In order to investigate the compaction and re-crushing characteristics of crushed coal gangue with different gradation, the compaction fractal test was carried out for crushed coal gangue with different Talbol power index n. The compaction deformation parameters such as displacement, stress-strain were analyzed according to the test results. And according to the test results, the compaction deformation relationship of the lateral limit crushed coal rock body considering the gradation is obtained. The test results show that: the crushing of coal rock particles exists in almost the whole compaction process of the crushed coal rock body, and the crushing of coal rock particles has a non-negligible influence on the deformation of the whole crushed rock body; the structural stability of large-size coal rock particles is not as good as that of small-size coal rock particles, and the large-size coal rock is more likely to be crushed under the same stress conditions; the distribution coefficient r of the crushed coal rock body decreases with the increase of axial stress; and before the Before the axial stress reaches 8 MPa, the distribution coefficient r of crushed rock samples tends to increase with n in general, and after reaching 8 MPa and later, the distribution coefficient r of crushed rock samples tends to decrease with n in general; the difference value of particle crushing increases with the increase of axial stress, and the weight value of particle crushing decreases with the increase of axial stress, and the changes of both are non-linear; according to the stress recovery in the compaction process of crushed coal rock body The compaction deformation model of the crushed coal rock body is constructed according to the crushing characteristics of coal rock particles in the stage of compaction, which effectively combines its fine action mechanism with macroscopic physical phenomena in a simple form and has certain practical engineering significance. In recent years, the fine view research of bulk filling materials has gradually received attention, and the compaction mechanical properties of bulk filling materials as the key factor of rock control in the coal mining technology of filling directly determine the effect and quality of filling mining , 2. The In terms of rock particle fragmentation characteristics, Liu Hanlong et al.  obtainedThe compressive strength characteristics of the filling material and the law of re-fragmentation are extremely important in the filling and mining technology. The above studies have done a lot of research on the compaction and crushing characteristics of crushed rock masses from macroscopic and microscopic perspectives, but there is less analysis on the intrinsic mechanism between compaction and re-crushing of graded crushed rock masses, and the mechanical intrinsic relationships in the compaction and crushing process need further depth. In this paper, the compression and fractal test of graded crushed gangue is based on fractal theory. The re-crushing characteristics of the graded crushed gangue in the compaction process are studied, and its fine action mechanism and macroscopic physical phenomena are effectively combined, so that the mechanical ontological model in the compaction process is established.Based on the crushing mechanism of crushed coal and rock mass under pressure . A self-The test system mainly includes a DDL600 electronic universal testing machine, crushed rock compression device, computer acquisition instrument, control system, vibration sorting screen and control cabinet. Among these components, the DDL600 electronic universal testing machine provides axial pressure for the cylinder under the setting and control of the computer control system. The computer controls the testing machine and collects the required data. The vibrating sorting screen device screens and separates particles into various particle size ranges in a controlled manner. The cabinet can display data changes such as the displacement and stress. In addition, tools such as steel rulers and electronic scales were required. The test device is shown in 3. The raw materials were crushed in a crusher, and 2.5~5 mm, 5~10 mm, 10~15 mm and 15~20 mm particles were screened with the sorting screen, as shown in The test raw materials were collected from the Cuijiagou Coal Mine in Shaanxi Province, and the density was measured as 1930 kg/md is the particle size of the broken coal gangue, mm; D is the largest particle size of the broken gangue, mm; Md is the mass of broken gangue in the rock sample with a particle size smaller than or equal to d, g; Mt is the total mass of broken gangue in the sample, g; and n is the Talbol power exponent.According to the Talbol grading equation  Eq (1))Eq (1)), The above crushed rock mass compaction system with a DDL600 electronic universal testing machine as the loading device was implemented to determine the compaction characteristics of crushed coal gangue samples, and crushed coal gangue particles with the same gradation structure were loaded into the compression cylinder under confinement. The press was activated, and the target axial stress was applied to the broken coal and rock mass sample through the computer control system in a gradually increasing loading manner. When the load on the broken rock samples of the same grades reached the target stress value, the sample was maintained under the target stress for 30 minutes. This ensured that the broken coal and rock particles attained equilibrium, and the piston height changes after each axial stress loading step were recorded to calculate the sample porosity. Apply the next target stress until all target stress loads are completed, and then change to the next set of specimens. After sample measurement, stress and strain data were obtained through the computer data acquisition system and stored, the previous steps were repeated with other samples, and data were recorded until the test was completed. The test flow chart is shown in Pressure-bearing broken coal rock masses exhibit different mechanical properties and behaviours to those of complete rock masses in the compression process. In the literature , crushedn and various axial stress levels were analysed. According to the test displacement data collected during the tests, a diagram of the change in the axial displacement with the axial stress was generated, as shown in To study the mechanical properties of pressure-bearing broken coal and rock masses, the compaction changes in broken coal and rock masses under different Talbol power exponent values n exhibited an overall downward trend under the action of the various axial stress levels, but with increasing axial stress, the axial displacement rate decreased, and it was found that the relationship between the axial displacement and stress could be expressed with a power function. The sample axial displacement changed the fastest from 0~2 MPa, and all broken samples with different Talbol power exponent values n were reduced by more than 20 mm. At this point, the main deformation mechanism of the coal particles under stress was particle adjustment. After the stress reached 4 MPa, the filling process of the larger pores in the broken coal rock mass sample was basically completed due to particle filling. The process could no longer overcome the internal friction to reduce the porosity through inter-particle movement. At this point, under the higher axial stress, the coal and rock particles were increasingly broken and ground, which caused the large particles to decrease and fill any remaining voids. Therefore, the porosity of the broken coal and rock mass sample decreased, and the sample was further compacted when the inter-particle spaces were reduced to a certain extent. Under the action of a high stress, the broken coal rock mass sample experienced phenomena such as consolidation and bonding, which compacted the broken coal rock mass sample into a proto-like rock mass similar to the original rock mass.n in the compaction process, a stress–strain relationship diagram was generated, as shown in To study the characteristics of the broken coal and rock masses with different Talbol power exponent values et al. [Ma et al.  proposeda and b in To determine the test parameters a and b in a and b and Talbol index n, it was found that fitting parameters a and b could be described with exponential and linear functions, respectively, of the Talbol index n, as follows:The values of test parameters a1, a2, b1, b2, and b3 are the fitting parameters of the functional relationships. The fitting parameters and correlation coefficients of the functional relationships are summarized in In the above equations, a and b and Talbol index n for the crushed coal rock mass samples was determined, as shown in n under confinement constraints is as follows:a1, a2, b1, b2, b3 and the other main parameters can be experimentally measured.In summary, based on the compaction stress–strain relationship of the crushed coal and rock masses under pressure, the relationship between fitting parameters n under strains of 0.066 and 0.116 were obtained, and the test results were coupled with the constitutive relationship. A comparison chart of the calculation results is shown in To verify the accuracy and rationality of the model expressed by n under the two different strain conditions was higher than 0.995 for the broken coal rock mass samples, and the correlation degree reached as high as 0.999 under a strain of 0.116. In the constitutive model for the compacted and broken coal mass samples considering the gradation structure, the calculation results were consistent with the test results, which verifies the rationality and accuracy of the proposed constitutive model.Broken coal and rock particles can deform under the action of axial stress, and the particle arrangement can accordingly vary. Coal and rock particles can move, rotate, break, crack, be ground, fill, and become consolidated under the action of contact forces. A series of complex effects results in a new particle arrangement balance. Each stress loading step can cause reorganization of broken coal and rock particles, thereby causing the entire broken coal and rock mass to undergo compaction deformation. The most intuitive manifestation in the experimental process entails the change in axial displacement.In the establishment process of the confined compaction constitutive relationship in the study of the compaction stability features of crushed coal and rock mass samples under pressure, an exponential relationship could suitably represent the stress–strain relationship of the crushed coal and rock mass samples under pressure, and the fitting degree was very high. However, this relationship is only a mathematical expression and lacks physical meaning. In the analysis of the compaction process of the broken coal rock mass samples, it was found that the compaction and crushing process entails irreversible plastic deformation. Even if the final compaction degree were the same, the final rock mass differed from the original rock mass. Hence, the final rock mass could only be referred to as a proto-like rock mass. The essential reason for the irreversibility of the compaction and crushing process is energy consumption. The work achieved by external forces was consumed not only in the coal and rock particle crushing process but also in the particle filling and movement processes, mutual rubbing and mutual friction. Based on the above studies, it could be found that coal and rock particle crushing occurred throughout nearly the entire compaction process of the crushed coal and rock mass samples, and the crushing process of coal and rock particles imposed a non-negligible effect on the deformation of the entire crushed body.In addition to the influence of external forces, the impact of the particle crushing degree mainly depended on the particle nature, such as the material hardness. The size of broken particles of the same material can differ, which can also affect the re-crushing state. The structural stability of large particles is typically not as high as that of small particles, and large particles may contain more defects. Compared to small particles under the same stress conditions, large particles are more readily broken.To facilitate further analysis and study of the fractal pattern and mass distribution of the broken coal and rock masses with the different gradation structures under the various axial stresses, the measurement and calculation data obtained during the tests