Nevertheless, the performance of retrofitted slabs is governed by debonding failure due to the low bond strength between CFRP and concrete overlay. Thus, this study investigates the behavior of flexural strengthened slabs with FRP retrofit systems and the effect of bond–slip laws on debonding failure. Firstly, two full-scale RC slabs with and without a retrofit system were tested in a four-point bending setup as the control specimens. Then, the same retrofitted slab was simulated by utilizing the commercial program ABAQUS. A sensitivity analysis was conducted to consider the influence of bond–slip laws to predict the failure mechanism of the retrofitted slabs based on load–deflection relationships. The results showed that the strengthened slab enhanced the load-carrying capacity by 59%, stiffness by 111%, and toughness by 29%. The initial stiffness of 0.1K Strengthening structures with external bonded materials has become one of the popular choices for rehabilitation and upgrading existing reinforced concrete (RC) structures ,2. ReseaThe conventional application in strengthening RC slab techniques focuses on attaching FRP to their tensile zone to maximize the tensile strength of these composite materials, which have been widely gaining acceptance in practice ,18,19. HConsequently, a hybrid retrofit system consists of carbon fiber reinforced polymer (CFRP) and concrete overlay applied on the top surface of the RC slab to overcome the shortcomings of traditionally strengthened solutions rather than exploit the high tensile strength of CFRP, as shown in In response to this need, the effect of bond–slip laws on the debonding failure of flexural strengthened RC slabs with a hybrid FRP retrofit system is investigated. To the best of our knowledge, previous studies were limited in evaluating the behavior of the externally bonded FRP on a concrete surface without considering cases of CFRP located between two concrete layers of the strengthened slab with a retrofit system ,35,36,37In this study, the behavior of a strengthened RC slab with a hybrid FRP retrofit system is described with particular attention to debonding failure. The effectiveness of the hybrid retrofit system in improving the flexural carrying capacity, stiffness, and toughness is investigated and compared with the full-scale experimental results. A three-dimensional FE model is developed with various bond–slip models for evaluating their accuracy by comparing numerical predictions with experimental measurements. Numerical models are also analyzed for sensitivity to the bond–slip parameters to assess their effects on the debonding failure. Following the validation of the model, the various parameters that strongly influence the behavior of retrofitted slabs are identified and discussed.cu) is 0.003, and steel yields at yield stress of fy. Without considering the tension force of concrete, the force equilibrium is calculated as follows:In some cases, strengthening the underside of the reinforced concrete slabs may not be possible due to logistical constraints and hindrances by other utilities. A hybrid retrofit system combining the tensile strength of FRP and compressive strength of the concrete overlay was applied on top of the existing RC slabs to improve their strength and ductility, as shown in These internal forces are computed as follows:F) as follows:An evaluation of strain compatibility conditions can determine FRP strain at strength limit (εThe moment capacity equilibrium condition can be defined as follows:2 cross-section. The tensile reinforcement was spaced at 185 mm spacing with five No.13 bars (ϕ12.7 mm) at the bottom of the RC slab. Transverse steel consisted of six No.10 bars (ϕ9.5 mm) spaced at 305 mm center on the center, as shown in The reference slab had a 2440 mm total length, 2290 mm clear span, and 130 × 900 mmA hybrid retrofit system was constructed utilizing a combination of CFRP wet layup laminate with a concrete overlay, as shown in Two full-scale RC slabs with and without a hybrid retrofit system were tested in a four-point bending setup with two concentrated loads, as shown in The experimental data were checked with the predicted analytical moment capacity and deflection at the mid-span due to the applied load of the reference and strengthened RC slabs, as recommended by ACI 440.2R . Based oc = 2.81 N/mm and Ws = 3.46 N/mm.The distributed load due to the self-weight of the reference slab and strengthened slab is determined as follows: WThe prediction of the load-carrying capacity corresponding to deflection at mid-span due to the applied load for the reference RC slab is given in The RC slab and CFRP laminate were assumed to be in perfect bond until the load-carrying capacity was reached. The prediction of the load-carrying capacity corresponding to deflection at mid-span for the CFRP-retrofitted slab due to the applied load is shown in x- and z-axis by restricting translation in directions 1 and 3. The concrete slab and concrete overlay with reduced integration were constructed with eight-node solid elements (C3D8R). Two-node linear truss elements (T3D2) and four-node shell elements (S4R) were used to model the reinforcements and CFRP laminate, respectively. A mesh size of 20 mm was suggested based on preliminary mesh refinement studies, ranging from 10 mm to 25 mm, for balancing accuracy and computational cost. The embedded function in ABAQUS/CAE 2022 was implemented to simulate concrete–steel bonding. The models were studied using static analysis in ABAQUS/standard. The models under monotonic displacement loads surveyed the behavior of a slab subjected to a four-point bending test. The steel bar was discretized as a rigid part to prevent manufactured stress concentrations under the loading points, as shown in The simulations were conducted using one-quarter of the control and strengthened slab specimens, based on specimen symmetry. The symmetric pane was simulated for the ct) and elastic modulus (Ec) of concrete, as recommended by ACI 318M [A model of concrete plastic damage was used with a failure mechanism of compressive crushing and tensile cracking. The stress–strain curve for concrete under uniaxial compression and tension was calculated by using the CEB–FIP model . CompresACI 318M .(6)fct=cr) is an inelastic parameter associated with the softening part of the curve. In mode I, fracture energy is defined as the area under the softening curve.Fracture energy (Gc0). In the plastic regime, the response is generally characterized by stress hardening, followed by strain softening beyond the ultimate compressive stress [Under uniaxial compression, a linear response is observed until reaching the initial value (σe stress .(9)σc=ERE=Rσ=4  .It is assumed that the reinforcement transmits force axially, and the most common perfectly linear–elastic model for reinforcement was used. Steel’s Poisson’s ratio is 0.3, and the other mechanical properties are listed in For strengthening, CFRP laminate is supposed to be linear and exert isotropic behavior. CFRP failure criteria were not considered for this study because tensile stress in CFRP is too low, compared with its ultimate strength, and therefore CFRP laminate hardly ruptures for these types of structures. The material properties of CFRP, including its thickness, elastic modulus, and ultimate strength, as shown in The FRP-to-concrete interface was modeled using cohesive zones. In this study, a bilinear traction–separation model was employed to depict the bonding characteristics of linear adhesive at the interface, as shown in The initiation and evolution of damage in the available traction–separation model were initially assumed to be a linear–elastic behavior ,51, whicFor specifying the damage initiation, quadratic nominal stress and maximum nominal stress between the CFRP and concrete interface were employed. The damage criterion for quadratic stress may be depicted as follows:The damage criterion for maximum stress can be described as follows:For specifying the damage evolution, the influence of power law and the Benzeggagh–Kenane (BK) fracture criteria on the structure behavior was further evaluated . The powThe BK fracture criterion may be defined as follows:In this study, the quantities 0), maximum shear stress (τmax), and fracture energy (Gf) [It is easily conceptualized that the traction–separation law, as described in rgy (Gf) . Based oA three-dimensional FE model using ABAQUS/CAE 2022 software was performed to simulate the behavior of retrofitted slabs. In the initial phase, an accurate FE model of the reference slab was assessed by comparison with the experimental results. In the second phase, a retrofitted slab model was developed based on the reference slab model, with the addition of FRP laminate and concrete overlay. The well-known bond–slip models were used to simulate the FRP-to-concrete interface to accurately simulate the behavior of the retrofitted slab validated by experiment results. If the available bond–slip models were not appropriate, the bond–slip parameters were revised to show good agreement with the experimental data. A flowchart of the analysis procedure for the FE model is shown in The experimental data from the reference slab showed a good agreement with those of the theoretical analysis. The RC slab behaved linearly and elastically until the first crack appeared at 17 kN of the applied load. The observed result revealed a higher load-carrying capacity of 43.5 kN than the theoretical prediction of 39.9 kN. The ultimate loading (50.8 kN) was 27.3% greater than the predicted load. The linearly elastic–perfectly plastic behavior of the steel can be the reason for the lower strength of the prediction because strain-hardened steel shows higher tensile strength in practice. The mid-span deflection reached a maximum value of 72 mm, as shown in For the retrofit slab, the cracks emerged and expanded most strongly at the mid-span zone—between the loading points, as shown in In this part of the study, the FE model of the retrofitted slab was used to examine the failure mode and delamination load after obtaining a good agreement between the FE model of the reference slab and the experimental results using the commercial ABAQUS program. A perfect