These methods predicted pentagastrin as a potential antiviral drug against ASFVs. Finally, it was also observed that the compound had an inhibitory effect on AsfvPolX activity. Results from the present study suggest that molecular docking and machine learning models can play an important role in identifying potential antiviral drugs against ASFVs.African swine fever virus (ASFV) causes a highly contagious and severe hemorrhagic viral disease with high mortality in domestic pigs of all ages. Although the virus is harmless to humans, the ongoing ASFV epidemic could have severe economic consequences for global food security. Recent studies have found a few antiviral agents that can inhibit ASFV infections. However, currently, there are no vaccines or antiviral drugs. Hence, there is an urgent need to identify new drugs to treat ASFV. Based on the structural information data on the targets of ASFV, we used molecular docking and machine learning models to identify novel antiviral agents. We confirmed that compounds with high affinity present in the region of interest belonged to subsets in the chemical space using principal component analysis and  Asfarviridae family (genus Asfivirus), which contains a genome measuring 170–193 kbp, and replicates predominantly in the cytoplasm of macrophages [African swine fever (ASFV) is an enveloped and double-stranded DNA virus belonging to the rophages . It has rophages . Recentlrophages ,6,7. Vacrophages . Thereforophages ,11,12,13rophages ,21,22,23AsfvAP), a repair DNA polymerase (AsfvPolX), and a DNA ligase (AsfvLIG) [AsfvPolX and AsfvLIG are very low. Therefore, in addition to their critical role in genome stability maintenance, these enzymes play an important role in the strategic mutagenesis and genotype formation of ASFV [ASFV is primarily replicated in the cytoplasm of swine macrophages, which is a major source of reactive oxygen species that cause constant damage to the ASFV genome ,25,26,27AsfvLIG) ,29,30. U of ASFV ,31,32,33 of ASFV ,35.AsfvPolX, to identify novel targets for the prevention and treatment of ASFV. AsfvPolX plays a role in the DNA repair process of the ASFV genome, and viral replication is partially dependent on the function of AsfvPolX. In addition, AsfvPolX has several unique structural features, including a 5′-phosphate (5′-P) binding pocket, a His115-Arg127 platform, and hydrophobic residues Val120 and Leu123, which can all affect the catalytic efficiency of AsfvPolX. It can be assumed that blocking the binding pocket with therapeutics may inhibit AsfvPolX activity and thereby impair the DNA repair process of the viral genome [AsfvPolX protein structures (AsfvPolX apo and AsfvPolX/DNA structures) to identify potential antiviral drugs using a novel integrated strategy that combines molecular docking and machine learning models [k-means clustering. Pentagastrin, which had the highest binding affinity in both the apo and DNA complex structures of AsfvPolX, was chosen as an antiviral candidate drug. We also identified the dose-dependent inhibitory effect of pentagastrin on AsfvPolX polymerase activity.In this study, we focused on an enzyme that helps synthesize DNA, the ASFV DNA polymerase X or l genome . In the g models ,37. Artig models ,40,41,42The use of this combination strategy can provide an alternative approach to identifying potential antiviral drugs against ASFVs from molecules that have already been tested or approved for other diseases. Furthermore, the region of interest that only appears in groups with high binding affinity to ASFV targets identified based on eight molecular descriptors used in this study can provide key information regarding the unique chemical characteristics of antiviral drugs against ASFVs. These results demonstrate that active compounds with regions of interest in the chemical space may be expected to provide insights into potential ASFV inhibitors.AsfvPolX protein were identified using SiteMap, with an applied force field of OPLS_2005 in the Schrödinger suite. SiteMap uses the hydrophobicity and accessibility of a detected binding site to assess the likelihood of a small-molecule inhibitor binding to it. We denoted the four top-ranking surface clefts identified by the SiteMap module on the structure of AsfvPolX protein in complex with DNA (PDB ID: 5HRB) from site 1 to site 4 , using FDA-approved drugs from the DrugBank database. The reported eight AsfvPolX complex structures demonstrated that AsfvPolX has one unique 5′-P binding pocket that can inhibit the activity of AsfvPolX and disrupt the DNA repair process of the ASFV genome [AsfvPolX as a potential binding pocket for antiviral inhibitors. To determine which conformations of the apo and DNA complex structures are structurally proximal conformations as template structures for virtual screening of antiviral compounds with high binding affinity, we implemented a molecular docking program using both the crystal structure of apo (PDB ID:1JAJ) and DNA complex structure (PDB ID:5HRB). We obtained 1729 drugs from the DrugBank database. After performing molecular docking, the 1JAJ and 5HRB datasets consisted of 1714 and 1712 compounds. AsfvPolX-DNA complex structure is a suitable template for determining compounds with high binding affinity and can also provide important guidance for the design and discovery of potential antiviral inhibitors for AsfvPolX.To discover new antiviral drugs capable of inhibiting the activity of V genome . PreviouTo investigate the distinct features of molecules based on docking results for apo and DNA complex structures, eight molecular descriptors of each molecule were calculated and applied to PCA . The 1JAk-means clustering for three datasets consisting of compounds with binding affinities ≤−4.0 kcal/mol based on eight molecular descriptors -RIL cells carrying pET28-AsfvPolX plasmid were cultured in the presence of isopropyl β-d-1-thiogalactopyranoside, and then the AsfvPolX protein was purified using a Ni2+-nitrilotriacetic acid (Ni-NTA) column  column A. AsfvPogradient B. We firy by 60% D, indicaAsfvPolX protein. The hydroxyl group of pentagastrin formed hydrogen bonds with Lys85 and Lys136 at the binding site , showed that pentagastrin was fitted into a groove at the same DNA binding pocket, and that it had a similar conformation as that of 1nt-gap DNA1 A. In theing site B. The prAsfvPolX, in complex with DNA  [The binding site was characterized using the SiteMap module of the Schrödinger software package  . The SitAsfvPolX-ligand complexes, molecular docking studies were performed using Glide software , which uses an optimized potential for liquid simulations (OPLS)-2005 force field, and refinement was carried out according to the recommendations of the Schrödinger Protein Preparation Wizard. The ligand database consists of 2413 FDA-approved drugs obtained from DrugBank. Prior to screening, the database was filtered to select compounds with molecular weights >200 Da and 1729 compounds were obtained. LigPrep  was used to generate three-dimensional ligand structures. The active grid was generated using the Receptor grid application in the Glide module. On a defined receptor grid, flexible docking was performed using the standard precision mode of Glide [To examine the binding interactions of the of Glide ,45. The https://go.drugbank.com ), consisting of more than 11,000 drugs. Drugs in this database are classified into different types as follows: trial stage drugs, approved drugs, withdrawn drugs, and investigational drugs. In this study, we focused only on FDA-approved drugs (around 2413 compounds) . These compounds were downloaded in the form of a structure data file from the DrugBank database. We removed compounds with a molecular weight less than approximately 200, and thus obtained a dataset comprising 1729 drugs. To perform molecular docking, we used apo and DNA complex structures of the AsfvPolX target to obtain sets of apo (PDB ID:1JAJ) and DNA complex structures (PDB ID:5HRB), which consisted of 1714 and 1712 compounds, respectively, based on the docking score. Finally, 1JAJ and 5HRB datasets consisting of 675 and 1035 compounds with binding affinities ≤−4.0 kcal/mol based on docking score were subjected to further PCA analysis.The dataset was generated from the DrugBank database . The calculated descriptors included molecular weight, number of hydrogen bond acceptors, number of hydrogen bond donors, ALogP, number of rotatable bonds, number of aromatic rings, number of rings, and polar surface area. The frequency distribution of physicochemical properties for the datasets is shown in O174L) was manually synthesized (BIONICS) and cloned into the BamHI and HindIII sites of the pET28 vector.To confirm the in vitro antiviral activity for the candidate compounds identified in this study, the full-length PolX gene (Escherichia coli BL21 (DE3)-RIL, and cells were grown at 37 °C until an A600 value of 0.5 was reached. Cells were then induced with 1 mM isopropyl β-d-1-thiogalactopyranoside for 3 h and collected by centrifugation. The cell pellet was resuspended in buffer T300 . The subscript number in T300 indicates the concentration of NaCl (mM). Cells were then lysed by sonication, and the cell extract was centrifuged for 30 min at 13,000 rpm. The supernatant was loaded onto a Ni-NTA column  column and washed successively with low concentrations of imidazole (20 mM and 60 mM) in T300 until no protein was eluted, and the protein was eluted with 250 mM imidazole in the same buffer. The peak protein fractions were pooled and diluted in 50 mM NaCl. The pooled protein was then loaded onto a HiTrap SP HP column  that was eluted with a linear gradient (50–600 mM) of NaCl in buffer T. The eluted fractions containing the highest concentration of protein were collected (eluting between 350 and 450 mM of NaCl), and an aliquot was subjected to glycerol gradient centrifugation (15–35% glycerol) at 54,000 rpm for 26 h in a Beckman SW55Ti rotor. Gradient fractions containing active PolX were stored at −70 °C until use.The pET28-PolX plasmid was transformed into Polymerase activity of PolX was measured using the polymerase activity assay kit , according to the manufacturer’s instructions, with minor modifications. Briefly, PolX protein was mixed with pentagastrin  for 10 min at 4 °C and then further mixed with 2× reaction buffer and primed substrate. For PolX induction, the mixture was incubated for 30 min at 37 °C. Following inactivation of polymerase activity, the synthesized DNA was denatured at 95 °C for 20 min. For annealing DNA, the temperature was decreased at 65 °C for 10 min, 45 °C for 5 min, and 25 °C for 5 min. One hundred cycles of 5 s, with 0.5 °C temperature increments from 45 °C to 95 °C, were used for the melting curves to measure synthesized DNA contents using a CFX Connect real-time system (Bio-Rad Laboratories). The 2× reaction mixture was incubated with a standard substrate  to create a standard curve.k-means clustering method was used to establish classification models for predicting whether compounds with high binding affinity are inhibitors of ASFVs. For the datasets, we identified the optimal clustering consisting of six clusters. In particular, compounds in Cluster 2 were found to have a much higher binding affinity on average compared to the other clusters. Therefore, we selected the top-ranked (with the top score) pentagastrin on both apo and DNA complex structures for further in vitro studies. Pentagastrin inhibited AsfvPolX activity by 60% at 100 μM concentrations, indicating that the strategy used in this study can reduce the processing time and cost required to identify potential antiviral drugs against ASFVs from molecules already tested or approved for other diseases. These studies to identify drugs with antiviral activity against ASFV have helped in determining active antiviral drugs available for the treatment of ASFV infections. The present study, performed through molecular docking and machine learning models, can play an important role in identifying potential antiviral drugs against ASFVs.In this study, we applied molecular docking and machine learning to build models from publicly available screening data (DrugBank) to predict antiviral drugs that could potentially inhibit ASFV. To determine which conformations of the apo and DNA complex structures are structurally proximal conformations as template structures for virtual screening, we first conducted a docking simulation using both structures. To investigate and compare molecules based on the docking scores for apo and DNA complex structures, eight molecular descriptors of each molecule were calculated. From molecular descriptor-based PCA, we determined that 5HRB and a duplicated dataset containing compounds with high binding affinity were generally well distributed throughout the chemical space. Conversely, the three datasets of compounds with low binding affinity were distributed concentrically around the center of the chemical space. In this work, the


