Although immunoglobulin E (IgE) increases significantly in tears and serum during seasonal allergic conjunctivitis (SAC), it is unclear whether tear total IgE can reflect the severity and prognosis of SAC more accurately than serum total IgE. We aimed to investigate the usefulness of measuring the total IgE in tears to evaluate the severity and determine the treatment of SAC.This prospective, nonrandomized study involved 55 patients with SAC and 10 age‐ and sex‐matched healthy controls. Serum and tears were collected before and after treatment to analyze the total IgE. SAC patients received the same topical anti‐allergy treatment and were followed‐up every 2 weeks for 1 month. The relationship of tear and serum total IgE concentrations with pollen concentrations and symptom severity before and after treatment was assessed.The total IgE concentration in tears was higher in SAC patients than in healthy participants with significant correlations between tear and serum total IgE concentrations. The total IgE concentration in tears, but not in serum, correlated with the pollen concentration and severity of ocular symptoms and reactions in SAC. Treatment‐associated improvements in symptoms and reactions in SAC correlated with decreased concentrations of the tear total IgE. Patients with disease recurrence following treatment demonstrated significantly higher tear total IgE concentrations than patients with no recurrence.The total tear IgE level can indicate the severity and predict the prognosis of SAC more accurately than the serum total IgE. The study was approved by the Institutional Review Committee of the hospital and carried out in accordance with the Helsinki Declaration of 1975 and the 1983 amendments (ethics number: HX‐015). Each individual provided written informed consent before participating in the study.2.2Fifty‐five patients  with SA2.3Pollen concentrations in areas of patients' residence were recorded using the Durham pollen collector,2.4The diagnosis of AC was based on the results of a slit‐lamp examination and an allergen test. The diagnosis was made by an ophthalmologist based on published guidelines, and successful treatment of conjunctivitis was noted when a patient reported the symptom of ocular itching to have disappeared or to occur only occasionally (score = 1) which was judged to be significantly improved.2.5All participants completed a questionnaire at the first visit and at each follow‐up visit, and their symptoms were scored with a doctor's assistance. The symptom scores were obtained for both eyes for each patient; however, for the purpose of this study, only the score from the right eye was used for analysis. The doctor who assisted patients with their symptom score also scored the ophthalmic results of each patient's slit‐lamp examination ; the tear fluids were stored in a 0.5 ml centrifuge tube, and the sample was frozen immediately at −80°C. A total of 2.2 μL of the sample was dispensed into the test card , and three full drops of buffer were added. The test card was allowed to rest undisturbed for 10 min, and then the i‐ImmunDx Analyzer was used for reading. The test was performed separately for each eye.2.7A total of 2 ml of blood was collected from each patient at the first visit. The serum was separated and stored by freezing at −80°C until it was analyzed. The serum total IgE concentration in the serum sample was detected using an ELISA kit  and quantified according to the manufacturer's instructions.2.8All patients received the same topical anti‐allergy treatment. The drugs used were fluorometholone eye drops, olopatadine eye drops, and sodium hyaluronate eye drops. All drugs were used according to the manufacturer's instructions. After the first visit, the participants were instructed to return to the consulting room for two further visits (one visit every 2 weeks). Thereafter, they were followed‐up via three telephone consultations every other month. Recurrence was defined as recurrence of the disease during the follow‐up period.2.9U test. Any associations between variables were calculated using Spearman's correlation coefficient. The level of significance was set at p < 0.05. Data are presented as the mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.GraphPad 8.0 software and SPSS 18.0 software were used for analysis and mapping. The differences between groups were compared using the two‐tailed Mann–Whitney 33.1p < 0.01; Figure r = 0.44, p < 0.01; Figure r = 0.55, p < 0.01) but not between pollen concentration and serum total IgE , whereas serum total IgE concentrations were not significantly different between the two groups . It is possible that this is because SAC is an allergic disease, which is dominated by local immunity with more pronounced local symptoms, whereas serum total IgE levels usually reflect the state of the immune response of the whole body. However, other studiesr = 0.71) between the total IgE in tears and serum. However, as SAC is a disease with predominantly ocular symptoms, testing for the tear total IgE is more relevant, and future studies on SAC should concentrate on the localized immune response.In our study, the tear total IgE levels in patients with SAC were substantially higher than those in healthy participants, suggesting that the tear total IgE levels reflect the immune response of patients similar to the serum total IgE levels. In patients with SAC, allergens can enter the eyes via the conjunctiva and produce local IgE, which infiltrates the tears through the blood tear barrier, resulting in increased IgE levels in tears.r = 0.55). It is possible that this is because we analyzed the total IgE in tears and not specifically pollen IgE. Recently, Yamana et al.The current study also investigated any associations between pollen concentration and total IgE in tears and serum. Interestingly, the tear total IgE positively correlated with the pollen concentration, indicating that higher pollen concentrations are likely to lead to the presence of higher total IgE concentrations in tears. Furthermore, there was no correlation between the pollen concentration and serum total IgE, indicating that allergens stimulate the conjunctiva to produce local total IgE rather than lead to increased serum total IgE. Indeed, there is evidence showing that stimulation of the conjunctiva by pollen results in various inflammatory cells infiltrating the conjunctiva and releasing different cytokines that regulate the synthesis of total IgE.This study demonstrated that tear total IgE was also able to better reflect the severity of SAC than serum total IgE, particularly as an increase in symptom scores and reactions, including itching, tearing, and edema coinciding with significantly increased concentrations of tear total IgE. This suggests that the tear total IgE is a key factor in aggravating ocular symptoms. When mast cells in close proximity to the itch‐sensory nerve fibers are activated by IgE and have undergone degranulation, a variety of itch‐inducing molecules or pruritogens, such as histamine, are released and bound to their respective receptors on terminal axons. This leads to signals that pass through the central nervous system to the brain, where these signals are perceived as an itch.r = 0.69). This might be attributed to the small sample size and the fact that each patient has a different level of sensitivity to drug treatment. Moreover, as improvement during the follow‐up period was associated with a significantly decreased concentration of tear total IgE, it is likely that the total IgE concentration in tears may also be useful in determining the prognosis of patients with SAC. Thus, it might be possible to predict the development of the disease by monitoring changes in the tear total IgE concentration. Concurrently, we also noted that the correlation coefficient for the change in tear total IgE concentration and the difference in clinical score at the every 2‐week follow‐up was relatively low (r = 0.49). It is possible that with the increase in the follow‐up time, the patient's condition significantly improved. We failed to collect tears immediately after the patient's condition improved, resulting in an incomplete analysis.Although SAC is a mostly self‐limiting disease, it often recurs and can persist for many years. In our study, we found that 52.72% (29/55) of patients had recurrent disease and that the total IgE concentration in tears of patients with recurrent illness was significantly higher than that in patients without recurrent disease. Interestingly, a higher tear total IgE concentration at the initial visit was also associated with a longer delay in improvement following treatment. The correlation coefficient for the correlation between the tear total IgE and days until symptom improvement was relatively low (The diagnosis of SAC currently includes the evaluation of the symptoms and signs of the patient, as well as laboratory examination. To properly diagnose the disease, the clinician needs to review the history, observe the symptoms and signs, and then supplement these findings with the results of laboratory examination prior to initiating treatment. However, the current laboratory examination methods cannot conveniently obtain the results or be used to evaluate the severity of SAC. Measurement of the tear total IgE levels of patients with SAC could be used as a rapid and immediate diagnostic method. The current study demonstrated the convenience and accuracy of measuring the tear total IgE. Indeed, this measurement has several advantages over measurement of the serum total IgE. In particular, immunochromatography and double antibody Sandwich method are quicker to perform than the standard conventional method and, therefore, may be of considerable benefit to the patient, as SAC may be diagnosed at the patient's first visit. Furthermore, this is a non‐invasive technique that requires only 3 μL of tears to diagnose SAC. Compared with serum, tears also have better stability, are easy to transport and store, and have a specific field application value. Additionally, compared with that of serum total IgE, sensitivity of tear total IgE is higher and can be detected at a low dose and used in micro amounts. Another simple and fast technology called “Allerwatch” can also detect total IgE levels in AC.One of the limitations of this study was that we excluded patients with atopic keratoconjunctivitis because the study focused on SAC. In the future, we will study the relationship between the total tear IgE levels and specific serum IgE levels in patients with atopic keratoconjunctivitis. Further, considering the favorable principle of bioethics, we only collected serum samples of the participants at the first visit to detect the serum total IgE and did not measure its dynamic change. Furthermore, in line with the principle of bioethics, only10 healthy volunteers were included in our study. In future research, we will pay more attention to the consistency of the numbers of the control and experimental groups. Moreover, all included patients had mild to moderate SAC. Therefore, further evaluation is needed for patients with severe AC.5This study demonstrated that the measurement of total IgE levels in tears over serum of patients with SAC could be used as a rapid and immediate diagnostic method, to better evaluate the severity of SAC and predict the prognosis of the disease.Lei Tian: Data curation; Methodology. Yifan Meng: Data curation; Formal analysis; Methodology. Binge Wu: Data curation. Jingyi Wang: Data curation; Methodology. Jiayu Bao: Data curation; Methodology. Jing He: Data curation; Methodology. Qiyan Shao: Data curation; Methodology. Chengshuo Wang: Conceptualization; Project administration; Writing – review & editing. Ying Jie: Funding acquisition; Project administration; Writing – review & editing; Luo Zhang: Conceptualization; Methodology; Project administration; Supervision; Writing – review & editing.Supplementary MaterialClick here for additional data file.


