Malignant nervous system cancers in children are the most devastating and worrisome diseases, specifically due to their aggressive nature and, in some cases, inoperable location in critical regions of the brain and spinal cord, and the impermeable blood-brain barrier that hinders delivery of pharmaco-therapeutic compounds into the tumor site. Moreover, the delicate developmental processes of the nervous system throughout the childhood years adds another limitation to the therapeutic modalities and doses used to treat these malignant cancers. Therefore, pediatric oncologists are charged with the daunting responsibility of attempting to deliver effective cures to these children, yet with limited doses of the currently available therapeutic options in order to mitigate the imminent neurotoxicity of radio- and chemotherapy on the developing nervous system. Various studies reported that c-Met/HGF signaling is affiliated with increased malignancy and stem cell enrichment in various cancers such as high-grade gliomas, high-risk medulloblastomas, and MYCN-amplified, high-risk neuroblastomas. Therapeutic interventions that are utilized to target c-Met signaling in these malignant nervous system cancers have shown benefits in basic translational studies and preclinical trials, but failed to yield significant clinical benefits in patients. While numerous pre-clinical data reported promising results with the use of combinatorial therapy that targets c-Met with other tumorigenic pathways, therapeutic resistance remains a problem, and long-term cures are rare. The possible mechanisms, including the overexpression and activation of compensatory tumorigenic mechanisms within the tumors or ineffective drug delivery methods that may contribute to therapeutic resistance observed in clinical trials are elaborated in this review. Gliomasmor site .The standard of care therapy for the high-risk brain tumors includes surgical resection followed by radio- and/or chemotherapy, whereas the low-risk neuroblastomas and low-grade gliomas require only surgical resection. Moreover, the more aggressive gliomas, such as the diffuse midline gliomas, and the CNS germline tumors generally cannot undergo surgical resection  due to tThe hepatocyte growth factor/scatter factor (HGF/SF) and its receptor tyrosine kinase (RTK) protein c-Met activate a signal transduction cascade that ultimately induces cellular proliferation, migration, invasion, and evasion of apoptosis. This signaling cascade is essential during development and tissue regeneration, such as when hepatocytes undergo regeneration ; howevervia the PI3K-AKT and the rat sarcoma oncogene homolog (RAS)-mitogen activated protein kinase (MAPK) pathways that induce activation of cell division control protein 42 (CDC42), which in turn modulates cell survival. CDC42 also regulates cell migration and adhesion via interaction with RAP1 and RAC1 that act on integrins and cadherins , and its ligand, HGF/SF , play cradherins .c-Met signaling is reported to be essential in epithelial-to-mesenchymal transition (EMT) required during the developmental process where extensive cellular migration occurs . In cancThe delicate balance of critical biologic processes, such as cell proliferation, migration, and differentiation is largely controlled by RTKs. Abnormalities in protein expression of these RTKs in tumor cells dysregulate multiple downstream signaling cascades that drive cell proliferation, invasion, metastasis, and angiogenesis . This dyCross-talk between c-Met signaling with other tumorigenic pathways has been reported, including direct interactions with the insulin-like growth factor receptor-1 (IGF1R) in pancreatic cancer , transfoin vivo clonogenic growth. The combined targeting of c-Met and EGFR pathways could be a promising tool for GBM and a possible treatment option to explore for the aggressive and malignant nervous system tumors.Overexpression or mutation in the WNT/β-catenin pathway is known to play a significant role in cancer transformation; c-Met is a direct transcriptional target of WNT/β-catenin in colon cancer cell lines . Moreovevia a paracrine and autocrine loop signaling  is used in order to delay the need for radiotherapy until absolutely necessary . DespiteMYCN expression, the WNT-subgroup and subgroup-3 tumors exhibit high levels of MYC expression, whereas subgroup-4 tumors exhibit relatively low expression of both MYC and MYCN -subgroup (named after the pathway believed to be the prime driver of tumor initiation in this subgroup) that arises from granule precursors of the external granule layer, WNT-subgroup that arises from the progenitor cells in the lower rhombic lip, subgroup-3 that arises from the neural stem cells, and subgroup-4 that arises from the unipolar brush cells . The WNTand MYCN .via MAP4K4 leading to increased cell migration. This increase in metastatic potential was dependent on the c-Jun N-terminal kinase (JNK) pathway , and the other designated as WT-C and driven by PDGFRA/MET. The WT-C subgroup, which harbored the PDGFRA and MET amplifications also expressed enrichment for chromosome 1p and 20q loss and 17q gain. The gene signature of this group highly resembled that of the adult GBM-defined “Proneural” group, and the median survival of patients within this group was approximately 18 months , and NTRK1  expression indicating that this may lead to permanent gene silencing of critical developmental loci. Whether or not silencing of a single gene or entire pathways is required for NB initiation and progression is still under investigation. However, several hypermethylated genes have been consistently identified in NB, including HIC1, putative tumor suppressor genes , differentiation genes (HOXA9), and apoptosis genes , which is associated with enrichment of H3K27me3 at the promoters of tumor suppressor genes such as K1 TrkA; . In addiK1 TrkA; . Moreoveg allele . Genome-n of DNA . String n of DNA , which wn of DNA .in vivo tumor growth models and resist conventional therapeutic approaches.CSCs comprise a small sub-population of self-renewing, multi-potent cells that reside within the bulk of the tumor. This sub-population is phenotypically and functionally similar to normal stem cells, but can drive tumor growth and recurrence . CSCs arStem cell markers in nervous system tumors have been utilized to enrich the CSC population for functional studies and they include: CD15, CD133, CD49f, CD36, CD44, L1CAM, A2B5, SOX2, musashi-1, BMI1, EGFR, and Nestin . MoreoveThe interplay demonstrated between c-Met and these pathways  implicatin vivo tumorigenicity . genicity . As mentgenicity . Moreove-renewal . In supp-renewal .in vitro and in vivo, indicating that c-Met activation may be required for GSC function /MYCN-driven NB, activation through the downstream signaling pathways of ALK  may enhance the propagation and maintenance of the stem cell-like sub-population in NB  bearing the NTRK1-G667C mutation. Specifically, foretinib inhibited tyrosine receptor kinase A (TRK-A) phosphorylation in cells bearing the NTRK1-G667C mutation  competitive c-Met inhibitor. To date, there are no reports of clinical trials using tivantinib in children with brain tumors. Only one report of a phase I trial in children with relapsed/refractory solid tumors reported no objective response with tivantinib treatment . Foretinmutation , implica6-methylguanine-DNA methyltransferase  that targets the HGF/SF, exhibited anti-tumor activity against a xenograft model of U-87 malignant glioma model . A phasein vitro using combination therapy versus single therapy  in an intracranial mouse model of GBM using U87 cells. The synergistic effect between dP@mVLP/RNAic-MET and TMZ was identified at multiple doses with complete cancer eradication with 5 mg/kg of dP@mVLP/RNAic-MET at a normal TMZ patient dose. This combination provides the potential for glioblastoma patients to have an enhanced, genetically regulated, and cost-effective treatment compared with ultrasound use or nanotherapeutic agents, which are currently used alongside TMZ. Animal trials, including the analysis of the liver and kidneys, which showed no signs of antigenicity or inflammation, solidified the therapeutic benefits of dP@mVLP/RNAic-MET pretreatment followed by oral TMZ compared with another synthesized molecule mVLP/RNAic-MET pretreatment with TMZ, which proved ineffective, confirming gene therapy and chemotherapy combination as a unique and considerable approach to brain tumor treatment, which should be further explored  induced apoptosis and reduced proliferation in a panel of NB cell lines tivation . The idetivation .via specific MET inhibition. To address the therapeutic efficacy of highly selective MET inhibitors, a highly selective MET kinase inhibitor, SAR125844, was tested in a phase I trial in patients with advanced solid tumors that harbored MET amplification. The maximum tolerated dose (MTD) was determined to be 570 mg/m2 administered once a week and showed significant anti-tumor activity in MET-amplified non-small cell lung carcinoma patients  was tested in a phase I trial of patients with advanced solid tumors. AMG 337 was well tolerated with an MTD of 300 mg administered once daily that showed promising response rate in patient with MET-amplified solid tumors . Moreovein vivo and in vitro activity of altiratinib, which inhibits MET/TIE2/VEGFR2 in human GSCs. Using GSCs (GSC17 and GSC267), altiratinib was shown to effectively reduce HGF-stimulated MET phosphorylation, which significantly reduced cell viability of GSC lines in vitro. More importantly, tumor volume and invasive potential were significantly reduced when altiratinib was combined with bevacizumab in an in vivo, xenograft model. In addition, the protein expression of mesenchymal markers as well as microvessel density, and TIE2-expressing monocyte infiltration were all significantly reduced after combination therapy compared with single therapy with bevacizumab. Furthermore, to determine the in vivo effect of combined therapy using altiratinib and bevacizumab in the GSC17 xenograft model, the authors reported significant increase in animal survival compared with bevacizumab mono-therapy , and AKT1 via TNF. This analysis revealed highly tumorigenic, pro-angiogenic, pro-invasive/pro-metastatic/pro-proliferative/anti-apoptotic, and stem cell pluripotency pathways. Therefore, while adult and pediatric brain cancers do differ, the common players that cross-talk with HGF/MET are potential avenues for further investigation in order to delineate the cooperative or synergistic interactions between the said molecules in pediatric brain cancers and develop new treatment approaches. Future attempts at therapeutic and genetic intervention should consider the simultaneous, multi-modality targeted approaches to reach better prognostic outcomes in children with these aggressive tumors. As illustrated in While the majority of these studies were conducted on adult GBM models, and pediatric brain cancers differ vastly from adult brain cancers, there are common targets between the two. For example, while pediatric brain