Beta-lactam antibiotics remain one of the most preferred groups of antibiotics in critical care due to their excellent safety profiles and their activity against a wide spectrum of pathogens. The cornerstone of appropriate therapy with beta-lactams is to achieve an adequate plasmatic concentration of a given antibiotic, which is derived primarily from the minimum inhibitory concentration (MIC) of the specific pathogen. In a critically ill patient, the plasmatic levels of drugs could be affected by many significant changes in the patient’s physiology, such as hypoalbuminemia, endothelial dysfunction with the leakage of intravascular fluid into interstitial space and acute kidney injury. Predicting antibiotic concentration from models based on non-critically ill populations may be misleading. Therapeutic drug monitoring (TDM) has been shown to be effective in achieving adequate concentrations of many drugs, including beta-lactam antibiotics. Reliable methods, such as high-performance liquid chromatography, provide the accurate testing of a wide range of beta-lactam antibiotics. Long turnaround times remain the main drawback limiting their widespread use, although progress has been made recently in the implementation of different novel methods of antibiotic testing. However, whether the TDM approach can effectively improve clinically relevant patient outcomes must be proved in future clinical trials. Antibiotics are one of the most prescribed types of medication in intensive care. In a large European observational study, 64% of patients were exposed to antibiotics during their ICU stay . There aKey components of adequate antibiotic treatment are the selection of the appropriate antibiotic (or combination of antibiotics), timely administration and adequate dosing. Both delays in antibiotic therapy and insufficient dosing could result in increased mortality ,4. DurinSeveral aspects of administering beta-lactam antibiotics should be considered, such as the site of infection, the immune status of the patient, the severity of infection, the level of renal impairment and the use of renal replacement therapy (RRT) or extracorporeal membrane oxygenation (ECMO). Antibiotic dosing adjustments in such patients are particularly challenging. The underdosing of beta-lactam antibiotics is reported in many critically ill patients ,10,11. TOur review describes various aspects of beta-lactam use in critical care, pharmacokinetics/pharmacodynamics (PK/PD), the characteristics of beta-lactams and the main factors of PK/PD variability in critically ill patients. Different modes of application, such as prolonged infusions and continuous infusions, are discussed. Current evidence supporting antibiotic guidance based on therapeutic drug monitoring (TDM) in critically ill patients is analyzed and, finally, recommendations on beta-lactam dosing utilizing target drug monitoring are given.d) characterized by tissue distribution limited to the extracellular space, i.e., plasma and interstitial compartment. This results in limited penetration across biological barriers. Binding to plasma proteins is significant in cephalosporins except for cefotaxime, ceftaroline, ceftolozane and cefepime. Ertapenem, flucloxacillin and oxacillin also display high binding to plasma proteins (>30–50%), while the free fraction of the remaining beta-lactams exceeds 80%. Apart from ceftazidime, ceftriaxone, ceftaroline and ceftolozane, the elimination half-life of beta-lactams is less than 2 h. The PK/PD characteristics of beta-lactam antibiotics are summarized in Table 1. Since most of these antibiotics are excreted primarily via glomerular filtration in kidneys, renal functions are critical factors affecting antimicrobial concentration [Achieving the adequate concentration of any antibiotic at the site of infection and preventing bacterial resistance is crucial for good clinical practice. The knowledge of the pharmacodynamics and pharmacokinetics of any antibiotic is essential for formulating an optimal dosing regimen. Different groups of antibiotics demonstrate various PK/PD properties. The clinical effectiveness of beta-lactams is based on the time that their unbound fraction spent above the minimum inhibitory concentrations (MICs) of the susceptible microorganisms ,15. Thisntration . d, decreased plasma binding capacity due to hypoalbuminemia and altered excretion by the kidneys and, to a lesser extent, by the liver [cr) greater than 130 mL/min, has been described in a considerable proportion of early septic patients [Specific dosing regimens are often derived from the pharmacokinetic studies of a population of healthy volunteers, although the physiology of critically and non-critically ill patients differ. Thus, the standard dosing of antimicrobials may be inadequate, not reaching the PK/PD target, particularly in earlier phases of critical illness . Criticahe liver ,20. The he liver . An impohe liver ,22. ARC,patients .d of any hydrophilic drug including beta-lactams [d [The systemic inflammation and following management of sepsis have several important pathophysiological consequences affecting drug metabolism. First, endothelial dysfunction results in large quantities of intravascular fluid moving into interstitial tissue, increasing the V-lactams . Second,-lactams . Third, -lactams . Fourth,ctams [d ,28. Thesctams [d .In septic shock, microvascular failure may decrease antibiotic concentrations at the infection site . MeasuriThese changes from normal physiology are often superimposed on a chronic condition, further complicating the predictability of drug metabolism and clearance . SimultaHypoalbuminemia is present in 40–50% of critically ill patients and may be associated with an increased free fraction of highly protein-bound beta-lactams ,31. AparThe glomerular filtration rate (GFR), particularly in patients with sepsis/septic shock, can be significantly altered in both directions. A significant proportion of critically ill patients show signs of ARC, but on the other hand, acute kidney injury (AKI) with decreased GFR is one of the most common organ dysfunctions in an ICU setting. This makes antibiotic dosing in critically ill patients difficult . Antibiocr) still represents clinically the most adequate estimation of the true GFR value. Even when based on shorter periods of urine sampling, it reflects GFR variation well [Acute kidney injury (AKI) is one of the most important organ dysfunctions. In critically ill patients, the incidence of AKI ranges from 30 to 60% . Its cliion well .About 5%  of critiThe main mechanisms of drug clearance by RRT are convection, diffusion and adsorption, all of which play a role in beta-lactam clearance due to their small molecule and low protein binding. The dialysate flow rate, blood flow rate, material and surface area of the hemofilter and the ultrafiltration rate and length of the procedure play role in beta-lactam removal . Apart fcr, can lead to suboptimal serum levels of beta-lactams, [cr may overestimate the true value of GFR [On the other hand, ,55 ARC, lactams,  althoughe of GFR .Likewise, in AKI, serum creatinine is an unreliable marker of these changes , so the As a result, the PK of beta-lactams in patients with renal disease, and particularly in those receiving any form of RRT, is highly unpredictable. As shown recently, the dosing regimens of beta-lactams in critically ill patients treated with CRRT are highly variable, reflecting the need for fitting the therapy to the actual needs of the patient . To achiExtracorporeal membrane oxygenation (ECMO) is important organ support provided to critically ill patients with severe respiratory or circulatory failure. Its use has expanded in the past several years especially, due to the COVID-19 pandemic . The sigRoutine antibiotic analysis in clinical laboratories is usually limited to aminoglycosides and vancomycin to prevent their nephro- and oto- toxic effects . These aAn increased range of antibiotics for TDM may be attained by utilizing chromatographic methods, predominantly high-performance liquid chromatography (HPLC) coupled with ultraviolet (UV)  or mass Quite recently, the analysis of ceftazidime and piperacillin via immunoassay on a tabletop analyzer was introduced, ensuring short TAT and allowing a point-of-care (POC) setting. Another feasible approach may be the automation of the HPLC methods coupled with a mass analyzer . The determination of MIC has gained a reputation as the golden standard of antibiotic stewardship over the past decades . When trThe PK/PD objective of beta-lactams is expressed as the percentage of time, during which the free antimicrobial’s fraction in plasma exceeds a certain concentration (% fT > concentration). Hypothetically, the target concentration should be equal to the MIC of the treated pathogen. Considering MIC variability, the use of one single MIC value obtained by MIC determination for achieving 50–100% fT > MIC is rather inappropriate and higher PK/PD targets seem necessary. To prevent antibiotic underdosing regarding MIC variability, an individualized approach was suggested. If MIC falls into a low-level resistance area, indicating that the strain is within the WT distribution range, the upper ECOFF value should be taken as a target. If the MIC is above the upper ECOFF value but still below the clinical resistance breakpoint, the PK/PD target should be set as MIC + two-fold dilution, meaning four times the MIC value for the 100% of the dosing interval (fT ≥ 4 × MIC = 100%). If the MIC value that is clearly above the clinical resistance breakpoint, switching to a different antibiotic is a clear option . Assays using a modified MIC approach were developed to assess serum antibiotic levels. The main advantage comes with lower costs and technological requirements. From their introduction in voriconazole , microbiClostridoides difficile infection (CDI) [Clinically relevant beta-lactam toxicity comprises effects on the central nervous system, hepatotoxicity, myelosuppression, nephrotoxicity and on (CDI) . Over thon (CDI) ,15. The on (CDI) . Neverthon (CDI) . The greon (CDI) . As betaon (CDI) ,81,82. Pon (CDI) ,15,83,84The nephrotoxic effects of beta-lactams remain underrated but still debated. The risk of nephrotoxicity is even higher when combined with certain known nephrotoxic drugs, e.g., vancomycin, especially in patients with premorbid kidney disease or older age ,85. AlthAcute interstitial nephritis is the usual underlying mechanism with non-IgE mediated hypersensitivity reaction and T-lymphocyte involvement ,92. WhenMyelosuppression with severe neutropenia is a rare but potentially fatal complication of beta-lactam exposure, usually resolving after discontinuation of the treatment . Cross-rAs the toxic effects of beta-lactams are directly related to their plasmatic concentration, the upper limit of plasmatic concentration 8 × MIC should not be exceeded ,15. As mentioned earlier, the PK/PD target directly connected to the bactericidal effect of beta-lactams is expressed as the percentage of time during which the free antimicrobial’s fraction in plasma spends above a certain level (% fT > concentration). As the post-antibiotic effect of beta-lactams is variable, the peak plasma concentration has no significant benefits ,32,95 anPseudomonas aeruginosa [For beta-lactams, as typical time-dependent killing antibiotics, optimal PK/PD targets of beta-lactams are achieved by keeping the plasmatic concentration within certain concentration limits without major fluctuations. Based on population pharmacokinetic studies, extended-length  or continuous infusions following a loading dose provide better attainment of PK targets than standard infusions ,99,100. ruginosa ,107. Proruginosa ,108. Theruginosa .The chemical stability of beta-lactam infusions lasting more than several hours has been questioned. This becomes an issue with imipenem (2 h), meropenem and ertapenem (6 h). Other beta-lactams remain stable after reconstitution in a 0.9% NaCl solution for more than 8–12 h, enabling their safe use in form of a continuous infusion with several changes of a new antibiotic solution per day ,110.d and should not be modified according to the degree of organ dysfunction. The administration of a loading dose identical to the dose used in intermittent application seems to be a reasonable approach, see Table 1.When applying a beta-lactam antibiotic in the form of a continuous or prolonged infusion, the application of a loading dose is crucial ,106. TheAntibiotics are one of the most used medications in hospitalized pediatric patients. The reported use in a pediatric setting is prevalent in between 40 to 79% of patients/cases ,114,115 Besides PK changes caused by critical illness or therapeutic processes , developmental and maturational changes  during childhood also significantly affect the PK antibiotics profile . These fStandard β-lactams dosing in PICU settings has been associated with a 95% incidence of underdosing , which iDespite large body of data suggesting the better attainment of PK/PD targets using the monitoring of the plasmatic levels of beta-lactam antibiotics ,136,137,At present, the measurement of plasmatic beta-lactam concentrations should be considered in patients with sepsis, patients with pneumonia and in patients infected with Gram-negative pathogens and with bacteria with decreased susceptibility ,15,142. A pragmatic approach to the target drug management of beta-lactam antibiotics is outlined in Despite significant advances in sepsis treatment and improvements in the care of critically ill patients over the last few decades, sepsis mortality remains well above 20% . Antibio