Ampelopsis grossedentata extract (AGE) displayed the most potent hPL inhibition activity. After that, the major constituents in AGE were identified and purified, while their anti-hPL effects were assayed in vitro. The results clearly showed that two abundant constituents in AGE (dihydromyricetin and iso-dihydromyricetin) were moderate hPL inhibitors, while myricetin and quercetin were strong hPL inhibitors [half-maximal inhibitory concentration (IC50) values were around 1.5 μM]. Inhibition kinetic analyses demonstrated that myricetin and quercetin potently inhibited hPL-catalyzed near-infrared fluorogenic substrate of human pancreatic lipase (DDAO-ol) hydrolysis in a non-competitive inhibition manner, with Ki values of 2.04 and 2.33 μM, respectively. Molecular dynamics simulations indicated that myricetin and quercetin could stably bind on an allosteric site of hPL. Collectively, this study reveals the key anti-obesity constituents in AGE and elucidates their inhibitory mechanisms against hPL, which offers convincing evidence to support the anti-obesity and lipid-lowering effects of this edible herb.Pancreatic lipase (PL) inhibitor therapy has been validated as an efficacious way for preventing and treating obesity and overweight. In the past few decades, porcine PL (pPL) is widely used as the enzyme source for screening the PL inhibitors, which generates a wide range of pPL inhibitors. By contrast, the efficacious inhibitors against human PL (hPL) are rarely reported. This study aims to discover the naturally occurring hPL inhibitors from edible herbal medicines (HMs) and to characterize the inhibitory mechanisms of the newly identified hPL inhibitors. Following the screening of the inhibition potentials of more than 100 HMs against hPL,  Obesity and obesity-associated metabolic disorders are serious and global public health problems , 2. Althvia inhibiting hPL that meet the above-mentioned requirements are rarely reported. Therefore, it is urgent and necessary to discover more efficacious anti-PL agents or products with good safety profiles for preventing and treating obesity-associated complications.Pancreatic lipase (PL), a key digestive enzyme responsible for the hydrolysis of dietary triglycerides in the gastrointestinal tract, has gained considerable attention as an important anti-obesity target . In the A. grossedentata extract (AGE), displayed the most potent anti-hPL effect. A. grossedentata has traditionally been used as a health tea and folk HM by ethnic minorities in China and displayed a wide variety of beneficial properties, such as clearing away heat, detoxification, activating blood circulation, and dissipating blood stasis  was used as probe substrate . With thd stasis . In thisA. grossedentata extract was provided by Eastsign Foods Co., Ltd. , other HMs were provided by Tianjiang Pharmaceutical Co., Ltd . Dihydromyricetin, iso-dihydromyricetin, myricitrin, and myricetin were isolated by reverse-phase liquid chromatography. Taxifolin was purchased from Sichuan Weikeqi Biotechnology. Quercetin was purchased from the Shanghai Standard Technology Co., Ltd . Reynoutrin was obtained from the BioBioPha Co., Ltd. The positive inhibitors (sanggenone C and orlistat) were purchased from the Dalian Meilun Biotech Co., Ltd . The purity of all compounds was higher than 98%. The stock solutions of each inhibitor and positive inhibitors were solubilized in grade dimethyl sulfoxide . The specific substrate (DDAO-ol) and its hydrolytic product (DDAO) were obtained from the previous study (2) was prepared by using Milli-Q Water . LC grade formic acid, acetonitrile, and methanol were purchased from Tedia Company . All the other chemicals, such as NaCl and CaCl2, were of analytical grade and obtained from the Sinopharm Chemical Reagent Co., Ltd .us study . Expressus study . PorcineThe inhibitory activity of each inhibitor against hPL was measured by using DDAO-ol as substrate, while orlistat and sanggenone C were used as positive inhibitors against hPL. Briefly, the incubation mixtures  contained 66 μl Tris-HCl buffer, 10 μl hPL solution , 20 μl porcine bile salt , and 2 μl each inhibitor. After pre-incubating at 37°C for 3 min, the substrate of DDAO-ol  was added to the initial reaction. Subsequently, the fluorescent signals of the hydrolytic product of DDAO-ol (DDAO) were quantified by using the microplate reader , with the wavelength of 600 and 660 nm for excitation and emission, respectively. The standard curve was plotted using DDAO as product standard . The con18 column  with a flow rate of 0.4 ml/min. The mobile phase was made up of water (0.1% formic acid) (A) and acetonitrile (B). The mobile phase program was shown in via LC–TOF–MS/MS equipped with an electrospray ionization (ESI) source in negative ion mode. The MS parameters settings were shown in To identify and characterize the naturally occurring hPL inhibitors in AGE, a UFLC system  coupling with a Triple TOF 5600 Mass Spectrometer system  was performed to analyze predominant constituents. The chromatographic separation was employed by the Shimadzu VP-ODS CKi) of two flavonoids (such as myricetin and quercetin) against hPL, the inhibition kinetics were carefully investigated via performing a series of kinetic analyses (Ki) were calculated as depicted in previous studies   simulations were conducted. Prior to MD simulations, each ligand was emplaced into the allosteric pocket of hPL that was predicted by Cavityplus . The lipsb.org/) , 41. Firsb.org/) . Second,via Discovery Studio Visualizer .The MD simulations of hPL-allosteric agent complexes were operated by GROMACS . SystemsIC50 values and Ki values were determined by GraphPad Prism 8.0 .All inhibition assays were performed in triplicate, while the data were expressed as mean ± SD. IC50 value of 6.15 ± 0.71 μg/ml. Meanwhile, the time-dependent inhibition assay showed that the hPL inhibition potential of AGE was not changed following long pre-incubation time .First, the hPL inhibitory activities of more than 100 HMs were assayed under identical conditions by using a high dosage  , 45. As ion time , suggestO-α-L-rhamnopyranoside, myricetin, and quercetin, were tentatively identified and their fragment ions were carefully characterized (1H NMR and 13C nuclear magnetic resonance (NMR) spectra , a previously reported naturally occurring reversible hPL inhibitor. Meanwhile, the IC50 values of dihydromyricetin, iso-dihydromyricetin, and taxifolin . The results showed that myricetin and quercetin displayed potent anti-hPL effects , with thectively , Table 2axifolin  were alsaxifolin , Table 2Subsequently, the contents of four major constituents  in AGE with hPL inhibition activity were determined by LC–UV. Prior to quantitative determination, the standard curves of these four ingredients in AGE were plotted and depicted in Ki) of two flavonoids (myricetin and quercetin) with strong hPL inhibition activity were investigated. Prior to inhibition kinetic analyses, the time-dependent inhibition assays of myricetin and quercetin were performed. As shown in Ki values of 2.04 ± 0.75 μM and 2.33 ± 0.71 μM, respectively  are beneficial for the treatment of obesity and obesity-related metabolic diseases (It is well-known that hPL is one of the most important enzymes responsible for the digestion of dietary lipid (such as triacylglycerols) in the intestinal tract , 49, whidiseases –56. MeanIC50 value of 6.15 μg/ml. A. grossedentata is known as the “king of flavonoids”, while the content of total flavonoids in AGE is up to 45% , following oral administration with AGE at the daily dosage of this herbal product recommended by Chinese Materia Medica . Moreover, although myricitrin and reynoutrin in AGE display weak inhibitory effects against hPL, these two glycosides can be readily hydrolyzed by gut microbial to form myricetin and quercetin in the gastrointestinal tract, which can potently inhibit hPL  in the stomach and small intestine is much higher than that in other deep organs  following oral administration, suggesting that dihydromyricetin has very poor oral bioavailability and this agent hardly affects the endogenous metabolism in the liver  in the future. It is noteworthy that there are significant differences in the flavonoid content from different batches of edentata , the posyricetin , while tIC50 values were around 1.5 μM. A panel of inhibition kinetic assays demonstrated that myricetin and quercetin potently inhibited hPL in a non-competitive manner, with the Ki values of <2.5 μM. MD simulations suggested that myricetin and quercetin could tightly bind on hPL at an allosteric site. Collectively, our findings reveal the key anti-obesity constituents in AGE and elucidate their inhibitory mechanisms against hPL, all these data offer solid evidence to support the anti-obesity and the lipid-lowering effects of AGE and suggest that this edible herb can be developed as dietary supplements for preventing and treating obesity or obesity-associated complications.In summary, this work reported the discovery of the bioactive anti-hPL substances from AGE, edible folk medicine in South China, and characterized the inhibitory mechanisms of the newly identified anti-hPL substances from this edible herb. Our results clearly demonstrated that almost all identified flavonoids in AGE were naturally occurring hPL inhibitors in which two abundant flavonoids in AGE (dihydromyricetin and iso-dihydromyricetin) were moderate hPL inhibitors, while myricetin and quercetin were identified as potent hPL inhibitors, with the The original contributions presented in the study are included in the article/X-YQ: methodology, validation, data curation, and writing–original draft. X-DH: validation, data curation, and writing–original draft. G-HZ and YX: methodology and data curation. Y-QS: investigation. LZ: methodology. D-FZ: formal analysis. S-NJ: resources and funding acquisition. JH: writing–review and editing and project administration. HT: conceptualization, supervision, and project administration. G-BG: conceptualization, supervision, writing–review and editing, and project administration. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China , Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine (No. ZYYCXTD-D-202004), Shuguang Program (No. 18SG40) supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission, the Program for Innovative Leading Talents of Qinghai Province (2018 and 2019), the Key R&D and Transformation Science and Technology Cooperation Project of Qinghai Province (2019-HZ-819).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.