were compiled, as summarized in r was introduced to describe the particle size distribution of the broken coal and rock particles [Considering that crushed coal and rock masses can deform in the pressure-bearing process, large particles can experience cracking, crushing, grinding and other re-crushing effects, which can affect the original particle size distribution of the crushed particles. To analyse the changes in the particle size distribution of the graded broken coal and rock masses under the different axial stresses, the distribution coefficient articles :r=∑n=1k is the number of rock sample groups in a given particle size interval; Dn is the maximum particle size in the n-th particle size interval, mm; and Wn is the percentage of the mass in the n-th particle size interval to the total mass of the rock sample, %. A smaller r value indicates that after rock sample deformation under loading, the proportion of small-size particles in the rock sample is greater.In the above equation, n were obtained, and the distribution coefficient r of the broken coal rock masses with the different Talbol power exponent values n could be determined by substituting the data into n, as shown in According to the test results, the mass proportions in the broken coal and rock masses with the different Talbol power exponent values r of the broken coal rock masses decreased with increasing axial stress before the axial stress reached 8 MPa. The distribution coefficient r of the broken rock samples exhibited an overall increasing trend with n. The distribution coefficient r of the fractured rock samples decreased up to 8 MPa and subsequently exhibited a decreasing trend with n. However, the distribution coefficient r of the broken coal and rock masses with the different Talbol power exponent values n fluctuated locally. In the loading process of the broken rock samples, large particles were continuously crushed, and small particles gradually increased. The particles in the broken rock samples became increasingly interlaced. The main skeleton was broken, producing more small particles, and the relationship between the distribution coefficient r and n values of the broken rock samples fluctuated.n under the various axial pressures were analysed in great detail. Based on the test results, the percentage of 0~2.5 mm particles was defined as p1, the percentage of 2.5~5 mm particles was denoted as p2, the percentage of 5~10 mm particles was defined as p3, the percentage of 10~15 mm particles was denoted p4, and the percentage of 15~20 mm particles was defined as p5.The distributions of the coal and rock particles in the samples with the different Talbol power exponent values p1 of the smallest particle size range, i.e., 0~2.5 mm, to p5 of the largest particle size range, i.e., 15~20 mm, was defined as the particle breakage difference mp. The ratio of the sum of p2, p3 and p4 to that of p1 and p5 was defined as the particle crushing weight m, and the evolution of the mean values of mp and m was explored.The ratio of mp and m is as follows:The calculation method of the mean values of mp increased with increasing axial stress, and the particle crushing weight m decreased with increasing axial stress. The changes in both parameters occurred in a non-linear manner with the Talbol power exponent. Under an index value n of 0.8, the mp value was larger than that under an n value of 0.2. In the process of gradually increasing the axial stress, the edges and corners of the large coal sample particles were broken under force application, resulting in a continuous increase in fine coal sample particles. Moreover, movement occurred to fill the spaces between the coal sample particles. Therefore, mp increased with increasing axial stress. The particles of the broken coal sample with a Talbol power exponent of 0.8 were relatively large. Consequently, the phenomenon of large-particle coal fragmentation occurred more notable. Moreover, the mp value was larger than that under an n value of 0.2. The change in the mean values of mp and m could reflect the crushing degree of the mixed coal samples in the loading process to a certain extent.et al. [In the compaction testing process of the crushed coal and rock masses under pressure, the particles of the broken samples with the different gradation structures could be regarded as spherical particles of varying sizes. The particles could be broken before they exhibited re-crushing behaviour and became more powdery particles. The compaction deformation process of each coal and rock mass sample as a whole was considered to be caused by the mesoscopic contact and movement phenomena of the particles. The mesoscopic mechanical analytical model proposed by Chang et al. , 21 was In summary, in the establishment process of the constitutive compaction equation for the graded, crushed coal and rock mass samples, it was necessary to fully consider the mesoscopic compaction mechanism. In the study of the compaction process of the broken coal and rock mass samples, the compaction process was divided into three stages: plastic instability stage, stress recovery stage and proto-rock stress stage. According to the stress characteristics of the crushed coal and rock mass samples in the compression process, the deformation of the broken coal and rock mass samples at the stress recovery stage could be divided into two mechanisms: displacement reorganization and broken particle reorganization. Displacement reorganization occurred in two ways, namely, filling and sliding. At the stress recovery stage, the crushed coal and rock mass samples experienced compaction and deformation under the action of confinement constraints and pressure, and the bearing capacity was gradually recovered. The stress–strain relationship for the broken coal and rock mass samples at this stage can be expressed as :σ=aebεε is the total axial strain, ε1 is the strain caused by particle filling, ε2 is the strain caused by particle sliding, and ε3 is the strain caused by particle breakage.Therefore, in the construction process of the constitutive compaction equation for the graded crushed coal and rock mass samples, the strain , 23 ε waIn the compaction and reorganization process of the graded broken coal particles, deformation was mainly attributed to the instability of the local pore structure between the broken coal particles. At the initial near-linear stage, the work achieved by the axial pressure was largely consumed to overcome the friction between the particles. The stress was concentrated near the pores. Once the stress near the pores exceeded the static friction force, local instability occurred near the pores. Subsequently, the particles overcame the internal friction and filled any gaps. Alternatively, inter-particle interlocking could occur. The deformation process at this stage could be approximated as a pre-sliding system. The deformation caused by local instability could be regarded as pre-slip displacement, which mainly includes plastic deformation and non-linear elastic deformation . Since tTherefore, considering all graded broken coal and rock mass samples, a model with a damper and spring in parallel could be adopted to intuitively define a non-linear elastic model of the compaction deformation phenomena attributed to particle filling. The compaction caused by particle filling can be expressed as:ζ1 is the friction factor between the particles, E1 is the buckling deformation modulus, and σ1 is the axial stress.In the above equation, At the particle sliding stage, the work achieved by the axial pressure was also mainly consumed to overcome the internal friction between the coal and rock particles, including static and sliding friction forces. According to the characteristics of the dynamic strength, the deformation process of the broken coal and rock mass samples could also be regarded as the dynamic process of the friction strength. In cohesive soils, the deformation process mostly includes the friction and bonding strengths . As a frThere exists a significant non-linear correlation between the internal friction angle of the samples and the plastic strain at the plastic deformation stage of the bulk particles , 29, andζ2 is the coefficient of friction between the particles , and E2 is the sliding deformation modulus between the broken particles.In the above equation, In regard to crushed coal and rock particles, under the action of axial loading, the crushing strength of the particles can reveal notable time-dependent characteristics . The higζ3 is the particle crushing viscosity coefficient.In the above equation, Based on the above discussion, it could be considered that the crushed coal and rock mass compaction model comprised two Kelvin composite elements and one viscous pot element, as shown Although particle packing and particle sliding were both described with Kelvin components, the two Kelvin components exhibit different stages and methods. The particle packing phenomenon described with a Kelvin component mainly occurred at the initial near-linear stage, i.e., the compaction deformation rate was relatively low at the beginning. At the rapid stage, the other Kelvin component was mainly considered, and the compaction deformation rate decreased. In contrast, the viscous pot element mainly functioned at the compaction stage where the compaction deformation rate tended to stabilize. By connecting the two Kelvin components and viscous pot element in series to establish a compaction model for the broken coal and rock mass samples, the compaction equation can be expressed as:This equation can effectively reflect the three-stage characteristics of the compaction and deformation process of broken coal and rock masses. This model effectively combines mesoscopic mechanical and macro-physical phenomena. The proposed equation is simple in form and provides a certain practical engineering significance.After the stress reaches 4 MPa, the filling process of the large pores in the broken coal rock mass samples is basically completed under particle filling. When the inter-particle pores are reduced to a certain extent, the broken coal rock mass experiences consolidation and bonding under the action of high stress, which further compacts the broken coal rock mass into a proto-like rock mass very similar to the original rock mass. Coal and rock particle crushing occurs throughout nearly the entire compaction process of the crushed coal and rock mass samples, and coal and rock particle crushing imposes a non-negligible effect on the deformation process of the entire crushed body.n and the various parameters of the stress–strain relationship for the crushed coal and rock mass samples under confinement constraints is obtained through interpolation. A constitutive compaction relationship for the crushed coal samples is established considering gradation. Under the same pressure conditions, the larger the n of the broken coal and rock mass, the higher the strain rate, and the larger the n, the easier it is to compact the broken coal rock mass. The test data are substituted into this constitutive relationship, which verifies its accuracy and rationality.A relationship between the Talbol power exponent r of the broken rock masses decreases with increasing axial stress. Before the axial stress reaches 8 MPa, the distribution coefficient r of the broken rock samples generally increases with n. However, when the axial stress exceeds 8 MPa, the distribution coefficient r of the rock samples tends to decrease with n in general. The particle breakage difference increases with increasing axial stress, while the particle breakage weight decreases with increasing axial stress. Both changes are non-linear changes. When the Talbol power index n is 0.8, the mp value is greater than that when n = 0.2, and the change rate of the mean m value is higher than that when n = 0.2The distribution coefficient According to the crushing characteristics of the coal particles at the stress recovery stage during the compaction of the crushed coal and rock mass samples, two Kelvin components are connected in series to establish a compaction model for the crushed coal and rock mass samples. This model can effectively reflect the three-stage characteristics of the compaction and deformation process of the broken coal and rock mass samples, and the model effectively combines mesoscopic mechanical and macro-physical phenomena. The proposed model is simple in form and provides a certain practical engineering significance.S1 File(DOCX)Click here for additional data file.