Antimicrobial stewardship (AMS) is imperative in addressing the menace of antimicrobial resistance (AMR) in health systems. Commonwealth Partnerships for Antimicrobial Stewardship uses a health partnership model to establish AMS in Commonwealth countries. The Hospital of Kwame Nkrumah University of Science and Technology in partnership with Ulster University, Northern Ireland, undertook an AMS project from November 2021 to May 2022. We report on implementation of the AMS, its impact on antibiotic use and infections management at the University Hospital; Kumasi, Ghana.The Global-Point Prevalence Survey (PPS) protocol was used to assess antibiotics use at the hospital at baseline, midpoint and end of the project. Feedback on each PPS was given to the hospital to inform practice, behavioural change and improve antibiotic use.Antibiotic use reduced from 65% at baseline to 59.7% at the end of the project. The rate of healthcare-associated infections also reduced from 17.5% at baseline to 6.5%. Use of antibiotics from the WHO Access group was 40% at baseline but increased to 50% at the endpoint. Watch antibiotics reduced from 60% to 50% from baseline. Culture and susceptibility requests increased from baseline of 111 total requests to 330 requests in the intervention period to inform antimicrobial therapyThe model AMS instituted improved antibiotic use and quality of antimicrobial therapy within the study period. Continuous staff education and training in AMS, and use of standard tools for assessment and application of local data to inform infections management will ensure sustenance and improvement in the gains made. The associated increase in healthcare costs in relation to the menace of AMR also places a great economic burden on the already constrained finances of households in LMICs.Low- and middle-income countries (LMICs) such as Ghana are known to have a high burden of antimicrobial resistance (AMR), which adversely impacts on healthcare delivery and patients’ outcomes.3 Subsequently, the development of Ghana’s first policy and National Action Plan guided by the Global Action Plan of WHO and partners, outlined strategies and interventions required to fight AMR in Ghana.5 The action plan was based on the one health concept, which takes a holistic approach to fight AMR. It advocates for increased surveillance on AMR and antimicrobial use (AMU), infection prevention and optimized AMU across the human, animal and food production, and environmental health sectors.6In such LMICs, interventions aimed at improving antibiotic use need to be cost-effective to achieve a sustainable impact. A situational analysis of the AMR challenge in Ghana led to the identification of many gaps regarding the use of antibiotics in the health system and measures to combat this challenge.7–10 AMS programmes enable health facilities to optimize antibiotic use and improve health outcomes from infectious disease management. Impactful AMS programmes should possess important core elements and structure to be successful and ensure a long-term and sustainable impact. The WHO has developed a practical toolkit based on which institutions in LMICs can model their programmes.11 Additionally, the Commonwealth Partnerships for AMS (CwPAMS), managed by the Commonwealth Pharmacists Association and Tropical Health and Education Trust, uses a health partnership model shown to be effective in establishing AMS programmes in LMICs in Commonwealth countries.12 This model leverages expertise from the National Health Service facilities in the UK to support the implementation of AMS in LMIC Health facilities. CwPAMS, based on this model, has also developed a toolkit to support institutions to set up sustainable cost-effective programmes to improve antibiotic use.13Antimicrobial stewardship (AMS) is known to be a cost-effective means of reducing the progression of AMR especially in resource-limited settings with a high AMR burden.15 Point prevalence surveys (PPS) on antibiotic use is an established standard means of measuring antibiotic use among inpatients. Their relatively low cost of delivery means they are easy to conduct in LMIC health facilities.16 PPSs support AMS programmes to assess their impact on antibiotic use through multiple evaluations as well as identifying targets for improvement in antibiotic use.18Core among AMS activities are prevalence surveillance of antibiotic use.18 On the basis of the health partnership model established and gaps identified in the earlier study, the University Hospital of Kwame Nkrumah University of Science and Technology (KNUST) in collaboration with the School of Pharmacy, Ulster University, Coleraine Campus in Northern Ireland, and other researchers at KNUST implemented and monitored an AMS programme over a period of 7 months. The purpose of this paper is to report on the outcome of the AMS model intervention implemented and its impact on antibiotic use.A previous assessment of antibiotic use through a PPS in hospitals in the Ashanti region including the University Hospital found antibiotic use to be relatively high among inpatients with potential implications for quality of patient care in infectious disease management.The University Hospital is a 135-bed district-level hospital located on the campus of the KNUST, Kumasi. It has a catchment population of more than 300 000. It provides general and specialist services to students of the university, staff and their dependents as well as private patients within the Oforikrom Municipality and greater Kumasi.The hospital as part of this project set up a multidisciplinary committee responsible for undertaking activities in AMS made up of three pharmacists, a nurse , three medical doctors , a hospital administrator and two laboratory scientists. The committee met at least monthly to discuss AMS activities conducted in the hospital. The AMS team that met at least twice a month consisted of two pharmacists, one nurse, two medical doctors and one laboratory scientist.The health partnership was set up between KNUST and Ulster University to implement AMS in the University Hospital. Ulster University provided technical support and expertise in AMS implementation based on experience from the National Health Service in the UK. Lead partners from both institutions met regularly through online meetings before, during and after the implementation of the project that facilitated knowledge exchange and success. There was no AMS in the hospital before this.The AMS intervention run from 1st November 2021 to 31st May, 2022 and targeted staff from all health cadres in the hospital through hybrid training and education seminars to enable staff identify ways in which they could include AMS in their routine activities while creating awareness on AMR, AMS and IPC.Audits of antibiotic use (PPS) were done and feedback was given to prescribers and pharmacists in the hospital through face-to-face meetings and by sharing on their various WhatsApp platforms. A report on AMS activities including audits were also shared with the hospital management as feedback.The AMS team conducted and provided feedback to the hospital staff  on antibiotic use through PPSs after the beginning, midpoint (fourth month after project start) and towards the end of the project (sixth month after project) to determine the impact of the interventions.th November 2021, 22nd March 2022 and 5th May 2022 at the University Hospital. The Global-PPS (G-PPS)  protocol was used to measure antibiotic use at the hospital, with all patients who were already admitted early in the morning (before 8:00 a.m.) on the survey day being surveyed using the data collection forms. The Global-PPS protocol was used to conduct the PPS due to its ease of use and quick feedback that could be promptly used to assess stewardship activities as well as provide information to prescribers especially on prescribing patterns. Data were collected anonymously from both paper-based medical records as well as the Electronic Medical Records system on the wards. No patients or prescriber’s identifiers were collected during survey points. There was no direct interaction with patients. Unless a patient did not meet the inclusion criteria, every patient on the ward at the time of the survey was included in the PPS. All acute care inpatient wards, admitted patients in the ward as at 8:00 a.m. of survey day and antibiotics administered via oral, parenteral, rectal or inhalational routes were included in the study.The PPS was a cross-sectional survey that took place on the 1620 This module collects additional variables in addition to the main protocol variables to survey HAIs. The online data entry platform of the G-PPS analyses all these data to ascertain the HAI rate for each participating hospital.The G-PPS has an optional healthcare-associated infection (HAI) module that enables a hospital to survey HAIs in greater detail.Data collected after each survey were sorted and structured to avoid data entry mix-ups. All data collected were stored on a password-protected and encrypted hospital desktop computer with access restricted to only the AMS team conducting this study. The study's data was put into the Global-PPS programme, an internet-based application for anonymized data entry, validation and reporting. Data were analysed and presented using median, frequencies, percentages and tables.Approval for this project was obtained from the management of the University Hospital, KNUST. Ethical clearance was sought and obtained from the Committee on Human Research, Publications, and Ethics of KNUST. (CHRPE/AP/470/22 of 15 August 2022). Regarding data confidentiality, the electronic databases are kept in a password-protected computer belonging to the principal investigator. No patient identifiers were used in this study.As this was a record review study with no patient identifiers, the issue of informed patient consent did not apply.Requests to access these data should be sent to the corresponding author. Additionally, data collected in this study were recorded using the G-PPS online tool and anonymously accessible on registration.13The intervention comprised of a bundled stewardship programme embedded with infection prevention and control involving an education, audit and feedback (information) strategy for improving antibiotic use at the University Hospital, based on the WHO AMS and Commonwealth Partnerships for Antimicrobial Stewardship (CwPAMs) toolkits. These are practical toolkits on which AMS programme can be modelled for impact and sustainability.The bundled stewardship programme involved the following;Education (capacity building)a. AMS/AMR/optimal antibiotic use training seminarsb. IPC training seminarAudit and feedback (information)c. PPSs to assess antibiotic useAdvocacy on rational prescribingd. Culture and drug susceptibility testinge. Therapy review by pharmacists and AMS teamIn conjunction with the management of the University Hospital, a multidisciplinary AMS committee was formed as a stand-alone committee.Members of this committee constituted the AMS team that implemented various components of AMS and also conducted surveillance (PPS) on antibiotic use at the hospital.The team led by a Family Medicine Consultant and infectious disease specialist and a clinical pharmacist/researcher subsequently carried out daily reviews of antibiotic use on the ward by pharmacists, monthly AMS team meetings and quarterly stewardship activities to achieve the objectives of the project. AMS activities carried out included the following:Monitoring to ensure indications for antibiotic therapy are appropriately documented.Educating clinicians to take samples for culture and susceptibility analyses before empiric antibiotic therapy is initiated.Conduct training seminars to provide counselling and recommendations to other clinicians to improve prescribing behaviours through seminars and ward rounds.In consultation with infectious disease specialist and other consultants in the hospital, selecting antibiotics to place on restricted access (requiring preauthorization before use) to reduce their inappropriate use.A total of 152 