bond model and well-known bond–slip models were applied to simulate the slab behavior through the load–deflection relationship. The material properties of the experimental structure were used to calculate the input parameters for the bond–slip models, as shown in f to Gf for the interfacial fracture energy. There was no evidence that Gf had a notable effect on the numerical results, as shown in The existing bond–slip models tended to overestimate the bond strength of the CFRP-to-concrete interface. In this part of the study, a sensitivity analysis of the bond–slip input parameters was performed to compare its results with the structural behavior obtained from the experimental data. The bond–slip model of Neubauer and Rostasy was used to illustrate the numerical results from the strengthened slabs. Initially, the CFRP and concrete bonding interface was assumed to fail using quadratic traction. A cohesive property coefficient of η = 1 was applied to depict the influence of the opening and sliding failure modes on fracture energy. The authors investigated the sensitivity of the results in the interfacial fracture energy between CFRP and concrete. For this case, a change was made from 0.01G0 to K0 to evaluate its effect. It had a substantial influence on the load–deflection curve’s slope of the structure. Based on the simulated results, the stiffness of the retrofitted slab decreased with decreasing the initial stiffness but was not proportional. With the initial stiffness of 0.1K0, the global response of the slab within the elastic limits could match the experimental results, as shown in The initial stiffness of the interface elements was changed from 0.01Kmax to τmax. The shear stress values had a considerable impact on the debonding failure. The delamination load of the retrofitted slab was sensitive to changes in the ranges of maximum shear stress from 0.1τmax to 0.15τmax, whereas a perfect bond was shown for values greater than or equal to 0.2τmax. It could also be easily seen that Neubauer’s and Rostasy’s bond–slip law, along with other equivalent models, overestimated the ultimate shear strength of the concrete overlay–CFRP interface. As mentioned above, the low bond strength between the concrete overlay and CFRP was the main reason for the delamination failure of the retrofit slab. It was possible to obtain better explanations for the differences between the simulated and experimental results by modifying the maximum shear stress. Therefore, the maximum interfacial stress of the concrete overlay/CFRP was further investigated using sensitivity analysis. An appropriate value for this parameter was crucial for the accurate prediction of CFRP debonding failures. As shown in max and 0.15τmax, respectively. The numerical results also indicated that the debonding failure emerged earlier, at 0.1τmax and later at 0.15τmax, compared with the experimental results. Moreover, the overestimation of the initial stiffness should also be highlighted in this case. The maximum shear stress ranged from 0.1τmax to 0.15τmax, combined with the initial stiffness of 0.1K0; thus, the case was further evaluated. The delamination load and mid-span deflection of the retrofitted slab increased with the increase in the maximum shear stress but was not proportional; however, there were no notable differences in the delamination load after the yield in steel. The global response of the strengthened slab was displayed as a function of the shear stress values. As a result, it was possible to reasonably simulate the behavior of the retrofitted slab and compare it with its experimental counterpart with the initial stiffness of 0.1K0 and maximum shear stress of 0.13τmax, as shown in According to the simulations, the delamination loads were 66.2 kN and 74 kN, corresponding to 10.1 mm and 48 mm of the mid-span deflection, respectively, occurring with the maximum shear stresses of 0.1τHere, we describe the effects of damage evolution based on energy. The load–deflection relationship was derived from the power law or BK law, as shown in In brief, these results were consistent with the previous literature regarding the influential factors on debonding failure, which is significantly affected by the maximum interfacial stress ,56,57 buIn this paper, a hybrid retrofit system considering the difficulty of accessing and installing CFRP laminates to the underside of RC slabs to enhance the flexural carrying capacity, stiffness, and toughness of the existing RC slabs was proposed. The proposed retrofitting mechanism proved suitable through a good agreement between the experimental results and theoretical analyses at the mid-span section. The global response and the effect of the bond–slip law in predicting the debonding failure of the retrofitted RC slabs were presented. The FE model using a sensitivity analysis based on the load–deflection relationship was performed to evaluate the parameters influencing the interfacial behavior between CFRP and concrete. Based on the obtained results, the following conclusions could be drawn as follows: The efficiency of the retrofit system was verified through the experimental test of the strengthened slab in the positive moment section. The retrofit system enhanced the load-carrying capacity of the slab by 59%, stiffness by 111%, and toughness by 29%.The neutral axis of the retrofitted slab was located within the overlay, and CFRP held tension at the mid-span section at the ultimate failure state.0 and maximum shear stress of 0.13τmax were compared with the corresponding values of Neubauer’s and Rostasy’s bond–slip law and showed a good agreement with the experimental data. The bond–slip models overestimated the criteria regarding the damage initiation between CFRP and the concrete overlay. The numerical analysis results with the initial stiffness of 0.1KThe stiffness of structures is notably impacted by the initial stiffness of the interface elements, while the delamination load and failure load can be decided by the maximum interfacial stress. Quadratic nominal stress is recommended as a criterion for damage initiation based on the considerable interaction between stresses.The global response of the retrofitted slab was not sensitive to changes in the interfacial fracture energy and damage evolution regarding the mix-mode failure criteria and the cohesive coefficient of the interface.Our study contributes to the evaluation of the impact of bond–slip parameters on the behavior of retrofitted slabs. Nonetheless, further research into an appropriate bond–slip model for retrofitted RC structures should consider the influence of factors regarding materials properties and geometric dimensions.


Autoimmune diseases (AIDs) are often co-associated, and about 25% of patients with one AID tend to develop other comorbid AIDs. Here, we employ the power of datamining to predict the comorbidity of AIDs based on their normalized co-citation in PubMed. First, we validate our technique in a test dataset using earlier-reported comorbidities of seven knowns AIDs. Notably, the prediction correlates well with comorbidity (R = 0.91) and validates our methodology. Then, we predict the association of 100 AIDs and classify them using principal component analysis. Our results are helpful in classifying AIDs into one of the following systems: (1) gastrointestinal, (2) neuronal, (3) eye, (4) cutaneous, (5) musculoskeletal, (6) kidneys and lungs, (7) cardiovascular, (8) hematopoietic, (9) endocrine, and (10) multiple. Our classification agrees with experimentally based taxonomy and ranks AID according to affected systems and gender. Some AIDs are unclassified and do not associate well with other AIDs. Interestingly, Alzheimer’s disease correlates well with other AIDs such as multiple sclerosis. Finally, our results generate a network classification of autoimmune diseases based on PubMed text mining and help map this medical universe. Our results are expected to assist healthcare workers in diagnosing comorbidity in patients with an autoimmune disease, and to help researchers in identifying common genetic, environmental, and autoimmune mechanisms. Autoimmune diseases (AIDs) have a multifactorial etiology including genetic and environmental components. The incidence of AIDs is estimated at 3–5% worldwide. Autoimmunity is known to have a genetic component; yet, concordance rates of AIDs in monozygotic twins are incomplete, indicating a multifactorial etiology. The differences in autoimmunity incidence rates in different ethnic groups and geographical locations suggest the involvement of environmental factors such as lifestyle, exposure to infection, and nutrition, among others.Autoimmune diseases (AIDs) occur when the immune system attacks self-molecules as a result of a breakdown of immunologic tolerance to autoreactive immune cells . These cThe idea that different AIDs are associated is not new, and ~25% of patients with one AID tend to develop other comorbid AIDs . The staSeveral approaches have been used to classify human AIDs ,8. TheseBioinformatics uses the wealth of information to analyze biological data. Text mining and citation counts are often used to identify trends and patterns in medicine . Several1 and AID2 correspond to AID pairs:To mine for AIDs associations, we counted the number of co-citations on PubMed. The association of two AIDs was taken as the number of PubMed citations of the AID pair , divided and normalized by the number of PubMed citations for each disease alone  according to the equation below, in which AIDThe nominator of this equation is biased towards highly cited AIDs. To counter this bias, the association is divided and normalized by the denominator, which reduces the association of highly cited AIDs and increases the association of poorly cited AIDs. This equation also assumes random noise, and signal to noise is expected to grow by the square root of the signal, similar to random walk. To further reduce the chance of random co-occurrence, we queried only the title, abstract, and other terms of PubMed citations, but not the full text paper. Finally, to facilitate the laborious process of counting citations and to prevent scribe copy errors, a Python script was used. The script uses the “wget” function