The literature on LAN is mostly published in Chinese language, restricting its international accessibility. We, therefore, wanted to generate a complete English bibliography of all LAN publications and then use it for a comprehensive formalised literature review. Altogether, 379 LAN publications (322 in Chinese and 57 in English) were retrieved from PubMed and Science and Technology Paper Citation Database. Indications covered are motor (257), glandular (16), pain (32) and aesthetics (48). Topics are neurological (250), aesthetic (48), paediatric (38), ophthalmological (18), urological (9), methodological (6), gastroenterological (5), ear, nose and throat (4) and surgical (1). Seventy-one publications are randomised controlled trials, forty-one publications are interventional studies and observational studies, fifteen publications are case studies, eighteen publications are reviews, and two publications are guidelines. LAN publications cover all relevant topics of BT therapy throughout a period of more than 20 years. This constitutes a publication basis resembling those of other BT drugs. None of the LAN publications presents data contradictory to those generated with other BT type-A drugs. LAN seems to have a similar efficacy and safety features when compared to onabotulinumtoxinA using a 1:1 LAN– onabotulinumtoxinA conversion ratio. Large controlled multicentre studies will become necessary for LAN’s registrations in Europe and North America.In 1997, lanbotulinumtoxinA (LAN) was introduced in China. It is now available in Asia, Latin America and Eastern Europe under various brand names including Hengli Clostridium botulinum. In the early 19th century, Justinus Kerner first recognised BT as the cause of botulism. In 1980, Alan B. Scott invented BT’s therapeutic use by applying it as a muscle relaxant for the treatment of strabismus in children [Botulinum toxin (BT) is produced by the anaerobic bacterium ®, A; Abbvie, Chicago, IL, USA), abobotulinumtoxinA  and incobotulinumtoxinA  [Globally, there are three major BT drugs available: onabotulinumtoxinA  . They ar®; Lanzhou Institute of Biological Products, Lanzhou, China) as a drug for the treatment of strabismus, blepharospasm and hemifacial spasm. In 2012, this license was extended to moderate to severe glabellar frown lines. In 2002, LAN was successfully registered in South Korea. Since then, it has spread over Asia, Latin America and Eastern Europe, including major countries such as Brazil, India, Mexico and Russia [®, Prosigne®, Lantox®, Lazox®, Redux®, Liftox®, HBTX-A and CBTX-A. In this review, we will use LAN as the abbreviation of the generic name lanbotulinumtoxinA.In China, the development of therapeutic BT started in 1985 by Professor Yinchun Wang at the Lanzhou Institute of Biological Products. Wang was trained by Edward J. Schantz at the University of Wisconsin in botulism research. In 1993, his product was approved by the Ministry of Health of the People’s Republic of China. This made China—after the USA and the UK—the third country in the world producing therapeutic BT. In 1997, the Lanzhou product was licensed by the Chinese Food and Drug Administration under the name lanbotulinumtoxinA  as their active pharmaceutical ingredient, complexing proteins and excipients . In LAN,The literature on LAN is mostly published in Chinese language and in Chinese journals restricting its international accessibility. We, therefore, retrieved the entire LAN literature from Chinese and English databases. The English translations of the publications allowed us to generate a complete English bibliography and to perform a formalised review using commonly accepted classification criteria.As shown in Two-hundred and fifty-seven publications cover motor indications, sixteen publications include glandular indications, thirty-two publications include pain, forty-eight publications include aesthetic indications, six publications include methodological aspects, and twenty publications are reviews and guidelines.Two-hundred and fifty publications are research in the neurological field, forty-eight publications are in the aesthetic field, thirty-eight publications are in the paediatric field, eighteen publications are in ophthalmologic field, nine publications are about urologic indications, five publications are about gastroenterologic indications, four publications are regarding ear, nose and throat, and one publication is regarding surgical indications.Two-hundred and thirty-one publications are about observational studies, seventy-one publications are about randomised controlled trials, forty-one publications are about interventional studies, eighteen publications are reviews, fifteen publications are case studies, and two publications are guidelines.In the following, we will summarise the number of publications and discuss in detail some selected randomised controlled trials, interventional studies, reviews and guidelines for each indication.Motor indications are indications where the therapeutic effect is based on the blockade of the cholinergic innervation of hyperactive muscle tissue. Those are the first indications of BT therapy. Whether the entire therapeutic effect relies on a direct peripheral effect alone is not entirely clear. Retrograde axonal transport  may alsoDystonia was—after strabismus and together with hemifacial spasm—the second group of indications developed. It is still the largest indication group for BT’s therapeutic use. Altogether, 106 LAN publications refer to the treatment of dystonia. In most publications, more than one dystonia manifestations are covered. Publications on blepharospasm also include the treatment of Meige syndrome, hemifacial spasm and sometimes cervical dystonia.Thirty-five publications (thirty-two as primary topic publications and three as secondary topic publication) cover blepharospasm. They deal with the efficacy and safety of LAN therapy, injection sites, injection patterns and its comparison to that of surgical treatment. Sometimes, large cohorts are reported, and long-term experience is shared. Thirty-two publications  cover cervical dystonia. They deal with injection guidance by electromyography or ultrasound, variations of LAN concentrations, drug comparisons between LAN, ONA and ABO, combinations of LAN and orthoses, combinations of LAN and thermocoagulation of peripheral nerves, the treatment of tardive cervical dystonia, as well as long-term studies and reports on larger cohorts. Five publications  cover craniocervical dystonia, two publications are about writer’s cramp , tweleve publications are about dystonias (eleven as primary topic publications and one as a secondary topic publication), and three are about spasmodic dysphonia . They mainly report larger cohorts, long-term follow-ups and general efficacy and safety data. Three publications refer to the treatment of temporomandibular joint dysfunction, with all as primary topics. The aetiology of the condition is mixed , and potential therapeutic mechanisms remain unclear. Hemifacial spasm is together with dystonia the second indication developed for BT therapy following strabismus. As shown in Tics have been tried successfully on several occasions, because of their similarity with dystonia, especially cranial dystonia. As shown in Spasticity is another main indication for BT therapy. Historically, it was developed after strabismus, dystonia and hemifacial spasm. As shown in As shown in Cerebral palsy is a syndrome consisting of various neurologic deficits all caused by perinatal brain damage. Motor deficits are the hallmark, frequently including dystonic and spastic elements which have been treated successfully with BT therapy for many years. Economically, cerebral palsy turns out to be one of the most important BT indications, not only because of its prevalence, but also because of the usually high BT doses applied. As shown in Strabismus is the first indication developed by Alan B. Scott for BT therapy. Its relevance today is limited. As shown in BT therapy for bladder disorders is one of the established indications for BT therapy. Altogether, eight LAN publications refer to bladder dysfunction . They cover different aetiologies , different LAN doses and different BT drugs. Different terminologies are used, such as overactive bladder, detrusor hyperreflexia, interstitial cystitis, bladder pain syndrome and neurogenic incontinence. Gastroenterological indications are a collection of mainly experimental indications with achalasia being the most robust one studied. As shown in There are publications dealing with Raynaud syndrome (as a primary topic publication) , AnismusGlandular indications are indications where the therapeutic effect is based on an activity reduction of exocrine glands innervated by the cholinergic autonomic nervous system. Affected glands may be sweat glands, salivary glands and lacrimal glands. Additional exocrine glands may be used for therapeutic purposes in the future. Injections into smooth muscles which are also innervated by the cholinergic autonomic nervous system will be covered in the section “Muscle indications”. Glandular indications are very rewarding indications for BT therapy. Especially, hyperhidrosis is an important indication because of its prevalence, the lack of successful treatment alternatives and the excellent results of BT therapy. As shown in Eleven publications  cover hyperhidrosis. Topics include general data on efficacy and safety, different LAN doses, different regions affected  and different aetiologies (primary and secondary). Often, unusual terminology is applied, such as bromhidrosis, osmidrosis and hircismus.As shown in Four studies  deal with sialorrhea or hypersalivation. Topics cover general data on efficacy and safety, ultrasound guidance of parotid and submandibular gland injections and different aetiologies . As shown in As shown in Pain indications are indications where the therapeutic effect is based upon modulations of nociception, pain transmission and pain processing. Involved neural structures are not entirely understood. Where and how BT might interfere is not well investigated. Central nervous system effects may exist. BT can also achieve pain reduction by reduction excessive muscle hyperactivity. Additional nociceptive effects may be caused by potentially anti-inflammatory effects.Pain indications are the latest major extension of BT therapy with chronic migraine being all dominant. As shown in Three randomised controlled trials examined LAN in trigeminal neuralgia: one of them showed efficacy and safety against placebo , one 5858 showedOne randomised controlled trial  found thOne randomised controlled trial  demonstrAesthetic indications are indications where BT is not used to treat a disease, but to improve appearance. All indications presented here are medical procedures reserved for physicians. They are generally not covered by reimbursement systems. Aesthetic indications were developed very early on by neurologists treating blepharospasm. Soon, their potential for aesthetic medicine was realised and strategically developed. Around 50% of global medical BT use is in the field of aesthetic medicine. The contouring of the masseter and the calf muscles is specific to Asian countries. Altogether, 48 LAN publications refer to aesthetic indications. Thirty-eight publications (37 as primary topic publications and one as a secondary topic publication) cover wrinkles, six publications refer to calf reduction , two publications