Denosumab has been suggested as a first-line therapy for osteoporotic patients. However, a standardized protocol for the prevention of denosumab induced medication-related osteonecrosis of the jaw (MRONJ) has not yet been established. The purpose of this study was to report denosumab induced MRONJ cases, and investigate the factors affecting the occurrence of MRONJ in patients who underwent denosumab and invasive dental treatment  between October 2016 and March 2020. Four of the 98 patients developed MRONJ before and after tooth extraction. The participants were divided into two groups: receiving only denosumab (n = 51) and receiving bisphosphonate as first treatment and denosumab as second treatment (n = 47). There was no significant difference between groups in the occurrence of MRONJ and factors affecting MRONJ. Two out of 4 patients developed MRONJ regardless of invasive treatment after denosumab administration and proceeded with extraction; one patient developed MRONJ after denosumab administration and extraction. The other patient underwent a tooth extraction without osteoporosis treatment, and non-identified MRONJ developed after denosumab administration. MRONJ cases reported in this study show that MRONJ can develop as chronic inflammation without invasive dental treatment; therefore, implementing preventive dental treatment before initiating denosumab treatment is necessary to reduce the occurrence of MRONJ. Denosumab is suggested as a first-line therapy for postmenopausal osteoporosis patients with high risk of fracture5. Compared to bisphosphonate’s mechanism of reducing bone resorption by inhibiting differentiation and maturation of osteoclasts and inducing apoptosis6, denosumab reduces bone resorption and increases bone strength by inhibiting the differentiation and function of osteoclasts as a single cell antibody against RANKL7. However, denosumab must also be evaluated in light of the issues surrounding the use of bone resorption inhibitors, including bisphosphonate. Particularly, medication-related osteonecrosis of the jaw (MRONJ) is one of the most controversial side effects of bone resorption inhibitors9.Bisphosphonates and the receptor activator of nuclear factor-κB ligand (RANKL) inhibitor denosumab are the most common anti-resorptive agents used in the treatment of osteoporosis10. Since bisphosphonate-related osteonecrosis of the jaw (BRONJ) was first reported in 2003, it continues to be of interest to dental clinicians and researchers12. However, due to independent reports of denosumab-related bone necrosis, the American Association of Oral and Maxillofacial Surgeons (AAOMS) revised the term BRONJ to MRONJ in 201410.MRONJ is defined based on three criteria: (1) current or previous treatment with antiresorptive or antiangiogenic agents; (2) exposed bone or bone that can be probed through an intraoral or extraoral fistula(e) in the maxillofacial region that has persisted for more than eight weeks; and (3) no history of radiation therapy to the jaws or obvious metastatic disease to the jaws14. However, there is insufficient evidence regarding the exact pathogenesis of MRONJ15. The most important systemic risk factor for MRONJ is administration of a powerful anti-resorptive agent, such as a nitrogen-containing bisphosphonate or a RANKL inhibitor. For the management of bone metastases in cancer patients, subcutaneous denosumab (120 mg every 4 weeks) was associated with an overall 1.7% risk of MRONJ and an increased risk of developing MRONJ in comparison to high intravenous doses of bisphosphonate (3 mg every 3–4 weeks)16. Local risk factors include dentoalveolar surgery , ill-fitting dentures, and existing inflammatory dental diseases 10.Various hypotheses have been put forth to explain why bone necrosis occurring after the administration of antiresorptive agents such as bisphosphonate or denosumab is limited to the jaws17. However, the AAOMS and American Dental Association (ADA) Council on Scientific Affairs found no evidence that interrupting bisphosphonate and denosumab therapy alters the risk of MRONJ in patients after tooth extraction18. Accordingly, given the lack of standard duration of the drug holiday for MRONJ prevention in osteoporotic patients with a history of denosumab therapy, there is a need for retrospective clinical cohort studies to develop prevention guidelines and protocols for MRONJ in these patients.Previous studies have noted that a 2-month drug holiday before invasive dental procedures should be adequateTherefore, we investigated patients diagnosed with osteoporosis who were administered denosumab (Prolia®) at the Department of Endocrinology at Severance Hospital , and underwent invasive dental treatment (tooth extraction) at the Department of Advanced General Dentistry and Oral and Maxillofacial Surgery at Yonsei University Dental Hospital . Accordingly, the aim of this study was to report denosumab induced MRONJ occurrence cases, and the factors affecting the occurrence of MRONJ in patients who underwent denosumab and invasive dental treatment at our institution.This study protocol was approved by the Institutional Review Board of the Yonsei University Dental Hospital . Written informed consent was waived, because of the retrospective nature of the study and use of de-identified participant data. This study was performed in accordance with the Declaration of Helsinki.We screened a total of 159 patients who received denosumab therapy at the Department of Endocrinology at Severance Hospital  and underwent invasive dental treatment at the Department of Advanced General Dentistry and Oral and Maxillofacial Surgery at Yonsei University Dental Hospital  from October 2016 to March 2020.The inclusion criteria were as follows: age > 20 years, diagnosis of osteoporosis, history of treatment with denosumab (60 mg administered as a single subcutaneous injection once every 6 months) or denosumab combined with bisphosphonate , and a history of invasive dental treatment  at the Department of Advanced General Dentistry and Oral and Maxillofacial Surgery at Yonsei University Dental Hospital for teeth with hopeless prognosis due to various reasons, including caries, periodontitis, and impaction. The exclusion criteria were as follows: no history of denosumab therapy before and after invasive dental treatment, administration of denosumab 120 mg (Xgeva®) instead of denosumab 60 mg (Prolia®), history of other bone metabolism disorders or administered drugs, secondary osteoporosis due to oncological dose administration in cancer patients, history of denosumab therapy for diseases other than osteoporosis , and a history of denosumab or invasive dental therapy at a different institution, preventing accurate evaluation.Of the 159 patients (297 teeth) screened, 98 patients (189 teeth) were included in the study and 61 patients (108 teeth) were excluded. The following patients were excluded: 29 patients (53 teeth) who received drugs other than denosumab both immediately before and after invasive dental treatment; 24 patients (43 teeth) who had received other drugs , except denosumab, immediately before invasive dental treatment; 5 patients (9 teeth) who received denosumab treatment or underwent invasive dental therapy at a different institution; 2 patients (2 teeth) with no history of elective tooth extraction (loss of teeth); and 1 patient (1 teeth) who received prophylactic treatment for osteoporosis.Participants’ age, sex, comorbidities, bisphosphonate administration history, bisphosphonate administration period before denosumab administration, number of denosumab dose before extraction, type of drug administered before and after tooth extraction, period from drug cessation to extraction, period from extraction to drug initiation/resumption, reason for extraction, location of extraction (maxillary/mandibular/multiple), and presence or absence of MRONJ were investigated retrospectively.For patients with MRONJ, location, staging, associated local factors, and the type of MRONJ treatment  were additionally investigated. Information on denosumab administration was obtained through prescription records, and electronic medical records were used to obtain information on the invasive dental treatments.2 test was used to compare proportions across levels of categorical variables. Due to the low incidence of MRONJ, 40 cases were randomly sampled to obtain stable results. In order to investigate the relationship between MRONJ and comorbidities, multiple logistic regression was used. P-value < 0.05 was considered statistically significant for all analyses. Statistical tests were performed using SPSS statistical software .Patient demographics are expressed as n (%) and mean ± standard deviation. The χThe baseline characteristics of the 98 participants surveyed in this study, including the average age, sex, and the comorbidities of the patients before tooth extraction are presented in Table The participants were divided into two groups, one receiving only denosumab  and another that received bisphosphonate as the first treatment and denosumab as the second treatment   of the 52 patients received denosumab after tooth extraction, 2 (3.8%) received bisphosphonate, 2 (3.8%) received selective estrogen receptor modulator (SERM), and 11 (21.2%) did not receive any drug. The mean period from tooth extraction to drug initiation or resumption was 7.7 ± 9.1 months. Residual roots, dental caries and other endodontic lesions were the main reasons for tooth extraction, the extraction site was maxillary in 26 patients (50.0%), mandibular in 18 (34.6%), and both in 8 (15.4%).In BP + Dmab group, the mean age was 72.04 ± 8.13 years which was higher than that in the Dmab group. Bisphosphonate was administered for an average of 43.85 ± 47.80 months before denosumab administration. Denosumab was administered on average 2.50 ± 1.61 times before tooth extraction. The mean period from drug cessation to extraction was 6.28 ± 3.86 months. Sixteen (34.8%) of the 46 patients received denosumab after tooth extraction, 2 (4.3%) received bisphosphonate, 6 (13.0%) received an SERM, 3 (6.5%) received teriparatide, and 19 (41.3%) did not receive any drug. The mean period from tooth extraction to drug initiation or resumption was 2.56 ± 2.03 months. The main reason of tooth extraction was residual root, dental caries, and other endodontic lesions in 23 (50.0%) patients, followed by periodontitis in 20 (43.5%) patients. The extraction site was