patient records were included in the three PPSs conducted. Most of the patients involved in the study were female (63.2%). The median age was 26.5 years  therapy reduced from 70.8% to 64.7%. The HAIs rate also reduced from 17.5% at baseline to 6.5% Table .At baseline and endpoint, third-generation cephalosporins were the most used antibiotics. At midpoint, penicillin with beta-lactamase inhibitors were the most used at 21.4% Table .Table Antibiotic use according to WHO’s AWaRe classification in the University Hospital, KNUST is presented in Figure Culture and drug susceptibility testing (CDST) requests increased from the beginning of the intervention in November 2021 with 111 total requests to 330 requests as at April 2022. The pattern of requests is provided in Figure 21 Over the period of the intervention, the prevalence of antibiotic use initially reduced by 8% but went up by 2.8% at the end, which was still lower than at baseline. This is comparable to a national AMS project in China over a year (2011–2012), which reported a reduction in antibiotic use from 68% to 58% among inpatients.22 Prevalence of antibiotic use in this project was, however, higher at the endpoint than in a project from Nigeria where antibiotic use reduced from 82.5% at baseline, 68.3% at midpoint and 51.1% at endpoint.23 The Nigerian project, however, ran between 2015 and 2018 compared to the current project that implemented AMS over a 7-month period. Continued AMS activities may further reduce the prevalence of antibiotic use in the hospital. The prevalence of antibiotic use in this study was also higher than that from other Ghanaian hospitals (55%) where PPS was done and subsequently efforts have been made to implement AMS.24 Similar studies in Ghana evaluating antibiotic use in hospitals were conducted to assess opportunities for AMS, unlike the current study that assessed the impact of post-AMS deployment on antibiotic use.26 A high baseline prevalence of antibiotic use could be attributed to the COVID-19 pandemic that resulted in increased antibiotic use worldwide among COVID-19 patients.27 The pandemic may have affected prescriber behaviours relating to antibiotic use, although this may not always have been in a negative way due to differing health system characteristics.28 The initial reduction in antibiotic use at the hospital (midpoint) is likely to be a result of the feedback provided to prescribers after each PPS as a means of creating awareness to improve prescribing. Additionally, the novelty of the AMS programme as well as the engagement surrounding it may have driven the initial drop in antibiotic use. The marginal increase between the second and third PPS could be attributed to a possible increase in infectious diseases during that time leading to increased antibiotic use. To ensure sustainability of the AMS activities at the University Hospital, further research is needed to understand the possible mechanisms behind the change in antibiotic use. This may include a qualitative study to assess perceptions and behavioural factors among prescribers relating to antibiotic use. Although this is higher than WHO’s optimum, it is a demonstration of the potential of AMS programmes to improve antibiotic use in hospitals. Antibiotic use overall seemed to have improved during this period of the surveys. The AMS programme at KNUST needs to be sustained to further improve antibiotic use in the long term. Continuous and regular audits of antibiotic use, training of staff and implementation of evidence-based guidelines can help to sustain the programme.The multiple PPSs demonstrated the impact of the AMS programme instituted at the University Hospital. To the best of our knowledge, this is the first report from Ghana on the impact of AMS on antibiotic use among inpatients at a hospital. At baseline, about two-thirds of patients were on antibiotics. This is more than twice the WHO optimum for rational antibiotic use in hospitals (20%–27%).29 Watch antibiotics are at increased risk of selection and development of resistance compared to Access antibiotics. Reducing their use among inpatients can objectively improve antibiotic prescribing. This is a key target for improvement in antibiotic use at the University Hospital as part of the ongoing AMS. This could be addressed by the implementation of AMS policies and guidelines for antibiotic use as well as the development and use of facility-specific antibiotic formularies.The use of WHO’s Access antibiotics increased at the midpoint of the project by 22 points and decreased by 12 points at the endpoint. Watch antibiotic use decreased by 10 points to 50% of all antibiotic prescriptions at the end compared to baseline. Although improved from baseline, there is room for improvement as WHO recommends Access antibiotics to be at least 60% and other categories below 40%.An encouraging finding during the period was the increased number of CDST requests between the project start date and the end point. Requests for CDST more than doubled. Increased reliance on microbiology data from CDSTs are essential to improve antibiotic use and reduce AMR. They are also invaluable to supporting the implementation of AMS. Culture positivity rate as well as turnaround times for requests were not assessed. Such assessments could help to identify quality improvement of microbiological services as well as assess the capacity of the microbiology lab to handle such increased requests.30 At baseline, about a fifth of infections were considered healthcare associated whereas at the endpoint, HAIs reduced by 11 points. Such infections are more difficult to treat requiring improved IPC to prevent and control their spread.31 Training provided during the intervention period on AMR and IPC may have contributed to the reduction in HAIs observed.IV therapy was high but reduced at the endpoint. IV therapy requires the use of invasive devices, a potential source of HAI among inpatients. To optimize their use in the hospital, an IV to oral switch by pharmacists could be included in the bundled programme as it contributes to reduced use of IV therapy and consequent reduction in HAIs.To sustain the impact of this programme, a long-term action plan on AMS was developed as a result of this partnership to ensure continuity. Continuous AMS activities such as those used in this report are planned to ensure this.32 However, there are some limitations to this study; no assessment of patient outcomes was conducted and the period over which these assessments were done was too short to assess the effectiveness of the programme. Antimicrobial consumption assessment was undertaken by using mainly the Global-PPS protocol. Future assessments could include antimicrobial consumption by a defined daily dose in addition to assessing patient outcomes and the AMR situation to allow further improvements to the AMS at the University Hospital, KNUST.33–35 Additionally, only three time points were evaluated at different months of the year that may be confounded by seasonal variations in antibiotic use. This may, however, not be significant as a recent assessment of antibiotic prescribing in the outpatient department of the hospital showed no seasonal variations in antibiotic use.36 Furthermore, the baseline demographic characteristics of patients as well as differing sites of infection, which may be a result of the intervention, precluded further analysis from being carried out.The study has the following strengths. This intervention was implemented based on WHO’s AMS toolkit, which is based on international best practice for implementing AMS. The study was conducted and reported in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines statement.The AMS programme instituted at the University Hospital contributed to significant reduction in antibiotic use among inpatients over the intervention period. However, there was a marginal increase of about 3% in the prevalence of antibiotic use between the midpoint and end of the study evaluation. There was also an improvement in reduction of the rate of HAI as well as the consumption of antibiotics belonging to the Watch group in the WHO’s AWaRe classification at the University Hospital compared to baseline. Continuous staff education and training in AMS, regular use of quality improvement tools such as those from WHO for impact assessment and the application of local data to inform efficient therapy of infections will ensure sustenance in the gains made. This will thus minimize the risk of the emergence and spread of AMR with optimal health outcomes from infectious disease therapy.


NMR (Nuclear Magnetic Resonance) tests and uniaxial compression tests of concrete, sandstone, and sandstone-concrete composite after different numbers of freeze–thaw cycles were carried out to analyze the pore structure development and uniaxial compression mechanical properties of the three types of specimens under different numbers of freeze–thaw cycles. The results show that freeze–thaw cycling promotes the development of pores in sandstone and concrete, and sandstone is more sensitive to low-temperature freeze–thaw than concrete. The UCS  of the sandstone-concrete binary changed in a V-shaped with the increase of the dip angle of the cemented interface, and the angle had no obvious effect on the microscopic pores. The freeze–thaw effect on the deterioration of the microscopic pore structure and mechanical properties of the sandstone-concrete binary has a similar effect pattern, in which the deterioration rate of porosity and compressive strength is faster in the early freeze–thaw period, slower in the middle period, and increases in the later period compared with the middle period, but the increase is smaller than that in the early period of freeze–thaw. In addition, the relationship between the porosity and UCS of the sandstone-concrete binary under the freeze–thaw cycle environment is a quadratic parabola.In China's cold region water conservancy and hydropower projects, the contact interface between the dam and the reservoir bank rock is prone to cracking under external loading and freeze–thaw action, which may lead to dam-bank failure and damage and cause engineering disasters. The  Therefore, the study of freeze–thaw on the microstructure and mechanical behavior of rock-supported concrete can provide a theoretical basis for the safe construction and operation of engineering in cold areas.In recent years, China has accelerated the construction of a strategic system of clean, efficient, safe and sustainable development, especially in the western region, as an important support for the national energy, and has successively developed and constructed a large number of water conservancy and hydropower projects, mining and energy extraction and underground energy storage projects involving large-scale rock engineering. While the geological structure of the western region is complex, and there are many cold areas. Repeated freezing and thawing lead to changes in the physical properties of the rock and soil, thus affecting the stability of the project8. Zhu et al.9 proposed a numerical calculation method based on granular flow expansion and simulated the deformation and rupture evolution of sandstone under the coupled stress-freezing–thawing action using the freeze-thawing deformation law of sandstone obtained from indoor tests. Jiang et al.10 conducted uniaxial compression tests on red sandstone under different freeze–thaw cycles and established the relationship between mechanical parameters and the number of freeze–thaw cycles. Shen et al.12 prepared sandstone-concrete composite material, conducted freeze–thaw cycle and NMR (nuclear magnetic resonance) tests and studied the pore change rule and micro-stripping process of the sandstone-concrete interface. For rock-concrete binary materials, current research has focused on shear mechanical properties and interfacial contact properties, and a few researchers have studied the compressive strength and other properties of rock-concrete composite specimens14. Selcuk et al.15 investigated the strength and damage behavior of sandstone-concrete composite specimens by uniaxial compression tests and the influence of the interface tilt angle on the strength and damage of the composite layer and the influence law of the interface tilt angle on the strength and damage pattern of the composite layer. Huang et al.16 investigated the fatigue damage and energy evolution process of rock-concrete composite specimens under cyclic loading. Vladimir et al.17 analyzed the interfacial shear characteristics of concrete-rock mass, established the functional analytic form of shear deformation and shear strength of rock mass with normal direction and shear load, and deduced the mathematical model which can be used for structural analysis in the preliminary design stage of a concrete dam. Yang et al.18 conducted