and rapidly looped over all AIDs pairs. The search was performed on a standard PC equipped with 16 GB RAM and operating under Microsoft OS. At an internet speed of 40 Mbps, one query took less than a second, and a complete run of the script took ~6 h. https://autoimmune.org/disease-information, accessed on 12 July 2021), the Autoimmune Institute website , and the Autoimmune Registry . Inadvertently, some of the 150 diseases were not AIDs and were omitted from our classification. Furthermore, temporary diseases were also excluded because they do not fit with the progressive nature of AIDs. Finally, indistinct AIDs with low PubMed citation counts were also omitted. The final list included only 100 AIDs. To validate our technique, we calculated the association values of a limited dataset of 7 AIDs reported by Cifuentes et al. . The traTo classify AIDs, we use heatmap clustering based on association values. In particular, the ClustVis website was used for visual classification . In addiTo validate our technique, we calculated the association values of seven AIDs. Notably, the association values reported in Notably, most AIDs are comorbid, as shown in Retroperitoneal fibrosis, which is caused by the buildup of inflammatory and fibrous tissue (collagen type 1) in the retroperineum , is assoCastleman disease is a lymphoproliferative condition, which presents either idiopathically, or in association with herpesvirus , or a maGuillain Barré syndrome occurs upon demyelination of peripheral nerves (collagen type 6), and is often induced by an infection or a drug . GuillaiRelapsing polychondritis is characterized by recurrent inflammation of cartilaginous tissues (collagen type 2) throughout the body . Figure Goodpasture’s syndrome occurs through the deposition of autoantibodies in basement membranes (collagen type 4) of kidneys and lungs, eliciting rapidly progressive glomerulonephritis and pulmonary hemorrhage . Figure Neonatal lupus and congenital heart block can lead to atrioventricular conduction abnormalities diagnosed in utero or within the first month of life . MaternaEosinophilic fasciitis involves inflammation of tissue under the skin and over the muscle, called fascia (collagen type 1), and it tops the list of AIDs associated with collagen and male predominance . Next, stiff person syndrome is caused by autoantibodies to glutamic acid decarboxylase (GAD), which lead to muscle weakness. Surprisingly, undifferentiated connective tissue (UCTD) is not clustered with other connective tissue diseases, and Cold agglutinin disease is a form of autoimmune hemolytic anemia following exposure to cold temperature. Interestingly, the onset age depends on the climate, and chilly weather precipitates the disease. As such, as seen in Parry Romberg syndrome (PRS) is characterized by the progressive degeneration of tissues of one side of the face, leading to hemifacial atrophy. Cogan’s syndrome is an autoimmune inner ear disease, and Lambert–Eaton myasthenic syndrome is an autoimmune disorder characterized by muscle weakness of the limbs . NotablySusac’s syndrome is a rare autoimmune disease that mainly affects young women. It is characterized by endotheliopathy, which presents as encephalopathy, retinal vasoocclusive disease, and hearing loss . Figure Remarkably, our PubMed clustering differentiates between male and female AIDs. Here too, autoimmune oophoritis and autoimmune orchitis are gonad AIDs, and antibodies bind to both testicular and ovarian target antigens during their development .Mucha–Habermann disease (MHD), also known as “Pityriasis lichenoides et varioliformis acuta” is a skin disease characterized by rashes and small lesions. Progesterone dermatitis (PD) is a skin disease due to progesterone toxicity, for example during the menstrual cycle . Both PDGraves’ disease is the most common cause of hyperthyroidism. It is an autoimmune disorder with systemic manifestations that primarily affect the heart, skeletal muscle, eyes, skin, bone, and liver. Pernicious anemia is commonly caused by deficiency of vitamin B12 . Sometimes, it is also classified as an autoimmune disease, as it is comprised of salient features of autoimmune chronic atrophic gastritis and cobalamin deficiency . Figure Trivially, polyglandular syndromes (PGS) type 1, 2, and 3 are highly associated . PGS typDermatitis herpetiformis (formerly known as Duhring’s disease) is associated with other skin diseases such as bullous pemphigoid and pemphigus , as showCicatricial pemphigoid , and ocular cicatricial pemphigoid, are different manifestations of the same bullous disease, which is manifested through blisters all over the body . Figure Discoid lupus is a dermatological AID that can lead to rashes, scarring, hair loss, and hyperpigmentation of the skin, which tends to get worse when exposed to sunlight. Vitiligo is associated with several comorbid autoimmune, systemic, and dermatological diseases, primarily thyroid disease, alopecia areata, diabetes mellitus, pernicious anemia, systemic lupus erythematosus, rheumatoid arthritis, Addison’s disease, inflammatory bowel disease, Sjögren’s syndrome, dermatomyositis, scleroderma, psoriasis, and atopic dermatitis, among others  59]. . 59]. Alopecia areata is characterized by a well-defined non-scarring alopecic patch or patches that can extend to the entire scalp or lead to total body hair loss . Figure Primary biliary cirrhosis and primary sclerosing cholangitis are associated in Myositis is an inflammation of muscles responsible for movement. Trivially, myositis, polymyositis, dermatomyositis, and inclusion body myositis are highly associated in Scleroderma is another connective tissue disorder, both local and systemic, and can further be classified as limited systemic sclerosis, formerly known as the CREST syndrome , which cPsoriasis and psoriatic arthritis are trivially associated in Primary Sjögren’s syndrome is a systemic AID that is characterized by a triad of symptoms, namely, dryness, pain and fatigue . Figure Rheumatoid arthritis is a chronic inflammatory joint disease, predominantly of autoimmune origin. Rheumatoid arthritis autoantibodies include the rheumatoid factor and antibodies against citrullinated proteins .Systemic lupus erythematosus (SLE) is a multisystem AID with varied clinical manifestations and a complex pathogenesis . SLE is Glomerulonephritis, IgA nephropathy, and amyloidosis are highly and often associated with arthritis, as shown in Next are cardiovascular AIDs, and Although, vasculitis is not specifically autoimmune, inflammation of the blood vessels is associated with AIDs. IgA vasculitis , and IgG and IgM vasculitis are three types of leukocystoclastic vasculitis, and their high association is trivial . Rarer vMounting evidence suggest that narcolepsy is a neuronal AID, and it is associated with restless legs syndrome  is often due to chemical exposure. Benign mucosal pemphigoid is mostly associated with external noxious stimuli, unlike the AID mucous membrane pemphigoid , and as In addition, some of the names are not necessarily associated with AIDs, further confounding their classification. Palindromic rheumatism and autoimmune urticaria are temporary rheumatisms and do not fit with the progressive nature of AIDs, and are not included in Some of the potential AIDs result from injury and are not included in Postpericardiotomy syndrome is a subgroup of post-cardiac injury syndromes together with postmyocardial infarction syndrome (Dressler’s syndrome) and posttraumatic pericarditis and are omitted from Giant cell myocarditis (GCM) is not an autoimmune disease in more than 80% of cases  and was Parsonage–Turner syndrome, also known as neuralgic amyotrophy, is a poorly understood neuromuscular disorder affecting peripheral nerves mostly within the brachial plexus distribution but can also involve other sites, including the phrenic nerve . The etiAlzheimer’s disease (AD) is an irredeemable chronic neurodegenerative disorder and the leading cause of dementia in the elderly. Despite rigorous multinational efforts and decades of costly research, no scientific consensus regarding the causes of AD has been achieved. Still, an exclusive pathological event leading to AD development remains a mystery. Growing evidence indicates that AD results from several intertwining pathologies ,103. It Several groups tried to provide evidence linking autoimmune diseases with dementia. Wotton and Goldacre performed a retrospective, record-linkage cohort study to determine whether hospital admission for autoimmune disease is associated with an elevated risk of future dementia . ResearcAnother recent investigation demonstrated a significant decrease in the total and resting regulatory T cells in AD patients. Surprisingly, a similar phenotype was detected in MS patients. The authors suggest that alterations in regulatory T cells number and activity observed in both diseases play a role in the T cell-mediated immunological tolerance impairment, which indicates a link between these pathologies . Of noteHere, we support this hypothesis and show a high association between AD and MS . MoreoveUndoubtedly, there is primary clinical importance in revealing the putative autoimmune components of AD. A novel complex approach to the disease treatment and diagnosis, which considers several intricate mechanisms, may pave the way to efficient therapy.Here, we quantify the association of some 100 different AIDs. The affected systems include: (1) gastrointestinal, (2) neuronal, (3) eye, (4) cutaneous, (5) musculoskeletal, (6) kidneys and lungs, (7) cardiovascular, (8) hematopoietic, (9) endocrine, and (10) multiple. Interestingly, our clustering differentiates between predominantly male and female AIDs. The male/female classification does not explain why most AIDs are more prevalent among women, while others are more prevalent among men. One reason could be the type of tissue affected, and if collagen is targeted, then female predominance could be masked by the high collagen level in male skin. In addition, our clustering of AIDs follows average onset age and systems affected. Our results are expected to assist the diagnosis of comorbidity, and identification of common genetic and environmental factors.