maxillary in 24 patients (52.2%), mandibular in 18 (39.1%), and both in 4 (8.7%).Four of the 98 (4.1%) patients developed MRONJ in this study. Two patients belonged to the Dmab group and 2 patients belonged to the BP + Dmab group. Two of 4 patients developed MRONJ before tooth extraction, and 2 patients developed MRONJ after tooth extraction.The factors affecting MRONJ development are presented in Table Among 4 patients with MRONJ, 2 had hypertension, 2 had diabetes, and 1 had rheumatoid arthritis . Two of 46 patients with a history of bisphosphonate administration developed MRONJ, and 2 of 52 patients without a history of bisphosphonate administration developed MRONJ. Among 98 patients, 73 (74.5%) patients administered denosumab before tooth extraction and 53 (54.1%) patients administered denosumab after tooth extraction. One of 3 patients who developed MRONJ had tooth extraction within 1 month after denosumab administration, 1 patient had tooth extraction 6 months later, and 1 patient had tooth extraction after > 6 months. Among the patients who received denosumab after tooth extraction, one developed MRONJ > 6 months after tooth extraction. There was no significant difference between the occurrence of MRONJ and factors affecting MRONJ.A multiple logistic regression showed no association between the comorbidities of cancer, hypertension, and diabetes and the occurrence of MRONJ Table . HoweverFour patients were diagnosed with osteoporosis in this study population. Following bisphosphonate and denosumab therapy, MRONJ developed before extraction in Cases 1 and 2. In Case 3, after tooth extraction following only denosumab therapy, MRONJ had occurred. In Case 4, the patient had no history of osteoporosis treatment at the time of extraction, and developed MRONJ afterwards, following denosumab administration 1 year after extraction  therapy from March 2015 to March 2016 due to osteoporosis. From October 2017 to October 2018, she received a total of three denosumab doses. In December 2018, there was severe tooth mobility due to localized chronic advanced periodontitis, and extraction was planned to be performed after ≥ 3 to 6 months. Non‐surgical periodontal therapy  was performed before extraction; however, MRONJ was diagnosed in the right mandibular region 6 months after the last denosumab administration. The drug was changed to Calcitriol [0.25 mcg/Soft Cap], and extraction and sequestrectomy were performed after lesion localization . One year later, denosumab was administered once. Six months thereafter, MRONJ occurred in the left maxillary region in the presence of localized chronic advanced periodontitis, and bone necrosis was observed up to the mesial root site from the first premolar to the second molar in the radiographic image. Approximately 4 weeks later, tooth extraction and sequestrectomy of the affected area were performed, followed by prosthetic rehabilitation of the missing teeth  was found on the lingual side of the extraction site. With denosumab clearing up around October 2019, the area was observed during follow-up and treated conservatively  in June 2020  of MRONJ occurred22. In animal studies, lesions similar to MRONJ have been reported in cases of periapical inflammation and periodontitis23. However, there is still a lack of clinical research on denosumab-induced MRONJ and its incidence, and no protocol has been established yet for invasive dental treatment of the patients receiving this drug.Various factors have been implicated as causes of MRONJ, but recently, it was suggested that anti-resorptive agents affect the immune function of the bone. Tooth extraction has been reported as a predisposing factor of MRONJ in approximately 45–61% cases, but the prevalence and incidence rates of other spontaneous occurrences, which are the second largest factor and present as periodontal or periapical lesions, implants, or dentures, as well as extractions, have not been reported24, and existing inflammatory lesion such as periodontal and apical disease are known risk factors25, but that for Dmab-induced MRONJ has not yet been reported.In this study, we reported four cases of MRONJ in patients using denosumab, out of 98 patients who received either only denosumab injections or with bisphosphonate and who underwent tooth extractions. Although all four cases have something in common, they are cases of MRONJ occurring after denosumab administration, each situation is distinct. Two of the four MRONJ cases (Cases 1–2) had a history of bisphosphonate treatment. As for predisposing local factors of MRONJ occurrence, Case 1 and 2 are due to worsening of the chronic inflammation of the existing lesion after the denosumab administration, Case 3 was due to tooth extraction, and Case 4 as a non-identified occurrence. This means that chronic active periodontitis may have been a significant risk factor in the two cases, suggesting the importance of preventive dental treatment. Marx et al. reported that advanced periodontitis was the cause of bone exposure in 28.6%27. In previous studies, it was reported that osteonecrosis occurred when anti-resorptive agents were administered to animal models that induced pulpal and periodontal inflammation31. Cheong et al. observed an increase in bisphosphonate uptake at the apex in mice with periapical disease, and suggested that this may be associated with ONJ development32. Recently, Kim et al. reported that the incidence of osteonecrosis decreases if pre-inflammatory conditions are removed prior to extraction and emphasized the importance of pulpal and periodontal disease as local factors in the occurrence of MRONJ23. Osteonecrosis occurs when bisphosphonate or RANKL inhibitors are administered in the presence of inflamed pulpal or periodontal tissue, because anti-resorptive agents affect the function of various immune cells, such as of neutrophils and polymorphonuclear leukocytes, macrophages, and dendritic cells38.Several hypotheses have been raised regarding the occurrence of MRONJ in sites of chronic inflammatory lesions, and an M1 macrophage shift is considered the most likely explanation40. Zhang et al. reported that IL-17 mediated M1/M2 macrophage alteration is related to the development of BRONJ35, and Tamaki et al. reported that dynamic M1/M2 macrophage polarization was induced by the anti-RANKL antibody in a MRONJ model41. In this context, Hoefert et al. reported that patients with MRONJ had a compromised macrophage function when compared to patients with osteoradionecrosis and osteomyelitis37, and Paschalidi et al. found M1/M2 macrophage polarization in macrophages of patient tissues in MRONJ, and that macrophages shift to the M1 phenotype at stages 2 and 3, as compared to stage 1 of MRONJ26.The most recently studied immune cells are macrophages, and a particular focus on macrophage polarization. Polarization refers to the different ratio of M1 macrophages having pro-inflammatory and antimicrobial properties and M2 macrophages having anti-inflammatory propertiesIn Case 3, the extraction was performed one month after the administration of a single denosumab dose injection. MRONJ developed four months after surgery. The lesion was classified as grade 2 MRONJ, and cured with a five-month conservative treatment. This case suggests a one month time between the denosumab injection and extraction may be too short.24. Non-identified occurrence is the third most common group of MRONJ cases reported in the literature, with an estimated prevalence between 16 and 70%42. Most of these non-identified occurrences were found to occur in relation to the lower posterior tooth region, but the lesion described in Case 4 developed anteriorly. In this case, the teeth with severe mobility and periodontic-endodontic lesion had been extracted. Although the lesion was completely removed during the operation, MRONJ occurred at the surgical site 21 months after extraction. Therefore, we classified this case as a non-identified occurred MRONJ, not an occurrence in an existing lesion. Importantly, a non-identified MRONJ occurrence poses the risk of a pathological fracture of the jaw after denosumab administration.The patient in Case 4 had no history of bisphosphonate administration, and the development of MRONJ was “non-identified” after two administrations of denosumab a year after extraction. The most common causes of MRONJ are tooth extraction and obvious periodontal disease, followed by “non-identified” occurrences with no identifiable causeIn this study, the incidence of MRONJ after denosumab administration was 4.1% and no statistically significant factor was found that influenced the incidence of MRONJ in patients receiving denosumab treatment. To prevent MRONJ, which degrades the patient's quality of life, both the risk of fracture and osteonecrosis of the jaw risk should be considered. In case of the risk of fracture and ONJ are high but invasive dental treatment is required, the discontinuation of denosumab should be considered. However, denosumab increases fracture risk upon discontinuation of treatment, so teriparatide can be prescribed if not contraindicated or alternative osteoporosis drugs should be prescribed during discontinuation of treatment, and monitoring is required.This study provides information on the incidence of MRONJ in patients with denosumab but has limitations. First, it was a retrospective study using data from a single institution. In addition, bone turnover markers (BTM) were not investigated because all participants' BTMs were not measured at the same time. However, clinical data only makes it difficult to predict the occurrence of MRONJ and requires another predictive factor, therefore molecular biomarkers need to be included in future research.Because it is difficult to predict MRONJ occurrence in patients under denosumab therapy, oral healthcare before, during, and after denosumab treatment is essential. Before the initiation of denosumab therapy, it is necessary to control existing inflammatory lesions through the preventive dental care. During denosumab therapy, it is important for both physicians and dentists to have common consensus of drug changes according to the risk of fracture and osteonecrosis when performing invasive dental treatment. In addition, continuous periodic dental management needs to be performed during denosumab treatment to prevent “non-identified” MRONJ. In the future, further multi-center or molecular biological studies are needed.Supplementary Figures.