compression tests of rock-fiber concrete composite structures with the help of 3D-DIC full-field strain technology and analyzed the deformation characteristics, damage characteristics, and failure modes of composite layers. In order to study the damage evolution of shotcrete lining under the action of the freeze–thaw cycle in cold area tunnels, Zhang et al.19 proposed an improved general state weekly dynamics (OSBPD) model by introducing the freeze–thaw damage function, considering the difference of mechanical behavior of shotcrete under tensile and compressive action. In terms of freeze–thaw microstructure, Xue et al.20 investigated the influence of service load and dry and wet freeze–thaw environment on the initial damage of concrete, and found that with the increase of dry and wet freeze–thaw cycles, micro and small holes in concrete evolved into medium and large holes. Liu et al.21 conducted several freeze–thaw cycle tests on sandstone samples and revealed the evolution law of microscopic damage during the freeze–thaw cycle using NMR.At present, scholars at home and abroad have conducted in-depth studies on the mechanical behavior of freeze–thaw rocksNMR and mechanical properties tests were conducted using T2 spectra, porosity, and compressive strength.In summary, there are fewer existing studies on the microscopic pore structure and mechanical behavior of rock-concrete binary materials under freeze–thaw environments, which are prevalent in the engineering community. Studying microscopic pore structure and mechanical properties of rock-concrete binary materials under a freeze–thaw environment has important practical significance for engineering design, safe construction, and operation in cold regions. In view of this, this study quantifies and evaluates the effects of freeze–thaw cycles on the pore structure and mechanical properties of rock-concrete composite specimens by making rock-concrete binary specimens with different inclination cementation interfaces indoors from the viewpoint of the cementation contact surfaces of hydroelectric dams and dams and reservoir banks. Furthermore, freeze–thaw cycles, The sandstone used for the test was selected from Sichuan Province, China, which is grayish-white in color, coarse-grained in structure, with moderate porosity and uniform distribution. The collected sandstone was processed into a standard cylindrical specimen of 50 mm × 100 mm (diameter × height) according to the International Society of Rock Mechanics (ISRM) test protocol. Rock-concrete binary composite specimens were made from C30 grade concrete with sandstone specimens of different inclination angles. The test was designed to produce sandstone, concrete, and sandstone-concrete binary body with 0°, 45°, and 90° dip angles. Five types of specimens and 42 samples were prepared. The specimens with similar ultrasonic wave velocities were then selected by manually eliminating the appearance defects , the interface inclination of 0° is the middle, and the interface inclination of 90° is the minimum (11.49 × 10–3). There are too few test results from specimens with different interfacial inclinations to distinguish whether this law exists in itself or is caused by the dispersion of specimens. Hence, there is no obvious pattern in the effect of cementation interface inclination angle on the change of T2 spectral area, but there is an obvious pattern in the effect of the number of freeze–thaw cycles on the change of T2 spectral area.Tables T2 spectral area of sandstone specimens and sandstone-concrete composite specimens were significantly higher than those of 20–40 and 40–60 freeze–thaw cycles during 0–20 freeze–thaw cycles, while the growth rate of the T2 spectral area of concrete was the smallest for the whole cycle. The growth rate of the T2 spectral area of sandstone-concrete composite specimens slowed down during 20–40 freeze–thaw cycles compared with 0–20 cycles, but the T2 spectral area of 40–60 cycles showed a circular growth trend. The result indicates that concrete specimens have better freezing resistance than sandstone specimens and sandstone-concrete composite specimens, and the low temperature at the beginning of freeze–thaw can cause irreversible damage to sandstone specimens and sandstone-concrete composite specimens. The damage continues to occur as the freeze-thawing continues, and when the number of cycles exceeds a certain threshold, the porosity develops again and the damage increases.The growth rates of the Figure The uniaxial compressive stress–strain curves and the variation of strength and porosity with the number of freeze–thaw cycles of various specimens after freeze–thaw are shown in Figs. UCS  of sandstone-concrete composite specimens in the dry state is between the strength of sandstone and concrete and changes with the change of the dip angle of the cemented interface. The UCS of the sandstone-concrete composite specimens in the dry state or after freeze-thawing in the water-saturated state showed a V-shaped variation pattern at the three angles of 0°, 45°, and 90° of the cementation interface inclination. To analyze the reason, when the specimen containing interface inclination is subjected to vertical load, the interface inclination changes the direction of vertical load transfer, part of which is transformed into normal stress perpendicular to the inclined plane, and the other part is transformed into downward shear force parallel to the inclined plane, at which time, the interface shear force causes the interface to produce slip damage. Therefore, the interface inclination angle has a large deterioration effect on the compressive strength of the sandstone-concrete binary. With the increase in the number of freeze–thaw cycles, the depression of the compressive phase of the stress–strain curve of the specimen is more obvious, indicating that the pores of the specimen are more easily compressed by the freeze–thaw cycles.Figure UCS of the sandstone-concrete composite specimens under the action of freeze–thaw cycles is basically consistent with the variation pattern of NMR porosity, and the increase of the number of freeze–thaw cycles has a deterioration effect on the strength of the specimens. The uniaxial strength decreases linearly with the increase in freeze–thaw cycles. The UCS decreases linearly with the increase of freeze–thaw cycles. The UCS of sand-concrete composite specimens with 0°, 45° and 90° interface inclination angles decreases to 29.72 MPa, 20.86 MPa and 23.14 MPa, respectively, which decreases by 44.98%, 45.39%, and 43.26% compared with the compressive strength after 0 freeze–thaw cycles. During the whole freeze–thaw cycle, the first 20 cycles led to the most obvious deterioration of compressive strength, and the strength loss rate of the composite specimens with 0°, 45°, and 90° bonding interface inclination reached 17.07%, 16.92%, and 14.49%, respectively. The 20–40 freeze–thaw cycles resulted in a slower strength loss rate, and the 40–60 freeze–thaw cycles resulted in an increased strength loss rate in the ring, but the increase was smaller than that of the first 20 freeze–thaw cycles.As shown in Figs. UCS of sandstone-concrete composite specimens with different cementation interface inclination angles and the fitted curves are shown in Fig. UCS of sandstone-concrete composite specimens with different cementation interface inclination angles decreases parabolically with increased porosity. The variation is consistent with the change of mechanical indexes of sandstone and concrete during freeze–thaw cycles.The relationship between microscopic porosity and macroscopic Figure The above analysis indicates that the change of pore structure of sandstone-concrete binary, i.e., microscopic damage, is closely related to the number of freeze–thaw cycles. The concrete has relatively few pores, so saturated free water entering the pores and condensing into ice has little effect on the overall pore structure of the sandstone-concrete binary. Whereas the sandstone itself is rich in micro-fractures and micro-porosity, and its structure is loose, so free water is more likely to infiltrate, making the sandstone gather a large amount of pore water inside, which plays a dominant role in the microstructural damage of the sandstone-concrete binary under freeze–thaw conditions. Concrete, although less strong than sandstone, is prone to cracking when subjected to expansion force, and theoretically, porosity development should be more pronounced. However, the total pore volume of sandstone is larger than that of concrete. The relative content of large pores is high, and the good water stability of concrete is not easy to react with water. In contrast, the free water enters the sandstone interior to soften the sandstone mineral particles, which promotes the development and expansion of pore space and connectivity. Therefore, in the process of a continuous freeze–thaw cycle, the sandstone has a more pronounced accumulated damage than concrete and a higher sensitivity to freeze–thaw temperature difference than concrete. Due to the different sensitivity of sandstone and concrete to freeze–thaw temperature difference, when in the freeze–thaw environment, the expansion force at the cemented interface is of different magnitudes, and to achieve internal equilibrium, the sandstone-concrete material shows uncoordinated deformation. As a result, the freeze–thaw cycle further reduces the bond strength of the cemented interface and forms a structurally weak surface.The mechanical properties of the composite sandstone-concrete specimens under a freeze–thaw environment are developed comprehensively, and a generalized model of the microscopic frost damage mechanism of the sandstone-concrete binary shown in Fig. As can be seen from Fig. T2 spectra of concrete had two spectral peaks, while sandstone and sandstone-concrete binary had three spectral peaks, and the signal intensity amplitude of sandstone-concrete composite specimens was between the two materials of sandstone and concrete. The freeze–thaw cycles did not change the morphology of the T2 spectral curves of various specimens but promoted the pore development of various specimens.The NMR T2 spectral area of all types of specimens tended to increase with more freeze–thaw cycles. After 60 freeze–thaw cycles, the T2 spectral area of sandstone specimens increased by 28.92%, while that of concrete specimens increased by only 8.71%. The results indicate that the rock is more sensitive to temperature freeze–thaw cycles than concrete. In the process of 0–20 freeze–thaw cycles, T2 spectral area growth rate is the largest, 40–60 cycles next, and 20–40 cycles in the process of the smallest, indicating that the initial low temperature of freeze–thaw can cause obvious irreversible damage to the specimens, with freeze–thaw continued damage will also continue to occur, and when the number of cycles exceeds a certain threshold, the specimen porosity development speeds up and the irreversible damage intensifies.The UCS of sandstone-concrete composite specimens is between the strength of sandstone and concrete and is affected by the dip angle of the cementation interface, which varied in a V-shape with the increase of the dip angle. During the freeze–thaw cycles, the change of compressive strength of sandstone-concrete composite specimens with the number of freeze–thaw cycles is basically consistent with the deterioration of microscopic porosity with the number of freeze–thaw cycles. The compressive strength deterioration was most obvious in the first 20 freeze–thaw cycles, and the rate of strength loss slowed down in 40–60 cycles, and the ring increase of strength loss rate in 40–60 cycles was smaller than that in the first 20 cycles.The