cancers are largely driven by epigenetic dysregulation and micro-environmental influences exert a greater effect on adult brain cancers, common molecular drivers between the two include MYCN, NOTCH, PI3K/AKT/MAPK/mTOR, WNT/−catenin, and SHH. In addition, common epigenetic aberrations between adult and pediatric brain cancers include SOX2, BMI1, EZH2, FOXG1, and STAT3 , which aWhile a plethora of pre-clinical studies have shown the efficacy of combined therapy to target various signaling pathways in nervous system tumors, acquired resistance to tyrosine kinase inhibitors or radiotherapy remain a hurdle in therapeutic intervention. Often the targeting of one signaling cascade in cancers was shown to induce the upregulation and activation of yet another tumorigenic pathway in a compensatory fashion. Examples include upregulation and activation of MAPK after mTOR inhibition in melanoma ; activatAn alternative approach to c-Met-targeted treatment was the downregulation of the c-Met-induced DNA repair pathway to allow improved effect of chemotherapeutic agents. A more recent investigation revealed that metabolic reprograming may lead to therapeutic resistance to c-Met-targeted therapy observed in clinical trials. More importantly, the authors were able to suppress this response to c-Met inhibition (using crizotinib) by combining it with the potent mitochondrial matrix chaperone inhibitor, gamitrinib. The combination therapy reduced oxidative stress and led to potent anti-tumor activity evidenced by synergistic cell death compared with single-dose treatment with either compound alone . This unApplying c-Met inhibition on pediatric cancers has been slightly more challenging. Malignant nervous system tumors in children are devastating diseases with current therapies being largely ineffective, possibly due to the highly invasive, treatment-resistant, and recurrence-prone stem cell sub-populations within the bulk tumors. Epigenetic dysregulation, aberrant signaling, therapeutic resistance, and the intricate interplay between c-Met and other tumorigenic drivers may fuel the aggressive nature of these cancers . The abiTA-A conceptualized the study, designed the figures, and wrote the original draft of the study. TA-A and AK reviewed and initially screened the literature and performed the revisions and editing on the final manuscript. Both the authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Coral concrete has low cost and convenient materials, making it an excellent raw material for processing. However, its lower strength limits the application of coral concrete. Surface modification is expected to increase the properties of porous coral concrete. In this study, single and compound modification treatments were applied to the surface of a coral aggregate to improve its properties for promoting the mechanical performance of coral concrete. The results showed that the micro-aggregate effect and pozzolanic activity of granulated blast furnace slag (GBFS) and the permeability and polycondensation of sodium silicate (SS) could be mutually promoted. The GBFS and SS could effectively fill the pores of the coral aggregate, enhancing the properties of the aggregate, such as density and load-bearing capacity, and reducing the water absorption and crushing index by more than 50%. GBFS and SS could intensify and accelerate the hydration of cement, and generate a large number of hard hydration products at the interfacial transition zone (ITZ), which could strengthen the bonding between the aggregate and mortar, improving the strength of the ITZ. The compressive strength of the coral concrete was significantly increased. The utilization of efflorescent coral aggregates can effectively save both time and expense regarding the transportation of aggregates from inland to the island. It can also successfully avoid the issues of inconvenient aggregate stacking and storage, construction constraints, and construction difficulties due to the narrow space on a sea island . HoweverTo improve the strength of coral concrete, most researchers have been concentrating on changing the amount or type of cementitious material components. In order to improve the strength of coral concrete, researchers usually adopt methods such as adding auxiliary cementing materials, reducing the water-to-binder ratio, and adding fibers ,4. The mBacillus Pasteurii) bacteria for the surface treatment of recycled aggregate to precipitate calcium carbonate under specific conditions and improve the performance of recycled aggregate.Many researchers used inorganic modification , organic2 emissions. This study mainly chose granulated blast furnace slag (GBFS) and sodium silicate (SS) as surface modifiers. The GBFS-slurry-impregnated coral aggregate can effectively physically block the pores of coral aggregate, effectively reduce the water absorption rate of coral aggregate, and improve the crushing performance of coral aggregate. At the same time, the reduction in coral aggregate water absorption can effectively reduce construction water use, reduce the water–cement ratio, and enhance the concrete strength. In addition, the GBFS slurry that is attached to the surface of the coral aggregate can chemically react with the cement hydration product, which can promote the cement hydration reaction and increase the strength of the interface between the aggregate and the cement slurry. Sodium silicate solution impregnates coral aggregates, and after solidification, it can also block the pores of coral aggregates to a certain extent and improve the basic performance of the aggregates. At the same time, the water glass that is attached to the coral aggregate can effectively promote the hydration of the cement and enhance the interface strength of the coral aggregate.It was found in the review of previous research that few studies took into account the surface modification of the coral aggregate; furthermore, there is no report on the compound modification of coral aggregate. Applying a surface modification approach instead of increasing the dosage of cement can significantly reduce the cost of preparing the concrete and the COIn this study, three kinds of treatment methods, namely, granulated blast furnace slag (GBFS) slurry steeping, sodium silicate (SS) impregnated, and GBFS and SS compound modification were used for the surface treatment of the coral aggregate to investigate the change rule of the crushing index and water absorption of the coral aggregate under different modification conditions. Comparisons of the concrete slump, microhardness of the ITZ, and compressive strength of the concrete with original coral aggregate and modified coral aggregate were performed. The micro-morphologies of untreated and treated coral aggregate and coral concrete were identified using SEM. The strengthening mechanisms of aggregate and concrete under different modification conditions were analyzed and compared.The raw materials that were used in this study were ordinary Portland cement (OPC) (P.O 42.5R), GBFS, SS, coarse coral aggregates, fine coral aggregates, and a superplasticizer. The chemical compositions of the OPC and GBFS are presented in The coral aggregates that were used in this study were collected from an oceanic island in China. The coarse coral aggregates were screened with an electric sieve shaker, which had nominal particle sizes of 5–20 mm. The concrete was prepared using the mechanical mixing method as follows: First, the cement, coarse aggregate, and fine aggregate were mixed evenly and stirred for 3 min; at the same time, the water-reducing agent and water were mixed evenly; then, the water-reducing agent mixture was added while stirring, and stirred for 2 min; finally, the mixture was stirred for 5 min to form the concrete. The rotation speed was 60–80 r/min during stirring. After the mixing was completed, it was poured into the mold, and a concrete shaker was used to form (vibration frequency was 50 ± 3 Hz) and perform standard curing after vibrating. The water temperature was 20–30 °C. After pouring into the sample, it was cured for 1 day to remove the mold and then cured for 28 days to test the performance strength of the sample.m0; the sample was then sieved using a square screen with an aperture of 2.36 mm and the mass was recorded as m1.According to the Chinese National Standard GB/T 14685-2011 , 3 kg agσ (%) represents the crushing indicator of the coarse coral aggregates, m0 (g) is the sample mass, and m1 (g) is the sample mass after crushing and screening. The arithmetic mean value of three measured values was adopted as the test result.The crushing indicator of the coral aggregates could be calculated using Equation (1):The coral aggregates have a strong influence on the flowability and workability of concrete. The saturated surface-dry water absorption of coarse coral aggregates was measured ,25. WhenmW (%) represents the water absorption of the coarse coral aggregate, G0 (g) is the sample mass in the saturated surface-dry state, and G1 (g) is the dried sample mass. The arithmetic mean value of three measured values was adopted as the test result.The water absorption of the samples can be calculated using Equation (2) (accurate to 0.1%):The coral concrete slump test was performed according to the Chinese National Standard GB/T 50080-2002 . Each weThe hardness of the interface between the aggregate and cement stone is a direct reflection of the combination of the aggregate and cement stone. An HV-1000 semi-automatic microhardness testing system was employed to measure the hardness of the interface between the coral aggregate and cement stone before and after modification. The sample was collected from the interface between the coral aggregate and cement stone in the sample concrete block after 28 days of standard maintenance. It was first soaked in absolute ethanol for 1 day to prevent hydration and cut into a 40 mm × 40 mm × 10 mm specimen. The prepared specimen was dried to a constant weight. The top and bottom surfaces of the specimen were then polished. The contact surface between the aggregate and cement stone was set as the zero-point to measure the internal aggregate and the basal body of the cement stone every 20 μm (220 μm in length). The measurement of each test point was repeated three to five times, and the mean value of the measured values was considered to be the microhardness value of the test point.The compressive strength test was conducted using a microcomputer-controlled electro-hydraulic servo universal testing machine according to Chinese National Standard GB/T 50081-2002 . The samA scanning electron microscope was employed to observe the microscopic structures of the coral aggregates and coral concrete before and after modification, especially the interface between the aggregates and mortar. The voltage was 12 kV and the working distance was about 12 mm.For the GBFS slurry steeping, the coarse coral aggregate was steeped in 5, 10, and 20% GBFS slurry. The aggregate during the steeping process was not superimposed. The water surface was 10 mm higher than the upper layer of the aggregate. For the SS impregnation, the coarse coral aggregate was impregnated in 5, 10, and 20% SS solution. For the GBFS–SS compound modification, the aggregate was first steeped in GBFS slurry for a fixed time and then impregnated in SS solution for a certain time. The specific mixing and soaking parameters were as follows:(1)Modification of the granulated blast furnace slag solutionThe granulated blast furnace slag was mixed with water according to a certain mass percentage to form a certain concentration of granulated blast furnace slag solution. Then, the coral aggregate was added to the granulated blast furnace slag solution, stirred using mechanical stirring at a speed of 50–100 r/min for 1 h, and then left to stand for 12 h. After standing still, the coral aggregate was taken out and placed in a blast drying oven at 100 ± 5 °C to dry to a constant weight.