The automatization of behavioral tests assessing motor activity in rodent models is important for providing robust and reproducible results and evaluating new therapeutics. The CatWalk system is an observer-independent, automated and computerized technique for the assessment of gait performance in rodents. This method has previously been used in adult rodent models of CNS-based movement disorders such as Parkinson’s and Huntington’s diseases. As motor and gait abnormalities in neuropsychiatric disorders are observed during infancy and adolescence, it became important to validate the CatWalk XT in the gait analysis of adolescent mice and unravel factors that may cause variations in gait performance. Three adolescent wild-type inbred mouse strains, C57BL/6N, DBA/2 and FVB/N, were tested using the CatWalk XT (Version 10.6) for suitable detection settings to characterize several gait parameters at P32 and P42. The same detection settings being suitable for C57BL/6N and DBA/2 mice allowed a direct comparison between the two strains. On the other hand, due to their increased body weight and size, FVB/N mice required different detection settings. The CatWalk XT reliably measured the temporal, spatial, and interlimb coordination parameters in the investigated strains during adolescence. Additionally, significant effects of sex, development, speed and body weight within each strain confirmed the sensitivity of motor and gait functions to these factors. The CatWalk gait analysis of rodents during adolescence, taking the effect of age, strain, sex, speed and body weight into consideration, will decrease intra-laboratory discrepancies and increase the face validity of rodent models of neuropsychiatric disorders. Therefore, qualitative and quantitative behavioral experiments of motor deficits including the rotarod, grip strength, inverted screen, and beam balance rod tests are often used in rodent models based on the wide range of parallels between rodents and human locomotion. Additionally, these assays are a quantifiable measure of the effectiveness of various therapeutic strategies. However, these motor tests share the limitation of being mainly observer dependent and of detecting only gross deficits in coordination and motor function12. For gait analysis, observational scoring as a simple low-technology tool was widely performed 20 years ago. It used rank-order structured scales to grade the severity of rodent gait abnormalities from video recordings using visual observations13. Since these scales have been developed by individual laboratories, scoring systems were relatively inconsistent and observer biased, and provided only semi-quantitative gait descriptions13. On the other hand, several gait parameters have been studied using different experimental setups including the electric grid14 and footprint analysis18. In recent years, technological advances and automated systems provide observer-independent quantitative assessments of spatiotemporal patterns. Although quadrupedal gait patterns used by rodents vary significantly from bipedal human patterns, the conceptual basis for gait analysis remains analogous. Therefore, advanced technologies for rodent gait analysis are critical for better modeling human disorders.Motor and coordination dysfunctions including gait abnormalities are the main diagnostic criteria for many CNS disorders such as Parkinson’s, Huntington’s and Alzheimer’s diseases, amyotrophic lateral sclerosis, and stroke  and was carried out in compliance with the ARRIVE guidelines.Animals and housing conditions were similar to our previous studiesThe habituation and experimental procedures of the CatWalk XT were performed during the same period of the day (from 7 am to 3 pm). Mice were brought into the behavioral room half an hour before the behavioral testing. We analyzed the behavior in 2 cohorts of group-housed mice of both sexes at P32 and P42. The numbers of male and female mice per cohort and strain are listed in Table 46 with modifications. First, the walkway area was set according to the company’s recommendations. Mice were then habituated to the CatWalk XT and allowed to cross the corridor voluntarily for three accomplished runs at P31 and P41. To achieve an unforced type of locomotion, we did not include in our method any light stimulus, food enticement or even using the home cage as bait at the end of the walkway. On the following day, mice were tested by crossing the corridor three times. A compliant run is described as a mouse walking across the runway without stopping, turning around, or changing direction. We set up the minimum and maximum run durations to be 0.5 and 12 s, respectively. Three compliant runs made up the testing run. We have configurated different detection settings for the 3 strains. For all C57BL/6N and DBA/2 mice, the same detection settings were used . Due to their larger body size and weight, all FVB/N mice required other detection settings . For the analysis, gait parameters were automatically generated after each footprint being manually checked and respectively labeled LF (Left Front), LH (Left Hind), RF (Right Front), and RH (Right Hind) paws. For the presentation of the data, we categorized the gait parameters into 4 major groups: (a) run characteristics and kinetic parameters, (b) temporal parameters, (c) spatial parameters, (d) interlimb coordination parameters. These parameters include body and swing speeds, stand and swing times, step cycle, maximum contact at (% of stand time), print area, maximum intensity, base of support, stride length, print position, support, phase dispersion, and step sequences  gait analysis system  consisted of a 1.3-m black corridor on a glass plate with a green LED lit inside, and placed in a dark and silent room (< 20 lx of illumination). Using the Illuminated footprints technology, paws were captured by a high-speed video camera (100 frames per second) that was positioned underneath the glass. The experiment was performed as previously described0.6 gait p value ≤ 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 7 and Microsoft Office Excel software. The respective numbers of male and female mice per cohort are described in Table For the comparison of the body weights of mice in Fig. Most of the gait parameters that we focused on in our study are shown for each paw, namely the left front (LF), left hind (LH), right front (RF), and right hind (RH) paw. The definitions and explanations for the investigated gait parameters are shown in Fig. p < 0.0001)    Fig. . The bodb01 Fig. For the presentation of the data, we categorized the gait parameters into 4 major groups: (1) Run characteristics and kinetic parameters, 2) temporal parameters, (3) spatial parameters, (4) interlimb coordination parameters. The same applied detection settings for both C57BL/6N and DBA/2 mice allowed a direct comparison of their gait characteristics. DBA/2 mice showed increased average speed and cadence and a reduced number of steps compared to C57BL/6N mice at P32  (Supplementary Table p = 0.041) and female DBA/2 mice showing more support percentage on two paws (girdle) than males (p = 0.037)  , with only the RF paw of male DBA/2 mice showing an increase in the maximum intensity (p = 0.005) (Supplementary Table p < 0.0001). The support percentage showed sex-biased results with females C57BL/6N mice showing more support on two paws  than males (p = 0.049). For the regularity index, female C57BL/6N mice showed an increased regularity index than males (p = 0.008). For DBA/2 mice, only the phase dispersion of LF with RF as the anchor paw showed a sex difference with females showing a higher percentage than males (p = 0.022)   and increased maximum intensity of the RF paw (p = 0.047). For interlimb coordination parameters, the base of support of the hind paws at P32 and P42 showed sex difference with a higher base of support in male than female FVB/N mice   are dependent on the locomotion speed of the mouse during the run. In a comprehensive study, the relationship between speed and 162 gait parameters in a group of 16 wild-type mice was investigated and revealed that over 90% of these parameters, reported by the CatWalk software, were dependent on speed19. Notably, that study investigated female B6SJLF1/J mice, generated by breeding C57BL/6J female with an SJL/J male, in comparison to our study that investigated both male and female mice of three different inbred strains. Our study was very similar to another study using male C57BL/6 mice revealing a negative correlation between speed and temporal parameters and a positive correlation with kinetic parameters such as swing and body speed with certain correlations to the spatial and interlimb coordination groups46. Although we showed a direct correlation of the speed with several gait parameters, we did not normalize our results comparing C57BL/6N and DBA/2 mice to speed since we were interested to examine the general gait performance of these strains including the effect of their speed as an intrinsic factor characterizing the strain. However, based on our study and the aforementioned ones, the speed of mice should be taken into consideration when characterizing the gait performance of rodents by a simple normalization of the speed or including it as a covariate during the statistical analysis of gait parameters. For mice modeling brain disorders that are expected to cause a shift in the gait performance irrelevant from their hyper/hypoactivity, it may be better to investigate the gait characteristics in a system that uses a controlled speed treadmill.In our study, we aimed at evaluating the suitability and sensitivity of the CatWalk XT for gait characterization of three adolescent mouse strains, C57BL/6N, DBA/2, and FVB/N. To the best of our knowledge, the present study is the first to objectively and reliably quantify a large number of gait parameters of different mouse strains during adolescence. The same detection setting used for C57BL/6N and DBA/2 mice enabled a direct comparison between the two strains to provide a general idea about the difference in their gait performance during adolescence. A similar pattern of differences between the two strains was found at two ages during adolescence (P32 and P42) including the increased average, body and swing speed, enhanced spatial parameters, and decreased temporal ones in DBA/2 mice. Additionally, a similar pattern of differences in all interlimb coordination parameters except the number of patterns was found between the two strains at the two ages, suggesting a continuous strain effect at different ages during adolescence. Notably, the average speed of DBA/2 mice was higher than that of C57BL/6N mice at both ages. Since locomotor speed is known to affect several gait parameters in quadruples causing intraindividual and interindividual variability that are invisible in the statistical analysis57, which was absent at P42. Still, other factors may play a role in the similar increase in the maximum intensity of both front and hind paws. On the other hand, the increased print area of paws in DBA/2 mice may suggest a less efficient and incomplete clearance of the paw during locomotion compared to C57BL/6N mice, causing an increased hind paw contact with the floor rather than the normal contact of only the front part of the hind paw to the surface58. Since the CatWalk XT can not measure the absolute durations of paw clearance, the aforementioned assumption needs to be further validated using other gait analysis systems.The high maximum intensity of all paws in DBA/2 mice at P32 indicates their increased propulsion, especially in hind paws58. Our study revealed that the main step sequence pattern, Ab, was the same for the DBA/2 and FVB/N strains at both investigated ages. These results are in line with a previous study showing that intact rodents tend to prefer the Ab regular step pattern most of the time (80–95%)59. Interestingly, in our study, C57BL/6N mice showed a similar percentage of alternate and cruciate step sequences at P32 and P42. These results highlight the big effect of strain on interlimb coordination. For the support percentage, the longest simultaneous usage of paws during floor contact in all tested strains was with two diagonal paws, followed by three paws. These results indicate that interlimb coordination is a very stable gait characteristic that does not differ between mouse strains. This finding is in line with a previous study revealing no difference in interlimb coordination parameters in three rat strains, Sprague–Dawley, Wistar, and Lewis55. Notably, phase dispersions may be related to the support percentage since LF- > LH and RF- > RH dispersions are near 50%, especially in DBA/2 mice, matching the high percentage of their diagonal support.Interlimb coordination is a key characteristic of locomotion and, therefore, the parameters concerning coordination are of particular interest. There are six step-sequence patterns described in rodents, and the used pattern by rodents is known to differ because of either stress or a motor disease60, which add another layer of complexity in comparing the behavioral results of different strains. Still, sex hormones and genes on X and Y chromosomes may have an impact on the behavioral outcome. In conclusion, our data confirm the strong intercorrelation between sex and strain on gait performance and highlight the importance of considering sex during