At present, the pedicle screw is the most commonly used internal fixation device. However, there are many kinds of common posterior pedicle screw insertion techniques performed to reconstruct the lumbar stability. Therefore, spinal surgeons often face a difficult choice. The stress distribution of internal fixation system is an important index for evaluating safety. Unfortunately, little had been known about the difference of stress distribution of screw-rod systems that established by Roy-Camille, Magerl and Krag insertion techniques. Here, combination of finite element analysis and model measurement research was adopted to evaluate the difference of stress. Following different pedicle screw insertion techniques, three lumbar posterior surgery models were established after modeling and validation of the L1–S1 vertebrae finite element model. By analyzing the data, we found that stress concentration phenomenon was in all the postoperative models. Roy-Camille and Magerl insertion techniques led to the great stress on screw-rod systems. Then, fresh frozen calf spines were selected as a model for subsequent measurements. Fitted with a specially designed test pedicle screw, L5–L6 vertebrae were selected to repeat and verify the results of the finite element analysis. With the aid of universal testing machine and digital torque wrench, models simulated flexion, extension, lateral bending and rotation. Finally, the strain value was captured by the strain gauge and was then calculated as the stress value. Krag and Magerl were found to be the safer choice for pedicle screw insertion. Overall, our combination method obtained the reliable result that Krag insertion technique was the safer approach for pedicle screw implantation due to its relatively dispersive stress. Therefore, without the consideration of screw size, pedicle fill, bone density, and bone structures, we recommend the Krag insertion technique as the first choice to reconstruction of lumbar stability. Additionally, the combination method of finite element analysis and strain gauge measurement can provide a feasible way to study the stress distribution of spinal internal fixation. Trauma, Congenital disease, tumor, infections and other degenerative reason may cause lower back pain. Due to a fracture, the lumbar spine may become unstable. And operations on most of the diseases often destroy the stability of lumbar spine. Therefore, reconstrucion of lumbar stability is the key to treatment. Due to the great loading conditions, lumbar region reconstruction is usually accomplished with the posterior pedicle screw-rod system.Lower back pain, the second common ailment, happens to almost 80% people all over the world2. After a long period of development and improvement, they have been widely used for providing immediate stability, correcting deformity, enhancing bony fusion and maintaining normal spinal segments. Based on this, the pedicle fixation technique was able to treat deformations, tumors, unstable fractures, tuberculosis and degenerative disorders of the spine. With the increasement of clinical applications of pedicle screws, postoperative problems including breakage, loosening, improper placement, spinal cord injury, nerve root injury, dural tears, pseudarthrosis and instrumentation infection gradually appear7. The most common postoperative problem is breakage, which resulted from screw fracture due to torsion or bending. The previous studies show that screw breakage often occurs around the thread-shank region and has an incidence of 2.6% to 60%10. In recent years, the safety of pedicle screw and connecting rods have been improved by application of new shape, hard material and motorization, but case reports of breakage are not rare12. Protecting pedicle screws and connecting rod from breakage is still needed to be resolved.In 1959, Boucher first described the pedicle screw instrumentations13. Furthermore, the retrospective study has achieved the same result14. Although researchers have shown that screw size, pedicle fill, bone density, bone structures, and insertion technique are important factors for influencing internal fixation stability17. Unfortunately, there is still no adequate evaluation on the biomechanical features of different pedicle screw insertion techniques. Under circumstance of using the same pedicle screw, the choice of the insertion technique varies with different surgeons. They prefer for paying more attention for intraoperative safety during selecting insertion technique. But the resulting breakage risk of internal fixation system will be unconsciously neglected. In fact, insertion technique has three key elements including position, orientation and depth. As mentioned in the previous literature, different techniques with different elements will have effect on the stress state of inserted screw instrumentations18. Therefore, from the perspective of biomechanics, performing the low stress technique may reduce the breakage risk as well as ensure the success of the operation19. With simulation, veracity and repeatability, the finite element (FE) analysis has been viewed as a reliable approach for evaluating the biomechanical behavior of different internal fixation system. More intuitive data can be obtained from traditional cadaver research, which is also very important for biomechanical study. In the following research, three pedicle screw insertion techniques will selected as the research objects. Roy-Camille, Magerl, and Krag are three common and typical insertion techniques that are performed for stabilizing the lumbar spine22 . The study was carried out in accordance with the relevant guidelines and regulations, and an informed consent was obtained from the subject. Geometrical details were obtained from a thirty-year-old healthy male volunteer in an unloaded neutral position by high-resolution computed tomography. Including five lumbar vertebrae, parts of sacral vertebrae and five intervertebral disks, the Dicom data, was imported into the Mimics 10.01 software . A threshold was set to differentiate bone and soft tissue. Boolean calculation and interactive three-dimensional manual/automatic cutting operations were performed to establish a rough three-dimensional (3D) model of L1–S1 vertebrae . Range of motion (ROM) was the only parameter chosen for validation. Model validation was accomplished by comparing with in vitro biomechanical test data28 and simulation results obtained from four well-validated FE models in the literature31.For most FE analysis studies, simulation results of the intact FE model were compared with the data reported in the previous literatures. Here, simulation results in this study were compared with both existing well-validated FE models reported in the literature and the data of biomechanical test. Generally, it is accepted that greater number and diversity of comparisons between a model and experimental data increases the reliability of validationIn Geomagic Studio 12.0 software, simulation models including simplified pedicle screws  and connecting rods  were designed respectively. Because of the higher incidence of spine diseases, L3–S1 vertebrae of the intact FE model were adopted to simulate the posterior lumbar surgery. With the combination of bilateral connecting rods, four pedicle screws were inserted into the pedicle of vertebral arches according to different methods . Therefore, three different postoperative models of L4–L5 vertebrae could be achieved.The same as the simulation method of intact FE model, postoperative models also underwent a series procedure of remeshing, defining material properties, and setting boundary and loading conditions Fig. . ConsideNine calf spines (L4–S2) were purchased from the butcher. Radiological examination was performed to exclude spinal disease for these spines. Following muscle removal, soft tissue structures like ligament and joint capsule was retained. Then, the ends of the head and tail was smoothed. Four Kirschner wires were implanted along the longitudinal and transverse axes of L5 and L6 to assist to determine whether intervertebral activities occurred. Finally, the models were randomly grouped and stored at low temperature.Stress measurement of internal fixation of spine in vitro was rarely studied. Based on common internal fixation, special screws and connecting rods were designed and manufactured. They had the advantage of having a smooth platform, which could fit with strain gauges. According to the FE analysis results, the design position of the platform was set to the stress concentration area included the screw tail and the middle part of the connecting rod. For biomechanical experiments with high stress, fixture that could connect model and test instruments was also designed. The stable clamped model could also ensure the normal measurement of strain value.Meanwhile, the special fixture could cooperate with the testing machine and torque wrench to produce different working conditions. The parameter of compressive preload and pure moment was consistent with the computer simulation of FE analysis. When the working condition reached the maximum, the strain value on the internal fixation would be measured. Static strain test analysis system , universal testing machine  Co., Ltd., Shenzhen, China) and torque wrench  were selected and used.SPSS 17.0 software  was performed for statistical analysis. Data were statistically analyzed based on the analysis of variance for repeated measures. Data were expressed as the mean ± standard deviation, and the normality of the data distribution was assessed using the Shapiro–Wilk test.Concerning the results of the intact L1–S1 vertebrae FE model, we took full advantage of Mimics 10.01, Geomagic Studio 12.0, Solidworks 2012, HyperMesh 13.0, and Patran/Nastran 2012 softwares. The high-quality FE model contained six vertebrae, five intervertebral discs and six ligaments; which also consisted of 368,233 tetrahedron elements and 79,722 nodes.Figure Based on the validated intact FE model, the L3–S1 model was selected to simulate posterior screw-rod system fixation surgery of L4–L5 vertebrae. The simplified pedicle screws and connecting rods were designed in Geomagic Studio 12.0 software and were assembled into different internal fixation systems on three same FE models. Here, the contact surfaces between screws and trajectories were defined as an infinite friction coefficient. Additionally, all components of the internal fixation system were recognized as a whole. Then, re-meshed models were established by using Hypermesh 13.0 software. With different points and orientations of pedicle screws, the Roy-Camille, Magerl, and Krag surgery FE models were ready for biomechanical studies.After the internal fixation of surgery, L4–L5 intervertebral ROMs of the different FE models were all reduced in comparison with the intact FE model  were serially processed to removal muscle as well as keep soft tissue structures. Finally, intact vertebral bone, intervertebral discs, anterior longitudinal ligament, post longitudinal ligament, ligamentum flavum, interspinous ligament, supraspinous ligament, and articular capsule were retained. For the convenience of assembly, the ends of the head and tail was ground down by an angle grinder. Then, the models were grouped by the sortition randomization method. Low temperature storage of the models were reserved for further experiments. Then models would be assembly internal fixation following three different insertion techniques Fig. .Figure 1According to the results of the FE analysis, the stress concentration area was found. This provided a possibility for carrying out the measured test. Here, the special internal fixation were produced. Different from the previous internal fixation, special screws and connecting rods had a platform structure in the rear of screw and central region of rod Fig. . These sOn the other hand, the previous experiments were analyzed and evaluated comprehensively to assist to complete the design of special fixture. This study also brought a whole new tool Fig. . Stable After internal fixation implantation and fixture assembly on the prepared calf spine, twelve strain gauges were attached to unilateral two screws and a connecting rod. Under the joint drive of fixture and testing machine, the model simulated different working conditions of normal physiological activities Fig. . InterveThe formulas for calculating stress is below..32. The diagnosis and treatment of lower back pain have been the hot research fields that draw more and more attention. With the rapid development of radiological technology, the diagnosis of lower back pain becomes easy to be determined. Meanwhile, internal fixation techniques including implants and operation methods are being constantly improved. However, for those cases that undergo the spinal surgery, postoperative complications of fixation