IL7) and mal, T cell differentiation protein 2 (MAL2), which conferred resistance to Sorafenib by activating the pro-survival JAK/STAT and PI3K/AKT signaling pathways. Additionally, autophagy inducing stapled peptides exhibited an anti-proliferative effect on both wild-type and drug-resistant HCC cells. When combined with Sorafenib, these peptides demonstrated efficacy against drug-resistant cells. Therefore, targeting autophagy could present a novel approach for overcoming Sorafenib-associated drug resistance in HCC induced by IL7- and MAL2-overexpression.Drug resistance remains a major challenge in the treatment of hepatocellular carcinoma (HCC). Our objective was to investigate the genetic mechanisms involved in the development of Sorafenib-associated drug resistance in HCC cells. To achieve this, transposon insertional mutagenesis was utilized as a forward genetic tool to generate Sorafenib-resistant HCC cell lines and identify potential drug resistant genes. Our studies identified two genes, interleukin 7 (IL7) and mal, T cell differentiation protein 2 (MAL2) were identified as candidate genes that promote survival by activating JAK/STAT and PI3K/AKT signaling pathways. Sorafenib-resistant cells exhibited higher clonogenic survival and lower drug sensitivity due to IL7 and MAL2 upregulation. Higher anti-apoptotic effect, clonogenic survival and increased PI3K/AKT/STAT3 activities were observed in IL7 and MAL2 co-overexpressing cells compared with controls or cells overexpressing IL7 or MAL2 individually. Given the critical role of MAL2 in endocytosis, we propose that MAL2 might facilitate the endocytic trafficking of IL7 and its cognate receptors to the plasma membrane, which leads to upregulated JAK/STAT and PI3K/AKT signaling pathways and Sorafenib resistance. Additionally, our previous studies showed that an autophagy-inducing stapled peptide promoted the endolysosomal degradation of c-MET oncogene and overcame adaptive Sorafenib resistance in c-MET+ HCC cells. In this study, we demonstrate that these stapled peptides readily induced autophagy and inhibited the proliferation of both wild-type and Sorafenib-resistant HCC cells co-overexpressing both IL7 and MAL2. Furthermore, these peptides showed synergistic cytotoxicity with Sorafenib in drug-resistant HCC cells co-overexpressing both IL7 and MAL2. Our studies suggest that targeting autophagy may be a novel strategy to overcome IL7/MAL2-mediated Sorafenib resistance in HCC.Drug resistance poses a great challenge in systemic therapy for hepatocellular carcinoma (HCC). However, the underlying molecular mechanisms associated with resistance to anti-cancer drugs, such as Sorafenib, remain unclear. In this study, we use transposon insertional mutagenesis to generate Sorafenib-resistant HCC cell lines in order to identify potential drug resistant causative genes. Interleukin 7 ( Although human hepatocellular carcinoma (HCC), i.e., liver cancer, is the sixth most commonly diagnosed cancer and the fourth leading cause of cancer death worldwide, there are only a few Food and Drug Administration (FDA)-approved drugs for the treatment of this disease. To ensure better treatment regimens and prognosis of HCC, it is imperative to detect the disease as early as possible. At early stages of the disease, surgical removal of the tumor is the standard therapy for HCC patients with well-preserved liver functions . As a rep < 0.001) and longer median time to progression (TTP) (5.5 vs. 2.8 months). After 1 year, 44% of patients who received Sorafenib were still alive, while 33% of patients who received the placebo were still alive . Mo. Mon = 3and TCGA . Taken t by qPCR A. In add by qPCR B.PB)-based transposon vector system was used as a gene delivery tool to stably overexpress IL7 and/or MAL2 in parental PLC/PRF/5 cells to determine their effect on Sorafenib resistance by two parameters: colony formation ability and anti-apoptotic effect. Overexpression of IL7 and/or MAL2 were confirmed in transfected PLC/PRF/5 cells by qPCR , STAT1  and STAT2   and phosphoinositide-3-kinase regulatory subunit 3  and SFN   and CDKN1A   ; and the 10−171) . Our RNA× 10−97) . Upregul pathway . In corr/5 cells A.IL7 and/or MAL2 PLC/PRF/5 cells by Western blot analyses , to represent the Sorafenib-sensitive type, or overexpressing both IL7 and MAL2 (IL&MAL2) to represent the Sorafenib-resistant type. Treatment by Rapamycin, a known autophagy inducer, failed to trigger an autophagic response in GFP cells as the autophagy markers LC3-II and p62 showed little change A,B. In cWe then treated the GFP and IL7&MAL2 cells with Tat-SP4 and Tat-SP9, two autophagy-inducing stapled peptides developed in our previous studies ,25. Both50 of ~69.71 μM and 69.89 μM respectively (50 of ~12.63 μM and 12.85 μM respectively (To assess whether our autophagy-inducing stapled peptides could overcome Sorafenib resistance, we treated both GFP and IL7&MAL2 cells with Tat-SP4 and Tat-SP9 and measured cell viability using the trypan blue exclusion assay. Our results show Tat-SP4 exerted mild anti-proliferative effect on both GFP and IL7&MAL2 cells with ICectively G. In comectively G.50 values obtained from the Trypan Blue assay (We also used clonogenic survival assay to further assess the anti-proliferative efficacy of our designed peptides in GFP and IL7&MAL2 cells. We decided to focus on Tat-SP9 only given its better potency than Tat-SP4 as measured by the Trypan Blue assay. Our clonogenic data shows that Tat-SP9 significantly reduced the colony numbers of both GFP and IL7&MAL2 cells in a dosage-dependent manner, further corroborating the ICue assay H. Additiue assay I. This dSB insertional mutagenesis was used in vitro to generate Sorafenib-resistant HCC cells. This method of generating drug-resistant cell lines allows for an effective way to generate various resistant cell line models rapidly by high dosage drug selection after SB transposon mutagenesis (IL7 and MAL2 in contributing to PLC/PRF/5Sor-R resistant cells. Clinically, IL7 and MAL2 were found to be consistently dysregulated in both HCC and Sorafenib-resistant HCC patients using TCGA and GEO databases, respectively. In addition, there is significant tendency for co-occurrence between IL7 and MAL2. Compensatory activation of different survival pathways in Sorafenib treatment has been shown to occur as a mechanism for chemoresistance [IL7 and MAL2 in contributing to Sorafenib resistance were validated by overexpressing experiments in parental PLC/PRF/5 cells and observing for colony formation ability and anti-apoptotic effect. Interestingly, both IL7 and MAL2 were required to induce Sorafenib resistance in PLC/PRF/5 cells, while overexpressing either gene alone does not significantly induce Sorafenib resistance (IL7 and/or MAL2 were more anti-apoptotic compared with wild-type or GFP-transfected cells when treated with Sorafenib (In the current study, agenesis . Geneticsistance . The rolsistance C. Cells orafenib D,E. IL7 IL7 and MAL2 overexpressing PLC/PRF/5 cells compared with GFP control PLC/PRF/5 cells (IL7 and MAL2-overexpressing PLC/PRF/5 cells (IL7 and/or MAL2 overexpressing PLC/PRF/5 cells under Sorafenib treatment (IL7 and MAL2-overexpressing PLC/PRF/5 cells compared with GFP control PLC/PRF/5 cells under Sorafenib treatment in a dose dependent manner (IL7 and MAL2-overexpressing PLC/PRF/5 cells. Finally, it is also hypothesized that MAL2, a transmembrane protein, contributes to intracellular trafficking and secretion of IL7 to the plasma membrane for the binding of cytokine receptors in order to trigger downstream signaling pathways that contributes to drug resistance (It is hypothesized that the binding of self-secreted IL7 and its cytokine receptors leads to the activation of JAK/STAT and PI3K/AKT signaling pathways to sustain HCC cellular proliferation and survival during Sorafenib treatment ,15,16. O/5 cells A. Activa/5 cells B. In addreatment C. A prevreatment D. Interet manner D. The casistance E.With IL7 and MAL2 identified as two candidate genes that contribute to Sorafenib resistance in HCC cells, we set out to search for pharmacological interventions that may override such resistance. In our search, we have turned to autophagy-inducing stapled peptides developed in our previous studies based on the following considerations. First, as an evolutionarily conserved process that degrades and recycles cytosolic content in lysosome-dependent manner, autophagy is intricately involved in cancer and acts as a double-edged sword playing both pro- and anti-survival roles with context-dependent manner ,56. SecoIL7/MAL2-overexpressing HCC cells to overcome drug resistance. These results further validate autophagy as a potential target for novel anti-cancer therapies, especially to overcome IL7/MAL2-mediated Sorafenib resistance.Our results demonstrated that two autophagy-inducing stapled peptides, Tat-SP4 and Tat-SP9, readily induced autophagy in both wild-type and Sorafenib-resistant HCC cells and inhibited their proliferation with comparable potency. Additionally, our stapled peptides showed synergy with Sorafenib in IL7/MAL2-mediated drug resistance and enhance the therapeutic effect of existing drugs like Sorafenib in devastating diseases like HCC.Taken together, our in vitro screen identified novel genetic interactions that contribute to Sorafenib resistance in HCC and may play an important role in primary and acquired drug-resistances. Additionally, our autophagy-inducing peptides offer possible pharmacological strategies to overcome such IL7 and MAL2 as candidate drug resistance genes. Their roles were validated in vitro and the resistance mechanism involved the activation of pro-survival JAK/STAT and PI3K/AKT signaling pathways. IL7 and MAL2 co-overexpressing cells also showed higher anti-apoptotic effect, clonogenic survival, and increased PI3K/AKT/STAT3 activities. In addition, autophagy inducing stapled peptides could induce autophagy and inhibited the proliferation of both wild-type and Sorafenib-resistant HCC cells that co-overexpress both IL7 and MAL2. Furthermore, these peptides exhibited synergistic cytotoxicity with Sorafenib in drug-resistant HCC cells co-overexpressing IL7 and MAL2. Therefore, targeting autophagy could serve as a promising novel approach to overcome IL7/MAL2-mediated Sorafenib resistance in HCC.Transposon insertional mutagenesis was used to generate Sorafenib-resistant HCC cells and identified


Freeze–thaw (F-T) is one of the principal perils afflicting concrete pavements. A remedial strategy used during construction encompasses the integration of hybrid fibers into the concrete matrix. An extant research gap persists in elucidating the damage mechanism inherent in hybrid steel fiber (SF)- and basalt fiber (BF)-reinforced concrete subjected to F-T conditions. This paper empirically investigated the durability performance of hybrid fiber-reinforced concrete (HFRC) subjected to F-T cycles. The impact of SF/BF hybridization on mass loss, abrasion resistance, compressive strength, flexural strength, damaged layer thickness, and the relative dynamic modulus of elasticity (RDME) was examined. The damage mechanism was explored using micro-hardness and SEM analysis. The results indicate that incorporating hybrid SF/BF effectively enhances the F-T resistance of concrete and prolongs the service life of concrete pavement. The mechanisms underlying these trends can be traced back to robust bonding at the fiber/matrix interface. Randomly dispersed SFs and BFs contribute to forming a three-dimensional spatial structure within the concrete matrix, suppressing the expansion of internal cracks caused by accumulated hydrostatic pressure during the F-T cycle. This research outcome establishes a theoretical foundation for the application of HFRC to concrete pavements in cold regions. In the frigid region, diurnal temperature variations engender deleterious effects on rigid materials such as concrete. These deleterious consequences are particularly manifest in the context of concrete road pavements, where they manifest as premature cracking, diminished wear resistance, and a concomitant reduction in load-bearing capacity ,2,3. HenThe incorporation of fibers into concrete can effectively mitigate, regulate, and retard the onset, progression, and merging of minute and substantial cracks ,8,9, theAmong fiber hybridizations, the combination of steel fiber (SF) and basalt fiber (BF) stands as an innovative prospect with vast applicability . Guler eIn light of the antecedent literature survey, there exists a dearth of investigations dedicated to the comprehensive evaluation of the durability of hybrid SF/BF-reinforced concrete subsequent to exposure to F-T cycles, especially when juxtaposed with conventional concrete for utilization in rigid pavement applications. Furthermore, the explication of the damage mechanism and the laws governing damage evolution have not been sufficiently elucidated, leading to an insufficient comprehension of the durability and predictive lifespan of HFRC within F-T regions.This study aims to develop a composite fiber-reinforced concrete made of hybrid SF/BF. The intention is to deploy this composite in road engineering projects in frigid climates, endowing it with superior resistance to F-T cycles compared with conventional concrete road surfaces. Specifically, the F-T deterioration patterns in the HFRC were analyzed comprehensively, encompassing both macroscopic and microscopic dimensions, while also engaging in an in-depth exploration of the damage mechanisms inherent to HFRC during the F-T process. Finally, based on the test results, a Grey–Markov model is used to establish a law governing the evolution of damage in HFRC, ultimately facilitating the characterization of its damage degree and prediction of the service life of HFRC materials within the context of F-T cycle environments. The findings of this study hold promise in offering valuable insights into the selection of concrete pavement materials in cold regions, concurrently