This study aimed to determine the impact of ancient wheat varieties  and spontaneous sourdough fermentation on the bioaccessibility of total phenolic content (TPC) and the DPPH antioxidant capacity evolution during breadmaking and in vitro digestion. Sourdough and yeast-fermented modern wheat breads were used as controls. After 6 h of fermentation, the total titrable acidity of the sourdough increased from 139 to 167%. The wheat variety, type of fermentation and processing affected TPC, antioxidant activity and bioaccessibility. Antioxidant activity and TPC were reduced by dough mixing, increased after sourdough fermentation and slightly decreased or remained the same after baking. Although wheat flour had the highest TPC, the modeling of TPC kinetic revealed that emmer and spelt sourdough exhibited a higher bound phenolics release rate due to the higher acidity, which contributed to increased phenolics solubility. Although wheat bread, both before and after digestion, had the lowest TPC, especially the one prepared with yeast, high TPC bioaccessibilities and antioxidant activities after the digestion suggested that, except phenolics, digestion process improved the release of additional compounds with different bioaccessibility and biological activity. The results of this study proved that the application of sourdough fermentation can increase the potential of ancient wheats in the developing of functional bakery products. Triticum aestivum) represents one of the major cereal crops in the world with the annual global production in 2020 of approximately 760 million tons [Common wheat  to bread. The investigation was performed on both modern and ancient wheats  in order to evaluate the impact of variety on the fate of polyphenols and antioxidant capacity during breadmaking. The role of sourdough on polyphenols and antioxidants distribution during processing was studied by comparing wheat sourdough bread with bakery yeast fermented bread.Triticum aestivum L.) cultivar “Pobeda” served as a modern wheat variety. Interviews with a local organic producer revealed that emmer, spelt and khorasan seeds were non-certified, and were self-produced by farmers. All wheat samples were grown in the same location and weather conditions. Grains were stored for 6 months in the storage and then dehulled using a Heger’s large-scale friction de-huller  and finally milled by a large-scale stone mill Osttiroler Getreidemühlen . Moisture, protein, total dietary fiber and ash content of the obtained flour samples were determined according to ISO 712:2012, ISO 20483:2013, AOAC 985.29 and ISO 2171:2012, respectively. The obtained results are presented in Emmer, spelt and khorasan grains were donated from a local certified organic producer , while Serbian winter wheat  and salt which were used in the breadmaking procedure were obtained from the local market.Spontaneous fermentation of tested wheat flours was carried out using a backslopping procedure  in a laboratory incubator  at 25 °C. Mature sourdough was subsequently used as a “starter” instead of bakery yeast in the further breadmaking steps. In order to prepare the starter for breadmaking and further analysis, mature starter was mixed with flour and demineralized water in a 1:2:2 starter: flour: water ratio and incubated in a laboratory incubator at 25 °C for 6 h. This period of time was previously determined as a time needed for a preparation a mature sourdough starter. Dough samples were taken every 2 h during this 6 h of fermentation procedure. Samples at 0 h and 6 h were subjected to TTA measurements. Briefly, after the homogenization of dough samples (10 g) with distilled water (90 g), TTA was determined and expressed as the amount (mL) of 0.1N NaOH to achieve a pH of 8.5. Subsequently, dough samples  for each flour variety  were collected and frozen at −80 °C before the further freeze-drying procedure.The scheme of the experimental procedure used in this study is illustrated in Dough samples containing sourdough instead of bakery yeast as a leavening agent were prepared by replacing 25% of the flour with the appropriate type of sourdough in order to keep the total flour amount at 300 g required for the Farinograph mixing bowl . An amount of 5.4 of salt and demineralized water up to 400 BU consistency were added and mixed in a Farinograph mixing bowl during the seven minutes. Control dough sample was prepared with 300 g of modern wheat flour, 7.5 g of bakery yeast, 5.4 g of salt and demineralized water in the amount required to achieve a consistency of 400 BU. Prepared dough samples were then left for fermentation in a cabinet at 30 °C during the 30 min, following by the punching down step. Dough samples were then rested and punched down an additional 3 times. Subsequently, 120 g of dough samples were hand-molded and transferred into teflon pans (L × W × H: 80 mm × 50 mm × 50 mm) for the final proofing step which was carried out at 30 °C and a relative humidity of 85% up to the optimum volume increase, which was 65 min for control dough and approximately 3 h for sourdough-containing samples.Baking was performed in a modular deck oven  at 220 °C until a mass loss of 8% was reached. The obtained bread samples were then left to cool down to room temperature during the 2 h, cut to 10-mm thickness slices and placed in a laboratory freezer at –80 °C for further analysis. Two batches per each tested bread sample were prepared.To simulate the physiological process in the digestive tract , 5 g of ground fresh bread sample was subjected to GI digestion conditions in vivo  according to assay described by Brodkorb et al. . BrieflyThe lyophilization of previously freezed dough and bread samples (–80 °C) was carried out using an Alpha 1-2 LDplus freeze dryer  for 24 h for bread samples and 48 h for dough samples. Dried samples were homogenized using laboratory mortar, transferred in sealed polyethylene bags and stored at −18 °C prior further analyses.For the determination of total phenolic content and antioxidant activity gallic acid, Folin–Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid (Trolox) were purchased from Sigma Aldrich, Germany. All other used chemicals were of analytical grade.Prior to extraction, samples were milled in a kitchen blender in order to obtain homogenous sample. Ethanol was selected as a green solvent and as it was previously reported by several authors it shows the highest efficacy in the extraction of the total phenolic compounds from cereals ,26,27.Extraction from flour, dough and bread samples was performed using a two-step extraction assisted with the ultrasound. One gram of the sample was mixed with 20 mL of solvent  in extraction tubes using ultrasonic bath , operating at a frequency of 45 kHz for 10 min as was previously suggested in the research conducted by Milićević et al. . The supThe total phenolic content in prepared extracts and digests was determined using colorimetric assay with Folin-Ciocalteu reagent . The mixThe antioxidant activity of samples was evaluated by DPPH radical scavenging activity assay. The absorbance of samples was measured at 515 nm after 60 min of standing in the dark. The antioxidant capacity was calculated from the Trolox calibration curve, and results were expressed as a Trolox equivalent (μmol TE/g dw). All experiments were performed in three replicates.A is total phenolic content or DPPH antioxidant capacity of sourdough, A0 indicates the initial value of the parameter A, t is the fermentation time and k is the rate constant for each wheat variety. The individual measurements were used instead of mean values of triplicate experiments and the results were expressed as mean values of A0 and k.The kinetics of phenolic content and antioxidant capacity change during 6 h of sourdough fermentation were described by fitting a first-order kinetic model (Equation (1)) to the experimental data:D represents total phenolic content or antioxidant capacity after in vitro digestion, while B represents total phenolic content or antioxidant capacity before in vitro digestion. The bioaccessibility of total phenols and antioxidant capacity was evaluated in triplicate.The bioaccessibility index of total phenols and antioxidant capacity was calculated according to Andrade et al.  using thFactorial ANOVA combined with post hoc Tukey HSD test (α = 0.05) were performed using Statistica 10 software .W. confusa and Ped. pentosaceus/acidilactici, emmer flour harbored a few species of lactic acid bacteria, with the predominance of Lact. plantarum isolates. According to Van der Meulen et al. [L. fermentum was found in stable wheat sourdough ecosystems, whereas it was not encountered in spelt sourdoughs.In order to get insight into the growth and metabolic activity of the microbiota during spontaneous sourdough fermentation, progress of the TTA from initial (0 h) to final (6 h) fermentation point was monitored . The mosn et al. , L. fermThe evolution of the phenolic content during the whole process of the sourdough breadmaking from different wheat varieties is presented in In general, some of the authors underlined that variations in TPC are much more according to growth location, agronomic practices and environmental conditions than wheat variety , which eUpon mixing the flour with water and part of the mature sourdough , a decreDuring sourdough fermentation from 0 h to 6 h, a significant increase in TPC was recorded in all samples  which coA0 parameter in Furthermore, the extent of the increase in TPC during 6 h of sourdough fermentation depended on the wheat variety, where emmer exhibited the highest rate constant (k) according to the results of the modelling of kinetics of phenolic content change during 6 h of sourdough fermentation . In geneUpon baking, a decrease in TPC was observed compared to 6 h fermented sourdough in all the tested samples . HoweverThe evolution of DPPH antioxidant capacity from flour to bread is presented in In general, the trend of DPPH antioxidant capacity change during breadmaking quite resembled the one observed for TPC content . HoweverThe bioaccessibility assay of TPC and antioxidant capacity, performed in this study, has been used to enhance the knowledge of the behaviour of these compounds. After the simulated in vitro digestion process, the TPC and antioxidant activity for the DPPH method ranged from 3788 to 5302 μg GAE/g dw and 14.26 to 15.17 μmol TE/g dw, respectively , which wIn addition, a study performed by Venturi et al.  revealedThe fact that the control bread, prepared in this study, exhibited the lowest TPC both before and after digestion suggested that sourdough fermentation represents a useful tool in increasing polyphenols release thus leading to creation of functional bakery products.The results of this study revealed that sourdough fermentation, along with the incorporation of ancient wheat varieties, represents an important tool in the development of functional bakery products with increased antioxidant capacity and phenolics bioaccessibility. Sourdough fermentation improved the release of bound phenolics, even from the flours in which the initial free phenolics content was lower. This could potentially contribute to the avoiding of antioxidants addition in food formulation. In general, antioxidants are food additives which are negatively preserved by consumers. Therefore, their replacement would result in “clean label” product design without compromising its oxidative stability. Moreover, high phenolics bioaccessibilities and antioxidant activities after in vitro digestion suggested that, except phenolics, the digestion process improved the release of additional compounds with different bioaccessibility and biological activity, which is beneficial for health and well-being.