(2)Modification of the sodium silicate solutionA certain mass percentage of sodium silicate solution was prepared and the coral aggregate was put into the sodium silicate solution and left to stand for 5 h. After reaching the soaking time, it was removed and dried in a blast-drying box at 100 ± 5 °C to a constant weight.(3)Compound modificationFirst, the coral aggregate was put into the granulated blast furnace slag solution, stirred using mechanical stirring at a speed of 50–100 r/min for 1 h, and then left to stand for 12 h. Then, the coral aggregate was taken out and placed in the sodium silicate solution to stand for 5 h. Finally, it was taken out and dried to a constant weight at 100 ± 5 °C in a blast-drying oven.Both the GBFS and sodium silicate solutions were calculated using the mass percentage.Compared with the untreated coral aggregate with the polyporous and non-compacted structure pictured in The change in the macroscopic crushing index exhibited the improvement of the aggregate’s microstructure properties. It can be seen from 2 in the air, generating silica sol:It is worth noting, however, that SS can have a hardening reaction through self-polycondensation. It is slow without external promotion. Nevertheless, SS can also be hardened by COMoreover, the hardening reaction is also faster than the self polycondensation of SS ,34. On tAdditionally, an optimal SS solution concentration and treating time were observed from the experimental results. Overall, the 10% SS solution had a better reduction effect on the water absorption of aggregate compared to the other two SS solutions used in the experiment. The optimal steeping time was 5 h.The two single inorganic modification methods demonstrated effective respective utilities regarding improving the properties of the coral aggregate and concrete. It was notable that the GBFS treatment had a better reduction effect on the crushing index than that SS treatment. In contrast, the SS treatment had a better reduction effect on the water absorption than the GBFS treatment. In this section, based on their complementary performances, the effects of the GBFS–SS compound modification on the properties of the coral aggregate were investigated. The aggregates were first steeped in the GBFS slurry for 5 h, and then they were impregnated in the SS solution for another 5 h. 2+, Si4+, Al3+, and SiO44− are rapidly dissolved and form a C-S-H gel after a series of polymerization reactions [2+ in GBFS can promote the hydration process of the adhesive film of SS [The substantial reduction in water absorption and crushing index of the modified aggregate was mainly due to the chemical reaction between the compound materials. The active substance of GBFS can be activated in a high-alkali environment that can be generated from an SS solution on account of its pozzolanic performance ,36. The eactions ,38. The eactions . During eactions . On the lm of SS ,41, wherThus, more and more “adhesive film” is attached to the surface of the aggregate to block water infiltration. The compound modification with GBFS slurry and SS solution had the effect of mutual promotion. The best modification condition in our present testing was first steeping the aggregate in 20% GBFS slurry for 5 h, then steeping it in 10% SS solution for another 5 h, and finally drying it to a constant weight.Under the same mix proportion, the performances of the ordinary concrete, unmodified coral concrete, and coral concrete with different modification treatments were studied. It can be seen from x-axis “0” is the position of the interface between the concrete aggregate and the cement paste, the positive axis represents the cement paste, and the negative axis is the concrete aggregate. Due to the high hardness of ordinary concrete aggregates, the microhardness was slightly higher than the interface hardness and the hardness of cement paste. However, due to the lower hardness of the coral aggregates and the lower interface hardness, the microhardness value in the negative range of the x-axis showed a downward trend.Hardness is an important mechanical property for the study of materials, which reflects the ability of the material to resist local plastic deformation and the ability of the tested object to resist pressure from another hard object. The microhardness test of an ITZ is an important indicator that can characterize the hardness of an ITZ of concrete and the strength of a crystal component. 2O3 and SiO2 of GBFS particles were attached to the aggregate surface. They could react with the calcium hydroxide generated by the hydration of cement, which promoted the hydration process, strengthening the interface strength [It can be seen from strength . For thestrength ,46:(8)NaIt promoted the hydration of cement and produced a hydraulic calcium silicate colloid, which improved the mechanical occlusion of the coral aggregate and cement, enhancing the interface strength . For theThe micro-morphologies of the unmodified coral concrete and coral concrete with compound modification (in the A4 condition) are depicted in 2+, in the GBFS promoted SS polycondensation to form a film. The two promoted each other and significantly enhanced the filling effect of the aggregate pores.In this study, GBFS slurry, SS solution modification, and GBFS–SS compound modifications were used for the surface treatment of a coral aggregate to study the changes in properties of the coral aggregate and coral concrete. The results indicated that GBFS slurry had no significant reduction effect on the water absorption of the aggregate, and it could significantly reduce the crushing index. In contrast, the SS solution had significantly reduced the water absorption, and it had no significant impact on the reduction of the crushing index. There existed an optimal steeping time, above which a further increase in time had a negative effect. Through the GBFS–SS compound modification method, the water absorption and crushing index of the coral aggregate were greatly reduced. The water absorption and crushing index were decreased by 55.56 and 56.25%, respectively. The slump of coral concrete with the GBFS–SS modification treatment was increased on account of the effective filling of the pores of the aggregate. The compressive strength of such concrete was 26.88% higher than that of the untreated coral concrete and was only 2.2 MPa lower than that of ordinary concrete under the same mix proportion. The reason for this phenomenon was that the SS stimulated the active substance in GBFS to depolymerize and polymerize to form a C-S-H gel, and metal ions, such as Mg


GRIN3B (p = 0.021), rs3983721 in GRIN3A (p = 0.00326), rs2129575 in TPH2 (p = 0.033), rs6583954 in CYP2C19 (p = 0.033), and rs174699 in COMT (p = 0.036). These were all associated with heroin-using and craving scores with and without adjustments for age, sex, and body mass index. We combined five variants, and the ensuing dose-response effect indicated that heroin-craving scores increased with the numbers of risk alleles (p for trend = 0.0008). These findings will likely help us to understand the genetic mechanism of craving, which will help in predicting the risk of relapse in clinical practice and the potential for therapies to target craving in heroin addiction.Addiction is characterized by drug-craving, compulsive drug-taking, and relapse, and results from the interaction between multiple genetic and environmental factors. Reward pathways play an important role in mediating drug-seeking and drug-taking behaviors, and relapse. The objective of this study was to identify heroin addicts who carry specific genetic variants in their dopaminergic reward systems. A total of 326 heroin-dependent patients undergoing methadone maintenance therapy (MMT) were recruited from the Addiction Center of the China Medical University Hospital. A heroin-use and craving questionnaire was used to evaluate the urge for heroin, the daily or weekly frequency of heroin usage, daily life disturbance, anxiety, and the ability to overcome heroin use. A general linear regression model was used to assess the associations of genetic polymorphisms in one’s dopaminergic reward system with heroin-use and craving scores. Results: The most significant results were obtained for rs2240158 in  Heroin addiction, a chronic relapsing disease characterized by compulsive drug-seeking, drug abuse, tolerance, and physical dependence, is a major public health concern worldwide . It is aCOMT) [POMC), tryptophan hydroxylase 2 (TPH2), and brain-derived neurotrophic factor (BDNF) [OPRK1, OPRL1, PDYN, cannabinoid receptor 1 [COMT), tryptophan hydroxylases 1 (TPH1 and 2 TPH2), methadone-metabolizing enzymes , and cognitive function-related genes . Previous linkage and association studies of polymorphisms have reported several genetic variants associated with drug abuse, addiction, and related phenotypes ,6. TheseCOMT) ,11, proor (BDNF) . Physiolr (BDNF) . To idenCraving is an integral symptom that is considered to be central to the motivational drive in heroin addiction . We explThe study protocol was reviewed and approved by the Institutional Review Board of China Medical University Hospital (CMUH) (DMR101-IRB1-218) and was in compliance with the Declaration of Helsinki. Informed consent was obtained from all subjects involved in the study. In total, 316 heroin-dependent patients undergoing methadone maintenance therapy (MMT) were recruited from the Addiction Center of the CMUH. As assessed by senior psychiatrists experienced in heroin dependence, all the recruited patients met the criteria from the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, for heroin abuse and dependence, and had been receiving MMT for at least 6 months at a dosage that had been unchanged for at least 4 weeks at the CMUH. We selected from the extreme margin of the specific phenotype range  to maximize the power of the study. For each patient, the following clinical information was recorded: sex, weight (in kg), height (in cm), liver function test results, comorbidities, and daily methadone dose.We designed a 14-item heroin-use and craving (HUC) questionnaire to evaluate the behavior of heroin users undergoing MMT. Each question was scored from 0 to 