Identification and clearance of human immunodeficiency virus (HIV) reservoirs is a major challenge in achieving a cure for HIV. This is further complicated by comorbidities that may alter the size of the reservoirs. + microglia/macrophages in morphine-dependent RMs. These results suggest that in the proposed model, morphine plays a differential role in SIV reservoirs by reducing the CD4+ T-cell reservoir in lymphoid tissues while increasing the microglia/macrophage reservoir size in CNS tissue. The findings from this preclinical model will serve as a tool for screening therapeutic strategies to reduce/eliminate HIV reservoirs in opioid-dependent PLWH.Human immunodeficiency virus (HIV) persists in cellular reservoirs despite effective combined antiretroviral therapy (cART), and there is viremia flare-up upon therapy interruption. Opioids modulate the immune system and suppress antiviral gene responses, which significantly impact people living with HIV (PLWH). However, the effect of opioids on viral reservoir dynamics remains elusive. Here, we developed a morphine-dependent simian immunodeficiency virus SIVmac251-infected rhesus macaque (RM) model to study the impact of opioids on HIV reservoirs. RMs on a morphine  regimen were infected with SIVmac251. cART was initiated in approximately half the animals 5 weeks postinfection, and morphine/saline administration continued until the end of the study. Among the untreated RMs, we did not find any difference in plasma/cerebrospinal fluid (CSF) viral loads or in cell-associated DNA/RNA viral loads in anatomical tissues. On the other hand, within the cART-suppressed macaques, there was a reduction in cell-associated DNA load, intact proviral DNA levels, and in inducible SIV reservoirs in lymph nodes (LNs) of morphine-administered RMs. In distinction to those in LNs, in the central nervous system (CNS), the size of latent SIV reservoirs was larger in the CD11bIMPORTANCE Identification and clearance of human immunodeficiency virus (HIV) reservoirs is a major challenge in achieving a cure for HIV. This is further complicated by comorbidities that may alter the size of the reservoirs. There is an overlap between the risk factors for HIV and opioid abuse. Opiate abuse has been recognized as a prominent comorbidity in HIV-infected populations. People infected with HIV also abusing opioids have immune modulatory effects and more severe neurological disease. However, the impact of opioid abuse on HIV reservoirs remains unclear. In this study, we used a morphine-dependent simian immunodeficiency virus SIVmac251-infected rhesus macaque (RM) model to study the impact of opioids on HIV reservoirs. Our studies suggested that people with HIV who abuse opioids have higher reservoirs in central nervous system (CNS) than in the lymphoid system. Extrapolating the macaque findings to humans suggests that such differential modulation of HIV reservoirs among people living with HIV abusing opioids could be considered for future HIV cure research efforts. Opioid use disorder (OUD) is a chronic illness characterized by persistent use of opioids to the detriment of the user . Globall6–OUD is important in the context of HIV infection, as several studies have reported that people living with HIV (PLWH) who use opioids are at a higher risk of developing neurological disorders than PLWH without OUD –20. Opio–21–25–+ T cells are the best characterized, though persistence of viral reservoirs in perivascular macrophages and microglia in the central nervous system (CNS) has also been reported  and subsequent cellular activation  of SIVmac251 by intravenous injection. Five weeks postinoculation, cART was initiated in six of ten RMs in the morphine group and in five of nine RMs in the control group. Four monkeys from each group were left untreated until the end of the study (n = 9), the median peak plasma viral load was 1.25 × 107 SIV copies/ml and median peak cerebrospinal fluid (CSF) viral load was 1.02 × 105 SIV copies/ml. On the other hand, in the morphine-administered group (n = 10), the median peak plasma and CSF viral loads were 8.68 × 107 SIV copies/ml and 4.62 × 104 SIV copies/ml, respectively  of ART-naive RMs, the median cell-associated SIV DNA load was 15,898 copies/106 CD4+ T cells in the morphine-administered group versus 8,547 copies/106 CD4+ T cells in saline controls (P = 0.600). In the PBMCs of ART-treated RMs, the median cell-associated SIV DNA load was 3,332 copies/106 CD4+ T cells in the morphine-administered group versus 4,583 copies/106 CD4+ T cells in saline controls (P = 0.055) . In the PBMCs of the ART-treated group, the median cell-associated SIV RNA load was 94 copies/106 CD4+ T cells in the morphine-administered group versus 446 copies/106 CD4+ T cells in controls (P = 0.120)  . Similar= 0.120) .+ T cells purified from LNs. In LNs of ART-naive RMs, the median cell-associated SIV DNA load was 11,295 copies/106 CD4+ T cells in the morphine-administered group versus 14,962 copies/106 CD4+ T cells in saline controls (P = 0.600). On the other hand, in LNs of the ART group, a significant difference was found, with a median cell-associated SIV DNA load of 4,392 copies/106 CD4+ T cells in the morphine-administered group versus 7,847 copies/106 CD4+ T cells in saline controls (P = 0.008) . In the LNs of the ART-treated group, the median cell-associated SIV RNA load was 143 copies/106 CD4+ T cells in the morphine-administered group versus 260 copies/106 CD4+ T cells in saline controls (P = 0.360)  . In LNs = 0.360) .6 cells in the morphine-administered group versus 10,961 copies/106 cells in saline controls (P = 0.342). In the ART-treated group, a significant difference was found, with a median cell-associated SIV DNA load of 70 copies/106 cells in the morphine-administered group versus 272 copies/106 cells in saline controls (P = 0.036) . In the ART-treated group, the median cell-associated SIV RNA load was 11 copies/106 cells in the morphine-administered group versus 39 copies/106 cells in saline controls (P = 0.080)  . In ART-= 0.080) .We also measured the cell-associated DNA/RNA in both the spleens and lungs of macaques. There was no difference in cell-associated SIV DNA/RNA levels in the lungs or spleens of the morphine-administered versus saline controls in the presence of ART treatment as well as in the ART-naive RMs . In summ+ T cells producing inducible multiply spliced (ms)-Tat/rev transcript per million of CD4+ T cells was 4.54 in the morphine-administered group versus 9.11 in the saline control group (P = 0.067)  (We used the Tat/rev induced limiting dilution assay (TILDA) to measure the size of inducible SIV reservoirs in the blood and LNs of morphine/saline-administered, SIV-infected cART-treated RMs . In peri= 0.067) . In the = 0.036) .+ T cells purified from peripheral blood, the median number of intact SIV genomes per million cells was 1,480 for the morphine-administered group versus 2,521 in the saline control group (P = 0.080)   to estimate the size of replication-competent SIV reservoirs in both the blood and LNs. Recently Bruner et al.  develope= 0.080) . However= 0.036) .+ T cells  from LNs to identify Th1-like Tfh (CXCR3+), Th2-like Tfh (CCR4+), and Th17-like Tfh (CCR6+) subpopulations (+ Th1 polarized cells was measured by quantifying levels of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) cytokine-secreting cells. In morphine-administered RMs, IFN-γ-secreting cells were a median of 0.87% versus 1.55% (P = 0.536) for controls (P = 0.017)     . The med= 0.305) . The lev= 0.930) . Remarka= 0.004) .P = 0.017) (+ FoxP3+ Tfh) cells was elevated, with a median of 8.78% versus 2.87% in saline control group (P = 0.030) (+ CXCR5− Treg (CD25+ FoxP3+) cells in the morphine-administered group, with a median of 4.57% versus 1.25% in saline control group (P = 0.004) (+ Tfh cells (Th1), with a median of 47.70% versus 56.30% in the saline control group (P = 0.004) (+ Tfh cells (Th2), with a median of 18.70% in the morphine-administered group versus 8.49% in the saline control group (P = 0.004) (+ Tfh cells (Th17) were a median of 55.95% in the morphine-administered RMs versus 60.40% in saline control RMs (P = 0.792) (+ T cell activation. For CD4+ T cell activation (denoted by CD4+ CD38+ HLA-DR+), the morphine-administered RMs had a significantly lower frequency, with a median of 3.37% versus 6.13% in saline controls (P = 0.017) (+ CD38+ HLA-DR+), the morphine-administered macaques had a significantly reduced frequency, with a median of 27.10% versus 53.50% in saline controls (P = 0.017) (− CD8α/CD38+ HLA-DR+), the morphine-administered group had a median frequency of 11.45% versus 9.16% in the saline control group (P = 0.428)  expressing PD-1, CXCR5, and CD95, with a median value of 7.61% versus 13.49% in saline control groups (= 0.017) . On the = 0.030) . Similar= 0.004) . Evaluat= 0.004) , there w= 0.004) . Further= 0.792) . Finally= 0.017) . Similar= 0.017) . On the = 0.428) .