tract (n = 21). Seventy-seven patients received temocillin as first-line therapy and 106 received it empirically, with temocillin prescribed in combination with another antibiotic in 82% of the empirically treated cases. Clinical success was observed in 88.9% of cases with no difference between patients treated empirically and others (89.6% versus 85%) or in efficacy among abdominal (91%), pulmonary (87.5%) and urinary (81%) infections. One case of Clostridioides difficile infection was reported in a patient treated with four different antibiotics. During the shortage period, the hospital’s standardized mortality ratio was significantly lower when compared with the same period of the preceding year (85 versus 96).Temocillin was used in 126 patients with median age of 73 years. Infection episodes mostly originated from the abdomen (Using temocillin as part of an empirical strategy is feasible and safe as long as appropriate antibiotic combination is recommended based upon the indication and the likely bacterial pathogen. Temocillin was advised as monotherapy for pyelonephritis/urosepsis, in combination with metronidazole for complicated intra-abdominal infections (IAIs) and with amoxicillin and metronidazole for aspiration pneumonia. Combination therapy was advised to extend the limited bacterial coverage of temocillin and careful consideration of the likelihood of pseudomonal infection was advised in latter situations, including analysis of prior microbiology results, and gentamicin was added whenever P. aeruginosa was suspected.In October 2016, the explosion of a manufacturing site in China led to a major shortage of piperacillin/tazobactam across the globe. In response, the Department for Health and Public Health England (PHE) jointly issued guidance restricting the use of this antibiotic to specific indications only  and to place piperacillin/tazobactam use under the supervision of hospital microbiologists and Infectious Disease specialists.Pseudomonas was not expected, for hospital-acquired pneumonia (HAP) in combination with amoxicillin (1 g three times a day), for SUS in combination with amoxicillin (1 g three times a day) and metronidazole (500 mg IV three times a day) and, for IAI, in combination with metronidazole (500 mg IV three times a day). After the antibiotic formulary was changed, an audit was conducted to assess the efficacy and safety of this new local guidance.In consequence, a formulary decision was taken in Watford General Hospital (520 beds) to replace piperacillin/tazobactam with temocillin  alone for the empirical treatment of urinary tract infections (UTIs) and, where The audit was a service evaluation initiated at the request of the trust antimicrobial committee and conducted retrospectively for internal hospital guidance. According to the UK National Research Ethics Service, neither consent nor ethical approval was required.During a 4 month period (May 2017 to August 2017), data were collected from each episode of infection treated with at least 3 days of temocillin. Patient demographics, renal function, microbiological data, prior and concomitant antibiotics, hospitalization time and clinical outcome were recorded. For analysis purposes, temocillin was categorized as first-line treatment if no previous antibiotic treatment was given to the patient and as second-line treatment if another antibiotic was given before starting temocillin treatment. Clinical success was defined as clinical and biochemical improvement with no need for escalation in antibiotics and no relapse requiring repeat treatment within 7 days of completing the antibiotic course, as assessed by a single reviewer, unless efficacy was unclear, in which case an assessment was also made by a second independent reviewer. Clinical failure was defined as the lack of clinical response as judged by the treating clinician and as evidenced by the NEWS score (with or without rising of inflammatory marker).Clostridioides difficile infections (HA-CDI) and hospital standardized mortality ratio (HSMR) were calculated during the shortage period and compared with the same period in the preceding year. The HSMR was calculated by dividing the observed number of deaths at the hospital by the expected number of deaths, based on patient characteristics such as age, sex and diagnosis. A ratio above 100 means that more deaths are occurring at the hospital than would be expected, while a ratio below 100 means fewer deaths are occurring.Antibiotic consumption (DDD/1000 bed days), healthcare-associated t-test and Fisher’s exact test, respectively.Statistical analyses were performed using JMP 14.0.0 (SAS Institute); continuous and nominal variables were compared using Student’s 2). Further patient demographics are presented in Table One hundred and twenty-six episodes of infection from 126 patients were retrieved and included in the analysis. The mean age of patients was 70 years (SD 