4. The questionnaire was divided into two parts: Part I , assessing the urge for heroin and whether the individual can shift their attention away from heroin, and Part II , investigating the daily or weekly frequency of heroin usage, daily life disturbance, anxiety, and ability to overcome heroin use. In essence, the higher the score, the more severe the heroin craving was. The score of each item was calculated according to the manual.OPRK1, OPRL1, PDYN, CNR1, COMT, TPH1, TPH2, CYP1A2, CYP2B6, CYP2C19, GRIN3A, GRIN3B, and GRM6. Single nucleotide polymorphism (SNPs) with minor allele frequency >0.05 were selected on the basis of the previous findings and population data in the SNP databases of the National Center for Biotechnology Information (Han Chinese) in Beijing, China. In total, 44 SNPs were genotyped at the National Center for Genome Medicine, Taiwan, by using Sequenom iPLEX matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry technology. DNA was extracted from 3 to 10 mL of whole blood using a QIAamp DNA Blood Mini Kit  according to the manufacturer’s protocol. Duplicate samples were randomly selected for quality control, and the concordance rate was >0.99 for all SNPs assayed.This study focused on the 13 aforementioned genes encoding various metabolic components: t test for continuous variables in both sexes. Single SNP association analyses were conducted with a general linear model, under dominant or recessive model assumptions. We performed QTL mapping for HUC. The general linear model was used to assess associations of genetic polymorphisms in the reward pathway with heroin-use and craving scores, making adjustments for sex, age, and body mass index (BMI). Each SNP genotype frequency distribution was examined for Hardy-Weinberg equilibrium using the chi-square one degree of freedom goodness-of-fit test. Demographic characteristics and clinical parameters were evaluated with a chi-squared contingency table for categorical variables and a The demographic details and heroin use history of the subjects are summarized in GRIN3B, rs3983721 in GRIN3A, rs2129575 in TPH2, rs174699 in COMT and rs6583954 in CYP2C19) were significantly associated with HUC questionnaire scores. Two SNPs (rs174699 in COMT and rs6583954 in CYP2C19) were significantly associated with urge for heroin. Another two SNPs (rs2240158 in GRIN3B and rs3983721 in GRIN3A) were significantly associated with ability to overcome heroin use . The genetic risk allele scores (combined gene effects) were significantly associated with HUC questionnaire scores, urges for heroin, and ability to overcome heroin use . We found that five SNPs were significantly associated with HUC questionnaire score, as well as the urge for heroin and the ability to overcome heroin use; in addition, GRIN3A, GRIN3B, CYP2C19, TPH2, and COMT, contributing to susceptibility to heroin addiction. All the variants identified are from non-coding regions. As expected for a complex genetic disorder, each variant was shown to have a small effect on the risk of heroin addiction. The R-squared ranged from 3% to 5% for each genetic variant. This explained a variance in the combined effects of multiple genes on heroin-using and craving scores, and ability to overcome heroin use, of 6.3%  receptors, which belong to the superfamily of glutamate-regulated ion channels, and are involved in the physiological and pathological processes of the central nervous system. Some genetic variations in the glutamatergic system have been reported to contribute to vulnerability to drug addiction [GRIN3A and GRIN3B may play roles in heroin addiction, acting as dominant-negative modulators of NMDA receptors.The variants in the ehaviors ,18,19. Wddiction ,20. GRINTPH2 have previously been associated with heroin addiction in African Americans and in Hispanics, and are also associated with phenotypes related to smoking status, neuropsychiatric disorders, higher reward dependence, and personality traits [Several variants in y traits ,21. TPH COMT gene is associated with urge for heroin, which plays a critical role in heroin dependence [COMT gene SNP rs174699 was associated with urge for heroin. Individuals with the CC genotype would thus be expected to exhibit a stronger urge for heroin use than individuals with the CT or TT genotypes. COMT plays an important role in the regulation of synaptic dopamine levels, and in the reinforcement mechanism of drug dependence. The COMT SNP rs174699 was selected as a possible marker of genetic predisposition to addiction.Our findings indicate that the pendence ,22. The CYP2C19 gene encodes a CYP450 enzyme that contributes to methadone metabolism, dependent on treatment dose, plasma concentration, and the side effects of methadone [CYP2C19 gene SNP rs6583954 was found to be associated with heroin-using and craving score, and the ability to overcome heroin use. Individuals with the T allele would thus be expected to exhibit a stronger ability to overcome heroin use than individuals with the C allele. Variations in the CYP2C19 gene may influence the steady-state plasma concentrations of methadone and the plasma concentrations of methadone [CYP2C19 gene may potentially serve as an indicator for ability to overcome heroin use.The ethadone . Methadoethadone , while tThere are some limitations in this study that need to be considered. The results were compared to those of previous studies. Some variants from previous reports were not genotyped, and the phenotype and population were often different. We cannot determine if the negative findings in this study are true negatives, because the SNP coverage of some of the genes may be insufficient to exclude real associations. Based on known or inferred biologically functional genes, we used a candidate gene approach that allows for scanning a limited number of SNPs and is limited by current knowledge. Our study had only limited phenotypic and genotypic information. It is thus necessary to genotype more novel susceptibility genes identified by genome-wide association studies (GWAS) ,25,26,27This study suggests an extension to the list of susceptibility genes and variants underlying heroin addiction. Although the variants identified in this study are suggested for assessing the degree of urge for heroin and the ability to overcome heroin use, they may uncover a genetic basis for craving and heroin-addictive behavior. These findings will likely help us understand the genetic mechanism of craving, which will be helpful in predicting the risk of relapse in clinical practice as well as the potential applicability of therapies for targeting craving in heroin addiction.


Since 2017, an increasing number of opioid overdoses in Oslo, Norway, has been categorized as involving unspecified opioids, as noted in the patient records by the doctor treating the patient. In this study we compare the characteristics of overdoses involving unspecified opioids, long-acting opioids, and heroin. Data on patients presenting with opioid overdose was retrospectively collected from 1 October 2013 to 31 December 2019 at the Oslo Accident and Emergency Outpatient Clinic.Among 2381 included cases, 459 (19.3%) involved unspecified opioids, 134 (5.6%) long-acting opioids, and 1788 (75.1%) heroin. Overdoses involving unspecified opioids needed longer observation, median 5 h 29 min vs. 4 h 54 min (long-acting opioids) and 4 h 49 min (heroin) (p < 0.001), and had a lower Glasgow coma scale score, median 10 vs. 13 in both the other groups (p < 0.001). Naloxone was given in 23.3% of cases involving unspecified opioids, vs. 12.7% involving long-acting opioids and 30.2% involving heroin (p < 0.001). A larger proportion of patients were transferred to hospital care when unspecified or long-acting opioids were involved compared to heroin, 16.3% and 18.7% respectively vs. 10.1% (p < 0.001). Our results indicate that the category “unspecified opioids” encompasses a substantial proportion of opioids acting longer than heroin. In Europe there were 1.3 million high-risk opioid users in 2018, and opioids were involved in 82% of drug overdose deaths [The main danger of opioid overdose is respiratory depression , 5. NaloOpioids were involved in 83% of the 275 registered drug overdose deaths in Norway in 2019 . If thisData on patients presenting with opioid overdose at the OAEOC was retrospectively collected from 1 October 2013 to 31 December 2019, using the case definition and data registration tool developed by the European Drug Emergencies Network (Euro-DEN) . We inclThe diagnosis of drugs taken was based on the clinical assessment noted in the patient records by the doctor treating the patient, in its turn based on the clinical presentation and all available information from the patient and the patient’s companions. The category “unspecified opioid” is used by OAEOC doctors when the patient has an obvious opioid toxidrome (miosis and reduced level of consciousness) and there is no specific information on which opioids have been taken. The OAEOC has limited diagnostic and treatment resources available, and patients needing more intensive treatment are transferred to hospital care . ArteriaAmong 5236 opioid overdoses in the inclusion period, 2808 were excluded as other drugs or ethanol also had been taken. The included cases were categorized as involving unspecified opioids (only), long-acting opioids , and heroin (only). Another 47 overdoses with opioids or combinations of opioids not categorizable into these three groups were excluded.The three opioid groups were compared on observation time, Glasgow Coma Scale (GCS), vital signs, naloxone administration, and transfer to hospital. Statistical analyses were done using SPSS 27. Continuous variables were compared using Kruskal–Wallis test. Categorical variables were compared using chi-square test, or Fisher’s exact test when > 20% of the cells had an expected count < 5.In total 2381 opioid overdoses were included. Median age among the patients was 38 years (interquartile range 31–46), 1892 (79.5%) were males. There were 459 (19.3%) cases involving unspecified opioids, 134 (5.6%) involving long-acting opioids, and 1788 (75.1%) involving heroin  and 4 h 49 min (heroin) p < 0.001), and lower GCS than both other groups, median 13 vs. 14 at presentation (p < 0.001) and 10 vs. 13 at the lowest (p < 0.001) (Table , and lowPatients treated for opioid overdose categorized as involving unspecified opioids had a lower level of consciousness and needed longer observation time than other opioid overdose patients, and as many were transferred from outpatient care to hospital admission as when long-acting opioids were involved. This indicates that the category of unspecified opioids among opioid overdoses in Oslo, Norway, encompasses a substantial proportion of opioids acting longer than heroin. The increase in the number of overdoses involving opioids other than heroin is in line with trends seen both elsewhere in Europe and in the USA –3 and alThe increase in the number of overdoses involving opioids acting longer than heroin also underscores the importance of the recommended