Bothrops lanceolatus, whereas Bothrops atrox is responsible for the majority of snakebites in French Guiana, although other venomous snake species also occur in this South American territory. This review summarizes some of the key aspects of the natural history of these species, as well as of their venom composition, the main clinical manifestations of envenomations, and their treatment by antivenoms. B. atrox venom induces the typical set of clinical manifestations characteristic of Bothrops sp. venoms, i.e., local tissue damage and systemic alterations associated with coagulopathies, hemorrhage, hemodynamic alterations, and acute kidney injury. In the case of B. lanceolatus venom, in addition to some typical features of bothropic envenomation, a unique and severe thrombotic effect occurs in some patients. The pathogenesis of this effect remains unknown but may be related to the action of venom components and inflammatory mediators on endothelial cells in the vasculature. A monospecific antivenom has been successfully used in Martinique to treat envenomations by B. lanceolatus. In the case of French Guiana, a polyvalent antivenom has been used for some years, but it is necessary to assess the preclinical and clinical efficacy against viperid venoms in this country of other antivenoms manufactured in the Americas.Snakebite envenomation is a relevant medical hazard in French Guiana and Martinique, two French territories in the Americas. All snakebite envenomations in Martinique are inflicted by the endemic viperid species  Bothrops species are responsible for the vast majority of snakebite envenomations [Bothrops, accounting for 80% to 90% of all snakebite cases, mainly from Bothrops atrox [Bothrops lanceolatus, a venomous pit-viper species, is endemic to this French Caribbean island [B. atrox and B. lanceolatus may be considered the species responsible for most medically significant snakebite envenomations in these French overseas territories of the Americas.Overseas France includes island territories in the Atlantic, Pacific Indian Oceans and peri-Antarctic region, and French Guiana in South America. Among these territories, French Guiana and Martinique, a French Lesser Antilles island in the Caribbean Sea, are habitats for many animal species, including hundreds of snake species . In Frenomations –3. Amongps atrox . In MartB. atrox is the most abundant venomous snake in French Guiana [B. atrox probably represents a complex of several lineages across the species distribution [B. atrox mitochondrial DNA lineages was recently confirmed by other studies based on a more comprehensive geographic sampling [B. atrox is mostly endemic to the Amazon rainforest [B. atrox inhabits forested areas, although it may be found in disturbed habitats around human settlements, including pastures and crops, and urban areas [B. atrox is especially active at night when adults are often seen on the ground hunting, while juveniles frequently hunt in vegetation (up to heights of 1.5 m) [h Guiana . There iribution . The nonsampling . Consideinforest . The speinforest . In Frenan areas . B. atrof 1.5 m) . Regardif 1.5 m) . Juvenilf 1.5 m) .B. lanceolatus is the only venomous snake in Martinique [B. caribbaeus, endemic to the neighboring island of Saint Lucia [B. asper/B. atrox, with which they form a sister clade [B. lanceolatus and B. caribbaeus form a monophyletic group divergent at the molecular level from all other South American species of Bothrops. It is estimated that the timing of the split of these Lesser Caribbean species from the B. asper/B. atrox clade occurred during the late Miocene or early Pliocene 4.2 to 8.9 million years ago [B. lanceolatus is an inhabitant of tropical moist forest and tropical wet forest, primarily in upland regions. It also occupies rocky hillsides and tends to be arboreal, having been found as high as 20 m above ground [B. lanceolatus feeds chiefly on lizards, later on birds, and when adults, mostly on rats. In the dense woods, it lies quiet, seldom disturbed but by the singing of some birds that live in the wilderness. The night is the time of its wandering, and it has been seen in the roads crossed by people during the day [B. lanceolatus has most recently been assessed for The IUCN Red List of Threatened Species in 2015 [rtinique . This spnt Lucia , originaer clade . B. lancears ago . The geoears ago . B. lance ground . When yo the day . B. lanc in 2015 .Bothrops sp., indicate that the predominant components playing a role in toxicity are zinc-dependent metalloproteinases (SVMPS), phospholipases A2 (PLA2s), and serine proteinases. Other components include vasoactive peptides, disintegrins, L-amino acid oxidases, cysteine-rich secretory proteins (CRISPs), and C-type lectin-like components [Over the past decades, proteomic studies of viperid snake venoms, including those of mponents .B. atrox along the Amazon, which are likely to have implications in the pathophysiology and clinical manifestations of envenomations [B. atrox venom variability occurs among wild specimens from different geographical areas and different habitats, life span of individuals (juvenile versus adults), body size, and gender [Bothrops species of the same genus, likely caused by differences in the feeding habits of juveniles and adults [B. atrox snakes, suggesting that small snakes (juveniles) are associated primarily with moderate local pathology and with systemic hemorrhagic and coagulopathic manifestations. In contrast, larger snakes were responsible for severe cases and characterized by major local effects  [There are important regional differences in the composition of the venoms of omations . Indeed,d gender . Venom pd adults . A studyfection) .B. atrox captured in the Amazonas State of Brazil and maintained in captivity revealed that the major classes of transcripts expressed in venom gland are those of class III metalloproteinases and PLA2 [B. atrox from specimens collected in different localities in South America [B. atrox venom composition, from Colombia, Brazil, Ecuador, and Peru [2s [An initial study of the transcriptome of a young male adult specimen of and PLA2 . Proteom America –23. Protand Peru ,21–23  since this location is close to the Territory of French Guiana. This venom has a predominance of PI and PIII SVMPs over other protein families [Notably, proteomic characterization of venom of families  , which directly hydrolyze fibrinogen, generating feeble fibrin clots [B. atrox venom hemorrhagic toxins are well characterized and are involved in the local and systemic effects of human victims of snakebite [Bothrops venoms on human plasma and fibrinogen have recently deciphered coagulotoxic effects of these venoms [B. atrox venom [Hemorrhagic syndrome and its pathophysiological consequences remains the most important cause of lethality associated with nomation ,25. Sincin clots . Adding in clots ,24,27. Bnakebite –31. Throe venoms ,32. Signox venom ,32.B. atrox venom PLA2s also contribute to local tissue damage due to their myotoxic activity, causing lysis of muscle cells and release of intracellular proteins that also enhance the reactive proinflammatory status of the tissues close to the bite [B. atrox venom contains toxins that directly induce inflammatory effects, leading to plasma exudation and accumulation of polymorphonuclear and mononuclear leukocytes and macrophages to the injury site [Hemorrhage is primarily attributed to PI- and PIII-class SVMPs that induce vascular lesions. The different degrees of hemorrhagic activity of SVMPs have been attributed to their hydrolytic activity of extracellular matrix components such as laminin and type IV collagen, and also to the fact that PIII-class SVMPs bind to targets present on the basement membrane, allowing the concentration of the enzyme adjacent to the capillaries and venules, making more productive the hydrolysis and rupture of the vessel ,33. The the bite . In addiury site ,36.B. atrox venoms can elicit coagulotoxic effects in prey captured by snakes, in which hemorrhage, plasma extravasation, and the rapid formation of endogenous thrombin can result in prey incapacitation through circulatory shock or intravascular thrombosis. It is clear that, in the evolution of the venom of B. atrox, there has been a strong selection for generating coagulotoxic proteins. At the same time, the venom lacks neurotoxic and cardiotoxic components. B. atrox venom is weakly lethal in mice, with relatively high Median Lethal Dose (LD50). The LD50 of B. atrox venom from French Guiana in mice is 131 μg/20 g body weight [In line with the well-known hemostatic disturbances, y weight . In humay weight .B. lanceolatus venom contains a variety of toxins, which belong to SVMPs, SVSPs, and PLA2, and L-amino acid oxidases, among others [B. lanceolatus induce local tissue damage similar to that of B. atrox but infrequently elicit systemic bleeding and incoagulability. In sharp contrast, B. lanceolatus envenomations may be complicated by multiple systemic thrombotic events within 48 h after the bite [2s are, however, less abundant in B. lanceolatus venom mixture. In line with this proteomic profile, this venom induces hemorrhage, whereas the content of PLA2s is low, and PLA2-induced myotoxic activity is relatively weak, especially compared to other Bothrops species [B. lanceolatus venom PLA2 activity has been previously found when determined in the absence of detergent with egg yolk as substrate. Activation of PLA2 from B. lanceolatus venom has been attributed to the catalytic property of the enzyme itself rather than the dissociation of PLA2 inhibitors such as in the cases of other Bothrops species [B. lanceolatus venom [g others   living in rural areas of the Brazilian agricultural frontier, who are bitten during work activities [B. atrox is responsible for the majority of snakebites in French Guiana [In South America, epidemiological studies suggest that the majority of snakebite cases are caused by different species of snakes of the genus tivities . A highetivities ,52. LikeB. atrox envenomation in French Guiana resemble the classic syndrome associated with bites from viperids, including local effects at the bite site  and systemic effects  [Bothrops are hemorrhagic, damaging the vascular endothelium and consuming coagulation factors, i.e., inducing venom-induced consumption coagulopathy, and eliciting inflammation [B. atrox and has been related to inflammatory processes and poor local prognosis as they increase the risk of infection and necrosis [B. atrox envenomation may also include compartment syndrome of the bitten muscle, wound infection, hemodynamic instability and shock, and acute kidney injury (AKI) [Clinical features of rrhages) ,8,53–55.ammation . Blisternecrosis . Complicry (AKI) .B. atrox was identified in 43.6% of cases. The main clinical manifestations were edema (98.5%), pain (97.7%), systemic hemorrhage (18%), blisters (14.3%), and local hemorrhage (14.3%). Circulatory shock (1.5%), AKI (15%), and systemic hemorrhage (18%) were the most frequent complications. The elapsed time from snakebite to the development of systemic hemorrhage was 4.5 h. Infections such as abscesses (65%), necrotizing fasciitis (18.6%), and cellulitis (27.9%) were recorded in this group of the patients. In this series, overall mortality was 1.5% [Though the precise number of snakebites is unknown, an estimated annual incidence of envenomation in French Guiana exceeded 20 cases per 100,000 inhabitants, with a case fatality rate of 3% ,61. FromBothrops envenomation [B. atrox to 38.5% in B. asper [Bothrops venom-induced AKI include the direct action of venom on the kidney, the hemodynamic alterations, myoglobinuria, hemoglobinuria, glomerular microthrombi deposit