enriching the variety of ingredients in HFRC.In this study, CEM 42.5 Portland cement, possessing a 28-day compressive strength of 49.8 MPa, was used alongside Grade I fly ash, characterized by an activity index surpassing 95%. The corresponding chemical compositions are shown in This experiment ascertained the mixture proportions using the JTG D40-2011 standard , as presThe internal time–temperature curve of concrete during F-T cycles is depicted in nW is the mass loss rate of the specimen that underwent F-T cycles, nW is the mass of the specimen after F-T cycles (g), and W0 is the initial mass of the specimen (g).The mass loss of the specimen was determined using a precision electronic balance with a precision of 0.1 g. Measurements were conducted at regular intervals of 25 F-T cycles. The mass loss rate was utilized to characterize the specimen’s mass loss; the calculation formula is as follows: nG is the abrasion loss of the specimen that underwent F-T cycles, M0 is the mass of the specimen before abrasion (g), nM is the mass of the specimen after abrasion (g), and A is the specimen abrasion area (m2).According to JTG 3420-2020 , the precR is the compressive strength loss ratio of the specimen that underwent F-T cycles, cR0 is the initial compressive strength of the specimen (MPa), and cnR is the compressive strength of the specimen after F-T cycles (MPa).The compressive strength of the concrete specimens was determined using a hydraulic testing machine. A load application rate of 0.5 MPa/s was steadily and continuously applied until the specimens were destroyed. The mechanical assessment index was acquired with the utilization of the compressive strength loss ratio, as per Equation (3), to facilitate subsequent calculations.fR is the flexural strength loss ratio of the specimen that underwent F-T cycles, fR0 is the initial flexural strength of the specimen (MPa), and fnR is the flexural strength of the specimen after F-T cycles (MPa).The flexural performance of concrete was scrutinized through a four-point bending test, as illustrated in nP is the RDME value of the specimen subject to n times cyclic F-T, nV is the UPV value after n times cyclic F-T (m/s), and V0 is the UPV value without any F-T cycle (m/s).Following the completion of each F-T cycle, an ultrasonic testing device a was useBased on a micro-hardness tester a, the meA scanning electron microscope (SEM) was used to conduct a microscopic analysis of the concrete specimens. Tiny particle samples from the crushed specimens, both before and after F-T cycles, were extracted. A gold sprayer was used to apply a fine gold coating to the sample surfaces to enhance image quality. Subsequently, these samples were placed onto a specimen holder, ensuring electrical connectivity with the holder with the use of conductive tape. Once the sample surfaces dried, the specimens were introduced into the SEM for examination.The apparent morphological transformations of the concrete specimens under varying F-T cycles are depicted in In relation to the extent of surface damage to the concrete, the mass loss rate serves as a quantitative analytical method. The mass loss rate outcomes for each group are presented in 2 to 1.45 kg/m2. As the F-T cycles progressed, Group A witnessed a sharp increase in abrasion loss, whereas the abrasion loss rate for the three fiber-reinforced groups notably remained lower than that of the control group. Finally, after 200 F-T cycles, Group A exhibited an abrasion loss of 4.13 kg/m2 under a 200 N load. In contrast, the wear values for Groups B, C, and D ranged from 2.07 kg/m2 to 2.75 kg/m2, significantly lower than Group A. Notably, Group D displayed the most substantial divergence in abrasion loss when compared to the control group, with a difference of 2.06 kg/m2. This observation underscores that, when abrasion resistance serves as a reference criterion, HFRC exhibits at least twice the F-T resistance of conventional concrete.The abrasion loss of each group of concrete specimens measured after each F-T cycle is presented in Furthermore, as discerned from The compressive strength of specimens as a function of F-T cycles is displayed in Further analyzing the compressive strength durability of HFRC, the correlation between the rate of compressive strength loss and the number of F-T cycles can be discerned b. As depDuring stage (a), when the number of F-T cycles was fewer than 50, the compressive strength of each group concrete increased by approximately 1.12% to 3.76%. This elucidates that, during the early stages of F-T cycles, cement particles in the concrete were still capable of initiating hydration reactions, thereby leading to an elevation in the compactness of the cementitious matrix and an augmentation in compressive strength. In stage (b), under the expansion pressure upon F-T exposure, internal defects within each group of specimens gradually enlarged, leading to a decrease in the compactness of concrete, and consequently, reducing compressive strength. In stage (c), the rate of compressive strength loss for all groups was significantly higher than in the previous two stages. Group A exhibited an earlier failure compared with the other three groups with fiber incorporated, reaching the specified failure threshold after 175 F-T cycles. Additionally, after 200 cycles, the compressive strength loss rates for the fiber-incorporated Groups B, C, and D ranged from 9.48% to 16.59%. Among them, Group D exhibited the lowest compressive strength loss rate.Apart from compressive strength, the flexural strength of concrete is typically designated as a crucial mechanical parameter for pavement design. The curve depicting the flexural strength of concrete in relation to the number of F-T cycles obtained from a four-point bending test is illustrated in The flexural strength loss rates of specimens from Group A to Group D are depicted in fR values for Groups B to D ranged between 19.38% and 24.18%. Notably, Group D displayed the lowest flexural strength loss rate, indicating that the interwoven SF/BF within the concrete forms a “chaotic supportive system,” effectively impeding crack propagation induced by F-T damage and enhancing the concrete’s toughness [In stage (a), the increase in flexural strength loss rate is gradual. Group A’s Rf was increased by 6.11% after 50 F-T cycles, while the magnitude of flexural strength loss for specimens from Group B to Group D ranged between 2.03% and 4.61%. In stage (b), the flexural strength loss rate for Group A specimens experienced a rapid escalation, surpassing the failure threshold of 25% at 175 F-T cycles (29.17%). In contrast, although the flexural strength loss rates from Group B to Group D exhibited an increase compared with that of stage (a), the extent of damage remained lower than that of Group A. After 200 F-T cycles, the Based on the aforementioned observations, it becomes evident that repeated F-T cycles expedite the degradation of the internal structure of concrete. Therefore, the incorporation of hybrid SF/BF into concrete serves to enhance its internal structural integrity, making it a promising solution for widespread use in cold-region concrete pavement applications.From Therefore, the micro-hardness value measured in this study for HFRC significantly surpasses the other specimens, thereby implying the superior bonding achieved with the incorporation of hybrid fibers and the consequential enhancement in F-T durability, as evidenced by mass loss, compressive strength, and RDME.In this study, the micro-structure images of Group A and Group D specimens, after 0 and 200 F-T cycles, were assessed with SEM analysis. The interior structure images obtained using the SEM analysis of Group A and Group D with hybrid SF/BF-reinforced specimens are shown in Based on the aforementioned analysis, the essence of the damage and deterioration of concrete pavement under freeze–thaw action lies in the evolutionary process of microcrack initiation, propagation, penetration, and ultimately fracture. The incorporation of hybrid SF/BF can enhance the flexural and fracture toughness of concrete, mitigating the rate of crack propagation.Drawing upon the theoretical framework presented by Jabeen et al. , this seX(0)(t) = {X(0)(1), X(0)(2), …, X(0)(n)} represents the irregularly distributed raw data sequence, the application of an Accumulated Generation Operation (AGO) yields X(1)(t) as follows:Assuming a represents the growth coefficient, b denotes the grey input coefficient, and Z(1)(t) pertains to the background value concerning X(1)(t).The GM model can be expressed by the first-order differential equation as shown in Equation (7).Z(1)(t) can be calculated using the following equation:Substituting Equation (8) into Equation (7) and using the Laplace inverse transform yields the general solution:Ultimately, by using Equation (9) for cumulative reduction and restoration, the GM prediction values can be obtained.Due to the influence of various stochastic factors on the durability degradation trend in concrete pavements in F-T environments, experimental data exhibit significant randomness. This randomness, in turn, affects the predictive accuracy of GM, leading to less than ideal results. Therefore, to enhance predictive precision, a Markov chain with GM was amalgamated.ε may have negative values, after applying the absolute value transformation, the residual ε(0)(t) was obtained.For the original data sequence, fitting values can be obtained using GM. Since the residual original values ε(0)(t) yields the residual prediction model and residual prediction values.Furthermore, applying the steps delineated in Equations (6)–(9) to ε, we can establish a Markov transition matrix to define their states: when the residual original value ε is positive, it corresponds to state 1, and when it is negative, it corresponds to state 2. Based on the polarity of the states, we can determine the state transition probabilities.Regarding systematic information within the residual original values The state transition probabilities lead to the derivation of the state probability transition matrix.The remaining computational steps, following the guidelines outlined in Ref. , ultimatThe computational flow of the Grey–Markov model described above is summarized in R2) lie within the range of 0.9791 to 0.9942, thereby attesting to a commendable concurrence between the projected values and experimental outcomes. Hence, it can be inferred that the utilization of the Grey–Markov model contributes to the anticipation and assessment of the detrimental ramifications inflicted upon HFRC pavement in F-T environments. The corresponding predictive curve is depicted in According to the results in In (1)F-T cycling exerts detrimental effects on the durability characteristics of concrete. Compressive strength, flexural strength, abrasion resistance, and RDME typically exhibit a declining trend with increasing F-T cycles, while the mass loss and damage layer thickness increase with the augmentation of F-T cycles. A copious assembly of randomly dispersed hybrid SFs/BFs within the concrete matrix engenders a three-dimensional constraining framework, thereby efficaciously enhancing the F-T durability of the concrete.(2)The SEM analysis reveals that the fibers dissipate the energy required for crack propagation by means of friction with the cementitious matrix, as well as the pull-out energy and fracture energy of the fibers, thereby serving to toughen and impede crack propagation, consequently enhancing F-T resistance. The microhardness test results indicate that the ITZ strength is lowest in the control group, whereas in the HFRC, the impact of F-T cycles on the ITZ is relatively minimal due to the robust bonding between fibers and the surrounding matrix.(3)A Grey–Markov model, built upon the results obtained from the RDME test, is formulated to predict the service life of each group of specimens. The hybrid method affects the concrete’s service life. Under F-T cycles, the predicted life of each group in the sequence is Group D > Group B > Group C > Group A.This study examined the influence of hybrid SF/BF on the durability performance of concrete pavement under varying F-T cycles. The significant achievements of this paper can be summarized as follows:This study conducted initial endeavors in the application of hybrid steel fibers and basalt fibers in concrete pavement. Prior to on-site implementation, further concrete mix experiments, primarily encompassing the design optimization of fiber volume fractions, should be undertaken. Considering aspects such as mechanical performance, crack width control, economic viability, and environmental conditions, the ideal blend involves steel fiber volume fractions below 2% and basalt fiber fractions below 0.2%. Additionally, the potential mechanisms by which the hybrid fibers control cracks warrant further investigation.