MET) proto-oncogene alterations, including the exon 14 (METex14) skipping mutations as well as the amplification and overexpression of MET gene, which are associated with molecularly targeted therapy for PSC. METex14 skipping mutation is the most common and well-studied mutation type, occurring in about 22-31.8% of PSC patients, while the prevalence of MET amplification is reported as 4.8-13.6% and MET ovexpression is about 20.2%. Molecular pathology tests, including IHC and NGS, are valuable in determining the prognosis of patients with PSC and helping to determine the treatment. The existing clinical data have confirmed the efficacy of MET-TKI in PSC patients with MET alteration, among which the clinical study of Savolitinib has enrolled the largest proportion of PSC patients and achieved relatively good efficacy, but more clinical researches are still needed. The multi-disciplinary team may maximize the optimal treatment options for patients with the advanced PSC.Pulmonary sarcomatoid carcinoma (PSC) is a rare subset of NSCLC that accounts for about 0.5-1% of all primary lung carcinoma, and its malignant biological behavior is more aggressive than other pathological types of lung cancer. Recent studies have reported a variety of gene mutations associated with the occurrence, development and treatment of PSC, especially the mesenchymal-epithelial transition ( ALK, BRAF, EGFR, KRAS, MET, NTRK1/2/3, RET and ROS1. The broad genomic testing can be used to assess mechanisms of drug resistance in patients who have relapsed after the targeted therapies, to distinguish primary lung cancer from intrapulmonary metastasis, and to help determining suitability for certain molecular-driven clinical trials  is the most common type of lung cancer, accounting for approximately 85% of all lung cancer with significant heterogeneity and it may be associated with some known and/or unknown driver gene changes . AccordiPulmonary sarcomatoid carcinoma (PSC) is a rare subset of NSCLC that accounts for about 0.5-1% of all primary lung carcinoma, and its malignant biological behavior is more aggressive than other pathological types of lung cancer . AccordiPSC usually occurs in older men with a history of smoking, and its clinical symptoms are non-specific compared with other types of NSCLC, such as cough, chest pain, hemoptysis, and dyspnea, which makes early detection and diagnosis difficult to some extent , 6. PSC MET) proto-oncogene alterations in PSC, including MET exon 14 (METex14) skipping mutations and MET amplification, with an incidence of approximately 22% and association with molecular-targeted therapy for PSC  and contains 21 exons. The MET proto-oncogene encodes a membrane MET tyrosine kinase receptor that mainly expressed in epithelial cells, also known as hepatocyte growth factor (HGF) receptors, which have been identified as a tumor driver gene and potential target of NSCLC /AKT (protein kinase B), signal transducer and activator of transcription protegins (STAT), mitogen-activated protein kinase (MAPK), Wnt/beta-catenin, extracellular signal regulated kinase (ERK), mammalian target of rapamycin (mTOR)and nuclear factor-kB (NF-kB)  , 12.HGF is the only known natural ligand of MET, and its binding to MET leads to receptor dimerization and phosphorylation of Y1234 and Y1235 tyrosine residues in the kinase domain’s catalytic loop and autophosphorylation of the carboxy-terminal bidentate substrate-binding sites 1349 and 1356, which can activates RTK-mediated downstream signaling pathways mentioned above . These sMET gene fusions  and occur more frequently in PSC, and METex14 skipping mutation is the most common and well-studied mutation type, occurring in about 3%-4% of adenocarcinoma patients and 22% of PSC patients  and Sanger sequencing have both been used to detect MET mutations, but these targeted methods are rarely used because of the efficiency of detection  amplification . This me dropout . In addid method . Anotherd method . These pd method . Chen etd method  reportedMETex13 to 15, which is a common consequence of any altered splicing mechanism or deletion . But there are also problems with RNA NGS. RNA is less stable than DNA, which limits the shelf life of the tissue. In addition, interpretation of RNA NGS results poses challenges due to the high variability of mRNA expression between nonmalignant and tumor tissues . Therefore, the efficacy of molecular targeted therapy for MET is a hot topic at present, and many clinical studies of drugs are being carried out. Crizotinib, a multi-target TKI covering MET, has been reported in some small retrospective studies to treat advanced PSC with METex14 mutation. Unfortunately, these data have not been analyzed independently from other non-small cells  enrolled four PSC patients with METex14 mutations, including one with stable disease and three with partial response , but the trial data were not analyzed by pathological type . The mPFS of PSC subgroup was 5.5 months, mOS was 10.6 months, ORR was 50%, and disease control rate (DCR) was 90% , fluorescence in situ hybridisation (FISH), quantitative Real-Time reverse transcriptase-PCR (qRT-PCR), or NGS (Next Generation Sequencing). However, IHC appears to be a poor screen for MET amplification since MET IHC-positive cases may be MET amplification negative and vice versa  or the ratio of MET to chromosome enumerating probe against chromosome 7 (CEP7). Generally, MET GCN≥5 or MET : CEP7≥2.0 is used as the FISH criteria for MET amplification , intermediate (>2.2 to <5) and high (≥5) or low (≥1.8 to ≤2.2), intermediate (>2.2 to <4) and high (≥4)  , 35. In n tumors , 36. Adde sample .De novo MET amplification (primary MET amplification) occurs in about 1-5% of non-small-cell lung cancers (NSCLC), and acquired MET amplification (secondary MET amplification) are typically identified in about 5-20% patients with oncogene-positive NSCLC following resistance to tyrosine kinase inhibitors (TKIs), such as EGFR TKIs , antibodies and ADCs combinations.igenesis . MET oveMET expression is IHC with kinds of antibodies, consisted of monoclonal antibodies, polyclonal antibodies and antibodies to phosphorylated MET (MET overexpression is defined as IHC 2+ and 3+ (MET overexpression, but different thresholds vary between different clinical studies (The most commonly used detection method of ated MET . Generalated MET . IHC 1+ + and 3+ . The H-s+ and 3+ . The sco studies .MET overexpression has been reported with high frequencies in NSCLC, ranging from 22.2-74.5%, and seems to portend poorer prognosis (MET overexpression in PSCs are limited. Liu et al. (MET overexpression in 20.2% PSCs and carried survival analysis of MET gene alterations and protein expression in Chinese PSCs with only surgery but no MET TKI treatment. They found that MET amplification suffered shorter mOS, while METex14 skipping mutation and overexpression didn’t affect patients’ survival. The studies showed different prognostic value of MET overexpression between PSCs and NSCLCs.u et al.  found MEMET overexpression as a poor predictor of MET amplifications or exon 14 mutations in PSCs. They reported that MET exon 14 mutations could cause loss of ubiquitination and improve the presence of c-MET membrane, while lack of association between them and MET overexpression remains to be understood. These results may be induced by the different genomic backgrounds between PSCs and NSCLCs, or other mechanisms in the oncogenesis of MET exon 14 mutations. The lack of clinical trials about PSCs results in the limited understanding of the biological mechanisms, development, diagnosis, prognosis and treatment of the tumors (Xavier Mignard et al.  found MEe tumors . We are MET alterations occur more frequently in PSC than in other types of NSCLC, and METex14 skipping mutation is the most common type. The existing clinical data have preliminarily confirmed the efficacy of MET-TKI in PSC patients with MET alteration, among which the clinical study of Savolitinib has enrolled the largest proportion of PSC patients and achieved relatively good efficacy, but more clinical research is still needed. The multi-disciplinary team may maximize the optimal treatment options for patients with advanced PSC.PSC is a subtype of NSCLC with unique malignant biological behavior. The symptoms are not specific, and PSC is often found in the advanced stage. It is not sensitive to traditional chemotherapy and radiotherapy, and the expected survival time is short, and the prognosis is poor. It is most likely that the treatment of PSC should continue to follow the treatment guidelines for advanced NSCLC. Molecular pathology tests, including IHC and NGS, are valuable in determining the prognosis of patients with PSC and helping to determine the treatment of NSCLC patients. MET mutation. HX and JZhang designed the review and revised the manuscript. KQ wrote the section of wild-type MET. JW wrote the section of MET amplification. JZhao wrote the section of MET overexpression. YC wrote the section of conclusion. All authors contributed to the article and approved the submitted version.CG wrote the section of introduction and The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study aimed to assess the financial burden of out-of-pocket (OOP) payments to purchase antidiabetic medicines for type 2 patients in Iran.The “budget share” and “capacity to pay” approaches were employed to assess the catastrophic pharmaceutical expenditures of antidiabetic medication therapies. The catastrophic thresholds were adjusted for pharmaceutical sectors. The data was 2019 monthly household expenditures in rural and urban areas, insurance coverages of antidiabetic medicines and patients' out-of-pocket (OOP) payments in 30-day treatment schedules.The results show that expenditure on diabetes medication therapies in the form of mono-dual therapy and some cases triple oral therapies were not catastrophic even for rural households. Insulin puts patients at risk of catastrophic pharmaceutical expenditures when added to the treatment schedules, and lack of financial protection intensifies it. In general, the poorer households and those resistant to first-line treatments were at increased risk of catastrophic pharmaceutical expenditures. The number of treatments that put patients at risk of catastrophic pharmaceutical expenditure in "budget share" was higher than the "capacity to pay" approach.Assessing medication