+ myeloid cells (microglia and perivascular macrophages) from the brains of the cART-treated morphine-administered and saline control SIV-infected monkeys, all of which had complete viral suppression in the plasma and CSF (<50 copies/ml). Immediately after necropsy, CD11b+ cells were purified from brains. After purification, we obtained more than 95% cells positive for CD11b   (+ microglia/macrophages using macrophage QVOA (MØ-QVOA). The number of CD11b+ macrophages used for the MØ-QVOA for each animal is described in + T cells within the enriched CD11b+ cells from brain, which is in line with 0.06% reported by Avalos et al. , the probability of the presence of CD4+ T cells with intact proviral genome in any sample of macrophage quantitative outgrowth assay was found to be less than 1. The CD11b+ microglia/macrophages obtained from morphine-administered macaques had a median infectious unit per million (IUPM) value of 0.89 versus 0.19 in saline control group (P = 0.014)  . The med= 0.710) . We nexts et al. . Based o= 0.014) , indicat+ T cells and in lymphoid tissues. Several lines of evidence suggest that within 3 to 7 days of infection, HIV enters the CNS as well as tissue resident macrophages and microglia, which likely serve as viral reservoirs and are a source of viral rebound in the cases of therapeutic interruptions , the possible effects of opioids on viral tropism is unknown.The major caveat of the present study is the number of RMs included in each group due to the prohibitive cost of conducting long-term studies with multiple daily injections. Other issues include the experimental design, in which animals were initially ramped-up with morphine, then infected, and then treated with cART. In a patient cohort, this may not be an ideal scenario, as most of the people suffering from substance use disorders take multiple forms of drugs. Furthermore, opioid use may follow infection; for example, in PLWH, opioids are prescribed to treat chronic pain, and subsequently they may become addicted to these substances. For this population, how opioid use modulates the dynamics of different viral reservoirs needs to be explored. In addition, given the different cellular nature of the reservoir  on HIV reservoirs. Our results suggest that morphine differentially modulates SIV reservoirs in LNs and rectal mucosal tissue versus SIV reservoirs in myeloid lineage of cells within the CNS. Chronic morphine administration reduces the size of viral reservoirs in CD4l-glutamine , and 100 U/ml penicillin and 100 μg/ml streptomycin  at 37°C and 5% CO2.Antiretroviral drugs tenofovir alafenamide (TFV) and emtricitabine (FTC) were obtained from Gilead Sciences, Foster City, CA, USA, while dolutegravir (DTG) was procured from ViiV Healthcare, Research Triangle Park, NC, USA, as a material transfer agreement (MTA) with S. N. Byrareddy. All other molecular biology-grade fine chemicals used in the study were purchased from Sigma-Aldrich, St. Louis, MO, USA, unless otherwise mentioned. CEMx174 is a hybrid human lymphoid cell line generated from human B721.174 and T-CEM cell lines that was used for expansion of virus in quantitative viral outgrowth assays due to its enhanced susceptibility to SIV infection . This ceMacaca mulatta; mean age, 4.67 years; range, 4.1 to 7.0 years) were used in this study  and the Institutional biosafety Committee (IBC) under protocol number 16-073-07-FC titled “The effect of cART and drug of abuse on the establishment of CNS viral reservoirs” and 15-113-01-FC titled “The combinatorial effects of opiates and promoter-variant strains of HIV-1 subtype C on neuropathogenesis and latency.” UNMC has been accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.n = 9) received a similar dose of normal saline (control group). At this point, all the macaques were intravenously inoculated with 200 TCID50 of SIVmac251 , while the administration of morphine/saline continued until the end of the study. At 5 weeks postinoculation, daily ART was initiated in six macaques from the morphine group and five macaques in the control group and continued until the end of the study. ART regimen consisted of two reverse transcriptase inhibitors  and one integrase inhibitor . All the drugs were dissolved in a vehicle made up of 15% Kleptose HPB  (wt/wt) in 0.1 N NaOH. The antiretroviral drugs were administered subcutaneously once daily at 1 ml/kg body weight. Peripheral blood from the femoral vein, CSF by direct puncture of the cisterna magna or by lumbar puncture, LNs, and colorectal mucosa biopsy specimens were collected longitudinally at different time points of the study after anesthetizing the monkeys with ketamine-HCl (5 to 20 mg/kg) or tiletamine and zolazepam  to monitor SIV viral loads and a series of immunologic and virologic parameters as described in the experimental schema.The overall design of the study is illustrated in a schematic form in GTCTGCGTCATCTGGTGCATTC-3′; SIVGAGR, 5′-CACTAGGTGTCTCTGCACTATCTGTTTTG-3′; and SIVP, 5′-/6-carboxyfluorescein (FAM)/CTTCCTCAG/ZEN/TGTGTTTCACTTTCTCTTCTGCG/3IABkFQ-3′.SIV RNA concentration in the plasma and CSF samples were measured by quantitative reverse transcription-PCR (qRT-PCR) as previously described . In brie+ T cells using the EasySep NHP CD4+ T cell isolation kit from STEMCELL Technologies Canada Inc. (cat. no. 19582) as per the manufacturer’s instructions. Briefly, frozen cells were thawed at 37°C, washed with complete RPMI (composition described above), centrifuged for 6 min at 1,200 rpm, and resuspended in 1 ml of recommended medium . The cell suspension was transferred to a 12-by-75-mm polystyrene tube, 50 µl of EasySep negative selection cocktail was added and mixed well, and the cell suspension was incubated at room temperature (RT) for 10 min. Then 100 µl of EasySep magnetic particles were added, and the cells were incubated at RT for 5 min. After incubation, 2 ml of recommended medium was added to the solution, and then the tubes were placed in an EasySep magnet for 5 min. The negatively selected enriched CD4+ T cells were collected for downstream applications.Peripheral blood mononuclear cells (PBMCs) and LN cells were enriched for CD4Myeloid-enriched brain isolation used a modification of the procedure described by Marcondes et al. . In brie+ cells were collected, and cells were counted on Coulter Counter Z1. Isolates were used for downstream processing and analyzed for epitope expression using a fluorescence-activated cell sorter (FACS).Cell isolates were washed in PBS, and reconsituted in MACS buffer with 0.1% bovine serum albumin (BSA) . Cells were counted and the volume was adjusted for staining with nonhuman primate CD11b microbeads (Miltenyi). Forty million cells were reconstituted in 160 µl of MACS buffer and reacted with 80 µl of CD11b microbeads at 4°C for 15 min. After incubation, cells were washed with MACS buffer with 0.1% BSA, reconstituted into 500 µl of MACS buffer, and loaded onto MACS Separator LD columns. Both the negative flowthrough and the positve CD11bg for 10 min at 4°C. The cell pellets were washed twice with PBS containing 2% FBS and 2 mM EDTA. Cells were resuspended in complete RPMI (composition described above) and used for downstream applications.At necropsy, the lung was lavaged with sterile saline, and the bronchoalveolar lavage (BAL) fluid was collected. Cells were collected through centrifugation of the BAL fluid at 250 × + T cells and CD11b+ macrophages were pelleted by centrifugation for 6 min at 1,200 rpm and stored at −80°C until time of processing. To extract the DNA/RNA from the cells, first, the pellets were thawed on ice and thoroughly loosened by flicking the tube. Then, 600 µl Buffer RLT Plus with 2-mercaptoethanol (β-ME) at 10 µl/1 ml concentration was added and thoroughly mixed by vortexing. Following lysis, the mixture was transferred to a QIAshredder , and the AllPrep DNA/RNA minikit  was used for DNA/RNA isolation. The remaining protocol followed was per the kit’s instructions. RNA was eluted in 30 µl of RNase-free water after performing column DNase digestion, while DNA was eluted in 50 µl of elution buffer provided in the kit. DNA/RNA concentrations were measured using a SimpliNano spectrophotometer  and stored in a −80°C deep freezer for future use.Enriched CD4For DNA/RNA isolation from tissues, the AllPrep DNA/RNA minikit  was used. Approximately 30 mg of frozen tissue was placed into a 2-ml flat-bottom centrifuge tube on dry ice with a 5-mm