gait analysis, even in young mice.The effect of sex on the body weights of mice was shown at P42 for C57BL/6N, DBA/2 and FVB/N mice. Regarding the effect of sex on gait performance, it was obvious mostly at P42 in the three investigated strains. Although these differences can be accounted by a change in the body weights of mice, especially the print area, base of support and regularity index that are shown to be correlated with body weight . These variabilities in different studies need to be reduced or, at least, taken into consideration for achieving reproducible results between laboratories.The correlation between body weight and gait performance has been previously investigated in several studies in adult rodents. In two studies in rodents with middle cerebral artery occlusion (MCAO), body weight showed a strong correlation with several parameters including stand, step cycle, and print area of paws41. The results of that study showed that most of these experiments can be performed reliably on young mice with DBA/2 mice having less motor and coordination abilities41. How these results can be correlated with gait performance needs to be further investigated. The increased average, body and swing speeds of DBA/2 mice may have a negative effect on coordination. Additionally, the increased print areas of all paws can be a sign of incomplete clearance of paws during locomotion, which decreases the efficiency of the whole process. This hypothesis can be strengthened by the increased print areas of the right paws in male DBA/2 mice compared to females at P42, which is consistent with the better performance of female mice on the rotarod41. Still, the reduced body weight of DBA/2 mice can result in a decrease in the muscle strength as shown previously in the grip strength test41 and may have an impact on the motor ability in the rotarod that requires high endurance.In our previous study, we compared the motor and coordination functions of these three inbred strains in six behavioral tests: grip strength, beam balance rod, inverted screen, cliff avoidance reaction, rotarod, and voluntary wheel running63. DigiGait and TreadScan have similar equipment, including a treadmill driving rodents into passive walking/running, which requires extra balance, and thus shorter strides and increased stance times62. In contrast, the CatWalk system uses a stable track that enables rodents to actively move forward. Differences in results using different devices are reported64, which can be explained by differences in the relevant task (passive vs active movements), equipment size, color, and lighting. This highlights that even small methodological factors play an important role in the gait analysis of rodents. Recently, new high-level software with an intuitive, easy-to-use interface built on DeepLabCut™ was used for the analysis of gait performance of mice and named Visual Gait Lab (VGL)65. The main advantage of VGL over the CatWalk system is the visual tracking of absolute positions of the body and the paws, rather than differentiating paw prints compared to back-ground intensity, which can be unreliable with injured mouse models where paw contact with the ground is compromised66. Therefore, it will be of interest to validate the results of our study using another gait analysis system.Other devices including DigiGait , TreadScan , and Dynamic Weight Bearing test are well known to study the gait of rodents73. Recently, Timotius et al. developed a CatWalk gait parameter combination for sensitive and efficient gait recovery assessment in a rat thoracic spinal cord injury model by combining nine of the topmost parameters into a gait index score based on linear discriminant analysis74. The analysis of different combinations of gait parameters more efficiently represents the fully-coordinated motion of gait than single parameters and enhances the evaluation power of gait performance in rodent models of neuropsychiatric disorders.There are some limitations of the CatWalk system including the high dependence on the rodent’s behavior during examination  and their motivation to complete the task. These limitations can be aggravated by using adolescent mice that are known to show an intrinsic higher activity, to be more anxious, sensitive to social isolation, and have a lower capability to learn the task. Therefore, data acquisition of gait performance can be affected due to interrupted walk and heterogeneous velocity, demanding for taking care of the data interpretation. Another limitation of the CatWalk system is the difficulty to make a complete interpretation of all gait parameters, especially when they are evaluated individually. For unraveling the effect of neuropsychiatric disorders on motor functions, understanding the definition of every gait parameter is required, and the reason for each change needs a comprehensive determination and consideration of how multiple different parameters contribute to an altered gait pattern. Indeed, simple combinations of CatWalk parameters have been employed in a variety of disorders to assess gait, such as baseline parameter ratio, left–right-parameter ratio, left–right-parameter averaging, and subtractionOur results can serve as a template for studying changes in locomotor and gait performance in C57BL/6N, DBA/2, and FVB/N adolescent mice. Moreover, the present report confirmed that genetic strain differences are important determinants of motor and gait functions. Having unraveled age-, sex-, speed- and weight-dependence of gait performance, we recommend littermates of the same delivery day, and alike male/female ratios for the behavioral characterization of knockout/knockin mice, as well as correcting for the speed and body weight during the statistical analysis. Keeping these factors in mind will allow a reliable characterization of the motor activity and coordination functions in rodent models of neuropsychiatric disorders.Supplementary Information.


Equal dosage of X-linked genes between males and females is maintained by the X-inactivation of the second X chromosome in females through epigenetic mechanisms. Boys with aneuploidy of the X chromosome exhibit a host of symptoms such as low fertility, musculoskeletal anomalies, and cognitive and behavioral deficits that are presumed to be caused by the abnormal dosage of these genes. The objective of this pilot study is to assess the relationship between CpG methylation, an epigenetic modification, at several genes on the X chromosome and behavioral dysfunction in boys with supernumerary X chromosomes.AR and MAOA of boys with 49,XXXXY whose methylation levels were skewed from levels predicted from having one active (Xa) and three inactive (Xi) X chromosomes. Further, methylation levels of multiple CpGs in MAOA showed nominally significant association with externalizing behavior on the CBCL, and the methylation level of one CpG in AR showed nominally significant association with the BRIEF Regulation Index.Two parental questionnaires, the Behavior Rating Inventory of Executive Function (BRIEF) and Child Behavior Checklist (CBCL), were analyzed, and they showed expected differences in both internal and external behaviors between neurotypical  boys and boys with 49,XXXXY. There were several CpGs in AR) and monoamine oxidase A (MAOA), compared to that in boys with 47,XXY and neurotypical boys. Our pilot study results suggest a link between CpG methylation levels and behavior in boys with 49,XXXXY.Boys with 49,XXXXY displayed higher levels of CpG methylation at regulatory intronic regions in X-linked genes encoding the androgen receptor (The online version contains supplementary material available at 10.1186/s13148-021-01123-4. Pediatric endocrinologists currently prescribe HRT, specifically testosterone, to males with 47,XXY and 49,XXXXY based on physical examination. Neurodevelopmental investigations on these males showed that infants with 47,XXY who receive early hormonal treatment (EHT) showed improvement in Full-Scale IQ, Verbal IQ, and speech and language development when compared to untreated boys . In addiXIST (X-inactive specific transcript) RNA from the inactive X chromosome  based on the experimentally obtained CpG-2 methylation (52.5%) in the 47,XXY samples. These derived values for Xa and Xi for CpG-2 were then used to assign hypothetical values to each X chromosome in the 48,XXXY and 49,XXXXY groups to assess whether the observed methylation levels corresponded to specific numbers of Xa and Xi in each group. For CpG-2, the estimated methylation values for 47,XaXiXiY and 49, XaXiXiXiY were 67.7% and 75.3%, respectively. Notably, these estimated values were similar to the observed pyrosequencing values at CpG-2 for 47,XXXY and 49,XXXXY groups at 67.5% and 70.8%, respectively. However, some CpGs showed methylation levels that were consistent with a scenario where all of the X chromosomes have been inactivated, i.e., 47,XiXiXiY or 49,XiXiXiXiY. For instance, the observed methylation levels at CpG-1 of MAOA in 49,XXXXY was 54.3%, which showed no statistically significant deviation from the 56.4% methylation predicted from all four X chromosomes being inactive /GABA receptor 3 (GABRB3) and SHANK3 loci that are located on chromosomes 15q12 and 22q13, respectively. These loci were chosen based on their association with neurodevelopmental disorders such as autism [GABRA5 was slightly lower in methylation for 49,XXXXY , CpG-2 (P = 0.06), and CpG-7 (P = 0.08) on the CBCL total scale. There were no statistically significant associations between MAOA methylation and any of the BRIEF subscales. For the CpGs in AR, only CpG-1 was significantly associated (P = 0.03) with the BRIEF Behavioral Regulation Index, with a 10% increase in methylation correlating to a 15.9-point  increase in this scale. Similar to the MAOA CpG sites, there was only marginal significance with the CBCL total scale for CpG-1  and CpG-2 .To assess whether behavioral differences between individuals with 49,XXXXY and neurotypical 46,XY were associated with methylation, BRIEF and CBCL scores were compared against T) to promote optimal sexual and neurocognitive development [T and 49,XXXXY T groups. The 49,XXXXY T group showed a lower average score on the CBCL Total and the Externalizing Problems subscale, but not with any of the three BRIEF subscales (Table P = 0.04). History of testosterone treatment was then compared with MAOA methylation. When examining the effect of different testosterone doses on changes in methylation, there were two nominally significant.Males with 49,XXXXY typically undergo testosterone treatment . Specifically, CpGs 2 and 4 both showed an increase of 6.5% (P = 0.01) and 6.4% (P = 0.047) in methylation, respectively. In contrast, individuals undergoing hormonal treatment later in life (> 60 months of age) did not show any significant MAOA CpG methylation differences compared to those that did not receive treatment . Although there was a general trend of increasing MAOA DNA methylation in individuals receiving testosterone treatment after 6 years of age, none of the increases were significant with the exception of CpG-3 . Despite several promising CpGs that increase in DNA methylation with treatment, none of the P values for the HBT and TRT groups compared to the EHT group were statistically significant following correction for the multiple CpGs tested. When modeling the effect of testosterone and DNA methylation on CBCL or BRIEF outcomes, there was no statistically significant interactive effect between the two variables for any CpG sites. P = 0.04 in methyThe objective of this pilot study was to assess the relationship among the number of supernumerary X chromosomes, CpG methylation at different loci on the X chromosome, and parent report of behavior in males. In two parental questionnaires, the BRIEF and CBCL, significant differences were observed in both internalizing and externalizing behaviors between the 49,XXXXY group and both the 47,XXY and neurotypical 46,XY groups, which is consistent with a previous study by our group . HoweverMeCP2 gene have been described in male patients that present with severe developmental delay and neurological effects [For males with supernumerary X chromosomes, increased gene expression could be a result of incomplete inactivation of the X chromosomes. Elevated expression of genes on the X chromosome due to duplication at different X-linked loci is associated with neurodevelopmental disorders. For example, duplications of Xq28 that include the  effects . Another effects . We studMAOA is a noteworthy candidate to investigate the role of DNA methylation in behavior. It has been shown that genetic variants linked to low MAOA activity have been associated with increases in aggression and antisocial behavior in neurotypical 46, XY males exposed to childhood trauma [MAOA in attention deficit hyperactivity disorder (ADHD) [MAOA may be one of the genes targeted by methylphenidate or Ritalin [MAOA hypomethylation in female patients with panic disorder when compared to neurotypical counterparts [MAOA intronic region in the 49,XXXXY group compared to the 46,XY controls. This finding was expected due to the extra number of X chromosomes that are presumed to