due to coagulation abnormalities, and immunologic mechanisms [AKI is the main systemic complication and a common cause of death in nomation . SeveralB. asper . Proposechanisms –64. In echanisms . OverallBothrops snakebite envenomation [2 fragments, although some antivenoms are made of whole IgG molecules. There are two main types of antivenom, namely monospecific  antivenom, which is generated against the venom of a single snake species, and polyspecific  antivenom, which is made using the venom of multiple snake species as immunogens [Administration of antivenom is the only specific treatment to counteract nomation ,66. AntiBothrops envenomations are treated with Antivipmyn-Tri, a polyvalent antivenom for Bothrops and other viperid species  [B. atrox endemic of French Guiana [Bothrops envenomations in French Guiana  failed to demonstrate evidence of clinical benefit [Bothrops snakebite to achieve normal dosages of coagulation parameters was shorter in patients who received antivenom than in those who did not [In French Guyana,  Mexico) ,67. Thish Guiana . In thish Guiana ,53,68–70 did not . These cB. atrox venom endemic of French Guiana [B. atrox venom, while in contrast, Antivipmyn-Tri antivenom did not neutralize lethal activity even at the highest antivenom level tested [B. atrox from Colombia, Peru, Brazil, and Ecuador [B. atrox from various localities in South America [Bothrops venoms has been shown in the neutralization of the procoagulant activity B. atrox from French Guiana [B. atrox venom [Immunological and functional in vitro assays and preclinical efficacy of a freeze-dried antivenom manufactured in Costa Rica  against the lethal, hemorrhagic, coagulant, and myotoxic effects have recently highlighted the therapeutic potential of this antivenom against h Guiana . Regardil tested . In thisl tested . These r Ecuador ,72. More America . Interesh Guiana , and thiox venom . OverallB. lanceolatus is the only venomous snake, responsible for about 15 envenomations treated in hospitals yearly  [B. lanceolatus venom induces local and systemic effects comparable to the typical bothropic syndrome, but the envenomation is characterized by a unique predominant prothrombotic profile. Envenomation is characterized by intense local reactions featured by extensive swelling, erythema, bleeding at the bite site, ecchymosis, and pain [In Martinique, and pain . About 4and pain . These eand pain ,74–76. Tand pain ,76–79. Cand pain ,77,78. Aand pain .B. lanceolatus develop thrombocytopenia, while disseminated intravascular coagulation is rare [B. lanceolatus envenomed patients who will further develop thrombosis [B. lanceolatus envenomed patients, since such systemic inflammation in the absence of defibrinogenation might induce a procoagulant phenotype in endothelial cells.Up to 50% of the patients envenomed by  is rare ,78,79. Trombosis ,78. The rombosis . As prevrombosis ,47, as eB. lanceolatus venom using various in vitro assays and in vivo mouse studies, the efficacy of monospecific Bothrofav antivenom against this venom currently in use in Martinique has been demonstrated. The monospecific antivenom, Bothrofav, manufactured by Sanofi-Pasteur, was introduced in Martinique in 1991 and has proven highly effective in preventing the development of life-threatening disturbances in these envenomations [B. lanceolatus [B. lanceolatus, thus underscoring the high neutralizing efficacy of this antivenom [Following the toxicological characterization of omations ,80. Expeceolatus ,81. Thirntivenom .B. lanceolatus envenomations and a significant reduction in thrombosis and other systemic disturbances in these envenomations [B. lanceolatus antigens exhibited by the venom. In line with this, WHO recommends a collection of at least 20 to 50 snakes of different sizes and geographical locations for the production of snake antivenom [The systematic use of Bothrofav in Martinique has led to the almost complete disappearance of serious complications of omations ,78,80. Homations . Severalntivenom . Hence, ntivenom .B. lanceolatus and B. caribbaeus snakes. Immunodepletion findings with Polival-ICP correlate well with neutralization results, since it cross reacts with B. lanceolatus SVMPs, L-amino acid oxidases, and PLA2s, and since a majority of venom components are recognized by antivenom antibodies in western blots and immunodepletion experiments [B. lanceolatus venom has not been reproduced in the mouse model, it is not known whether Polival-ICP is able to neutralize this clinically relevant effect.Besides Bothrofav, Polival-ICP polyvalent antivenom has also been evaluated for its neutralizing ability after the characterization of venom proteomes of eriments . HoweverB. lanceolatus, particularly regarding the severe thrombotic effect described in human patients? Which SVMP is responsible for endothelial lesions of the microcirculation, which may lead to thrombosis? What is the role of inflammation in the thrombotic effect? What is the role of coronary angiography in case of thrombotic damage in patients suffering envenomations by B. lanceolatus? Will it be possible one day to consider the manufacture of a bivalent Bothrofav, specific for B. lanceolatus and B. caribbaeus? Would reducing the time in administering the antivenom following bites allow better control of complications? What is the benefit of using the ELISA test for assessing venom concentration in case of clinical worsening despite well-conducted antivenom therapy? What is the benefit of using metalloproteinase inhibitors in treating thrombotic stroke in these envenomations? Will Bothrofav continue to be available in Martinique?Several issues related to snakebite envenomation in French Guiana and Martinique remain unresolved to date and demand further experimental and clinical research. Some of these issues in Martinique are as follows: What are the pathophysiological mechanisms of envenomation by B. atrox? What is the optimum dosage regime for antivenoms used in French Guiana? How can the time lapse between the bite and access to the hospital be reduced in French Guiana in order to avoid complications? Is it feasible to prevent local and systemic complications of these envenomations by the use of enzyme inhibitors? Would it be possible to correlate these experimental findings with the clinical observations and also with the dosage of antivenom needed in human cases?In the case of French Guiana, some pending issues are as follows: Which is the most appropriate antivenom for the treatment of envenomations by Bothrops but also on envenomations inflicted by other tropical venomous snakes. The question of research funding by the French health authorities, with the support of international organizations such as the Pan American Health Organization (PAHO) and the World Health Organization (WHO), is therefore crucial. There is also an urgent need to develop regional collaborative and multidisciplinary approaches for the improvement of snakebite in the region and to allow access to safe, affordable antivenoms.It is therefore urgent to set up fundamental, translational, and clinical studies to better understand the mechanisms of these envenomations and treat their clinical consequences, with the identification of effective therapies: effective and safe antivenoms, antithrombotic treatments, inhibitors of the action of venom on the vascular endothelium, and aftercare active not only on B. atrox envenomation in French Guiana. Moreover, protocols for the management of snakebites should be evaluated and updated, especially regarging antivenom dosage and management of complications. Another aspect that needs to be considered has to do with the availability and accessibility of antivenoms in remote rural regions and on the training of medical and nursing staff in the treatment of envenomations. Strategies for the rapid transportation of patients to health facilities need to be carefully considered, as well as the promotion of regional cooperation platforms with neighbouring countries and with regional health authorities, such as PAHO.Snakebite envenomation in French Guiana poses a significant challenge from the public health perspective. In the absence of a specific antivenom for French Guiana, it is necessary to undertake a detailed preclinical assessment of the various antivenoms available in Central and South America in order to identify the best option for the treatment of B. lanceolatus are nevertheless an important public health problem in Martinique. The clinical picture and management principles are now well established, but the search for specific therapeutics and antivenom drugs still pose unresolved problems. Preliminary clinical data available at the University Hospital of Martinique show the excellent efficacy and tolerance of an early infusion of the antivenom Bothrofav, to prevent complications of envenomation. We have thus been able to establish a harmonized protocol for managing envenomation in Martinique and include all patients in a specialized toxicology program. A joint project for Bothrofav to treat envenomations by B. lanceolatus and B. caribbaeus is also under development and will mark the beginning of a medical collaboration between the two islands, Martinique and Saint Lucia. Further studies are still needed to optimize the modalities of administration of this antivenom.Although rare, envenomations by Snakebite envenomation is a relevant medical hazard in French Guiana and Martinique, two French territories in the Americas.B. atrox is responsible for the majority of snakebites in French Guiana, while B. lanceolatus is the only venomous snake in Martinique.B. atrox venom typically induces coagulopathies and hemorrhage, while B. lanceolatus venom has a thrombotic effect in some patients.Bothrofav, a monospecific antivenom, has been successfully used in Martinique to treat envenomations.B. atrox envenomings.It is necessary to evaluate several antivenoms for their efficacy and safety in 10.1038/nrdp.2017.63Gutiérrez JM, Calvete JJ, Habib AG, Harrison RA, Williams DJ, Warrell DA. Snakebite Envenoming. Nat Rev Dis Primers. 2017;3:17063. doi: 10.3390/toxins12100662Resiere D, Houcke S, Pujo JM, Mayence C, Mathien C, NkontCho F, et al. Clinical Features and Management of Snakebite Envenoming in French Guiana. Toxins. 2020;12:662. doi: 10.3390/toxins14050297Bourke LA, Zdenek CN, Tanaka-Azevedo AM, Silveira GPM, Sant’Anna SS, Grego KF, et al. Clinical and Evolutionary Implications of Dynamic Coagulotoxicity Divergences in Bothrops (Lancehead Pit Viper) Venoms. Toxins. 2022;14:297. doi: Bothrops atrox, the Most Important Snake Involved in Human Envenomings in the Amazon: How Venomics Contributes to the Knowledge of Snake Biology and Clinical Toxinology. Toxicon X. 2020;6:100037. doi: 10.1016/j.toxcx.2020.100037Monteiro WM, Contreras-Bernal JC, Bisneto PF, Sachett J, Mendonça da Silva I, Lacerda M, et al. Bothrops atrox of Two Polyspecific Antivenoms. Toxicon. 2020;173:1–4. doi: 10.1016/j.toxicon.2019.11.001Resiere D, Villalta M, Arias AS, Kallel H, Nèviére R, Vidal N, et al. Snakebite Envenoming in French Guiana: Assessment of the Preclinical Efficacy against the Venom of