stainless steel bead  at the bottom. Then, 600 µl buffer RLT plus with 2-mercaptoethanol (βME) at 10 µl βME/ml of RLT plus was added before placing the tube into a TissueLyser LT  set at 50 oscillations/s for 5 min. The lysate was checked for homogeneity and transferred to a QIAshredder . DNA/RNA was then isolated per the kit’s instructions. RNA was eluted in 30 µl of RNase-free water after performing column DNase digestion, while DNA was eluted in 50 µl of buffer EB. DNA/RNA concentrations were measured using a SimpliNano spectrophotometer  and stored in a −80°C deep freezer for future use.Total cell-associated SIV RNA was quantified using RT-ddPCR using 100 ng of RNA, a 1-Step RT-ddPCR Advanced kit for probes , and the same set of primers and probe used for SIV plasma viral load quantification. ddPCR was carried out on a Bio-Rad QX200 AutoDG digital droplet PCR system. In brief, 22 μl of reaction mix was used for droplet generation using a QX200 droplet generator, the ddPCR plate having the emulsified samples was heat sealed with foil , and amplification occurred in a C1000 Touch thermal cycler . After thermal cycling, ddPCR plates were transferred to the QX200 droplet reader (Bio-Rad) for droplet count and fluorescence measurement. Positive droplets with amplified products were separated from negative droplets without target amplicon by applying a fluorescence amplitude threshold, and the absolute quantity of RNA per sample (copies/µl) was determined using QuantaSoft software. For quantification of cell-associated SIV DNA, the same methodology was used as described above without using the reverse transcription step and with 2× ddPCR Supermix for probes (no dUTP)  instead of the 1-Step RT-ddPCR Advanced kit.+ T cells from frozen PBMCs or LN total cells were suspended in complete RPMI at a 2-million cells/ml concentration and then rested at 37°C and 5% CO2 for a minimum of 2 h. After resting, cells were divided into two groups: one was stimulated with 100 ng/ml phorbol myristate acetate (PMA) and 1 μg/ml ionomycin, and the other group was left untreated as the control. After 16 h, the cells were counted and then centrifuged at 2,000 rpm for 10 min to stop the reaction. Cells were then serially diluted in complete RPMI medium to the following concentrations: 18,000, 9,000, 3,000, and 1,000 cells/μl. A 96-well PCR plate was divided column-wise in half for the two treatments and then into quarters for the four-dilution sets in 8 replicates. In each well of the plate, 5 µl of master mix was added, and then 1 µl of the appropriate dilution for the corresponding treatment was added to each well. The first PCR mix contained 0.2 µl of Super Script III RT/Platinum Taq mix , 0.1 µl of Superase RNase inhibitor , 2.25 µl of nuclease-free H2O , 2.2 µl of tris-EDTA (TE) buffer, 0.125 µl of 10 µM SIVF , and 0.125 μl 10 µM SIVR . To each well of the plate, 5 µl of reaction mix was added. Preamplification settings included reverse transcription for 15 min at 50°C followed by denaturation for 2 min at 95°C and then 24 cycles of amplification at 95°C for 14 s and 60°C for 4 min. Preamplification was conducted in a LifePro thermal cycler (Bioer Technology). After preamplification, 9 µl of qPCR mix containing 5 μl LightCycler 480 Probes Master , 3.4 μl of nuclease-free H2O , 0.2 µl of 20 µM nested FW primer , 0.2 µl of 20 µM SIVR , and 0.2 µl of 5 µM probe (5′-/56-FAM/AAACCCATA/ZEN/TCCAACAGGACCCGG/3IABkFQ/-3′) was added to each well of a new 96-well PCR plate. Then, 1 µl of PCR product from preamplification was added to the corresponding well in the new plate. Real-time PCR was performed in QuantStudio 3  with the following settings: preincubation at 95°C for 10 min followed by 45 cycles at 95°C for 10 s, 60°C for 30 s, and 72°C for 1 s, and then finally a cooling period at 40°C for 30 s.The Tat/rev induced limiting dilution assay (TILDA) was performed as described earlier , 86. In The intact proviral DNA assay was performed as described by Bender et al. , includi+ macrophages isolated from brain, lung, and liver as described by Avalos et al.  as described above. The presence of the contaminating CD3+ T cells within the enriched CD11b+ cells was estimated by quantification of TCRβ as described by Avalos et al.  for 30 min and then washed twice with PBS. Purified cells were plated in triplicates and in a 10-fold serial dilution in the presence of 10 µM zidovudine (Sigma), 25 nM darunavir , and 5 nM ritonavir . BrMφ medium  was used for brain cells, and complete RPMI medium was used for lung and liver. The culture was then placed in a 37°C 5% CO2 incubator for 72 h to permit proper cell adherence. After the 3-day incubation, the antiretroviral-containing medium was removed, and cells were washed with PBS (1×) and then replenished with BrMφ/complete RPMI medium containing 10 ng/ml tumor necrosis factor alpha (TNF-α) , 1 µg/ml Pam3CSK4 , and 1 µg/ml prostaglandin . Additionally, approximately 105 CEMX-174 feeder cells were added to each well. Supernatants were collected on days 5, 7, 10, and 14 post-culture activation and replenished with medium containing activating agents TNF-α, Pam3CSK4, and prostaglandin. RNA was extracted from the supernatants using the QIAamp Viral RNA minikit . Prior to extraction, the supernatants were centrifuged at 2,000 rpm for 5 min, and then 140 µl was aliquoted and used per the kit’s instructions to isolate RNA. The RNA was eluted in 50 µl of AVE buffer. Viral RNA was quantified in culture supernatant using RT-ddPCR as described above. The frequency of replication-competent latent reservoir cells was estimated using the IUPMStats v1.0 infection frequency calculator  QVOA was performed on CD11bs et al. . In bries et al. . Prior tlculator .n = 11) during necropsy. Two million to four million PBMCs were stimulated with PMA and ionomycin at final concentrations of 50 ng/ml and 500 ng/ml, respectively, in complete RPMI 1640 medium (composition described above). An unstimulated condition was included as an experimental control. After 2 h of incubation at 37°C in a humidified 5% CO2 incubator, 1 μl of GolgiPlug  containing brefeldin A was added to each well together with 1× of monensin  and incubated for an additional 4 h. After that, a 1:1,000 dilution of zombie aqua amine reactive dye  was added to the cell suspensions and incubated for 30 min in the dark. The cells were then washed and Fc blockade was carried out using polyclonal anti-human Fc receptor binding inhibitor . Surface staining was performed using anti-CD45, anti-CD8, anti-CD4, and anti-NKG2A antibodies for panel 1 and using anti-CD25 and anti-CD127 antibodies for panel 2 (Treg panel); cells were incubated for 1 h at room temperature in the dark. Thereafter, for panel 1, cells were fixed using a 2% paraformaldehyde (PFA) solution for 30 min, and permeabilization was performed using a 1× BD perm/wash solution  for 15 min at 4°C. Then, anti-CD3, anti-IFN-γ, anti-TNF-α, and anti-IL-17 antibodies were added, and cells were incubated at 4°C for 15 min, washed, and resuspended in PBS. For the Treg panel, fixation and permeabilization were performed using a 1× solution of FoxP3/transcription factor Fix/Perm concentrate (4×)  that was diluted using the 1× FoxP3/transcription factor Fix/Perm diluent . Following this, anti-CD3, anti-IL-4, and anti-FoxP3 antibodies were added, and cells were incubated at 4°C for 15 min, washed, and resuspended in PBS.The details of all fluorochrome-conjugated monoclonal antibodies used in the flow cytometry experiments are listed in For LN cell suspensions, 2 million to 4 million cells were taken, zombie UV viability dye was added, and Fc receptor blockade was performed as described above. Then, in the T cell polarization panel, anti-CXCR5, anti-CXCR3, anti-CCR4, and anti-CCR6 antibodies were added, and in the Treg panel, anti-CXCR5 antibody was added. After that, cells in both panels were incubated at 37°C for 30 min. Following this, ani-CD45, anti-CD3, anti-CD4, anti-CD8, anti-CD20, anti-CD95, anti-CD38, anti-PD1, and anti-human HLA-DR antibodies were added in the polarization panel, while in the Treg panel, anti-CD25 and anti-PD1 antibodies were added. After that, cells in both panels were incubated at room temperature for 30 min, washed, and fixed. For the Treg panel, intracellular staining was performed as described above for PBMCs using anti-FoxP3 and anti-CTLA4 antibodies. All events were acquired using the BD Fortessa X450, and data were analyzed using FlowJo version 10.6. Fluorescence-minus-one (FMO) controls were included as cutoffs for placement of gates when handling nondiscriminatory variables.P values of less than 0.05 are considered statistically significant.Graph Pad Prism 8.0 software was used to carryout statistical comparisons and plot the figures. Descriptive statistics were presented to summarize continuous variables. Wilcoxon rank sum tests were used to determine the statistically significant difference in continuous outcomes between morphine and saline groups for flow cytometry data and other experimental data. All


Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae collected from across China were determined. Monte-Carlo simulations were performed for various dosing regimens (0.42–1.5 mg/kg/12 h via 1 or 2-h infusion). The probability of target attainment, PK/PD breakpoints and cumulative fraction of response were determined for each bacterial species. MIC90 of polymyxin B was 1 mg/L for P. aeruginosa and 0.5 mg/L for A. baumannii and K. pneumoniae. With the recommended polymyxin B dose of 1.5–2.5 mg/kg/day, the PK/PD susceptible breakpoints for P. aeruginosa, A. baumannii and K. pneumoniae were 2, 1 and 1 mg/L respectively for bloodstream infection. For Chinese patients, polymyxin B dosing regimens of 0.75–1.5 mg/kg/12 h for P. aeruginosa and 1–1.5 mg/kg/12 h for A. baumannii and K. pneumoniae were appropriate. Breakpoint determination should consider the antimicrobial PK/PD at infection site and delivery route. The recent withdrawal of polymyxin susceptible breakpoint by CLSI primarily based on poor efficacy against lung infections needs to be reconsidered for bloodstream infections.The latest PK/PD findings have demonstrated negligible efficacy of intravenous polymyxins against pulmonary infections. We investigated pharmacokinetic/pharmacodynamic (PK/PD)-based breakpoints of polymyxin B for bloodstream infections and the rationality of the recent withdrawal of polymyxin susceptibility breakpoints by the CLSI. Polymyxin B pharmacokinetic data were obtained from a phase I clinical trial in healthy Chinese subjects and population pharmacokinetic parameters were employed to determine the exposure of polymyxin B at steady state. MICs of 1,431 recent clinical isolates of  Pseudomonas aeruginosa and Klebsiella spp. and >70% of Acinetobacter spp. are now carbapenem resistant (http://www.chinets.com/). Elsewhere, the World Health Organization has reported high median resistance rates to carbapenems in several bacterial species globally, exemplified by resistance rates in Klebsiella spp. and Acinetobacter spp. isolated from bloodstream infections of ∼20 and ∼60%, respectively . Although the newly developed β-lactam/β-lactamase inhibitor combinations (such as ceftazidime-avibactam and ceftolozane-tazobactam) have mitigated the situation, none of them is active against metallo-β-lactamase-producing Enterobacterales, P. aeruginosa or carbapenemase-producing A. baumannii  that bridge adjacent LPS molecules and resulting in OM leaflet expansion, disruption of the membrane integrity, and increased membrane permeability  . Recentl0-inf of polymyxin B in WinNonlin , which was linear with dose according to a power model with a 95% confidence interval . Population pharmacokinetic (PPK) analysis was conducted using NONMEM 7.4  with G77 FORTRAN complier and FOCEI algorithm. The base model of polymyxin B was fit into a three-compartment model. The interindividual and residual variabilities were best described by an exponential model and a proportional model, respectively. Age and gender were included in the final PK model  were conducted in healthy Chinese subjects . The liqPK model .P. aeruginosa, 262 of A. baumannii and 652 of K. pneumoniae) were collected across 2017–2019 from more than 30 teaching hospitals in 23 provinces across China. Polymyxin B and meropenem minimum inhibitory concentrations (MICs) were determined by broth microdilution and interpreted according to the CLSI breakpoints . Escherichia coli ATCC 25922, E. coli ATCC 35218 and K. pneumoniae ATCC 700603 acted as quality control strains. Statistical analysis was performed using WHONET (version 5.6). The study protocol was approved by the Institutional Review Board of Huashan Hospital, Fudan University .A total of 1,431 clinical isolates (517 strains of fAUC0–24h/MIC) is the most predictive PK/PD index for polymyxins (10 CFU and 2-log10 CFU reductions in colony forming units (CFU)/thigh were derived from dose-fractionation studies of polymyxin B (K. pneumoniae) and colistin (P. aeruginosa and A. baumannii) in murine thigh infection models  of polymyxin B in plasma of 42% was applied . The add-in macro permitted the MATLAB program to perform Monte-Carlo simulations for 10,000 simulated data sets . The upper limit of the MIC range was taken as the PK/PD breakpoint when PTA was >90%. MIC distributions of polymyxin B for each bacterial species were used to determine the CFR, with a CFR of 90% considered effective.Four thousand data sets were generated with the final PPK model based on the estimated PPK parameters for each dosage regimen ranging from 0.42 to 1.5 mg/kg/12 h (administered via a 1- or 2-h infusion). Mean and SD of AUC50/MIC90 values for the 1,431 clinical isolates are shown in P. aeruginosa, 0.25 to >32 mg/L for A. baumannii, and 0.25–16 mg/L for K. pneumoniae. Meropenem resistance was detected in 25.9, 85.5 and 37.0% of P. aeruginosa, A. baumannii and K. pneumoniae isolates, respectively. The AUC0–24h,ss achieved with different polymyxin B dosing regimens (0.42–1.5 mg/kg/12 h) and infusion times (1 or 2 h) are shown in Polymyxin B MIC distributions and MICP. aeruginosa, the fAUC0–24h/MIC required to achieve 1- and 2-log10 CFU killing  were achieved in 100% of cases for MICs ≤0.5 and ≤1 mg/L with the lower-dose regimens . However, with these regimens the PTA decreased dramatically as MICs increased above these values. For an MIC of 2 mg/L, the 1 mg/kg/12 h dosing regimen was effective with a PTA for 1-log10 CFU killing of 99% and the PTA for the 1.25 mg/kg/12 h dosing regimen was 95.1% to achieve 2- log10 CFU killing  against strains with MICs ≤0.5 mg/L (PTA = 97.5 and 100%), while the remaining dosing regimens were additionally effective against strains with an MIC ≤1 mg/L (PTA = 100%). No dosing regimen achieved the fAUC0–24h/MIC target of 17.6 when the MIC was ≥2 mg/L.Against K. pneumoniae, 0.42 and 0.75 mg/kg/12 h achieved the fAUC0–24h/MIC target of 17.4 in 98.1 and 100% of cases against strains with MICs ≤0.5 mg/L, respectively. All higher-dose regimens were additionally effective against strains with an MIC of ≤1 mg/L  against in China , indicatCss,avg) achieved with intravenously administered polymyxin B (0.45–3.38 mg/kg/day) or CMS (2–18 MIU/day) are 2–3 mg/L (polymyxin B and colistin) in critically-ill patients , A. baumannii or A. calcoaceticus  and Enterobacteriaceae  was 2, 2, and >4 mg/L, respectively. Given substantial regional susceptibility differences, effective therapeutic regimens must consider the regional susceptibility of the bacterial species involved.In China, the MICIn a recent Chinese study investigating the clinical efficacy and safety of polymyxin B-based regimens  for treatment of bloodstream infection caused by extensively-drug resistant Gram-negative bacteria, the clearance rate of microorganisms was 65.2%, overall effectiveness (cure or improvement) 59.0%, and 28-day all-cause mortality 41.0% . Importa0–24h,ss for polymyxin B should be 50–100 mg h/L. In our study, the polymyxin B AUC0–24h,ss achieved with the 0.42 and 0.75 mg/kg/12 h dosing regimens fell below this target range  are employed, the recent MIC data collected in Chinese hospitals showed that most isolates of P. aeruginosa, A. baumannii, and K. pneumoniae  would have intermediate susceptibility to polymyxin B. However, further PK/PD analysis showed that increasing doses could be effective against strains with MICs up to 2 mg/L. Taking decreases in bacterial counts of 1-log10 CFU (for K. pneumoniae) or 2-log10 CFU (for P. aeruginosa and A. baumannii) kill as the target values, dosing regimens of ≥0.75 mg/kg/12 h could be effective against P. aeruginosa with MIC of 1 mg/L; for A. baumannii and K. pneumoniae, all dosing regimens were effective against isolates with MIC ≤0.5 mg/L. One PK/PD study on polymyxin B for bloodstream infections has been conducted though the rationality of the present CLSI breakpoints were not well explained which is a valuable reference in clinical practice . The breakpoints for P. aeruginosa, A. baumannii and K. pneumoniae were 2, 2 and 1 mg/L, respectively. The variable protein binding may affect the PK/PD breakpoints as well. According to the reported protein binding of 78.5–92.4%  should be utilized in terms of Chinese recommended dosing range. The revival of CLSI susceptibility breakpoint of polymyxins for bloodstream infections is potential in the future.