have been inactivated, in part, by DNA methylation. However, we found evidence of multiple loci where methylation levels were different from expected. For instance, several intronic CpGs in MAOA did not display the expected greater methylation levels in the 49,XXXXY group compared to the 46,XY controls, whereas others reflected methylation levels that were consistent with all four of the X chromosomes being methylated and inactive. A study investigating the same intronic CpGs as ours reported a significant inverse correlation between methylation levels and MAOA enzymatic activity in the brain [MAOA gene activity, consistent with the above studies that examined low MAOA activity, aggression, and antisocial behavior. Studies of additional regulatory regions as well as gene expression are needed to delineate the functional consequences of DNA methylation in MAOA.Due to its involvement in the oxidative deamination of monoamines such as dopamine, norepinephrine, and serotonin, d trauma , 42. Ther (ADHD) , a disorr (ADHD) . In fact Ritalin . Other sterparts . In the he brain . If thesAR were also investigated in our study due to its reported influence on male behavior. The androgen receptor is necessary for mediating androgen signaling to shape the male sexual phenotype. Polymorphisms in the androgen receptor gene have also been associated with aggression [MAOA locus, methylation levels at one region in AR displayed the expected increased levels of methylation in boys with extra X chromosomes but another region did not. The less-than-expected methylation levels evident at some loci of the AR gene may be associated with the upregulation of AR expression as a compensatory response to hypogonadism.Methylation levels at gression , 49. SimMeCP2 gene encodes a protein that regulates gene expression and maintains normal CNS functioning by binding to methylated CpGs [MeCP2 causes Rett syndrome, which is characterized by impairments in language, loss of motor coordination, and severe autistic features [MeCP2 gene was also associated with an increase in anxiety-driven behavior in males [MeCP2 locus also did not display drastically elevated methylation levels in the 49,XXXXY group. However, only two CpGs in an intronic GRE were tested to demonstrate that not all X-linked regions show X chromosome number-dependent increase in DNA methylation. A thorough investigation of additional CpGs is needed.The ted CpGs . Mutatiofeatures . A recenin males . The MeCMAOA and AR expression [AR gene. Individuals who transitioned from female to male and underwent 12 months of testosterone treatment showed an increase in estrogen receptor alpha (ESR1) methylation [MAOA and AR are preliminary and warrant further interrogation in a larger study of boys with 47,XXY as well as 48,XXXY and 49,XXXXY.The effect of testosterone treatment on behavior and DNA methylation was also assessed. Receiving testosterone was associated with lower average CBCL total scores and CBCL externalizing subscales, suggesting that treatment may have a positive effect on mental health. The beneficial effects associated with testosterone and behavior have been reported for more than a decade. Notably, testosterone treatment regulates both pression –55 with pression . Generalhylation . Our finSeveral notable studies have investigated DNA methylation levels in individuals with Klinefelter syndrome , 58–60. MAOA locus, have been linked with MAOA activity, suggesting that they may play a functional role in the transcriptional activity of MAOA. Second, we cannot conclude from statistical association that MAOA or AR methylation is responsible for the behavioral deficits observed in the 49,XXXXY boys, although it is intriguing to consider. On the X chromosome alone, there are a myriad of genes that can affect behavior, and there are numerous CpGs that may potentially regulate the function of any single gene. Given the relatively small number of CpGs tested in this study, it is probable that other loci may play a role on behavior as well, which will be investigated in future studies. An animal model such as one that employs the disruption of MAOA activity may contribute to augmenting our understanding of these very important interactions. Third, another limitation stems from the subjective BRIEF and CBCL scores provided by the participants’ parents. More objective assessments, such as those administered for neurocognitive evaluations, are needed to better understand correlative relationships between behavioral scores and biological variables such as DNA methylation. We estimated the values of Xa and Xi based on experimental data from 46,XY and 47,XXY with the assumption that the Xa and Xi had the identical methylation levels across the different groups. It may be the case that both active and inactive X chromosomes harbor variable methylation levels in the different groups tested. Such a finding would undermine our assumption. However, our results show that methylation levels of many of the CpGs obey our assumption. Given the large number of neurodevelopmental genes that reside on the X chromosome and their potential to affect the transcription of genes across the genome, it is likely that autosomal CpGs undergo epigenetic changes with extra number of X chromosomes. A comprehensive genome-wide approach is needed and will be utilized in future investigations. We acknowledge the limitations associated with using saliva for molecular analysis, as it does not provide a sterile medium for reliably extracting full-length mRNA [MAOA, AR, or MeCP2, which is an endeavor much more easily accomplished with blood samples. Finally, we were unable to determine whether there was a statistical interaction between testosterone and MAOA methylation on the behavioral outcomes, as we were underpowered due to low sample size. However, these limitations provide fertile ground for future investigations into the intriguing relationship between genes, hormones, and behavior in the supernumerary X disorders.The current study has several limitations. First, although DNA methylation can provide a mechanistic understanding of how likely a gene is to be expressed, it is unclear whether any of the specific CpG methylation differences observed among the groups may account for differential gene expression. However, we state that the methylation levels, especially at the gth mRNA . This meOur study provides preliminary evidence indicating that boys with multiple supernumerary X chromosomes and their resulting behavioral issues likely involve some genes on the X chromosome that are aberrantly methylated due to incomplete X chromosome inactivation. These findings warrant a more comprehensive and detailed investigation to understand the relationship between specific genes, biological underpinnings of the testosterone deficiency, the inactivation of X chromosomes, and the behavioral presentations.MAOA. Males who were found to be born premature , have mosaicism, copy-number variants, and/or other co-existing genetic disorders were excluded from the study. Participants were thoroughly evaluated by their pediatric endocrinologist including necessary laboratory tests and physical examination. The endocrinologist then administered HRT to the participant on an individual basis. Fourteen neurotypical boys  were also included in this study to serve as controls. Not all participants submitted saliva samples for DNA methylation analysis.In this study, there were 29 boys with 47,XXY and 27 boys with 49,XXXXY who were referred by their physicians or ancillary healthcare providers for comprehensive neurodevelopmental evaluations. Inclusion for this study required confirmation of 47,XXY or 49,XXXXY diagnosis via karyotype. Three individuals with three X chromosomes (two with XXXY and one with XXXYY) were also included only for the epigenetic analysis at The school-aged version of the Child Behavior Checklist (CBCL)  is a behT-scores between 60 and 64 are considered slightly elevated, T-scores from 65 to 69 are considered at risk of clinical elevation, and T-scores above 70 are considered clinically elevated in terms of executive function.The school-aged edition of the Behavior Rating Inventory of Executive Function-2 (BRIEF-2)  is an 86g, the supernatant was discarded, and the DNA pellet was washed once with 70% EtOH. The dried DNA pellet was resuspended in 100 μL of TE buffer, and DNA quantity was obtained by a Qubit 2 Fluorometer . For each sample, 500 ng of DNA was used for subsequent bisulfite conversion, PCR amplification, and methylation analysis by pyrosequencing.Each of the participants provided ~ 1 mL of saliva sample in an Oragene Discover salivette tube . DNA was extracted using the prepIT-L2P saliva DNA isolation kit according to the manufacturer’s instructions (DNAGenotek). Briefly, saliva samples were incubated at 50 °C for 1 h, after which the PT-L2P solution was added at 1/25 of the saliva volume, and samples were vortexed and incubated on ice for 10 min. After the centrifugation of impurities at 15,000 × g, the supernatant was mixed with 1.2 times the volume of 100% EtOH, and saliva DNA was precipitated at room temperature. Following centrifugation at 15,000 × DNA (500 ng) was first bisulfite-converted using the EZ DNA Methylation-Gold Kit, according to the manufacturer's protocol . Primers were designed against regions as described in Additional file 2: Table 1. Whenever possible, we included regions experimentally verified as binding sites for the glucocorticoid receptor, or glucocorticoid response elements (GREs) , since aMAOA and AR CpG sites by using the observed methylation in XY and multiplying it by the potential number of inactivated X chromosomes: mj = n * m1, where m is the estimated average level of methylation at the CpG site, j is the total number of X chromosomes in a given sample, n is the number of inactivated X chromosomes (max(n) = j), and m1 is the average level of methylation observed among samples from neurotypical 46,XY individuals. We then determined the predicted methylation levels from the number of inactive X chromosomes that were closest to those experimentally obtained by bisulfite pyrosequencing. The observed vs. expected values were tested using the Kolmogorov–Smirnov test, with lack of statistical significance indicating that the observed was similar to the expected values. Finally, observed values were also adjusted by testosterone treatment. Same conclusions were reached by both sets of values, and therefore, only the adjusted methylation values are reported.We estimated the expected level of DNA methylation at We tested whether methylation was correlated with behavior across the CBCL: (1) total problems and (2) internalizing and (3) externalizing subscales using ordinary least-squares regression. This was also done across the BRIEF subscales: (1) the behavioral regulation index, (2) the metacognition index, and (3) the global executive functioning composite. These models were conducted without adjustment and with adjustment for aneuploidy status, with status in the model defined as having 2 X chromosomes  to having 4  to assess whether this differential aneuploidy accounted for the relationship between differential methylation and behavior.MAOA due to its prior association with behavior [T group), whereas three individuals with 47,XXY did not . Fifteen individuals with 49,XXXXY received testosterone treatment , and twelve individuals with 49,XXXXY did not . Individuals with no definitive treatment status were excluded from analysis. Testosterone treatment (T) includes early hormonal treatment (EHT), hormonal booster treatment (HBT), and testosterone replacement therapy (TRT). EHT is defined as three intramuscular injections of 25 mg testosterone enanthate, typically administered to boys between 4 and 60 months of age. HBT encompasses three intramuscular injections of 50 mg testosterone enanthate administered to boys between 5 and 8 years of age. TRT, beginning at puberty and continuing for life, involves either daily application of testosterone using Androgel or weekly or monthly injections.We assessed whether testosterone treatment had an impact on methylation, focusing on behavior –28. SeveT and differential methylation at the MAOA CpG sites.We tested for an association between testosterone treatment and scores on the CBCL and BRIEF subscales using ordinary least squares regression. In a separate model, we also tested whether there was an interactive effect between receiving any We used a Bonferroni correction for methylation analyses using the number of CpG sites to control for the Type I error rate. With an intended alpha threshold of 0.05, across 11 CpG sites, we used a corrected alpha threshold of 0.005. Given prior studies showing overlap in the psychodevelopmental constructs the BRIEF and CBCL measure , we consAdditional file 1: Supplementary Table 1. Primers used for bisulfite pyrosequencing.Additional file 2: Supplementary Table 2. P-values of one-sample Wilcoxon Signed rank test (non-parametric test of means) between the predicted and observed methylation levels for 46,XY, 47,XXY, 48,XXXY, and 49,XXXXY at the MAOA locus.Additional file 3. Behavioral and epigenetic database used for analysis.