refer to masseter hypertrophy (both as primary topic publications), two publications are about scars (both as primary topic publications) and one publication is about acne (as a primary topic publication). As shown in As shown in Altogether, 20 publications are reviews and guidelines. Eighteen reviews cover BT use in China in areas of neurology, cervical dystonia, autonomic disorders, hyperhidrosis, urology, overactive bladder, prostate hyperplasia, scars, aesthetic applications, calf hypertrophy and depression. Two guidelines deal with BT therapy in plastic surgery and adult limb spasticity.This paper presents the first complete English bibliography of all LAN publications until 1 July 2018. It is based on the English database PubMed and the Chinese data base Science and Technology Paper Citation Database (STPCD). It shows, for the first time, all authors’ names, publication titles and source names in English, thus making them accessible to the international English-speaking audience. This paper also presents the first comprehensive review of all LAN publications until 1 July 2018. It is based on the English bibliography above and additional English translations of all LAN articles originally published in Chinese. It is structured as a formalised literature review.LAN publications have been produced over a period of more than 20 years, making LAN one of the three longest used BT drugs worldwide.LAN is used for motor, glandular, pain and aesthetic indications. Medical specialties involved are neurology, aesthetics, paediatrics, ophthalmology, urology, gastroenterology, ENT and surgery. Therefore, LAN is used in all major BT indications other BT drugs have been used for. This also documents the widespread use of LAN in China.Of the 379 publications retrieved, 71 are randomised controlled trials, 41 are interventional studies, 231 are observational studies, 15 are case studies, 18 are reviews, and 2 are guidelines. This constitutes a publication basis similar to the ones of other international BT drugs. Relatively low percentages of randomised controlled trials and interventional studies are also typical for other BT drugs.None of the publications presents data contradictory to those generated for other BT type-A drugs. Based on a 1:1 LAN–ONA conversion ratio, LAN seems to have a similar efficacy and safety features compared to ONA. A recent publication studying the biological potency of LAN, ONA and INCO in a direct comparison did not detect differences in their potency labelling , thus coLarge controlled multicentre studies will eventually become necessary for LAN’s registrations in Europe and North America. Further studies on LAN’s product details, including its specific biological potency, its production consistency and its stability, will then become necessary. LAN’s increased international presence will increase the number of international studies published in international journals.This review is based upon comprehensive literature searches in the English data base “PubMed”  and the Chinese data base “STPCD” within Wanfang data. The searches include all references published until 1 July 2018. They were performed along two axes with the following search words:Axis 1: Botulinum toxin and China or Chinese or Lanzhou, Prosigne, Lantox, Hengli, HBTX-A, Lanzox, Redux, Liftox, CBTX-A.Axis 2: Strabismus, ophthalmology, blepharospasm, hemifacial spasm, Meige syndrome, spasmodic dysphonia, torticollis, cervical dystonia, craniocervical dystonia spasticity, writer’s cramp, dystonias, hemifacial spasm, tics, spasmodic dysphonia, spasticity, cerebral palsy, strabismus, bladder dysfunction, temporomandibular joint disorder, gastroenterology, gastroparesis, achalasia, dysphagia, anismus, tinnitus, hyperhidrosis, sialorrhea, Raynaud syndrome, headache, trigeminus neuralgia, postherpetic neuralgia, urology, bladder dysfunction, aesthetic, masseter, wrinkles, gastrocnemius, masseter, scars, adverse effects, allergic reaction.All publications contain at least one item on each axis. All retrieved publications appearing in Chinese were first translated into English, before they were referenced with their authors’ names, publication year, study design, title and source. Within their sections, all references were ordered by their authors’ names using the English alphabet and by their publication years. The complete bibliography is presented as


The c-MET receptor is a receptor tyrosine kinase (RTK) that plays essential roles in normal cell development and motility. Aberrant activation of c-MET can lead to both tumors growth and metastatic progression of cancer cells. C-MET can be activated by either hepatocyte growth factor (HGF), or its natural isoform NK1. Here, we report the cryo-EM structures of c-MET/HGF and c-MET/NK1 complexes in the active state. The c-MET/HGF complex structure reveals that, by utilizing two distinct interfaces, one HGF molecule is sufficient to induce a specific dimerization mode of c-MET for receptor activation. The binding of heparin as well as a second HGF to the 2:1 c-MET:HGF complex further stabilize this active conformation. Distinct to HGF, NK1 forms a stable dimer, and bridges two c-METs in a symmetrical manner for activation. Collectively, our studies provide structural insights into the activation mechanisms of c-MET, and reveal how two isoforms of the same ligand use dramatically different mechanisms to activate the receptor. Activation of c-MET receptor tyrosine kinase involves hepatocyte growth factor (HGF) and glycosaminoglycans, but the molecular mechanism is still under debate. Here, the authors present cryoEM structures of c-MET bound to two HGF splice variants and heparin, revealing the structural basis for c-MET activation. Aberrant activation of c-MET can lead to both tumor growth and metastatic progression of cancer cells, making it an important drug target for cancer treatments4. C-MET can be activated by its cognate ligand—hepatocyte growth factor (HGF)6. It has been proposed that the binding of HGF to c-MET induces the dimerization of c-MET that enables its intracellular kinase domains (KDs) to undergo autophosphorylation7. The phosphorylated KD recruits cytosolic effector proteins, leading to activation of downstream signaling pathways.C-MET receptor, which belongs to the receptor tyrosine kinase (RTK) family, plays essential roles in controlling a number of critical cellular processes such as cell proliferation, survival, motility, and morphogenesis8. The mature form contains the α- (32 kDa) and β-subunits (120 kDa) that are linked by at least three disulfide bonds  domain, a plexin-semaphorin-integrin (PSI) domain, and four consecutive immunoglobulin-plexin-transcription factors (IPT1-4) domains in the extracellular region, a single transmembrane helix (TM), and an intracellular KD Fig. . Notablynds Fig. . HGF, thnds Fig. . Similarnds Fig. . Interes13. One is mapped to the α-subunit of HGF10, and the other is located in the SPH domain, the only domain existing in the β-subunit of HGF. The interaction between the SPH domain of HGF and c-MET has been well characterized by X-ray crystallography, showing HGF-SPH makes close contact with the bottom surface of c-MET-SEMA14. Although the SPH domain alone cannot activate c-MET, mutations that disrupting the interaction between HGF-SPH and c-MET-SEMA significantly reduce HGF-induced c-MET activation, validating the functional importance of this interaction15. Nevertheless, the detailed interaction pattern between the α-subunit of HGF and c-MET remains unknown; thus, the complete picture of how full-length HGF recruits and dimerizes c-MET has not been depicted, hindering our understanding of the exact activation mechanism of c-MET. Furthermore, it has been shown that glycosaminoglycan, such as heparin, heparin sulfate, and dermatan sulfate, can promote HGF-induced c-MET activation through enhancing the binding affinity between HGF and c-MET18. The heparin-binding site at HGF has been mapped to the N domain by mutagenesis and NMR studies20. However, the exact heparin-binding site on c-MET has not been identified; therefore, how heparin facilitates the binding of HGF to c-MET is unclear. Finally, c-MET could also be activated by NK1, a naturally occurring splicing variant of HGF that contains only the N and K1 domains of HGF21. Different from HGF that exists mainly in a monomeric form, NK1 itself forms a stable dimer that can recruit two c-MET for activation. However, structural details of how NK1 engages and activates c-MET have not been revealed.Previous biochemical and structural studies have established that there are at least two distinct c-MET binding sites on HGFHere, we determine the cryo-EM structures of c-MET-HGF and c-MET-NK1 complexes mimicking their active states. The c-MET-HGF complex structure reveals an asymmetric 2:2 c-MET:HGF assembly, in which one of the HGF molecules is able to bridge two c-MET molecules together for receptor activation. This minimal 2:1 c-MET:HGF active complex is further stabilized by the second HGF molecule and heparin, leading to a more stable 2:2 complex and enhanced activation of c-MET. Finally, our structural model of the 2:2 c-MET:NK1 complex shows that distinct from the way that HGF activates c-MET, the NK1 dimer recruits two c-METs in a symmetric manner. The two c-MET molecules bound with the NK1 dimer are placed in close proximity that is ideal for receptor activation. Taken together, our results reveal the activation mechanism of c-MET receptor in response to different types of ligands.22. We reasoned that introducing a leucine zipper motif to the C-terminus of c-MET927 with a short flexible loop (denoted as c-MET927-LZ) could potentially stabilize the c-MET/HGF complex in the active state  and full-length HGF, and reconstituted the c-MET/HGF complex in vitro, showing excellent behavior in size exclusion chromatography (SEC). However, the resulting cryo-EM map from this sample only showed low-resolution features, indicating the structural flexibility of this complex. This low-resolution structure however showed that the membrane-proximal IPT2 domains of the two c-MET molecules are arranged in close proximity, consistent with the common ligand-induced dimerization mechanism for RTKsate Fig. 23. As exAfter the initial 3D classification of c-MET927-LZ/HGF particles set, two major classes were identified , which was resolved at 4 Å resolution  suggests that, upon the tethering of the N-terminal segment to the PSI and IPT1 domain of c-MET, the entire stalk region of c-MET rotates outward by ~20° (the pivot point is located at the linker between PSI and IPT1 domains), resulting in a more extended conformation  with an elongated shape simultaneously recruits two c-METs to its opposite ends. In one-half of the holo-complex, N, K2, K3, K4, and SPH domains of HGF I are arranged as a C-clamp shape that packs tightly against the SEMA domain of c-MET I Fig. . Totally28. To further confirm the functional significance of interface I, we mutated the residues Lys47, Lys91, Phe112, and His114 in the N domain of HGF to either glutamate or alanine. HGF WT and mutants were then purified and applied to H1299 cells for 10 min at 37 °C to test their abilities in inducing c-MET activation. As expected, K47E, K91E, F112A, and H114E mutants showed various degrees of reduction in activation of c-MET , double mutation (W321R/Y376A), and triple mutation (W321R/E361R/Y376A), designed to weaken the interface III. Although most of the point mutants exhibited