The levels of both reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) were reduced after RAW264.7 macrophages were treated with BBSLP after LPS induction. After RAW264.7 macrophage treatment with BBSLP and induction by LPS, the levels of inflammatory molecules, including nitric oxide (NO), prostaglandin E2 (PGE2), IL-1α, IL-6 and TNF-α, decreased. NF-κB signaling activity was inhibited by reductions in IκB phosphorylation and NF-κB DNA-binding activity after RAW264.7 macrophages were treated with BBSLP after LPS induction. In conclusion, BBSLP has antioxidant and anti-inflammatory capabilities and can be a supplement material for functional food.To assess the potential of by-products of the black bean fermented soybean sauce manufacturing process as new functional food materials, we prepared black bean steamed liquid lyophilized product (BBSLP) and analysed its antioxidant effects in vitro. RAW264.7 macrophages were cultured and treated with BBSLP for 24 h, and 1 μg/mL lipopolysaccharide (LPS) was then used for another 24 h to induce inflammation. The cellular antioxidant capacity and inflammatory response were then analysed. Activation of nuclear factor kappa B (NF-κB) signaling in RAW264.7 macrophages was also analysed. Results showed BBSLP had 2,2-diphenyl-1-hydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium (ABTS Glycine max (L.) Merr.) as the major raw material is one of the most favored. During the soybean sauce manufacturing process, raw soybean steaming is an important step before yeast inoculation [Soybean sauces are commonly used as seasonings and sauces in Asia. Among them, black bean-fermented soybean sauce which uses black beans (culation . Howeverculation . Therefoculation .The black soybean cultivar has abundant polyphenols in its seed coating . During 2 (PGE2) act as messengers for different physiological functions and pathological processes [The inflammatory response is a physiological protective mechanism of the body that occurs in response to infection; however, the occurrence of chronic inflammation with chronic inflammatory cells, including macrophages, lymphocytes and plasma cells, among others, is increasing . During rocesses . Conversrocesses . These crocesses . Excess rocesses .Some inflammatory response factors, such as IL-1β, IL-6, and TNF-α, and inflammation-mediated molecular enzymes, including inducible nitroxide synthase (iNOS) and cyclooxygenase-2 (COX-2), are primarily controlled by nuclear factor kappa B (NF-κB), which is well recognized to play an important role in inflammation ,20,21,22Currently, the food supply chain has many challenges due to decreased natural resources and increased food waste . Thus, rTo know the application of fresh BBSL, the pH was measured in this study. As shown in In the 2,2-diphenyl-1-hydrazyl (DPPH) radical-reducing ability test the scavenging abilities of 0.5, 1 and 2 mg/mL BBSLP were 42.5 ± 8.7%, 54.4 ± 0.4% and 79.6 ± 0.2%, respectively. The observed scavenging ability in the vitamin C-treated group was 88.0 ± 5.8% A. Althou50 value of BBSLP for DPPH radical-scavenging ability was 0.81 mg/mL. +) radical scavenging. The ABTS+ radical scavenging abilities in the 0.5, 1 and 2 mg/mL BBSL groups were 7.8 ± 2.3%, 20.5 ± 6.3% and 50.2 ± 8.3%, respectively, and showed a dose-dependent increase (p < 0.05). The effect concentration (EC50) value of BBSLP for ABTS+ radical scavenging ability was 1.50 mg/mL. In addition, the reducing power of 0.5, 1 and 2 mg/mL BBSLP reached 42.5 ± 84.6% in a dose-dependent manner (p < 0.05). The vitamin C-treated group showed 88.7 ± 9.6% reducing power . For reasons related to BBSLP yield and solubility, we used 0.1, 0.5 and 1 μg/mL BBSLP as the experimental doses in the following study.In the present study, the cell viability of RAW264.7 macrophages did not significantly differ between the 0.1, 0.5 or 1 μg/mL BBSLP (approximately 97–101%) under LPS induction or the control group (100%) A. Based p < 0.05) (p < 0.05) (p < 0.05) (p < 0.05).When RAW264.7 macrophages were treated with LPS alone, the thiobarbituric acid reactive substances (TBARS) level was significantly increased by 229% ( < 0.05) A. Howeve < 0.05) A. The TB < 0.05) B. Howeve2 production was significantly increased after RAW264.7 macrophages were induced with LPS compared with the control group . A 60–68% decrease in PGE2 was noted in cells cotreated with 0.1 to 1 μg/mL BBSLP and LPS (p < 0.05). Immunoblot analysis showed that the iNOS levels in RAW264.7 macrophages treated with 0.1, 0.5 and 1 μg/mL BBSLP were 82%, 77% and 69% that of cells treated with LPS alone, respectively ; however, when RAW264.7 macrophages were treated with 0.5 or 1 μg/mL BBSLP combined with LPS, the IL-1β levels were 59.9 ± 8.7% and 32.0 ± 8.7%, they were significantly lower than the LPS induction alone group . However, BBSLP did not affect the protein contents of cytosolic I-κB in EA.hy926 cells  analysis results have also shown that the black soybean seed coat is a more efficient reducing agent than dehulled black soybeans and yellow soybean coats [Oxidative stress usually triggers the inflammatory response in various cells and tissues . Macrophan coats . In addian coats . Previouan coats  showed tan coats . Additioan coats .In addition, resveratrol, a polyphenol constituent of grapes, acts as a COX suppressor, reducing the inflammatory response similarly to a nonsteroidal anti-inflammatory drug (NSAID). A molecular basis for the mutually beneficial relationship between plants and humans has been speculated . HussainIn the present study, BBSLP significantly inhibited NF-κB signalling activity by reducing I-κB phosphorylation and NF-κB-DNA binding activity. NF-κB signalling plays an important role in iNOS, COX-2, IL-1β, IL-6 and TNF-α transcription ,20,21,22Currently, the food supply chain is facing substantial pressures, including the availability of fewer natural resources and increased food waste . One impBBSL was a gift from Ta-Tung Soya Sauce Co., Ltd., located in Siluo Town . BBSL is collected from the soybean sauce manufacturing process. After fresh black beans are washed and steamed by 120 °C streams in a closed steam tank for 1 h. BBSL were directly collected as experimental materials. LPS was purchased from Sigma-Aldrich Co. .w/v).According to our previous methods , BBSLP w2O). Then, 500 µL of 7.5% Na2CO3 solution was added to react for 30 min, and the absorbance of each sample was measured at an optical density (OD) of 760 nm in a Biokinetics microplate reader . Calibration curves were constructed using 0, 0.125, 0.25 and 0.5 mg/mL gallic acid (GA). The total phenolic content is represented as mg GAE/mL BBSL or mg GAE/g BBSLP.The pH values of fresh BBSL were measured using a pH metre . The total phenol contents were analysed using a colorimetric method according to Padmavati et al. . One hun3 solution was added to 100 μL of the BBSL or BBSLP solution to react for 5 min. Then, 50 μL of 10% AlCl3 solution was added. Finally, 600 μL of 4% NaOH solution was added to the mixture for 30 min. The absorbance of the mixture was measured at OD 510 nm on the Biokinetics microplate reader. Calibration curves were constructed with 0, 0.02, 0.06, 0.08 and 0.1 mg/mL rutin (RU) as the standard. The total flavonoid content is represented as mg RUE/mL BBSL or mg RUE/g BBSLP.The flavonoid content was analysed using the colorimetric method according to Jia et al. . One hung for 20 min at 4 °C. Then, the supernatant was discarded. The precipitate was dissolved in 1 mL of NaOH and standing for reacted for 30 min at room temperature. Then, 1.0 mL of modified Lowry Reagent (Sigma-Aldrich Co.) was added, then mixing and incubation at room temperature for 10 min. Five hundred µL Prepared 1X Folin-Ciocalteu’s phenol reagent (Sigma-Aldrich Co.) was added, and the mixture was standing in a water bath at 37 °C for 30 min. After 30 min, the absorbance was measured at 660 nm on the Biokinetics microplate reader.Crude protein contents were analysed according to Lowry et al. . Fifty mBBSLP or Avitamin C/Ablank) × 100.In this study, DPPH (Sigma-Aldrich Co.) radical scavenging activity by BBSLP was analysed according to the method of Shimada et al. . For theg for 10 min. The supernatant (2.5 mL) was mixed with 2.5 mL of distilled water and 2.5 mL of 0.1% ferric chloride, and the absorbance at 700 nm was read using the microplate reader. The reducing power was calculated as (ABBSLP or Avitamin C − Ablank)/Avitamin C × 100. A vitamin C (1 mg/mL) was used as a control.According to the method of Shimada et al.  to analy+ radical scavenging ability of 0.5, 1, 2 or 4 mg/mL BBSLP was analysed according to the method described by Re et al. [•+) solution. The mixture was mixed and incubated at room temperature in the dark for 10 min. The optical density at 737 nm was measured using the microplate reader. The ABTS+ radical-scavenging ability was calculated as (Ablank or ABBSLP − ATrolox)/Ablank × 100. In this test, 1 mg/mL Trolox was used as a control.The ABTSe et al. . Use a 1v/v) heat-inactivated fetal bovine serum  was used as the culture medium. All cultured cells were incubated in an atmosphere of 5% CO2/95% air at 37 °C.RAW264.7 macrophages and mouse monocyte macrophages were purchased from the Bioresource Collection and Research Center . Dulbecco’s modified Eagle’s medium containing 42 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin and 10%  for another 24 h. LPS was used to induce inflammation [2O, and cells treated with sterilized H2O alone served as the control group. BBSLP (1 µg/mL) without LPS treatment for 48 h made up another control group.In this study, 1 × 10ammation . The indTo determine the optimum test concentration of BBSLP for use in this study, the cell viability of RAW264.7 macrophages was analysed according to the method of Denizot and Lang . After RThe effect of BBSLP on lipid peroxidation in RAW264.7 macrophages induced by LPS was determined according to the method of Fraga et al. . The lip3PO4) was mixed with an equal part of the cell culture medium of control or various experimental groups RAW 264.7 cells. In this test, the NO2 content was used as an indicator of NO content in RAW364.7 macrophages. The OD 550 nm was determined and calibrated using a standard curve of NaNO2 prepared in culture medium.To determine the inflammation level in RAW 264.7 cells, the Griess assay was used . Griess 2 levels in RAW264.7 macrophages were determined using an enzyme immunoassay (EIA) kit . The cell culture supernatants were collected after experimental treatment and centrifuged at 1000× g for 15 min to remove the particulate matter. The medium and PGE2 EIA conjugate was added to a 96-well plate pre-coated with goat anti-mouse IgG and left to react for 1 h, followed by a final wash to remove any unbound antibody-enzyme reagent. A substrate solution was added and the intensity of the color produced was measured at 412 nm. The concentration of PGE2 in each sample was calculated according to PGE2 standards.The PGEThe levels of IL-1β, IL-6 and TNF-α in RAW264.7 macrophages were analysed using rat IL-1β/IL-1F2 DuoSet ELISA , rat IL-6 DuoSet ELISA , and rat TNF-α DuoSet ELISA  kits , respectively, according to the manufacturer’s instructions. In brief, capture antibodies, cultured medium supernatants, detection antibodies, streptavidin-conjugated horseradish-peroxidase were processed on the plate in order, and the color subtract tetramethylbenzidine was used. The absorbance was measured and the concentration was calculated according to the standard.The iNOS, COX-2 and NF-κB signalling molecule expression was analysed using the method described by Hsieh et al. . At the Equal amounts (approximately 10–20 μg per sample) of cellular protein were separated by 10% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) , after wp-value less than 0.05 was used to indicate a statistically significant result.The SPSS Statistical Analysis Software for Windows, version 20.0  was used to analyse the experimental data in the present study. One-way analysis of variance (ANOVA) and Duncan’s or Tukey’s multiple-range test were used to evaluate the significance of differences between each mean value. A In conclusion, the presented data in this study demonstrate that BBSLP could reduce oxidative stress and pro-inflammation factors in LPS-induced RAW264.7 cells through the inhibition of the NF-κB signaling pathway, indicating that it may have potent antioxidant and anti-inflammatory capabilities, suggesting that BBSLP could be developed as a supplement material for functional foods.


