Application of rubberised concrete in earthquake prone areas is of significant importance. Although investigations have been conducted to research on the ductility of rubberised concrete, the behaviour of rubberised concrete with Burnt Clay Brick Powder (BCBP) is not well understood. This paper captures the ductility behaviour of rubberised concrete containing BCBP. In this study, 3 beams were investigated in flexure while the other 3 beams were made to fail in shear and flexure. For the beams that failed in flexure, ductility of concrete beams containing 5% BCBP and 10% Waste Tire Rubber (WTR) improved by 23.47% compared to control beam. This increase in ductility was evidenced with only 15.31% reduction in flexural load. Moreover, the beam containing 5% BCBP and 10% WTR failing in shear and flexure exhibited 14.59% ductility improvement with 16.33% load reduction in comparison to the control beam. Eventually, the study concluded that it is possible to achieve improved ductility without substantial loss in ultimate failure load by using 5% BCBP and 10% WTR. Such properties demonstrated that this rubberised concrete with 5% BCBP can be used in seismic applications. Burnt clay brick powder, Ductility, Rubberised concrete, Shear, Flexure, Seismic application. For instance, nearly 1000 million tires conclude their life each year of which more than 50% are disposed without any treatment , 3. The Displacement ductility is measured using ductility index from load-deflection graphs. This index is the ratio of deflection at ultimate load to deflection at yield point of steel . Other sThe use of pozzolanic materials in concrete production is becoming an essential approach towards mitigating environmental concerns due to cement production , 17. StoThis research investigated the influence of WTR on flexure, shear, ductility and cracking behaviour of reinforced concrete beams especially with addition of BCBP. The construction industry should consider the improvement of ductility properties of reinforced beams while achieving sustainability in construction. The inclusion of WTR and BCBP could present potential enhancement of ductility performance of concrete beams apart from reducing environmental pollution. This sustainable construction is also an economically sound construction method.22.12 + Fe2O3 + Al2O3) of greater than 70% in BCBP conformed to the requirements in the code [Ordinary Portland Cement (OPC) class 42.5 meeting specifications in the standard , was usethe code , as Clas2.2Class 30 concrete mix in accordance to British Research Environment (BRE) was selected for the beams. The mix design procedure has been presented in another study . Before 2.32.3.1The flexural, shear and ductility properties of rubberised concrete containing BCBP were assessed by testing 9 beams of 150 × 200 × 2000 mm dimensions in accordance with the code . The desTo facilitate strain gauge fixing, the tension bars at mid-span were smoothened using a bench grinder. The strain gauges were carefully embedded on steel bars using glue followed by covering with water proof material for protection. Steel reinforcement bars were transferred in the timber moulds. While keeping the moulds horizontally, the casting of the samples into the moulds was performed. Correspondingly, several cubes were cast to establish the compressive strength properties of concrete. This was followed by compaction which used a poker vibrator. After 24 h, the specimens were de-moulded without damage to either concrete or the moulds. They were then moist-cured by covering using wet gunny bags which ensured uniform curing. The samples were kept inside the laboratory having humidified air and room temperature for 28 days.2.3.2The specimen for flexural testing was mounted on rollers which provided supports as schematically illustrated in cf is the flexural strength in megapascal (MPa), F is the maximum load in Newton (N), b is the thickness of the specimen in mm and d is the depth of the specimen in mm.According to 3Several aspects relating to beam failures such as flexural, shear, ductility and cracking behaviour are discussed in two parts. The first part Section  focuses 3.13.1.1The findings of compressive strength were used to verify the characteristic strength of concrete used during casting beams. It is clear from 3.1.2Findings on loads and the number of cracks for each beam can be found in As expected, the reduction of the first crack load and the ultimate load was observed when cement was replaced by 5% BCBP. This might be due to the limited curing period. Visual observation of beam failure showed that the introduction of tire rubber resulted in a reduction of first crack load. The suggested reason for this behaviour is the significant reduction of flexural strength of rubberised concrete . Weak boFrom 3.1.3−6, -938.755 × 10−6 and 960.765 × 10−6 for B1/0P0T, B2/5P0T and B3/5P10T respectively. The tensile zone gave maximum strains of 155.024 × 10−6, 20.096 × 10−6 and 59.33 × 10−6 for B4/0P0T, B5/5P0T and B6/5P10T respectively. These strains were monitored during the entire duration of loading using strain gauges positioned at the top and bottom surfaces of the beams. The findings generally indicate increase in strains as the loading increased. This increase was more pronounced in strain gauges embedded at the top surface.Load-strain curves for mid-points of top and bottom concrete surfaces have been presented in In 3.1.4Crack development was enhanced in rubberised concrete with BCBP compared to the control concrete. An increase in mid-span deflection c due to The number of cracks increased in rubberised concrete containing BCBP compared to normal concrete. Figures 3.1.5It is clear from 3.1.6−6 for rubberised concrete with BCBP compared to 837.32 × 10−6 for control concrete confirmed that tire rubber contributed to enhancing the strains. Stress concentrations from tire rubber with low stiffness are thought to lead to increased strains [The compression zone of rubberised concrete containing BCBP heavily suffered from a large extent of damage compared to the control concrete c. The lo strains . Also, o strains , rubberi strains .3.23.2.1The load plotted against the deflection graph for the tested beams  is shown in A closer look at From 3.2.2The effect of tire rubber and brick powder inclusion in concrete beams is summarised in The reduced number of cracks in the control beam compared to the modified beams implied that modified beams had better distribution of strains. This phenomenon was also observed in beams that failed in flexure discussed previously. Larger stiffness of the control beam was suggested to result in a reduced number of cracks. However, the existence of the same number of cracks between B5/5P0T and B6/5P10T was surprising considering the discussion about deflection. From this observation, it seemed that positioning the loads at 202 mm from supports had negligible effect on the number of cracks compared to the deflection. The lower range of 2 cracks suggested that the inclusion of BCBP and WTR did not illustrate big differences in crack number among the beams compared to the beams that failed in flexure.The incorporation of tire rubber allowed the beams to undergo large deflections and increased the number of cracks ahead of ultimate failure. A probable justification for this behaviour is the reduction of modulus of elasticity due to the inclusion of tire rubber . Such be3.2.3The influence of WTR on a number of cracks and ductility ratio of concrete containing BCBP was investigated. The findings are illustrated in The reduction of load yielding failure was attributed to reduced section stiffness of B6/5P10T in comparison with the control concrete. Such phenomenon has been explained in the preceding section of beams that failed in flexure. In comparison with beams that failed in flexure, it is clear that under the same reinforcement conditions, the failure load of beams is likely to increase following positioning the loads close to the supports.3.2.4The load-strain graph for rubberised concrete containing BCBP illustrates an increased negative slope attributed to its low modulus of elasticity. By comparing Figures 3.2.5pred) for all tested beams subjected to loading were computed using BS [c is the cylinder compressive strength (MPa); fcu is the cube compressive strength (MPa); As is the area of tension reinforcement (mm2), bv is the beam width (mm) and d is the beam effective depth (mm). Because of the increased densities of all concrete mixes, the beams were not in the category of lightweight concrete. As such, the proposed interpolation expression following clause 8.6.1 of the ACI code [In this section, the shear capacities (Vusing BS  (Equatiousing BS  (EquatioACI code  was not ACI code .exp/Vpred) for all beams are presented. In general, both ACI and BS enormously underestimated the shear capacity of the beams. The underestimation was more pronounced in ACI compared to BS. For both standards, the Vexp/Vpred ratios were observed to decrease for B4/0P0T, B5/5P0T and B6/5P10T respectively. The ratios attained in rubberised concrete incorporated with BCBP surpassed that of control concrete by 10.82% for both ACI and BS. In another study [exp/Vpred ratio of 2.21 using ACI.er study , concret3) to 0.135 kcf (2161.35 kg/m3) as specified in the standard [As mentioned earlier, both ACI and BS showed limitations in the computation of predicted shear capacity values. Both standards neglect the contributions of BCBP and WTR on the shear behaviour of beams. Interpolation of concrete density modification factor in ACI is only applicable for concrete densities ranging from 0.1 kcf (1601 kg/mstandard . However41.For the beams that failed in flexure, the ductility of rubberised concrete beams containing 5% BCBP and 10% WTR improved by 23.47% compared to control concrete. This increase in ductility was evidenced with only a 15.31% reduction in flexural load. The existence of 5% BCBP in concrete demonstrated marginal improvement in ductility of 3.38% with just 2.78% load reduction compared to the control concrete.2.The beam containing 5% BCBP and 10% WTR failing in shear and flexure exhibited 14.59% ductility improvement with 16.33% load reduction in comparison to the control concrete. Beam with 5% BCBP failing in shear and flexure gave 5.28% ductility enhancement with 7.93% load reduction compared to the control beam.3.It is possible to achieve improved ductility without substantial loss in ultimate failure load by using 5% BCBP and 10% WTR. As such, concrete with 5% BCBP and 10% WTR has been established as a potential candidate in seismic applications.4.Failure of rubberised concrete beams containing 5 % BCBP was dominated by branched cracks compared to normal concrete beams especially in beams that failed in flexure.5.Numerical simulations were not combined with experimental findings in this study. The findings from this research could be used in future research to formulate numerical models. The models can be useful in the prediction of the performance of rubberised concrete incorporated with BCBP.Based on the outcomes from this study, these conclusions have been reached.David Sinkhonde: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Richard Ocharo Onchiri, Walter Odhiambo Oyawa & John Nyiro Mwero: Conceived and designed the experiments; Analyzed and interpreted the data.10.13039/501100015776African Union Commission.This work was supported by the Data will be made available on request.The authors declare no conflict of interest.No additional information is available for this paper.