Epithelial tissue is at the forefront of innate immunity, playing a crucial role in the recognition and elimination of pathogens. Met is a receptor tyrosine kinase that is necessary for epithelial cell survival, proliferation, and regeneration. Here, we showed that Met is essential for the induction of cytokine production by cytosolic nonself double-stranded RNA through retinoic acid–inducible gene-I-like receptors (RLRs) in epithelial cells. Surprisingly, the tyrosine kinase activity of Met was dispensable for promoting cytokine production. Rather, the intracellular carboxy terminus of Met interacted with mitochondrial antiviral-signaling protein (MAVS) in RLR-mediated signaling to directly promote MAVS signalosome formation. These studies revealed a kinase activity–independent function of Met in the promotion of antiviral innate immune responses, defining dual roles of Met in both regeneration and immune responses in the epithelium. Emerging viral infections continue to pose a major threat to global public health. The intracellular viral RNAs are detected by host cellular pattern recognition receptors, a retinoic acid–inducible gene I (RIG-I), melanoma differentiation–associated gene 5 (MDA5) , and TLR33The Met receptor is a transmembrane tyrosine kinase receptor expressed predominantly in epithelial cells and neurons and plays a pivotal role in tissue regeneration after injury . Upon heMet RNAi knockdown carcinoma cells and normal human epithelial cells displayed an unexpected loss in their ability to secrete TNF-α, IL-6, and IFN-β, specifically in response to short and long intracellular poly(I:C) stimulations that mimic intracellular virus dsRNA (double-stranded RNA)  Fig. 1  Fig. 1B.Intracellular short poly(I:C) could be recognized by RIG-I and TLR3 , 2, 8. TUpper) restored poly(I:C)-induced TNF-α production to levels comparable to that of wild-type (WT) Met and parental cells (Lower). As the intracellular domain (ICD) of Met is known to be targeted to the mitochondria , recognized mainly by MDA5 , Met is 10SI Appendix.The concentrations of cytokines were measured using ELISA MAX Standard kits for human TNF-α (BioLegend), OptEIA ELISA kit for human IL-6 (BD Pharmingen), and DuoSet ELISA for human IFN-β (R&D systems) according to the manufacturer’s protocol. Details are provided in Appendix 01 (PDF)Click here for additional data file.


The service was developed, standardized, piloted and agreed. The AMPT visited medical, surgical and older people service wards daily and screened prescribing charts for the following: (i) documented allergy status, (ii) documented antibiotic stop/review dates, (iii) completion of ‘protected’ antimicrobial order forms, and (iv) management of antibiotic cation interactions. AS issues identified were shared prospectively with the ward team and data displayed on run charts to measure improvement.Following introduction of the AMPT service documentation of allergy status and oral antibiotic stop dates improved from baseline in all wards. IV antibiotic stop/review documentation increased in all downstream medical and surgical wards with the greatest improvement achieved in the respiratory ward, increasing from a 0% baseline to a median of 37% after service introduction. Awareness and management of antibiotic cation interactions increased from a 41% baseline to a median of 86% and total antibiotic doses compromised by this interaction reduced from 66% at baseline to 21% post change. Completion of ‘protected’ antimicrobial forms improved from a baseline median of 53% to a median of 85% after service introduction.Introduction of an AMPT service resulted in improved documentation of allergy status, oral and IV antibiotic stop/review dates, recognition and management of antibiotic cation interactions and compliance with the local ‘protected’ antimicrobial policy. These results demonstrate that introduction of an AMPT to the ward team can improve AS, patient care and safety and reduce the risk of AMR.


Culture and susceptibility results are essential to identify the offending organism and optimize antibiotic treatment; hence, decrease the emergence of antibiotic resistance and achieve better clinical outcomes. Antibiotic prescribers should learn how to interpret MIC breakpoints to prescribe a suitable antibiotic as different antibiotics have different MIC breakpoints against different microorganisms. No study to date has evaluated the impact of MIC suppression in culture reports while keeping interpretation results on the appropriateness of antibiotics choice by non-infectious diseases prescribers. We assessed the impact of MIC suppression on the appropriateness of antibiotic prescriptions.This was a quasi-experimental study (pre-post intervention) that was conducted over 14 months on hospitalized patients in the medical floor who had a symptomatic infection with a positive culture and were prescribed a full course of definitive antibiotics by prescribers not specialized in infectious diseases pre and post MIC suppression in culture reports. The primary outcome was appropriateness of antibiotic selection . Secondary outcomes were clinical cure , length of stay (LOS), and antibiotics expenditure as defined daily dose (DDD) per 1,000 patient days and days of therapy (DOT) per 1,000 patient days.P=0.028). LOS was shorter in the post-intervention phase compared to the pre-intervention phase . No difference was reported in the other outcomes.100 patients were included, 50 in each phase. No difference was observed in the baseline characteristics, including age, sex, infection site, isolated organism, and prescribers’ departments. Appropriate antibiotic selection was significantly higher in the post-intervention phase compared to the pre-intervention phase (62% vs. 40%; Our study showed that suppressing MIC values from cultures reports was helpful in choosing the appropriate antibiotic and minimizing the LOS. Future research is needed to confirm these findings.All Authors: No reported disclosures


Breast carcinoma (BRCA) is one of the most common, fatal, and aggressive cancers, with increasing morbidity that has a major impact on human health. PIK3CD appears to have important roles in the beginning and advancement of various forms of human cancer, according to mounting data. However,the particular role and mechanism of PIK3CD in BRCA remains not fully identified.https://portal.gdc.cancer.gov/), Genotype-Tissue Expression (GTEx) data and the UCSC Xena browser (https://xenabrowser.net) data were used in this study’s initial pan-cancer analysis of PIK3CD expression and prognosis. Circular RNAs (circRNAs) that regulated the expression of PIK3CD were subsequently found using a combination of in silico investigations of expression, correlation, and survival. Measurements of PIK3CD expression and an analysis of the in vitro function of PIK3CD in BRCA cells were performed using real-time RT-PCR, Western blotting and Transwell assays.The Cancer Genome Atlas (TCGA, In BRCA GLI2, RAB32, LAMB1, MGAT2, ITGA8, CHF, COL6A3 and PRRX1-miR-30b-5p axis was identified as the most likely upstream CircRNA-related route of PIK3CD. PIK3CD was correlated with the expression of EMT markers. The PIK3CD cDNA improved the capacity for invasion and migration. The expression of PIK3CD was linked to some of the m1A/m5C/m6A regulators. Additionally, it was discovered that the expression of PIK3CD was found to be highly connected to the expression of immunological checkpoints, immune cell biomarkers, and tumor immune cell invasion.Our findings reveal that PIK3CD expression is associated with prognosis, EMT, and tumor immune infiltration in BRCA patients. The most frequent primary cancer and the second greatest cause of deaths due to cancer in women in the US is breast cancer (BRCA).Breast cancer is still difficult to prevent globally because it is a multi-step procedure that involves many types of cells. One of the best ways to avoid breast cancer is through early diagnosis . SeveralThe phosphoinositide 3-kinase (PI3K) is one of the most prevalent regulatory mechanisms in a range of human malignancies. Based on their substrate specificity and mammalian structural characteristics, PI3K is split into three types. The PI3K subfamily of class I appears to be the one most associated with human malignancies. According to their adaptors, Class I PI3K is further separated into subclasses IA and IB. Class IA catalytic isoforms p110α, p110β and p110δ are encoded three distinct genes termed PIK3CA, PIK3CB and PIK3CD. The functions and fundamental mechanisms involving PIK3CD in cancer, however, are poorly understood .The epithelial–mesenchymal transition (EMT) can promote metastasis in a variety of cancer types. EMT was linked to the exclusion of immune cells important in the immune reaction to cancer as well as the expression of several immunosuppressive cytokines. There may be a link between EMT and tumor immune infiltration in cancer .In our study, we first examined the expression and survival of PIK3CD in diverse human cancers. Following that, the CircRNA-related regulation of PIK3CD in BRCA was investigated. Subsequently, we examined the connection between PIK3CD expression and influx of immunological cells, biological markers, or immunological checkpoints in BRCA. These results suggest that PIK3CD is associated with tumor immune infiltration and prognosis in BRCA individuals.https://genome-cancer.ucsc.edu/), Genotype-Tissue Expression (GTEx) data and the UCSC Xena browser (https://xenabrowser.net) and then differential expression analysis for PIK3CD was conducted using the R package limma after normalization. Statistical significance was determined by a p value of < 0.05.The mRNA expression data of 24 tumor types  were retrieved from the TCGA database (http://gepia.cancer-pku.cn/) is a digital platform based on TCGA and Genotype-Tissue Expression (GTEx) data for cancer and normal gene expression profiling and interactive analytics. PIK3CD and circRNA expression in various types of human cancer were determined using GEPIA. The p value of < 0.05 was considered statistically significant. GEPIA was utilized to conduct PIK3CD survival study in different cancer types, including OS and RFS. GEPIA was also used to analyze the predictive relevance of putative BRCA circRNAs. The