devices usually cause surgeons to worry.With the increasing and aging trends of the population, low back pain has been the leading cause to disability2. As the main force for multi-plane stability reconstruction of lumbar vertebra, pedicle screws are used in most of posterior lumbar surgery. There's no avoiding the fact that postoperative problems mainly including loosening and breakage become frequent and tricky. With more in-depth studies, pedicle screw loosening has been proved to closely related to the decrease of pullout strength and the change of insertion torque. The main influencing factors of pullout strength are density, insertion angle, insertion depth and reinsertion33. And bone density of vertebra is an important factor in pedicle screw instrumentation and contributes highest toward insertion torque (82%) and pull out strength (76%)34. Pull out strength increases with an increase in density and insertion depth. Whereas, insertion angle has no significant effect on both pull out strength and insertion torque. In the bilateral screw-rod system, reinsertion also has no significant effect on pullout strength35. A new research suggested that the roughness of surface is another important factor for insertion torque36. Incidence of screw breakage ranges between 2.6 and 60%10 and its occurrence represents a serious problem. A few of methods like the modified screw and dynamic stabilization system are created to cope with it. However, these attempts still are not able to completely remove the risk of breakage37. Meanwhile, cadaveric research and clinical retrospective study predict the uncertain results because of obvious limitations mainly including nonrepeatability, proof heterogenicity and large consumption. Therefore, there has been limited progress in biomechanical research. There are many frequently-used kinds of pedicle screw insertion techniques. The answer about relativity between insertion techniques and risk of breakage is not given throughout. Based on this, FE analysis combined with model measurement research can be regarded as a reliable approach for evaluating the biomechanical characteristics of different pedicle screw insertion techniques.The use of the pedicle screw has been more than half a century31. ROM, commonly used in the step of comparison, was adopted by us. The values of flexion, extension, left–right lateral bending and left–right axial rotation movements of this intact FE model were in accordance with the numerical range of the included studies. Additionally, the mesh quantity with 368,233 tetrahedron elements and 79,722 nodes was moderate and acceptable. Therefore, our validated FE model could be used for further biomechanical researches and was different from previous reports. The reconstructed model was based on data from one healthy adult volunteer. This ensured the authenticity of the model. Combined with the processing advantages of different engineering software, the process of model establishment was optimized. This FE model had appropriate quantity of elements and nodes that reducing the computing burden will be beneficial to the study of stress distribution of the internal fixation system. Therefore, in this study, the accuracy of FE analysis results was guaranteed while the efficiency of model processing was improved.We drawn insights of FE analysis from previous literature and selected a healthy young volunteer to complete collection of imaging database. Mimics 10.01, Geomagic Studio 12.0, Solidworks 2012, Hypermesh 13.0, and Patran/Nastran 2012 softwares were performed to establish this intact FE model. To eliminate the difference of actual measurements and FE studies, five typical biomechanics data including in-vitro and FE studies were used for model validation38. Here, ROMs of L3–L4 and L5–S1 in three different postoperative models increased, compared to the intact model. The stiffness-increasing mechanism as the potential reason for the fixation-induced compensation is widely accepted. The stiffness-increasing effect protects the bridged segment from deformation, and transfers the load to the adjacent segments. This phenomenon can account for the higher kinematic and kinetic demand at the adjacent segments after implantation of internal fixation. Hsieh indicated that adjacent discs were subjected to the transferred loads from the instrumented segment38.Because of L4, L5 and S1 vertebra enduring the largest gravity in the lumbosacral vertebrae, L4–L5 intervertebral disc is often troubled by illness. Moreover, posterior lumbar surgery that mainly applied pedicle screws is widely used, now. Therefore, bilateral posterior pedicle screw-rod system fixation on L4 and L5 vertebra is the ideal surgery model for comparing different pedicle screw insertion techniques. Then, we extracted L3–S1 lumbosacral vertebrae from the intact FE model to perform this biomechanics study. By surgery simulation, mesh generation, materials properties definition, load applying and condition setting, three different models about pedicle screw insertion techniques following single-segment internal fixation were ready. The biomechanical characteristics of different models were compared. Previous studies have shown that lumbar interbody fixation are able to increase in ROM of adjacent segments of both sides40. When the different points and orientations were selected, stress distribution of pedicle screws and rods would be changed consequently. After lumbar posterior surgery, screw-rod system became the center of stress. As we know, stress concentration might directly lead to breakage of the fixation system19. Because the pedicle screw belongs to the posterior internal fixation instrument, it is like the "crane" force structure that determines the stress concentration on the junction of thread and smooth. Screw breakage described by previous literatures often happen in this area41. This study result tied well with previous studies wherein the same was observed from all the three different postoperative models. Similarly, the rods connecting two pairs of pedicle screws up and down were under the major stress39. In the FE study section, the mean stress values of the three groups  were 80.12 MPa, 92.77 MPa and 66.80 MPa, respectively. Krag with the minimum mean stress value seemed better than the other two insertion methods. To verify the results of FE analysis, biomechanical test with nine calf spines were performed. According to the characteristics of concentrated stress areas, special internal fixation and fixture were designed to well simulate six working conditions. Choosing the same load conditions as the FE study, the strain value was obtained and calculated into stress value. Finally, results of biomechanical test showed that the mean stress values of Roy-Camille, Magerl, and Krag group were 57.30 MPa, 32.92 MPa and 32.58 MPa, respectively. It was not difficult to see that Krag and Magerl were two more secure insertion method for protecting the posterior pedicle screw-rod system. Additionally, flexing, extension, left lateral bending and right lateral bending easily caused stress concentration. Taken together FE analysis and biomechanical test results, Krag insertion method was better than the other two with a lower risk of breakage. Krag insertion technique emphasizes on the trajectory that stands in sharp contrast to other insertion techniques. Screw-rod system based on Krag insertion technique transfers the stress to the lower part. This allows stress to be dispersed in the lumbar spine with internal fixation. In contrast, screws implanted by Roy-Camille and Magerl insertion technique parallel to the upper and lower endplates in the sagittal position. At that time, the tail of the screw is at right angles to the connecting rod. This may be the cause of relatively high stress of screw-rod system. Flexion and extension were still the most dangerous conditions after lumbar posterior surgery. Although the data was not sufficient, lateral bending and rotation could not be underestimated, too. Processed by software, FE model was made as idealized as possible, but biological simulations of tiny structures were difficult to complete, such as the motion of the joint capsule. The inconsistencies in the two experiments were related to the nuances of different models.For the stress of internal fixation system, flexing, extension, left axial rotating and right axial rotating were the noteworthy working conditions that caused stress concentration. The difference of the pedicle screws’ points and orientations contributed mostly to the difference in stress distribution42. A few of studies were performed by the similar method and continuously improved its reliability45. In recent years, this method began to be transplanted into orthopedic stress research. Bone surface strains of the radius and ulna in mouse has been studied by Begonia et al. via using the similar method46. Then, Gao et al. believed this combination method was a feasible way and showed the strain distribution of axial compressive load of rat tibia47. These satisfactory results proved the validity again. In this study, we adopted this research method, but the object of study was no longer human tissues, but internal fixations with metallic material properties. Strain gauge placement is a difficult point in this kind of measurement. Therefore, special screws and connecting rods with smooth platform was designed. Then, it is possible to measure the strain of internal fixations. In this study, the results of FE analysis were mostly in agreement with the measured results of strain gauge.It should be noted that the comprehensive analysis method adopted in this study was an innovation. As we known, FE analysis is widely used to complete mechanical analysis of internal fixation and bone. High emulation, low cost and time saving are its great advantages. However, biomechanical test is in stark contrast to the former. Its advantage is that repeated tests can be carried out with multiple specimens. Strain gauge is a common method of stress measurement and often used for mechanical and material testing. But, in the previous researches, combination with FE analysis and strain gauge measurement is rare. Oral biomechanics was the first to adopt this approach. Palamara et al. investigated the variations in strains in enamel under different patterns of occlusal loading. Strains predicted from the FE model were in excellent agreement with the strain gauge measurementsThe final results of this combination test will make some difference in the clinical choice of pedicle screw insertion technique. Based on the Krag technique, the much safer insertion techniques will be designed and promoted. Additionally, developing decision support system based on this study for pedicle screw fixation can supply the personalized therapy and precision medicine for patients suffering from lumbar disease.There were still some limitations in the present studies. Limited to the tremendous computing workload, elements and nodes of the intact model were moderate rather than elaborate. Based on data from different individuals, multiple FE model of lumbar spine would help to verify the results of FE studies. This might be a better representation compared to the combination of FE analysis and biomechanical test. Current pedicle screw insertion techniques are diverse. The differences are mainly in entry point and insertion orientations. Only three common and typical pedicle screw insertion techniques were selected for the comparison. This limitation of selection had impact on the research comprehensiveness. Thus, the results of other techniques were unknown and needed further studies. In the biomechanical test section, the sample size was still insufficient. There were differences from selected calf vertebra and human vertebra in the number, shape and size. The simulation of the working condition should be more ideal, more close to the FE study, more in line with the physiological state. There were some differences in the stress values measured from the FE analysis and the biomechanical test, which was related to the experimental environment, the model difference, experimental measurement and calculation method. The most authentic and reliable results could be attained by combining with large sample clinical data. Additionally, more efforts should be done to explore the influences caused by screw size, pedicle fill, bone density, and bone structures.In summary, compared with others, Krag insertion technique could reduce the stress concentration of screw-rod system. Without the consideration of other factors, Krag insertion technique was safer for reconstructing the stability of lumbar vertebra, due to its potentially low risk of fixation breakage. Additonally, combination of FE analysis and strain gauge test was a good selection for biomechanical study of spinal internal fixation.