Body condition scoring (BCS) and body weight (BW) are observations associated with labile tissue reserves, health, and reproduction efficiency of dairy cows. The effect of parity (1 through to ≥5) and feeding system (pasture-based and TMR) on BCS and BW were evaluated utilizing raw data sets from 16 retrospective studies that totaled 24,807 Holstein cows across 3 nations . Linear regression models were used to investigate the 5 outcome variables of precalving BCS, peak milk BCS, change in BCS from precalving to peak milk, and peak milk BW and their respective associations with parity and feeding system. To help control for the influence of calendar time, study treatment protocols when applicable, and genetic change, all outcome variables were center-transformed around each study group mean. Including feeding system as a covariate improved model fit for most outcome variables; however, the relative effect size of parity was generally much greater than feeding system effect size. Parity 2 cows had the lowest precalving BCS of −0.087  less than the mean, whereas parity 1 cows had the greatest, 0.068  above mean, regardless of feeding system. Peak milk BCS overall decreased with increasing parity  and BCS change during the transition period monotonically decreased with increasing parity . Peak milk BW monotonically increased with increased parity . A waffle plot was used to present the proportions of cows, by parity, that were partitioned into “low BCS and low BW,” “low BCS and high BW,” “high BCS and low BW,” or “high BCS and high BW” groups. Cows were assigned either a high or low status by being above or below their specific centered study group means, respectively. Considering a null hypothesis of 25% per BCS-BW category, there was a striking change in category from parity 1 cows that were predominantly in the “high BCS and low BW” category (61.2%) to parity ≥5 cows that were predominantly in the “low BCS and high BW” category (55.5%). The study supports studies showing increased weight and change in BCS with increased parity. We highlight the associations among production system, BCS, BW, and parity. Body condition score in dairy cattle is a rapid visual assessment that is highly associated with available energy reserves, primarily mobile fat reserves . Though Body weight also reflects the protein, fat, and energy reserves of cattle . There iIn this study we examine associations of parity with precalving and peak milk BCS, changes of BCS between precalving and peak milk observations, and peak milk BW for Holstein cows in different production systems (pasture-based or TMR) across 3 countries using retrospectively collected data. We hypothesized that BCS and BW of dairy cows are associated with cow parity and that BCS and BW are different between the management system types of TMR or pasture-based systems.The studies used to produce the database for this study were approved by the relevant Animal Care and Use Committees at the time of conduct.A convenience sample of 16 amalgamated data sets from previous studies were used in this study. In general, commercial dairies were purposively selected for use in the original studies based on good record keeping and a history of performance that suggested capability of maintaining attention to detail congruent with successful trial conduct. The study contributors had conducted large, prospective studies that allowed a rigorous evaluation of the original study hypotheses. Only Holstein or Holstein-Friesian cattle  were included, as there were few other cow breeds in the database.Individual study inclusion criteria were being an observational or randomized controlled trial that provided details on parity and either BCS or BW or both. Further details of individual studies that met the criteria are included in The outcome variables investigated were precalving BCS, peak milk BCS and BW, and change in BCS between precalving and peak milk observations. One precalving (−30 to 0 DIM) BCS data point and one peak milk (30 to 110 DIM) BW and BCS data point were entered into the database per cow per study, with only one cow per study. When there were multiple precalving BCS measures per cow, the closest data point to calving was chosen. The peak milk BW and BCS values were determined by the observations obtained at the closest postcalving time point to peak milk, as estimated from herd test or weekly milk yield data. Cows were not required to have a data point for all BW and BCS variables. When an intervention was applied in a herd according to the original study's protocol, treatments within the herd were considered as separate groups for statistical analysis purposes. All outcome variables were center-transformed around the study group mean to control for individual study treatment effects on study outcomes and help mitigate the effects of temporal, genetic, and other clustered, unobserved covariates between each study group.All statistical analysis was conducted using Stata Version 16 (StataCorp). Initial data evaluation included tabulation of data and visual appraisal of BCS and BW for normality of distribution.power twomeans). The unit of interest in this study was the cow and statistical significance was set at an α of 0.05.As the database constructed for this study utilized retrospective data, it was possible to compute the statistical power achieved post hoc. Adjustments were made for DIM and feeding system and clustering of study groups, with α set to 0.05. Centered peak milk BCS in heifers was 0.08  and −0.07 for parity ≥5 cows . Post-hoc power was calculated as 99.98%  were used to evaluate the effects of parity and system on BCS, BW, and BCS change between precalving and peak milk. The effects of group were controlled by either assigning group as a random effect in mixed or clustered-robust standard errors in regress, with the model structure being ultimately determined by minimizing Akaike information criterion (AIC). Nesting of group within feeding system was investigated but did not explain group variation beyond a 2-level model. Days prepartum or DIM at time of BCS assignment/BW observation were investigated as covariates, as either continuous, polynomial, categorical, or centered-mean variables. Interactions between system, parity, and time of observation were evaluated. A manual, forward stepwise model was used with model selection determined by minimum AIC. Regression residuals were formally tested for normality with the Shapiro-Francia normality test and heteroskedasticity visually assessed with residual versus fitted value figures.Ordinary least-squares linear regression and multilevel mixed-effect linear regression , with a total of 24,807 cows. There were 89.1%  fed in TMR systems and 10.9%  pasture based. For the TMR herds, there were 34.7% parity 1 , 29.9% parity 2 , 18.8% parity 3  9.4% parity 4 , and 7.2% parity ≥5 . In the pasture-based herds, there were 25.6% parity 1 (n = 691), 22.3% parity 2 (n = 600), 21.1% parity 3 (n = 569), 12.7% parity 4 (n = 341), and 18.3% parity ≥5 (n = 494). There were 12.9%  enrolled cows in AU, 22.5%  in CAN, and 64.6%  in the United States. In all cases, the intraclass correlation of study group was best controlled with clustered-robust standard errors in simple linear regression models, according to AIC.The precalving BCS  was evaluated in 12,168 cows across 212 study groups. Parity 1 cows had significantly greater BCS than all other parities, whereas parity 2 cows had significantly less BCS than all other parities, regardless of system of management . Parity P = 0.06) in peak milk BCS from parity 1 to parity 2 cows observed in TMR herds  compared with pasture-based herds  was assessed in 4,694 cows in 32 study groups. Body weight increased monotonically with increasing parity and was significantly different for all pairwise parity comparisons. Despite parity 1 cows not being different with regard to their predicted centered BW across management systems , the interaction of parity and management system shows a greater spread around the centered BW in TMR herds  compared with pasture-based herds  .The distributions of cows with observations for both BCS and BW at peak lactation were categorized as being above or below the centered group mean for each respective variable and graphically depicted in a waffle plot . GeneralThe finding of increased proportion of BW and lower BCS, with increased parity may have importance in identifying pathways that influence reproduction and health. The labile protein reserves are considered important determinants of health, reproduction, and production . CharactLimitations to this study include using single time point observations for BCS and BW. It is known that the nadir of body condition loss during early lactation is different across parities  and is nBody condition loss during the transition period is strongly associated with cow parity, with older cows losing a much greater amount of labile energy reserves than younger cattle. Parity 2 cows had lower BCS results than expected at both precalving and peak milk observations. The use of waffle plots to visualize BCS-BW is unique and easily highlights the dramatic change in the body as cows age.


Polyurethane (PU) is an indispensable part of people’s lives. With the development of polyurethane, the disposal of polyurethane waste has become a significant issue around the world. Conventional degradation catalysts have poor dispersion and low degradation efficiency when used in the process of solid degradation into liquid. Therefore, this paper innovatively adopts self-made core–shell nanoscale titanium catalysis, traditional alkali metal catalyst (KOH), and polyol to carry out the glycolysis of waste polyurethane (PU) pipeline foam. The homogenized nanoscale titanium catalyst coated with alcohol gel has an obvious core–shell structure. The alcohol gel not only protects the catalyst but also dissolves with the alcoholysis agent in the process of glycolysis and disperses more evenly into the alcoholysis agent to avoid the phenomenon of nanocatalyst agglomeration, so as to facilitate catalytic cracking without reducing catalyst activity. In this study, investigated and compared the production of renewable polyurethane foam via a one-step method based on use of a homogeneous core–shell nanostructured titanium catalyst vs. a traditional alkaline catalyst in terms of the properties of regenerated polyether polyols as well as of the foams produced from these polyols. The physicochemical properties of regenerated polyether polyols that were analyzed included viscosity, hydroxyl value, and average molecular weight. The regenerated polyurethane foams were characterized based on water absorption, TG, SEM, and thermal conductivity analyses. The results show that, when the addition of homogeneous titanium catalyst was T2 0.050 wt.%, the viscosity of regenerated polyether polyols was the lowest, at 5356.7 mPa·s, which was reduced by 9.97% compared with those obtained using the alkali metal catalyst (KOH). When the amount of titanium catalyst was T3 0.075 wt.%, the hard foam made of regenerated polyurethane prepared by the catalyst showed the best properties, with a compressive strength of 0.168 MPa, which is 4.76% higher than that of the foam prepared using KOH catalyst. Polyurethane (PU) materials first emerged in the 1930s in Germany ,3. In thIzotz Amundarain et al.  studied Catalysts play an important role in polyurethane degradation and recycling process as a bridge between grafted raw materials and products , and catIn this paper, a homemade titanium nanocatalyst was used to explore the degradation of waste polyurethane pipe foam, which has the characteristics of high efficiency and high activity, good dispersion, few side reactions, nanoscale size, and high catalytic efficiency . In addiIn this study, the chemical recovery of waste polyurethane foam was carried out using the alcoholysis method, and the source of the regenerated polyether polyols was waste polyurethane foam. As they have certain rigid components (MDI/TDI/PAPI), the recovered polyether polyols also have highly rigid structures. With substitution (30 wt.