19 years) and ranged from 21 to 100 years. Forty-five patients had impaired renal function, i.e. estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 mn = 25), treatment escalation (n = 17) and de-escalation (n = 7). The antibiotic regimen was empirical in 106 episodes and pathogen-guided in 20, and when prescribed empirically, temocillin was combined with another antibiotic in 82% of cases  in 90 episodes (71%). Guidance from a microbiologist initiated the use in 26 episodes and the remaining 10 episodes were out of the scope of the local guidance without advice from the Microbiology Department.Mean duration of treatment was of 6.2 ± 3.7 days, ranging from 3 to 34 days. Median duration is 5 days, ranging from 4–7 days and IQR were 5 ± 3 days. Treatment duration differed slightly per indication, without reaching statistical significance (Table Escherichia coli (n = 23), Staphylococcus spp. (6), other coliforms (n = 5), Klebsiella pneumoniae (n = 4), anaerobes (n = 3), Candida spp. (n = 3), other Gram-positive aerobes (n = 2) and Pseudomonas spp. (n = 1).Clinically significant positive microbial identification was obtained in 45 out of 126 episodes (36%) with the most common pathogens being P. aeruginosa and one IAI caused by Staphylococcus epidermidis. We found no treatment failures in the 10 episodes that were out of the scope of the guidance. In patients treated empirically with temocillin, clinical success rate was 89.6% and on univariate analysis no significant alteration of the clinical outcome was related to any parameter .Clinical success was observed in 112 episodes (88.9%), clinical failure in 12 episodes (9.5%) and 2 episodes (1.6%) were assessed as undetermined .Overall the drug was well tolerated, with just one case of HA-CDI. This instance occurred 4 weeks after the end of temocillin treatment in a patient who first received piperacillin/tazobactam and cefotaxime before temocillin combined with metronidazole. At the hospital level, a reduction of 50% was observed in the HA-CDI rate during the shortage period plus 3 months thereafter, compared with the same period in the preceding year.During the 4 month-long shortage period, there was a 29% increase in the total antibiotic consumption (DDD/1000 bed days) compared with the same period in the preceding year. At the same time, there was a 44% and 4858% increase in the use of meropenem and temocillin respectively, whilst there was a 64% decrease in the use of piperacillin/tazobactam.During the shortage the increase in antibiotic consumption that we observed was due to the use of antibiotic combinations. Although meropenem was only recommended in neutropenic sepsis and in penicillin-allergic patients, its consumption rose, probably due to a lack of experience with temocillin. The implementation of our guidance had no detrimental effect on the HSMR. However, guidance documents were not always followed, which could have been due to lack of confidence in the recommended regimens, miscommunication of the contents of our guidance or the short notice period between their publication and actual implementation.et al.2 reported a clinical efficacy of 82.0% (50/61) for patients treated empirically with temocillin . In a recent prospective randomized controlled trial, temocillin was compared with cefotaxime for the treatment of febrile UTI, which showed that the regimens were of comparable effectiveness, with both similar early  and late  clinical responses.3 This latter study is in line with the current Belgian guidelines, which recommend the empirical use of temocillin for several types of UTI.4When used empirically, clinical success was reached in 90% of the patients, which is comparable to other studies. Heard 5–7 For this indication our study shows a clinical success rate of 91.3% when combined with metronidazole, which is comparable to the results of one study looking at temocillin use in critically unwell patients.8Our dataset includes 46 IAI, an indication poorly described in literature for the use of temocillin despite a few studies on its tissue penetration.9 Interestingly temocillin is known for its low ecological impact,10–12 which might have helped in this regard.Out data showed a 50% decrease in HA-CDI, which may have been due to the fact that penicillin combination antibiotics (e.g. co-amoxiclav and piperacillin/tazobactam) are associated with a significant increase in the risk of HA-CDI.This audit has several limitations. The retrospective nature of the data may have introduced bias and no comparator group was included, though the HSMR and antibiotic consumption could be compared with the same period of the previous year. Furthermore, only a limited number of patients included in the dataset had a positive microbiological culture; this could have been due to starting treatment prior to sample collection or to the unavailability of accessible sample types from which to obtain an isolate (e.g. in the case of IAIs).In conclusion, the shortage of piperacillin/tazobactam was an opportunity to implement new antibiotic regimens in our hospital. Though guidance was not always followed, using temocillin as part of an empirical strategy seems feasible provided that an appropriate antibiotic combination is recommended taking into account the indication and appropriate pathogen coverage. Further prospective trials are needed to confirm these preliminary data, particularly if IAI is to be accepted as an indication for this agent. In the meantime, this audit allows sharing of lessons from hospital practice that could be helpful, particularly during future periods of antibiotic shortages.