IntroductionInterbody spacers are necessary for achieving disc height restoration when surgical intervention is used for the treatment of severe degenerative disc disease. Minimally invasive lateral lumbar interbody fusion (MIS LLIF) is a popular surgical approach that historically uses large static interbody spacers through a lateral approach. However, static spacers have been associated with iatrogenic distraction and excessive impaction forces, which may increase the risk of subsidence and loss of lordosis, compromising stability. Expandable interbody spacers with or without adjustable lordosis may help address these concerns by maximizing segmental lordosis and aiding in sagittal balance correction. This study describes the clinical and radiographic outcomes of patients treated with expandable interbody spacers with or without adjustable lordosis, for MIS LLIF.Materials and methodsThis is retrospective, single-surgeon Institutional Review Board-exempt chart review was of 103 consecutive patients who had undergone MIS LLIF at one to two contiguous level(s) utilizing expandable interbody spacers with or without adjustable lordosis (66/103 patients had adjustable lordosis spacers). Collection of clinical and radiographic functional outcomes occurred at preoperative and postoperative time points through 24 months.Resultsp<0.001). Oswestry Disability Index (ODI) scores significantly decreased by a mean of 67.4 ± 8.9 points at 24 months (p<0.001). Lumbar lordosis significantly improved by a mean of 3.1 ± 8.8° at 24 months (p=0.001). Anterior, middle, and posterior disc height significantly increased at 24 months by averages of 4.7 ± 3.1, 4.0 ± 3.0, and 2.1 ± 2.2mm, respectively (p<0.001). Neuroforaminal height had significantly increased at 24 months by a mean of 3.0 ± 3.6mm (p<0.001). Segmental lordosis significantly improved by 3.7 ± 2.9° at 24 months (p<0.001). There were 51 patients with abnormal preoperative Pelvic Incidence-Lumbar Lordosis (PI-LL) measurements that significantly improved by 9.1 ± 4.9° (p<0.001) and 52 patients with normal preoperative PI-LL measurements that improved by 0.2 ± 4.6° (p=0.748) at 24 months. One-hundred percent fusion occurred at all levels, and no findings of radiolucency were observed. One case of subsidence  was reported at 24 months. No implanted-related complications were reported, with 0% pseudoarthrosis and no secondary surgery required at the operative levels.One-hundred and three consecutive patients were evaluated-average age, 58.2 ± 12.1 years; 42.1% (45/107) were female. There were 78.6% (81/103) one-level cases and 21.4% (22/103) two-level cases for a total of 125 levels; 44.8% (56/125) were performed at L4-5 and 34.4% (43/125) at L3-4. The average estimated blood loss was 24.6 ± 12.3cc. Mean operative time was 61.0 ± 19.1 min, and mean fluoroscopic time was 28.2 ± 14.6 sec. Visual Analog Scale (VAS) back and leg pain scores decreased significantly by an average of 7.1 ± 1.0 points at 24 months (ConclusionIndirect decompression and sagittal correction were achieved and maintained through a 24-month follow-up. Functional clinical outcomes significantly improved based on decreased VAS pain and ODI scores at 24 months. This study resulted in positive clinical and radiographic outcomes for patients who underwent MIS LLIF with expandable interbody spacers with or without adjustable lordosis. A significant contributing factor for low back pain is the progressive irreversible condition known as degenerative lumbar disc disease (DDD), the leading cause of chronic back pain for the aging population. Life-altering disability may result from associated radiculopathy, spinal stenosis, myelopathy, degenerative spondylolisthesis, and herniation manifested from loss of disc height and lordosis . When coIn 2006, Ozgur first described LLIF as extreme lateral interbody fusion (XLIF) and since then, the lateral approach has gained popularity -7. AchiePromising clinical outcomes on the LLIF approach as a reliable approach to achieving the aforementioned surgical goals have been reported. LLIF permits the use of large spacers while preserving primary stabilizing structures that aid in stability and sagittal correction . Due to To achieve optimal decompression and sagittal correction, large static interbody spacers have traditionally been the standard of care for lumbar fusion . HoweverPatient populationThis is a retrospective, single-surgeon, Institutional Review Board (The University of Rochester Office for Human Subject Protection)-exempt evaluation of a prospectively collected cohort of 103 patients. The patient population was diagnosed with DDD at one or two contiguous levels from L1 to L5 with or without Grade 1 spondylolisthesis. Some were also diagnosed with spinal stenosis (30) or degenerative spondylolisthesis (3). All patients underwent an MIS LLIF surgery using a novel titanium expandable interbody spacer with  Figure  adjustabSurgical techniqueAfter administrating general anesthesia, patients were put in the lateral decubitus position and secured by adhesive tape to a radiolucent table. The break was positioned at the greater trochanter with the iliac crest above it. With fluoroscopic guidance, an oblique incision was made at the symptomatic disc segment allowing for direct visualization through subcutaneous tissue, external and internal oblique muscles, and transversus abdominis. A blunt dissection was performed and retroperitoneal fat was mobilized anteriorly; this exposed underlying psoas muscle, which was then palpated. After fluoroscopic confirmation of the level and location of the spinal marker, another blunt dissection was performed anteriorly; this was done to or at the very anterior part of the psoas muscle to the operative intervertebral disc level.Neuromonitoring stimulation was done in the initial step and whenever retractors were relocated. After the appropriate level was confirmed by fluoroscopy, a minimally invasive retractor was docked, dilated at the segment, and secured to the table-mounted arm. An annulotomy was next performed, which was followed by discectomy and endplate preparation. Using fluoroscopic imaging, sufficient endplate preparation was then completed and confirmed, allowing for trials to be inserted into the disc space for gradual distraction and determination of the appropriate spacer size for the patient. Once the appropriate expandable interbody spacer was selected and packed with local autograft, it was then implanted at the middle or slightly anterior middle of the disc space Figure . The spaPosterior decompression was performed for cases of severe spinal stenosis with neurological deficits, or in cases in which preoperative disc height had not increased by over 100% due to the LLIF procedure. Pedicle rods and screws were utilized for supplemental fixation. When the rods were properly positioned, locking caps were set. Intraoperative fluoroscopy images were taken of the final construct for confirmation and final approval, followed by cleaning and closure of the surgical incisions in the standard fashion. Antibiotic prophylaxis was administered to prevent infection.