Belamcanda chinensis (L.) DC. Previous studies indicated that both compounds could be metabolized in vivo; however, the kinetic parameters of enzymes involved in the metabolization of tectorigenin and irigenin have not been identified. The aim of this study was to investigate UGTs involved in the glucuronidation of tectorigenin and irigenin and determine enzyme kinetic parameters using pooled human liver microsomes (HLMs) and recombinant UGTs. Glucuronides of tectorigenin and irigenin were identified using high-performance liquid chromatography (HPLC) coupled with mass spectrometry and quantified by HPLC using a response factor method. The results showed that tectorigenin and irigenin were modified by glucuronidation in HLMs. One metabolite of tectorigenin (M) and two metabolites of irigenin (M1 and M2) were detected. Chemical inhibition and recombinant enzyme experiments revealed that several enzymes could catalyze tectorigenin and irigenin glucuronidation. Among them, UGT1A1 and UGT1A9 were the primary enzymes for both tectorigenin and irigenin; however, the former mostly produced irigenin glucuronide M1, while the latter mostly produced irigenin glucuronide M2. These findings suggest that UGT1A1 and UGT1A9 were the primary isoforms metabolizing tectorigenin and irigenin in HLMs, which could be involved in drug–drug interactions and, therefore, should be monitored in clinical practice.Tectorigenin and irigenin are biologically active isoflavones of  Belamcanda chinensis (L.) DC. is a perennial herbaceous plant [Belamcanda chinensis, the sole species in the genus Belamcanda [Iris and renamed Iris domestica. Its Chinese name is Shegan, and it is widely used in China and other East Asian countries [us plant . In 2005ountries  because ountries .To date, more than 100 compounds, including isoflavonoids, quinones, and terpenoids, have been identified in Shegan . IsoflavDrug metabolism is an important part of pharmacokinetic research and is considered one of the key factors affecting the bioavailability of target compounds . UnderstThe aim of this study was to identify the enzymes involved the metabolism of tectorigenin and irigenin and their kinetic parameters. The results should promote further research in this area and contribute to the development of new drugs based on tectorigenin and irigenin.+), which was 176.0322 Da higher than that of tectorigenin, with a product ion at m/z 301.0703 (When tectorigenin was incubated with human liver microsomes (HLMs) in the presence of uridine diphosphoglucuronic acid (UDPGA), a new obvious peak (M) appeared at 6.7 min, which was absent in the control (no UDPGA) A,B. Mass301.0703 C. These +), which was 176.03 Da higher than that of irigenin. The most abundant product ions of M1 and M2 were both 361.09 Da and were generated by a neutral loss of 176 Da  were detected A,B. M1  method was used to determine the concentration of the produced metabolites ,18. RF vThe glucuronide metabolites were quantified using the calibration curve of tectorigenin or irigenin, and the concentrations of these metabolites were corrected using the RF values. The quantification method was validated. The accuracies were represented by relative error (RE), and the precisions were represented by relative standard deviation (RSD). As a result, for both tectorigenin and irigenin, the lower quantification limit was 0.03 μM, and the calibration range was 0.03–10 μM. The precision represented by RSD was lower than 6.21% and 4.37% for tectorigenin and irigenin, respectively. The accuracies of tectorigenin and irigenin ranged from −3.70% to 2.13% and from −2.23% to 4.31%, respectively. The mean extraction recoveries of tectorigenin and irigenin were both greater than 97.3%.In the stability experiment, we evaluated the stability of tectorigenin and irigenin, as well as their metabolites, under different conditions. The RE and RSD values for tectorigenin and irigenin in pretreated samples placed in the autosampler for 12 h at 4 °C were both below ±5%, which indicated that tectorigenin and irigenin were both stable. The stability results of metabolites of the experiment are presented in In case of irigenin, silibinin significantly inhibited the production of M1 B, whereaNext, UGTs were used to determine the subtypes participated in the metabolic reaction of tectorigenin and irigenin. In glucuronidation of tectorigenin, UGT1A1 and UGT1A9 had the highest activity, and UGT1A3, UGT1A4, UGT1A6, and UGT2B7 had some activity, whereas UGT2B4, UGT2B15, and UGT2B17 had none A. In glumK), maximum velocity (maxV), and intrinsic clearance (intCL = maxV/mK) for M formation were 32.79 ± 4.79 μM, 5.87 ± 0.25 nmol/min/mg, and 180 μL/min/mg, respectively, whereas those for M1 and M2 formation were 16.86 ± 2.96 μM, 6.09 ± 0.27 nmol/min/mg, and 360 μL/min/mg, and 27.37 ± 2.46 μM, 1.16 ± 0.03 nmol/min/mg, and 420 μL/min/mg, respectively.Kinetic studies on the glucuronidation metabolites of tectorigenin and irigenin were conducted using HLMs. Kinetic profiling revealed that the glucuronidation of tectorigenin and irigenin was best fitted using the Michaelis–Menten equation. The results of an Eadie–Hofstee plot supported the Michaelis–Menten kinetic characteristics . The enzmK and maxV values for tectorigenin glucuronidation with UGT1A1 and UGT1A9 were higher and lower, respectively, than those with HLMs, indicating that UGT1A1 and UGT1A9 co-mediated tectorigenin metabolization. The results also revealed that UGT1A1 had better affinity to irigenin and higher catalytic activity in producing M1 than UGT1A9, suggesting that M1 formation was mainly mediated by UGT1A1. In addition, the enzyme activity of UGT1A9 in producing M2 was similar to that of HLMs.The detailed kinetics parameters of UGT1A1 and 1A9 for M, M1, and M2 are shown in Metabolic biotransformation is the main pathway of drug elimination from the body and can be subdivided into phase I and phase II. Phase I metabolism is mainly mediated by cytochrome P450 systems and comprises functional group reactions, including oxidation–reduction and hydrolysis . Phase ITectorigenin   and irigO-glucuronide and tectorigenin-7-O-sulfate are the primary metabolites, the C-7 position might be the main site targeted by glucuronidation, even though there are three phenolic hydroxyl groups available. Therefore, we speculated that, in the present study, M was tectorigenin-7-O-glucuronide.After the coincubation of tectorigenin with HLMs, one major glucuronidation metabolite was confirmed and detected by high-performance liquid chromatography coupled with TripleTOF 5600 quadrupole-time-of-flight mass spectrometry (Q-TOF-MS) and quantified using the RF method. RF values were stable, which demonstrated that using a prototype drug with its respective RF is a valid means to accurately calculate the parameters of tectorigenin. Chemical inhibition and recombinant enzyme experiments showed that multiple UGT subtypes exhibited glucuronidation activity toward tectorigenin; among them, UGT1A1 and UGT1A9 had the strongest catalytic capacity. Since both UGT1A1 and UGT1A9 are highly expressed in the liver   were detected. These results are consistent with a previous in vivo study showing that irigenin, the deglycosylation product of iridin, is metabolized into two glucuronide conjugates . SimilarTectorigenin and irigenin show good pharmacological activity and have been identified as candidate bioactive compounds ; howeverd-glucaric acid 1,4-lactone monohydrate were purchased from Meilunbio . Pooled HLMs  were purchased from the Research Institute for Liver Diseases . Nine recombinant human UGT enzymes (UGT1A1/1A3/1A4/1A6/1A9/2B4/2B7/2B15/2B17) were obtained from BD Biosciences . HLMs and recombinant UGTs were stored in a freezer (−80 °C) until use. All other reagents were supplied by the Sinopharm Group .Tectorigenin and irigenin were supplied by Yuanye Biotechnology Co., Ltd. . UDPGA, silibinin, 1-naphtol, fluconazole, trifluoperazine, and β-glycosidase were obtained from Sigma-Aldrich . Alamethicin and 2 (10 mM), HLMs (0.5 mg/mL), alamethicin (50 μg/g protein), 1,4-lactone monohydrate (5 mM), and UDPGA (1 mM). After the mixture was incubated for 30 min (37 °C), the reaction was stopped by ice-cold methanol. After centrifugation, the supernatant was used to qualitatively analyze the metabolites.To analyze metabolic stability, tectorigenin and irigenin were incubated with HLMs to explore their metabolic stability, as previously described ,29. In s18 column ; mobile phase A was 0.1% acetic acid and 2 mM ammonium acetate in water, while mobile phase B was acetonitrile. Gradient elution was performed as follows: 0–5 min, 90% to 80% A; 5–20 min, 80% to 50% A; 20–21 min, 50% to 40% A; 21–25 min, 40% to 10% A. Q-TOF-MS in positive electrospray ionization source (ESI mode) was used with a generic method for data acquisition. The mass range from m/z 100 to m/z 1200 was fully scanned. Data were acquired and processed using Analyst and Peakview (AB Sciex), respectively. The molecular weight of tectorigenin and irigenin glucuronides was determined using the HPLC–Q-TOF-MS method. Chromatographic analysis was conducted using an ODS CAs it is difficult to obtain sufficient amounts of tectorigenin and irigenin glucuronides for quantitative analysis, the RF method was used to calculate their concentrations. Tectorigenin and irigenin glucuronides were prepared with HLMs (1.0 mg/mL) in the reaction described in prototype drug), whereas the remainder was directly injected into the column to determine the peak areas of the metabolite (Ametabolite). The RF was expected be similar at different concentrations, and was calculated as follows:metabolites and Aprototype drug are the peak areas of metabolites and prototype drugs (tectorigenin and irigenin), respectively.A part of each fraction (200 μL) was co-incubated with β-glucuronidase (1000 U/mL) for 12 h at 37 °C to hydrolyze the metabolites and determine the peak areas of tectorigenin or irigenin , microsomes (0.5 mg/mL), 1,4-lactone monohydrate (5 mM), alamethicin (50 μg/g protein), UDPGA (1 mM), and different concentrations of tectorigenin or irigenin (1–300 μM) in Tris-HCl buffer . The mixture was incubated at 37 °C for 30 min; then, the reaction was stopped by adding ice-cold methanol. After the mixtures were centrifuged , and the supernatant was analyzed by HPLC. Michaelis–Menten parameters (maxV and mK) were determined on the basis of the nonlinear regression of the plot of the substrate concentration vs. rate of metabolite formation.To obtain suitable incubation conditions, we first evaluated the linearity of metabolite formation at different times (10–60 min), the UDPGA concentrations (0.5–5 mM), and the microsome protein concentrations in HLMs (0.2–2 mg/mL). The final incubation medium used in the experiment contained MgClSeveral well-characterized UGT substrates were used as “inhibitors” to examine the involvement of UGTs  in the gUGT reaction screening of tectorigenin and irigenin was conducted using nine commercially available human UGTs (UGT1A1/1A3/1A4/1A6/1A9/2B4/2B7/2B15/2B17). Furthermore, we studied the enzymatic kinetic characteristics of tectorigenin and irigenin using UGT1A1 and UGT1A9. Six concentrations of tectorigenin and irigenin  were incubated in triplicate in order to obtain the enzyme kinetic characteristics of tectorigenin and irigenin glucuronide.Km and Vmax of enzymes were calculated as follows:p-value < 0.05 was considered statistically significant.For both HLMs and UGTs, metabolic kinetic analysis was performed by fitting the data to the Michaelis-Menten model. The This study provides important information on the glucuronidation profiles of tectorigenin and irigenin, two important isoflavones in Shegan. In HLMs, tectorigenin and irigenin were converted to one and two glucuronides, respectively. UGT1A1 and UGT1A9 were identified as the main enzymes catalyzing glucuronidation of tectorigenin and irigenin. Both enzymes could produce tectorigenin glucuronide M, whereas UGT1A1 mostly generated irigenin glucuronide M1, while UGT1A9 mostly generated irigenin glucuronide M2. These results point to the need of monitoring drug pharmacokinetics when a Chinese medicine containing these two isoflavones is co-administered with clinical drugs sensitive to UGT1A1- and/or UGT1A9-mediated glucuronidation.