Neoadjuvant immunotherapy is a novel approach for lung cancer patients in stages where curative intent treatment is possible. The rationale is a based on the idea that recognition by the immune system is activated by the entire tumor prior to surgical resection. Promising pathologic response rates have been reported and the impact on survival is currently investigated in ongoing studies. In early-stage lung cancer, recurrences are observed even after curative resection. Neoadjuvant immunotherapy might be a promising approach to eliminate micrometastasis and to potentially reduce recurrence rates and improve survival. Early trials have shown encouraging rates of pathologic response to neoadjuvant therapy and have demonstrated that surgery can be safely performed after neoadjuvant immunotherapy with various agents and in combination with chemo-(radio)therapy. However, whether these response rates translate into improved disease-free survival rates and overall survival rates remains to be determined by ongoing phase III studies.  Lung cancer is the leading cause of cancer death with a 5-year overall survival (OS) after diagnosis below 20% and is concurrently one of the most preventable cancer types . SurvivaWith promising results in the use of cancer immune modulators in advanced stage NSCLC patients, some focus has shifted to the use of immune checkpoint inhibitors (ICI) in early-stage NSCLC. The rationale for neoadjuvant ICI treatment is based on the hypothesis that the antigen load of the entire cancer might enhance immune recognition leading to a response of the primary tumor as well as potential micrometastases prior to surgical resection. This could be an advantage compared to adjuvant therapy. Studies exploring the use of neoajduvant immunotherapy or combinations of chemo-immunotherapy have shown the feasibility of this approach and promising results regarding pathologic response rates; however, the effect on disease-free survival (DFS) and OS remain to be elucidated in phase III studies. Surgical resection is the most effective single modality and the primary treatment of choice for NSCLC patients in stage I and combined with adjuvant treatment in stage II. Minimally invasive techniques such as video-assisted thoracic surgery (VATS) or robotic-assisted thoracic surgery (RATS) are an established standard for lung cancer surgery and lead to a faster recovery and reduced perioperative morbidity  in the sProgrammed cell death protein 1 (PD-1) antibodies  as well The mechanisms of the PD-1/PD-L1 and CTLA-4 pathways have been extensively studied for therapeutic checkpoint inhibition in NSCLC. Briefly, T cells express the surface protein PD-1 to regulate immune mechanisms, whereas tumor cells express PD-L1. Antigen presentation by the tumor cells is required to kill tumor cells by the inhibition of the PD-1/PD-L1 interaction. This antigen presentation activates host T cell who release cytokines after the blockade of PD-1/PD-L1 and kill the tumor cell . The PD-CTLA-4 is a glycoprotein expressed by both cluster of differentiation (CD)4+ and CD8+ T cells on their cell walls and are activated by tumor cells. Their activation leads to a direct inhibitory effect on the T cell expression. Furthermore, extrinsic CTLA-4 signals affect T cell adhesion to antigen-presenting cells (APCs), the T cell motility, and decrease residence time on the APC. Additionally, it prevents CD28, located in the T cell wall, from stimulating T cell proliferation or interleukin-2 production. In general, a hyperreaction of the T cell reaction is downregulated and prevented by CTLA-4. CD28 and CTLA-4 are opposing immune regulators . CTLA-4,The rationale for using neoadjuvant ICI treatment rather than adjuvant treatment is based on the idea that the antigen load of the entire tumor enhances the immune recognition and thus the efficacy of the therapy compared to an adjuvant application . The assessment of the response for restaging after neoadjuvant immunotherapy has not yet been fully evaluated. Due to immune cell infiltration, there is a heterogeneity in published trials regarding whether tumor appearance on imaging correlates with pathological response.OS is the most important primary endpoint in the assessment of any neoadjuvant therapy . HoweverRadiologic and pathologic responses can be documented within a short timeframe after treatment. Objective response rate (ORR) on imaging including both partial and complete responses is an established endpoint for evaluating treatment effects after neoadjuvant chemo(-radio)therapy. However, there is heterogeneity in trials as to whether response imaging correlates to the pathological response due to the pseudoprogression of the primary tumor as well as the nodal immune flare. Thus, neither CT nor PET criteria for downstaging after neoadjuvant immunotherapy are currently established ,30,31,32Major pathologic response (MPR) is defined as a residual viable tumor of less than or equal to 10% that has been established as a surrogate endpoint for survival after neoadjuvant chemotherapy leading to a shortened time required to evaluate neoadjuvant trials with novel chemotherapeutics ,35,36,37Pathologic complete response (PCR) of the primary tumor and the lymph nodes after classic neoadjuvant chemotherapy correlate to improved OS, but nodal response is dependent on the accuracy of nodal staging and is only applicable to patients with documented nodal disease .The standardization of MPR evaluation standards has been controversial ; howeverThe correlation of MPR and PCR with DFS and OS after neoadjuvant immunotherapy is however not yet known . A consiDiscrepancies between radiographic and pathologic responses occur after neoadjuvant immunotherapy, and there are conflicting data in published studies as to whether a correlation between imaging and pathologic responses is observed. Imaging can underestimate the degree of pathologic tumor regression  and persCurrently, no criteria as to when to proceed with surgery after complete radiological response after immunotherapy are established. A detailed assessment of CT and PET criteria to evaluate indicators of pathological downstaging should accompany neoadjuvant immunotherapy trials. An evaluation based on immune-related response criteria (irRC) rather than response evaluation criteria in solid tumor (RECIST) has been postulated . An initAlgorithms to differentiate pseudoprogression from true progression or hyperprogression are required. Several biomarkers including PD-L1 status, tumor mutational burden, and baseline lymphocyte infiltration counts or immunoscore are under investigation; however, none have been established . The impNo biomarkers for therapy response have been established in clinical routine in the neoadjuvant setting. However, biomarkers are an important part in virtually all ongoing clinical trials, and a detailed discussion of all potential markers is beyond the scope of this review. Based on data from advanced stage NSCLC, PD-L1 and tumor mutational burden (TMB) ,48,49 arTMB describes the number of mutations in cancer tissue. High TMB might be a predictive of response to ICI therapy in NSCLC and SCLC . The LCMp = 0.042) [A high PD-L1 expression level could be identified as another independent biomarker  for prol= 0.042) . However= 0.042) .In summary, the prognostic and predictive value of PD-L1, TMB, and other biomarkers for neoadjuvant immunotherapy has not yet been established. Circulating tumor DNA (ctDNA) after curative-intent first-line therapies might develop as a marker to detect residual disease and disease recurrence for lung cancer earlier than imaging. This was shown in more than 70% of the patients, with a median lead time of >5 months being highly prognostic in the detection of molecular residual disease. Detecting multiple mutations improved the sensitivity . HoweverComplication rates, blood loss, procedure time, and perioperative course after neoadjuvant ICI therapy are so far comparable to patients receiving neoadjuvant chemotherapy or chemoradiation ,55. InitImmunotherapy has a different spectrum of side effects compared to other neoadjuvant therapies. These side effects can impact operability and affect the perioperative course. The increased use of immunotherapy in patients in a curative setting might lead to a more granular picture of possible therapy induced side effects compared to stage IV patients and might even highlight previously unknown long-term effects of immunotherapy due to prolonged survival times. Side effects mainly derive from the activation of the immune system and comprise a wide spectrum like pneumonitis, acute kidney injury, cardiotoxicity, hepatitis, dermatological reactions, neuropathy, nausea, muscular weakness, adrenal insufficiency, hepatitis, and gastroenteritis. .Immune-related adverse events might arise immediately after therapy initiation; however, late onset months after the start of therapy has also been observed. Thus, onset can occur after the completion of the neoadjuvant therapy period, for example, in the early postoperative phase or during postoperative recovery . PneumonAnother severe adverse event is cardiotoxicity. A 0.06% myocarditis rate was observed after ICI treatment in patients with PD-1 antibodies with a higher incidence in patients receiving a dual ICI therapy together with a CTLA-4 inhibitor of 0.27%, shortly after application and partly after first dose administration . CardiotAwareness of the potential side effects and accurate diagnosis throughout the perioperative period is important to initiate appropriate treatment. Several randomized phase II studies comparing neoadjuvant immunotherapy or immuno-chemotherapy have been conducted. Some trial designs include adjuvant therapy as well . These sCurrently published trials were initiated when TNM7 was still effective. According to TNM, stage IB shifted to stage II in TNM8. This must be considered when interpreting trial outcomes. While a clinical paper on neoadjuvant chemoimmunotherapy in 2017 purely focused on immune profiling , the firA phase II trial published by Shu and colleagues investigated i neoadjuvant atezolizumab and carboplatin/paclitaxel in patients with NSCLC stage IB-IIIA n an open label, multicenter, single arm setting . No treaThe randomized NEOSTAR trial compared three cycles of neoadjuvant nivolumab with three cycles of nivolumab plus one cycle of ipilimumab in 44 operable stage I–IIIA NSCLC patients . Out of The IONESCO trial  investigThe PRINCEPS  trial inThe LCMC3 trial looked at MPR after two cycles of atezolizumab in stage IB- selected IIIB patients . MPR wasInterim safety results of the NEOMUN trial ,69 reporThe TOP 1501  trial loOnly a few studies have focused exclusively on stage III patients. The NADIM trial investigated neoadjuvant chemoimmunotherapy with nivolumab and paclitaxel/carbolatin in a single arm design in stage IIIA (N2) patients followed by adjuvant intravenous nivolumab monotherapy for one year every two to four weeks . SurgeryThe SAKK 16/14 trial  includedInterim data on 14 patients of the ongoing ACTS 30 trial investigating neoadjuvant chemoradiotherapy and durvalumab in potentially resectable stage III NSCLC were presented at ASCO 2021 . NeoadjuSeveral additional trials are in progress with no data yet published. A phase II trial comparing ICI alone and ICI with platinum-based chemotherapy in a neoadjuvant setting is the ongoing NAJSCR trial for resectable lung cancer treated with the new PD-1 antibody toripalimab . The feaKEYNOTE-671 , IMpowerp < 0.0001) and a MPR rate of 36.9% versus 8.9%, respectively. The benefit expands across the key subgroups: disease stage , PD-L1 status , and TMB  [In a first presentation of the trial results during the Annual meeting of the American Association of Cancer Research (AACR) held virtually on April 10th 2021, it was determined that nivolumab plus chemotherapy shows statistically significant improvement compared to neoadjuvant platinum-based chemotherapy alone in the pathologic complete response as a neoadjuvant treatment for resectable non-small cell lung cancer in the ongoing phase III CheckMate 816 trial by 24% versus 2.2% (us 2.7%) ,83. Neoaus 2.7%) . Pembrolizumab in the neoadjuvant setting is under investigation in the KEYNOTE-671 trial . The endAtezolizumab plus chemotherapy versus chemotherapy alone is currently being explored in the neoadjuvant setting, and atezolizumab versus placebo is being investigated in the adjuvant setting for stages IB to IIIB (cT3 N2) in the IMpower 030 trial , the endBoth durvalumab and chemotherapy versus chemotherapy neoadjuvant and durvalumab or placebo adjuvant is currently being tested both in the AEGEAN trial  with thePhase III trials of adjuvant immunotherapy or chemo-immunotherapy compared to neoadjuvant immunotherapy or chemo-immunotherapy do not yet exist; however, trials in this area would be warranted .Depending on the long-term results of the ongoing phase III trials, immune checkpoint inhibitors might become part of standard therapy for localized NSCLC . HoweverBlood or exhalate based biomarkers with sufficient prognostic or predictive power have not yet been sufficiently evaluated in the neoadjuvant setting. TMB testing may complement PD-L1 testing in identifying patients who are likely to have good outcomes with immunotherapy . Novel targets for immunotherapy are currently being explored. As an example, lymphocyte activation gene 3 (LAG-3), an immune regulatory molecule for the T cells is being used in the NEOPREDICT-Lung trial. LAG-3 is expressed only on TILs and correlates with the expression of PD-1 and PD-L1 and might be related to tumor recurrence and prognosis . LAG-3 hInterdisciplinary cooperation and active participation or thoracic surgeons in the design and implementation of neoadjuvant ICI trials is crucial for the development of optimal strategies for patients with early stage NSCLC . The outNeoadjuvant ICI therapy in lung cancer treatment might be a promising approach to reduce recurrence rates and to improve outcome for early-stage lung cancer patients. To draw any definitive conclusions results of ongoing phase III trials have to be awaited. Available data show that surgery after ICI treatment can be conducted safely. There is a considerable rate of pathologic responses after neoadjuvant immunotherapy and an even higher response after neoadjuvant immuno-chemotherapy. How these results translate into DFS and OS remains to be seen. Interdisciplinary cooperation with the active involvement of thoracic surgeons is crucial for the development of optimal strategies for patients with early-stage NSCLC and provides an excellent opportunity for translational research.


Antibiotics are used to control infectious diseases in both animals and humans. They can be life-saving compounds but excessive use in animal husbandry leads to the development of antibiotic resistance which can impact the public health. Since similar antibiotics are used in both animal and human healthcare, it is important to reduce the use of antibiotics in production animals. In the Netherlands policies have been developed aiming for a decrease of antibiotic usage in animals, and alternatives to antibiotics are investigated. Currently, a one-on-one relationship between farmer and veterinarian is successfully implemented and  registration of antibiotic usage is mandatory. Unfortunately, after a 70% decrease in antibiotic usage since 2009, this decrease is now stagnating in most sectors. Innovative strategies are required to facilitate a further reduction. One promising option is a focus on farm management and natural alternatives to antibiotics. The Dutch government has invested in the spread of knowledge of natural remedies and good animal management to support animal health via so called Barnbooks for farmers and veterinarians. Another option is the analysis of on-farm antibiotic use to prevent unregistered applications. New (bio)analytical strategies to monitor the correct and complete registration of antibiotic usage have been developed and trial-tested in the Netherlands. Such strategies support a risk-based monitoring and allow effective selection of high-risk  users. Both effective monitoring and the availability and knowledge of alternatives is a prerequisite to achieve a further significant decrease in antibiotic veterinary usage. In the Netherlands, strategies have been developed aiming to decrease use of antibiotics in animals. In response to several outbreaks of multi-resistant bacteria and the resulting societal and political pressure, several compulsory and voluntary measures were initiated to reduce antibiotic usage by farmers and prescription by veterinarians , 2. ThesStaphylococcus aureus and extended spectrum beta-lactamase-producing bacteria , an independent body, was established in 2010 with the main aim of reducing antimicrobial use in the farm industry . The SDaAdditional legislation was set in 2014 which prescribed that only veterinarians were allowed to administer antimicrobials. Exceptions are possible under strictly regulated and monitored conditions. The goal was to create closer interaction between veterinarian and farmer, compulsory herd health checks and the presence of a farm health plan and farm therapeutic plan. Currently, a one-on-one relationship between farmer and veterinarian has been successfully implemented and  registration of antibiotic usage is mandatory.The current figures  show thaPhytotherapeutics are plant-based medicine that were traditionally used to prevent and cure disease in humans and livestock –10. NowaPhytogenics can be a huge resource for improving animal health since bioactive plant components can work on gut flora, modulate the immune system, affect digestion and many other systems, exerting a multi-target action –13. The The Dutch government invested in providing information for farmers and veterinarians concerning alternatives in management and use of health supporting products. Medicinal plants have been used worldwide for prevention and treatment of diseases in animals and humans for ages. The broad spectrum of plant metabolites represents a huge potential for medication of herbivore and omnivore livestock. While there is a large amount of evidence-based knowledge about medicinal plants published in literature in both English and German language , this isTo make the information on alternatives and their possible application accessible for a wider public, so-called “Barn books” per animal category were published by WFSR in a project funded by the Dutch Ministry of Agriculture, Nature and Food Quality. First books were aimed at organic farmers and focussed on dairy cattle, poultry and pigs. These books are also translated into English as Natural Dairy Cow Health, Natural Swine health, Natural Poultry Health, Guide to keeping your animals healthy with herbs, and other natural products –17. ThesThe books consist of chapters per life stage of the animal with general management advice and natural products that can support the animals' health or prevent the animals from becoming sick. The products described are complementary feeds or feed additives. In the annexes there is a list of herbs with their main constituents and use, and per product general information and information from research with references is provided. The barn books give information on products for which the supplier provided the full composition of the product and results from field trials and literature. Products with no full disclosure of the composition or without underlying substantiation are not included. The aim was to provide both farmers and their veterinarians with objective information on products and their expected effects.Natural products used as feed additives have shown to improve zootechnical parameters such as growth performance, feed conversion and gut health in livestock, and so reduce the need for antibiotics –31. The Next to management and phytogenics, (on-site) analysis of antibiotic residues to check for correct registration of antibiotic usage will prevent unregistered application and thus contribute to the prudent use of antibiotics.Compulsory registration of antibiotic use is an important step toward monitoring antibiotic use on farm level . HoweverRoutine monitoring of veterinary drug residues is most commonly carried out in the slaughter phase. Animal tissue is sampled, transported to the laboratory and analyzed for violation of the maximum residue limit. Sampling is also done at the farm. Usually urine, milk or eggs depending on animal type and production use are collected. Taking urine samples can be quite time-consuming. Excretion studies have shown that many antibiotics have a relative high excretion rate in urine and that residues can only be detected shortly after administration. Therefore, the analysis of urine samples to check legally correct antibiotic use, is not an economically feasible and practical strategy.Studies have demonstrated that antibiotic residues are present in hairs and feathers in relatively high concentrations . As resiAs hair and feather samples are rather easy to collect on site and contain as well as retain information about the history of antibiotic use in the animal , these sAdditional research (not published) demonstrated that, for screening of antibiotics, cotton pads could also be used to obtain samples from calves. Instead of shaving hair (which is an elaborate procedure), these cotton pads were wiped over the animal's neck or back and stored in a tube. The  analytical results of the cotton pad samples proved to be comparable to the results of the hair samples taken from the same animals. This demonstrated that also a quick and easy sample taking procedure can be applied for cattle and swine.Even though the above presented approach has been proven successful in finding non-registered use of antibiotics, a main disadvantage of this approach is that the whole procedure  is time consuming and relatively expensive. We