treatment affordability instead of a single medicine assessment is needed. Assessment could be done by utilizing a macro-level data approach and applying adjusted pharmaceutical sector threshold values. Considering the variation between treatment schedules that put patients at risk of catastrophic pharmaceutical expenditures, targeted pharmaceutical policies and reimbursement decisions are recommended to promote Universal Health Coverage (UHC) and to protect vulnerable populations from hardship. Defining specific adjusted catastrophic expenditure for the pharmaceutical sector based on national health systems whining countries is so critical.Using Macro-level data instead of conducting a household survey is recommended due to less expensive than the survey and feasibility of assessment in different time periods and facilitating international comparison between countries.Targeted pharmaceutical policies and reimbursement decisions are recommended to promote Universal Health Coverage (UHC) and protect vulnerable populations from hardship due to catastrophic pharmaceutical expenditures.Many countries depend on the patients' out-of-pocket (OOP) payments in a healthcare financing system . ExcessiPrevious studies have shown that different components of health expenditures could create a financial burden for households and increase catastrophic expenditures, including household OOP payments on inpatient services, outpatient services, and medicines , 5. EvidConcerns regarding the lack of protection against catastrophic drug costs led political authorities and policymakers to assess the affordability of healthcare, improve access, and achieve Universal Health Coverage (UHC) . InvestiBased on a systematic review, the incidence of CHEs in Iran was 7.5%  to measure CEs, which is difficult and costly. Previous studies have examined only one or a limited number of medicines –15. The The present cross-sectional study assessed Iran's financial burden of OOP payments for diabetes medicines in 2019. This was done by utilizing well-accepted and commonly applied approaches and adjusting the thresholds for the pharmaceutical sector .Catastrophic Health Expenditures (CHEs) refers to any medical treatment expenditure that can threaten a household's financial ability to maintain its subsistence needs . In the Different components of health expenditure could create a financial burden for households and drive catastrophic expenditures. Most literature investigated catastrophic health expenditures as total -how much you spend on health during the last month- , and limBy considering that pharmaceutical expenditure constitutes 20% of total health expenditure (THE) in Iran, we set new thresholds by multiplying this percentage by the predominantly used thresholds in literature for estimating Catastrophic Health expenditure (CHE) 10% or 40%). See Table 0% or 40%Most international clinical guidelines recommend monotherapy as the first-line treatment, including Metformin, Glibenclamide and Gliclazide. In the absence of optimal (appropriate) blood glucose levels, dual therapy was prescribed, i.e., Metformin plus one oral medication including Acarbose, Repaglinide, Glibenclamide or Pioglitazone. Triple therapy initiatives were prescribed when mono and dual-therapy failed, including Metformin plus two oral medications; Metformin plus basal Insulins or Insulin therapy.This study employs the macro method suggested by Niëns et al. . This meThe needed data on monthly household expenditure in rural and urban areas was obtained from the Statistical Center of Iran (SCI) . The SCITo estimate monthly patients' out-of-pocket (OOP) payments, first, the medication therapy costs in each treatment scenario were estimated regarding treatment schedules in Table The analysis was conducted using the budget share (formula 1) and capacity to pay (formula 2) methods for different rural and urban income deciles.If the CPEs were more than 2% in budget share approach or more than 8% in capacity to pay approach, it was considered a catastrophic pharmaceutical expenditure.The cost of treatment schedules, household out-of-pocket (OOP) while seeking those treatments, the catastrophic treatments, and the proportion of households to be confronted with CPEs in rural and urban areas for the year 2019 are presented in this section.Generally, clinicians choose three standard medication therapy regimens for managing type 2 diabetes. Table In case of initial treatment failure, clinicians may adjust treatment dosage or switch to the next medication therapy option. Figures Figure It was observed that the catastrophe medication varied by treatment schedule and available household resources. The assessment showed that oral medication therapies were affordable, and the household incurred catastrophic expenditure when Insulin was recommended for disease management. Furthermore, there is a considerable difference between the shares of OOP payments between the households. The highest OOP payment share belongs to rural and urban households in deciles 1 to 3 for treatment schedules, including only insulins. By moving to wealthier households, some treatments were not catastrophic or catastrophic, with the share close to the benchmarked thresholds. Glulisine, detemir, and glargine insulin contributed significantly to increasing OOP payments due to high prices and lower insurance coverage compared with the other Insulin such as isophane insulin . However, it is essential to note that exclusively insulin therapy  is prescribed for advanced type 2 diabetes patients when monotherapy, dual oral therapy, oral therapy and Insulin regimens in combination with oral medications fail. As a result, only a tiny percentage of patients with type 2 -almost 2%- were switched by clinicians to these treatment schedules.The poorest households in deciles 1, 2, and 3 had the highest share of catastrophic medication therapy expenditures. Moreover, pharmaceutical expenditures for rural households were more catastrophic.Figure As mentioned later, catastrophic treatment schedules are exclusively insulin therapy prescribed to patients when there is not enough response to initial oral therapy with Insulin. The number of catastrophic therapies would further decrease by excluding this small group of patients. Generally, the number of catastrophic treatments was lower than the budget share approach (with a 2% threshold).The proportion of households facing catastrophic payments for procuring antidiabetic medication is shown in Table Finally, in the "capacity to pay approach," the proportion of households with catastrophic expenditure is lesser than the budget share. This measurement approach difference should come to attention while making the policies because they may be over or under-estimated.In this study, the catastrophic pharmaceutical expenditures of diabetes medicines are being comprehensively investigated in different treatment schedules. The budget share and capacity to pay approaches were used to explore the catastrophic treatments and the incidence of catastrophic financial effects among rural and urban Iranian households. The new thresholds for the pharmaceutical sector were set considering the share of pharmaceutical expenditures (PE) to total health expenditures in Iran.To the best of our knowledge, the present study can be mentioned as the first to assess medication therapy's catastrophic expenditures in different mono-and combination treatment schedules. In the previous studies, only a limited number of medicines were assessed , 24, 25,The results showed that the OOP payment increased with increasing doses of medicines in initial treatment or when switched to second-line options. However, the treatment is still affordable. No one has encountered catastrophe expenditures with mono-and dual and sometimes triple oral therapies, even in rural areas. These findings align with Amiresmaili and Emrani's work which showed that only a tiny fraction, 0.007% of Iranian families, were required to pay over 40% of their income for Metformin . In addiTreatment schedules were catastrophic when Insulin was added to oral medication therapies. In line with the previous studies, Insulin is a significant contributor to catastrophic expenditures –29. AccoThe lowest-income households in rural areas are at most risk of being pushed toward a catastrophic border . This shDifferent studies confirmed that rural residences were significantly at risk of CHEs –33. ThisThe results showed that more catastrophic treatments were observed in the budget share approach, and a higher proportion of households faced catastrophic expenditures. Cylus et al.  showed tFurthermore, Niëns et al.  showed tMszar et al. revealed that although having diabetes increases the odds of financial hardship, individuals with concurrent atherosclerotic cardiovascular disease and diabetes had the highest relative odds of expressing an inability to pay at all when compared with those with neither of the condition  .In using macro-data, the mean expenditure in each income decile was used while assuming the linearity of income. While the distribution of income in each income group could be skewed since most individuals in the group could be potentially in the lower income bracket than the average. The average income for each income may be overestimated, and thus, the catastrophic effects of the medicines may be underestimated in the analysis. In addition, although using macro-level data is simple and generalizable, there is no access to individual-level data, so it is not possible to provide further analysis based on the actual status of each household like population income status, household size etc.Poorer households and patients who are resistant to first-line therapies probably suffer more from catastrophic expenditures as they will not be able to afford the Second- and third-line antidiabetic treatment expenses. Though this could be a small group of patients in Iran, making more efficient reimbursement decisions is required to ensure the affordability of antidiabetic medications.At a policy level, to provide policymakers with a better understanding and more profound insights toward catastrophe expenditure within health systems and across countries, defining threshold value for assessing catastrophic expenditure for each component of health expenditure and using the conventional approach of aggregated data is strongly recommended.