Vibrionaceae, Aeromonadaceae) were more prevalent, and probiotic taxa  were less frequent in the guts of individuals from the dump site, than those from the reference site. The differences in vulnerability of various bacterial taxa inhabiting cod gastrointestinal tracts to CWAs were hypothesised to be responsible for the observed microbiota dysregulation.Several hundred thousand tonnes of munitions containing chemical warfare agents (CWAs) are lying on the seafloor worldwide. CWAs have started leaking from corroded munitions, and their presence in the environment and in organisms inhabiting dump sites has been detected. The presence of CWAs in the water negatively affects fish, macrobenthos and free-living bacteria. It can be expected that the presence of CWAs would also affect the gut-associated bacteria in fish, which are vital for their condition. The main aim of this study was to test if the microbiota of cod collected in the Baltic Bornholm Deep (highly polluted with CWAs) is dysregulated. To investigate this, we conducted metagenomic studies based on 16S rRNA gene sequencing. We found that the microbiota of cod inhabiting the dump site was significantly less taxonomically diverse compared to those from a non-polluted reference site. Moreover, taxa associated with fish diseases (e.g.,  Chemical warfare agents (CWAs) are the toxic components of chemical weapons. They include choking agents designed to impede a victim’s ability to breathe , vesicant agents designed to inflict chemical burn injuries upon contact with the victim’s skin  and nerve agents designed to fatally interfere with the victim’s nervous system . During the past century, due to the enormous military potential of CWAs, they were mass produced and often exploited in numerous international conflicts.The mass disposal of several hundred thousand tons of unwanted, obsolete or captured chemical munitions was costly and problematic. Thus, the most common means of getting rid of unused chemical munitions was dumping them into the seas and the oceans. Sea-dumping operations took place worldwide . SeveralThe sea-dumping of chemical munitions continued until the early 1970s. Nowadays, they pose an immense threat to numerous aquatic ecosystems and human well-being ,7,8. RecMytilus trossulus), fish from the Mediterranean Sea (Blackbelly rosefish Helicolenus dactylopterus and European conger Conger conger) and fish from the Baltic Sea  [The CWAs toxicity to demersal fish has long been estimated using mathematical modelling, which screened the risk profiles of various CWAs based on their chemical structure  . Accordillarias) ,16,21.Besides geno- and cytotoxicity, CWAs could exert other negative effects on the aquatic organisms, i.e., the putative effect on the microbiota  inhabiting the gastrointestinal (GI) tracts of fish. The composition and diversity of GI microbiota play an important role as indicators of both water contamination  and the The presence of various anthropogenic pollutants in the aquatic environment, such as copper , lead 2 and micrGadus morhua callarias, Linnaeus, 1758), a keystone species in the Baltic ecosystem and an economically important resource. The major spawning site of the vast majority of the G. morhua callarias population, the Bornholm Deep [G. morhua callarias is especially prone to CWAs exposure: individuals of this species live in the vicinity of submerged military objects [Among the fish species that are particularly threatened by CWAs is the eastern Baltic cod  and to compare it to the GI microbiota of cod collected at a chemical warfare dump site . We tested two hypotheses: first, that the taxonomic compositions  of the GI microbiota of cod collected at a CWAs polluted site and at reference site are different and, second, that the taxonomic diversity of the GI microbiota of cod collected at a chemical warfare dump site is altered in comparison to that of cod collected at a reference site.The aim of this study was to analyze the hitherto undescribed GI microbiota of eastern Baltic cod . Two sampling sites were chosen: (1) the designated dumping area in the Bornholm Deep, around 55°18.949′N, 15°34.756′ E, where residual cod were previously confirmed to bioaccumulate CWAs-related compounds, which signifies considerable exposure to CWAs contamination [Specimens of mination , and (2)Upon capture and length measurements, individuals of adequate length were stunned by a blow on the head and afterwards put to death by severing the spinal cord and placed on ice. Next, in order to collect the GI tracts, the peritoneal cavity of each fish was aseptically opened using a scalpel, and the intestine was freed from the connective tissue, as well as adjacent internal organs. Afterwards, the esophagus was cut, and the whole GI tract  was isolated. In order to extract its contents , the GI tract was cut lengthwise using a same scalpel. Then, the entire contents were gently transferred to separate sterile “falcon”-type tubes (15 mL), and the samples were homogenized using a hand-held homogenizer. The samples were flash-frozen and kept at −80 °C until further processing. The whole extraction procedure was conducted in sterile conditions, using a new scalpel blade for each individual GI tract.In order to isolate the total DNA from the fecal matter, the FastDNA™ Spin Kit for Feces  was used, according to the manufacturer’s protocol, with 500 mg (wet mass) of each homogenized fecal matter sample representing individual fish.The V3–V4 hypervariable region of the 16S rRNA encoding gene was amplified in the samples using universal primers 341f (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGnGGCwGCAG-3′) and 785r (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTAChvGGGTATCTAATCC-3′), synthetized by Sigma-Aldrich, Saint Louis, Missouri, USA. The master mixes were prepared using the KAPA HiFi PCR Kit , and the PCR was run using the Eppendorf™ Mastercycler nexus X2 . The PCRs were performed in a 25 µL final reaction volume with 26 cycles of 98 °C (20 s), 55 °C (15 s) and 72 °C (15 s). The quality of DNA at each step was checked (including negative controls) by agarose gel electrophoresis and by measuring the double-stranded DNA concentration using the Qubit™ dsDNA HS Assay Kit on the Qubit™ 2.0 fluorometer . Finally, the samples containing PCR products were frozen at −20 °C until further analyses. The PCR was run in two repetitions for every individual sample while preparing new DNA template dilutions and new master mixes for each repetition. Before sequencing, two samples containing amplicons representing the microbiota of an individual GI tract (replicates) were pooled.The amplicons were sequenced using an MiSeq (Illumina) platform on a single run using the MiSeq Reagent Kit v2  and the paired-end method (2 × 300 bp), according to the standard protocols by Genomed .Demultiplexing and trimming of Illumina adapter sequences (cutadapt software v3.5 ) was perα and β diversities, as well as the mean relative abundances of dominant ASVs, were analyzed between the studied variants. To measure α diversity, the Chao1 estimator and Shannon index were calculated. Chao1 is an estimator measuring the total richness, which is particularly useful because of a valid variance, which can be used to calculate associated confidence intervals [β diversity, Jaccard, Bray–Curtis, unweighted UniFrac and weighted UniFrac indices were calculated. The Jaccard and unweighted UniFrac indices take into account only the number of observed ASVs, whereas the Bray–Curtis and weighted UniFrac indices consider both the number of observed ASVs and their relative abundance. The UniFrac indices additionally incorporate phylogenetic distances between observed ASVs. Basing on the mean relative abundance of ASVs in the samples, dominant phyla (top five most abundant ASVs in either group), as well as dominant families and genera (top ten most abundant ASVs in either group), were calculated. α diversity indices’ values were statistically compared using the Mann–Whitney U Test . The bootstrap resampling method (1000 iterations) was used to calculate the confidence intervals for the differences between the mean relative abundances of dominant ASVs . β diversity indices’ values were statistically compared using the permutational analysis of variance (PERMANOVA). The statistical analyses were performed in R Studio [The diversity of ASVs between the samples originating from the GI tracts of cod from the reference site and the CW dump site was analyzed. Both ntervals , whereasntervals . For theR Studio  and usinR Studio .Firmicutes, Fusobacteriota, Proteobacteria, Actinobacteriota and Spirochaetota were the predominant bacteria in the GI tracts of the studied G. morhua callarias (in either group) at the level of the phylum classification. Significant differences in the mean relative abundances between the reference site and the CW dump site were observed for two phyla, Actinobacteriota (whose abundance was decreased) and Spirochaetota (whose abundance was increased), in the gut content of fish from the CW dump site  comprised Cetobacterium, Aeromonas, Macellibacteroides, Sulfitobacter, Tyzzerella, Escherichia-Shigella, Photobacterium, Brevinema, Aliivibrio and Candidatus Bacilloplasma. Among them, the relative abundances of Aeromonas, Macellibacteroides, Brevinema and Aliivibrio were higher, while the relative abundance of Sulfitobacter were lower  and Shannon index  (The = 0.015) .G. morhua callarias originating from the CW dump site was notably distinct compared to those from the reference site, as evidenced by dissimilarity indices: Jaccard , Bray–Curtis , unweighted UniFrac  and weighted UniFrac . The NMDS  plots revealed clear clustering of samples corresponding to the two studied groups . The dominant taxa observed in our study mostly reflected typical microbiota of marine fish [The metagenomic analysis, based on 16S rRNA gene sequencing, allowed the indication of the main phyla, families and genera in the GI microbiota of ine fish ,48, withine fish .Actinobacteriota, known for their probiotic mode of action in the guts of animals [Rhodobacteraceae, which are symbionts known for synthesizing vitamin B12 and their probiotic properties [Clostridiaceae, solely associated with plant-based diets in fish [Sulfitobacter, which comprise probiotic bacteria that are capable of inhibiting the growth of bacterial fish pathogens [Aeromonadaceae, Brachyspiraceae and Vibrionaceae, as well as the genera Aeromonas, Brevinema and Aliivibrio, whose various species are opportunistic pathogens associated with fish intestine diseases [We found significant differences between the taxonomic compositions of cod GI microbiota from the CW dump site versus reference site, which allowed us to confirm the first hypothesis of this study (that the taxonomic structure of the GI microbiota of cod collected at the two sites are different). Generally, probiotic bacteria were less abundant, and pathogenic bacteria were more abundant, in the GI tracts of cod from the CW dump site, compared with the reference site. With regard to microorganisms considered to be probiotic, the following groups of bacteria at different taxonomic levels were found to be highly reduced in the GI microbiota of cod from the CW dump site: (i) at the phylum level,  animals ,50; (ii)operties , and Clo in fish ,53,54;  were notably lower for the GI microbiota of cod collected at the CW dump site, as compared to cod collected at the reference site, indicating a significant decrease in the microbial diversity in the GI tracts of fish living in the vicinity of sea-dumped chemical munitions. These differences of the microbial diversity together with a significant change in the taxonomic composition of GI