Maniola jurtina as an example, we investigate how a specific habitat type, lowland calcareous grassland, can affect the timing of flight dates. Although protracted flight periods have previously been reported in populations on chalk grassland sites in the south of England, no attempt has yet been made to quantify this at a national level, or to assess links with population genetics and drought tolerance. Using data from 539 sites across the UK, these differences in phenology are quantified, and M. jurtina phenology is found to be strongly associated with both site geology and topography, independent of levels of abundance. Further investigation into aspects of M. jurtina ecology at a subset of sites finds no genetic structuring or drought tolerance associated with these same site conditions.The influence of large‐scale variables such as climate change on phenology has received a great deal of research attention. However, local environmental factors also play a key role in determining the timing of species life cycles. Using the meadow brown butterfly  Maniola jurtina phenology is found to be strongly associated with both site geology and topography, independent of mean abundance across 539 monitoring sites. Across a subset of sites, no genetic structuring was associated with these site characteristics. Populations displayed high levels of genetic diversity but low genetic differentiation. Across a subset of sites, drought tolerance was not associated with site geology.  UKBMS data are collected by volunteers using the “Pollard walk” method  are dominated (>50% cover) by lowland calcareous grassland and that 25% of these sites (n = 70) have less than 4.3% cover. All of the chalk sites used in the analysis fall within the interquartile range of chalk cover across all UKBMS sites.For the population genetics analyses, distinct categories of sites were required. Sites were defined as either chalk or nonchalk sites based upon the presence of lowland calcareous grassland Appendix . The low2.2Poa trivialis) were grown under standard conditions, with each plant watered via individual trays. Once mature, plants were randomly assigned to the treatment groups—drought‐stressed or control. Control plants were watered daily from 20 days prior to larval hatching and then throughout the experiment. Plants were never oversaturated but watered enough to prevent soil drying and wilting. Drought‐stressed plants received no water from 20 days prior to larval hatching and were then only watered every six days throughout the experiment. This treatment meant that green leaves were available at all stages of the experiment but ensured moderate drought stress occurred. At the end of the experiment, green leaves were still present on all plants. This ensured that food availability was not a factor limiting larval growth and survival. Rainwater was used in both treatments.All drought experimentation was carried out following the methodology described in Gibbs et al. . A summaM. jurtina larvae were selected from populations originating from nine of the 15 sites used in the molecular analysis , 0.625 μl tagged forward primer, 0.625 μl reverse primer, 1.25 μl QIAGEN Q solution, and 2.25 μl RNase‐free water. Multiplex set 1 contained Mj7232, Mj5522, and Mj0247, all at 3 μM. Multiplex set 2 contained Mj4870 at 1.5 μM, Mj7132 at 5 μM, and Mj5331 at 4.5 μM. PCRs were carried out in an Eppendorf Mastercycler nexus eco with an initial denaturation for 15:00 at 95℃, followed by 40 cycles of 00:30 at 94℃, 01:30 at 56℃, and 01:00 at 72℃, and a final extension 10:00 at 72℃. All PCR products were diluted by 100× and run on an Applied Biosystems 3730 DNA Analyser. Allele peaks were then scored by using GeneMarker®, version 1.5 by SoftGenetics, using the microsatellite calibration settings.We conducted a molecular analysis of 287 7 Figure , were us2.42.4.1M. jurtina abundance were summed per UKBMS site in each year, and the day number of the recording period at which 10% of the total occurred was recorded as the flight period start date. The day at which 90% of the total occurred was recorded as the flight period end date. We used 10th and 90th percentiles to avoid the effect of outliers as in WallisDeVries et al.  were each fitted as response variables into separate linear mixed effects models, against the percentage cover of chalk grassland and mean slope angle of each site. The additional factors of mean abundance, northing (km north on Ordnance Survey grid), easting (km east on Ordinance Survey grid), mean site altitude, and mean site aspect (cos((aspect × π)/180), such that 1 = due north, −1 = due south) were included as fixed effects and site and year as random effects. We included mean annual abundance as a covariate in these models because larger populations are likely to have a greater flight period range due to mathematical mean–variance relationship  and topography of the site predicted Equation . The fou Taylor, . Northinlmer function from the lme4 package , C is the percentage cover of chalk grassland per site, S is the mean slope angle per site, A is the site total abundance, N is the site northing, E is the site easting, H is the mean altitude per site, F is the mean aspect of each site, i is a random intercept for site, y is a random intercept for year, and ε indicates error term with zero mean and normal distribution.All mixed‐effects models were carried out in R , G is the geology of the origin site , p is a random intercept for the origin population of the larvae, and ε indicates error term with zero mean and normal distribution.A generalized linear mixed‐effects model was used to determine whether larval survival rates varied between sites in association with site characteristics. The model was fitted with a binomial error structure and with host plant drought treatment and percentage chalk cover (geology) as fixed effects with an interaction term, and population as a random intercept Equation . The slomodel.sel function from the R package MuMIn , and all versions of the model were compared using the  Barton, .2.4.3F statistics, heterozygosity, allelic richness, and effective population sizes were carried out using GenePop v4.7.0  was tested from one to six, with 20 chains run per K. The likeliest K within the sample sets was estimated using the program STRUCTURE Harvester , (b) all chalk site and all nonchalk sites grouped into two populations , (c) only the individuals from the eight chalk sites (n = 137), and iv) only the individuals from the seven nonchalk sites (n = 150).Population structure was estimated using STRUCTURE v.2.3.4  both chalk, (b) both nonchalk, (c) one chalk, and the other nonchalk. The slope angle was not included owing to all nonchalk sites being shallow and all but one of the chalk sites steep. Pairwise FST values were fitted into a linear regression with geology and Euclidean distance between sites as a fixed effect , D is the Euclidian distance between sites, and ε indicates error term with zero mean and normal distribution.Individuals were then pooled by site to generate allele frequencies for genetic distance analysis. Weir and Cockerham pairwise Equation . (3)F=FST values between sites are not independent, Mantel randomization tests with 999 permutations were conducted to assess whether the predictor variable (geology) was significant following the methodology described in Powney et al. . The residuals from each model showed no evidence of spatial autocorrelation using Moran's I test (start day model I: observed (O) = 0.001, expected (E) = −0.0001, SD = 0.001, p = .259; Mean day model I: O = 0.0007, E = −0.0002, SD = 0.001, p = .476; End day model I: O = 0.0008, E = −0.0002, SD = 0.001, p = .421; Range model I: O = 0.001, E = −0.0002, SD = 0.001, p = .277).All phenology measures were significantly positively associated with differences in chalk cover , 341.4 (treatment and geology interaction), and 346.1 (geology only)], that is, larval survival rates were significantly affected by host plant drought treatment , and no locus displayed significant heterozygote excess or deficit , with a mean allelic richness of 8.2 for chalk sites and 8.3 for nonchalk sites , and none were significantly greater than zero  were also associated with the same landscape attributes. We found significant, positive, associations between the phenology of M. jurtina and geology  and topography , that is, key flight dates are delayed with increasing chalk cover and slope angle. These associations remained after accounting for abundance, therefore, aspects of geology and topography are associated with phenology independent of mean local abundance. We found no strong evidence of genetic structuring of M. jurtina populations linked to geology, and only very weak evidence of genetic differentiation among populations. Finally, we found no effect of geology on larval survival (drought response).In this study, we quantified characteristics of Hesperia comma), absent from other habitat types  chalk grasslands. Habitat and topographic diversity can allow species to persist in areas of suitable microclimate when the surrounding climate is no longer favorable   are extremely low, being below the 0.05% threshold typically viewed as indicative of genetic differentiation ; formal analysis (lead); investigation (lead); methodology ; project administration ; writing‐original draft (lead); writing‐review & editing . Marc S. Botham: Conceptualization (supporting); data curation ; writing‐review & editing . Michael W. Bruford: Conceptualization ; formal analysis (supporting); investigation (supporting); methodology (supporting); supervision ; writing‐review & editing . John C. Day: Conceptualization ; formal analysis (supporting); investigation (supporting); methodology ; supervision (supporting); writing‐review & editing . Luke C. Evans: Formal analysis (supporting); methodology (supporting); writing‐review & editing . Melanie Gibbs: data curation ; formal analysis ; investigation ; methodology ; writing‐review & editing . Ian Middlebrook: Data curation ; writing‐review & editing . David B. Roy: Conceptualization (supporting); data curation ; writing‐review & editing . Kevin Watts: Conceptualization ; funding acquisition (supporting); investigation (supporting); methodology ; supervision ; writing‐review & editing . Tom H. Oliver: Conceptualization (lead); formal analysis (supporting); funding acquisition (lead); investigation (supporting); methodology (supporting); project administration (supporting); supervision (lead); writing‐original draft (supporting); writing‐review & editing .Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.