Background: The influence of increased use of telehealth during the emergence of COVID-19 on antibiotic prescriptions in outpatient settings is unknown. The VA Northeast Ohio Healthcare System has 13 community-based outpatient clinics (CBOCs) that provide primary and preventive care. We assessed changes in antibiotic prescriptions that occurred as care shifted from in-person to telehealth visits. Methods: Using VHA administrative databases, we identified all primary care CBOC visits between January 1, 2019, and December 31, 2020, that included a diagnosis for an acute respiratory infection (ARI), a urinary tract infection (UTI), or a skin or soft-tissue infection (SSTI), excluding visits with >1 of these diagnoses or with additional infectious diagnoses . We summarized the proportion of telehealth visits and the proportion of patients prescribed antibiotics at quarterly intervals. We specifically assessed outpatient visits from April to December 2019 compared to the same months in 2020 to account for seasonality while analyzing diagnosis and antibiotic trends in the emergence of the COVID-19 pandemic. Results: The patients receiving care in April–December 2019 compared to April–December 2020 were similar (Table Conclusions: Among the CBOCs at our healthcare system, an increase in the proportion of telehealth visits and a reduction in ARI diagnoses occurred after the emergence of COVID-19. In this setting, we observed a reduction in the proportion of visits for ARIs, UTIs, and SSTIs that included an antibiotic prescription.Funding: MerckDisclosures: None


V72I was more stable structurally and more active in the ATPase activity than the wildtype. Analysis of our recently solved cryo-EM structure of mHsp60 revealed allosteric roles of V72I in structural stability and ATPase activity, which were supported by studies including those using the V72A mutation. Despite with the increases in structural stability and ATPase activity, mHsp60V72I was less efficient in folding malate dehydrogenase, a putative mHsp60 substrate protein in mitochondria and also commonly used in chaperonin studies. In addition, although mHsp60V72I along with its cochaperonin mHsp10 was able to substitute the E. coli chaperonin system in supporting cell growth under normal temperature of 37 °C, it was unable under heat shock temperature of 42 °C. Our results support the importance of structural dynamics and an optimal ATP turnover that mHsp60 has evolved for its function and physiology. We propose that unproductive energy utilization, or hyperactive ATPase activity and compromised folding function, not mutually exclusive, are responsible for the V72I pathology in neurodegenerative disease.Human mitochondrial chaperonin mHsp60 is broadly associated with various human health conditions and the V72I mutation in mHsp60 causes a form of hereditary spastic paraplegia, a neurodegenerative disease. The main function of mHsp60 is to assist folding of mitochondrial proteins in an ATP-dependent manner. In this study, we unexpectedly found that mutant mHsp60 Another essential function is to coordinate metabolic processes. Mitochondria produce metabolites for biosynthesis of amino acids, nucleotides, and fatty acids that are essential for both cell growth and communication3. A third essential function is to maintain Ca2+ homeostasis4: Ca2+ as an universal second messenger regulates a range of cellular activities including the synaptic activity in neurons5. These three essential mitochondrial functions are particularly important to neurons, as such, impaired mitochondrial function has been found associated with neurodegenerative disorders6.Mitochondria are essential for eukaryotic cells. One of the essential functions is to generate energy in the form of ATP: as the cellular power house, mitochondria provide more than 90% of the energy the human body needs to function7 and are required for cellular viability under both normal and stress conditions8. The main function of chaperonins is to assist folding and assembly of cellular proteins using an ATP mechanism13. Broadly, mHsp60 has been associated with human health conditions including Alzheimer’s disease14, diabetes15, cancers16 and cardiovascular conditions17. Mutations in mHsp60 are responsible for neurodegenerative diseases18 including hereditary spastic paraplegia19 and MitCHAP60 disease20. The hereditary spastic paraplegias (HSP) are a large group of inherited neurologic disorders with the primary symptom of difficulty in walking due to muscle weakness and muscle tightness (spasticity) in the legs. More than 80 different genetic loci with over 60 gene products identified, naming spastic paraplegia gene (SPG) 1–8322.Normal mitochondrial function relies on mitochondrial protein homeostasis, which is maintained by a range of molecular chaperones, for example, including mHsp60. mHsp60 belongs to a group of proteins called chaperonins that are highly conserved among the three kingdoms of lifemhsp60 gene19. mHsp60 is homo-heptameric with the seven subunits forming a ring-like structure . Unexpectedly, mHsp60V72I was more stable than the wildtype mHsp60 because the mutant had a higher melting temperature (Tm) and a larger unfolding enthalpy (∆Hunfolding) than mHsp60  is presumably the mHsp60 substrate because it is one of the proteins identified that interact with mHsp60E. coli chaperonin GroEL and cochaperonin GroES in supporting E. coli cell growth under the normal (37 °C) and heat stress (42 °C) conditions. mHsp60/mHsp10 could substitute GroEL/GroES to support cell growth under both normal and stress conditions. At 37 °C, both V72I and V72A mutants, in either the full length or truncated form, supported cell growth just like the wildtype mHsp60  in assisting folding of MDH, a presumptive endogenous substrate of mHsp60 in mitochondria33. In addition, we showed that mHsp60V72I lost the chaperone function in supporting cell growth under heat stress. These compromised in vitro and in vivo mHsp60 functions, together with the HSP SPG13 pathology caused by the V72I mutation19, underscore the critical role of subunit association dynamics in mechanism, function and physiology of mHsp60.Our studies highlight the unique properties, structural dynamics and optimal ATP turnover efficiency, that human mitochondrial mHsp60 has evolved. Unlike the paradigm bacterial chaperonin GroEL, mHsp60 is intrinsically dynamic as evident by its irreversible dissociation to monomers37. The extra-mitochondrial locations may impart novel, location-specific functions to mHsp60. Although information on how mHsp60 reaches these new locations and what function-structure of mHsp60 is limited, monomeric mHsp60 has been proposed as a functional conformation for the new functions38. Thus, in addition to the importance in the main function in assisting protein folding, dynamics of mHsp60 subunit association may have mechanistic implications for its extra-mitochondrial functions, adding another complexity to the physiology of mHsp60.Subunit association dynamics has been proposed to account for the new locations and functions implicated for mHsp60, which may contribute to the V72I pathology. Besides its conventional location of mitochondrion, mHsp60 has been found on the outer surface of cells, in cellular compartments such as peroxisome and endoplasmic reticulum, and in biological fluids including bloodE. coli GroEL-GroES28; however, the efficiency and effectiveness of mHsp60-mHsp10 in assisting MDH folding are the same as GroEL-GroES26. In our previous studies to activate single-ring GroEL mutant GroELSR via mutations in a concatenated GroES GroES7, we found that the ATPase activities of the single-ring GroELSR-GroES7 systems are not directly correlated with the folding activities of MDH39. For example, the ATPase activities of the systems with most active MDH folding activities are ~ 25% of the highest ATPase activities tested. Clearly, an optimal, energy efficient chaperonin system should assist protein folding at fast kinetics, with high yields, and at low ATP activity (ATP turnover rate). As such, mHsp60V72I is biochemically an inefficient chaperonin in two folds. Like the conventional mutants, it is compromised in assisting protein folding; unlike the conventional mutants, it is unproductive in using more ATP molecules. Physiologically, the V72I mutation is pathological in two folds. Due to mHsp60’s essential role in maintaining protein homeostasis in mitochondria thereby mitochondrial function, the mutant’s compromised chaperone function impairs the overall mitochondrial function. In addition, its hyperactive ATP turnover drains the cellular ATP supplies including those in neuron cells. Neurons have high demand for ATP: they spend ~ 50% of the total consumed ATPs on the synaptic activity. Together, these two mechanisms, not mutually exclusive, may account for the V72I-associated HSP SPG13 neurodegenerative disease. To provide structural rational to intervene the pathological effects caused by the V72I mutation, we are investigating structural basis for how the V72I mutation increases ATPase activity as well as to confirm the V72I effect on structural stability as proposed our current structural analysis.Our studies show that mHsp60 has evolved to utilize ATP optimally in assisting protein folding. The ATPase activity of mHsp60-mHsp10 is ~ 25% of that of E. coli, and was inserted into pBbE5a with NdeI/BamHI sites. To remove the C-terminal 26 a.a. Gly-Met repeats, primers (5’) AAACATATGGCTAAAGATGTGAAGTTT and (3’) TTTCTTAAGTTATGGGTCCTTCTCT were used to generate mHsp60GMless. mHsp60 mutants, mHsp60F72I, mHsp60V72A and their truncated counterparts were generated using QuickChange (Qiagen). E. coli BL21DE3 cells were used to express wildtype and mutant mHsp60 proteins. Conditions for cell growth and protein purification were published previously23.Gene encoding the mature mHsp60 sequence  was synthesized with codons optimized for protein expression in 40. Briefly, mHsp60 proteins were added to TEA buffer  to a final concentration of 0.25 μM heptameric mHsp60s. The final concentration of mHsp10 was 0.3 μM. The solution was incubated at 25 °C for 10 min. The hydrolysis was initiated by addition of 100 mM pH 7.0 ATP to a final concentration of 2 mM and followed every 3 min for 21 min using the malachite green assay. Measurements were repeated multiple times for each sample as indicated in Table The steady-state ATP hydrolysis rate was measured using the malachite green assay, as described in our previous work+, as described in our previous work40. Briefly, MDH was unfolded in TEA buffer including 3 M GdmHCl to a final concentration of 36.7 μM (monomeric MDH). To refold MDH, 2.75 μl of unfolded