microbiota, with probiotic taxa diminishing and pathogenic taxa proliferating, are a symptom of dysbiosis, which is known to have severe consequences for the host. Dysregulation of the taxonomic composition of the GI microbiome in fish impairs their performance, as it is correlated with intestinal inflammation and chronic diseases [The second hypothesis of this study  was also confirmed: the values of the diseases ,29,30,31β diversity , indicated notable differences in the taxonomic composition  of the GI microbiota of G. morhua callarias originating from the CW dump site, as compared to those originating from the reference site. The similarity of the GI microbiota composition of cod originating from the same site and the dissimilarity of the GI microbiota composition of cod originating from different sites  additionally confirmed that the fish have been living in these sites for a time long enough to develop a site-specific microbiota, originating from adaptation to local habitats .The values of G. morhua callarias collected from the studied dump site were earlier confirmed to come into contact with, as well as bioaccumulate, CWAs-related compounds [Despite the fact that both sites were slightly different in various physico–chemical parameters , family (Rhodobacteraceae) and genera (Sulfitobacter) levels, and (2) cod from the CW dump site had significantly higher abundances of pathogenic bacteria, as seen at the phylum , family  and genus  levels. Moreover, the GI microbiota of cods collected at the CW dump site expressed significantly reduced taxonomic diversity, as well as an overall distinct taxonomic composition  when compared to those collected at the reference site.The results of this study were the first to describe the GI microbiota of eastern Baltic cod. The GI microbiota was typical for marine fish and very similar to the GI microbiota of Atlantic cod. However, the taxonomic structure of the GI microbiota of cod collected at the two studied sites was significantly distinct. The two most important differences were (1) cod from the CW dump site had significantly lower abundances of probiotic bacteria, as seen at the phylum (The obtained results could become an important starting point for the future studies on the evanescing populations of this species in the Baltic Sea.


To compare the breakage risk of lengthened sacroiliac screws and ordinary sacroiliac screws to treat unilateral vertical sacral fractures and provide a reference for clinical application.A finite element model of Tile C pelvic ring injury  was produced. The sacral fractures were fixed with a lengthened sacroiliac screw and ordinary sacroiliac screw in 6 types of models. The maximal von Mises stresses and stress distributions of the two kinds of screws when standing on both feet were measured and compared.The maximal von Mises stress of the lengthened screw was less than that of the ordinary screw. Compared with ordinary screw, the stress distribution in the lengthened screw was more homogeneous.The breakage risk of screws fixed in double segments is lower than that of screws fixed in single segments, the breakage risk of lengthened screws is lower than that of ordinary screws, and the breakage risk of screws fixed in S2 segments is lower than that of screws fixed in S1 segments. Unilateral sacral fractures caused by high-energy injuries are rare , and mosVarious methods for vertically unstable sacral injuries have been advocated, including transiliac rods , transilTo maximize the effect of sacroiliac screws, we performed research –20 on orThis study aims to make a biomechanical comparison of two kinds of sacroiliac screws in various modes for fixing unilateral longitudinal sacral fractures using the three-dimensional finite element technique, assess factors involved in screw breakage and provide a theoretical basis for clinical practice.The finite element method  was adopFinite element models were constructed from computed tomography ) images of a normal female . The slices were 1 mm thick. A virtual 3D model of the sacrum and innominate was created from CT data in DICOM format with image processing software (mimics 10.0). The geometric extents of pelvic cortical and trabecular bones were defined based on the surface mesh. By using four noded linear solid tetrahedral elements with an average edge length of 2 mm, we created an unstructured mesh of bone trabeculae in Abaqus/CAE. A triangular shell element represents the cortical bone surrounding the trabecular bone. The thickness of the shell element is 2 mm . This se2 and 0.2 for trabecular bone and 18,000 N/mm2 and 0.3 for cortical bone [We assumed a tied condition between the inner surface of cortical bone and the surface of the trabecular bone. The Young’s modulus and Poisson’s ratio were taken to be 150 N/mmcal bone . The bilWe cut the sacrum halfway through the right sacral foramina to construct a unilateral sacral fracture  model. The final finite element normal pelvis and unilateral sacral fracture are shown in Fig. Six fixation cases were imitated for finite element analysis: ① one lengthened sacroiliac screw fixation in S1 segment (LS1) ② one lengthened sacroiliac screw fixation in S2 segment (LS2) ③ one lengthened sacroiliac screw fixation in S1 and S2 segments respectively(LS12) ④ one ordinary sacroiliac fixation in S1 segment (OS1) ⑤one ordinary sacroiliac fixation in S2 segment (OS2) ⑥ one ordinary sacroiliac fixation in S1 and S2 segments respectively (OS12) Fig. .Assembly was accomplished by placing constraints between interacting surfaces. These interaction surfaces were located at the bone–implant interface between the sacrum, the sacroiliac cartilage and the ilium, the pubic rami and interpubic disc, and the bone–implant interfaces in the screw thread. In the screw stem regions, frictionless sliding contact was used between the interaction surfaces of the bone–implant interfaces. We apply a penalty contact with a coefficient of friction of 0.3 on the fracture interaction surface. They were used to simulate sliding patterns between fracture interaction surfaces. The acetabular rotation centre was taken as the fixed point to fix the acetabulum to simulate the mechanical transfer of bipedal standing.The results of this study show the following:In either model, the stress concentration area of the screw was found in the corresponding screw area of the sacral fracture area.The stress distribution of the lengthened sacroiliac screw was more homogeneous than that of the ordinary sacroiliac screw.The screw stress distribution in the model of different fixed segments of the same screw was more concentrated in the fixed single sacral segment than in the fixed two sacral segments.When both sacral segments were fixed, the distribution of screw stress in the upper segment was more concentrated, and the distribution of screw stress in the next stage was more uniform.The maximal von Mises stress of one sacroiliac screw fixation in the S1 or S2 segment was greater than that of one lengthened sacroiliac screw fixation in the same sacral segment;The maximal von Mises stress of one sacroiliac screw fixation in the S1 and S2 segments was greater than that of one lengthened sacroiliac screw fixation in the S1 and S2 segments;The maximal von Mises stress of one lengthened sacroiliac screw fixation in the S1 or S2 segments was greater than that of one lengthened sacroiliac screw fixation in the S1 and S2 segments;The maximal von Mises stress of one sacroiliac screw fixation in S1 or S2 segments was greater than that of one sacroiliac screw fixation in S1 and S2 segments Table .Sacral fracture is a common occurrence in pelvic ring injury. The most common type is a unilateral sacral injury with anterior impaction of the sacrum, a lateral compression type 1 (LC-1) injury, which is usually associated with posterior ring instability and requires clinical treatment. A sacroiliac screw is a conventional internal fixation technique for posterior pelvic ring injury . SacroilThe maximum von Mises stress, one of the fundamental safety indicators of screws, increased with increasing screw fracture risk. The higher the stress is, the greater the likelihood of screw failure. The following results can be summarized from this study. When comparing different fixation modes with the same kind of screws, we found that the maximum von Mises stress was the largest, with only fixed S1 segments, and the minor model, with only fixed S2 segments. At least in the model, S1 and S2 are both fixed. Second, when fixing the S1 segment and S2 segment simultaneously, the maximum von Mises stress of the S1 segment screw in the same model was similar to that of the S2 segment screw, regardless of whether it was a lengthened screw model or an ordinary screw model. Third, if considered from the fixed segment, the screw fracture risk of double-segment fixation was lower than that of single-segment fixation. In double-segment fixation, the fracture risk of the two screws was similar. When different screws were used to compare the same fixed segment, we found that the maximal von Mises stress of the lengthened screw was lower than that of the ordinary screw, and the lengthened screw had a lower fracture risk than the ordinary screw.In summary, from the perspective of screw safety, it is recommended to use lengthened screws for fixation. The safest method is to fix the S1 and S2 segments with extended sacroiliac screws. Normal sacroiliac screws are recommended for S1 and S2, even in the absence of lengthening sacroiliac screws. If only one screw can be used, S2 segment fixation is recommended regardless of the sacroiliac screw. The results of the screw safety analysis are consistent with those of the stability analysis.The risk of a fatigue fracture in internal fixation can be reduced by avoiding excessive concentration in certain parts through the uniform distribution of stress in internal fixation. However, in this study, when we compared the stress distribution, we found that the stress distribution of screws was not uniform in any model. Compared with the different kinds of screws we used, the stress distribution of the lengthened sacroiliac screw was more uniform. Compared with different fixed segments, the stress distribution of double-segment fixed screws was more uniform. Similarly, these findings are consistent with the stability analysis results and the maximum von Mises stress analysis.The following points need to be noted in this study. First, although anterior ring instability is characteristic of type C pelvic ring injuries, considering the multiple fixation methods of anterior ring fractures may affect the stability of the posterior pelvic ring. This study did not imitate the anterior pelvic ring’s injury and fixation but only maintained the anterior pelvic ring’s normal state. The anterior pelvic ring had a slight effect on the stability of the posterior pelvic ring and did not affect our comparison of several study models. Second, to best simulate pelvic stability, we reserved multiple important pelvic ligaments in our research. Meanwhile, to eliminate any unpredictable forces that might affect the measurements, we did not simulate muscles to simulate the extra stability they would cause. Muscle parameters, joint data parameters, and joint flexibility settings will qualitatively influence the results. However, the calculation is too complicated to complete the experiment, so they are simplified , 27. ThiThe breakage risk of screws fixed in double segments is lower than that of screws fixed in single segments, the breakage risk of lengthened screws is lower than that of ordinary screws, and the breakage risk of screws fixed in S2 segments is lower than that of screws fixed in S1 segments.