The objective was to evaluate the influence of different dietary crude protein (CP) levels during the growth phase on reproductive characteristics and reproductive efficiency as well as the body development of adult male Japanese quail.Three hundred one-day-old male quails were distributed into five treatments with diets containing different CP levels  in a completely randomized design, with six replicates of ten birds each. The CP diets were applied only during the growth phase (1 to 35 days). At 36 days of age, the birds were transferred to 30 laying cages with three males and nine females each, and all birds received the same diet formulated to meet production-phase requirements until 96 days of age.The growth rate of the birds increased linearly (p<0.01) with increasing dietary CP, but the age of maximum growth decreased (p<0.05). At growth maturity, all birds had the same body weight (p>0.05). At 35 days of age, higher weight gain was obtained (p<0.05) with diets containing 22% CP or higher. No effects on feed conversion were observed in this phase. The increase in dietary CP enhanced (p<0.01) nitrogen intake and nitrogen excretion but did not affect (p>0.05) nitrogen retention. Testis size, seminiferous tubular area, number of spermatogonia, and germinal epithelial height at 35 days of age increased linearly (p<0.05) with dietary CP, while the number of Leydig cells decreased (p<0.01). The Sertoli cell number at 60 days of age increased linearly (p<0.01) with dietary CP. Dietary CP levels did not affect cloacal gland size, foam weight, foam protein concentration, semen volume, or flock fertility at 90 days of age.Dietary CP concentration affected body and testicular development in male Japanese quails but did not affect reproductive efficiency. Quails are bred worldwide for both meat and egg production because of their ease of handling, small size compared with chickens and broilers, precocious development, low space requirements, low investment requirement, and high egg production capacity and rates. In addition, quail breeding has been used as a good experimental model for bird research because of the birds’ rapid growth, greater egg production, and shorter intervals between generations relative to other species.Nutrition is a main factor affecting quail productive efficiency. Dietary crude protein (CP) is an important source of amino acids that are used for maintenance, growth, and production demands . During Regarding quails’ nutritional requirements, most current recommendations ,5 are baThis experiment was performed between March and June 2018 at the Department of Animal Science of the Federal University of Lavras (UFLA), Lavras, state of Minas Gerais, Brazil. The Animal Research Ethics Committee approved all experimental procedures under protocol no. 057/2017.Coturnix coturnix japonica) in the experiment were housed in a masonry shed in 30 cages (50 cm wide×70 cm deep×25.5 cm high) during the growth phase (1 to 35 days). For the production phase (36 to 96 days), the three males with the live weight closest to the cage average were selected, transferred to a screened laying shed, and housed together with nine females in laying cages (32 cm wide×38 cm deep×16 cm high). The temperature and humidity in the sheds were monitored using thermohygrometers placed at bird height, which recorded the minimum and maximum temperature and humidity. The temperature was kept at 38°C during the first three days using a wood-burning stove and was decreased by 0.5°C each day until the birds were 28 days old /LW)×100, where WTl is the left testis weight, WTr is the right testis weight, and LW is the live weight. After measuring these parameters, the testes were fixed in Bouin’s solution for approximately 12 hours at ambient temperature and then washed in 70% alcohol for subsequent histological analysis   23] can Male fertility is essential to one-day-old chick production systems and is directly related to spermatogenesis. Spermatozoid production, in turn, depends on the Sertoli cell number and activity and the number of proliferating spermatogonia . In the During the growth phase, rapid body development is directly related to reproductive organ development . Thus, tOverall, the amino acid supply should first meet the maintenance requirements, followed by the body development and reproductive activity requirements . In birdIn this study, the live weights of 15-day-old males increased linearly with increasing dietary CP, reaching the optimal point at 23.9% CP . This isThe protein requirements for bird body weight gain decreased until 36 days of age, decreasing from 24% over the first two weeks to 20% at the end of the growth phase , which wImproved knowledge of CP requirements for different animal species and categories through applying the concept of dietary formulations with ideal protein composition to optimize amino acid use is also needed. A decrease of greater than three or four percent in dietary CP, even if the feed meets all the amino acid requirements, may result in low productive performance . Thus, dIn this study, levels of 23% CP or higher accelerated bodily and reproductive organ development during the growth phase, affecting the testis up to reproductive age. However, requirements for the chick and grower phases may differ, and further studies are needed to determine the viability of using specific formulations for each rearing phase. In addition, no need for specific dietary formulations in males was evident in this study.In conclusion, dietary CP concentration affected body and testicular development in male Japanese quails but did not affect reproductive efficiency.