®-L) and lordotically actuated (RISE®-AL) spacers. RISE®-L and RISE®-AL spacers are expandable LLIF devices manufactured from titanium alloy, designed to maximize indirect decompression, provide a large graft space for optimal fusion, and minimize impaction forces. These devices are inserted at a contracted height and expanded up to 7mm in situ. The footprint options include 18 and 22mm widths, five lengths , 7-17mm height (height range dependent on lordosis), and in parallel or lordotic profiles. The height-actuated spacer is a fixed lordotic endplate of either 6° or 10°. The lordotically actuated spacer allows up to 3-15° of adjustable lordosis. The 18mm-wide implant was most commonly used in this study at 90%, versus the 22mm.The two lateral interbody spacers used in the current study were height-actuated  of antibiotics were administered at preoperative, intraoperative, and immediate postoperative time points.Outcome measuresFluoroscopy and operative times were recorded during LLIF procedures. Primary outcomes included self-assessment patient questionnaires such as Visual Analog Scale (VAS) for back and leg pain and Oswestry Disability Index (ODI), which were evaluated preoperatively and at six weeks, three, six, 12, and 24 months postoperatively.Radiographic assessments were also considered primary outcomes. Radiographic parameters such as disc height , segmental lordosis, neuroforaminal height, Pelvic Incidence-Lumbar Lordosis (PI-LL), and lumbar lordosis were evaluated at six weeks, three, six, 12, and 24 months. Radiographic lumbosacral parameters were measured on upright standing, lateral radiographs using imaging software  , radiolucency, ASD, and pseudoarthrosis were reported at 24-month follow-up for each patient. ASD was assessed clinically and in correlation with radiographic studies. Two independent observers assessed fusion with an agreed consensus using flexion/extension x-rays at two-year follow-up and cross-referenced by Fogel et al. for fusion grading following the Brantigan, Steffee, and Fraser (BSF) radiographic classification criteria . AccordiStatistical analysisIBM SPSS Statistics for Windows, Version 20.0  was used for statistical analysis. Frequency analyses and percentages were used for descriptive statistics. Paired sampled t-tests were used to calculate significant changes in ordinal and interval variables from preoperative to each postoperative follow-up time. Independent t-tests were used to determine significant differences between expandable groups, and between one- and two-level procedures. Statistical significance was found at p<0.05.Patient demographic and operative dataFrom August 2016 to November 2017, 103 consecutive patients underwent MIS LLIF, with 66/103 (64.1%) patients implanted with an expandable interbody spacer with adjustable lordosis (AL) and 37/103 (35.9%) patients implanted with an expandable interbody spacer without adjustable lordosis (L). There was an average age of 58.2 ± 12.1 years and 43.7% (45/103) were female  were one-level (1L) cases  and 21.4% (22/103) underwent a two-level (2L) MIS LLIF , for a total of 125 treated spinal fusion levels . Of the 125 levels, 44.8% (56/125) occurred at L4-L5, and 34.4% (43/125) occurred at L3-L4. Neuromonitoring did not show any nerve conduction abnormalities (lumbar plexus) or signal changes. Mean operative time was 54.4 ± 12.6 min for 1L fusions and 85.5 ± 19.2 min for 2L fusions. Mean fluoroscopic times were 27.4 ± 13.6 sec for 1L fusions and 30.9 ± 17.8 sec for 2L fusions. Length of hospital stay was 3.4 ± 1.9 days for 1L fusions and 4.4 ± 2.6 days for 2L fusions. Mean estimated blood loss for 1L fusions was 23.6 ± 13.2 cc and for 2L fusions 28.0 ± 7.7 cc , 62.9% (5.1 ±1.2), 72.2% (5.8 ± 1.3), 81.1% (6.5 ± 1.3), and 88.0% (7.1 ± 1.0) points, respectively (p<0.001) , 14.4% 5.8 ± 7.9°), 12.0% (4.9 ± 8.5°), 10.7% (4.3 ± 8.6°), and 7.6% (3.1 ± 8.8°), respectively (high standard deviation due to variance in each patient’s preoperative lordosis) (p<0.05). Mean segmental lordosis improved from baseline at six weeks and three, six, and 12 months, and sustained at 24 months by 103.5% (5.0 ± 2.9°), 95.1% (4.6 ± 3.1°), 89.9% (4.4 ± 3.1°), 80.7% (3.9 ± 3.1°), and 77.0% (3.8 ± 2.9°), respectively (p<0.001) . There was 100% fusion and no secondary surgical procedures were required at the index or adjacent levels reported. There were only one  case of subsidence and three cases of suspected ASD reported at two years postoperatively. ASD was observed as suspected due to radiographic assessments but was asymptomatic for patients. However, there were no revision surgeries through the 24-month follow-up.Expandable interbody spacers, with or without adjustable lordosis, have recently been adopted into the orthopedic practice with limited reported clinical data. With any new technology, clinical studies are necessary to determine its impact via post-market surveillance studies. Moreover, evaluation of novel insertion and expansion mechanisms (with various engineering designs) and their effects on clinical outcomes can be more thoroughly assessed through varying, objective, multi-source literature.DDD with loss of lordosis and disc height is the most common condition for debilitating back pain frequently associated with referred and radicular leg pain . The goaThe current study showed that the mean VAS pain scores decreased significantly from baseline at 24 months postoperatively by 88% (7.1 ± 1.0 points) and ODI scores significantly decreased by 87.7% (67.4 ± 8.9 points) (p<0.001). These observed clinical outcomes of the current study demonstrate that the use of expandable interbody spacers improves pain and disability scores by two to five times the minimal clinically important difference (MCID) at two-year follow-up .Advantages associated with MIS techniques include less blood loss, low infection rates, and decreased postoperative pain, compared to open approaches . These aIncreases in disc height, neuroforaminal height, lumbar and segmental lordosis were significantly achieved and maintained from preoperative to final follow-up. These significant radiographic improvements demonstrated the efficacy of expandable interbody spacers when used with a lateral lumbar interbody fusion, up to 24-month follow-up. The radiographic and clinical results of the current study compare favorably to findings in the expandable interbody spacer literature. A two-year follow-up study by Boktor et al. included 54 patients and 62 expandable TLIF interbody spacers between December 2013 to December 2015. Boktor et al. also reported low complication rates, short hospital stay, and significant restoration and maintenance of disc height, neuroforaminal height, and segmental lordosis [A systematic review by Uribe et al. reported a weighted average for segmental lordosis restoration of 3.9° in 23 studies, which compares satisfactorily to the average segmental improvement of 3.8° in the current study . PreviouThe current study showed minimal complications with implant subsidence observed in only one of the 125 operative levels (0.8%) and no cases of subsequent revision surgery were reported. Other studies have shown similar subsidence results. A systematic review on subsidence after LLIF conducted by Macki et al.  reportedLimitations of this study included its lack of a control group and being a single surgeon’s single-site experience. Nevertheless, it provides clinical evidence on the use of lateral expandable interbody spacers for lumbar interbody fusion, which is sparse in the literature. A minimum two-year follow-up is required to assess pseudoarthrosis, which was achieved in this study. This study provides clinical evidence for the sparse, existing literature for expandable lordotic interbody spacers with and without adjustable lordosis for MIS LLIF.Correction of segmental lordosis and disc height was achieved in a single-site patient cohort utilizing lateral expandable interbody spacers with and without adjustable lordosis. Significant positive improvements in segmental lordosis, disc height, and indirect decompression were observed at two years postoperative. There was 100% fusion with no revision surgeries reported. Functional outcomes also significantly improved. The use of expandable spacers resulted in postoperative improvements in pain and function at two-year follow-up with few complications.