two-hour observation following naloxone administration. Patients overdosing on longer acting opioids have a greater risk of recurrent respiratory depression following naloxone treatment and may become in need of repeated naloxone administration. Hence, they should be observed long enough for this potential need to have manifested itself . To avoiThe study was based on clinical diagnosis of toxic agents. Toxicological laboratory analyses could have provided specific information on which opioids constitute the category of unspecified opioids. However, toxicological laboratory analyses are not regularly done at the OAEOC and were not available to us. Though the opioid toxidrome with miosis and reduced level of consciousness is easily recognizable, the lack of laboratory support for the diagnosis of specific opioids is a limitation in our study.It is possible that some patients were observed longer because they had recurring respiratory depression. However, we did not have information on respiratory function over time. It is highly likely that patients with recurring respiratory depression also had prolonged or recurring reduced level of consciousness, which in itself would lead to longer observation. Hence, we do not think that the lack of this information alters the interpretation of our findings.Combinations of heroin and undiagnosed benzodiazepines could also be an explanation for the longer observation time seen for unspecified opioids. In a study done at the OAEOC in 2015 with toxicological analysis of saliva samples from patients with recreational drug overdose, benzodiazepines were found in more than half the patients, though not clinically suspected . In the


The test results revealed a slight decrease in the workability of the fibre-reinforced concrete, while all the mechanical and microstructural properties were enhanced significantly. It was observed that the compressive, splitting tensile and bonding strength of the concrete with 6 kg/m3 fibre dosage increased by 19.4%, 41.9% and 17.8% compared to the plain concrete specimens, respectively. Although there was no impact of the fibres on the modulus of rupture, they significantly increased the toughness, resulting in a progressive type of failure instead of the sudden and brittle type. Moreover, the macroporosity was reduced by the fibre addition, thus increasing the concrete compressive strength. Finally, simplified empirical formulas were developed to predict the mechanical properties of the concrete with fibre addition. The outcome of this study will help to increase the implementation of the recycled plastic waste in concrete mix design and promote a circular economy in the waste industry.Adding fibers to concrete helps enhance its tensile strength and ductility. Synthetic fibres are preferable to steel ones which suffer from corrosion that reduces their functionality with time. More consideration is given to synthetic fibres as they can be sourced from waste plastics and their incorporation in concrete is considered a new recycling pathway. Thus, this work investigates the potential engineering benefits of a pioneering application using extruded macro polyfibres in concrete. Two different fiber dosages, 4 kg/m Concrete is the most widely used material for constructing commercial and industrial infrastructure. Despite showing high strength in compression, it has very low tensile strength, which is why it is reinforced with steel bars. The steel reinforcing bars carry the tensile stress and provide more ductility and strength to concrete structures. However, steel reinforcements will eventually corrode and expand, which degrades their functionality and causes microcracks in the concrete affecting the durability of the concrete itself. Corrosion is thus a major concern in the construction industry as in many instances, contractors are unable to maintain the minimum cover required to inhibit further corrosion ,2. MoreoSteel fibres improve the compressive, tensile and flexural strength of concrete due to their high energy absorption and ability to control crack propagation ,5,6. HowKumar and Naik  examinedThe structural behaviour of concrete containing synthetic fibre under cyclic loading was investigated by Ghosni et al.  with the3 and 6 kg/m3 PP fibre dosages added to the control mix were conducted to obtain a better understanding on the effect of PP fibres on physical and mechanical properties  test, and pull-out test and bond strength. Further investigation was conducted on the effect of PP fibres on the microstructure and porosity. This research will advance the knowledge and help to promote the use of macro poly fibre in some civil engineering applications.There has been significant progress in the knowledge of using different fibre lengths and fiber ratios for specific physical and mechanical properties. Despite this, none of the previous studies have examined the overall impact of macro polypropylene fibres on concrete properties. Therefore, the study investigated various mechanical. The novelty of the study is to introduce a low-cost fibre system in concrete and to understand how will it affect the mechanical properties. Thus, in this study a comprehensive experimental test considering the use of 4 kg/mThe methods used to select the aggregate for the experiments in this study are specified in Australian Standard (AS 1726-1994) . The moiMacro polyfibres  are a macro synthetic polypropylene fibre (PP) type designed and manufactured in a novel continuously deformed shape to achieve high performance and perfect anchorage to the concrete making them more structurally compliant, as shown in The concrete was prepared based on AS 1012.2 ; the tarSlump tests of the three batches were carefully performed to ensure the same conditions when measuring the drop of the fresh concrete. All other constituents were kept at the same quantity to obtain accurate slump results. A slump test was carried out at the USQ laboratory according to AS 1012.3.1 as shown in 3 to 6 kg/m3) [3, 4 kg/m3 and 6 kg/m3 of macro polyfibre dosages were carried out. These tests followed the procedures prescribed in AS-1012.9 [Concrete with fibre volume fractions of 1% to 2% is considered ideal  . Higher S-1012.9 . The speThe modulus of elasticity test was carried out after 28-days for concrete with different fibre dosages. A compressometer was used to measure the axial shortening of the concrete specimen with the increase of compression load. The readings were taken from the dial gauge for every 10 kN load increment, and the modulus of elasticity was calculated according to AS 1774.31.2 .Flexural tests were conducted using the 500 kN SANS machine shown in The concrete beams reinforced with and without fibres have been tested to investigate load–deflection as well as crack mouth opening displacement (CMOD) behaviour. This test has been conducted to determine the effect of the fibre addition on the crack initiation and propagation as well as the post-cracking behaviour. A notch placed at the bottom part of the beam was the main difference between conventional flexural test and the CMOD test to accurately capture the first and main crack. To create the notch, a 3D printed plastic strip see a with 2 A microscope  was used to examine the microstructure and measure the pores diameter at the internal fracture surface of the concrete cylinders. Moreover, the porosity of tested specimens was analysed using the Gwyddion software .3 and 6 kg/m3 concrete dropped by 2.7% and 8.1% respectively compared to the normal mix of concrete with 0 kg/m3 of fibre. The specimen with 6 kg/m3 recorded a reduction in workability by 8.1% compared to the specimen without fibres. Due to larger surface area, fibers need to adsorb a lot of cement paste to wrap around them, which increases the viscosity of the mixture. Fibres form a network structure in the concrete which is restraining the fresh concrete from flow [rom flow ,36,37.3 shows splitting cracks along the height and localised crushing at the top/bottom ends of the cylinders  is lower than that of concrete (around 2500 kg/m3) which negatively affects the total modulus of elasticity. Nevertheless, the volume of the added fibres was less than 0.7% at maximum, which showed insignificant reduction to the modulus of elasticity value as evidenced by the unchanged in the axial compression stiffness in The effect of macro polyfibre on the modulus of elasticity has been investigated, which is given by the slope of the stress-strain curve within the proportional limit of the concrete, as shown in 3, 4 kg/m3, and 6 kg/m3 of fibre dosage are presented in 3 of fibre showed the highest 3 and 0 kg/m3, respectively. This is due to the role of the PP fibres to uniformly distribute the internal stresses preventing the initiation of the cracks and reinforcing them once initiated against propagation. This led to an increase in the tensile resistance of the concrete. The results show that the increase in the fibre content from 4 kg/m3 to 6 kg/m3 achieved a higher increase rate of 3 to 4 kg/m3. This could be due to the increase of the volume occupation by adding 6 kg/m3 fibre dosage which reinforced more volume in the tri-axial space of the tested specimens leading to increase the The impact of macro polyfibres on the splitting-tensile strength has been investigated in this study. The test results of the splitting-tensile strength  is due to the high strength PP fibres used in this study (550 MPa), while the ordinary behaviour of the PP fibre-reinforced beams is different by showing gradual decrease in the load resistance until the final failure. Fibres have the ability to resist shear across cracks, an enhanced residual strength, and enhanced energy dissipation [All the concrete beams reinforced with and without macro PP fibres were tested until failure to determine the influence of the fibres on the flexural behaviour including peak flexural strength  of a structure by showing strain hardening behaviour . This isIn Equations (2) and (3), Flexural strength (modulus of rupture):Energy absorption (toughness):3, 4 kg/m3 and 6 kg/m3 of macro polyfibre. It can be seen that the concrete beam without fibres showed no cracking until reaching around 2 mm deflection. Then a crack appeared and suddenly propagated to the topmost height of the beam leading to split the beam into two pieces. On the other hand, the beams with 4 kg/m3 and 6 kg/m3 of fibre dosage showed crack initiation almost at the same deflection (2 mm) , where the slope value in this equation was the average of the two slope values of both lines. Interestingly, the slope value (0.765) in Equation (4), proposed by Ding [ by Ding  for stee3, 4 kg/m3 and 6 kg/m3) and three steel bar diameters . In 3 fibre dosage showed bar rupture  and massive concrete cracking in the case of 16 mm and 20 mm bar diameter. It was observed a number of PP fibres tying the cracked concrete, as seen in  rupture b, when u rupture c,d, when3 fibre dosage to the concrete specimens allowed the 12 mm steel bar to reach its ultimate strain limit and show complete elastic-plastic pseudo behaviour, as