%), the regenerated polyurethane foam prepared using conventional commercially available polyether polyols has a higher compressive strength and higher modulus than that prepared using conventional foams. The considerable amount of replacement not only reduces the cost of the production process but also realizes global recycling strategy concepts of turning plastic waste into useful material and industrial waste recycling .The main reagents used in this experiment for recycling waste polyurethane are as follows: waste polyurethane direct buried pipe rigid foam, Daqing direct buried pipe foam, refrigerator waste foam; ethylene glycol, Tianjin Chemical Reagent No.1 Factory, analytically pure (AR); propylene glycol, Tianjin Chemical Reagent No.1 Factory, analytically pure (AR); butylene glycol, Tianjin Kaitong Chemical Reagent Co. Chemical Reagent Company , analytically pure (AR); ethanolamine, Langfang Quanzhen Chemical Co., Ltd., (Langfang China), AR; potassium hydroxide, alkaline catalyst, Tianjin Tianli Chemical Reagent Co., (Tianjin China), Tetrabutyl titanate, Nanjing Youpu Chemical Co., Ltd., , AR; anhydrous ethanol, deionized water.The main reagents used in the foaming experiment of recycled polyether polyol from degraded waste polyurethane are as follows: silicone oil stabilizer, Guangzhou Feirui Chemical Co., , Triethanolamine, Shanghai Demao Chemical Co., Ltd., ; tin solution, Beijing Zhongnuo Taian Technology Co., Ltd. ; blowing agent HCFC-141b, Shenzhen Huachang Chemical Co., Ltd. ; polyether 4110, Shandong Lianhao Yao New Material Co Ltd., ; tin solution, Beijing Zhongnuo Taian Technology Co., Ltd. ; blowing agent HCFC-141b, Shenzhen Huachang Chemical Co., Ltd. ; polyether 4110, Shandong Lianhao Yao New Material Co.In this experiment, 43.2 g (0.152 mol) of tetrabutyl titanate and 260 g (5.643 mol) of anhydrous ethanol were added into a 2 L three-necked flask, and a mixture of 13.5 g of deionized water and 43 g of anhydrous ethanol was slowly added dropwise via a dosing tube under the conditions of incubation in an oil bath at 30 °C and high-speed stirring, and the addition was controlled such that the desired amount was added dropwise over a period of about 3 h. The crude intermediate product was obtained, which was then refluxed at the appropriate temperature for 3 h and added to ethylene glycol followed by resting; finally, vacuum distillation was carried out at a certain temperature to obtain the colorless or light yellow titanium nano catalyst liquid. The route for synthesis of the titanium nanodiol catalyst is shown in According to our previous research, the optimal three-component alcoholic solubilizer ratio for degrading polyurethane foam is ethylene glycol (EG)/propylene glycol (PDO)/butylene glycol (BDO) = 35:35:30. The recycled waste polyurethane foam was crushed into 2–10 mm polyurethane foam chips using a shredder, and then 35 g of ethylene glycol, 35 g of propylene glycol, and 30 g of butylene glycol , as well as different ratios of KOH/titanium glycol catalyst , were added into the glass reactor at 90 °C, and the catalyst was left to dissolve completely at this temperature (the temperature was raised from room temperature to 90 °C after the liquid titanium glycol was added). After the catalyst had completely dissolved, the alcoholic solubilizer was added to the pre-crushed waste polyurethane foam scraps at a mass ratio of 1:1 for further reaction, and the temperature of the reaction at this stage was increased to 180 °C. The reaction ran for 3–5 h to allow complete degradation of the waste polyurethane scraps and to obtain recycled polyether polyol, which was cooled to room temperature and transferred to a disposable plastic cup for collection. The alcoholytic agent breaks the isocyanate in the polyurethane chain segment, thus generating relatively short chain segments, which produces recycled polyether polyols that can be further recycled for use.The recycled polyurethane rigid foam was prepared using a one-step method, and the specific operation formula is shown in Viscosity analysis: An NDJ-5S digital viscometer was used for viscosity testing; a number of degradation products were taken and placed in the container while a suitable rotor was selected as well as a suitable rotational speed, and viscosity testing was carried out at 25 °C.Compression strength test: We referred to the GB/T8813-2008 test standard—we took a sample size of 50 mm × 50 mm × 50 mm and used an EFS-24RE universal testing machine for the compression strength test.Water absorption test: We referred to the GB/T8810-1988 test standard—the sample size was 50 mm × 10 mm × 10 mm, and we used distilled water to determine the water absorption by weighing the mass of the sample before and after immersion to calculate its water absorption.Hydroxyl value determination: We referred to the GB/T12008.3-2009 standard, taking appropriate amounts of oligomeric regenerated polyether polyol in a 10 mL conical flask, and used the ester anhydride method with pyridine for hydroxyl value determination.Thermal conductivity analysis: Referring to the QB/T3806-1999 standard, the sample size was 200 mm × 200 mm × 20 mm, and we used the FEHC-S thermal conductivity tester of Changzhou Hua’ao Instrument Manufacturing Co Ltd., .SEM analysis: We cut the regenerated polyurethane rigid foam specimens into appropriately thin slices (without squeezing) and then used SEM to observe the microstructure of the bubble pores of the foam, magnified 20 times, observing the clear, complete and uniform area of the bubble pore structure and recording the observation results.−1.FT-IR test: GR-285 IR produced by Dalian Precision Scientific Instruments Co. Ltd., . was used to analyze the structure of the foam samples, and the samples were formed by the KBr pressing method; the test wavelength range was 500–4000 cmTG analysis: TG analysis was performed using TG under a nitrogen atmosphere at a rate of 30 °C/min from room temperature (25 °C) to 500 °C. The gas flow rate was set at 50 mL/min, the carrier gas was air, and alumina was used as the reference object.−1. The samples were first dissolved in N,N-dimethylformamide(DMF) followed by 1.6 wt.% THF, filtered through a nylon filter with a pore size of 2 mm, and then prepared for use.Determination of molecular weight distribution coefficient: The molecular weight distribution of the regenerated polyether polyol was determined by gel permeation chromatography (GPC). Measurements were performed using a thermal scientific chromatograph equipped with an isocratic DionexUltra3000 pump and a RefrtoMax521 refractive index detector. Separations were performed in four Phenomenex Phenogel GPC columns with a separation temperature of 30 °C, a particle size of 5 µm, and different porosities of 105, 103, 102, and 50 while placed in an ultimate thermostatic column at 3000 °C. The mobile phase was tetrahydrofuran (THF) set at a flow rate of 1 mL·minTEM test: The prepared ethylene glycol titanium samples were diluted and dispersed in anhydrous ethanol solution and then ultrasonically stirred using ultrasonic equipment to obtain a uniform suspended liquid. The drops were placed on a copper net with a supporting film, and the dispersion was tested after volatilizing in a dry environment.Waste polyurethane rigid foam is mainly composed of carbamate bonds. Under certain temperature conditions, the carbamate bonds are broken under the joint action of alcoholic solvents and catalysts, then replaced by alcoholic solvent chain segments to generate renewable polyether polyols, and the specific main reaction mechanism is shown in Equation (3) in Ethylene glycol titanium was prepared by adding a large amount of ethylene glycol, which led to a complexation reaction in the system, resulting in an ethylene glycol titanium catalyst with an outer layer covered with an alcohol gel. The alcoholytic agent used in this experiment was ethylene glycol (EG)/propylene glycol (PDO)/butylene glycol (BDO) = 35:35:30. When the ethylene glycol contained in the alcoholic solubilizer encountered the ethylene glycol titanium catalyst, the alcoholic gel covering the outer layer of the catalyst acted similarly to the alcoholic solubilizer in that it steadily released the catalyst and promoted good dispersion of the titanium nanoparticles without agglomeration, maximizing the catalyst activity through release of the ethylene glycol titanium catalyst in the alcoholic solubilizer. Following this, the crushed polyurethane foam was added. When the polyurethane foam met the alcoholizing agent, the alcoholizing agent and catalyst were absorbed into the foam. As the temperature increases, the titanium glycol catalyst attached to the polyurethane foam skeleton catalyzes the cracking of the polyurethane rigid section at the appropriate temperature, which eventually completely breaks the rigid section of the polyurethane foam to produce free small-molecule polyether polyols and amines by substitution with the small-molecule alcohols in the alcoholytic agent.The mechanism polyurethane alcoholysis degradation usually generates the following products  shown inThe flow of the process of recycling waste polyurethane foam, through its degradation by alcoholysis and subsequent use of the recycled polyether polyols to replace commercially available polyether polyols in the production of recycled polyurethane foam, is shown in the −1 are mainly Ti-O bond stretching vibrations; the peaks at 2930 and 2871 cm−1 are characteristic absorption peaks of sp3 C-H stretching vibrations; the peaks at 1085 and 1036 cm−1 are the peaks of C-H in-plane bending vibrations and C-O stretching vibration, respectively [ectively . The cha2CH2O)2 catalyst, and polyether polyol 4110 from polyurethane feedstock, respectively, and the results are shown in −1, which is the characteristic absorption peak of alcohol hydroxyl group (–OH) [−1 correspond to sp C−H stretching vibration; through comparison with the spectrum for polyether 4110 IR, a clear absorption peak appears at 1611 cm−1, which is a phenyl broadband peak [−1, confirming the successful degradation of waste polyurethane foam via the alcoholysis reaction [−1 [−1 [up (–OH) ,37. The and peak . Polyethreaction . The chation [−1 , and theOther experimental conditions were fixed, and the used polyurethane direct buried pipe rigid foam was degraded with an alkali metal KOH catalyst and homemade nanosized ethylene glycol titanium catalyst in appropriate alcoholic solvents. The viscosity of degraded products was then assessed using an NDJ-5S digital viscometer. The test results are shown in The good interfacial compatibility of titanium glycol and polyurethane leads to a mechanism under which the polyurethane and the catalyst have satisfactory compatibility, resulting in the good interfacial compatibility of the catalyst of titanium glycol in the solvent used in the process of degradation into small molecules. This ensures the good dispersion of the small titanium glycol molecules in the alcoholic solubilizer, allowing the highest possible effect and activity of the catalyst and, therefore, complete degradation of polyurethane. A polyether polyol with satisfactory properties is obtained, so the resulting regenerated polyether polyol also has satisfactory properties, and the polymer foam obtained after the application of re-polymerization will have strong compression properties. The titanium ethylene glycol catalyst yields foams with higher compression strengths when prepared from degraded polyether polyol compared with the alkaline earth catalyst.The hydroxyl values of the regenerated polyether polyols recovered from the used polyurethane foam degraded under different catalyst systems were determined using the ester-anhydride method with pyridine under the same experimental conditions.As can be seen from From the From the macroscopic point of view, the value of thermal conductivity is closely related to the integrity of the polyurethane foam’s bubble pores, the degree of fit of the bubble pore wall, and the skeleton thickness. From the microscopic point of view, the value of thermal conductivity is closely related to the selection and proportion of alcoholic solvents, catalyst activity, temperature, degradation time, degree of urethane bond breakage during degradation, amount of foaming agent added during foaming, amount of catalyst used in foaming. All macroscopic and microscopic factors have either direct or indirect influences on the polyurethane pore shape, which further affect the thermal insulation performance of polyurethane foam and thus cause the thermal conductivity to vary. Generally speaking, more complete degradation of the used polyurethane foam is achieved under appropriate pressure, temperature, and time conditions, and this leads to the more thorough urethane bond breakage and more uniform molecular weight distributions of the generated short-chain materials, such as the polyether polyol recovered from the degradation material, which is more favorable for the foaming reaction. Therefore, the more thorough the reaction of the degraded waste polyurethane foam, and the higher the amount of appropriate foaming agent and catalyst added during the foaming process, the more complete the pores of the regenerated polyurethane foam, the closer the pore walls are to each other, and the stronger the skeleton. The polyurethane foam generated under the optimal conditions will achieve the best heat preservation performance.Polyurethane foam achieves better thermal insulation performance and a lower thermal conductivity because the movement of gas entering the foam is obstructed, and the complete pores and thick pore walls of the foam prevent the convective heat transfer enabled by air flow. These pore walls act