Cystatin C, an inhibitor of cysteine protease, has been used as a biomarker for estimating glomerular filtration rate. However, the causal relation between cystatin C and diabetic nephropathy remains uncertain.GWAS=361194) on diabetic nephropathy (Ncase/Ncontrol up to 3283/210463). Multivariable MR (MVMR) was performed to assess the stability of cystatin C’s causal relationship. Two-step MR was used to assess the mediation effect of BMI and SBP.We assessed the causal effect of cystatin C together with other five serum biomarkers including KIM-1, GDF-15, TBIL, uric acid, and Scr on diabetic nephropathy by Mendelian randomization (MR) analysis. 234 genetic variants were selected as instrumental variables to evaluate the causal effect of cystatin C . After adjusting for the potential confounders BMI and SBP, cystatin C maintained its causal effect on the DN , which means that the risk of DN increased by 17% with an approximate 1 standard deviation (SD) increment of serum cystatin C level. Two-step MR results indicated that BMI might mediate the causal effect of cystatin C on diabetic nephropathy.Our findings discovered that cystatin C was a risk factor for diabetic nephropathy independent of BMI and SBP in diabetes mellitus patients. Future research is required to illustrate the underlying mechanism and prove targeting circulating cystatin C could be a potential therapy method. DN i2 . The theMany risk factors have been recognized to be related with the development and progression of DN in the recent decade . SeveralMendelian randomization (MR) can explore whether risk factors are causally linked to the outcome by analyzing genetic variants as instrumental variables, which represents with single nucleotide polymorphisms (SNPs). Since the gene randomly distributed at conception, MR can mimic randomized trials and minimize the effect of confounders biasing observational studies . Benefitvia serum biomarkers. Two-sample univariable Mendelian randomization was implemented to evaluate the causal association between multiple serum biomarkers  and DN. Biomarkers with significant causal effects on the outcome will be further searched for potential confounders from published articles and Phenoscanner V2 (http://www.phenoscanner.medschl.cam.ac.uk/). Multivariable Mendelian randomization that included biomarkers with significant causal effect and their confounders will be implemented to validate their causal association. Once a causal relationship was established, two-step MR was used to investigate whether confounder plays as a mediator between cystatin C and DN . Genetic variants that were highly associated with cystatin C, creatinine, urate, and total bilirubin were selected from a GWAS cohort conducted by the Neale Lab Consortium including 361194 individuals of European ancestry . IVs for KIM-1 and GDF-15 were obtained from a meta-analysis including up to 21,758 individuals of European ancestry . The BMI GWAS used for two-step MR was obtained from the Genetic Investigation of Anthropometric Traits (GIANT) Consortium including 681,275 samples of European ancestry  were extracted from GWAS data and SNPs with high linkage disequilibrium were removed. Independent SNP is defined by rancestry . Geneticancestry . Genetic-8). Genetic variants of GDF-15 were obtained with a lower threshold (p<1E-5) since few IVs were identified with the original threshold. Palindromic SNPs were further excluded from the IV list. For those instruments that are missing in the outcome, proxy SNP with LD score>0.8 was used. In order to satisfy MR assumption three, SNPs with significant association with the outcome were excluded. The F statistic was calculated using the formula: F = beta2/se2, where beta represents the effect of SNP on the exposure and se is the standard error of the beta, to assess whether there is a possibility of weak instrument bias  × beta2, where EAF represents the effect allele frequency of the SNP, represents the proportion of variance of the exposure explained by SNPs (Diabetic nephropathy as outcome was defined when there was glomerular disorders in the patients with diabetes mellitus with the criterion of ICD-10 (code: N08.3*), summary statistics of which was from FinnGen biobank including 213,746 European individuals (3283 cases and 210463 controls) . Except ent bias . R2 calc by SNPs .In this study, inverse variance weighted (IVW) analysis was utilized as the major statistic method. Meanwhile, MR-Egger regression, weighted median, and MR-PRESSO were also performed as complementary methods to validate the IVW result. The IVW method could combine each genetic variant’s Wald estimate in a meta-analysis model and produce unbiased result if horizontal pleiotropy is balanced . MR-Eggehttps://shiny.cnsgenomics.com/mRnd/) with a type 1 error rate of 0.05 (Multivariable MR (MVMR) was performed to assess the stability of the significant causal relationships, which could estimate the causal relationship between each exposure and a single outcome, producing a causal estimate of direct effect and adjusting pleiotropy caused by other exposures that were included in the MVMR analysis . Two-ste of 0.05 . Two-sidSummary information of selected IVs of 6 exposures was presented in Among 6 serum biomarkers, only cystatin C has a significant causal effect on diabetic nephropathy as a risk factor  Figure 2Although horizontal pleiotropy may exist with a p<0.05 as suggested with the global test of MR-PRESSO, the distortion test showed that there is no difference whether the pleiotropic outliers were removed or not (p=0.91). Therefore, we further removed all SNPs identified by MR-PRESSO which may cause horizontal pleiotropy . The resulting data showed that cystatin C has a greater causal association with diabetic nephropathy , and Cochran’s Q-test no longer showed evidence of heterogeneity (IVW p>0.05) Table S7–200), but not with DN   Table S1Next, we performed multivariable MR to further analyze the direct effect of cystatin C on diabetic nephropathy. Through online searching IVs selected for serum cystatin C with Phenoscanner V2, we found several potential confounding phenotypes . Finally, we chose BMI and SBP as adjusted confounders in the MR analysis for the following reasons: First, BMI and SBP were the most frequent phenotypes in the process of searching among all the potential confounding phenotypes. Second, BMI and SBP were reported to be risk factors in published MR analysis , 28. SubNext, we performed two-step MR to further investigate whether BMI, SBP functioned as mediator between cystatin C and DN. It showed that there was a casual association between cystatin C and BMI . After adjusted for cystatin C, BMI showed detrimental effect on DN . The proportion mediated by BMI was 15.2%  Figure 4In the present study, we took advantage of MR analysis to thoroughly examine the causal association between six serum biomarkers  and DN. Among the six biomarkers, only cystatin C positively associated with the risk of DN. There was no statistical significance for the genetic relationship among the other five biomarkers and DN.Several studies have shown that high levels of serum urate might cause CKD progression through a mechanism such as excessive production of nitric oxide, activating the renin-angiotensin system, stimulating the proliferation of vascular smooth muscle cell, and obstruction from urate crystals. However, a previous MR study showed that there is no causal relation between serum urate levels and CKD  and randAlthough KIM-1, GDF-15, and TBIL were previously reported to contribute to tubular injury , 32 and Cystatin C, a cysteine protease inhibitor, regulating the activity of cathepsins S and K, have multiple functions in human vascular pathophysiology , which iThe mechanisms underlying the casual relationship between cystatin C and DN are unclear. There are several potential explanations. Firstly, cystatin C function as a cysteine proteinase inhibitor, which regulated the protease-antiprotease activities of the vascular wall. Thus, the imbalance between cystatin C and cysteine cathepsins might lead to the remodeling of the vascular wall . Second,Our study has some limitations: First, since the GWAS derives from European ancestry, generalizability to other ethnicities is limited. Second, due to lacking of individual-level data of GWAS, we cannot explicitly present the baseline data of participants or further stratify serum cystatin C to calculate more detailed causal effect. Third, despite we adjust potential pleiotropy, there is still chance for other confounders to influence the causal estimate. Hence, future research is warranted for further validating our findings.Our results suggest that there was a causal relationship between serum cystatin C and DN in diabetic patients, which warns us that cystatin C is not only a biomarker bur also a risk factor for DN progression.https://gwas.mrcieu.ac.uk/).Summary statistics are available from each consortium  or via the MR-Base platform  and Natural Science Foundation for Distinguished Young Scholars of Hunan province (2021JJ10075).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.