only slightly reduced activity, both of double and triple mutants showed significant deficiency in inducing c-MET activation  adopts a flexible conformation with only its SPH domain, which contacts c-MET II-SEMA, being well resolved in the cryo-EM map. Nevertheless, after further 3D classification, a cryo-EM map reconstructed from a smaller class of particles reveals a complete version of HGF II that forms a tripartite interaction with c-MET II and HGF I Fig. . HoweverIn the 1:2 c-MET/HGF complex, the SPH and K1 domains of HGF II contact the SEMA and PSI domains of c-MET, respectively; while the K2 and K3 domains of HGF II also make weak interaction with the N domain of HGF I Fig. . Additio21. To understand how NK1 activates c-MET, we determined the cryo-EM structure of the 2:2 c-MET/NK1 complex at an overall 5 Å resolution  could further stabilize this interaction through the simultaneous binding to both HGF I and c-MET II  present in c-MET/HGF holo-complex have completely distinct roles. HGF I is the activating ligand that plays a vital role in c-MET dimerization and activation. HGF II, however, only plays a supplementary role in c-MET activation. Our cryo-EM analysis shows that, in most of c-MET/HGF particles, HGF II adopts a flexible conformation with only the SPH domain touching the SEMA domain of c-MET II. The HGF II with a flexible N-K1-K2-K3 fragment might be able to recruit a third c-MET through the K1–SEMA interaction. In such a structural configuration, a number of c-METs and HGFs molecules could, in principle, assemble into higher-ordered oligomers in a linear arrangement on the cell membrane. The formation of higher-order clusters might be important for the activation and regulation of c-MET, like that proposed for several other RTKsIn a small subset of particles, HGF II becomes rigid, and contacts both HGF I and c-MET II, with the help of a long heparin molecule. In this particular conformation, HGF II binding strengthens the interaction between HGF I and c-MET II and thereby stabilizes the entire c-MET I/HGF I/c-MET II complex Fig. . As mentIn this work, we provide the structural basis for the functional role of heparin-binding in HGF-induced c-MET activation. First, our model shows that a heparin molecule is sandwiched between the N domain of HGF I and the IPT1 domain of c-MET II, and acts as a “glue” to hold these two domains together. In this way, heparin contributes to the strong binding between HGF I and c-MET II. Furthermore, a long heparan molecule could potentially bind to both of HGF I and II simultaneously, thus stabilizing the 2:2 c-MET/HGF complex. It is also worth noting that a large cluster of long heparan polymers on the cell surface could be associated with multiple dimeric c-MET/HGF complexes and in principle induce the higher-order oligomerization of c-METs, and potentially lead to the formation of even larger biomolecular condensates on the cell membrane.35.It has been long known that HGF, and its natural splicing variant NK1, are both able to activate c-MET, but HGF exhibits higher biological activity than NK1. We determined the cryo-EM structures of both c-MET/HGF and c-MET/NK1 complexes in the active state. The comparison of these two complexes reveals several major structural differences. First, HGF recruits two c-METs in an asymmetrical manner that contrasts with the C2 symmetry observed in c-MET/NK1 complex Fig. . SecondlIn conclusion, our structural and functional analyses provide a large body of novel information for defining the molecular mechanism underlying the activation of c-MET in response to HGF and NK1 and paves the way for the eventual therapeutic intervention of diseases caused by aberrant activation of c-MET.All primers are listed in Supplementary Table 36, and the 6-histidine-tag (His6) tag, was cloned into the pEZT-BM, which is a BacMan vector that can infect both Sf9 insect and HEK293F cells (Supplementary Table Human cDNA encoding the c-MET extracellular region (amino acids 1–927) followed by the Human Rhinovirus 3 C (HR3C) recognition site, Tsi3 tagc-MET (amino acids 1–927) followed by a GGGGS linker, a GCN4 zipper sequence (RMKQL EDKVE ELLSK NYHLE NEVAR LKKLV GER), HR3C, Tsi3 tag, and His6 tag, was cloned into the pEZT-BM vector . For the phosphorylation assay, a full-length c-MET receptor followed by five or two repeats of the Myc tag sequence was cloned into pLVX-IRES-Puro. To introduce mutations to c-MET, short gene fragments (100–150 nt) containing mutations were synthesized (IDT Inc.), and the corresponding gene fragment in the wild-type c-MET was replaced by the synthesized gene fragment using HiFi DNA assembly enzyme (NEB).2.The plasmid was transformed into the DH10Bac bacteria (Geneva Biotech) for the production of bacmid DNA. Recombinant baculovirus was produced by transfecting Sf9 cells with the bacmid DNA using Cellfectin (Cellfectin Reagent Invitrogen). Protein was expressed with suspension-adapted HEK293F cells by infecting the virus at a ratio of 1:10 . The infected cells were supplemented with 5 mM sodium butyrate to increase protein expression. For HGF expression, 5% of FBS (Sigma) was supplemented to the culture media. The cells were cultured for 96 h at 37 °C and 8% COHuman HGF (amino acids 31–209) was tagged at the N-terminus with Thiol:disulfide interchange protein (DsBC) and His6 followed by HR3C recognition site cloned into pET22b vector. The plasmid was transformed into the Rosetta-gami 2 (DE3) (Milipore sigma) and cultured at 37 °C. When the OD600 reached 0.6, the cells induced with Isopropyl β-d-1-thiogalactopyranoside (IPTG), and expressed at 22 °C for 16 h.Culture media containing the secreted protein of interest was harvested by centrifugation and filtered using a glass fiber filter. The cleared media was incubated with the INDIGO-Ni resin (Cube Biotech Inc.) for 30 min on the bottle roller. The resin was collected using a chromatography column. The resin was washed with 10 resin volumes of the washing buffer  and eluted with the elution buffer . The eluted protein was dialyzed against the dialysis buffer  at 4 °C for 16 h. Tsi3 and His6 tags were cleaved with an HR3C protease during the dialysis. The protein was further purified by using SEC (Superose 6 increase 100/30 Cytiva) with the SEC buffer (20 mM Hepes at pH 7.5 and 400 mM NaCl).Culture media containing the secreted HGF was harvested by centrifugation and filtered using a glass fiber filter. The cleared media was adjusted to a final concentration of 50 mM sodium/potassium phosphate at pH 6.5 and applied to the cation exchange column . The protein was eluted with a linear gradient over 50% buffer B (20 mM Sodium/potassium phosphate buffer pH 6.5 and 2 M NaCl). The eluate was further purified using SEC (Superose 6 increase 100/30 Cytiva) with the SEC buffer (20 mM Hepes pH7.5 and 400 mM NaCl).Cells were harvested and lysed by sonication in sonication buffer . After centrifugation, the supernatant was applied to the Ni Sepharose resin (Cytiva). The DsBC and His6 tag were cleaved by adding HR3C protease to the resin and incubating for 16 h at 4 °C. Eluted protein was further purified with SEC  in the SEC buffer (20 mM Hepes pH 7.5 and 400 mM NaCl).c-MET927-LZ was mixed with HGF or NK1 in the presence of heparin  in a 1:1:4 molecular ratio. The complex sample was concentrated and loaded onto SEC (SRT SEC-500 Sepax) in the buffer containing 20 mM Hepes pH 7.5 and 100 mM NaCl. The fractions containing the complex were pooled and concentrated using an Amicon Ultra concentrator with a 100 kDa cut-off (Millipore) to ~5 mg/ml.37. A calibrated magnification of 46,296 was used for imaging, yielding a pixel size of 1.08 Å on images. The defocus range was set from −1.6 to −2.6 μm. Each micrograph was dose-fractionated to 30 frames under a dose rate of 18 e−/pixel/s, with a total exposure time of 4 s, resulting in a total dose of about 60 e−/Å2.Immediately before preparing the cryo-EM grids, 2 mM fluorinated Fos-Choline-8 (Anatrace) was added to the purified sample to prevent the protein from being damaged by the hydrophobic air–water interface on a cryo-EM grid. The cryo-EM grid was prepared by applying 3 μl of the protein samples to glow-discharged Quantifoil R1.2/1.3 300-mesh gold holey carbon grids . Grids were blotted for 4.0 s under 100% humidity at 4 °C before being plunged into the liquid ethane using a Mark IV Vitrobot (FEI). Micrographs were acquired on a Titan Krios microscope (FEI) operated at 300 kV with a K3 direct electron detector (Gatan), using a slit width of 20 eV on a GIF-Quantum energy filter. SerialEM was used for the data collection38. CTF correction was performed using GCTF39. The rest of the image processing steps were carried out using RELION 3.140. Particles were first roughly picked by using the Laplacian-of-Gaussian blob method and then subjected to 2D classification. Class averages representing projections of the c-MET/HGF in different orientations were used as templates for reference-based particle picking. A total of 3,287,093 particles were picked from 8100 micrographs. Particles were extracted and binned by three times (leading to 3.24 Å/pixel) and subjected to another round of 2D classification. Particles in good 2D classes were chosen  for 3D classification using an initial model generated from a subset of the particles in RELION. After the initial 3D classification c-MET927-LZ/HGF particles set, two major classes were identified showing good secondary structural features . The holo-complex comprises two c-MET and two HGF molecules; while the sub-complex only comprises one c-MET and two HGF. Particles for holo-complex or sub-complex were selected separately and re-extracted into the original pixel size of 1.08 Å. Subsequently, we performed finer 3D classification for each particle subset by using local search in combination with small angular sampling, resulting in improved density for both assembly states. The final reconstructions of holo-complex and sub-complex were resolved at 4.8 and 4 Å resolution, respectively. To improve the resolution for c-MET I and HGF I in holo-complex, we performed focused 3D refinement, by using a soft mask around c-MET I and HGF I, leading to a 3D reconstruction at an overall resolution of 4.5 Å. Finally, we performed another round of finer 3D classification on the particle set of sub-complex. After the further classification, we could identify a new class where HGF II adopts a rigid conformation. The subsequent 3D refinement of this class yielded a cryo-EM at 4.9 Å resolution, resolving a complete version of HGF II.Movie frames of c-MET927-LZ/HGF micrographs were motion-corrected and binned twofold, resulting in a pixel size of 1.08 Å, and dose-weighted using MotionCor2A total of 1,342,144 particles were picked from 3644 micrographs of c-MET/NK1. A total of 656,258 particles were selected by 2D classification. Subsequent 3D classification revealed one good class showing good density. Next, we performed two rounds of finer 3D classification by using local search in combination with small angular sampling, resulting in a new class showing improved density for the entire protein. 