are therefore, currently investigating the application of on-site lateral flow devices (LFD) for easy and quick analysis of antibiotics in hair, feathers and preferably cotton pad swipes.LFDs for the detection of antibiotics are commercially available  and PerkinElmer , and can also be easily prepared and adapted in-house, as long as residue specific binding molecules  are available. Samples can easily be extracted on-site with a buffer and a small volume of the extract is then transferred onto a LFD. After several minutes, a qualitative result is obtained. LFDs can detect a single specific compound or compound class, but multiplex devices are available. This development allows inspectors (after a short training) to carry out a quick assessment of antibiotic exposure on site. After screening multiple animals, results of the LFDs are compared with the farms antibiotic use registration system and within 15 min the inspector can obtain a good indication of the status of the farm with regard to antibiotic use and registration thereof. In case no discrepancies are observed, the inspector can quickly move on to the next farm. If differences are observed, the inspector can discuss this with the farmer and, if deemed necessary take additional samples for laboratory analysis. This approach is very promising and, as it is a very quick and easy approach, is expected to have a preventive effect of misuse of antibiotics in animal husbandry. The large scale implementation to LFD application on hairs and feathers at farm level will probably present new challenges. These especially regard the application of the test in a potentially contaminated environment and the legal status of the result. The latter includes the validation of the LFD under field conditions. Those hurdles are expected to be overcome in the coming years. Nonetheless, the currently proposed strategy can easily be applied at laboratory level already.In conclusion, in the Netherlands an important decrease of antibiotic use in animal husbandry has been achieved since 2009. However, it is currently plateauing even though a further reduction is urgent. New approaches are suggested to further decrease antibiotic usage. The differences between antimicrobial use reduction achieved per sector suggest that approaches should probably be sector and sometimes farm specific. These could include optimized management and raising awareness for the risk of antimicrobial resistance. Additionally, we advocate the public availability of knowledge on natural products as alternatives and an effective strategy to monitor and enforce correct use and registration of antibiotic applications. These are promising tools to achieve lower antibiotic inputs and additional monitoring of antibiotic use data must demonstrate their effectiveness.Publicly available datasets were analyzed in this study. This data can be found at: data available via the references.MG is the responsible author and wrote the part: use of phytogenics to keep animals healthy. NC wrote the part about the Dutch policy: Dutch reduction of antimicrobials by almost 70% now stagnant in most sectors. BB wrote the part: analytical strategies that support and stimulate prudent use of antibiotics. All authors have contributed the whole article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The Mycobacterium abscessus complex (MABC)is among the most drug-resistant mycobacterial species, and prompt diagnosis and effective eradication are burdensome. We present the clinical course of a 55-year-old female who was diagnosed with M. abscessus and explore her clinical diagnosis and possible treatment options. This case report emphasizes the challenges clinicians face in the prompt diagnosis of M. abscessus and discusses the treatment options in light of the recent guidelines.The incidence of infections by rapidly growing mycobacteria has increased in recent decades. nontuberculous mycobacteria(NTM)  Mycobacterium abscessus is the most common and rapidly growing NTM and is among the most drug-resistant pathogens, hence accounting for limited therapeutic options and a high treatment failure rate [M. abscessus can cause skin and soft tissue infections after trauma or surgical procedures, and pulmonary infections and disseminated diseases in immunocompromised patients.Over 190 species and subspecies of nontuberculous mycobacteria (NTM) have been documented, many of which are known to produce disease in humans. They can affect both pulmonary and extrapulmonary sites [ure rate . AlthougM. abscessus complex (MABC). She was then started on a four-month course of antibiotics and scheduled to follow up with an infectious disease (ID) clinic. The patient initiated her treatment regimen with improved symptoms but did not complete the full course of antibiotic treatment and was lost to follow-up until this visit. The patient states that her recent symptoms started within two months of stopping her antibiotic therapy.A 55-year-old White female with hypertension, chronic obstructive pulmonary disease (not on home oxygen), and a chronic cough presented to the emergency room (ER) with a worsening cough, low-grade fever, multiple episodes of small amounts of hemoptysis, and subjective weight loss over the past three months. She was an active smoker and reported that her symptoms had worsened for the past week before she arrived at the ER. The shortness of breath and cough (which was productive with white sputum) were also accompanied by diffuse, bandlike chest pain. The patient denied any significant weight loss, hemoptysis, or night sweats. The patient had a history of bronchoscopy performed nine months ago for bronchiectasis seen on the CT scan chest Figure , and herOn physical examination, the patient appeared cachectic but was hemodynamically stable without respiratory failure. A high-resolution computed tomography (HRCT) scan of the chest was done, which revealed a cavitary lesion Figure .. SputumM. abscessus complex is a complex of rapidly-growing mycobacteria (RGM) species of NTM which is second in prevalence after the M. avium complex (MAC). MABC often causes severe respiratory, skin, and mucosal infections in humans. This species is also considered the most antibiotic-resistant of all the mycobacteria [M. abscessus is the most common isolate, followed by M. massiliense and M. bolletii [The bacteria . As per bacteria . Out of bolletii . Howeverbolletii . In termbolletii ,7. IsolaM. abscessus-chelonae group. However, with time, the difference between MABC and M. chelonae in terms of genotype as well as pathogenicity and antibiotic resistance has become more and more clear. Therefore, there is a dire need to evolve the classification system of MABC based on pathogenetic characteristics. With the possibility of complete genetic sequencing, a consensus list of the targeted genes for classification needs to be adopted and followed by most diagnostic labs. Currently, there are no FDA-approved commercial DNA probes that completely sequence MABC species based on the 16StRNA gene. Furthermore, the ability to detect the erm41 gene to further classify MABC into subspecies is also essential to determine macrolide sensitivity [Different techniques are used in the detection of NTMs and the classification of them accordingly. As per the initial classification in the 1970s, there was no classification past the sitivity .M. abscessus and M. bolletii. M. massiliense subspecies is generally macrolide sensitive with better outcomes. Generally, in-vitro macrolide resistance may not be detected, but in-vivo Macrolide resistance is always present in the presence of the mentioned gene [Antibiotic resistance is also the biggest challenge in the treatment and eradication of all NTM infections, but most notably in MABC infections. It is important to never empirically start an NTM infection on antibiotic therapy as it can worsen the resistance and the empiric antibiotic therapy may not be appropriate. Appropriate sensitivities are always necessary in MABC cases. Macrolides remain the cornerstone for most NTM therapies, and the species sensitive to macrolides have higher treatment-response and clearance rates. The erm41 gene is responsible for macrolide resistance in two of the three subspecies of MABC, namely ned gene . AnotherClassically, for NTM treatment, long-term directed antibiotic therapy is given with serial monitoring for drug side effects and repeat sampling to confirm disease eradication. As mentioned, this strategy is often ineffective in MABC cases. An alternative strategy for the management of the disease can also be adopted. In asymptomatic diseases or those with minimal symptoms and low life expectancy, avoiding antibiotic therapy can be a feasible solution in certain cases. In other cases where the disease is quite localized with a particular lung area being affected, thoracotomy can be of benefit as long as the patient can tolerate the surgery. Another target for antibiotic therapy could be symptomatic relief, as severe symptoms can be eradicated by antibiotic therapy even without achieving disease eradication .M. abscessus chelonae complex" was identified. Empiric treatment with an antibiotic regimen was proposed, while surgical evaluation was also started. However, due to advanced COPD and deconditioning, plans for surgery were postponed. With empiric antibiotic therapy, improved symptoms were nevertheless observed.In our case, despite high suspicion and diagnosis of NTM in general, appropriate classification into MABC with macrolide sensitivity was not performed. Based on the lab report, the "M. abscessus chelonae" as a fast-growing, pathogenic nontuberculous mycobacterium species with significant challenges in diagnosis and management, thereby requiring a robust multidisciplinary approach. Long-term targeted antibiotic therapy, despite significant side effects, can prove inadequate in order to permanently clear the infection. However, it usually halts progression and provides symptomatic relief. The worsening structural disease often results in patients who are partially treated. Our case also throws light on the need for close outpatient follow-up and long-term management strategies.Our case aims to highlight "