CHEK2, HIF1A, HK2, HMGA1, SERPINA1, PTPN1, SLC2A1 and VEGFA. The comprehensive functional annotation was evaluated by the integrated functional enrichments analysis. We identified the activation of inflammatory-related pathways, metabolic reprogramming and angiogenesis, and the inhibition of cardiovascular development pathways in TAAD. Immune cell infiltration (ICI) analysis further demonstrated that innate immune-cells were more dominant than adaptive immune-cells in TAAD tissues, especially in macrophages, monocytes, activated-DC, NKT cells and CD56+dim NK cells. The cellular landscape was further validated by single-cell RNA sequence technology with significant associations with anoikis in TAAD patients. Four vital ARGs  were ultimately identified along with the changes of differentiation trajectory, and major expressions were conformably concentrated on Macro1-3, Mono1-2 and Mono4 subtypes. These findings provide a promising diagnostic biomarker for the accurately diagnosing the disease and would be helpful to further explore the potential pathogenesis with anoikis process for TAAD.Type-A aortic dissection (TAAD) is common life-threatening cardiovascular diseases with high-morbidity and mortality but the concrete etiology of disease remains unclear, which might disturb or delay the early diagnosis for TAAD. Anoikis is a special form of programmed cell-death (PCD) induced by detachment of anchorage-dependent cells from the extracellular matrix (ECM) or neighboring cells, and has been widely applied to identify anoikis-related biomarkers for the prediction and prognosis in oncological fields. However, the specific roles of anoikis-related genes (ARGs) in TAAD remain unclear. In this study, we first identified and validated eight diagnostic ARGs for TAAD based on multiple RNA-sequence datasets, including  Aortic dissection (AD) is one of the most common life-threatening cardiovascular diseases characterized by a tear and bleeding of inner aortic layer and wall. The annual incidence of AD is approximately 3 to 6 cases per 100,000 people, and the incidence has been dramatically increasing in recent years . During AS a special form of programmed cell death (PCD), anoikis is characterized by the detachment of extracellular matrix (ECM) to avoid cells’ abnormal proliferating and growing after attaching to inappropriate matrix, which is essential for apoptosis . CurrentIn this study, based on the datasets of transcriptome sequencing, we firstly identified variant transcript patterns and further screened diagnostic ARGs for the diagnosis of TAAD patients, which were verified in multiple external datasets. In addition, potential targeted regulatory micro-RNAs were predicted to construct the complex regulatory networks for the diagnostic ARGs, and corresponding expression of miRNAs was successfully validated in datasets of non-code RNA-seq. Moreover, we comprehensively evaluated immune-cell landscapes and successfully validated them at cellular aspect via single cell RNA sequence (scRNA-seq) technology. Finally, we investigated the potential relationships of diagnostic ARGs and differentiation of vital cellular subtypes and further identified fatal ARGs in TAAD patients. In summary, our findings generated an in-depth understanding of ARGs diagnostic worth and the exploration of potential correlations between immune microenvironment and ARGs at single-cell levels, to help interpret possible anoikis-related mechanism for TAAD patients.https://www.ncbi.nlm.nih.gov/geo/). These datasets included the sequencing data of aortic tissues samples from 27 TAAD and 24 HC cohorts, including GSE153434 (10 TAAD vs 10 HC), GSE98770 (6 TAAD vs 5 HC), GSE52093 (7 TAAD vs 5 HC) and GSE190635 (4 TAAD vs 4 HC) respectively. All these datasets were transformed into fragments per kilobase million/FPKM values and genes with mean expression<1 were eliminated. Subsequently, the probes were converted into corresponding symbols according to the platform annotation file and the data normalization was further conducted using the “normalizeBetweenArrays” function of Limma package [A total of four transcriptome files were obtained from the Gene Expression Omnibus (GEO) database by searching