3D refinement of this class, along with CTF refinement and particle polishing, yielded a structure at 5 Å resolution. The resolution was estimated by applying a soft mask around the protein density. The Fourier Shell Correlation (FSC) 0.143 criterion was used. Local resolution was calculated in RELION.30. The models of K3 and K4 of HGF were first built by homology modeling using the structures of K1 of HGF as a template , and then rigid-body fitted into the cryo-EM density in Chimera41. The manual building was carried out using the program Coot. The model was refined by using the real-space refinement module in the Phenix package (V1.17)42. Restraints on secondary structure, backbone Ramachandran angels, and residue sidechain rotamers were used during the refinement to improve the geometry of the model. MolProbity 4.5 as a part of the Phenix validation tools was used for model validation  were generated in HEK 293 T packaging cells following standard transfection protocol43. Titrations of lentiviruses were used to infect H1299 cells, and infected cells were selected using 2 μg/ml puromycin for 2–3 days. Cells stably express near-endogenous myc-c-MET (WT or mutant) were selected for the following assays.Naive H1299 cells were gifted from John Minna  and cultured in RPMI 1640 medium (Thermofisher) supplemented with 10% (vol/vol) FBS at 37 °C and 5% COTo examine the agonist activities of HGF mutants, naive H1299 cells were plated 200,000 cells/well on six-well plates (Corning) and starved in an FBS-free medium for 16 h. The starved cells were stimulated with 2 ml, 100 ng/ml HGF (WT or mutant) for 10 min at 37 °C. After stimulation, cells were washed once with 1x PBS and then collected in 150 µl 2x Laemmli Sample Buffer supplemented with 5% 2-Mercaptoethanol. SDS-PAGE and Western Blotting were used to probe c-MET , phospho-c-MET , and loading control β-actin . Blotting results were analyzed using ImageJ.To examine the phosphorylation capacities of c-MET mutants, 3′UTR siRNA (hs.Ri.MET.13.1 from IDT) was used for selective knockdown of endogenous c-MET in H1299 cell lines that stably express near-endogenous myc-c-MET (WT or mutant). Transfections of siRNA were carried out using Lipofectamine RNAi-Max (Life Technologies) following the manufacturer’s protocol. After depletion of endogenous c-MET, 100 ng/ml HGF WT was used to stimulate the phosphorylation of myc-c-MET following the protocol described above.Five micrograms of the bait protein c-MET927-TSI3-His was immobilized to TSE resin by incubating for 30 min on ice and washed with binding buffer PBS (Gibco) supplemented with10 µM Tinzaparin and 2 mM CaCl2. The resin was incubated with 5 µg of the prey proteins HGF wild type or mutants in 50 µl of binding buffer for 1 h at 4°. Each tube was tap gently but thoroughly every 10 min. The resin was washed with binding buffer to wash away prays. The resin was boiled in SDS loading buffer (Bio-rad) and loaded on the SDS-PAGE. The intensity of the bands was calculated using ImageJ.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The curing of concrete significantly influences the hydration process and its strength development. Inadequate curing leads to a loss of quality and has a negative effect on the durability of the concrete. Usually, the effects are not noticed until years later, when the first damage to the structure occurs because of the poor concrete quality. This paper presents a non-destructive measurement method for the determination of the curing quality of young concrete. Hyperspectral imaging in the near infrared is a contactless method that provides information about material properties in an electromagnetic wavelength range that cannot be seen with the human eye. Laboratory tests were carried out with samples with three different curing types at the age of 1, 7, and 27 days. The results showed that differences in the near infrared spectral signatures can be determined depending on the age of the concrete and the type of curing. The data was classified and analyzed by evaluating the results using k-means clustering. This method showed a high level of reliability for the differentiation between the different curing types and concrete ages. A recommendation for hyperspectral measurement and the evaluation of the curing quality of concrete could be made. The curing of concrete is largely responsible for the quality of concrete components. To achieve durability, concrete must be treated and protected. Professional curing focuses on the chemical processes during the hydration which is associated with the hardening of concrete. The hardening process and compaction of concrete starts immediately after its pouring, e.g., in its formwork. During this phase, the chemical hydration generates the cement stone, which ties together the grains of the composite material concrete . At thispremature drying of the surfacestrong temperature fluctuationsand excessive cooling or heating In order to avoid or minimize these negative processes for the lifespan of a reinforced concrete component, the following harmful processes are minimized for young concrete:by protecting the concrete surface with foils and protection mats by moistening the concrete surface with a water mistby applying a liquid protective film or after-treatment agentby leaving the concrete in the formwork ,9,10The primary goal of a good curing is therefore the creation of a dense concrete structure, avoiding capillary pores and cracks reaching from the surface to the reinforcement layer. A good curing can be obtained with the following measures:Checking or monitoring the correct curing of concrete components is not an easy task under construction conditions. Processes are required that are easily manageable, can be used over a wide area, are reproducible, and allow unambiguous statements. Among other things, the test procedures must not destroy or impair the properties of the concrete. A promising method that has already found its way into several evaluation processes is hyperspectral imaging. It has already been used in individual cases to check the developmental strength of concrete.Hyperspectral imaging is a non-destructive method for the determination of material properties. It provides data of reflectance information over a certain wavelength range in the electromagnetic spectrum that cannot be seen by the human eye, e.g., in the case of the near infrared. According to ,12, the Previous investigations showed that different concrete qualities can be determined with spectral imaging. The aim of this work was to evaluate the potential of near-infrared hyperspectral imaging for assessing the quality of curing for sufficient concrete hydration.In particular, it should be clarified: (a) whether an incorrect hydration process, and consequently, a non-optimal strength development can be recognized; (b) at which times the application can be used optimally; (c) if the spectral signature of the hyperspectral analysis differs dependent on the curing type; and (d) how the signatures change over the time of an optimally cured, a less well cured, and a poorly cured concrete.Xenics NIR camera ;Specim N17E spectrograph operating in the range of 950–1650 nm with an IR optimized objective lens;600 mm Y-table gear;stable diffuse 45/0 illumination created by halogen bulbsConcrete samples were scanned using a NIR hyperspectral imaging instrument  located at the University of Natural Resources and Life Sciences, Department of Material Sciences and Process Engineering, Institute of Physics and Materials Science, in Vienna, Austria with improved hard- and software components. The actual system components of the instrument are:The experimental setup used for the hyperspectral analyses is shown in The whole system was controlled using Argus software  . PrepariThe y-length needed to be defined in the Argus software according to the size of the sample. The final setup in the Argus software is shown in Test series were carried out on concrete samples with composition and properties as shown in As indicated in Five faces per sample (the labelled face was excluded from the measurements) were measured with the hyperspectral camera on the first day (day 1), one week (day 7), and 4 weeks (day 27) after concreting (day 0). The measurement data were subsequently processed and compared with one another to determine differences in the spectral signatures with respect to unequally treated concrete samples and the various days of measurement.The data from the samples of the same curing type and age all showed similar results. In the following, the results of three representative measurements per curing type and measurement day are used to compare the data.The entire hyperspectral imaging workflow, from sample preparation to data analysis and evaluation, is shown in ®  [The obtained hyperspectral data in the form of a spectral hypercube consisting of spatial and spectral information were treated with a noise reduction algorithm to remove the so called “salt and pepper” noise, which detects unserviceable bright or dark pixels. These pixels were replaced by a neighboring pixel algorithm script in MATLABMA, USA)  that detMA, USA) .The data were further processed with the data analysis software PLS-Toolbox in Matlab. The region of interest of each image was defined by selecting the concrete surface and removing the background in the picture.The spectral pre-processing of the data was done by normalization and Savitzky–Golay smoothing (SGS)  with PLSData analysis was carried out using k-means clustering. K-means clustering is an unsupervised data classification algorithm that divides the dataset into different groups. First, k cluster centers, called centroids, are randomly assigned, and each data point  is assigned to the cluster with the closest mean value ,25,26. AK-means clustering was used to determine whether samples with different curing and results from different measurement days could be differentiated from one another by means of pixel classification. Hence, images of the three different curing types and images with different concrete ages were merged to one single image for statistical analysis. Different combinations of images, as shown in Combinations 1–3 included measurements of all three curing types. Images of NB1, NB2, and NB3 were compared on day 1, day 7, and day 27 after concreting  shows the images of the three curing types on measurement day 1, and (b) shows the composition of images from NB 1 samples from day 1 to day 27. The merged images are displayed in false colors, based on the average reflectance value of each pixel. The other four combinations were created in the same way.In The optimal number of classes for k-means clustering resulted from the interpretability of the associated spectral signatures. In general, clear signature differences could be achieved with three clusters (in a few cases with four clusters), where the explanatory clusters contained relevant spectral information and the additional clusters contained only a comparably small number of pixels, representing artefact values near holes and edges or caused by direct reflection.Each pixel of a merged image was assigned to one of the three (or four) clusters, later referred to as classes. After k-means clustering, the pixels were shown in the colors red, blue, and green, depending on their class affiliation. Each class had its own characteristic spectral signature. The proportions of pixels per class for the respective samples or on the respective measurement day allowed conclusions to be drawn about the material properties of the various curing types or the hydration progress.The statistics showed that pixels assigned to classes A and B were especially predominant for all analyses. Class C and class D (only with the choice of four classes) contained only a few pixels and therefore were not representative. They