To investigate associations between isoniazid for latent tuberculosis and risk of severe hepatitis, affecting patients with rheumatoid arthritis or ankylosing spondylitis whose treatment includes tumor necrosis factor inhibitors. Our self-controlled case series study analyzed Taiwan’s National Health Insurance Database from 2003 to 2015 to identify RA or AS patients, aged ≥ 20 years, receiving TNF inhibitors and a 9-month single isoniazid treatment. The outcome of interest was hospitalization due to severe hepatitis. We defined risk periods by isoniazid exposure (days): 1–28, 29–56, 57–84, 85–168, 169–252, and 253–280. To compare risk of severe hepatitis in exposed and non-exposed periods, we performed conditional Poisson regressions to generate incidence rate ratios (IRR) and 95% confidence intervals, with adjustment of patients’ baseline covariates including age, sex, HBV, HCV and related medication. Of 54,267 RA patients and 137,889 AS patients identified between 2000 and 2015, 11,221 (20.7%) RA and 4,208 (3.1%) AS patients underwent TNFi therapy, with 722 (5%) receiving isoniazid for latent tuberculosis. We identified 31 incident cases (4.3%) of hospitalization due to severe hepatitis. Of these hospitalization events, 5 occurred in the exposed periods, 25 occurred in the INH unexposed periods, and 1 occurred in the pre-exposure period. Compared with non-exposure, the risk of severe hepatitis was higher in exposed periods , especially 57–84 days  and 85–168 days . The INH related fatal hepatotoxicity was not identified in our study. Our findings suggest an association between risk of severe hepatitis and exposure to isoniazid in patients with RA or AS under TNFi therapy, particularly within the exposed period 57–168 days. A close monitoring of liver function is mandatory to minimize the risk, especially within the first 6 months after initiation of 9 months isoniazid. Therapeutic targets of T2T are still emerging for AS3. Tumor necrosis factor inhibitors (TNFi) are the first class of biological DMARDs (bDMARDs) used in treating RA and AS, their anti-inflammatory effect mainly resulting from the binding of cytokines in order to block TNF-mediated signaling4.Rheumatoid arthritis (RA) and ankylosing spondylitis (AS) are chronic inflammatory rheumatic diseases involving peripheral joints and the axial skeleton. If they are left untreated or are unresponsive to therapy, progressive joint destruction may lead to loss of physical function, inability to perform activities of daily living, and difficulties in maintaining employment. It is widely accepted that clinical remission is the main therapeutic target for patients with RA, and a treat-to-target (T2T) strategy should be applied when treating patients with RA6 have implicated tumor necrosis factor inhibitors (TNFis) as increasing the risk of tuberculosis (TB), particularly in patients with a pre-existing latent tuberculosis infection (LTBI). Recent studies specifically demonstrate that RA patients receiving TNFi treatment are exposed to an increased risk of tuberculosis (TB) infection11. A risk management plan (RMP) for TNFi has been implemented in Taiwan since 2012, focusing on minimizing the risks of TB and hepatitis B/C virus infection. The RMP recommends that isoniazid treatment for latent TB infection (LTBI) should be given one month before TNFi therapy. The annual incidence rate of TB during TNFi treatment increased from 2008 to 2012 but has decreased since the year of RMP implementation12. The recommendation is for nine months of isoniazid (9H) prophylaxis in patients with LTBI who receive TNFi to prevent TB reactivation14.Both randomized controlled trials (RCTs) and real-world studies15. The long therapeutic period of isoniazid raised safety concerns regarding potentially serious or fatal hepatotoxicity16 and it is often asymptomatic. Although previous studies have provided information on this issue and investigated the risk of hepatitis in those receiving isoniazid for LTBI, patients with RA/AS represent a relatively small fraction of the cohorts in those studies20. In addition, many studies were subject to uncontrolled, unmeasured confounding factors, including patients’ genetic and family history, consumption of alcohol, underlying liver disease, and concurrently administered hepatotoxic drugs22. For example, Campbell et al. found that hepatotoxicity of INH was most common among patients with renal failure or use of TNFi. However, those results were based on only 64 participants completing 9H treatment19. Therefore, we used a population-based data to evaluate the association between risk of severe hepatitis and 9H for LTBI in patients with RA or AS under TNFi treatment. A self-controlled design was implemented to eliminate time-constant confounders.Isoniazid is the most commonly used medication for LTBI in Taiwan. Nine months of isoniazid (9H) is often associated with minor, transient and asymptomatic elevations in serum aminotransferase levels. However, serious adverse events with INH for prevention of LTBI are common in primary care23. The NHID compiles records from out-patient and in-patient departments, and from pharmacy services. Patients with RA were eligible to apply for a catastrophic illness certificate (CIC) offering exemption from copayment, and all applications were assessed by review committee based on relevant clinical and laboratory information. Consequently, a high degree of precision is associated with CIC diagnoses. To improve the validity of the study we linked the NHID to the catastrophic illness registry, confirming diagnosis of RA among the patient group24. This study was approved by the Research Ethics Committee of National Taiwan University Hospital (registration number 201803030RINC).This is a population-based study using the National Health Insurance Database (NHID) of Taiwan. Taiwan has maintained a single-payer National Health Insurance (NHI) program since March 1st 1995, with 99.9% of Taiwan’s population  enrolled by 201426. The SCCS included patients aged 20 years and older diagnosed with rheumatoid arthritis (RA) or ankylosing spondylitis (AS) and receiving as the first biologic a TNFi treatment of either etanercept  code L04AB01), adalimumab (ATC code L04AB04), or golimumab (ATC code L04AB06), between 2003 and 2015 under 9H with severe hepatitis. We did not include certolizumab and infliximab in the analysis because they were not available in Taiwan during the study period. The RA diagnosis was confirmed using both records of the International Classification of Disease, Ninth Revision; ICD-9-CM code 714.0 and CIC. Because patients with AS were not eligible to apply for a CIC, we confirmed the diagnosis by identifying a record of AS (ICD-9-CM code 720.0) from inpatient claims or at least 3 records of AS from out-patient claims. Following reimbursement guidelines of Taiwan’s National Health Insurance Administration, all TNFi users must pass through review by committee members; consequently, we consider diagnosis validity for both RA and AS to be very good.We used a self-controlled case series (SCCS) design for this study, whereby the relative risk is based on risk estimates for periods of exposure to isoniazid versus periods of non-exposure for the same individuals. The SCCS method allowed us to eliminate time-constant confounding factors, such as patient sex, ethnicity, and genetic factorsOur study outcome was severe hepatitis  identified from inpatient claims for the SCCS. To ensure patients were newly diagnosed with the event, patients with a history of hepatitis recorded between 2000 and 2002 were removed from the SCCS. We retrieved all medication records to identify those receiving isoniazid (ATC code J04AC01 and J04AC51) therapy while under TNFi treatment. The 9-month single isoniazid, also known as 9H, was the only LTBI treatment covered by the NHI during the study period. Not included in our study were the newer and shorter regimens of rifamycin  recommended by Taiwan’s CDC since 2017; 9H has remained the most commonly used regimen because of greater convenience for doctors and patients without directly observed therapy (DOT). We removed cases with any record of hepatitis  within 3 years before the starting date  to ensure all cases were new for hepatitis, without any history when they entered the SCCS.19. A 28-day pre-exposure period was added to account for the possibility that the outcome of interest may affect the likelihood of LTBI treatment, which in turn may introduce bias into the risk estimate during treatment26. We also designed a 28-day washout period after LTBI treatment to address the issue of residual effects of drugs after discontinuation. These two periods were analyzed independently and belong to neither exposed nor unexposed periods. All remaining time from 2003 to 2015 was considered as the unexposed period. Figure For each study participant we defined the risk periods according to duration of exposure to isoniazid. We categorized risk periods into 6 windows: 1–28 days, 29–56 days, 57–84 days, 85–168 days, 169–252 days, and 253–280 days, since most of the hepatotoxicity appeared in the first 3 monthsContinuous variables were determined by assessing standard deviations from the mean, and categorical variables by assessing number with proportion. We calculated the incidence rate ratio (IRR) and 95% confidence interval (95% CI) by conditional Poisson regression to evaluate the risk of severe incident hepatitis associated with isoniazid in different predefined risk windows compared to the non-exposed period. Several covariates were captured during the baseline period  and were considered in the regression model for risk estimation adjustments, including patients’ age, sex, HBV, HCV and co-prescribed traditional DMARD , selective COX-2 inhibitors, and non-selective COX inhibitors. Besides, we performed an additional analysis by logistic regression modeling to obtain odds ratios and 95% confidence intervals, to evaluate the association between these covariates and the outcome of severe hepatitis within the cohort of 722 patients receiving 9H.To evaluate the effects for patients who died and could not be observed in terms of further exposure to isoniazid, we conducted a sensitivity analysis by restricting our scope to surviving patients within the study period, then repeating the analysis. We used SAS version 9.4 for all statistical analyses.All data in the study was anonymous. This study was approved by the Research Ethics Committee of National Taiwan University Hospital , Taipei, Taiwan.We identified a total of 54,267 patients with RA and 137,889 patients with AS between 2000 and 2015, of which 11,221 (20.7%) RA patients and 4,208 (3.1%) AS patients initiated TNFi therapy. In total, 768 (5.2%) individuals initiated isoniazid LTBI treatment. We excluded 46 patients under 20 years of age  criteria of grade 3 or 4 events. None of the studies focus on severe hepatitis that requires hospitalization in specific groups of patients, such as RA and AS, who require polypharmacy which is itself hepatotoxic. Our results highlight the importance of close monitoring of liver function to prevent the occurrence of severe hepatic adverse events in these groups of patients.Although severe hepatitis has been identified as a known side effect of isoniazid, the risk profile has not been well-evaluated in patients with RA and AS who undergo TNFi therapy. In early studies of general population, the rate of hepatotoxicity due to isoniazid for LTBI treatment was 0.1% to 3.0%17. Concerning drug-drug interaction, 9H may have less effect compared with rifamycin regimens, especially for patients with RA and AS who usually require several kinds of medication. In our study the incidence rate of hospitalized severe hepatitis during isoniazid treatment was only 0.69%, a lower figure than identified in the aforementioned studies. We believe that these differences in results can be explained by different definitions of hepatotoxicity. Isoniazid-associated hepatotoxicity can be severe and indeed life threatening, thus of particular concern to clinicians. For a solid outcome that is clinically significant, we defined severe hepatitis as that which led to hospitalization, as recorded in the inpatient claims data. Consistent with a previous study18, the risk of hepatitis in our study was higher between 57 and 168 days after exposure to isoniazid. This finding suggests aggressive monitoring of liver function is mandatory to minimize the risk of severe hepatitis, especially in the initial 6 months of 9H treatment.In the biologic era, rheumatologists should take special care with the use and monitoring of long-term isoniazid treatment in combination with potentially hepatotoxic drugs such as cDMARDs and NSAIDs. RA patients in Taiwan require at least 2 conventional DMARDs with a refractory response before commencing TNFi therapy. Currently, isoniazid is the most commonly used medication for LTBI in Taiwan. It is associated with various levels of hepatitis in general population26. This method therefore controls for time-constant confounders and also unmeasured confounders42.To our knowledge, compared with previous studies, this is the largest study currently including incident cases of severe hepatitis in sufficient numbers of RA and AS patients who undergo TNFi treatment while on 9H; this study allows more definite conclusions regarding the association between 9H therapy for LTBI and severe hepatitis. A significant strength of our study is that we used a large population-based database to provide sufficient sample size and statistical power for analysis. This allowed us to evaluate events with a very low incidence rate which were nonetheless clinically significant to RA or AS patients under TNFi, conventional DMARD and NSAID therapies. Moreover, we used the SCCS design to minimize the unmeasured confounders. Each patient acted as his/her own control. Despite the inclusion of many potential confounders in the multivariate models, residual confounding by factors that are not measured in claims databases may remain. The SCCS design provides relative incidence estimates in high-risk periods compared with low-risk periods44. Consequently, HBV and HCV infection have been included in our regression models for adjustment. Because the recurrent severe hepatitis was not independent of the first occurrence, our analysis only considered the first events of severe hepatitis, as suggested by Irene Petersen et al.45. We may underestimate the risk of severe hepatitis for the exposed periods if patients had severe hepatitis before exposure. The analytic population in our study likely represents a lower-risk subset of the total RA/AS population and the effect estimate may be different for other groups of patients. Lastly, although we have examined a large population-based database with 23 million individuals for this study, only 31 patients entered the SCCS resulting in wide confidence intervals around the estimates.Our study presents some limitations. First, we do not know the overall numbers of LTBI patients in these cohorts. Second, there was no laboratory data available in the claims data to which our study referred; we thus could only use inpatient records to define severe hepatitis. However, to improve the validity of diagnosis, we have only captured acute hepatitis from the diagnosis in inpatient claims data. Consequently, the actual number of cases with severe hepatitis may be an underestimate. Our analysis only included patients with severe hepatitis requiring hospitalization. No inference can be made for the association between 9H treatment and relatively mild hepatic reactions from our analysis. Third, in using the NHID, we did not have access to information regarding the patient’s alcohol consumption or self-payment medication. Forth, as with all studies using a claims database, we were unable to confirm the drug compliance of patients. However, a post-hoc analysis found the overall proportion of drugs covered (PDC) was more than 89.29% for those who did not experience any severe hepatitis during the exposed period. Generally, however, any patients discontinuing treatment would be unable to refill their prescription for 9 months. Fifth, the generalizability of this study to Western populations should be discussed since Asian populations had a higher baseline risk of hepatitis, possibly due to higher rates in Asia of hepatitis B virus (HBV) and hepatitis C virus (HCV) infectionOur findings suggest an association between risk of severe hepatitis and exposure to isoniazid in patients with RA or AS, and the risk was especially high within the exposed period of 57–168 days. The findings warrant attention because liver impairment is usually asymptomatic and we may delay stopping isoniazid for those with liver impairment. A close monitoring of liver function is mandatory to minimize the risk, especially within the first 6 months after initiation of 9 months isoniazid.