shown in 3 fibre dosage  which agreed with the microscope observation. Increasing the amount of fibres decreased the macroporosity, A previous study conducted by Al-Harthi, Al-Amri [ Al-Amri  conclude Al-Amri  indicate3 to 6 kg/m3 slightly reduced the workability of the fresh concrete mix (up to 8.1%) due to the network structure formation in the concrete by the fibres which restrains the fresh concrete from flow.Increasing the fibre dose from 4 kg/m3 and 6 kg/m3 fibre dosages increased the compressive strength by 9.6% and 19.4% compared to the plain concrete, respectively. However, fibre addition has no significant effect on the modulus of elasticity value of the concrete due to the low volume of the added fibres by less than 0.7%Macro polyfibres changed the mode of failure of the concrete cylinders under compression from longitudinal splitting to shear failure. Moreover, the inclusion of the 4 kg/m3 and 6 kg/m3 fibre dosages to the concrete enhanced the splitting-tensile strength by 28.7% and 41.9%, respectively. This is due to the uniformly distribute of fibres which resist the internal tensile stresses as well as blocking the initiation of the cracks and reinforcing them once initiated against propagation.Adding 4 kg/m3 fibre dosage attained 228% and 857% increase in the toughness compared to the beams with 4 kg/m3 and 0 kg/m3 fibre dosage, respectively. Moreover, the fibre addition changed the final failure from sudden and brittle to progressive failure.The fibre addition has no influence on the modulus of rupture of the concrete specimens due to the flexural loading mechanism which allows the fibre to function only after initiating the crack. However, the fibre addition contributed significantly to the flexural toughness of the tested beams where the beams with 6 kg/mThe fibrous concrete beams showed progressive crack mouth opening displacement (CMOD) more than 5 mm, while the plain concrete beam failure once recorded CMOD around 0.5 mm. It was observed that there is a relationship between the CMOD and the deflection indicating the stable behaviour of the fibrous concrete even after cracking.It was observed that increasing the bar diameter increased the pull-out force and however decreased the bonding strength as the latter negatively proportioned to the bar diameter. Moreover, all the steel bar diameter showed a similar mode of failure by splitting the concrete cylinder in a brittle manner. In contrast, adding macro poly fibres increased the bonding strength compared to the plain concrete and changed the mode of failure making the concrete cylinder failing in progressive way for the bars with 16 mm and 20 mm and interestingly a bar rupture in the 12 mm bar diameter.Theoretical interpretations on the test results were proposed thus they can predict the enhancement in the mechanical properties (compression and splitting tensile) of the concrete. New formulas were developed to predict the bonding strength of the concrete considering the fibre addition accounting for the fibre dosage and the steel bar diameter. These interpretations and formulas resulted in a good agreement with the experimental results.The stereo microscopic analysis revealed the ability of the macro polyfibres to control the internal cracks and reduce the inner pores by occupying and filling their locations. Furthermore, the pore size distribution influenced significantly the development of compressive strength more than the total porosity. More specifically, the macroporosity (>30 μm) has a direct correlation with the compressive strength, as opposed to the micropores and mesopores. Thus, the microporosity porosity in the concrete specimens decreased as the amount of fibres increased by 31%, 8% and 6%, respectively.This study investigated the effect of different amounts of macro polyfibre inclusion on the physical, mechanical, and micro-structural properties of concrete. Based on the results obtained by the wide range of testing conducted in this study, the following conclusions can be draw:The outcome of this study will help to increase the implementation of the recycled plastic waste in the concrete mix design as a waste management pathway to minimise the environmental pollution. In addition, the result of this study will add to the existing knowledge of the functionality of these macro polyfibres in concrete structures and their ability to enhance structural performances. Therefore, it is recommended to utilise these macro polyfibres in full-scale concrete elements so that the holistic influence of these fibres in the concrete structures can be quantified.


This might lead to increasing trends in the consumption of broad-spectrum and last-resort antibiotics. For fiscal years 2014-2017, we assessed annual antibiotic consumption trends and patterns in total defined daily doses (DDDs). (2) Methods: We followed World Health Organization (WHO) methodology for surveillance of antimicrobial consumption based on hospital antibiotic procurement records (as a proxy). (3) Results: In 32% of all public hospitals where data were retrieved, total antibiotic consumption reduced by 19% between 2014  and 2017 . Consumption per 1000 inhabitants per day  also reduced from 0.6 to 0.3. Over 60% of procurement was for beta-lactam antibiotics and quinolones; quinolones decreased over time. Consumption of first-line antibiotics increased (42% in 2014 to 54% in 2017), whereas broad-spectrum antibiotics decreased (46% in 2014 to 38% in 2017). Linezolid was the only last-resort antibiotic procured. There was a progressive reduction in per capita government current health expenditure from approximately 9.2 US$ in 2014 to 8.3 US$ in 2017. (4) Conclusions: Antibiotic consumption decreased over time in public hospitals. This first study provides a baseline for developing an antibiotic consumption surveillance system in Myanmar. Antimicrobial resistance (AMR) has become a recognized threat to the effective prevention and treatment of bacterial infections globally ,2. ImporEscherichia coli (83%), Klebsiella pneumoniae (61%), Pseudomonas species (30%), methicillin-resistant Staphylococcus aureus (10%), extended spectrum beta-lactamase (ESBL) producing Escherichia coli (34%), Klebsiella pneumoniae (32%), carbapenem-resistant Acinetobacter spps. (44%), and Pseudomonas spps. (32%) [Appropriate and inappropriate antibiotic use creates a selection pressure for bacteria to select antibiotic resistance. Inappropriate use of antibiotics is one of the main drivers of the emergence and spread of AMR. This includes using antibiotics to treat conditions that are not caused by bacterial infections, the use of the wrong type or dosage of antibiotics, the wrong route of administration, or the duration of treatment. Use of prophylactic antibiotics in surgery, such as cesarean sections and gastrointestinal surgery, is common when not always justified. Almost half of all antibiotics used in human health care can be considered inappropriate . There is. (32%) .To improve access and, at the same time, preserve the effectiveness of existing antibiotics, particularly second-line and “last resort” antibiotics, WHO has categorized antibiotics into three categories, named AWaRe—Access, Watch, and Reserve antibiotics. The Access category includes antibiotics needed for common infections and should be available at all times, affordable, and quality-assured . The WatOne of the pillars of the World Health Organizations’ (WHO) Global Action Plan and Myanmar AMR Action Plan to tackle AMR is to optimize the use of antibiotics through antibiotic stewardship . In 2014A concern is that this policy change might be associated with increasing trends in antibiotic consumption after 2014. A shift could be to more broad-spectrum antibiotics (belonging to Watch and Reserve categories) as the demand for antibiotics could be more easily influenced by individual physician preferences and persuasive effects of pharmaceutical companies. Data on antimicrobial consumption using a standardized metric of defined daily doses (DDDs) and by AWaRe categories provide an important “handle” for countries like Myanmar to better understand the trends and amounts of antibiotics used at the national level. This could inform policies, regulations, and interventions to optimize the use of antibiotics. It could also serve as a surveillance baseline and a yardstick for future monitoring and evaluation.Myanmar is yet to report to WHO on surveillance for antibiotic consumption at the national level and at health facilities. We thus aimed to assess antibiotic procurement (as a proxy for consumption) in three categories of hospitals: procurement for hospitals with up to 200 beds, hospitals with 200 or more beds, and central  hospitals.The specific objectives were to assess for the fiscal years 2014–2015 to 2017–2018, (a) the overall trend in annual antibiotic consumption , (b) the proportion (%) of total consumption by pharmacological subgroup, AWaRe categories, and administration route, and (c) the top ten most consumed antibiotics by hospital category.This is a cross-sectional study involving drug procurement records from hospitals in Myanmar.Myanmar lies in South East Asia and has a population of 51 million, according to the 2014 census data, with a predominantly rural population (70%). Health services are provided by the public and private sectors and non-government organizations (NGOs). The Department of Medical Services of the Ministry of Health and Sports is responsible for the procurement, storage, and distribution of medicines to all public health institutions.In Myanmar, health care services are largely provided by the public sector, which had 1115 public hospitals, while the private sector had 187 hospitals  in 2016 . The pubDrugs are provided free of charge in the public sector, but patients are required to purchase from private pharmacies if the prescribed drugs are out-of-stock or not included in the list of drugs procured by the hospital. There are national treatment guidelines for infectious diseases, and some central hospitals have also developed their own treatment guidelines on antibiotic use. Microbiology laboratory facilities to guide antibiotic prescriptions are available only in central, regional, and state hospitals.Drug procurement is according to the budget allocation and implemented in line with the Myanmar fiscal year (April to March). Prior to decentralization before 2014), drugs were procured and distributed by the central medical store depot (CMSD) of the Division of Medical Care  and distributed to government health facilities by a “push” system . The budget allocation was estimated for each health facility according to the number of beds of the facility and records of antibiotic usage. Purchase by the CMSD was about 70% from government facilities  and 30% from outside companies. During this time, the needs were underestimated, and frequent stock-outs were common. Moreover, as government budgets for generic drugs were less than 0.2 USD/capita/year, medicines supplied by the CMSD were insufficient, leading to stock-outs and resulting in