were not considered for further evaluation of the data.Two characteristic absorption bands around 1340 nm and 1400 nm could be defined in the spectral signatures for classes A and B. The extent of absorption differed depending on the curing type and the measurement day. The water band was found in the wavelength range around 1400 nm. The absorption in this area was strongest in class A, which means that the pixels in class A had a higher water content compared to class B.Class A: spectral signatures showed the strongest absorption at 1400 nm compared to class B. A significant proportion of pixels was assigned to class A.Class B: spectral signatures showed less absorption at 1400 nm compared to class A. A significant proportion of pixels were assigned to class B.Class C (and D): The proportion of pixels was not representative, mainly representing artefact pixels near holes and at the edges.In general, classes A, B, and C could be distinguished as follows:The results of the analyses of all image combinations are shown in The results of combinations 4–6 are shown in The spectral signatures (a) differ, dependent on the curing type. The differences in the spectral signatures of class A and B are greatest for NB1  and smalNote: The dark areas on face 1 of NB1 Day 7 and Day 27 in The spectral signatures of the three classes  are given under point (a) in the figures. The distribution of the classes for the respective curing types or on the respective measurement days is shown under point (b).First, the combinations 1–3 are considered, where the curing types were compared with one another on measurement day 1 , day 7 , and dayRegarding the spectral signatures (a) in Looking at the class distribution statistics (b) of the three combinations in The influence of the lighting can be clearly seen in The results prove that differences in the hydration process according to the curing type and the duration of hydration can be determined by statistical analysis of hyperspectral data. The distribution of the corresponding classes for the samples or measurement days provides information about the quality of the hydration process and the developmental strength of concrete. Differences between the curing types and measurement days can be seen in the characteristic absorption bands at 1341 nm and 1398 nm (water band). The combinations 1–3 a–c show A similar development regarding the reflectance intensities can also be seen in the data of the different curing types in The analysis of the characteristic absorption bands of the spectral signatures allowed an evaluation of the classes and an assessment of the curing quality. A pronounced absorption at 1341 nm for class B and a clear absorption at 1398 nm for class A indicated good curing quality d. If theIt could be proven that the quality of concrete can be classified by comparing the hyperspectral data of samples with different curing treatments. The classification using k-means clustering enables differentiating concrete samples based on their curing quality. The samples could be analyzed in terms of their hydration process by interpreting the statistics and spectral signatures of the generated classes.The spectra of the analyzed images showed significant changes over the hydration time. The most significant developments were determined at the absorption bands at approx. 1340 and 1400 nm.The reflectance values for the water band at approx. 1400 nm showed that the optimally cured samples had the most extreme value and thus a higher water content compared to non-optimally cured samples. Consequently, non-optimally cured samples dry out early, which has a negative effect on the developmental strength of concrete.The class distribution of the image pixels as a result of the k-means clustering allowed a clear distinction between the optimally, less well, and poorly cured samples. The samples could be distinguished from one another at the ages of 1, 7, and 27 days. Since the classes were divided more and more evenly among the compared samples with increasing concrete age, the tests of young concrete at the ages of 1 and 7 days gave the clearest results.Hyperspectral analysis of young concrete enables obtaining valuable information about the hydration process, premature drying, and consequently the final quality of the concrete. It could be proven that this method is a reliable and promising application for evaluating the curing quality of concrete samples.Further research is needed on the application of the method directly on a structure. A reliable evaluation could be realized using reference samples, whereby the characteristic absorption bands could be used as limit values for the evaluation of the curing quality. The measurement results are heavily dependent on the concrete composition, as well as on environmental influences such as light intensity and humidity. Therefore, standardized on-site tests are necessary to use the method for a reliable evaluation. It would also be worthwhile to further investigate wavelength ranges, as this would provide additional information and even clearer results.In this respect, it is hoped that through further research in this area, the assessment of concrete curing will continue to gain in importance and that the hyperspectral method can find wide application in construction practice.


This research investigates the effectiveness of bio-sourced flax fiber-reinforced polymer in comparison with a traditional system based on carbon fiber-reinforced epoxy polymer in order to confine recycled aggregate concretes. The experimental investigation was conducted on two series of concrete including three mixtures with 30%, 50%, and 100% of recycled aggregates and a reference concrete made with natural aggregates. The concrete mixtures were intended for a frost environment where an air-entraining agent was added to the mixture of the second series to achieve 4% air content. The first part of the present work is experimental and aimed to characterize the compressive performance of confined materials. The results indicated that bio-sourced composites are efficient in strengthening recycled aggregates concrete, especially the air-entrained one. It was also found that the compressive strength and the strain enhancement obtained from FRP confinement are little affected by the replacement ratio. The second part was dedicated to the analytical modeling of mechanical properties and stress–strain curves under compression. With the most adequate ultimate strength and strain prediction relationships, the full behavior of FRP-confined concrete can be predicted using the model developed by Ghorbel et al. to account for the presence of recycled aggregates in concrete mixtures. Concrete structures are subjected to accidental loads such as seismic or mechanical shocks, excessive loads, temperature gradients, design flaws or defective implementation, which could lead to material damage and failure. Therefore, their strengthening is a possible issue and requires advanced research, and this is especially true if the aggregates come from construction and demolition wastes (CDWs) known as recycled concrete aggregates (RCA). Several studies have been conducted on recycled aggregates concretes (RAC) during these last decades, since the reuse of CDWs is defined as a priority area in the EU according to the Circular Economy Action Plan .It is currently admitted that the incorporation of RCA leads to recycled aggregates concretes RAC with higher porosity and ductility and lower permeability, durability resistance, and mechanical properties than those of natural aggregates concrete ,9,10,11.Many investigations have explored since 2012 the potential of confining RACs by fiber-reinforced polymers (FRPs) in order to enhance their compressive strength and to increase their ductility using both experimental and analytical approaches ,18,19,20Fiber-reinforced polymers (FRPs) became the most used materials for concrete structural elements repairing and strengthening since 1990  because The most common composites used are based on epoxy resins systems reinforced by fibers such as carbon (CFRP), glass (GFRP), aramid (AFRP), and basalt (BFRP). Carbon fibers as well as aramid ones are manufactured respectively from polyacrylonitrile (PAN) and para-phenyleneterephthalamide (PPD-T), while glass fibers and basalt ones are obtained respectively from silica and igneous rocks. Their production is responsible for toxic gas emission impacting the environment. Hence, these fibers are not biodegradable and non-sustainable materials. The matrix is generally a thermosetting resin. The most used are epoxy, polyester, and vinyl ester resins. These polymers contain bisphenol A (BPA), which leads to health damages and are obtained from nonrenewable fossil resources.The European directives enforce not to use materials produced from non-renewable resources, non-biodegradables, having unhealthy effects on humans and a high carbon footprint. Using RACs aims to diminish natural resources consumption and to achieve the European target for CDWs management. Confining RACS by conventional composites such as CFRP, GFRP, AFRP, or BFRP is not a sustainable solution and acts against European recommendations. Therefore, the use of bio-based composites for the reinforcement and repairing of concretes is recommended. Fewer studies have been carried out to investigate the efficiency of natural fibers reinforcing polymers, and the results are promising ,27,28,29This paper intends to demonstrate how far the bio-resourced composite can be developed based on the work of Limaiem et al. , replaci-The valorization of recycled sand in concrete mixtures without and with air entraining agent.-The formulation of concretes with a fully recycled granular skeleton.-An investigation on the effectiveness of unidirectional flax fiber-reinforced bio-sourced epoxy resin in confining air entrained recycled aggregates concrete.-A comparison between the effectiveness of bio-sourced FRP and the traditional CFRP.-A validation of the applicability of an analytical stress–strain model to confined air entrained recycled aggregates concrete with any type of fibers.Moreover, the target RACs of this research are formulated using both recycled sand and gravels, while the published research is limited to FRP-confined RACs incorporating only coarse recycled aggregates. Hence, four formulations have been studied differing in the replacement ratios: 0% (the reference), 30%, 50%, and 100%. In addition, the RACs were intended for a frost environment and should be resistant to frost exposure class (XF1–XF3). To hit this objective, the four mixtures were adjusted to contain 4% of entrained air. The confinement effectiveness by CFRP in comparison to bio-composites of RACs was experimentally studied, and the obtained stress–strain curves were modeled using an analytical approach. To this end, the main objectives of this work are:-Cement type CEM II/A-L 42.5N, Betocarb HP-OG,-Limestone fillers manufactured by OMYA SAS,-MC PowerFlow 3140 superplasticizer-Natural river sand called NS 0/4, two crushed natural gravels NG1 4/10 and NG2 6.3/20, and recycled aggregates provided from construction demolition wastes designed by RS0/4, RG1 4/10, and RG2 6.3/20.The raw materials used to formulate concrete were: m), was defined by Equation (1).RSM is the mass of RS in 1 m3 of concrete, RGM, NAM, and RAM are respectively, the mass of recycled gravels, the total mass of natural aggregates (NA), and the total mass of recycled aggregates RCA in 1 m3 for all mixtures. Four mix proportions initially proposed within the framework of the ANR ECOREB project  were elacmf and 8% and 20% for cE), while an increase is noticed for the peak strain “εc1” for non-entrained air formulation. However, it appears that the introduction of an air-entraining agent for a given formulation leads to the decrease in the mechanical properties.Compressive tests were conducted on cylindrical specimens (15 cm of diameter and 30 cm of length) at 90 days age, and the obtained properties for each concrete formulation are summarized in Three types of composites were used in order to investigate their effectiveness in improving the mechanical behavior and durability of recycled aggregates concretes:The first composite FOREVA TFC, called “CBF”, is a carbon bidirectional woven fabric with 0°/90° fiber orientation reinforced with epoxy resin commercialized by FREYSSINET.The second one “SIKAWRAP-230C”, designed as “CUS”, is unidirectional carbon fiber fabric reinforced with epoxy polymer delivered by SIKA.