Competitive tenders on pharmaceuticals are one of the most effective cost-containment instruments in healthcare systems. Its effectiveness has been demonstrated, among other things, in markets for generic medicine and biosimilars. In Denmark, an internationally unique model for competitive tenders on analogue substitutable pharmaceuticals has been developed and implemented for all public hospitals.We obtained data on all analogue competitive tenders carried out by the Danish Medicines Council from its foundation on January 1, 2017, to October 9, 2020. We calculated univariate descriptive statistics, pairwise correlations and made a multiple regression analysis on tender savings.Average annual saving on hospital pharmaceutical purchase prices was 44.1% ranging from 0.4% to 92.8% between therapeutic areas and areas of indication. There was a significant positive correlation between tender savings and the number of competitors participating in the tender, and a significant negative correlation between tender savings and the number of days since market authorization.This study finds analogue tenders to be similar in effect and mechanism to competitive tenders in markets for generic medicine and biosimilars. It supports the increasing number of empirical findings that competitive tendering has a high potential to generate substantial savings on healthcare budgets. Public expenditure on healthcare absorbs a significant and growing share of many countries' resources. Latest figures from the Organisation for Economic Co-operation and Development (OECD) shows that health spending in OECD countries has grown from around 7.9% in 2003 to 9.7% in 2020 measured as a share of gross domestic product (GDP) . This leMore than 70% of healthcare spending across OECD countries is funded from public sources . To counOne of the most effective cost-containment instruments is competitive tenders on pharmaceuticals. Competitive tendering is used when equivalents for a specific medicine are available and can be defined as: “The acquisition of pharmaceuticals based on a competitive bidding process where the contract is granted to the pharmaceutical supplier who offered the best bid following strict criteria” . CompetiWhile tenders can reduce acquisition costs, they may also expose the healthcare system to risks of negative consequences. Such negative consequences can stem from poor tender practice by the policy makers and purchasers, and from unwanted market responses from the pharmaceutical companies. Poor tender practice includes non-transparent tender practices, a lack of consistency, unclear tender award criteria, a focus on lowest price only, single-winner tendering, and a lack of impact monitoring , 16. RisMost OECD countries use competitive tendering for purchasing pharmaceuticals to some extent, however mostly in hospital settings on mature markets for generic medicine , 19, 24.Recently, competitive tendering has been applied with positive results outside the area of generic medicine. This includes biosimilar drugs, where empirical experiences show tender outcomes and mechanisms comparable to generic medicine tenders –27.In Denmark, a model for competitive tenders on analogue substitutable pharmaceuticals was formally implemented with the establishment of the Danish Medicines Council (DMC) in 2017 , 29. AnaMost international research literature on pharmaceutical tendering is exploratory in nature. Remarkably little empirical research has been conducted to date probably because data are difficult to collect . To our knowledge, the Danish competitive tendering system specially designed for analogue competition on hospital pharmaceuticals is internationally unique .The purpose of this study is therefore to describe and evaluate the experiences from competitive tenders on analogue substitutable pharmaceuticals in Denmark in the period 2017–2020.The Danish hospitals are publicly owned and run by the five Danish regions, and all in- and out-patient services are delivered free of charge to citizens. There are no charges for medicine provided to patients at the hospital. Almost all hospital pharmaceuticals (approximately 98%) are purchased by the regions’ central buyer organization, Amgros, who carries out tenders and negotiations with producers and wholesalers. Pharmaceutical pricing is free in Denmark, but historically there have been several attempts to control prices . At the We obtained data from Amgros on all analogue competitive tenders carried out by the DMC from its foundation on January 1, 2017, to October 9, 2020 . These aData were provided in excel format on a USB stick after signing the research agreement. The material included the tender results from nineteen tenders covering eight different therapeutic areas at Anatomical Therapeutic Chemical classification system (ATC) level five . The dataset contained information on the specific pharmaceuticals that were included in the tenders, application areas in the therapeutic area, tender price, number of competitors bidding, and promised market share for the winner. Tender prices were confidential. Amgros also provided data on the total annual consumption of each of the nineteen winning pharmaceuticals from the period May 2020 to April 2021.Date of marketing authorization for each winning pharmaceutical was obtained from European Medicines Agency (EMA) . OfficiaWe applied standard economic theory on tendering as analytical framework –15. The We calculated univariate descriptive statistics for all the variables including mean, median, minimum, and maximum as well as standard deviation. To assess associations, we calculated pairwise correlations between all our variables. Finally, to elaborate on the pairwise associations we made a multiple regression analysis (ordinary least squares) of the discount rate on all the remaining variables .Average annual saving on hospital pharmaceutical purchase prices was 44.1% obtained through competitive analogue tender  and the number of competitors participating in the tender Fig. . There wThe regression analysis explained approximately 72% of the variation in the savings at tenders Table . ControlThis study is the first to quantitatively assess the effect of competitive analogue tenders on pharmaceuticals for public hospitals. Our results indicate that analogue tenders are remarkably similar in effect and mechanism to competitive tenders in hospital markets for generic medicine and biosimilars.The calculated cost savings in Danish hospitals were 44% (0.4–92.8%) on pharmaceuticals purchased through competitive analogue tenders. In monetary terms, the annual savings were estimated to approximately DKK 1.1 billion corresponding to 6–7% of the annual total hospital consumption of pharmaceuticals in Denmark measured in pharmacy purchase prices (PPP) . These fThe results should not be interpreted as a causal analysis of competitive analogue tenders. The therapeutic areas in the sample were selected by the DMC, and selection criteria are unknown. We cannot conclude that analogue tenders create possibilities for large public savings in general, but it does in a number of situations.Results also show a huge variation in savings ranging from 0.4% to 92.8%. This appears to be a larger variation compared to tenders on generics and biosimilars . One posThis study supports the increasing number of findings that competitive tendering has a high potential to generate substantial savings in OECD health systems –7. PubliThere is no suggestion in economic theory that competitive tendering will lead to lower spending levels in the public healthcare system as a whole . The savThis study shows that competitive tendering on analogue substitutable pharmaceuticals can provide large savings on public healthcare budgets. Annual savings on hospital pharmaceuticals purchase prices were 44.1% obtained through competitive analogue tenders. For each additional competitor participating in a tender an extra saving of approximately 5% was obtained. Potential negative long-term effects of the analogue competition program, e.g., companies choosing not to participate in the tender or withdrawing their products from the market should be further investigated in the future.


Myosins belong to a large superfamily of actin-dependent molecular motors. Nonmuscle myosin II (NM II) is involved in the morphology and function of neurons, but little is known about how NM II activity is regulated. Brain-derived neurotrophic factor (BDNF) is a prevalent neurotrophic factor in the brain that encourages growth and differentiation of neurons and synapses. In this study, we report that BDNF upregulates the phosphorylation of myosin regulatory light chain (MLC2), to increases the activity of NM II. The role of BDNF on modulating the phosphorylation of MLC2 was validated by using Western blotting in primary cultured hippocampal neurons. This result was confirmed by injecting BDNF into the dorsal hippocampus of mice and detecting the phosphorylation level of MLC2 by Western blotting. We further perform coimmunoprecipitation assay to confirm that this process depends on the activation of the LYN kinase through binding with tyrosine kinase receptor B, the receptor of BDNF, in a kinase activity-dependent manner. LYN kinase subsequently phosphorylates MLCK, further promoting the phosphorylation of MLC2. Taken together, our results suggest a new molecular mechanism by which BDNF regulates MLC2 activity, which provides a new perspective for further understanding the functional regulation of NM II in the nervous system. We also demonstrate that this process does not depend on the three canonical signaling pathways downstream of TrkB; rather, it is mediated through LYN kinase in the Src family. This study contributes to an enhanced understanding of both the function and mechanism of NM II.in vitro [DIV]7), and B27 was then removed over a 6 h period. This step was followed by BDNF (50 ng/ml) treatment for various time periods. Cell lysates were then collected at different times. NM II activation was monitored by immunoblotting with phospho-MLC2–specific antibodies, and the ratio of p-MLC2 to MLC2 was calculated. As shown in Figure 1A, we found that the phosphorylation level of MLC2 began to increase 30 min after neurons were stimulated with BDNF and continued to increase with time (A and B). BDNF can bind to two receptors on the cell surface with different affinity  and tat-pep5 —30 min prior to BDNF treatment to inhibit the two signaling pathways (C and D). To verify the results in vivo, we injected K252a or BDNF into the dorsal hippocampus of mice and found that the level of p-MLC2 increased after injection of BDNF, whereas the addition of K252a decreased the level of p-MLC2 (E and F). From these in vivo and in vitro experiments, we found that BDNF can modulate NM II activity by regulating MLC2 phosphorylation and that this modulation depends on TrkB.To examine whether BDNF can modulate the activity of NM II, we assessed the phosphorylation level of MLC2 in primary cultured rat hippocampal neurons upon BDNF treatment. Hippocampal neurons were cultured for 7 days . To confirm this result, we synthesized peptides to specifically block the binding of MLCK and MLC2 (TAT-MLCK). When the TAT peptides were added to the medium, MLC2 no longer responded to BDNF (C and D). These results suggest that BDNF regulates the phosphorylation of MLC2 by activating MLCK.The phosphorylation of MLC2 is mainly regulated by two kinases, MLCK and ROCK , 21, and to BDNF , C and DA and B, we found that the phosphorylation level of MLC2 did not change significantly compared with that in the control group after pretreatment with the three signaling pathway inhibitors.After BDNF binds to TrkB, TrkB is dimerized and autophosphorylated to activate the three canonical signaling pathways MAPK, PI3K, and PLC-gamma, which thereby perform their corresponding functions . To addrC and D). We found that the addition of PP1 did not