It is well known that some pathological conditions, especially of autoimmune etiology, are associated with the HLA (human leukocyte antigen) phenotype. Among these diseases, we include celiac disease, inflammatory bowel disease, autoimmune enteropathy, autoimmune hepatitis, primary sclerosing cholangitis and primary biliary cholangitis. Immunoglobulin G4-related diseases (IgG4-related diseases) constitute a second group of autoimmune gastrointestinal, hepatobiliary and pancreatic illnesses. IgG4-related diseases are systemic and rare autoimmune illnesses. They often are connected with chronic inflammation and fibrotic reaction that can occur in any organ of the body. The most typical feature of these diseases is a mononuclear infiltrate with IgG4-positive plasma cells and self-sustaining inflammatory response. In this review, we focus especially upon the hepatopancreatobiliary system, autoimmune pancreatitis and IgG4-related sclerosing cholangitis. The cooperation of the gastroenterologist, radiologist, surgeon and histopathologist is crucial for establishing correct diagnoses and appropriate treatment, especially in IgG4 hepatopancreatobiliary diseases.  Autoimmune diseases of digestive organs can be divided into three groups: 1) autoimmune digestive diseases associated with human leukocyte antigen (HLA), 2) immunoglobulin G4-related diseases (IgG4-RD) and 3) other autoimmune gastrointestinal diseases. It is well known that some pathological conditions, especially of autoimmune etiology, are associated with different HLA phenotypes [ autoimmu immunogl other auCeliac disease is represented by a gluten-sensitive enteropathy. It develops in genetically susceptible individuals. The main role is played by T cell lymphocytes reactivity against gluten . The diaThe role of HLA in patients with inflammatory bowel disease (IBD) remains uncertain. In addition, unclear is the etiology of IBD, which is assumed to include a combination of an individual’s genetic background, alteration of gut microbiota, immune dysregulation and environmental factors . HLA typThe HLA system is also considered to be a major genetic marker and is associated with extraintestinal manifestation of IBD. For example, HLA-B27-positive patients with IBD have higher risk for developing ankylosing spondylitis. Primary sclerosing cholangitis, another autoimmune disease which is often coexisting with IBD, has also been associated with various HLA alleles. Generally, patients with IBD have increased an risk of various autoimmune and inflammatory diseases . TreatmeIBD is a diagnosis which offers wide scope for use of different biological drugs. They interfere with immune system on different levels. For example, in cytokine production, signaling pathway in T cell activation or inhibiting Januse kinase ,22. The We also have possibility to use some novel biologics for IBD treatment with promising therapeutic effects such as vedolizumab or ustekinumab ,27.Autoimmune enteropathy (AIE) in adults is a heterogeneous disease associated with a variety of circulating gut antibodies and predisposition to autoimmunity .+CD4+ regulatory T cells probably plays an important role [The pathophysiology of AIE is not exactly known. Dysfunction of CD25ant role . AIE is ant role . Anti-enant role . On the ant role . Antibodant role .AIE is a rare condition, clinically connected with refractory diarrhea and malnutrition, mainly in children. Other typical changes are histological changes in small intestinal biopsy. In many patients, immunosuppressive therapies are principally used. Diagnostic criteria are detailed in Criteria 1–4 are required for a definite diagnosis of AIE. Presence of anti-enterocyte antibodies is an important diagnostic support, but their absence does not exclude the diagnosis of AIE. In the light of study from Biagi et al., we dare to remove anti-goblet cell antibodies from previous proposed diagnostic criteria. They are more unspecific than expected .Autoimmune hepatitis (AIH) is a non-resolving inflammation of the liver. The disease reflects a complex interaction between triggering factors, autoantigens, genetic predisposition and immunoregulatory networks . The disAIH is a global disease, occurring most frequently as type 1. HLA typing was part of original and revised diagnostic criteria of AIH , but nowFormerly known as primary biliary cirrhosis, primary biliary cholangitis (PBC) is a chronic cholestatic disease of unknown etiology and affecting mainly females. Essential to positive diagnosis is a combination of serological markers of cholestasis, the presence of autoantibodies such as antimitochondrial antibodies (AMA) and PBC specific antinuclear antibodies (ANA) anti-gp210 and anti-sp100 and imaging of liver and bile ducts. In unclear cases, we can perform liver biopsy . The disGenetic factors are very important and documented by the high concordance rate of PBC among monozygotic twins . This grPrimary sclerosing cholangitis (PSC) is a rare, chronic cholestatic liver disease that can lead to liver fibrosis and cirrhosis. Pathologically, PSC is characterized by an inflammation and destruction of extra- and intrahepatic bile ducts. The etiology of this disease is unknown. Association between PSC and IBD has been described . IBD wasCFTR mutation (cystic fibrosis transmembrane receptor mutation)) [The pathogenetic mechanism is still unknown. Inflammatory changes are centered on the biliary epithelium and damage of the biliary tree is frequently observed. PSC is probably an immunologically mediated process with HLA association . Experimtation)) ,48. Subttation)) . ImagingThere is no effective pharmacological therapy. It remains unclear whether or not administering ursodeoxycholic acid can be effective. Liver transplantation is an effective treatment for end-stage liver disease .The second group of autoimmune gastrointestinal, hepatobiliary and pancreatic diseases is termed immunoglobulin G4-related diseases (IgG4-RD). IgG4-RD is a group of systemic and rare autoimmune diseases, often connected with chronic inflammation and fibrotic reaction that can occur in any organ of the body . EtiologIgG4 is an immunoglobulins fraction accounting for just 5.0% of the IgG pool. IgG4 is physiologically produced after a long-term exposure to food or environmental antigens. A current hypothesis is that the transformation from B cells to plasma cells and activation of eosinophilic granulocytes can probably be triggered by an initial Th1-type immune response via secretion of proinflammatory cytokines .It is important that the level of plasmablast correlates with the diagnosis and disease activity much better than it does with serum IgG4 level. Plasmablast seems to be a precursor of tissue-resident antibody-producing plasma cells . OligoclThe most frequently encountered manifestation of IgG4-RD in the gastrointestinal system is autoimmune pancreatitis (AIP). Two subtypes of AIP have been described. Type 1 AIP is a typical pancreatic manifestation of IgG4-RD, while type 2 AIP is an autoimmune disease of the pancreas.Type 1 AIP is known as lymphoplasmacytic sclerosing pancreatitis, or LPSP. There exists an international diagnostic consensus that can be followed when making the diagnosis according to the major IgG4-RD criteria. This consensus was published by Shimosegawa et al.  in 2011.Despite international guidelines for diagnosing AIP, its differentiation from pancreatic cancer is still challenging . In an iThe most common IgG4-RD among hepatobiliary diseases is IgG4-related sclerosing cholangitis. This disease is often associated with other organ manifestations of IgG4-related illnesses, most typically with type 1 AIP . The disIn cholangiography, four typical images have been described and are classified by Nakazawa et al. . The typIn differential diagnoses, we must also always consider PSC. In IgG4-related hepatopathy, five manifestations of liver involvement have been identified: (1) portal inflammation, (2) lobular hepatitis, (3) portal sclerosis, (4) lobular cholestasis and (5) bile duct obstruction . The impOnly a few clinical papers have been published about IgG4-related gastrointestinal diseases of the esophagus, stomach and bowel, with diffuse infiltration of the gastric mucosa by IgG4 + plasma cells being the most commonly described . This inIn IgG4-related gastric lesions, focal polypoid lesions or focal masses up to 3 cm, gastric ulcer, diffuse thickening of the wall and association with local lymphadenopathies have been observed .In colon biopsies from a set of 119 patients with IBD, Topal et al. found IgG4 positivity in 17.6% . Obiorah12 deficiency [Autoimmune gastritis (AIG) is an immune-mediated chronic disease with mostly mild or non-specific clinical manifestation. It affects corpuscular acid-producing mucosa, especially parietal and chief cells, and leads to intrinsic factor deficiency and hypo- or achlorhydria . AIG’s eficiency . Essentificiency . Treatmeficiency .Autoimmune diseases of the gastrointestinal organs constitute a huge gastroenterological challenge. Many gastrointestinal organs undergo biochemical and histopathological changes in connection with IgG4-RD or the HLA system. Diagnosis and therapy of these diseases require a multidisciplinary approach and cooperation among gastroenterologists, hepatologists, surgeons, immunologists, histopathologists and radiologists. There can be no doubt that autoimmune organ disorders have a place in the broad and multidisciplinary field of gastroenterology.


Antimicrobial resistance (AMR) is a threat to hospital patients. Antimicrobial stewardship programs (ASPs) can counteract AMR. To optimize ASPs, we need to understand what affects physicians’ antibiotic prescription from several contexts. In this study, we aimed to explore the factors affecting hospital physicians’ antibiotic choices in a low-resistance country to identify potential targets for future ASPs. We interviewed 14 physicians involved in antibiotic prescription in a Norwegian hospital. The interviews were audiotaped, transcribed verbatim, and analyzed using thematic analysis. The main factors affecting antibiotic prescription were a high work pressure, insufficient staff resources, and uncertainties regarding clinical decisions. Treatment expectations from patients and next of kin, benevolence towards the patients, suboptimal microbiological testing, and limited time for infectious disease specialists to offer advisory services also affected the antibiotic choices. Future ASP efforts should evaluate the system organization and prioritizations to address and manage potential time-pressure issues. To limit the use of broad-spectrum antibiotics, improving microbiology testing and the routines for consultations with infectious disease specialists seems beneficial. We also identified a need among the prescribing physicians for a debate on ethical antibiotic questions. Antibiotics are a cornerstone in the treatment of infections and critical to modern medicine . HoweverIn Norway, broad-spectrum antibiotics (BSAs) and AMR rates are lower than in most other countries. For instance, Norway has the lowest rate of methicillin-resistant staphylococcus aureus and a lower rate of other resistant bacteria, in company with Denmark and Finland, compared to southern and eastern Europe . The maiThe study aim of the present study was to explore factors affecting hospital physicians’ antibiotic prescribing practices, in a low-resistance country, to identify potential targets for improvement of future ASPs.The final study population included 14 respondents: five physicians from infectious diseases; three physicians from oncology; one medical resident; and one physician from anesthesiology, gastrointestinal surgery, gastroenterology, pulmonology, and hematology. The participants’ experience level and characteristics are presented in We identified four main themes affecting the physicians’ antibiotic prescribing choices: (i) Clinical workflow pressures, (ii) Clinical uncertainty, (iii) Decision support, and (iv) Benevolence. Visualization of the themes and subthemes is shown in The theme “Clinical workflow pressures” consists of the subthemes “time pressure” and “follow-up resources”. Both subthemes represent physicians’ experience of unfulfilled intentions of making thorough antibiotic decisions. The physicians constantly experienced being in a squeeze between providing optimal patient care, including making rational antibiotic decisions, and having a high workload and limited follow-up resources.