The presence of clinical signs was also more evident on the cervical site when using avian and bovine PPDs in the high prevalence herd (p < 0.01). In contrast, increases in higher skin fold thickness were observed on the scapular site when compared to the cervical site after the bovine and avian PPD inoculations were employed in the low prevalence herd (p < 0.01). These results suggest that the cervical injection of PPDs may improve the sensitivity of the intradermal tuberculin test in high TB prevalence caprine herds, mainly owing to the increased presence of local clinical signs and a better performance of the CIT test. Moreover, specificity was not affected when using standard interpretations, although further analyses in a great number of herds are required in order to confirm these findings.The single and comparative intradermal tuberculin (SIT and CIT) tests are used for the ante-mortem diagnosis of caprine tuberculosis (TB). The tuberculin injection site has been associated with a different performance of the test in cattle. In contrast to that required in cattle in Europe , it can be carried out in the scapular region in goats. Nevertheless, there are no previous data concerning the effect of the injection site on the performance of the test in goats. The aim of the present study was to evaluate the effect of two different inoculation sites  on the performance of the SIT/CIT tests. This was done by intradermally inoculating 309 goats from two infected herds and one TB-free herd with both avian and bovine PPDs in the mid-cervical and scapular regions. None of the animals from the TB-free herd had positive reactions, and the number of reactors was not significantly higher, regardless of the inoculation site, in the high and low prevalence herds. However, significantly higher increases in skin fold thickness were observed on the cervical site when compared to the scapular site after the avian and bovine PPD inoculations in the TB-free herd ( Mycobacterium tuberculosis complex (MTBC) (Mycobacterium bovis and Mycobacterium caprae (Tuberculosis (TB) is a zoonotic infection disease caused by members of the x (MTBC)  that affx (MTBC) –4. Caprim caprae , 6, withm caprae . Severalm caprae , 8. The m caprae . Moreovem caprae . The intm caprae –13.Mycobacterium avium subsp. paratuberculosis (MAP) ((PPDs)  , 18, theis (MAP) , the pot ((PPDs) , and the ((PPDs) , 21. Ind ((PPDs) , age . This had led to their classification as high  and low  prevalence herds on the basis of previous TB testing using the SIT test and IGRA in parallel. The TB-free herd  had no previous history of TB and had been tested annually for more than 10 years within the framework of a regional eradication programme. Moreover, a vaccination programme against MAP had been employed with these three herds, using the Gudair vaccine  in goats under 6 months of age.The study was performed with two TB-infected Guadarrama–breed dairy herds and one TB-free Murciano-granadina–breed dairy herd located in central Spain. Both TB-infected herds had previously been confirmed by means of bacteriological culture  for both PPDs in all three herds. Serum samples were also assayed for TB by using an indirect ELISA that detects antibodies against a protein complex purified from bovine PPD , denominated as P22 (P22 ELISA) . IGRA anThe animals included in the study were not experimental animals. All handling and sampling procedures were carried out in accordance with local and Spanish legislation .The SIT and CIT tests were performed in the mid-cervical and scapular regions on both sides of each animal  using a Blood samples were collected from the jugular vein using lithium heparin tubes  and stimulated with bovine and avian PPDs  at a final concentration of 20 μg/mL for the detection of IFN-γ production. The blood samples were then processed as described previously . IFN-γ pBlood samples were collected from the jugular vein using plastic serum tubes . Serum samples were stored at room temperature for 24 h and then centrifuged for 15 min at 650 g and conserved at −20°C until the assay. The ELISA was performed as described previously . The samSerum samples with E% values >150 or 100 were considered positive to the standard and severe interpretations, respectively.p-value of 0.05 was indicative of statistical significance. Wilson's 95% confidence intervals (CI) were calculated for the percentage of animals found positive to the different tests. Cohen's kappa coefficient (κ) was used to calculate the agreement between tests  and inoculation sites . The Kappa statistic (k) was interpreted as follows: ≤ 0 poor, 0.01–0.20 slight, 0.21–0.40 fair, 0.41–0.60 moderate, 0.61–0.80 substantial, and 0.81–1.00 almost perfect agreement. Quantitative differences in the skin fold thickness (expressed in mm), IFN-y levels (OD), and ELISA percentage (E%) within a herd were analyzed using the Wilcoxon signed-rank test. The proportion of reactors to the cervical and scapular inoculation sites within a herd was compared using McNemar's test. The proportions of test reactors in each herd were compared using Fisher's exact test. The skin fold thickness, IFN-γ OD, and ELISA E% obtained for each herd were analyzed employing the Kruskal–Wallis and Mann–Whitney U tests.All the statistical tests were carried out using SPSS Statistics 25  and R version 4.0.5 software  and intep = 0.003; p = 0.372; p > 0.05, k = 0.29 and k = 0.43, respectively).The median increases in skin fold thickness observed after the bovine PPD injection on the cervical and scapular sites were 6 mm (IQR = 4–7.75 mm) and 5 mm (IQR = 4–7 mm), respectively, and the differences observed were significant (p < 0.01) (k = 0.45) and severe (k = 0.44) interpretations.In contrast to the SIT test results, a significantly higher number of reactors to the CIT test was observed as regards the cervical site in comparison to the scapular site when using the standard interpretation ( < 0.01) . The agrp < 0.01). A higher number of goats with clinical signs on the cervical site when compared to the scapular site were similarly observed after the avian PPD injection (p < 0.01).Of those animals that obtained positive results to the SIT and CIT tests, seven had local clinical signs after the bovine PPD injection on the cervical site, and only one on the scapular site  were observed on the scapular site in comparison to the cervical site after the bovine and avian PPD inoculations . No local clinical signs were detected in this herd, regardless of the PPD (bovine or avian) or the inoculation site  used.A high proportion of animals in the low prevalence herd (61/94) did not undergo any increase in skin fold thickness on the cervical site when using the bovine PPD . No reactors to the severe CIT test were, however, detected for any of the inoculation sites  and scapular sites (IQR = 0–1 mm) when using the bovine PPD . The medon sites . No clinp < 0.01, In addition to the seven animals that were positive to the severe SIT test on the cervical site, another three different animals were positive to IGRA when using the severe cut-off point (0.05). When interpreting the P22 ELISA in combination with the IGRA (0.05 cut-off), the percentage of positive reactors increased significantly  were observed on the cervical site when compared to the scapular site. It is possible to hypothesize that this higher reactivity of the cervical site when compared to the scapular site could be related to the closer proximity to regional lymph nodes , which are in turn associated with the entrance route of TB infection , 41. AnoIn the low prevalence herd, the proportion of reactors to SIT test when using the severe interpretation criteria was significantly higher on the scapular site. However, most of the animals that were positive only on the scapular site were negative to the CIT test, IGRA, and P22 ELISA. Bearing in mind the low prevalence previously observed in this herd and the lack of agreement with the CIT test, IGRA, and P22 ELISA, the positives reactions on the scapular site could be related to a reduced specificity when compared to the cervical site in goats. In this respect, Casal et al. established that the probability of detecting a reactor to a skin test as regards any specific inoculation site was strongly associated with the prevalence, since higher values were observed in herds with a high proportion of reactors . It is aNo positive reactors to the intradermal tests were observed when using the recommended standard interpretation in the TB-free herd, which proved to have an excellent specificity. Although 7 out of 119 animals were detected as being positive in the case of the cervical site when using the severe interpretation of the SIT test, this criterion is not recommended in TB-free and MAP-vaccinated herds, particularly in the first months after vaccination. Moreover, none of these positive reactors to the severe SIT test were positive to IGRA or the P22 ELISA, regardless of the cut-off point used. These results are supported by previous studies in TB-free cattle, in which greater skin responsiveness was observed in the anterior neck when compared with the area nearer to the shoulder .The CIT test was used to evaluate the effect of the inoculation site after the avian PPD inoculation in MAP vaccination circumstances. In our study, all three herds were vaccinated against MAP, which could be associated with the reactivity to the avian PPD observed . DiffereM. bovis SB0121) by means of bacteriological culture, although the study population was not subjected to individual post-mortem examination. The use of in vitro cell-based and humoral tests, such as IGRA or the P22 ELISA, therefore, makes it possible to obtain better knowledge of the immunological status of the animals. It is necessary to state that none of the goats from the low prevalence and TB-free herd that reacted only on one of both sites (severe interpretation in the TB-free herd) attained a positive result to IGRA, and few were positive to the P22 ELISA. In this respect, a recent study showed that the sensitivity of IGRA is higher than that observed for the SIT/CIT test in goats . Written informed consent was obtained from the owners for the participation of their animals in this study.JO, ÁR, and JB wrote the manuscript, performed the literature search, and designed the figures. JO, JS-C, ML, and ÁR performed the experiments. JÁ, JO, JI-L, JB, LD, ÁR, LJ, JS, and BR interpreted the data. All authors reviewed and approved the manuscript.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.