the “type A aortic dissection” and “RNA sequencing” as the keywords ( package . The GSEhttps://cloud.r-project.org/package=factoextra/) to expound the integral transcript difference for TAAD patients and the DEGs were defined with absolute log(fold-change/FC) >1 and adjusted p-value <0.05. The functional annotations of TAAD were conducted by Gene Ontology (GO) enrichment with ClusterProfiler package [To investigate the difference of transcript patterns between TAAD and healthy cohorts, we performed the multiple comparative analysis including principal component analysis (PCA), differential expressional genes (DEGs) identification and comprehensive functional enrichment analysis. The PCA was performed by the “factoextra” package , and ARGs with the relevance score >1 was comprised in our study. KEGG functional enrichment analysis was applied to assess the function of these differential ARGs. Differential expressional ARGs were further screened via intersecting DEGs with above ARGs. Subsequently, we performed univariate logistical [A total of 338 anoikis-related genes (ARGs) were obtained from the GeneCards database  values . To furtTo further screen potential targeted-regulatory micro-RNA (miRNA) of these diagnostic ARGs, we applied a powerful R package, called multiMiR, which included 8 predicted databases, 3 validated databases and 3 drug/disease-related databases . ThroughSubsequently, we systematically evaluated the comprehensive characteristics of immune microenvironment in TAAD tissues via a single-sample gene set enrichment analysis (ssGSEA) algorithm of GSVA package based on 28 immune cells . The enrMoreover, to validate the immune microenvironment and further explore the potential correlation between immune cells and ARGs, we gained a current single-cell RNA sequencing (scRNA-seq) dataset (GSE213740) to perform related analysis and validation, including 6 TAAD patients and 3HC cohorts. We downloaded the normalized 10X files with expressional matrix, cell barcodes and features in each sample, and then created the seurat object by the Read10X and CreateSeuratObject function of Seurat package . Low-quahttps://github.com/xmc811/Scillus) and functional annotation of each cell in TAAD patients was performed by GSEA using the test_GSEA and plot_GSEA function.To identify cellular clusters, we selected 2000 highly-variable genes to perform the PCA dimensionality reduction and applied the RunUMAP function to conduct Uniform Manifold Approximation and Projection (UMAP) dimension reduction with 30 principal components. Then, the cellular clusters were identified by FindClusters function with resolution as 0.3. Cell clusters were further annotated based on the comparison between cluster’s markers and Zhang et al. annotated markers. The expression of cellular markers was exhibited in bubble diagram by DotPlot function and cellular proportions among groups were further compared to validate the results of ssGSEA. Based on the Wilcoxon rank sum test, DEGs of each cell were also identified between TAAD and HC groups using the “find_diff_genes” function of Scillus package . The continuous variables were exhibited as mean ± standard deviation and the comparison between groups was tested with Wilcox test. The statistical significance was considered with two-tailed adjusted https://www.ncbi.nlm.nih.gov/geo/) ; The scRNA-seq dataset was obtained from the GSE213740 datasets.Publicly available datasets were analyzed in this study. This data can be found here: Gene Expression Omnibus (GEO)  and fibroblasts) and 5 immune cells  . The topThe DEGs of each cell were further identified, and we found interferon-related genes  and immunoglobulin-related genes  were upregulated in macrophages and monocytes of TAAD patients respectively . The GSETo validate the landscape characteristics of T cells in TAAD, we further divided T cells into various cellular subtypes and compared the cellular ratio between TAAD and HC groups. After subjected to dimension reduction, T cell profiles were divided into 6 clusters and annotated as naïve T, CD8+ effector memory T (EMT), CD8+ exhausted T (EXT), CD8+ NKT, CD4+ S100A8+ T and CD8+ IFITM3+ T cells . The vioWe also compared the levels of anoikis scores between TAAD and HC groups in each cellular subtype and indicated the elevation of scores was primarily focused on macrophages and monocytes , 7B. MorNotably, most macrophage and monocyte subtypes of TAAD patients exhibited higher levels of anoikis scores than HC cohorts, especially for macro1-3 subtypes . The pseAs a life-threatening abrupt cardiovascular emergency, TAAD patients always exhibit poor prognosis with high mortality. However, the precise pathogenesis of TAAD remains still unclear, leading to delaying the early diagnosis and intervention of disease, thus identification of novel reliable objective indicators for accurate diagnosis for TAAD is urgently needed. Anoikis is a special