as a barrier to prevent the air from passing quickly through the foam, thus causing the gas to stay in the foam for a much longer time and thus achieving the effect of heat insulation. Therefore, the bubble pore integrity and pore wall thickness of the foam directly affect its thermal insulation and thermal conductivity properties .2CH2O)2 polyols are relatively low under different degradation systems but essentially similar and fully comply with the national standard, indicating that they are suitable for use in actual production.From 2CH2O)2 for two different catalyst systems. The temperature range of this test is from 25 °C (room temperature) to 500 °C. It can be seen from the figure that the starting decomposition temperatures of the two regenerated polyurethane foams are around 240 °C, and the reaction ends at 500 °C.2CH2O)2) initially lost weight slowly, and the maximum thermal weight loss rate was around 340 °C, which is 10 °C higher than that of the alkali metal catalyst. Therefore, it can be seen that the regenerated polyurethane foam prepared by the catalytic degradation of the new titanium catalyst (Ti(OCH2CH2O)2) exhibits higher thermal stability than that prepared using the conventional alkali metal catalyst (KOH).Thermogravimetric weight can reflect the relationship between the polyurethane foam mass and temperature. The decomposition of polyurethane foam is generally divided into three stages : the fir2CH2O)2 system reaches the optimal state when the catalyst addition amount is 0.075%.The standard water absorption of polyurethane rigid foam is generally ≤3.3%, which is a property closely linked to the foam’s bubble pore uniformity, bubble pore size, pore diameter, pore film, skeleton, and other factors, and it further affects the thermal conductivity of the foam as well as its thermal insulation performance. In general, the lower the water absorption rate, the higher the pore integrity of the bubble, resulting in elevation of the thermal insulation performance of the foam. As can be seen from 2CH2O)2 catalytic system.SEM analysis is an important and versatile tool for examining foam morphology because it reflects how bubbles grow through visualization of the tightly packed bubble network that forms the typical cellular structure of a foam . The str2CH2O)2 catalyst is thus higher than that of the traditional alkali metal catalyst, which causes “excessive” breakage of the carbamate bonds in the waste polyurethane chain segments during degradation. After degradation, these ultra-short chain segments will “self-polymerize” into new chain segments of different chain lengths, resulting in the uneven distribution of molecular weight in the degraded material, which leads to the serious fragmentation of pores in the prepared foam and greatly reduces the foam strength and insulation performance.It can be seen from In this paper, a prepared homogenized titanium catalyst and the traditional catalyst (KOH) were successfully used in the glycolysis of waste polyurethane pipeline material. The composition of the obtained regenerated polyether polyol was similar to that of the foam prepared from commercially available polyether 4110, and the reaction rate could be controlled by adjusting the amounts of different catalysts. The regenerated polyurethane rigid foam prepared using regenerated polyether polyol to replace part of the commercially available polyether 4110 (up to 30 wt.%) conforms to the national and industrial standards, and its compression strength is even higher than that of traditional foam, thus demonstrating it meets the requirements for actual application and production.2CH2O)2) to degrade polyurethane waste, the following trends were observed: Catalyst addition of T2 0.050 wt.% resulted in the lowest viscosity of regenerated polyether polyols of 5356.7 mPa·s, which is a decrease of 9.97% compared with the lowest viscosity of regenerated polyether polyols obtained using the alkali metal catalyst. Catalyst addition of T3 0.075 wt.% resulted in a molecular weight distribution coefficient of 1.2245 and a hydroxyl value of 495.7 mg KOH/g of the regenerated polyether polyols. Catalyst addition of T4 0.10 wt.% resulted in a viscosity of 6010.5 mPa·s and the most uniform molecular weight distribution coefficient of 1.2568. Catalyst addition of T5 0.20 wt.% resulted in an optimal hydroxyl value of 408.3 mgKOH/g, similar to the hydroxyl value when using the alkali metal catalyst (KOH). Titanium catalyst addition of T3 0.075 wt.% resulted in the preparation of regenerated polyurethane foam with the best properties with a compressive strength of 0.168 MPa, which is 4.76% higher than that of 0.168 MPa for the foam obtained using the KOH catalyst. The thermal conductivity was 0.01836 W/(m·K), the texture of the polyurethane rigid foam was uniform, the pores were not collapsed and were complete, the skeleton was thick and dense, and the foam demonstrated good thermal insulation performance.Using the homogenized titanium catalyst (Ti(OCH3, and the ratio of white material to black material was about 0.1–1:1.1–1.2. The values for density and the degree of network crosslinking in the obtained polyurethane pipe material are between those corresponding to the classifications of soft foam and high-hardness foam. Therefore, in this study, the experimental scheme was designed for waste components, and related experiments were carried out. The amount of catalyst, the experimental temperature, the pressure, and other experimental conditions were determined via continuous exploration and experiments. However, the high-hardness foam with high black material content and highly rigid structure struggled to reduce the solution under the same conditions in our study. In addition, in future work, our research team will focus on optimizing the removal of amine product impurities produced in the side reaction of glycolysis in the recycled polyether polyols obtained from waste polyurethane, employing chemical impurity removal and physical adsorption to purify the recycled polyether polyols, so as to prepare high-quality regenerated polyurethane foam.The density of the recycled waste polyurethane pipe material in this study was between 30 and 50 kg/m


Klebsiella pneumoniae susceptibility to CFDC.Cefiderocol (CFDC) is a novel siderophore-cephalosporin, effective against multidrug-resistant Gram-negative bacteria. As it has a siderophore side chain, it can utilize iron acquisition systems for penetration of the bacterial outer membrane. We aimed to elucidate the role of siderophores and iron uptake receptors in defining K. pneumoniae strains were characterized for susceptibility to different antibiotics including CFDC. CFDC minimum inhibitory concentrations (MIC) were determined in iron-depleted and iron-enriched conditions. Iron uptake genes including siderophores, their receptors, ferric citrate (fecA) and iron uptake (kfu) receptors were detected by PCR in all the strains. For 10 selected strains, gene expression was tested in iron-depleted media with or without CFDC treatment and compared to expression in iron-enriched conditions.Initially, 103 50: 0.5 and MIC90: 4 μg/ml). Only three strains (2.9%) were intermediately susceptible and a pandrug resistant strain (0.97%) was resistant to CFDC . The presence of kfu and fecA had a significant impact on CFDC MIC, especially when co-produced, and if coupled with yersiniabactin receptor (fyuA). CFDC MICs were negatively correlated with enterobactin receptor (fepA) expression and positively correlated with expression of kfu and fecA. Thus, fepA was associated with increased susceptibility to CFDC, while kfu and fecA were associated with reduced susceptibility to CFDC. CFDC MICs increased significantly in iron-enriched media, with reduced expression of siderophore receptors, hence, causing less drug uptake.CFDC exhibited 96.1% susceptibility, being superior to all the other antibiotics (MICIron acquisition systems have a significant impact on CFDC activity, and their altered expression is a factor leading to reduced susceptibility. Iron concentration is also a major player affecting CFDC susceptibility; therefore, it is essential to explore possible ways to improve the drug activity to facilitate its use to treat infections in iron-rich sites. Klebsiella pneumoniae is an important Gram-negative bacterial pathogen that represents a public health threat, coupled with the spread of antimicrobial resistance (AMR) across the globe. There is a continual rise in mortalities related to ineffective treatment as bacteria develop resistance mechanisms against novel antibiotics [Enterobacteriaceae, including K. pneumoniae [3+) and using the iron transport systems found on the outer membrane (OM) of Gram-negative bacteria (GNB) to enter the bacterial cells using trojan-horse approach [ibiotics . Cefidereumoniae . The unieumoniae . Naturalapproach . CFDC-irapproach . This isapproach .in vitro [K. pneumoniae can cause serious infections in the liver, including pyogenic abscesses triggered by hypervirulent strains [Iron is a fundamental microelement for bacteria, used as a cofactor with many enzymes involved in vital cellular processes, such as electron transport and cellular respiration . It is uin vitro . On the in vitro . It is i strains . To date strains .K. pneumoniae. Chen et al., 2020 tested bacterial growth under different iron concentrations for K. pneumoniae strains causing liver abscess compared to others that do not cause liver abscess. They have demonstrated that maximum bacterial growth was observed in iron rich media with the highest tested concentration (50 μM) of iron. Another important finding in their study was the siderophore expression rates. They established that all four siderophores, namely enterobactin, aerobactin, yersiniabactin, and salmochelin were expressed in iron depleted condition more than in iron rich media [More recent studies have focused on the importance of iron, its uptake mechanisms, and its relation to the pathogenicity of ch media .Klebsiella ferric iron uptake (kfu), and ferric iron citrate receptors (fecA). FecA transports ferric iron (Fe3+) across the outer membrane and one study identified that fecA system contributed to the virulence of enterobactin (entB) mutant E. coli [Klebsiella pneumoniae. Although CFDC is mostly dependent on the iron content at the site of infection, no study documented the effect of iron concentration on the expression of iron acquisition genes and their effect on CFDC activity in K. pneumoniae.Bacteria can also acquire iron through other pathways such as  E. coli . LimitedfepA, fyuA, iroN, and iutA), ferric iron uptake (kfu), and ferric citrate receptor (fecA) were not investigated in relation to the activity of CFDC. Therefore, this study aimed to investigate the effect of these iron acquisition genes on the activity of CFDC on a group of K. pneumoniae strains form diverse clinical sources. It also aimed to test the expression of these genes in response to treatment with CFDC in iron depleted condition compared to the expression obtained in iron enriched and iron depleted conditions without CFDC treatment.To the best of our knowledge, iron uptake genes including siderophore receptors . K. pneumoniae strains were isolated from multiple infection sites, including urinary specimens (n = 38), respiratory specimens (n = 29), wound or tissue specimens (n = 12), blood (n = 11) and body fluids (n = 1) as shown in S1 Table.A total of 103 non-duplicated S2 Table). All the bacteria were preserved in brain heart infusion broth  with 20% glycerol and stored at– 80°C. The strains were checked for purity before any experiment.Additional 12 strains were purchased from American Type Culture Collection  including strains with special characteristics and known resistance gene profiles . The plates were incubated overnight at 37°C. The experiments were performed and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [Disk diffusion test was used for antibiotic susceptibility screening using 14 antibiotics covering seven antibiotic categories. Antibiotic disks obtained from Mast, UK, including amoxicillin/ clavulanic acid (20/10 μg), piperacillin/ tazobactam (100/10 μg), cefiderocol (30 μg), cefotaxime (30 μg), cefoxitin (30 μg), ceftazidime (30 μg), cefepime (30 μg), cefpodoxime (10 μg), gentamicin (10 μg), ciprofloxacin (5 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), ertapenem (10 μg) and meropenem (10 μg) were applied on Muller Hinton Agar  using MastPhenotypic detection of extended spectrum beta-lactamase (ESBL) production was performed using double disk synergy test with cephalosporins and amoxicillin-clavulanate (AUG) . ModifieS1 Table.Determination of minimum inhibitory concentration (MIC) was performed as a quantitative measurement of antimicrobial sensitivity using broth microdilution test. As iron is depleted at sites of infection, iron depleted media was prepared as per the CLSI recommendations  in orderK. pneumoniae over a period of 24 hrs treatment. This was important in order to determine the duration of treatment to be used in the gene expression studies. To conduct the test, CFDC was diluted to the concentration of predetermined MIC for each strain. Concentrations prepared were equivalent to ¼, ½, 1, 2, 4, 8, and 16 X MIC, as well as a growth control (untreated culture) without the addition of any antimicrobial agent [5 CFU/ml was used. The test was done in a 96 well plate which was incubated at 37°C during the experiment. Every 2 hrs; starting from 0, 2, 4, 6, and finally 24 hrs after treatment, an inoculum of 5 μl was taken from each well, serially diluted in 0.85% NaCl and spotted on agar for colony counting [10 of the bacterial concentration at every time point for each concentration tested. Log10 coordinate was plotted on the Y axis against time on X axis [Selected strains (n = 8) were chosen to establish the time at which CFDC can kill al agent . An inoccounting . After on X axis , 20.3  stock solution was prepared to a concentration of 0.1 M, then added to the iron depleted media to reach a concentration of 100 μM. The level of ferric iron in the culture media was confirmed using a colorimetric iron assay kit . MICs were determined in iron enriched media for 24 selected strains and were compared to the MICs in iron depleted conditions (list of selected strains is shown in S3 Table).Activity of CFDC was tested in iron depleted and iron enriched media. FeClkfu) and ferric citrate receptor (fecA). In addition, as CFDC is siderophore-cephalosporin, PCR was used to detect the genes encoding siderophore and siderophore receptors, including enterobactin (entB), and its receptor (fepA); aerobactin (iucA) and its receptor (iutA); salmochelin (iroB), and its receptor (iroN); yersiniabactin (irp1) and its receptor (fyuA).DNA was extracted from pure bacterial colonies using boiling lysis method . The mec® Ready to Load Master Mix  was used with a primer concentration of 0.2 μM for each of the forward and reverse primers [For the amplification of each gene, 5x Hot FIREPol primers . List ofkfu and/or fecA) were included. The collection also composed of strains selected based on susceptibility to CFDC, including some susceptible strains (n = 6), all the intermediately susceptible strains (n = 3), and the only strain which was resistant to CFDC (n = 1).From the 103 strains investigated in this study, a total of 10 strains were chosen for gene expression experiments. Strains were selected to encompass a variety of iron acquisition genes; thus, strains harboring 1–4 siderophores and/or different iron transport system genes (8 CFU/ml) to allow for the expression of genes.Three types of treatment were applied, including growth in iron depleted media, iron enriched media and treatment with CFDC in iron depleted media. To test the effect of CFDC treatment on the expression of siderophores and iron acquisition genes, 2 X MIC of CFDC was used to treat bacterial cultures. The concentration was determined as subinhibitory and not bactericidal when used to treat a culture with density of 0.5 McFarland units  according to the manufacturer’s recommended protocol for RNA isolation from Gram negative bacteria. Briefly, fresh bacterial cultures  were prepared. RNA was extracted from 2 ml culture at the baseline (time 0), then after 2, 4 and 6 hours of incubation at 37°C in an orbital shaker. Bacterial suspensions were centrifuged at 14,000 rpm for 10 mins to pellet the cells. Cell lysis was done by enzymatic destruction by suspending the pellet in 200 μl of 3 mg/ml lysozyme  and incubation at 37°C for one hour. NanoDrop™ 1000 Spectrophotometer  was used for quantification of RNA and to check its purity. Extracted RNA was immediately stored at– 80°C until use . cDNA wa® EvaGreen® qPCR Supermix , adjusted to 20 μl with molecular biology grade water [Real-time assay was carried out to assess the expression of the genes listed in de water . Real-tirpoB was used a housekeeping gene for normalization as described before for K. pneumoniae [To analyse the generated data, cycle threshold (Ct) values were used to calculate ΔCt using mean CT values generated for each gene subtracted from mean Ct values of the housekeeping gene of the same sample . The geneumoniae . ExpressFold change (FC) equation used is:-ΔΔCt where ΔΔCt = ΔCt value of target gene–average ΔCt of control.FC = 2ΔCt of control was either for time 0 (bassline expression), or the values for untreated culture grown in iron depleted media at the same time point for the same target gene.p < 0.05. Graphs were generated using GraphPad Prism® Version 9.4.0 . R Project for Statistical Computing software (R version 4.1.2) was used for dendrogram generation and heatmap construction.Statistical analyses were performed using IBM SPSS Statistics 26.0 software . Data were expressed as mean ±SD. Mann-Whitney U test was used for the comparison of two independent groups. Two-tailed Spearman’s test was used to examine the correlation between MICs and gene expression in different groups, and to correlate the expression of various iron acquisition genes. Statistical significance was determined at Based on antibiotic resistance profiles, 31% (n = 32) strains were denoted as multidrug resistant (MDR) due to resistance to at least one antibiotic in ≥3 and <6 antimicrobial categories. In addition, 36.9% (n = 38) strains were found to be extensively drug resistant (XDR) due to resistance to one antibiotic in ≥ 6 antimicrobial categories . Carbape50 and MIC90 of the antibiotics used on the tested bacterial isolates. A full list of all the strains with results of antibiotic susceptibility testing are shown in S1 Table.CFDC was highly effective against the tested strains with 96.1% (n = 99) susceptibility, while only three strains (2.9%) were intermediately susceptible and one (0.97%) pandrug resistant strain (KPN 68) resistant to CFDC, with MICs of 8 and 256 μg/ml, respectively. CFDC inhibited 91% (n = 61) of strains resistant to one or both parent antibiotics, CPM and CAZ, and was highly effective on MDR and XDR strains including those resistant to carbapenems and colistin.MICs for CDFC were almost always lower than cefepime, ceftazidime, meropenem and colistin with mean fold difference ± SD, 44.3±75.5, 51.4±77.3, 14.8±39.2 and 3.2±5.3, respectively. This was true for all the strains except one (KPN 8) which was highly susceptible to CPM, CAZ, and MEM exhibiting extremely low MICs ; however, it showed a higher MIC to CFDC (2 μg/ml), but still in the susceptible range. This finding attracted our attention to the presence of other factors affecting the activity of the drug, namely, iron transport systems.p<0.001). CFDC lost its activity against 14/24 strains when compared to CLSI breakpoint with 2–32 folds increase in the MICs when cultured in iron enriched media. List of strains tested in both iron depleted and iron enriched media with their MIC values is shown in S3 Table.As shown in 10 CFU/mL) versus time for representative strains are demonstrated in Fig 1.Time-kill assay for CFDC was done to determine the time of killing for use in gene expression studies. Accordingly, bactericidal effect was observed at ≥ 1X MIC at 6 hrs across all tested strains (n = 8). Higher concentrations such as 8 and 16 X MIC had bactericidal effects at 4 hrs time point in 50% of the strains. CFDC showed little effect in the first 2 hrs then, the growth rate started declining rapidly after 4 hrs with concentration ≥ 1X MIC, till complete killing at the 6 hrs time point. Plots of colony count  was detected in all 103 strains, and 38 strains (36.9%) produced entB only without other siderophores. The co-production of other siderophores in addition to entB is shown in the overlapping areas between the labelled sections of the Venn diagram. As shown in fepA), with only 36 strains (34.95%) had this receptor alone, while the rest of the strains carried other receptors accompanied by fepA. Although no strain produced the four siderophores, a few strains  produced the four receptors.As shown in fecA) or iron uptake receptor (kfu) with enterobactin receptor (fepA) had a significant effect on CFDC MIC. They also caused a significant increase to the MIC of CFDC when they were co-produced with yersiniabactin receptor (fyuA). The presence of kfu with fyuA, even without fecA, also caused a significant increase in CFDC MICs in some strains.As shown in Gene expression (GE) of siderophores, siderophore receptors and iron transport genes was assessed in 10 strains, including CFDC resistant , intermediately susceptible , and susceptible  strains. GE in iron enriched media and CFDC treated cultures were compared and normalized to expressions detected in iron depleted media at the same time point of treatment. Folds change in expression are shown in Figs entB) and its receptor (fepA) were detected in all the 10 strains, while the rest of the siderophore genes and their receptors were expressed in some strains as shown in Figs It was observed that maximum expression of all iron acquisition genes was reached at 4 hrs in some strains and at 6 hrs in the others in different growth conditions Figs –6. As shentB siderophore was significantly higher in I group than S group in iron enriched media (p = 0.023) (fepA was significantly higher than the expression of entB when bacteria were cultured in iron depleted media with or without CFDC. As shown in fepA expression was significantly lower in bacteria grown in iron enriched media than bacteria treated with CFDC (p = 0.015). After checking GE patterns in heatmaps for entB . Also, fepA GE was unique in the latter strain, being expressed highly in iron depleted media without CFDC than iron enriched media. Generally, GE of fepA was lower when the bacteria were grown in iron enriched media, except for the KPN 23 (S) and KPN 8 (S), whereby the expression did not significantly vary across the different media. Strain KPN 8 was also unique as entB gene was downregulated in iron depleted media with or without CFDC. Same was noted for KNP 23 (S) although the expression was higher when grown in iron depleted media without CFDC. The resistant strain (KPN 68) also demonstrated different pattern as both entB and fepA were less expressed in all media, except with CFDC treatment. Other unique patterns for fepA were noted in strains CDC AR0066 (I) and CDC AR0039 (S) which have known outer membrane porin (OMP) mutations, and the gene was downregulated in all the media (GE of = 0.023) . GE of ffor entB  and fepAtB  was highly expressed in all strains except in KPN 8 at the end of CFDC treatment period (6 hrs), and the receptor (fyuA) was more expressed than its siderophore (irp1) in all media  whereby the expression was low when cultured in iron depleted media without CDFC, while the expression was increased in iron enriched media (p = 0.400) as shown in irp1 and fyuA, showing higher expression when treated with CFDC compared to other growth conditions.As shown in ll media . The expfecA was lower than the other receptors, and the difference was statistically significant in majority of the strains (p<0.05). As shown in fecA receptor was produced in all strains from I group; however, GE was low and non-significantly increased overtime in iron enriched media (p = 0.70). The expression of fecA in strain KPN 62 (S) was significantly higher than the expression of the I group cultured in iron enriched media (p = 0.009). On the contrary, the expression of fecA receptor in the S group was more in iron enriched media than in CFDC treated cultures. When checking GE patterns in the heatmap  in bacteria grown in iron enriched media (p = 0.009) as shown in Kfu was overexpressed in KPN 68 (R) and KPN 23 (S), and its expression was consistent when these two strains were cultured in all the media . As seen for fecA, strain CDC AR0066 exhibited a unique pattern as kfu was under expressed in all the three growth conditions . There was a strong positive correlation between the expression of irp1 and fyuA (p = 0.003). Also, there was a strong positive correlation between the expression of fecA and kfu at 2 and 