“Time pressure” reflects the pressures upon physicians’ to increase patient turnover. The respondents reported experiencing a pressure mainly from nursing staff due to bed pressure. The physicians also reported putting pressure on themselves, e.g., when they observed many patients waiting in the emergency room. This strain was reported to often result in unnecessary use of broad-spectrum antibiotics to compensate for short time to observe patients and to perform adequate diagnostics:“I think a lot is due to the pressure in the hospital; we hear all the time, ‘Discharge, discharge, discharge’ (…) We get whipped up like that, (…) and then I think the threshold (…) to give broader spectrum antibiotics is lower.”.“Follow-up resources” refers to the physicians’ experience of a lack of patient follow-up resources which made them scared of patient adverse outcomes and thus led to use of more antibiotics to ensure safety. A lack of nurses (and thus capacity) was, for instance, viewed as a challenge:“It is important that the patient is properly followed up; that is what’s scary in an understaffed hospital full of patients: a lack of nurses. Then, it might be more rational to use broader spectrum antibiotics than in a more orderly situation (…) A typical example is that we actually shift from penicillin to cefotaxime  on Fridays due to lesser capacity during weekends”.The theme “Clinical uncertainty” consisted of two subthemes: “just in case” and “knowledge and experience”. Both subthemes underscored that the most important aspect for the physicians when they prescribed antibiotics was to feel certain in their decisions, both regarding a favorable outcome for their patients and to justify their decisions to their peers later.“Just in case” reflects the finding that if the physicians were uncertain whether the clinical condition would deteriorate at home, they reported they would often continue to give antibiotics, even broad-spectrum antibiotics, to be on the safer side:“We know that, theoretically, they often don’t need antibiotics, but it is hard to select the patients who actually don’t need antibiotics and have the courage to send them home without any (…) you are scared the patient will come back in worse shape, and you will be asked why you didn’t continue antibiotics”.The physicians also considered their own “knowledge and experience” levels when making antibiotic decisions. The less experienced (under five years of experience) physicians more often reported that a feeling of inadequate knowledge led to less confidence to prescribe restrictively .“Decision support” consists of the subthemes “microbiological tests” and “collegial consulting”. These subthemes illustrated that the physicians viewed microbiological test and consultation as facilitators of rational prescription, but experienced that these facilitators were underused due to capacity constraints. Thus, they reported that underuse of available decision support negatively affected antibiotic prescription.The physicians considered microbiological tests to aid in rational prescription as long as the sampling was correct, which was reported to not always be the case due to time pressure:“Often the material is inadequate. It is an extreme pressure in the emergency ward sometimes, and you choose the easiest solution, which gives non-representative results”.The physicians experienced inadequate material as a lost opportunity to de-escalate to narrow-spectrum antibiotics.“Collegial consulting” is bipartite; on one side, consultations with ID specialists were considered an important measure to facilitate more rational prescription by providing support in decision-making:“What works well is to seek collegial support when you are in doubt, call an infectious disease specialist”.One the other side, the ID specialists highlighted that their antibiotic prescription advice was sometimes suboptimal due to limited time to engage, potentially hampering its effect on antibiotic prescription:“(…) If we had time for consultation, it would be fine, but when it (the consultation phone) buzzes on top of everything else you have to do, then you don’t have time (…), and the advice might be suboptimal”.The theme “Benevolence” consists of the subthemes “the patient’s wellbeing” and “treatment expectations”. This theme addresses the physicians’ benevolent efforts; they described a desire to improve their patients’ health and meet the patients’ or their relatives’ wishes, which often would include the prescription of antibiotics. Simultaneously, they expressed a desire to limit unnecessary use of antibiotics and AMR.“The patient’s wellbeing” refers to the physicians’ prioritization of the present patient’s potential improvement, a possible future increase in antibiotic resistance notwithstanding, as illustrated by respondent 7:“(…) Sometimes I think, ‘Why am I doing this?’ when I, e.g., give broad-spectrum antibiotics to an old patient who has dementia and has not improved on other antibiotics—it feels wrong, but at the same time, I feel I don’t have a choice when I stand in front of the patient”.The physicians expressed an unease knowing that such decisions often could result in a more inappropriate antibiotic prescription.The “treatment expectations” subtheme represents situations when the physicians saw it as routine and expected to prescribe antibiotics, even though they often had second thoughts whether the antibiotic prescription was correct, regarding the aspect of AMR. An example some of the respondents used was oncologic or transplant patients for whom prolonged antibiotic treatment is often routine beside chemotherapeutic regimes. The physicians reported they obviously complied in offering life-extending therapy to individual patients. Nevertheless, they questioned the price to pay in terms of antibiotic resistance development:“It is obvious that every patient, who can and wishes to get well, should receive all available treatment (…). For some patients, this would mean complicated antibiotic regimens to keep them alive during the treatment . (…) This is worrisome due to the development of resistant bacteria that may kill other individuals”.Another challenge the physicians experienced was the expectations of antibiotic treatment from the patient or the patient’s family in situations when the physicians did not intend to prescribe an antibiotic:“You often feel pressured; you know (when a relative asks), “Can you guarantee that my father doesn’t have this and this condition?”—in situations like that you might push the boundaries more than you intended to”.Our main finding was the physicians report of the clinical workflow pressure to commonly facilitate excess antibiotic prescription to be on the safe side. Workflow pressure was perceived to hamper (1) correct microbiological specimens and (2) the quality of advice that the ID specialists gave, potentially facilitating unnecessary antibiotic prescriptions. We also identified a need to debate ethical dilemmas of antibiotic prescribing in specific clinical encounters, particularly with terminally ill patients.The finding of clinical workflow pressure aligns with previous studies that have identified a high workload as a contributor to inappropriate prescriptions in hospitals ,17,18. IOur second most prominent finding was the physicians’ need for clinical certainty. This finding aligns with the findings of two systematic reviews that showed that fear of patient adverse outcomes causes’ antibiotic overtreatment ,19. ConsAnother finding was the physicians’ desire to do all they could for the present patient as a driver for an increased use of broad-spectrum antibiotics. However, the physicians sometimes had second thoughts about the benefit of their prescription , but they experienced that they did not have a choice when facing the patient. This sentiment echoes the ‘tragedy of the commons’ principle, which is known to lead to failure: If everyone thinks about themselves (misuse antibiotics now), all will suffer (increased AMR in the future) . In our In this study, we have aimed to identify factors, in a low-resistance context, that may help improve future ASPs. Factors identified in our context does not seem to diverge noteworthy from factors in other contexts , and thuAdditionally, our findings of structural deficiencies may be more tangible to implement in ASPs than changes in physicians’ behavior. Additionally, structure and behavior should be seen as integral to each other .For more long-term gain regarding AMR ethical questions in antibiotic prescriptions should be evaluated.The validity of our findings was strengthened as three researchers with different backgrounds  read the transcripts and agreed on the themes in the analytic process . To ensuOur study also has some limitations. First, one should always be careful to generalize the results from single center studies. Still, we believe the results from our hospital in a country with a low AMR represents a valuable supplement to similar exploratory studies from locations where AMR is far more prevalent. Several of the factors we identified may be relevant to aid in developing ASPs, particularly in settings of low antibiotic resistance rates and for high-intensity antibiotic prescribers in hospitals. Secondly, the recruitment of “frequent antibiotic prescribing physicians” resulted in a high representation of ID specialists and oncologists, indicating a potential selection bias. On the other hand, this has resulted in valuable viewpoints from two perspectives, i.e., from advice-seeking prescribers and advice-giving ID specialists. Finally, the principal investigator (IC) knew most of the respondents before the interviews, which may have affected their responses and her interpretations in the analytic process. We sought to limit such influence using reflexivity throughout the process .In conclusion, future ASP efforts should evaluate the system organization and prioritizations to address and manage potential time-pressure issues. To limit the use of broad-spectrum antibiotics, improving microbiology testing and the routines for consultations with infectious disease specialists seems beneficial. We also identified a need among the prescribing physicians for a debate