logrank p value of < 0.05 was used to evaluate statistical significance. Furthermore, the expression connection of PIK3CD with immunological checkpoints in BRCA was evaluated using the GEPIA database. The selection criteria for identifying it as statistically significant were R > 0.1 and p value < 0.05.GEPIA , an online resource able to evaluate gene or miRNA influence on survival in over 20 cancer types, including BRCA, was utilized to perform survival analysis for miRNA in BRCA. Log rank p value of < 0.05 was regarded as statistically significant.As initially noted, the Kaplan–Meier plotter (http://starbase.sysu.edu.cn/). In BRCA, starBase was used to conduct an expression analysis of correlation for miRNA-PIK3CD, circRNA-miRNA, or circRNA-PIK3CD. StarBase was also used to examine miRNA-30b-5p expression in BRCA and normal controls. Furthermore, starBase was employed to anticipate candidate circRNAs that might bind to miRNA-30b-5p.StarBase is a database for miRNA research -1640 medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen) and antibiotics (100 g/mL streptomycin and 100 U/mL penicillin) at 37 °C and 5% CO2 in a humidified tissue culture chamber. Cells from the period of logarithmic growth were employed in further tests.The NC (negative control) mRNA precursor sequences and the PIK3CD sequences (cDNA) were amplified and inserted into the pLV3 vector, both from GeneChem in Shanghai, China. For virus production, HEK293T cells were transfected with the packaging vectors pGag/Pol, pRev and pVSV-G in combination with the recombinant lentiviral plasmids. After 48 h of transfection, the lentivirus-containing medium was recovered and filtered. The viral supernatant containing polybrene was then introduced to the breast cancer MDA-MB-231 cells and MCF-7 cells to cause infection. The transduced cells were then chosen for 14 days of puromycin to identify those that were consistently producing the constructs. It was determined how much PIK3CD was present in these stable cells using quantitative real-time PCR [−ΔΔCt method was used to determine gene expression differences.Total RNA was isolated from cultivated cells using the Trizol reagent (Invitrogen) according to the instructions supplied by the manufacturer. SYBR®Green (Thermo Fisher Scientific) was used in quantitative RT-PCR to measure the mRNA levels of the listed genes.Internal standards for mRNA and miRNA were GAPDH and U6, respectively. Sequences for the PCR primers were as followed: PIK3CD (FW:5′-CATATGTGCTGGGCATTGGC-3′ RV:5′-TTTCACAGTAGCCCCGGAA C-3′), GAPDH (FW:5′-GAGTCAACGGATTTGGTCGT-3′ RV: 5′-GACAAGCTTCCC GTTCTCAG-3′). U6 . Dissociation curve analysis was used to confirm particular product amplification. The 2The migration experiment was conducted by seeding cells in the upper chambers without matrigel using inserts . In invasion assay (BD Bioscience), the inserts were completely covered with matrigel. Cells were resuspended in FBS-free culture and introduced to the upper Transwell chamber, including Matrigel, while the lower chamber received 800 µl of culture medium with 10% FBS.After the cells were incubated at 37 ℃ for 24 h, the inserts were fixed with methanol and stained with crystal violet at room temperature for 20 min. The cells were taken away from the upper side of the filter membrane, and the invading cells were stained for 10–15 min at room temperature with a 0.1% crystal violet solution. An inverted microscope was used to take the pictures, and ten random fields' worth of cells were counted. Displayed were the statistical analyses and illustrative pictures. Each group’s experiments were carried out in triplicate.Cell lysates were produced using a RIPA lysis solution containing a protease inhibitor. The proteins’ concentration was established using a BCA kit from Beijing, China’s Beyotime Biotechnology, before they were separated on polyacrylamide gels (PAGE) with 10% SDS and transferred onto polyvinylidene difluoride (PVDF) membranes. PIK3CD , E-cadherin , vimentin , SNAI1 and GAPDH  were then incubated with the hydrogels at 4 ℃. The membranes were incubated for 45 min with a second antibody  in combination with horseradish peroxidase following three TBST washes. We imagined and measured the protein bands.https://cistrome.shinyapps.io/timer/) is a web server for extensive research of tumor-infiltrating immune cells. TIMER was employed to look into the link between PIK3CD expression and infiltration of immune cells or immune-related checkpoint expression in BRCA. A p value of 0.05 was regarded as statistically significant.TIMER  hypothesis, miRNA regulates target gene expression and there should be an adverse correlation between miRNA and PIK3CD.As a result, the expression correlation analysis was carried out. In BRCA, PIK3CD was significantly negatively linked with hsa-miR-30b-5p|R|= 0.169) and favorably correlated with hsa-miR-30e-5p and hsa-miR-7-5p, as shown in Table 69 and faCircular RNAs (circRNAs) have been linked to the progression of cancer . We usedThe starBase database was then used to analyze the amounts of expression of these circRNAs in BRCA. Only CD99 was not significantly regulated in BRCA as compared to normal controls, as seen in Fig. Taking expression, survival, and correlation studies into account, CHF, GLI2, RAB32, LAMB1, MGAT2, ITGA8, COL6A3 and PRRX1 could be the eight most likely upstream circRNAs of the miR-30b-5p/PIK3CD axis in BRCA.The link between PIK3CD and EMT variables was then investigated. In the majority of the 5 cancer types, PIK3CD expression was linked favorably with the expression of mesenchymal markers such as VIM (Vimentin), TWIST1, SNAI1 (SNAIL), SNAI2 (SLUG), and CDH2 Fig. A. FurtheWe studied the impact of PIK3CD on cell migration, invasion, and EMT of BRCA cells after verifying its expression and prognostic value for BRCA. MCF-7 BRCA cell lines were transfected with PIK3CD cDNA. To evaluate the effect of PIK3CD on cell migration and invasion, Transwell Assay was utilized. BRCA cells that received PIK3CD cDNA displayed higher migration and invasion abilities Fig. B–D. AfteThese findings demonstrated that PIK3CD cDNA increased cell EMT, migration, and invasion, indicating that PIK3CD plays an active role in BRCA cell activities..The TIMER database was used to demonstrate the link between PIK3CD expression and immune cell infiltration levels in BRCA cells. The amount of immune cell infiltration significantly changed with different PIK3CD copy counts in BRCA, as illustrated in Fig. Clinical treatment sensitivity and disease diagnosis are significantly impacted by the microenvironment, which includes immune cells, extracellular matrix, inflammatory chemicals, and other growth factors. Using the CIBERSORT technique, the fraction of 22 immune cells was estimated using a histogram in this study. The results showed that the tumor group had considerably larger proportions of most immune cells than the control groups, with the exception of some immune cells such as CD56 dim natural killer cells in BRCA,LUAD, and STAD Fig. C.The GEPIA database was used to examine the expression connection of PIK3CD with immune cell indicators in BRCA, able to better investigate the function of PIK3CD in tumor immunity. According to the findings, PIK3CD expression was strongly positively linked with immunity-associated gene marker expression, such as biomarkers of B cells including CD19 and CD79A, and biomarkers of dendritic cells in BRCA  and Genotype-Tissue Expression (GTEx) to do a pan-cancer analysis of PIK3CD expression.PIK3CD was significantly downregulated in 10 tumor types, including BRCA, COAD, LIHC, LUAD and LUSC  were identified. Eight of the most likely elevated circRNAs, including CFH, GLI2, RAB32, LAMB1, MGAT2, ITGA8, COL6A3 and PRRX1, were discovered using expression, survival, and correlation analysis.These circRNAs, including COL6A3 , PRRX1 , CFH 1212, GLI2 PIK3CD expression correlated favorably with the expression of mesenchymal markers and was strongly inversely related to the epithelial marker. The correlation heatmaps revealed a strong relationship between PIK3CD and the EMT variables. PIK3CD cDNA increased cell EMT, migration, and invasion, indicating that PIK3CD played an active role in BRCA cell activities Fig. . These fTo our interest, PIK3CD enhanced the migration, invasion, and EMT of BRCA cells,but PIK3CD might be used as a favorable prognostic biomarker in BRCA patients. PIK3CD was shown to be related to the expression of SNAI1. Luminal A tumors with high SNAI1 expression had a higher OS marker, while luminal B, Her2-positive, and basal-like malignancies had a poorer OS marker . As a reSeveral studies have shown that immune cell infiltration in tumors can affect the efficacy of chemotherapy , radiothThe effectiveness of immunotherapy is also influenced by the expression of immunological checkpoints and the number of immune cells entering the tumor microenvironment . As a reWe discovered that PIK3CD expression had a positive relationship with the abundance of 28 different types of tumor-infiltrating lymphocytes and that PIK3CD methylation was strongly inversely linked with tumor-infiltrating lymphocyte abundance in the TISIDB database. We also discovered links between PIK3CD methylation and clinicopathological characteristics in BRCA patients Fig. . ReducedRNA modifications such as m6A, m5C and m1A have been shown in recent years to influence the immune response and cancer . CorrelaIn the present study, we systematically investigated PIK3CD's function in BRCA. We identified that PIK3CD had the potential to operate as a detection and prognosis biomarker for BRCA by examining its underlying mechanism. We found an upstream regulatory mechanism for PIK3CD in BRCA, which included GLI2, RAB32, LAMB1, MGAT2, ITGA8, CHF, COL6A3 and PRRX1/miR-30b-5p/PIK3CD. Furthermore, our current findings suggest that PIK3CD may have an important function by boosting EMT, immune checkpoint expression and tumor immune cell infiltration. However, these results require confirmation in the future through much more fundamental research and substantial clinical trials. To date, there have been relatively few studies on PIK3CD in BRCA. Our comprehensive examination of PIK3CD and its conceivable mechanisms may serve as the basis for further investigation.