Patients with chronic underlying diseases are more susceptible to coronavirus disease 2019 (COVID‐19) complications. Recent studies showed people living with HIV (PLWH) are not at greater risk than the general population. Few studies have reviewed the impacts of COVID‐19 on PLWH. The purpose of this systematic review was to investigate the impact of COVID‐19 on patients infected with HIV.We executed a systematic search using four databases of PubMed, Scopus, Science Direct, and Web of Science and screened the records in two steps based on their title/abstract and full text. This study follows the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) checklist to elevate the validity and reliability of its results.We reviewed 36 studies. The patients' age was above 20 years in all studies. In almost all studies, the inflammatory parameters were reported high. In most of the studies, all HIV patients completely recovered from the COVID 19 infection. Although CD4 count was not recorded in all studies, the minimum level was reported as 12 cells/µl.Based on the current review, we concluded that HIV patients at advanced stages (3 or 4) of the disease, whose CD4 counts are low, may show less severe COVID‐19 infection symptoms. Similarly, Interference can reduce the severity of immune reactions and subsequent cytokine storms and consequently mitigate the symptoms. Therefore, in most of the studies, the majority of HIV patients showed no severe symptoms and completely recovered from COVID 19 infection. There is yet no certain answer whether patients infected with HIV are at greater risk of severe illness and worse outcomes. We still need to know whether there are any differences between controlled HIV infection with undetectable viral load and a CD4 count ≥200 cells/µl and uncontrolled HIV infection or AIDS in COVID‐19 outcomes. Thus, the aim of this systematic review was to investigate the outcome of COVID‐19 among patients infected with HIV.22.1https://www.covidence.org). The screening consisted of two‐step title/abstract and full‐text screening processes. Utilizing this website eased settling of the discrepancies in the inclusion process. A third researcher (A. K.) addressed the remaining discrepancies. Two researchers (H. M. and A. R.) extracted and summarized the data of the included studies.We systematically searched four databases, including PubMed, Scopus, Science Direct, and Web of Science to retrieve the related articles based on the keywords used in our search strategy. Two researchers (S. P. M. and S. S.) screened the retrieved articles using the Covidence website (2.2A.[SARS‐CoV‐2] (Title/Abstract) OR [COVID‐19] (Title/Abstract) OR [2019‐nCoV] (Title/Abstract) OR[Novel Coronavirus] (Title/Abstract)B.[Human Immunodeficiency Virus] (Title/Abstract) OR [HIV] (Title/Abstract) OR [Acquired Immune Deficiency Syndrome] (Title/Abstract) OR [AIDS] (Title/Abstract)C.[A] AND [B].We included the search terms for HIV/AIDS and COVID‐19 as presented below:2.3(1)Review articles, letter to the editors, or other studies without original data.(2)Ongoing studies.(3)Irrelevant to the aims, settings, and design of this study.(4)Abstracts, conference abstracts, errata, or other studies lacking full‐texts.We included all the original articles discussing the COVID‐19 status in PLWH. The exclusion criteria are the following:2.4We utilized the National Institutes of Health (NIH) tool3Most of the studies were considered of good quality (25/37). Other studies 12/37) had a fair quality, and we did not classify any studies as poor  were male, 0.01% (11 patients) were transgender. Patients' gender was not available in two studies. Patients' age was above 20 years in all studies. As the review revealed, nine studies did not mention the ART regimen for HIV + patients. At the time of the COVID‐19 diagnosis, the most common symptoms were fever, cough, myalgia, and headache. Additionally, most of the patients had various comorbidities such as hypertension, diabetes mellitus, asthma, renal insufficiency, cardiovascular disease, etc.Laboratory values including CRP, ferritin, and Interleukins levels were available in about two‐third of the studies. But in almost all the studies with inflammatory tests results, the values were elevated. In most of the studies, all HIV patients completely recovered from the COVID‐19 infection. In 8 studies, mortality was reported ranging from 1% to 36%. Although CD4 count was not recorded in all the studies, the minimum level was reported as 12 cells/µl.4We found that HIV patients at advanced stages (3 or 4) of the disease with low CD4 count and weak immune systems show less severe COVID‐19 symptoms. However, some studies showed controversial results which contradict our primary hypothesis. The main reason for these contradictory results was the scarcity of existent literature and inconsistency of evidence that limited our ability to address and reasonably argue our main hypothesis. This may also be due to simultaneous symptoms and underlying comorbidities that come along at advanced stages of HIV infections and could perplex and obscure the typical presentation of COVID‐19 in such patients. Despite these contradictory results, the majority of studies included in this review indicate mild or no typical symptoms of COVID‐19 in HIV patients, particularly in those at the advanced stages of HIV disease. This review also found an unexpected high recovery rate in these patients after COVID‐19 infection, which contradicts the common knowledge of higher morbidity and mortality rate in immunocompromised patients.SARS‐CoV‐2 is a new strain of coronavirus, which is the causal agent of COVID‐19. The usual symptoms of COVID‐19 are fever, cough, headache, shortness of breath, tiredness, loss of taste or smell, and gastrointestinal symptoms such as diarrhea, anorexia, nausea, and abdominal pain,In the present review, a CD4 count less than 500 was assumed as a cutoff point. Thus, the patients with a mean CD4 count less than 500 were considered immune‐deficient and assumed to be at advanced stage patients. In the study conducted by Calza et al. all the nine HIV‐positive patients with CD4 count less than 258 fully recovered, which supports the hypothesis of our study.4.1Despite the limited available evidence, the findings of the present review authenticate the primary hypothesis arguing less severe clinical outcomes in HIV patients at the advanced stages. Although this could be mainly due to the inability of the immune system in HIV patients to provoke the cytokine storm, which is believed to be the main responsible event for severe clinical outcomes in COVID patients, the contradictory results inform future studies to explore further the possible underlying causes of such an observation.5In conclusion, the results of the present study suggest that HIV patients at advanced stages (3 or 4) of the disease, when CD4 counts are low and their immune system is compromised, manifest less severe symptoms and less mortality following COVID‐19. This has been attributed to the inability of HIV‐positive individuals' immune systems in provoking the cytokine storm that caused the severe clinical outcome in COVID patients. By a similar mechanism, it seems that corticosteroids mitigate the severity of symptoms in COVID patients with a healthy immune system.The authors declare that there are no conflict of interests.Conception and design of the study: Esmaeil Mehraeen, SeyedAhmad SeyedAlinaghi; acquisition of data: Amirali Karimi, Seyed Peyman Mirghaderi, Amirata Fakhfouri; analysis and interpretation of data: Hengameh Mojdeganlou, Alireza Barzegary; drafting the article: Esmaeil Mehraeen, Mehrzad MohsseniPour, Solmaz Saeidi; revising it critically for important intellectual content: SeyedAhmad SeyedAlinaghi, Armin Razi; final approval of the version to be submitted: Esmaeil Mehraeen, Amir Masoud Afsahi, Omid Dadras, Marcarious M. Tantuoyir.Not applicable. This research did not receive any specific grant from funding agencies in the public, commercial, or not‐for‐profit sectors.