The lateral habenula (LHb) is a phylogenetically primitive brain structure that plays a key role in learning to inhibit distinct responses to specific stimuli. This structure is activated by primary aversive stimuli, cues predicting an imminent aversive event, unexpected reward omissions, and cues associated with the omission of an expected reward. The most widely described physiological effect of LHb activation is acutely suppressing midbrain dopaminergic signaling. However, recent studies have identified multiple means by which the LHb promotes this effect as well as other mechanisms of action. These findings reveal the complex nature of LHb circuitry. The present paper reviews the role of this structure in learning from reward omission. We approach this topic from the perspective of computational models of behavioral change that account for inhibitory learning to frame key findings. Such findings are drawn from recent behavioral neuroscience studies that use novel brain imaging, stimulation, ablation, and reversible inactivation techniques. Further research and conceptual work are needed to clarify the nature of the mechanisms related to updating motivated behavior in which the LHb is involved. As yet, there is little understanding of whether such mechanisms are parallel or complementary to the well-known modulatory function of the more recently evolved prefrontal cortex. The lateral habenula (LHb) is a brain structure that has received a great deal of attention and has been a hot topic in the past decades. Consequently, this research field has been extensively reviewed. We review in detail some key recent findings that are pivotal in framing the role of the LHb in well-described associative learning phenomenon, conditioned inhibition. This specific topic has not been considered deeply enough in previous review articles. We also outline the possible mechanisms by which the LHb updates behavior by means of two identified pathway categories (inhibitory and excitatory). This provides a comprehensive account potentially embracing more issues than previously thought, refines our understanding of multiple reward-related mechanisms, and raises novel research questions.The lateral habenula (LHb) is a phylogenetically preserved brain structure located in the dorsomedial surface of the thalamus that participates in learning from aversive  experiences. These include primary aversive stimuli, reward omission, and cues associated with either aversive stimuli or reward omission . HoweverThe LHb has been characterized as a part of a “brake” mechanism to suppress firing in midbrain dopaminergic neurons . Such efNumerous reviews about the physiology of the LHb have already been published; most of them cover the participation of the LHb in primary aversive learning, stressing on the its putative implications for psychiatric conditions . Other sXΔV represents the change in the associative strength (V) of the target stimulus  in a given trial, λ represents the magnitude of reward (zero if the reward is omitted), NΣV represents the sum of the values of all cues that are present during a conditioning trial , and α, which is bound between 0 and 1, represents a learning rate parameter. To show how the model operates, let us first consider the simplest possible example. This requires assuming that, during conditioning trials, the only relevant input besides the reward is a single cue (thus being X ≈ ΣVNV). If λ (> 0) and α are held constant, XV will increase approaching λ throughout conditioning trials  in a decelerated fashion, as the discrepancy between these parameters  progressively declines  or aversive, and as positive or negative . Such dopamine bursts increase the probability of occurrence of the actions that took place priorly, whenever the organism is presented to a similar situation in the future . In addition, dopamine bursts backpropagate to reliable signals of reward, making them acquire signaling  and rewarding  properties in themselves (XV > 0) is triggered by inputs received by midbrain neurons from multiple nodes in a brain-wide network subsequent to sensory processing  trigger a phasic decrease (or dip) in dopamine activity  obtaining a negative value for XVwith XV would range from zero to λ, it can only vary in its degree of “rewardingness” (for lack of a better term). That is, one could conceive of a stimulus as more or less rewarding than the other, but hardly as more aversive in a general sense. However, a distinctiveness of the Rescorla–Wagner model allows to account for both net negative associative strength values and acquisition of opposed affective effects. Such feature is important because it captures aspects of key conditioning phenomena.When conceptualizing conditioning trials as if a single cue is relevant for reward learning , XV could take negative values under appropriate conditions. In order for X to end up with a net negative associative strength following reward omission requires that (1) its current associative strength to be sufficiently low and (2) the remaining of the coextensive stimuli have some associative value . Crucially, this model assumes that the context  could acquire associative strength and is thus capable of meaningfully impacting learning. For example, presenting X without the reward and the reward without X with time intervals in between would imbue that stimulus with net inhibitory properties . This protocol is known as the explicitly unpaired procedure. In such a condition, the context, C, acquires a positive associative strength (CV > 0), by being present whenever the reward is delivered. Importantly, the context is also present during the trials involving the X stimulus plus the omission of reward, being NΣV equal to CV + XV . The remaining type of trial consists of the same stimulus accompanied by another stimulus, X, followed by the omission of that outcome. Such a manipulation, according to the Rescorla–Wagner model, leads stimulus A to acquire a positive associative strength and stimulus X to acquire a deep negative associative strength .A convenient way to conceptually and empirically instantiate these attributes of the Rescorla–Wagner model is the ngth see . This hyThe omission of an expected reward seems to promote a similar backpropagation process to the one that takes place when a cue predicts reward. Interestingly, the LHb is likely to play a crucial role in this process see also . As mentFirst, not omitted. Thus, the inhibitory effects in the target cue must have arisen from the artificial dips in dopamine firing, complementing the findings of The second experiment was conducted recently by As we mentioned above, one of the most robust physiological effects of LHb activation is producing phasic dips in dopamine firing. Therefore, both The above section concluded that dopamine dips may function as “teaching” signals for gradually imbuing a stimulus with negative associative strength. The Rescorla–Wagner model states that the negative associative strength accrued by a stimulus that is paired with reward omission depends on concurrent reward cues see . TherefoA relevant consideration is the origin of the signals required for invigorating the LHb activity on signals of an impending reward. The entopeduncular nucleus , other protocols are also capable of doing so see . LikewisThe disruption of conditioned inhibition was first observed by performing an electrolytic lesion in the entire LHb. This result was replicated when the authors induced a selective ablation of neurons in the LHb that projected to the RMTg via a viral technique. Therefore, the authors concluded that this LHb-RMTg pathway is crucial for learning these specific negative predictions about rewards. Remarkably, electrolytic lesions in the LHb did not disrupt PIT performance for rats trained with traditional forward-conditioning (stimulus→reward) procedure. This suggests that the disruptive effect was confined to inhibitory learning see also . LaurentNotably, the findings reported by At the behavioral level, The findings reported by Both in backward conditioning and in explicitly unpaired conditioning preparations, the reward would not be expected to occur in any particular moment; specifically, a reward delivery is scheduled to occur at random intervals without discrete cues anticipating it, unlike in extinction and in feature-negative discrimination procedures. The ability of the LHb to modulate reward seeking, even in paradigms not involving explicit reward omissions, raise the question of what the mechanisms involved are. A recent study by in vivo real-time recording or special tests for conditioned inhibition in lesion/inactivation studies. In addition, it is not clear how the LHb would be more active in explicitly unpaired conditions than in control conditions involving equivalent reward density, which was the main result reported by The LHb is known to modulate other monoamines besides dopamine, such as serotonin . Tonic sThe glutamatergic LHb-VTA-mPFC pathway see  is less We should consider first that the mPFC is divided in functionally dissociable anatomic subregions, at least for some eutherian mammals see . A subreIt has been suggested that the opposing effects of the prelimbic division of the mPFC upon the reward systems is exerted via an aversion mechanism. This rationale is supported by evidence that this region is involved in fear learning  and exhiHowever, some findings e.g.,  seem to Furthermore, a recent finding has tilted the scale in favor of the aversive–appetitive modularity of the mPFC. There is an emerging literature suggesting that many psychiatric disorders can be characterized as alterations of the ability to predict rewards and aversive outcomes . TherefoSome authors have proposed that a pervasive reward learning process  can explSynthetizing behavioral and neurophysiological studies could serve to address the etiology and phenomenology of these psychiatric conditions. Such an enterprise would require integrating details regarding behavioral changes in response to environmental constraints and the underlying neurobiological mechanisms involved. This is precisely what we have attempted in the present article. If ongoing research continues to make advances, then this perspective could serve to guide clinical practice. An example would be determining whether psychotherapy would be enough to tackle a particular case or whether combining it with pharmacotherapy would be pertinent. Knowledge about neural and behavioral dynamics might serve to design combined intensive interventions to avoid long-term side effects of medications and dependency . AdditioSome recent studies have furthered our understanding of behavioral-physiological interactions involving the LHb from basic science to application. For example, Computational modeling of associative phenomena is just one possible approach to account for the relationship between LHb physiology and inhibitory learning. There are other alternatives for improving our understanding of this topic, even in a more detailed manner, such as neural network models. These are powerful tools for envisaging plausible mechanisms implemented by biological agents to interact adaptively with their environment. A clear advantage of these models is that they make physiological hypotheses more tenable . To our Another matter for future consideration is whether the role of the LHb in learning from reward omission is exclusive for edible rewards or it extends to other types of reward, such as sexual stimuli for receptive individuals. In addition, our understanding of the behavioral neuroscience regarding the LHb come from select model species. It has been hypothesized that the habenular complex evolved in an ancestor of vertebrates to enable circadian-determined movement modulation . In thisDrosophila flies  in associative strength, presumably through the omission of a reward at a precise moment following a cue. However, backward conditioning also induces conditioned inhibition and this effect is impaired by specific ablation of the LHb-RMTg pathway . In thisConverging lines of evidence suggest that LHb participates in the induction and expression of inhibitory learning under different conditions involving reward. This supports the intriguing idea that this structure is composed of several parallel circuits operating in different functionally equivalent situations . Unfortu