form related to apoptosis, characterized by anchorage cells’ detachment from ECM or adjacent cells, resulting in the incompatibility with the tissue’s microenvironment. Recently, studies have indicated the potential regulatory mechanism of anoikis on TAAD through multiple pathways, including ECM’s degradation, hypoxia, oxidative stress and proinflammatory factors. Therefore, we infer anoikis-related genes might be suitable as candidates for the precise diagnosis for TAAD and associated with the abnormal immune microenvironment of TAAD.Through variant transcript analysis, we identified massive DEGs for TAAD patients and these DEGs were significant enriched in the activation of inflammatory signaling and response to stimulus or stress, including TNF, JAK-STAT, IL-17 signalings, Cytokine-cytokine receptor interaction, NF-kappa B signaling pathway, and Fluid shear stress and atherosclerosis. In contrast, cardiomyocyte-related pathways were significantly inhibited in these patients, including Dilated cardiomyopathy, Adrenergic signaling in cardiomyocytes, Vascular smooth muscle contraction, Calcium and Wnt signaling pathway. Wu et al. demonstrated that degradation of ECM was associated with the development of AD and might serve as a potential target for the treatment of AD . MoreoveTo further screen diagnostic ARGs for TAAD, we combined DEGs with ARGs and performed the machine learning of logistical and LASSO regression based on the 51 different expressional ARGs. A total of 8 diagnostic ARGs were ultimately identified with significant diagnostic efficiency for TAAD, including CHEK2, HIF1A, HK2, HMGA1, SERPINA1, PTPN1, SLC2A1 and VEGFA. All these biomarkers were significantly upregulated in TAAD patients and their high-expressions were repetitively validated in multiple external datasets. Interestingly, there are some classical biomarkers reported to be involved in the acknowledged AD-associated mechanism. For example, by activating the HIF-1A pathway, hypoxia can promote the up-regulation of MMP-2/9 and further participate in the formation of AD and VEGFA also participates in the pathogenesis of AD via angiogenesis and remodeling . HMGA1 hNotably, we also screened vital targeted miRNAs to construct the complex regulatory networks for above 8 diagnostic ARGs based on the prediction of databases and validation in the dataset of non-code RNA-seq. The regulatory role of fatal miRNAs was extremely complicated with homodromous or reversed regulations. We found that five homodromous miRNAs  were significantly upregulated while four reversed miRNAs  were downregulated in TAAD patients.Immune cell infiltration has been raising the focus on the field of cardiovascular diseases in recent decades and macrophages have been found recalled and activated inside the aortic walls to be involved in the pathogenesis of TAAD through neoangiogenesis and matrix degradation . In addiMetabolic reprogramming refers to the adaptation of cellular metabolism in response to changes in environmental and physiological conditions, which is essential for maintaining cellular homeostasis in various pathological conditions, such as cancer, obesity, and diabetes . Besidesin vivo and in vitro.However, there are still several limitations in our study. For one thing, although we have identified and validated eight diagnostic ARGs with high diagnostic efficiency, the number of each dataset is still limited and they were obtained from public databases. Hence, their corresponding diagnostic efficiency remains to be further explored through more external researches or clinical practice. For another, multiple immune cells, particularly in macrophages, were found significant association with anoikis scores in TAAD, indicating that macrophages subtypes might be responsible for the difference of anoikis. However, the concrete mechanism of macrophages leading to the development of TAAD by anoikis -related pathways remains to be further investigated and validated by more functional experiments Our study first screens diagnostic ARGs as the novel biomarkers for the precise diagnosis of TAAD based on integrated analysis. A total of eight diagnostic ARGs were successfully identified with high diagnostic efficiency and their functional annotations were applied to expound potential mechanism of anoikis in TAAD. Integrated RNA-seq and scRNA-seq analysis revealed that inherent immunity-related cells, especially for macrophages and monocytes, were considered as primary pathogenic parts for the development of TAAD, with close connection to the anoikis process. These findings provide a promising diagnostic biomarker for the accurately diagnosing the disease and would be helpful to further explore the potential pathogenesis with anoikis process for TAAD.Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6Supplementary Table 7Supplementary Table 8Supplementary Table 9Supplementary Table 10Supplementary Table 11