on ethical antibiotic questions.This qualitative study, using semi-structured interviews, was conducted at Østfold Hospital Trust, a 380-bed, secondary, acute care hospital with a catchment area of 320,000 inhabitants. From 2016 all Norwegian hospitals began establishing ASPs and from 2017 and at the time of the study, the study hospital had an antibiotic stewardship team consisting of an infectious disease (ID) specialist, a clinical pharmacist, and a nurse trained in infectious diseases. This team mainly conducted surveillance of antibiotic use, revised antibiotic guidelines, and conducted education promoting rational antibiotic use. The national antibiotic guidelines were made available electronically and distributed as a pocket booklet. The recommendations are based on treatment tradition and national antibiotic resistance patterns. Narrow-spectrum antibiotics are recommended in several empirical regimens, e.g., benzylpenicillin is the first choice for community-acquired pneumonia, and benzylpenicillin or ampicillin combined with gentamicin is recommended for sepsis .In the study hospital, similar to most Norwegian secondary acute care hospitals, the physicians consult ID specialists by phone whenever they have complicated questions regarding infections, and antibiotic treatment.To participate in the study, we sought respondents that could give rich information on the factors affecting physicians’ antibiotic prescription. We purposively invited physicians that were prescribing antibiotics on a daily basis. Relevant respondents were chosen from lists over physicians in departments with high antibiotic use as documented by routine pharmacy data statistics and point-prevalence surveys of antibiotic prescriptions . We madeWe developed an interview guide based on recommendations by Kallio et al. , and seaThe interviews were conducted in a quiet room in the study hospital from November 2018 to February 2019. The objectives of the study were presented both orally and on a written consent form. All interviews were performed by IC, a female MD and Ph.D. student trained in qualitative methods and with clinical experience in surgery and oncology. The pilot interviews were conducted together with LPJJ, a professor of health science with prior extensive experience with qualitative research, to observe and give feedback. Since interviews were performed at the same hospital as IC was employed, both interviewer and interviewees were known to each other, either in person or as colleagues. Consequently, measures were taken to minimize the risk of bias .The interviews were audio-recorded, transcribed verbatim, and de-identified (I.C.). In the transcripts I.C. made remarks about the physicians’ body language, pauses in speech and other non-verbal communication, to provide more context to the transcripts. To minimize the risk of bias L.-P.J.-J. and J.B.H. consecutively reviewed and gave feedback on the interview transcriptions. The respondents were not automatically offered return of transcripts, unless they inquired it. One respondent did so and read and approved of the content. I.C. and L.-P.J.-J. coded the transcripts and themes derived from the data following the recommendations of Braun and Clarke . NVIVO s


Bone tissue engineering based on adipose‐derived stem cells (ASCs) is expected to become a new treatment for diabetic osteoporosis (DOP) patients with bone defects. However, compared with control ASCs (CON‐ASCs), osteogenic potential of DOP‐ASCs is decreased, which increased the difficulty of bone reconstruction in DOP patients. Moreover, the cause of the poor osteogenesis of ASCs in a hyperglycemic microenvironment has not been elucidated. Therefore, this study explored the molecular mechanism of the decline in the osteogenic potential of DOP‐ASCs from the perspective of epigenetics to provide a possible therapeutic target for bone repair in DOP patients with bone defects.AK137033 small interfering RNA (SiRNA) and an AK137033 overexpression plasmid were used to regulate the expression of AK137033 in CON‐ASCs and DOP‐ASCs in vitro. Lentiviruses that carried shRNA‐AK137033 or AK137033 cDNA were used to knockdown or overexpress AK137033, respectively, in CON‐ASCs and DOP‐ASCs in vivo. Hematoxylin and eosin (H&E), Masson's, alizarin red, and alkaline phosphatase (ALP) staining, micro‐computed tomography (Micro‐CT), flow cytometry, qPCR, western blotting, immunofluorescence, and bisulfite‐specific PCR (BSP) were used to analyze the functional changes of ASCs.An animal model of DOP was established in mice. CON‐ASCs and DOP‐ASCs were isolated from CON and DOP mice, respectively. AK137033 expression, the DNA methylation level of the sFrp2 promoter region, Wnt signaling pathway markers, and the osteogenic differentiation potential were decreased in DOP‐ASCs. In vitro experiments showed that AK137033 silencing inhibited the Wnt signaling pathway and osteogenic ability of CON‐ASCs by reducing the DNA methylation level in the sFrp2 promoter region. Additionally, overexpression of AK137033 in DOP‐ASCs rescued these changes caused by DOP. Moreover, the same results were obtained in vivo.The DOP mouse model was established successfully. Compared with CON‐ASCs, LncRNA‐AK137033 inhibits the osteogenic potential of DOP‐ASCs by regulating the Wnt signaling pathway via modulating the DNA methylation level in the sFrp2 promoter region. This study provides an important reference to find new targets for the treatment of bone defects in DOP patients. In this work, the diabetic osteoporosis (DOP) animal model was successfully constructed. DOP‐ASCs were successfully isolated and cultured from DOP mice. In vitro and in vivo studies have demonstrated that LncRNA‐AK137033 regulates the osteogenic potential of DOP‐ASCs by modulating the Wnt signaling pathway via DNA methylation in the sFrp2 promoter region. This systemic glucose metabolism disorder has a serious negative effect on the skeletal system by causing severe complications of the bone and joint system, ie, diabetic osteoporosis (DOP).Wnt signaling pathways are a group of multifunctional signal transduction pathways activated by the binding of the Wnt ligand to the cell membrane receptor. They participate in various physiological and pathological processes of cells, which include various bone tissue diseases such as osteoporosis and stem cell–related bone regeneration.Mammalian DNA methylation refers to methylation of the fifth carbon atom on cytosine in the CpG dinucleotide of DNA, which is catalyzed by four DNA methyltransferases, namely DNMT1, DNMT2, DNMT3a, and DNMT3b. It is generally believed that hypermethylation of DNA is related to the inhibition of gene expression, whereas DNA demethylation has the opposite effect.Protein coding genes have been studied extensively, but they account for only 1.5% of the human genome.In the present study, we subjected CON‐ASCs and DOP‐ASCs to mRNA/LncRNA expression profiling and MeDIP sequencing. The results showed a significant difference in the DNA methylation level of the promoter region of the Wnt signaling molecule sFrp2, which was related to LncRNA‐AK137033. Subsequently, we conducted functional studies of sFrp2 and LncRNA‐AK137033 in vivo and in vitro to explore the molecular mechanism that regulates bone differentiation of DOP‐ASCs from the perspective of epigenetics.22.1All procedures related to animal experiments were reviewed and approved by the Ethics Committee of Southwest Medical University (20180391222) and carried out in accordance with the guidelines of the Care and Use of Laboratory Animals . Four‐week‐old male C57BL/6 mice were purchased from the Experimental Animal Center of Southwest Medical University. Fifty mice were randomly divided into CON and DOP groups. The CON group was fed an ordinary diet, and the DOP group was fed a high fat and sugar (HFS) diet. The HFS diet consisted of 65% standard chow, 10% fat, 20% sucrose, 2.5% cholesterol, and other necessary additives . The weight and blood glucose of mice were measured every week. After 4 weeks, 50 mice were fasted for 12 h, followed by injection of streptozotocin  in the DOP group (140 mg/kg) or the same volume of citric acid‐sodium citrate buffer (140 ml/kg) in the CON group. After the injection, CON mice were raised on standard chow and DOP mice were still raised on the HFS diet. Additionally, all mice were fed under appropriate conditions (20–25°C with 65%–80% humidity) with free access to drinking water and food. The blood glucose and body weight of each group of mice were recorded every 3–5 days. After 4 months of feeding, the diabetic osteoporosis model was established. Until the end of the study, the mortality rate of STZ‐induced diabetic osteoporosis mice was 20%–33%.2.2Samples were fixed in 10% paraformaldehyde for 24 h, decalcified in a decalcification solution for about 1 month (this step was omitted for soft tissue), washed with tap water for 48 h, dehydrated with an alcohol gradient, embedded in paraffin, and sectioned. The samples were then stained with H&E and Masson's dye.2.3Samples were fixed with 10% paraformaldehyde for 24 h and then analyzed by a SCANCO Medical CT‐40 . Scanning parameters were as follows: voltage, 80 kV; current, 500 μA; exposure time, 200 ms; rotation angle, 220°; CT reconstruction method; and COBRA‐filtered back‐projection reconstruction.2.43 and evenly spread in a T25 culture flask. After covering the cap tightly, the flask was turned upside down and incubated at 37°C for about 5 min. Then, α‐modified Eagle's medium  with 10% FBS  was carefully added to the culture flask to completely immerse the tissue blocks. Subsequently, the tissue blocks were incubated at 37°C with 5% CO2 for 7–10 days to obtain passage 0 cells. The cells were passaged at 80%–90% confluence, and passage 3 cells were used in experiments.CON‐ASCs and DOP‐ASCs were obtained from inguinal subcutaneous adipose tissue of CON and DOP mice, respectively. An adipose block from the groin was cut into pieces of about 1 mm2.5Passage 3 ASCs was resuspended with PBS and stained for CD29, CD44, CD45, CD90, CD31, or CD34 with fluorophore‐conjugated antibodies at 4°C for 30 min. Unstained cells were used as a blank control. After washing with PBS, the cells were analyzed using a FACS Calibur flow cytometer .2.6AK137033, secreted frizzle‐related protein 2 (sFrp2), cadherin‐associated protein, delta 1 (β‐catenin), osteopontin (Opn), and runt‐related transcription factor 2 (Runx2) in ASCs after osteogenic induction. Primer sequences are presented in Table Quantitative polymerase chain reaction (qPCR) was used to measure the mRNA expression levels of 2.7Protein expression levels in ASCs after osteogenic induction were measured by western blotting. Total protein was extracted from ASCs using a total protein extraction kit  in accordance with the manufacturer's instructions. Protein samples were mixed with loading buffer and boiled for 5 min for denaturation. After separation by SDS‐PAGE, the protein samples were