Ginkgo biloba leaves (GBL) displayed the most potent hUGT1A1 inhibition in HeLa-UGT1A1 cells (Hela cells overexpressed hUGT1A1). Further investigations demonstrated that four biflavones including bilobetin, isoginkgetin, sciadopitysin and ginkgetin, are key constituents responsible for hUGT1A1 inhibition in living cells. These biflavones potently inhibit hUGT1A1 in both human liver microsomes (HLM) and living cells, with the IC50 values ranging from 0.075 to 0.41 μM in living cells. Inhibition kinetic analyses and docking simulations suggested that four tested biflavones potently inhibit hUGT1A1-catalyzed NHPN-O-glucuronidation in HLM via a mixed inhibition manner, showing the Ki values ranging from 0.07 to 0.74 μM. Collectively, our findings uncover the key constituents in GBL responsible for hUGT1A1 inhibition and decipher their inhibitory mechanisms against hUGT1A1, which will be very helpful for guiding the rational use of GBL-related herbal products in clinical settings.Human UDP-glucuronosyltransferase 1A1 (hUGT1A1) is one of the most essential phase II enzymes in humans. Dysfunction or strong inhibition of hUGT1A1 may result in hyperbilirubinaemia and clinically relevant drug/herb-drug interactions (DDIs/HDIs). Recently, a high-throughput fluorescence-based assay was constructed by us to find the compounds/herbal extracts with strong inhibition against intracellular hUGT1A1. Following screening of over one hundred of herbal products, the extract of  In adulractions   and herbal constituents (such as flavonoids and phenolic acids) have been reported (ely weak . The priGinkgo biloba leaves (GBL) displayed the most potent hUGT1A1 inhibition potency, with the inhibition rate exceeding 95% at the dose of 100 μg/ml. This finding encouraged us to further investigate the inhibition potentials of GBL and its major constituents against hUGT1A1 in various enzyme sources including HeLa-UGT1A1 cells and human liver microsomes, as well as to identify the key constituents in GBL responsible for hUGT1A1 inhibition. Furthermore, the inhibition potential and the inhibitory mechanisms of the newly identified naturally occurring hUGT1A1 inhibitors from GBL were also investigated by performing an array of inhibition kinetic assays and docking simulations. All these studies are very helpful for researchers to understand the interactions between the used GBL and human drug-metabolizing enzymes, and also provide the key data to assess potential risks or beneficial effects of the used GBL by inhibition of hUGT1A1.Recently, a high-throughput cell-based fluorescence assay was developed by using an artificial specific fluorescence substrate (termed N-butyl-4-(4-hydroxyphenyl)-1,8-naphthalimide (NHPN)) as the probe substrate and the hUGT1A1 overexpressed Hela cells (HeLa-UGT1A1 cells) as the enzyme sources . With thGinkgo biloba leaf extract (GBL) was obtained from Shanghai Sine Promod Pharmaceutical Co., Ltd. , and the chemical analysis of Ginkgo biloba leaf extract was analyzed by using ultra high performance liquid Chromatography-Q exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometric  (O-glucuronide (NHPNG) were chemically synthesized by one of the authors (Ping Wang) according to the previously reported scheme . UDPGA trisodium salt and polyethylene glycol hexadecyl ether (Brij 58) were purchased from Sigma-Aldrich . Cell culture medium and fetal bovine serum were obtained from Hylcone . Phosphate buffered saline , Millipore water and LC grade organic solvent  were ordered from Tedia company  and used for all experiments.The standard  States) . Other 1 States) . N-butyld scheme , with thO-glucuronidation activity of HeLa-UGT1A1 cells were determined by using NHPN as the probe substrate , equipped with a DGU-20A5R vacuum degasser, two LC-20ADXR pumps, a SIL-20ACXR autosampler and a CTO-20A column oven, was used to quantify NHPN and NHPNG. Chromatographic separation of analytes was carried out on a shim-pack Acchrom HC-C18 analytical column , while the column temperature was kept at 40°C. The flow rate was kept at 0.5 ml/min. The mobile phase consisted of acetonitrile (A) and water containing 0.2% formic acid (B). The gradient of the mobile phase was as follows: 0–0.5 min, 80–60% B; 0.5–3.5 min, 60–5% B; 3.5–5 min, 5% B; 5–7.5 min, equilibration with 80% B. The excitation wavelength and the emission wavelength were set at 370 and 520 nm, respectively for detection.2 in Modified Eagle’s Medium (MEM) containing 0.1% antibiotic-antimycoticmix, supplemented with 10% fetal bovine serum (FBS). HeLa-UGT1A1 cells were seeded in 96-well plates and cultured for 24 h. The cells were incubated in the medium containing different kinds of herbal products  or chemical constituents of GBL extract  for 60 min at 37°C under 5% CO2, and co-incubated with NHPN  for another 60 min. The reaction was terminated by adding an equal volume of ice-cold acetonitrile to each well, and then centrifuged at 20,000×g for 20 min at 4°C. After centrifugation, 5 μl aliquots of the supernatant was taken for LC-FD analysis. The validation data for this cell-based flourescence assay can be found in our previous publication  and the enzymes. Inhibitors  were serially diluted to the required concentration by DMSO. The final concentration of DMSO in the incubation mixture was less than 1%. When HLM was used as the enzyme source, HLM (10 μg/ml) and Brij 58  were pre-incubated for 20 min at 4°C to disrupt the ER membrane to remove the latency. A concentration of 5 μg/ml was used when recombinant UGT1A1 was the enzyme source. After pre-incubation for 3 min at 37°C, UDPGA was added to the incubation mixture to initiate the reaction. After 60 min incubation at 37°C, the reaction was terminated by adding 200 μl ice-cold acetonitrile, and the incubation mixture was kept on ice. After centrifugation at 20,000×g for 20 min at 4°C, 5 μl aliquots of the supernatant was taken for LC-FD analysis.NHPN was used for evaluating the inhibitory effects of GBL extract on HLM. A typical incubation mixture with a total volume of 200 μl consisted of 50 mM Tris-HCl (pH = 7.4), 5 mM MgCl50).The residual activity of hUGT1A1 was determined as follows: Residual activity (%) = (the fluorescence intensity of NHPNG generated in the incubations with inhibitor)/(the fluorescence intensity of NHPNG generated in the incubations without inhibitor) × 100%. A set of incubations with the same fluorescent substrate concentration and different inhibitor concentrations were performed to determine the half maximal inhibition concentration (ICKi) values. The incubation and analytical sample preparation were conducted as described previously. Goodness-of-fit parameter and Akaike’s Information Criterion (AIC) were employed to select the most appropriate inhibition kinetic modes from the following three inhibition modes including competitive inhibition A set of incubations by changing both the fluorescent substrate concentration and the inhibitor concentration were performed to determine the inhibition kinetic modes and the corresponding inhibition constant ; Ki is the inhibition constant that describes the affinity of the inhibitor towards hUGT1A1. The corresponding Ki value of each tested inhibitor is calculated by utilizing the slopes of second plot from Lineweaver-Burk plot versus inhibitor concentrations.Here, Docking simulations were used to explore the binding modes of each inhibitor into hUGT1A1 by using AutoDock Vina (1.1.2) . The 3D 50 and Ki values were determined by using GraphPad Prism 7.0 .All assays were conducted in triplicate. Data was expressed as mean ± SD. IC-O-glucuronidation in HeLa-UGT1A1 cells, with an apparent IC50 value as low as 12.41 μg/ml.First, 127 herbal products were collected and their inhibitory effects on hUGT1A1 were assayed in HeLa-UGT1A1 cells at the final concentration of 10 μg/ml. As shown in 50 values ranging from 1.38 μM (kaempferol) to 64.56 μM (myricetin)  to 0.41 μM (bilobetin)  . A totalricetin) . Notablylobetin) . The fivlobetin) , which c-O-glucuronidation in HLM, with the IC50 values ranging from 0.15 to 0.67 μM. Notably, the inhibition potentials of four tested bioflavones against hUGT1A1 in HLM are similar to that in Hela-UGT1A1 cells. However, the IC50 value of amentoflavone in HLM is much lower than that in living cells (0.41 vs. 3.29 μM)  and in Hela-UGT1A1 cells (12.41 μg/ml)   . For GBL1 μg/ml) . In addi0.67 μM) . The inhvia hydrophobic interactions  and a variety of clinical drugs  . InhibitGinkgo biloba is among the most widely consumed herbal supplements and phytopharmaceutical drugs used in the world. The primary active constituents of GBL are flavonoids and terpene lactones, which account for 26.8 and 9.8% of the tested GBL in the present study, respectively . It is worthy to be noted that, amentoflavone, also baring a biflavone structure, showed a relatively weak inhibition on hUGT1A1, with the IC50 value more than 10-fold higher than those of the other four biflavones. Although it only differs from the others in the number of the methoxy groups, a lower logP value (3.49), which indicates a poorer permeability compared to the other four with logP values of 5.23–5.38, could serve as an explanation for its decreased inhibition potency on hUGT1A1 observed in Hela-UGT1A1 cells. The much lower IC50 value in HLM (0.41 μM) than that in Hela-UGT1A1 cells (3.29 μM) for amentoflavone may further support the poor permeability of this compound  (ectively . Our curcompound . However1 μg/ml) .Cmax) of the specific four biflavones which were found to be the most potent hUGT1A1 inhibitors in the present study, the Ki values for these four compounds ranging from 0.15 to 0.67 μM were much higher than the measured plasma Cmax of their bioflavone analogs orally administrated in rats  in healthy volunteers , which may be a plausible explanation for the mixed-inhibition mode of these newly identified hUGT1A1 inhibitors. In conclusion, our findings revealed that the biflavones in GBL are potent hUGT1A1 inhibitors, which can be very helpful for guiding the rational use of GBL-related herbal products in clinical settings.In summary, with the help of a newly designed high-throughput cell-based fluorescence assay, the inhibitory potentials of a number of commonly used herbal medicines against hUGT1A1 were assayed in living cells. The results demonstrated that GBL extract used in the present study displayed the most potent hUGT1A1 inhibition potency, with the inhibition rate exceeding 95% at the dose of 100 μg/ml. Further investigations showed that four biflavones including bilobetin, isoginkgetin, sciadopitysin and ginkgetin, are key constituents responsible for hUGT1A1 inhibition in living cells, while the flavonoids in GBL contribute to a lesser extent for hUGT1A1 inhibition. Inhibition kinetic analyses suggested that the biflavones in GBL potently inhibit hUGT1A1-catalyzed NHPN-