affect the phosphorylation level of MLC2 compared to that in the control group under basal conditions; however, after PP1 pretreatment, BDNF no longer upregulated MLC2 phosphorylation (C and D). These results suggest that SFKs are involved in the phosphorylation of MLC2 triggered by BDNF. In vertebrates, the Src kinase family is composed of nine members: SRC, LCK, LYN, BLK, HCK, FYN, FGR, YES, and YRK. Among them, FYN, SRC, and LYN are distributed throughout the brain (A–D). However, when the expression of LYN was knocked down by siRNA, BDNF no longer triggered an increase in the p-MLC2 level (E and F). This result suggests that LYN is involved in BDNF-induced MLC2 phosphorylation. To further confirm the involvement of LYN in this process, DIV7 neurons were stimulated with BDNF for various times, and the phosphorylation level of LYN was determined. We found that the p-LYN level began to increase significantly 30 min after BDNF addition and returned to the basal level by 2 h (A and B). We further pretreated neurons with bafetinib (a Lyn-specific inhibitor) for 30 min and then stimulated them with BDNF for various times to detect p-MLC2. The results demonstrated that the phosphorylation of MLC2 was no longer regulated by BDNF after treatment with bafetinib to specifically inhibit LYN activity (C and D). Taken together, the aforementioned results show that BDNF activates the downstream LYN kinase to participate in the regulation of MLC2 phosphorylation after binding to TrkB.BDNF increases the activity of Src family kinases (SFKs) following TrkB activation , 28. SFKrylation , C and Dhe brain , 31. To he brain , A–D. HoC2 level , E and Fl by 2 h , A and Bactivity , C and DA and B).Furthermore, we sought to investigate whether LYN binds to TrkB. We generated constructs expressing Myc-tagged LYN, FLAG-tagged TrkBwt, and a FLAG-tagged TrkB–kinase-dead mutant (FLAG-TrkB-KD). We then overexpressed LYN-Myc, FLAG-TrkB, and FLAG-TrkB-KD in the HEK 293 cell line. Binding was detected by coimmunoprecipitation (co-IP). The anti-FLAG antibody M2 and an anti-Myc antibody were used for immunoprecipitation (IP). The results showed that LYN can bind to TrkB. After eliminating the kinase active site in TrkB (TrkB-KD), the binding rate of LYN to TrkB decreased significantly. The results suggest that the binding of Lyn to TrkB depends on the kinase activity of TrkB , A and Bin vitro. We transfected siRNA (si-LYN) into PC12 cells (a neuron-like cell line) to stably express TrkB for 72 h, and we then starved the cells overnight, stimulated them with BDNF for 2 h, and determined the phosphorylation levels of MLCK and MLC2 by WB. The results showed that MLCK phosphorylation decreased significantly after knockdown of LYN; moreover, MLC2 no longer responded to BDNF, and the level of p-MLC2 remained unchanged (A and B). We also sought to determine whether LYN kinase can interact with MLCK. We generated constructs expressing Myc-tagged LYN and HA-tagged MLCK. In HEK293 cells transfected with those constructs, we determined whether LYN and MLCK had a physiological interaction by co-IP. As shown in Figure 7C, HA-MLCK was pulled down by Myc-LYN. This finding suggests that MLCK is a downstream target of LYN.Next, we sought to address whether LYN kinase regulates MLCK activity directly. SRC can rapidly phosphorylate MLCK and increase its enzymatic activity by twofold to threefold nchanged , A and BNM II is an important intracellular motor protein. It has been reported that NM II is involved in the migration of central neurons, the growth and guidance of axon processes, the state and transport of microtubules in the growth cone of neurons, actin dynamics, the formation of dendritic spines, and the maintenance of normal neuronal morphology , 34, 35.BDNF is a neurotrophic factor that is widely expressed in the brain , 18, 19.In this paper, we report that BDNF/TrkB signaling pathway activity can upregulate the phosphorylation of regulatory MLC2 to increase MN II activity. Furthermore, we showed that this process depends on the LYN signaling pathways downstream of TrkB. The findings of this study can facilitate an improved understanding of both the function and mechanism of action of NM II.in vivo results were consistent with the in vitro results, meaning that BDNF could promote the phosphorylation of MLC2 through TrkB in the brain. Our study found for the first time that BDNF regulates the activity of MLC2 at both the cellular and systemic levels.Our results provide several new insights into the mechanism by which the activity of NM II is controlled. First, we demonstrated that BDNF can increase the level of p-MLC2 in primary cultured hippocampal neurons. It was further shown that this process depends on the high affinity receptor TrkB. Furthermore, we showed that the same phenomenon occurs in the brains of mice. We injected normal saline into the dorsal hippocampus in the control group and injected BDNF or K252a + BDNF into the dorsal hippocampus in the experimental groups. The Second, we reported that BDNF is dependent on LYN in the Src family to promote the phosphorylation of MLC2. We found that inhibitors of the three traditional TrkB signaling pathways  did not affect the level of p-MLC2 in cultured neurons and that the MLC2 phosphorylation level remained increased after BDNF stimulation. Next, we found that TrkB can upregulate the phosphorylation of MLC2 by activating SFK signaling. By treating neurons with a broad-spectrum SRC signaling inhibitor and siRNAs for different Src family members, we discovered that treatment with the SRC inhibitor PP1 or LYN siRNA inhibited the BDNF-induced changes in p-MLC2. Bafetinib, a specific inhibitor of LYN kinase, blocked the response of LYN to BDNF. Bafetinib is an antitumor drug used in clinical medicine. Our study provides important insights for further study of its physiological function. We demonstrated by co-IP assays that TrkB interacts with LYN and that this binding depends on the activity of TrkB. It is suggested that TrkB may activate LYN through direct binding, which is similar to the mechanism by which EGFR activates LYN.Third, we verified that LYN upregulates the phosphorylation of MLC2 by activating MLCK. SRC kinase can promote MLCK activity by directly binding to MLCK. We further demonstrated that LYN also binds to MLCK and that reducing the expression of LYN can inhibit MLCK activity, whereas exogenous overexpression of LYN can enhance MLCK activity.As mentioned, neuronal structure is stabilized by the cytoskeleton. The molecular motor protein (NM II) binds to actin filaments, whose contractile properties further endow the cytoskeletal network with highly dynamic control of neuron behavior and architecture. However, the upstream regulatory mechanism of NM II is not well known. Our study elucidated the regulatory network connecting BDNF/TrkB signaling, Lyn kinase signaling, and NM II. Because both BDNF/TrkB signaling and Lyn kinase have a variety of inhibitors used in clinical treatment, such as belizatinib and bafetinib, we should consider the effect of NM II activity and nervous system injury caused by related effects when using these drugs in the clinic. Targeted strategies to protect the activity of NM II in neurons have potential clinical significance.via activation of the SFK LYN downstream of TrkB and then via activation of MLCK. We identified the molecular mechanism by which BDNF regulates MLC2 activity, and this finding provides a new perspective for further understanding the functional regulation of NM II in the nervous system.Based on the aforementioned results, we confirmed that BDNF is involved in the regulation of MLC2 phosphorylation, which occurs myc (9E10) from Santa Cruz Biotechnology; rabbit anti–c-myc from Bethyl Laboratories; rabbit anti-MYL9 from ProteinTech; mouse anti-HA (HA.11) from Covance Research Products; and rabbit anti-p44/42 MAP kinase, mouse antiphospho-p44/42 , rabbit antiphospho-TrkA (pY490), and horseradish peroxidase–conjugated goat antimouse or rabbit IgG from Calbiochem. Human recombinant BDNF was obtained from PeproTech. Restriction enzymes were purchased from Ferments. Mounting medium was obtained from Vector Laboratories (H-1000). The other reagents were obtained from Sigma–Aldrich.ML-7 HCl (S8388), Y-27623 (S6390), U0126 (S1102), U73122 (S8011), LY294002 (S1105), PPI (S7060), and bafetinib (INNO-406) were purchased from Selleck . The following antibodies were purchased: rabbit anti-TrkB (#07-225) from Millipore; mouse anti-FLAG (M2), rabbit anti-HA, mouse IgG Sepharose, and mouse antitubulin from Sigma–Aldrich; rabbit anti-FLAG from Thermo Fisher Scientific; mouse or rabbit anti-p-MLCK and mouse anti–c-et al., 2011). In brief, embryos were removed from rats on embryonic day 18 and were then maintained in Hanks’ balanced salt solution without Ca2+ and Mg2+. Hippocampal tissue was isolated from embryos, digested with 0.05% trypsin-EDTA at 37 °C for 5 min, and then mechanically ground and separated in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM)/F-12 (Life) and 10% fetal bovine serum with a sterile, flame-polished glass Pasteur pipette. Neurons were cultured on a cover glass coated with 0.1 mg/ml poly-D-lysine (Sigma–Aldrich) in a 6-well plate in neural basic medium supplemented with 2% B27 and 0.5 mM glutamine. Cells were incubated in an incubator under conditions of saturated humidity, 5% CO2, and a constant temperature of 37 °C. Neurons cultured in vitro for 3 days (DIV3) were transferred to Opti–MEM (Invitrogen) using Lipofectamine 3000 transfection reagent (Invitrogen) according to the manufacturer's instructions. HEK293 cells were cultured in high-glucose DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Life), 100 U/ml penicillin, and 100 μg/ml streptomycin. Crosscontamination with other human cell lines was evaluated, and all cells tested negative. siRNA and overexpression plasmids were transfected using Lipofectamine RNAiMAX (#13778500) and Lipofectamine 2000 , respectively, according to the manufacturer’s instructions.Hippocampal neurons from pregnant Sprague–Dawley rats (E18) were cultured as previously described  overnight at 4 °C. The beads were then washed three times with IP buffer, eluted by boiling in SDS–PAGE sample buffer, and finally analyzed by immunoblotting with the indicated antibodies.Forty-eight hours after being electroporated with the indicated constructs with a Lonza Amaxa Biosystems electroporator, HEK293 cells were extracted with IP buffer . Cell lysates were clarified by centrifugation at 14,000The mice were cared for in strict accordance with the institutional guidelines for animal care and the experiment was approved by the Committee on the Ethics of Animal Care and Use, Institute of Basic Medicine, Shandong Academy of Medical Sciences. Before surgery, mice were anesthetized with 5% chloral hydrate . Agents were injected into the dorsal hippocampus of mice using a Hamilton microsyringe. The coordinates (in reference to the bregma) were as follows: dorsal hippocampus—anteroposterior(AP), 1.7 mm; lateral (L), 1.5 mm; dorsoventral (V), 2.3 mm. The infusions were performed in a volume of 1 μl over 2 min, and the infusion cannula was kept in place for diffusion for an additional 4 min. Mice were killed 2 h after microinjection. This research study was approved by the Institutional Review Board of Shandong First Medical University.t test, and the chi square test. All statistical results are expressed as the mean ± S.E.M. values. The ∗ symbol indicates statistical significance (p < 0.05). Student’s t test was used to compare pairs of interest.Data were obtained from three separate experiments. Experimental data were analyzed with SPSS software (IBM). The statistical methods used were one-way ANOVA, Student's The data underlying this article will be provided on reasonable request to the corresponding author.The authors declare that they have no conflicts of interest with the contents of this article.