MDH was diluted at 1:100 (v/v) to a final volume of 275 μl of refolding solution (at 30 °C) containing 50 mM TEA, pH 7.4, 50 mM KCl, 1 mM ATP, and 10 μM heptameric mHsp60s with 51 μM mHsp10. At desired time intervals, 20 μl of reaction solution was removed and mixed with 1 ml of NADH assay solution , and absorption at 340 nm was taken to monitor the decrease of NADH. As a positive control, activity of 36.7 μM native MDH (monomeric concentration) was measured at the same time intervals and was taken as 100% activity. Measurements were repeated more than three times for each sample.Refolded MDH was assayed by measuring its ability to convert NADH to NAD41:yN and yD are the derived signals for the native and denatured samples, and mN and mD are the derived baseline slopes for the native and denatured samples, T is the temperature, ∆Hvh is the van’ Hoff enthalpy, R is the gas constant and Tm is the temperature of the transition midpoint. Experiments were repeated at least twice.mHsp60 protein stocks were in 100 mM NaF, 10 mM KHEPES, pH 7.4, 1 mM EDTA, 1 mM DDT and 5% glycerol. Proteins were tenfold diluted to 100 mM NaF, 10 mM KHEPES, pH 7.4, 1 mM EDTA and 1 mM TCEP. Samples were heated from 20 to 86 °C with a heating rate of 2.0 °C/min by a Jasco Peltier thermo control device. The CD signal at 222 nm was recorded by a J-715 CD spectrometer every 2 °C with a 4-s averaging time. Measurements were repeated more than once for each sample. Thermal denaturation was analyzed by fitting the data to a two-state model according to the following equationE. coli cell strain , whose groE promoter is replaced by a pBAD promoter, was obtained from the E. coli Genetic Stock Center at Yale University. groEL and groELSR are in pTrc (ampicillin resistant AmpR) , a lac promoter-based vector, while all mhsp60 wildtype or mutants are in the lac-based vector pBbE5a (AmpR). mhsp10 and groES are in the lac-based vector pBbE5c . CaCl2 competent MGM100 cells were co-transformed with both plasmids and plated onto LB agar containing 50 μg/mL kanamycin, 100 μg/ mL ampicillin, 50 μg/mL chloramphenicol, and 0.2% w/v arabinose. Cells were serially diluted and plated, with added glucose to repress the chromosomal groE and added IPTG to express plasmid-encoded genes with lac promoters. Conditions for cell growth and titration are described previously40. Experiments were repeated at least three times.The MGM100


For most of the respondents , the education level was high school, while 23.58% (n = 454) of respondents were college graduates. One in three Romanians (33.3%) know the WHO predictions related to this topic. Overall, the Romanian population is less disciplined when it comes to completing antibiotic treatments, as 29.19% of the respondents stop the course of antibiotic administration if their symptoms improve. The key findings from the present study may help policy makers in designing targeted interventions to decrease confusion, ambiguity or misconceptions about antibiotic use.Considering the major limitations of the latest studies conducted in Romania on the knowledge, attitudes and practices (KAPs) of antibiotic use and antibiotic resistance, we conducted this study to assess this major public health threat. A cross-sectional survey based on a validated questionnaire was conducted among the general population of Romania for a period of 5 months, i.e., September 2021–January 2022. The questionnaire was distributed using Google Form and it covered demographic characteristics and KAP assessments consisting of 12 items on knowledge, 10 items on attitudes and 3 items on practices. Latent class analyses (LCAs) were conducted to group respondents based on their responses. The response rate was 77%, of which females responded in a greater number ( Antibiotics and their use in the fight against infectious diseases in both humans and animals represents one of the greatest discoveries of the 20th century ,2. HowevThe consumption of animal products obtained from animals treated with antimicrobial substances also contributes to the development and spread of AMR . Even thIn addition to food, the general population is exposed to antibiotics through various treatments throughout their lifetime. In general, penicillins, fluoroquinolones and macrolides are most commonly prescribed in humans, whereas in animals, the main antibiotics are tetracyclines and sulfonamides. In addition to the many side effects of these substances, AMR must be considered, as it leads to numerous annual deaths and increased public health costs. Raising public awareness of the side effects of antibiotics can prevent the spread of AMR and its effects, significantly reducing the costs to health systems in the uncertain future of human health. Even though maximum limits have been set for antibiotics in food, in the long term, the intake of antibiotics, even in quantities that do not exceed the maximum limits, can lead to the development of AMR. The excessive use of antibiotics should therefore be avoided by both veterinarians and human doctors, but also by the general population. The reduction in AMR should include measures to educate the public on the efficacy of these substances and their proper use, as demonstrated by numerous studies ,43,44,45To the best of our knowledge, no study has been conducted in Romania to highlight the aspects related to antibiotic use and knowledge in the general population. The latest studies conducted in Romania on the knowledge, attitudes and practices (KAPs) of antibiotic use and antibiotic resistance have had major limitations. One of these limits is the regional level at which the studies were conducted. Studies were set up in a single region—the eastern part of Romania , or in aThus, this study is designed to determine KAPs towards antibiotic use among the general public in Romania, as well as to provide important information about the population’s perceptions towards antibiotic resistance.A cross-sectional survey based on a validated questionnaire was conducted among the general population of Romania from September 2021 to January 2022 inclusive. The questionnaires used in previous studies ,44,49,50The questionnaire included two parts: 1. Demographic characteristics and 2. KAPs of antibiotic use and antibiotic resistance. The first part of the questionnaire included questions about demographic characteristics, such as gender, age, educational level, civil status and income. The second part included three sections that aimed to evaluate the Romanian population’s perception regarding the KAPs of antibiotic use and antibiotic resistance. The first section consisted of 12 statements aimed to evaluate the knowledge regarding antibiotic efficacy, mode of action and antibiotic resistance (AK01–AK12). Attitudes regarding antibiotic consumption were assessed in the second section of the questionnaire, which included 10 questions (AC01–AC10). The last section of the questionnaire contained 3 questions (AU01–AU03) that aimed to identify the respondents’ antibiotic use practices. The questionnaire was prepared in Romanian, and then translated into English. The invited participants filled in the questionnaire only in Romanian, so that, for each questionnaire, 15 min were sufficient to complete it.The data were first inspected and cleaned using Microsoft Excel 2016. From the original dataset comprised of 2067 answers, entries that were ambiguous  were discarded. The final dataset contained 1925 individual answers. All data analysis steps were performed in the R programming environment v.4.1.2 . Data wep < 0.05, with the answers to questions regarding personal details modeled as categorical predictors. The base R function glm was used to implement logistic regression. Results are reported as odds ratios (ORs) and 95% confidence intervals (CIs). The membership in the previously described LCA classes (classes 1 and 2) for each of the traits was used as a dependent variable in logistic regression, with class 1 used as reference for expressing the OR. Independent variables included the following demographic traits: age , sex , level of education , civil status  and income level .For the three KAPs studied traits, LCA class 1 comprised the respondents with the largest proportion of appropriate KAPs regarding antibiotics, whereas respondents from class 2 had a majority of answers demonstrating incorrect KAPs, or a lack of certainty (as signified by the answer “not sure”). Associations of respondents’ personal details with the antibiotic-related traits of focus in this study were evaluated via logistic regression at a significance level threshold of n = 1925) correctly completed the proposed questionnaire. Of the participants, 64.99% (n = 1251) were female and 35.01% were male. As in many other studies [n = 1332), were young and aged between 18 and 24 years. According to the acquired data (n = 1296) reported that their level of education was high school and 23.58% (n = 454) were college graduates; only 16.05% (n = 309) were married and 37.09% considered the monthly income to be confidential.Of the 2500 people invited to participate in this study, only 82.67% accessed the questionnaire link and 77% , while the percentage of people who demonstrated less accurate knowledge or uncertainty (LCA class 2) was 53.5%  were unsure about this subject. There was also a great confusion about the effectiveness of antibiotics on viruses, since only 32.88% correctly disagreed on the statement “Antibiotics treat viral infections”, and 37.51% believed that antibiotics treat viral infections and 29.61% were unsure. Furthermore, the majority of respondents incorrectly agreed on three statements regarding the effectiveness of antibiotics: “Antibiotics reduce pain and inflammation” (52.31%), “Antibiotics significantly reduce fever” (47.27%) and “Antibiotics help a lot with dental pain” (47.12%), and only one third disagreed . The respondents unsure of these statements did not exceed 25%. Over 1/2 of respondents (53.87%) responded correctly to the statement “Excessive antibiotic use leads to AMR”, while those unsure or those who did not agree with this statement were 24.31% and 21.82%, respectively. Of the respondents, 73.77% and 81.77% correctly stated that antibiotics can cause allergies and that antibiotics have side effects, respectively. A considerable percentage 57.35%) was aware that antibiotic treatment should not be stopped when symptoms have disappeared. However, 24.99% presented an incorrect answer for this statement, which is quite worrying in terms of the phenomenon of increased AMR. Respondents were asked to present their opinion on some information about antibiotic resistance and the severity of the phenomenon. The percentage