4 hrs, but the correlation was negative at 6 hrs. In iron enriched cultures, there was a strong positive correlation between fepA with irp1 and fyuA , while there was no correlation with entB (p = 0.254).Correlation analysis for GE of iron acquisition genes in iron depleted media revealed a strong positive correlation between the expression of entB and fepA receptor with the MICs of bacteria cultured in all three media at all the time points. Also, there was negative correlation between the expression of irp1 and fyuA receptor and the MIC during growth in iron depleted media and iron enriched media (significant at 6 hrs). On the contrary, there was positive correlation between the expression of fecA and the MIC of bacteria cultured in iron depleted media and iron enriched media. Lastly, positive correlation was seen between the expression of kfu and the MIC for CFDC treated bacteria at all the time point, and at the end of treatment time (6 hrs) in the other media.Although non-significant, a negative correlation was found between the expression of both K. pneumoniae strains from diverse clinical sources with various antibiotic susceptibility profiles. In agreement with other reports [Cefiderocol is the first in class siderophore-cephalosporin that is FDA approved for clinical use as an advanced-generation cephalosporin with a catechol iron-binding moiety . There a reports , 19, CFD reports . Howeverin vivo [Pseudomonas aeruginosa [kfu), and ferric citrate receptor (fecA) that might be related to CFDC activity. The genes were first detected by PCR, then their expression was tested by real-time PCR. To the best of our knowledge, this is the first study linking the selected siderophore receptors, and iron transporters to CFDC activity.The efficacy of CFDC was first tested in iron depleted media in order to mimic iron depleted conditions in vivo . Then, truginosa . The latruginosa . Taking fepA) expression was highest between 4 and 6 hrs of treatment, which can explain the killing time, considering that drug uptake can be facilitated by this siderophore receptor. As entB is the major catechol siderophore in K. pneumoniae [fepA can act as a major pathway of CFDC entry to the bacterial cell. In fact, enterobactin (entB) and its receptor (fepA) were found to be highly produced in K. pneumoniae [entB and fepA receptor were detected in 100% of our strain collection. Correlation analysis showed a negative relation between CFDC MICs and GE of entB and fepA receptors. Although the results were not statistically significant, possibly due to small sample size, they reflect that higher expression of these genes can lead to lower CFDC MICs. It is important to mention that we reported reduced susceptibility to CFDC in strains with downregulated fepA as in CDC AR0066 (I) and KPN 68 (R), suggesting a possible role of fepA in facilitating the drug uptake. This is also supported by the finding that expression of entB and fepA was higher in CFDC treated bacteria than in bacteria grown in iron enriched media in the majority of tested strains, which could explain the loss of drug activity in iron enriched media due to less drug uptake as the receptors are down regulated.Based on the results of the time-kill study, CFDC produced bactericidal effect at ≥ 1X MIC after 6 hrs exposure to the drug. Thus, gene expression studies were done with maximum treatment duration of 6 hrs. Our results showed that maximum expression of iron acquisition genes was reached after 4–6 hrs of treatment. Importantly, enterobactin siderophore receptor  exhibited GE that was strongly correlated with fepA. The expression of both fepA and fyuA was highest in CFDC treated bacteria, even higher than what was seen in iron depleted media without CFDC treatment, which means that the bacteria are actively expressing their siderophore receptors to acquire iron while being in an iron depleted environment. As CFDC was added to iron depleted media, it is expected that siderophores will be expressed more to acquire iron and their receptors will be expressed to assist in their uptake. As CFDC can chelate Fe3+, it can further starve the bacteria for iron and hence, can force greater expression of siderophore receptors to capture the siderophores loaded with iron.Another siderophore receptor (Klebsiella ferric iron uptake (kfu), a regulator of iron transport responsible for acquisition of iron in Klebsiella species. Previous studies reported a link between kfu and ability of K. pneumoniae to cause invasive infections such as liver abscess, that was attributed to its role in mediating ferric iron uptake and modulating virulence and hypermucoviscosity. Besides, its presence in the clinical strains has been shown important for capsule formation and purulent tissue infections [FecA usually binds to ferric citrate leading to conformational changes with subsequent transcriptional activation to uptake iron during iron starvation [fecA was lower than the other receptors in this study, but it is still expressed by some strains particularly from I group. It was noticed that the presence of the fecA receptor alone, in addition to fepA, caused a significant increase in the MIC, and its presence with other receptors, mainly kfu, had a significant impact on the MICs as all the strains which were intermediately susceptible to the drug (MIC = 8 μg/ml) carried this gene. The expression of kfu was also associated with significant increase in CFDC MIC, when it was present alone with fepA, or with other receptors as fecA and/or fyuA. Correlation analysis showed a positive relation between CFDC MICs and GE of fecA receptor and also kfu receptor. Although the results were not statistically significant, probably due to small sample size, these results confirm our conclusion that these genes are linked to reduced susceptibility to CFDC as higher expression was associated with higher MICs, for example, a significantly higher expression of kfu was seen in R compared to S groups.In addition to siderophore receptors, bacteria can acquire iron using other receptors, such as fections . Ferric arvation . OverallfepA, fyuA, and iutA) and did not harbour any iron transport genes (fecA and kfu). The expression of fepA receptor was low at the end of CFDC treatment (6 hrs). Our explanation to this phenomenon is that since there was very low expression of the receptor, CFDC was not able to penetrate the bacteria efficiently and therefore resulted in an increased MIC. This was supported by the finding that CFDC susceptible bacteria had higher expression of this gene, reflecting that this receptor is involved in the drug uptake. When KPN 8 was compared to another S strain (KPN 23), both of them had upregulated fepA and fyuA genes in iron enriched media, and therefore were able to maintain a non-resistant MIC in iron enriched conditions. Notably, KPN 23 had double MIC (8 μg/ml) compared to KPN 8 (4 μg/ml) in iron enriched media, while their MICs in iron depleted media was the same (2 μg/ml). As mentioned, KPN 8 devoid from iron uptake genes, while KPN 23 had kfu gene which was also overexpressed in all the growth conditions. In general, higher MIC in iron enriched media were found in strains expressing kfu and/or fecA, but it is important to consider other receptors that can be involved in iron uptake. Although the expression of kfu was very similar between KPN 23 (S) and KPN 68 (R), being consistent when these two strains were cultured in all the media, it is important to note that the increased susceptibility of KPN 23 can be due to the overexpression of fepA and the under expression of fepA is linked to reduced susceptibility in KPN 68, which exhibited unique patterns of expression for majority of receptors. This further signifies the importance of fepA receptor in the capturing of CFDC.Overall, some strains demonstrated very unique GE patterns, suggesting dysregulation of iron uptake genes that can probably contribute to their altered susceptibility to CFDC. This is mainly true for R and I strains and some S strains. For example, one strain in the collection (KPN 8) had lower susceptibility to CFDC than the parent cephalosporines (CAZ and CPM). This was surprising, as all the other strains exhibited higher susceptibility to CFDC than the other cephalosporins and even meropenem, which was also reported by other investigators , 43. We Cir , on susceptibility to CFDC in different species of GNB [CirA is known as an OM receptor for linear Fe3+-enterobactin degradation products, as compared to fepA which is responsible for the uptake of intact Fe3+-enterobactin [fepA receptor has high ligand affinity to Fe3+-enterobactin, while cirA is more specific to Fe3+- catecholates and their breakdown products containing Fe3+ [fepA was selected for testing in our study, and our results clearly showed its impact on drug entry to bacteria as CFDC MICs were negatively correlated with fepA expression.Considering other factors that might affect bacterial susceptibility to CFDC, it is important to mention that during the course of this study, some investigators reported the effect of mutations in the gene encoding colicin I receptor and catecholate siderophore receptor; s of GNB , 34, 44.robactin ; therefoing Fe3+ . For thiK. pneumoniae can be affected by multiple factors, particularly cirA mutation and metallo-β-lactamases that can cause drug degradation. Using whole-genome sequencing, the latter study proved that high-level CFDC resistance could be mediated by the presence of NDM gene coupled with cirA mutation. They have shown that either factor present alone was not sufficient to cause CFDC resistance, while their combined effect jointly conferred high-level CFDC resistance [CirA might not be the sole receptor for CFDC entry. Another study revealed that when only cirA was knocked out in E. coli, CFDC MIC was increased by 2-fold, whereas the MIC increased 16-fold by the double knockout of two receptors including both cirA and fiu; nevertheless, the bacteria remained susceptible [CirA, in iron-limiting condition and downregulation in iron-rich condition was reported by other investigators [CirA GE was not tested in this study. We focused on testing siderophores, siderophore receptors and iron uptake genes which were not tested before; however, the effect of the CirA gene can be investigated in our bacterial collection in future studies using whole genome sequencing to find out mutations that can affect CFDC activity in our strains. As shown, the results reflect highly variable expression in between the different strains which was also seen in another study testing the expression of siderophores in K. pneumoniae strains [It is worth mentioning that a very recent study by Lan et. al. in 2022 addressed the fact that susceptibility to CFDC in carbapenem-resistant sistance . Lan et.ceptible . These ftigators ; thus, C strains . Our finfepA) as a crucial receptor, mostly responsible for capturing of the drug and allowing its entry to the periplasm. The production of other iron uptake receptors (such as fecA and/or kfu) can reduce the requirement to express siderophores and hence, their uptake will be reduced. It is possible that when the bacteria harbour multiple iron acquisition systems, they do not rely on siderophores expression, and this could result in lowering the uptake of catechol-cephalosporin such as CFDC. Lastly, the variations and dysregulation in the GE of iron transport receptors in different growth conditions related to iron availability can contribute to the differences in the activity of CFDC. Alarmingly, one strain was highly resistant to CFDC, thus, the whole genome of this strain will be analysed through sequencing to identify causes of resistance. In addition to that, whole transcriptome sequencing is recommended in the future to provide a wholistic insight on different genes expressed during treatment with CFDC, for a better understanding of the diverse factors affecting the drug susceptibility in GNB with diverse characteristics. It will also help us to uncover underlying mechanisms of action that are not discovered and not understood about the drug.When considering factors that can affect the activity of CFDC, iron seems to have a major impact as CFDC lost its activity when tested in iron enriched media and this could explain the increase in mortalities in patients receiving CFDC for the treatment of BSIs. Therefore, iron acquisition receptors can play a major role in the activity of CFDC. We identified enterobactin receptor (S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.