transferred to a polyvinylidene fluoride membrane by the wet transfer method and blocked in 5% dry skim milk at 37°C for 1 h. The blots were then incubated with diluted primary antibodies against sFrp2 (abs134753) , GSK‐3β (12456), p‐GSK‐3β (5558) , GAPDH (ab181602), β‐catenin (ab32572), OPN (ab8448), or RUNX2 (ab92336)  with shaking at 4°C overnight. The following day, the membranes were washed three times with Tris‐buffered saline with Tween‐20 (TBST) and then incubated with diluted HRP‐labeled goat anti‐rabbit or anti‐mouse secondary antibodies for 1 h. Then, the membranes were washed three times with TBST and developed with an enhanced chemiluminescence detection system .2.8Immunofluorescence staining was used to analyze the relative expression of RUNX2 and OPN protein in ASCs. CON‐ASCs and DOP‐ASCs after osteogenic induction were washed with PBS for three times, fixed with 4% paraformaldehyde for 30 min, and then permeabilized with 0.5% Triton X‐100 for 10 min. Subsequently, the samples were incubated with 5% sheep serum at 37℃ for 1 h and then incubated with diluted primary antibodies (anit‐RUNX2 or ‐OPN) overnight. The following day, the samples were rewarmed at 37°C for 1 h and then incubated with a diluted fluorescent dye‐conjugated anti‐rabbit secondary antibody  at 37°C for 1 h. The cytoskeleton and nucleus of ASCs were stained with phalloidin and 4′6‐diamidino‐2‐phenylindole, respectively. Fluorescence images of each sample were obtained under a confocal laser microscope .2.9AK137033‐specific SiRNA was synthesized by GenePharma , SiRNA sequences are shown in Table AK137033 overexpression, the AK137033 cDNA sequence was amplified and subcloned into a pcDNA3.1 vector. An empty pGFP3.1 vector that carried eGFP was used as the negative control.For SiRNA and the plasmid were transfected into ASCs using the Auto Electroporator system  in accordance with the manufacturer's instructions for in vitro experiments.AK137033‐specific overexpression, lentivirus and shRNA lentivirus were synthesized by Obio Technology . For AK137033 overexpression, the AK137033 cNDA sequence was amplified and subcloned into the pSLenti‐EF1‐EGFP‐F2A‐Puro‐WPRE2‐CMV‐MCS lentiviral vector. For AK137033 knockdown, oligonucleotides with AK137033 splice variant RNA interference targets were annealed and ligated into the pSLenti‐U6‐shRNA‐CMV‐EGFP‐F2A‐Puro‐WPRE lentiviral vector. The oligonucleotide sequences of shRNA with AK137033 RNA interference targets are shown in Table For AK137033 overexpression or AK137033 knockdown lentiviruses were transfected into ASCs in accordance with the manufacturer's instructions for in vivo experiments.2.10After 3 or 5 days of osteoinduction, ASCs were rinsed with PBS three times and then fixed with 4% paraformaldehyde at 4°C for 30 min. ALP activity was detected by an Alkaline Phosphatase Assay Kit  in accordance with the manufacturer's guidelines. Similarly, after 14 days of osteoinduction, ASCs were rinsed with PBS for three times and then fixed with 4% paraformaldehyde at 4°C for 30 min. Fixed ASCs were incubated in a 0.1% alizarin red solution for 30 min at 37°C to assess the formation of calcium nodules.2.115/cm2 and cultured in osteogenic induction medium for 48 h for subsequent in vivo experiments.Biphasic calcium phosphate (BCP) scaffolds were provided by Sichuan University Research Center. After autoclaving the scaffolds, ASCs infected with lentiviruses were seeded on BCP scaffolds at 2 × 102.12AK137033 knockdown (CON‐shRNA), BCP scaffold seeded with DOP‐ASCs (DOP‐B), BCP scaffold seeded with DOP‐ASCs infected with the overexpression lentiviral vector (DOP‐NC), and BCP scaffold seeded with DOP‐ASCs overexpressed AK137033 (DOP‐LVRNA). Fifteen CON mice were randomly divided into three groups, and fifteen DOP mice were randomly divided into three groups. A circular defect of 4 mm in diameter was made on the right calvarium of each mouse under sterile conditions. The prepared scaffolds were implanted into the bone defects (five mice in each group), and then, the muscle layer and skin were sutured. After the operation, the mice were fed under appropriate conditions (20–25°C with 65%–80% humidity).ASC‐seeded BCP scaffolds were prepared as the following groups: BCP scaffold seeded with CON‐ASCs (CON‐B), BCP scaffold seeded with CON‐ASCs transfected with the knockdown lentiviral vector (CON‐NC), BCP scaffold seeded with CON‐ASCs with 2.13p < 0.05.SPSS 19.0 software was used for statistical analysis. Data were tested by the t‐test or one‐way analysis of variance. Each experiment was repeated at least three times. The results are expressed as the mean ± standard deviation (SD). Data were significantly different at 33.1After STZ injection, we continuously observed changes in body weight and blood glucose in mice. The blood glucose levels of CON mice were <9 M; the blood glucose levels of mice in the DOP group were maintained at >16.8 M; and the weight of mice in the DOP group was lower than that in the CON group Figure . Compare3.2p < 0.05)  Figure .3.3AK137033, β‐Catenin, Runx2, and Opn were measured by qPCR. Additionally, ALP and alizarin red staining was used to detect differences in the osteogenic abilities of CON‐ASCs and DOP‐ASCs after 3 and 14 days of osteogenic induction. Compared with CON‐ASCs, the protein and gene expression levels of Wnt signaling pathway makers and osteogenesis‐related molecules, and the expression of LncRNA‐AK137033 were downregulated in DOP‐ASCs  Figure . Interesn Figure . These r3.5AK137033 and changes in the DNA methylation level in the sFrp2 promoter region, and AK137033 was highly expressed in CON‐ASCs. Therefore, we silenced AK137033 by specific siRNAs in CON‐ASCs and then detected the expression of mRNAs and proteins related to the Wnt signaling pathway. CON‐ASCs in the SiRNA group were transfected with si‐AK137033, and CON‐ASCs in groups B and NC were treated with ordinary osteogenic induction medium and a siRNA negative control, respectively. At 3 days after osteogenic induction, we performed qPCR and western blot analyses. Compared with B and NC groups, the Wnt signaling pathway markers and downstream osteogenesis‐related molecules in the SiRNA group were suppressed after AK137033 silencing  group  and overexpressed AK137033 in DOP‐ASCs through a specific LVRNA lentivirus (LVRNA group). After lentivirus infection, the effect was assessed by qPCR and western blotting after 3 days of osteogenic induction. The results showed that the shRNA lentivirus knocked down the expression of AK137033 in CON‐ASCs and the LVRNA lentivirus overexpressed AK137033 in DOP‐ASCs ; and BCP scaffold seeded with DOP‐ASCs (DOP‐B), BCP scaffold seeded with DOP‐ASCs infected with the overexpression lentivirus vector (DOP‐NC), and BCP scaffold seeded with DOP‐ASCs that overexpressed AK137033 (DOP‐LVRNA) were implanted in DOP mice. At 8 weeks after transplantation, the formation of mouse calvarial bone was detected by micro‐CT and histochemistry. Micro‐CT scanning showed that the knockout of AK137033 decreased the bone volume/total volume (Tb.BV/TV) and trabecular thickness (Tb. Th) in CON mice, while the bone surface area/bone volume (Tb.BS/BV) was increased. Conversely, in DOP mice, overexpression of AK137033 reversed the decrease in bone formation caused by DOP  affect bone modeling and reconstruction by regulating the energy metabolism and osteogenesis of osteoblasts.AK137033 expression level, Wnt signaling pathway difference, and osteogenic differentiation potential of CON‐ASCs and DOP‐ASCs in vitro. The results showed that, compared with CON‐ASCs, the AK137033 expression level, Wnt signaling pathway, and osteogenic differentiation potential were inhibited in DOP‐ASCs. Moreover, Meth Primer analysis and BSP results demonstrated that the methylation degree of CON‐ASCs was higher than that of matched DOP‐ASCs in the sFrp2 promoter region. The above results supported the mRNA/LncRNA expression profiling and MeDIP sequencing results. However, further functional studies are needed to demonstrate the relationship between the DNA methylation level of the sFrp2 promoter region and AK137033.The Wnt signaling pathway plays an important role in the differentiation and development of stem cells because of high conservation, structural complexity, and the ability of the developmental cascade to integrate the signals of other pathways.AK137033 in CON‐ASCs decreased the DNA methylation level in the sFrp2 promoter region, inhibited the Wnt signaling pathway, and suppressed the osteogenic differentiation potential of CON‐ASCs. Additionally, overexpression of AK137033 in DOP‐ASCs increased the DNA methylation level in the sFrp2 promoter region, activated the Wnt signaling pathway, and restored the osteogenic differentiation potential of DOP‐ASCs. Interestingly, mRNA/LncRNA expression profiling and MeDIP sequencing of CON‐ASCs and DOP‐ASCs also showed that the difference in the DNA methylation level of the sFrp2 promoter region between the two groups was related to LncRNA‐AK137033. These results suggest that AK137033 inhibits the osteogenic potential of DOP‐ASCs by regulating the DNA methylation level in the sFrp2 promoter region.LncRNAs are a kind of long‐chain RNA (more than 500 nt) that lacks a protein‐coding ability.AK137033 in vivo, a critical‐sized calvarial bone defect model in mice was established and ASC‐seeded BCP scaffolds were implanted. At 8 weeks after transplantation, micro‐CT and histochemistry showed that the knockdown of AK137033 reduced bone formation in CON mice and overexpression of AK137077 rescued the reduced bone formation ability of DOP mice caused by the hyperglycemic microenvironment. Therefore, we conclude that AK137033 regulates osteogenesis of ASCs in vivo.Studies have shown that the diameter of a mouse calvarial bone defect that cannot heal itself is 4 mm.In summary, in vitro and in vivo experiments demonstrated that LncRNA‐AK137033 regulates the osteogenic potential of DOP‐ASCs by modulating the Wnt signaling pathway via DNA methylation in the sFrp2 promoter region. Our study provides an epigenetic explanation for the regulatory mechanism of the osteogenic potential in DOP‐ASCs and is an important reference for the treatment of bone defects in DOP patients.The authors declare that there are no competing interests.All authors have made important contributions to this study. Shuanglin Peng conducted in vitro and in vivo experiments, sorted and analyzed the data, and wrote the main manuscript. Yujin Gao established the diabetic osteoporosis mouse model and cultured CON‐ASCs and DOP‐ASCs. Sirong Shi completed mRNA/LncRNA expression profiling, MeDIP sequencing, and in vitro validation experiments. Dan Zhao carried out in vitro experiments and data collection. Huayue Cao and Ting Fu assisted in the in vivo experiments. Xiaoxiao Cai designed the study and revised the manuscript. Jingang Xiao conceived and initiated the study, analyzed the data, and provided funding. All authors have read and approved the manuscript.