patients having to purchase drugs from private pharmacies [, drugs wDecentralization was initiated during the 2014–2015 and became fully functional in the 2015–2016 fiscal year. The decision to decentralize drug procurement was taken to alleviate the high workload related to drug procurement and related activities at the Division of Medical Care . There are two ways to distribute and purchase medicines for hospitals. The first involves asking hospitals for a list of essential medicines they need. The Division of Medical Care submits this list to the Myanmar Pharmaceutical Industry (Ministry of Industry), which delivers the medicines. The second is for hospitals with 200 beds and more to be given a specific budget to purchase medicine based on need every six months. For hospitals with less than 200 beds , a budget is given to their respective region or state health authorities. Each hospital has a procurement committee under the leadership of the medical superintendent , who takes decisions on drug procurement issues. Consumption was estimated using the WHO methodology for surveillance of antimicrobial consumption and based on the ATC/DDD classification system . The ATCThe DDD is the assumed average maintenance dose per day of an antimicrobial substance(s) used for its main indication in adults and is assigned to the active ingredient with an existing ATC code. To adjust for population size, antibiotic consumption is usually presented as the number of DDDs per 1000 inhabitants per day (DID). This metric can be roughly interpreted as the number of individuals per 1000 inhabitants on antibiotic treatment per day .The study included three procurement categories of public hospitals in Myanmar for whom drug procurement data was available for the entire study period. These were hospitals with less than 200 beds in the same eight regions/states, hospitals with 200 or more beds, and central/specialist hospitals. For the estimation of consumption, we included antibiotics for systemic use, i.e., including all antibiotics categorized under the ATC group J01. Anti-tuberculosis drugs and antibiotics used for local therapy  are not included in the WHO surveillance methodology for consumption and were thus excluded . The stuData on public hospitals and coverage populations were obtained from annual hospital reports. Details of the antibiotics procured were obtained from the drug procurement reports available from the Procurement Division, Department of Medical Services, Naypyidaw. For DDD calculation, variables collected included International Non-proprietary Name (INN), the strength of the active ingredient(s) in the unit it is specified, route of administration, and the number of units procured of the product.Details on the procured antibiotic products were manually entered into a formatted Microsoft excel template provided by WHO with embedded macros to generate DDD per product and year. Annually aggregated DDDs were then stratified according to ATC level 3 and level 5, route of administration, and AWaRe categories.For hospitals with less than 200 beds, antibiotic consumption in DDDs was adjusted for population size in the hospital catchment area by standardization for 1000 inhabitants per day. However, as information on the catchment area was not available for hospitals with 200 beds or more and central hospitals, DDD standardization for 1000 inhabitants was not possible. Data were managed and analyzed using Stata 15.1 .The total number of public hospitals in Myanmar increased from 975 in the 2014–2015 fiscal year to 1122 in 2017–2018. Of these, an average of 32% had data on procurements for the entire study period and was included in the analysis. The included hospitals, stratified by hospital category, are shown in The antibiotic consumption stratified by hospital categories and years is shown in In hospitals with <200 beds, the DDD by 1000 inhabitants per day also declined from 0.6 in 2014–2015 to 0.3 in 2016–2017 and remained the same thereafter. During the study period, there was a progressive reduction in per capita government current health expenditure from 12,487 Myanmar Kyats (9.2 US$) in 2014–2015 to 11,293 Myanmar kyats (8.3 US$) in 2017–2018. The fiscal year 2016–2017 had the lowest health expenditure, which was 23% lower than in 2014–2015 .The proportion of antibiotics consumed by the pharmacological subgroup is shown in Antibiotic consumption by route of administration is shown in The top ten most consumed oral antibiotics stratified by hospital categories are shown in This is the first study from Myanmar showing that decentralization of drug procurement since 2014 was neither accompanied by an increasing trend in antibiotic consumption nor was a shift from Access to more broad-spectrum antibiotics belonging to the Watch and Reserve categories.The study findings are important and suggest that introducing a locally tailored pull system of drug procurement did not perversely affect antibiotic consumption due to possible factors, such as clinicians’ preferences and/or persuasive effects of pharmaceutical companies. Importantly, this data serves as a surveillance check (a yardstick) to inform and improve antibiotic consumption monitoring in Myanmar. The experience also suggests the operational feasibility of using antibiotic procurement data for estimating national antibiotic consumptions, which can be fed into the WHO report on surveillance of antibiotic consumption. On a broader level, such operational research could help build health system resilience and accelerate efforts towards achieving sustainable development goals.The study strengths are that we included over 30% of all public hospitals in 12 regions/states in the country; included three of the main categories of hospitals; assessed trends over four fiscal years using the same hospitals and used the WHO methodology for calculating and presenting the results . Data onThe main study limitation is that our procurement data did not cover public hospitals in all the states and regions, health facilities in the private sector, health facilities run by other ministries (like Railway), locally manufactured antibiotics, imported drugs, or drug donations received by specific hospitals. Antibiotic procurement through these channels, as well as community procurement, are aspects for future research. We also did not have pre-decentralization data to conduct a before-and-after comparison. Further, procured antibiotics may not reflect actual use, e.g., when in stock and not dispensed. Besides, when antibiotics are unavailable or out of stock, they may have to be purchased by patients, which may be particularly relevant to Watch and Reserve categories and will result in underestimations. We were also unable to assess consumption in relation to possible changes in patient numbers and infectious disease patterns over the years, which could influence antibiotics consumption. This is an area for future research, and these are important variables to be included while developing AMR surveillance systems in Myanmar. In addition, antibiotics that do not have assigned DDD were not included. This is an inherent limitation of the WHO ATC/DDD classification system. Understandably, our consumption figures are thus likely to be underestimated. However, our decision to use trend data in this study would allow such biases to be consistent over time. Finally, our analysis used a combination of levels of health care  and bed capacity (<200 beds or ≥200 beds), which are two different dimensions, and as such, there may be a limited correlation between the two.Despite these limitations, the study has a number of policy and practice implications. First, in comparison to 2014, total antibiotic consumptions (in DDD) dropped and were lowest in the 2016–2017 fiscal year. This seems to follow the pattern of similar declines in national recurrent expenditure for health. When standardized to 1000 inhabitants per day, antibiotic consumption in hospitals with <200 beds also reduced from 0.6 to 0.3. Similar reports from other countries reported to WHO ranged from 4.4 (Burundi) to 64.4 (Mongolia); however, those figures were for national consumption data . Our staSalmonella spp. and Escherichia coli infections. Further, this antibiotic class is vital for the treatment of serious urinary tract infections and for the construction of second-line drug regimens for multi-drug resistant tuberculosis (MDR-TB) in humans.Second, the quinolone group of antibiotics showed a reducing trend over the years. This is encouraging as this antibiotic, since 2017, has been classified by WHO as one of the “highest priority critically important antibiotics” that should be used prudently both in humans and animals . RestricThe finding that linezolid (used for tuberculosis treatment) was the only Reserve antibiotic available  implies that antibiotics in this category are not being procured, which points to an access issue. Reserve antibiotics are expensive, and in the light of budgetary cuts, the procurement choice might have been to allocate spending to commonly used Access and Watch categories of antibiotics. The National List of Essential Medicines of Myanmar has linezolid as the only Reserve antibiotic, and this may need to be reviewed . The lacThird, in hospitals with over 200 beds, we found fluctuations in proportions of parenteral antibiotics, reaching a high of 70% in 2017–2018. This is concerning, and although we do not know the exact reasons for this finding, it merits urgent investigation. The fact that metronidazole was the most common parenteral antibiotic used in hospitals with less than 200 beds also merits investigation. This antibiotic is commonly used for surgical prophylaxis prior to and during abdominal and gynecological surgery. It is also often used to cover anaerobic bacteria in maxillofacial and dental surgery. Verifying the rationale behind this finding and whether (or not) justified would require specific quantitative and qualitative research.With regards to cephalosporins, we observed a progressive increase in the use of third-generation cephalosporins in all hospital categories over the years. As this is a broad-spectrum antibiotic that is accessible, relatively cheap, and effective against monitoring several bacterial infections, its antibiotic resistance pattern would be useful to guide individualized treatment and empirical use.Finally, the field experience gained from this study would provide the impetus of the need for developing a national electronic logistic management information system to capture antibiotic procurement and consumption in a holistic manner in Myanmar. This should be inclusive of the private, public, and community sectors and would be vital to improve national AMR surveillance on antibiotic consumption in Myanmar and fluid reporting to WHO.In conclusion, this operational research study has provided useful insights into antibiotic consumption patterns in Myanmar and could provide impetus towards building a more robust antibiotic consumption surveillance system in the country. Steps are being taken in this direction.