®530 product cross-linked with Phenalkamine NX5619. The development of the bio-sourced epoxy resin and its hardener was done in the framework of the ANR MICRO [The last one named “FUB” is a unidirectional flax fiber fabric associated with EnviPOXYNR MICRO . The comCylindrical specimens with dimensions 15 × 30 cm were casted and stored in water at room temperature for 90 days. After wiping with a wet cloth, the resin was applied on all the side surface of the sample with a paintbrush, as shown in In order to examine the effect of FRP on the concrete behavior, cylindrical specimens were tested under monotonic compressive loading. Tests were conducted using a hydraulic press INSTRON SCHENCK with a capacity of 3000 kN by imposing a displacement rate of 1 mm/min. Axial strains in the middle portion of the specimens were measured using 3 LVDTs spaced 120° along the circumference of a crown. Splitting tensile and fracture tests were not carried out, although these properties are essential for the evaluation of the overall behavior of concrete structures. However, these properties can be predicted through the compressive characteristics as shown by Sucharda et al. . Using CFRP to strengthen recycled aggregates concrete is less efficient as the ratio of recycled aggregates increases . It shouThe failure mode of samples reinforced with three types of FRP is shown in The general stress–strain curves shown in The strengthening of air-entrained concrete is efficient regarding the compressive strength as well as ductility. CFRP is more efficacious than the bio-sourced composite “FUB”, as illustrated in lf of FRP to the compressive strength cmf of unconfined concrete) as a function of the equivalent replacement ratio Γm for both series of concrete. Concerning the confinement pressure, lf, it can be given by relation 2 as a function of the mechanical properties of the used FRP, the thickness, and the number of FRP layers as well as the diameter of the tested specimen:n, t, and tf are the number of FRP layers, the thickness of the FRP jacket, and the tensile strength, respectively, while D is the specimen’s diameter. It can be observed from cc/fcmf) against the replacement ratio for both series of concrete. It can be observed that the gain decreases with the replacement ratio for the series CxR-yR, while it increases for the series CxR-yR-4 containing an air-entraining agent, especially with CBF fiber. It can be also shown that the compressive strength enhancement obtained from FRP confinement is little affected by the replacement ratio. The same statement was found by Chen et al. [ccε, to the peak strain of unconfined concrete named cε1) follows the same variation of the strength gain with the variation of the replacement ratio, as shown in n et al. . The strccf, and the compressive strength of NAC for each series named cm,NACf. The variation of this ratio against the replacement ratio is illustrated in The stress recovery is defined as the ratio between the ultimate strength of confined concrete, cc/fcmf) as a function of the confining ratio (l/fcm)f, while cc/εcε1) against the confining pressure. It can be shown that the stress gain ratio and the strain gain ratio have a linear growth trend with the confining pressure regardless the type of the used FRP, the RCA replacement ratio, as well as the entrained air content. These trends, which are in perfect agreement with the results of the literature [terature ,18,36,37R2 given by Equation (3). A value of R2 close to one indicates that the experimental points approach the model, while a value near zero means that the experimental points are too scattered around the line describing the model.SSE: the residual sum of squaresSST: the total sum of squaresn: the number of experimental pointsiy: the ith experimental measurement The strength and strain enhancement ratios were calculated using analytical relationships presented in The obtained results in terms of R² show that the model of Lam and Teng  (Equatiocmf of ccf); then, the tangent elastic modulus can be calculated based on the mean compressive strength and the replacement ratio according to Equation (14). The peak strain for unconfined concrete can be calculated according to Equation (15), where it was found that this strain depends only on the compressive strength regardless of the replacement ratio [ciE is initial elastic modulus.The model given by Equation 13) was adopted to describe the full stress–strain behavior of both unconfined and FRP-confined NAC and RAC. This model proposed initially by Popovic and adopted later by Mander  was modi was adopnt ratio .(13)σfcmβ. Indeed, when the experimental peak strain of unconfined concrete is used instead of peak strain calculated using Equation (15), the agreement between analytical and experimental curves is better.In ®530 product called FUB. The obtained experimental results lead to the following concluding remarks:-Confining recycled aggregate concrete by the unidirectional flax fibers reinforced bio-sourced epoxy resin, FUB, is significant for 4% air-entrained recycled aggregates concrete, and its effectiveness is comparable to the effectiveness of traditional composites based on carbon fibers CBF and CUS.-For the same type of FRP and the same replacement ratio, the confining ratio is more important when an air-entraining agent is used.-The compressive strength and the strain enhancement obtained from FRP confinement are little affected by the replacement ratio.-Bio-based fiber, FUB, led to the recovery of the strength of the air-entrained concrete with an increase of 8% for the mixture C100R-100R-4. Moreover, for C100R-100R, a single layer of CUS and two layers of FUB fibers allowed restoring 100% and 91% of the NAC’s compressive strength.-The performance of the model developed by Ghorbel et al. is satisfactory to predict the full stress–strain curves for both series of studied concrete in unconfined and confined configurations.In this paper, the axial behavior of FRP-confined recycled aggregate concrete was investigated. Two series of concrete without and with 4% air-entraining agent and various replacement ratios of recycled aggregates were prepared and tested to examine the performance of three types of FRP. The used FRP were a carbon bidirectional woven fabric with 0°/90° fiber orientation reinforced with epoxy resin named CBF, a unidirectional carbon fiber fabric reinforced with epoxy polymer (CUS), and a unidirectional flax fiber fabric associated with EnviPOXYThe experimental findings of this paper are mainly based on the compressive behavior of laboratory-size specimens. For structural reinforced concrete elements, more research is needed to validate the confinement effectiveness of bio-based fibers for air-entrained recycled aggregates concrete in a harsh environment. One might think also that confining and strengthening would be also benefits for rebar preservation against the ingress of chemical substances.


Drug repurposing  is a strategy for identifying new uses for approved or investigational drugs that are outside the scope of the original medical indication . It has https://www.who.int/campaigns/world-antimicrobial-awareness-week/2021). Recent studies have shown the simmering public health crisis with increase in resistance against antibiotics of last resort, carbapenems and polymixins . It has become the most dreaded menace increasing at an alarming rate and threatening the global health. The emergence of antimicrobial resistant bacteria is one of the most important public health challenges worldwide . World Hlymixins . In 2019lymixins . So, theWhile the concept of drug repurposing sounds logical and a scientific way of dealing with hitherto untreatable diseases viz. Alzheimer’s, Cancer, Parkinson’s etc., the repurposing of antibiotic drugs has raised eyebrows over the use of antibiotics in treating such diseases given the rampant antimicrobial resistance that has wreaked havoc in contemporary times. AMR has been known to cause over 700,000 deaths globally and is estimated to kill around 10 million people worldwide by 2050 . There aH. pylori infection in AD patients as triggers for the release of inflammatory mediators (H. pylori is known for its resistance against levofloxacin (in vitro, their efficacy is not warranted in vivo. Further, the long-term exposure to an antibiotic either at clinical or sub-stoichiometric dose can induce drug resistance among the gut microbes. There is a need to administer these antibiotics chronically for the treatment or management of AD. Therefore, chronic use of antibiotics at clinical dose may impose antibiotic resistance as a result of which it would become difficult to treat the common infection. Additionally, the long-term use of antibiotics may make the patients more prone to infection. If any antibiotics are showing anti-AD potential either in vitro or clinical studies, then, long-term consequences of using these antibiotics need to be evaluated. There has been no substantial study to understand the long-term effects of using repurposed antibiotics on enhancing AMR but as prior data show a direct correlation between antibiotic consumption and AMR (V. cholerae and warranted strict restrictions on the indiscriminate use of antibiotics. Another facet is the limitation of unrestricted sale of these antibiotics without prescription especially in developing countries like India, one of the largest antibiotic consumer (The antibiotic levofloxacin has been recently suggested as a potential repurposed drug for Alzheimer’s disease  while seediators . H. pylofloxacin . The majfloxacin . At a hifloxacin . Though  and AMR , there m and AMR . The stuconsumer . So, theconsumer .During the COVID-19 pandemic, the health practitioners resorted to the prescription of azithromycin alone as well as in combination with drugs like hydroxychloroquine. It became a highly controversial repurposing of an antibiotic without any proof of its antiviral properties. However, its use still amplified with successive waves of COVID-19. Only time will tell as to what will be the ramifications of such an unregulated and unscientific use of such antibiotics. Its consequences on AMR will worsen the situation . Given tThere is utmost need to find alternatives of antibiotics in repurposing, especially those which have been already shown to cause resistance. The successful repurposing of non-antibiotic drugs against cancer has been reported  and the