of respondents who agreed with the statement “Animal products can contain antibiotic residues, which may increase AMR when entering the human body” was less than half, specifically 40.57%, while 47.95% were unsure and 11.48% disagreed. The data presented in .35% was as 53.5% .p-value = 0.014) to have inaccurate knowledge of antibiotics compared to people between 18–24 years old. Similarly, women were approximately twice less likely  than men to have inaccurate vs. accurate knowledge of antibiotics and antibiotic resistance, i.e., to belong to LCA class 2 vs. class 1. In regard to their education, higher levels of education were significantly associated with a reduced likelihood of having inaccurate knowledge about antibiotics and antibiotic resistance. People who had a postgraduate education were approximately 10 times less likely  to have inaccurate knowledge compared to people who only had a secondary education. There were no significant differences noticed in terms of civil status, whereas for income level, the only statistically significant difference was observed between people earning RON 1501–3000 and those earning <RON 1500, with the former having an increased likelihood  to belong to LCA class 2 vs. class 1, i.e., to have less accurate knowledge about antibiotics and antibiotic resistance on average  vs. class 1 (more accurate knowledge), compared to people from the 18–24 age group. The most significant difference was observed in the 55–64 age group, which was approximately 4 times less likely  incorrectly agreed with the statement ″I always try to have an antibiotic in the house″, which is considered a negative attitude. The majority (83.64%) had an appropriate attitude disagreeing with the statement ″If a family member feels unwell, I usually give them an antibiotic″, whereas 8.42% incorrectly agreed with this statement. The percentage of respondents who confidently used remaining antibiotics for reoccurring similar symptoms was 21.19%, while the vast majority (67.58%) disagreed. A total of 83.32% of the respondents agreed with the statement ″I use antibiotics according to the instructions for use″. Over half of the respondents (59.43%) disagreed with the statement ″I most often use antibiotics when the symptoms are cold/flu″, which is an appropriate attitude regarding antibiotics consumption. Similarly, 54.23% of respondents showed a correct attitude towards the statement ″I most often use antibiotics when the symptoms are related to toothache″, by disagreeing. Even so, almost 30% of the population participating in the present study agreed with these two statements, demonstrating a misguided attitude towards antibiotic consumption. Only 9.19% agreed with statement ″I most often use antibiotics when the symptoms are related to gastrointestinal disorders″, which means that the use of antibiotics is associated more often with toothaches and colds than with gastrointestinal diseases in the respondent population. Similar percentages of respondents gave appropriate answers to a question that appeared reformulated in both the knowledge and attitude sections, which suggests that respondents who had adequate knowledge of antibiotics were more likely to also have a positive attitude toward the use of antibiotics. If 57.35% correctly disagreed with the AK10 statement from the knowledge section, 58.96% of respondents disagreed with the same statement, which was reformulated as AC09 in the attitude section. The combination of drug–food (AC10), in this case antibiotic–food, is of particular importance to 57.3% of respondents. Moreover, 32% were unsure if the statement ″There are many interactions between antibiotics and food consumed″ was correct, while 10.7% disagreed. Among the demographic variables included in this study, the likelihood of respondents to belong to class 2 vs. class 1 in terms of attitudes towards antibiotic consumption was significantly associated with all five traits . Class 2p-value < 0.001) to have improper attitudes about antibiotic consumption rather than proper attitudes , compared to people of ages 18–24 years. A similar but stronger trend was noticed for people aged 35–44 and 45–54 years, where OR values of 0.256 and 0.227 were observed, respectively. Similar results were observed for gender, with females being less likely to have improper attitudes compared to males, and for education, where people with higher levels of education were less likely to have proper attitudes compared to those with the lowest level of education. Regarding civil status, single people were approximately twice as likely  than married people to belong to class 2. Surprisingly, a similar trend was observed for levels of income, with people with higher levels of income (as well as an undisclosed income) being more likely to have improper attitudes towards antibiotic consumption than people with the lowest income level.People of ages 25–34 years were half as likely  was “only on the doctor’s prescription”  of the respondents. The percentage of respondents who answered that they used an antibiotic when they fell sick, with or without prescription, was 10.29% (n = 198). The experience of friends/acquaintances in the use of antibiotics influenced the way to use such a drug only for a small number of respondents (n = 135). Regarding the first impulse when they fell sick, more than half of respondents (53.82%) said they went to see a doctor. Almost one-third (29.92%) reported that they were informed of the symptoms on the Internet. Less than 10% of respondents (8.52%) said they were based on their own experience when they feel sick, which implied self-medication. The pharmacist’s advice was sought by the fewest study participants when they felt unwell (7.74%). Among the respondents, 71.79% first went to the doctor following symptoms of a disease and only then used antibiotics. Only 10.39% (n = 200) of respondents considered that the passage of time did not help at all. This population adopted an inappropriate practice of using antibiotics when the symptoms of the disease appeared. They declared that, immediately after the onset of the symptoms of the disease, they used antibiotics. Less than 20% of respondents let at least 2 days elapse from the onset of the symptom and then used antibiotics, an inappropriate practice as in the case of those who immediately used antibiotics. In terms of demographics, fewer significant associations were observed for practices regarding antibiotic consumption compared to those for knowledge and those for attitudes  to perform improper practices rather than proper practices, compared to people from the lowest age group (18–24 years). In terms of civil status, people in a relationship and single people were around twice as likely to perform improper practices, as opposed to proper practices, compared to married people. Finally, people with a level of income of RON 1501–3000 were more likely to perform improper practices regarding antibiotic consumption compared to people with the lowest level of income (>RON 1500), with no significant associations found for people with other categories of income level.The multiple possible outcomes for the three questions in this scenario stem from the fact that people were not restricted to choosing a single answer from the possible choices listed in n = 1332). Similarly, the youngest age group responded in a larger proportion compared to other recent studies on the same subject [n = 1518) of respondents use antibiotics only when prescribed by the doctor. A previous study reported that self-medication is a common practice in Romania among students [Compared to the latest studies conducted in Romania ,47,48 re subject . In term subject ,65,69. T subject ,59,61. P subject ,49,64. Sstudents , but thestudents . The corstudents , 55.1% ostudents  or 51.5%students  stopped students ,75,76; bn = 2067) and the high response rate (77%), as well as the fact that the respondents were not selected from certain educational units or certain sectors  in which they could have interacted with antibiotics or could have come across theoretical knowledge about antibiotics.This study had several limitations. The main limitation of the study was the Internet access required to complete the questionnaire. Study participants received the questionnaire link via the Internet, which could have led to an overestimation of positive outcomes. Another limitation was that some key factors were not analyzed as covariates associated with the KAPs of antibiotic use and antibiotic resistance . A higher number of positive responses could also be due to the number of women who participated in the study, which was twice as large as that of men. Women are much more concerned with health than men. It is possible that respondents may have reported socially acceptable attitudes. In addition, it is possible that respondents reported more on their knowledge regarding the use of antibiotics and not their real attitudes and practices regarding the use of antibiotics. Another limitation was that our results were based on reported practices rather than measured practices. Despite these limitations, the present study provides important information for evaluating the KAPs of antibiotic use and antibiotic resistance among the general population of Romania. A strong point of our study is the large number of participants (Our results show that the knowledge of the general public in Romania regarding antibiotics efficacy, mode of action and antibiotic resistance is considerably limited. A significant percentage of the population is quite confused about the effectiveness of antibiotics on viruses or bacteria. There is also confusion when it comes to the diseases that antibiotics treat. A fairly high percentage of the population found antibiotics to be the solution for most diseases. Unfortunately, the general population in Romania often associates antibiotics with toothache, flu or cough. Our study found that older people appear to have more accurate knowledge than younger people, but more frequent improper practices. The present study did not show a direct association between monthly income and proper practices, nor between monthly income and knowledge related to efficacy, mode of action, use of antibiotics and AMR. Keeping in mind the limitations of this study, its key findings may help policy makers in Romania to plan actions and strategies in the fight against AMR and mitigate the effects of this phenomenon. The results obtained can lead to the improvement of the KAPs of antibiotic use and antibiotic resistance through specific actions targeted towards the groups of Romanian populations where confusion, ambiguity or misconceptions have been reported.