Bifidobacterium and Sutterella and a decrease in Akkermansia at genus level compared to the control group. Fecal microbiota transplantation (FMT) further confirmed that the microbiota altered by heroin dependence was sufficient to impair body weight and intestinal mucosal barrier integrity in recipient mice. Moreover, short-chain fatty acids (SCFAs) profiling revealed that microbiota-derived propionic acid significantly decreased in heroin dependent mice compared to controls. Overall, our study shows that heroin dependence significantly altered gut microbiota and impaired intestinal mucosal barrier integrity in mice, highlighting the role of the gut microbiota in substance use disorders and the pathophysiology of withdrawal symptoms.The gut microbiota is believed to play a significant role in psychological and gastrointestinal symptoms in heroin addicts. However, the underlying mechanism remains largely unknown. We show here that heroin addicts had a decrease in body mass index (BMI) and abnormal serum D-lactic acid (DLA), endotoxin (ET) and diamine oxidase (DAO) levels during their withdrawal stage, suggesting a potential intestinal injury. The gut microbial profiles in the mouse model with heroin dependence showed slightly decreased alpha diversity, as well as higher levels of  As a chronic and relapsing brain disease , 2, heroAlthough the mechanism of the gut microbiota in disease is not fully understood, the gut microbiota is a critical component of the brain-gut axis  potentiaIn this study, we investigated the serum indicators of intestinal mucosal barrier damage in heroin addicts during withdrawal stage and generated a mouse model mimicking heroin dependence. High-throughput sequencing of 16s rRNA gene and bioinformatic analysis was performed to determine the variations in gut microbiota in heroin dependent mice compared with saline-treated mice. To confirm the altered microbiota contributing to the intestinal mucosal barrier integrity, donor stool from mice with heroin dependence were transplanted into recipient mice by fecal microbiota transplantation (FMT). To assess intestinal mucosal barrier integrity, the serum level of DAO, DLA, and ET, as well as protein expression levels of Claudin-1 and ZO-1 were evaluated, respectively. The concentrations of metabolic products SCFAs in mouse fecal samples were analyzed using gas chromatography–mass spectrometry (GC-MS).We show that heroin dependence causes substantial changes in the composition of the gut microbiota contributes to pathological changes of intestinal mucosal barrier function. Furthermore, it offers a deep insight into the function of the gut microbiota in heroin dependence induced digestive diseases and a new paradigm for addiction treatment.Thirty-eight male heroin addicts (age ranging from 18 to 58) from the hospital of the Longchuan Drug Rehabilitation Center in Dehong, China were recruited as the heroin group; 38 age-matched males with no heroin-use history were recruited as healthy controls (HCs). The inclusion criteria were: (1) male sex and aged between 18 and 58, (2) diagnosed with heroin dependence, and (3) in withdrawal stage. Participants were excluded if (1) severe neurological disorders, (2) severe chronic illnesses, infections, (3) antibiotic treatments (<7 days prior to study start), (4) refuse or inability to give informed consent. All participants provided written informed consent prior to study participation. The participants' age, gender, body weight, and height were collected. 2 ml of blood was drawn from each participant to measure intestinal mucosal barrier damage indicator.Male C57BL/6 SPF mice  were obtained from the Kunming Medical University Center for Laboratory Animals, housed individually under controlled temperature (22 ± 2°C) and humidity (50 ± 5%), and maintained on a 12/12 h light/dark cycle (lights on at 7 a.m.). The mice had free access to tap water and a standard mouse chow diet. Animals were deeply anesthetized with pentobarbital sodium  then sacrificed, colonic tissues were perfused with 0.1 M PBS  followed by 4% (w/v) paraformaldehyde in 0.1 M PBS, prepared at 20 μm thickness by using a cryostat microtome . All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.The Narcotics Department of Yunnan Provincial Public Security Administration generously supplied the diacetylmorphine HCl (heroin), and the heroin was freshly dissolved in 0.9% sterile saline for intraperitoneal (10 mg/kg).n = 10); (2) heroin group (10 mg/kg heroin i.p. n = 10). The experimental design is showing in In order to generation the mouse model with heroin dependence, mice were randomly separated into two groups after 1 week of adaptation: (1) control group  and the ANY-maze software (Stoelting Co.) . Mice wen =1 0), recipient mice: .We performed FMT as described previously , 20: fecThe OFT assessed the effects of chronic psychological stress on anxious behavior. The mice were subjected to OFT for 5 min. The time spent in the center (inner 25% of the surface area) were recorded by ANY-maze software (Stoelting Co.) The open field arena was cleaned with 70% ethanol between each trial.The elevated plus-maze test was performed as previously described . The appA day before the test, a mouse was placed individually into a glass cylinder  filled with water to a depth of 10 cm at 25°C for 5 min. During experimentation, subjects were placed individually in a filled glass cylinder for a period of 6 min and the total duration of immobility was measured for 5 min by ANY-maze software as indicative of depressive-like behavior. Motionless floating was considered immobile behavior .The tail suspension test was conducted as described previously . Mouse wFecal samples were collected from the control and heroin dependent mice, frozen immediately and stored at −80°C. Microbial DNA was extracted from fecal samples fusing the MagPure Stool DNA KFKit B . The quantity of genomic DNA was verified using the Qubit dsDNA BR Assay kit .16S rRNA gene sequencing procedure was performed by BGI . Briefly, the V4 regions of the 16S rRNA gene in the DNA extracted from fecal samples were amplified using the following degenerate PCR primers: 515F (5′-GTGCCAGCMGCCGCGGTAA−3′), 806R (5′- GGACTACHVGGGTWTCTAAT-3′) and sequenced on the Illumina HiSeq2500 platform following the standard pipelines of Illumina and generating 2 × 250 bp paired-end reads. Taxonomic annotation was based on a customized version of SILVA reference database. Alpha and beta diversities were analyzed using QIIME. The relative abundance of bacteria was expressed as the percentage. Partial least-squares discrimination analysis (PLS-DA) was performed using the R package mix Omics, whereas principal coordinate analysis (PCoA) was performed using QIIME.The serum levels of DLA, ET, and DAO reflect intestinal mechanical barrier dysfunction, usually regarded as a very important evaluation of intestinal mucosal barrier damage . The indExpressions of Claudin-1 and ZO-1 were evaluated with immunofluorescence staining. Briefly, mouse colon tissue was fixed in 4% paraformaldehyde for 24 h, dehydrated using 30% sucrose, embedded in frozen section embedding agent and sliced (10-microns thick), permeabilized using 0.3% triton X-100 for 10 min, and blocked with 10% BSA for 60 min. The following primary antibodies was used: Claudin-1 , ZO-1 . The tissue sections were then incubated with the appropriate Anti-rabbit IgG (H+L) or F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) . Nuclei were stained with DAPI. The immunofluorescence quantification was analyzed by image J. Each experiment was repeated three times.http://www.metware.cn/) based on the Agilent 7890B-7000D GC-MS/MS platform. As previously described . Notable non-significant differences were indicated in the figures by “n.s.,” non-near significant (0.05 <p < 0.1) were indicated in the figures.The data were analyzed using the GraphPad Prism v.8 Software, data reported as means ± SD. Student's p < 0.05)  between the two groups. The serum levels of D-lactic acid (DLA), endotoxin (ET) and diamine oxidase (DAO) were the main indicators for evaluating the function of intestinal mucosal injury, intestinal wall permeability, and bacterial translocation  were included in the survey sample, and population demographics revealed that the distribution of the sample population by sex and age was not statistically different between the two groups . The meap < 0.0001 p < 0.05, p = 0.07, p < 0.0001, p < 0.001, After repeated 10 mg/kg heroin administrations for 14 days, the mouse model with heroin dependence was established and confirmed by behavior sensitization . The totp > 0.05). For better mimicking the heroin dependence and withdrawal syndrome in patients, we decided to profile the microbiota in the heroin dependent mouse model undergoing withdrawal stage to explore the associations with intestinal mucosal integrity.In our heroin-dependence mouse model, heroin-induced gut barrier dysfunction may occur at any time within the 21 days during heroin treatment or after the initiation of drug withdrawal. We thus compared the serum level of DLA, ET, and DAO, the expression of Claudin-1 at both day 21(Heroin-addicted stage) and at 48-h after initiation of withdrawal  . Compare(LEfSe) cladogram represents differentially abundant taxa . Moreover, the composition of the gut microbiota was significantly different from the two groups according to the LDA score . Mice with heroin dependence had a higher abundance of Bifidobacteriaceae, Planococcaceae, Streptococcaceae, Desulfovibrionaceae (the relative abundance of these bacteria <0.01%) compared to control mice . In addition, the level of Bifidobacterium, Sporosarcina, Streptococcus, and Alistipes (the relative abundance of these bacteria <0.01%) were increased more in heroin dependent group than that in the control group  at the family  and genecchaceae . The relrol mice . At the rol mice , of thesin group . The remol group .p = 0.0305) and ET (p = 0.0476) were observed in the FMT mice receiving microbiota from heroin dependent mice compared to control mice  and the gastrointestinal tract . Many CNAkkermansia, which belongs to the family of Verrucomicrobia, was significantly increased in the heroin dependent mouse group  and associated with higher susceptibility to chemically-induced colitis . In addition to the details described above, the altered bacterial such as Desulfovibrio, Prevotella, and Oscillibacter may utilize microbial exopolysaccharides synthesized by Bifidobacterium to produce SCFAs in the intestine ; Yunling Scholar (YLXL20170002); the Major project of Yunnan Provincial Bureau of Education ; Yunnan Engineering Technology Center of Digestive Disease (2018DH006); and Science and Technology Department of Yunnan Province .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.