Although the success rate of recanalization in acute intracranial artery occlusion is high, there is a poor rate of improvement in functional clinical outcome. The purpose of this study was to assess the functional outcome of mechanical thrombectomy for proximal M1 occlusion involving lenticulostriate arteries (LSAs) compared with distal M1 occlusion-sparing the LSAs.A retrospective analysis was conducted in patients with middle cerebral artery (MCA) M1 occlusions who had a successful recanalization subsequent to mechanical thrombectomy. The recanalization results were estimated using the thrombolysis in cerebral infarction grade assessed by digital subtraction angiography. To confirm the ischemic change resulting from the lenticulostriate artery occlusion, we reviewed the neuroimaging findings from magnetic resonance imaging 1 day after mechanical thrombectomy. The functional outcomes were then evaluated using the modified Rankin scale at 90 days.In total, 28 patients with MCA M1 occlusion had successful recanalization outcomes with thrombolysis in cerebral infarction grades IIa, IIb, and III. Among the 28 patients, 17 had proximal M1 occlusions and 11 had distal M1 occlusions. Demographic factors, including initial National Institutes of Health Stroke Scale score, time from symptom to recanalization, and recanalization rate did not differ considerably between patients with proximal and distal M1 occlusions. Regarding infarctions in the basal ganglia, internal capsule, and corona radiata, there were statistically significant differences between the proximal and distal M1 occlusions. However, there were no significant differences in good functional outcome  observed between the groups at 90 days after mechanical thrombectomy.Although proximal M1 occlusion had more frequent infarctions associated with the LSA territories, these were not related to poor functional outcomes. Both proximal and distal M1 occlusion demonstrated comparably good outcomes. However, in recent years, endovascular recanalization for an acute occlusion of a major intracranial artery by means of a stent retriever device has remarkably advanced, thus reducing the associated disastrous consequences to a significant degree.Acute middle cerebral artery (MCA) occlusion is a critical condition known to comprise 49.7% of all intracranial artery occlusion cases.Although great progress has been made in increasing the technical success rate of recanalization, this has not led to an equivalent improvement in functional clinical outcome. The factors influencing the functional outcome include the timing, the patient's age, the patients score on the National Institute of Health Stroke Scale (NIHSS) upon admission, the patient's systemic condition , and numerous vasculature factors. The vasculature factors are particularly important and include the extent of early ischemic changes before recanalization, the status of the collateral circulation which affects the degree of perfusion status and determines the infarct core and penumbra, the degree of recanalization, and the exact location of the occlusion. proposed for assessing the angiographic recanalization of acute occlusion of the intracranial artery has been widely used in the literature either in its original or modified form. Based on the TICI scale, many clinical trials have attempted to establish more effective and safer guidelines as well as to estimate the outcome of acute ischemic stroke patients.Various approaches have been used for predicting the outcome of patients with acute occlusion of a major intracranial artery following endovascular recanalization. Among them, thrombolysis in cerebral infarction (TICI), which Higashida et alHowever, there is a lack of documented reports on functional outcomes after recanalization among the different types of artery occlusions, especially MCA. Therefore, this study was designed to assess the functional outcomes of recanalization after M1 segment occlusion, which subdivides into the proximal half of the M1 (involving the lenticulostriate vessels) and the distal half of the M1 (sparing the lenticulostriate vessels).2We retrospectively reviewed our database of mechanical thrombectomy for acute occlusion of the intracranial artery from December 2010 to February 2016. Three reviewers independently extracted the clinical data from the electronic records including initial clinical status, patient demographics, thrombectomy procedures, radiological findings, treatment results, and clinical outcome. The radiological findings were confirmed by 2 experienced neuroradiologists. The NIHSS and modified Rankin scale (mRS) were used to assess clinical status.2.1Patients were selected as follows: time window within 6 hours from symptom onset to start of procedure of mechanical thrombectomy; M1 segment occlusion of MCA; only a stent retriever was used for the thrombectomy; brain computed tomography angiogram (CTA) was performed before the thrombectomy procedure; brain magnetic resonance imaging including MR diffusion weighted images (DWI) were checked 24 hours after the procedure; and the clinical outcome was measured with mRS at 90 days post procedure.The exclusion criteria included: patients with a tandem occlusion of the internal carotid artery (ICA) and terminal ICA; patients with underlying severe stenosis of M1; and patients with an unsatisfactory recanalization (TICI score of 0 and 1).2.2 as follows: grade 0 = absent collateral supply to the occluded MCA territory; grade 1 = collateral supply filling <50% but >0% of the occluded MCA territory; grade 2 = supply filling >50% but <100% of the MCA territory; and grade 3 = 100% collateral supply of the occluded MCA territory. All CTA images were 7-mm maximum intensity projection reconstructions and 4-mm axial reformats. We used the same CTA imaging technique and divided the grading system as poor collaterals (grades 0 and 1) and good collaterals (grades 2 and 3). We confirmed irreversible acute stroke after 24 hours on control MR DWI. Basal ganglia (BG), internal capsule (IC), corona radiata (CR), territory infarctions, and BG hemorrhagic transformation were regarded as key parameters that could result in moderate to severe disability. Figure The distal and proximal M1 occlusions were defined according to whether they spared the lenticulostriate arteries (LSAs) or not. The proximal M1 occlusions involved the LSAs that were not visualized on digital subtraction angiography, while the distal M1 occlusions spared the LSAs , underlying diseases , onset to recanalization time, and initial NIHSS score.2.3All procedures were performed under local anesthesia. Mechanical thrombectomy in this study was carried out using stentretriever—Solitaire AB , Solitaire FR , Trevo XP , and Trevo ProVue . Proximal suction aspiration through a guide catheter was performed, a balloon guide catheter was used since November 2014.2.4U test was used for the continuous parameters, including age, onset to recanalization time, and NIHSS score. A Student t test was used to compare the remaining non-continuity variables. A P value of <.05 was considered significant.Statistical analysis was performed using commercially available software . As a nonparametric test, the Mann–Whitney 3P = .320).In total, 28 patients with pure M1 occlusions and successful recanalization after mechanical thrombectomy met the inclusion and exclusion criteria in this study. Demographic data are summarized in Table We also analyzed the predictors that influenced a good clinical outcome at 90 days after treatment and found that there were no meaningful variables among the demographic factors, initial clinical status, recanalization time, and underlying diseases. Contrary to this expectation, the location of the occlusion site of M1  and BG infarction on 24 hours control MR DWI were not predictors of functional outcome. Instead, the strongest predictors of outcome were IC, CR, and territory infarctions Table .4–9 Among these important factors, the location of the occlusion and whether it is possible to recanalize without complication have remained the factors that physicians are most concerned about. Given that acute MCA occlusions are among the most common type of occlusions, it is worthwhile to analyze them in detail based upon their anatomical subdivision .Since the introduction of intra-arterial recanalization using either thrombolytic agents or mechanical thrombectomy, the most predictive factors for determining patients’ outcomes have been reported to be the initial NIHSS, the status of the collateral circulation, the success rate of recanalization, location of occlusion, infarct volume, and hemorrhagic transformation.–12 and are subdivided into medial LSAs, which are anterior to the cerebral artery, and LSAs which are lateral from the MCA. The lateral LSAs supply the posterolateral head of caudate nucleus, the anterior portion of the body of caudate nucleus, tail of caudate nucleus, anterior putamen, and external part of the globus pallidus.,13 Nevertheless, the globus pallidus, the lower part of the genu as well as the posterior limb of the IC, and the posteromedial portion of the lentiform nucleus are generally supplied by the anterior choroidal artery, the recurrent artery of Heubner, and perforators from the posterior communicating artery. A study by Behme et al found that proximal M1 occlusions showed an unfavorable clinical outcome in comparison with distal M1 occlusions, which was attributed to IC infarction. Obviously, the IC is an important location in connecting the pyramidal tract, but a proximal M1 occlusion  is not likely to incapacitate the entire IC because the basal IC genu and posterior limb of the IC are usually supplied through the anterior choroidal artery and the posterior communicating artery. In addition, Kleine et al reported that, in lateral LSAs occlusions, the IC involvement is much less consistent than involvement of the striatum due to the greater ischemic tolerance of white matter compared with gray matter.The present study demonstrates that functional outcomes after recanalization of M1 occlusions are not different based upon their location . The crucial factors that are important in determining the functional outcome in MCA occlusion seem to be the patency of LSAs emerging from the M1 segment of the MCA. The LSAs do not communicate with other perforators, known as “end arteries,” These results may be explained by the fact that baseline ischemic core had a greater effect on the clinical outcome than the occlusion site after mechanical thrombectomy. Another study reported no difference in the clinical outcome between proximal M1 and distal M1 occlusion, which could explain why thrombus first causes ischemic damage to the basal ganglia by obstructing the orifice of perforators originating from the mid-M1, then spontaneously migrating to the distal MCA causing distal M1 occlusion.In this study, there were no significant differences in initial NIHSS, the status of the collateral circulation, the success rate of recanalization, and hemorrhagic transformation between proximal and distal M1 occlusion patients. However, BG and CR infarctions occurred more frequently in proximal M1 occlusions than in distal M1 occlusions, which is in accordance with lateral LSAs occlusion. Nevertheless, the final functional outcomes of proximal M1 occlusions were similar to those of distal M1 occlusions because the ICs were spared in both proximal and distal M1 occlusion. Several studies have found results similar to ours. One prior study reported that there was no difference in the Alberta Stroke Program Early CT score and low density of IC on CT after mechanical thrombectomy for proximal M1 and distal M1 occlusion.Our study was intended to help physicians facing acute MCA occlusions to provide an estimation of their functional outcome along with M1 segmental subdivision. Importantly, both proximal and distal M1 occlusions demonstrated comparably favorable functional outcomes in terms of IC preservation.P = .320) in our study. Although the etiology of acute stroke has not previously been investigated between proximal M1 and distal M1 occlusions, Kim et al reported that proximal M1 occlusions were more likely to be caused by atherosclerosis, whereas distal M1 occlusions were more likely to be caused by cardio-embolism, and that a good clinical outcome (mRS ≤ 2) was achieved for atherosclerotic occlusion and cardioembolic occlusion. The clinical outcomes of atherosclerotic occlusion may be poor because of the possibility of reocclusion after mechanical thrombectomy; however, according to a previous report, with rescue management, clinical outcomes can be improved by reducing the rate of reocclusion in atherosclerotic occlusion. Another previous study has reported that there is no evidence of significant interaction between ischemic core and occlusion location. That is, the baseline ischemic core was shown to be a more powerful predictor of functional outcome than the occlusion location, but the relationship between ischemic core and outcome does not different by occlusion locations. Although acute ischemic stroke with a distal M1 occlusion have higher rate of favorable outcome than a proximal M1 occlusion, the baseline ischemic core, that is influenced by the physiology such as collateral status, vessel patency status, has a greater influence on functional outcome than the occlusion location associated with LSAs involvement.The final good functional outcomes were not significantly different between proximal M1 and distal M1 occlusions  were not compared, Lapergue et alThis preliminary study has some limitations. First, the population of patients was relatively small. However, we placed emphasis on analyzing single-vessel occlusions among all intracranial vessel occlusions. Thus, this report might still be sufficient for displaying functional outcomes between proximal M1 and distal M1 occlusions. Second, this study was retrospectively investigated, and data collection through medical records can increase the risk of selection bias. Therefore, studies incorporating larger sample sizes and a prospective study design are needed to resolve these limitations.5Although proximal M1 occlusions had more frequent infarctions associated with the LSAS territories, they were not found to have a poorer functional outcome than distal M1 occlusions. In fact, we were able to present comparably favorable outcomes for both proximal and distal M1 occlusions. Our study may reconfirm the importance of an initial NIHSS assessment, the time to recanalization from symptom onset, the status of collateral circulation, the success rate of recanalization, and the infarction volume after recanalization.www.editage.com) for English language editing.The authors would like to thank the patient who consented to this report and Editage (Conceptualization: Hak Cheol Ko.Data curation: Hak Cheol Ko.Investigation: Hak Cheol Ko.Writing – original draft: Hak Cheol Ko.Writing – review & editing: Hak Cheol Ko.