Escherichia coli lipopolysaccharide. The cell viability, cell cycle, and cell proliferation were evaluated by Cell Counting Kit-8, flow cytometry, and cell scratch test, respectively. The mRNA transcriptions of prostaglandin-endoperoxide synthase 1 (PTGS1) and PTGS2, Toll-like receptor 4, and proliferation factors were detected using quantitative reverse-transcription polymerase chain reaction. The activations of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Wnt/β-catenin pathways were determined using western blot and immunofluorescence. As a result, co-treatment of meloxicam and lipopolysaccharide inhibited (P < 0.05) the cell cycle progression and reduced (P < 0.05) the cell healing rate and the mRNA level of proliferation factors as compared with the cells treated with lipopolysaccharide alone. Meloxicam decreased (P < 0.05) the lipopolysaccharide-induced PTGS2 gene expression. Neither lipopolysaccharide nor meloxicam changed PTGS1 mRNA abundance (P > 0.05). Meloxicam inhibited (P < 0.05) the lipopolysaccharide-activated Wnt/β-catenin pathway by reducing (P < 0.05) the protein levels of β-catenin, c-Myc, cyclin D1, and glycogen synthase kinase-3β and prevented the lipopolysaccharide-induced β-catenin from entering the nucleus. Meloxicam suppressed (P < 0.05) the phosphorylation of PI3K and AKT. In conclusion, meloxicam alone did not influence the cell cycle progression or the cell proliferation in BEEC but caused cell cycle arrest and inhibited cell proliferation in lipopolysaccharide-stimulated BEEC. This inhibitory effect of meloxicam was probably mediated by Wnt/β-catenin and PI3K/AKT pathways.Meloxicam is a non-steroidal anti-inflammatory drug and has been used to relieve pain and control inflammation in cows with metritis and endometritis. Meloxicam has been found to be effective in inhibiting tissue or cell growth when it is used as an anti-inflammatory therapy. However, the influence of meloxicam on bovine endometrial regeneration has not been reported. This study was to research the effect of meloxicam (0.5 and 5 μM) on the proliferation of primary bovine endometrial epithelial cells (BEECs) stimulated by  Escherichia coli precede infection by other common pathogenic bacteria via the production of lipopolysaccharide (LPS)  , 3.E. coli and in repairing the epithelium  in combination with antibiotics are used increasingly in the treatment of metritis and endometritis . StudiesPTGS1) and PTGS2, Toll-like receptor 4 (TLR4), and growth factors and the key proteins of Wnt/β-catenin and PI3K/AKT pathways were determined.The goal of this study was to reveal the influence and mechanism of MEL on BEEC proliferation. The BEEC was treated with LPS. The changes in the cell cycle, cell scratch test, the mRNA transcriptions of prostaglandin-endoperoxide synthase 1  and 50 U/ml penicillin/streptomycin, and inoculated into a 25-cm2 bottle. The cells were cultured in the incubator at 37°C and 5% CO2 saturation humidity. The medium was changed every 1–2 days. The primary cells could be obtained after 3–4 days.The cells were isolated as described from Dong et al. . BrieflyMEL  was dissolved in DMSO and stored at −20°C. It has been reported that the injection dose of 0.5 mg/kg MEL could maintain the blood concentration of 0.2 μg/ml MEL in cows with endometritis . In addi3 cells per well and grew to 80% fusion in a 5% CO2 incubator at 37°C. Then the cells were treated with 5 × 10−2, 5 × 10−1, 5, or 5 × 101 μM MEL or with 10 μg/ml LPS. CCK-8 was added into each well and incubated at 37°C for 2–4 h. The optical density was then read at 450 nm using a microplate .The Cell Counting Kit-8  was used to determine the effect of MEL and LPS on cell viability. The cells were seeded on 96-well plates with a density of 1 × 10BEEC was treated with MEL or LPS or LPS–MEL for 24 h. The cells were washed twice with cold PBS and fixed with 70% ethanol at 4°C for 12–24 h. Then the cells were washed twice with cold PBS and resuspended with RNase A and propidium iodide  in the dark at 37°C for 30 min. Cell cycle was detected by flow cytometry  and was analyzed by FlowJo software 7.6  to obtain the relative number of cells in each phase of the cell cycle.6 cells per well and were cultured overnight in a cell incubator with 5% CO2 at 37°C. The cell scratch test started when the cells reached 90% fusion. The cells were scratched by using the tip of a 200 μl sterilized pipette. This scratching line was perpendicular to the previous three lines and was along the diameter of the well. The cells were quickly washed with PBS at least three times until no cell was visible at the scratch microscopically. Then the DMEM/F-12 containing LPS or MEL was added to the well. The cell treatments were as follows: LPS, MEL (0.5 or 5 μM), or LPS–MEL (0.5 or 5 μM).Before cell culture, three parallel lines, each 0.5 cm apart, were drawn on the back of a six-well plate using a marker pen. The cells were then seeded on this plate with a density of 1 × 10The six-well plate was observed under the inverted microscope at ×100 magnification. Photos were taken immediately after the scratching (0 h) and at 3, 6, 12, and 24 h at fixed positions, which were localized with the help of the three parallel lines . The phoScratch width = scratch area/scratch lengthHealing rate = (scratch width at 0 h – scratch width at various time points)/scratch width at 0 h × 100%ΔCT−Δ method was used to calculate the relative abundances of mRNA transcripts (ACTB) was used as the internal control. The primers were designed according to the mRNA sequences of bovine ACTB, PTGS1, PTGS2, TLR4, VEGFA, cellular communication network factor 2 (CCN2), insulin-like growth factor 1 receptor (IGF1R), TGFB1, and TGFB3 published in GenBank. A single product was amplified by each primer pair. All the polymerase chain reaction (PCR) products were purified and sequenced , and the sequence results were analyzed using BLAST and compared to the GenBank database (http://blast.ncbi.nlm.nih.gov/blast.cgi). The primer sequences are shown in The cells were seeded into a six-well plate and were treated with LPS, MEL (0.5 and 5 μM), or LPS–MEL (0.5 and 5 μM) for 0, 3, 12, and 24 h. Total RNA was extracted using TRIzol reagent  according to the manufacturer's instruction. A Nanodrop 2000 spectrophotometer  was used to detect the quantity and purity of the RNA. The absorption ratio A260/A280) was determined to be between 1.8 and 2.1. The total RNA was reverse-transcribed into cDNA by a PrimeScript RT regent kit gDNA eraser . The cycle conditions were as follows: 95°C for 2 min; 95°C for 5 s and 60°C for 34 s, 40 times; 95°C for 15 s; 60°C for 5 s; 60–95°C, 0.5°C gradient heating. The reaction system included 10 μl SYBR Premix Ex Taq™ II , 1 μl of each primer, 1 μl cDNA template in a final volume of 20 μl per reaction. The 20/A280 waBEECs were treated with LPS, MEL (0.5 and 5 μM), or LPS–MEL (0.5 and 5 μM). The total protein was extracted and quantified by a bicinchoninic acid protein assay kit . The protein (20–30 μg) was separated by 10% SDS–polyacrylamide gels and was transferred to the polyvinylidene difluoride (PVDF) membrane . The PVDF membrane was incubated in 5% skimmed milk diluted by TBST (0.05% Tween-20 Tris-HCl buffer) to prevent its binding to non-specific protein. The primary antibody used for incubation included β-catenin, c-Myc, cyclin D1, glycogen synthase kinase-3β (GSK-3β), p-PI3K, PI3K, p-AKT, AKT, and β-actin at 4°C overnight. Then the PVDF membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody  for 1 h at room temperature. Proteins were detected using a chemiluminescence assay. The antigen–antibody complex was visualized using a HRP substrate  and the ChemiScope 5300 Pro CCD camera . The band intensity quantification was analyzed by Quantity One software . The primary antibodies of c-Myc (#5605), cyclin D1 (#2978), p-PI3K (#4228), PI3K (#4292), p-AKT (#4060), AKT (#4691), and β-actin (#4970) were purchased from Cell Signaling Technology. The β-catenin primary antibody (#ab32572) was purchased from Abcam (UK), and the GSK-3β primary antibody (#A2081) was purchased from ABclonal (China).The cells were seeded on a 24-well cell culture plate and were treated with LPS, MEL (0.5 μM), or LPS–MEL (0.5 and 5 μM) for 15 min. Then the cells were fixed with 4% paraformaldehyde for 15 min. After washing with PBS, the membrane was penetrated with 0.4% Triton X-100  for 15 min. The cells were washed with PBS three times and were blocked by 5% BSA for 1.5 h at room temperature. The cells were incubated with anti-β-catenin  at 4°C overnight. After PBS washings for three times, the cells were incubated with an FITC-conjugated secondary antibody  for 1.5 h at room temperature. The nuclei were stained with DAPI . Finally, the cells were visualized with a fluorescence microscope .P < 0.05 was considered statistically significant.The experiment was repeated at least three times. All the data were analyzed using SPSS 21.0 software . Statistically significant differences throughout this study were calculated by one-way ANOVA, followed by Dunnett's test. The data were shown as means ± standard error of means (SEM). A bilateral P > 0.05) by various concentrations  of MEL (P > 0.05) was observed in the cell viability between the cells treated with only LPS and the cells co-treated with LPS and MEL (0.5 and 5 μM).The CCK-8 method was used to detect the effect of MEL on BEEC viability. The viability of BEEC was not influenced  of MEL . As showP > 0.05) in cell migration at 24 h in cells treated with 0.5 or 5 μM MEL alone (P < 0.01) than that of the blank control group. Compared with the LPS group, the cell healing rate decreased (P < 0.01) in the LPS–MEL groups  on the number of cells in each cell cycle. After treatment with LPS for 24 h, the number of cells increased (P < 0.01) in the S phase and decreased (P < 0.01) in the G2 phase, indicating the cell cycle arrest in the S phase. After treatment with LPS and MEL for 24 h, the number of cells decreased (P < 0.01) in the G1 phase and increased (P < 0.01) in the S phase, suggesting the cell cycle arrest in the G0/G1 phase  were detected using quantitative reverse-transcription PCR. As shown in PTGS2 and TLR4 increased (P < 0.01) after LPS stimulation at 3, 12, and 24 h. Compared with the LPS group, the PTGS2 gene expression decreased (P < 0.01) in LPS–MEL groups. There was no difference (P > 0.05) in the TLR4 transcription between the LPS group and the LPS–MEL groups. Compared with the blank control group, the gene expression of most growth factors showed no change (P > 0.05) or a downregulation (P < 0.05) after LPS treatment at observed time points, except TGFB3 at 12 and 24 h (P < 0.05) than those in the LPS group at 3 and 12 h. Other than the decreased mRNA abundance of TGFB3 (P < 0.05) in the LPS–MEL groups as compared with the LPS group, we found no other differences (P > 0.05) at 24 h. The mRNA abundance of PTGS1 was not influenced (P > 0.05) by LPS or LPS–MEL treatment (The effects of MEL on the gene expressions of and 24 h . The genreatment .P < 0.01) at 15 and 45 min and decreased (P < 0.01) at 60, 75, and 90 min. The levels of c-Myc and cyclin D1 increased (P < 0.05) at 15 and 30 min. The level of GSK-3β increased (P < 0.05) at 15, 30, and 45 min. Based on these results, we selected 15 min to detect the changes in the Wnt/β-catenin pathway in subsequent experiments.The effects of MEL on the protein levels of β-catenin, c-Myc, cyclin D1, and GSK-3β were detected using western blot. As shown in P > 0.05) the protein levels of β-catenin, c-Myc, cyclin D1, or GSK-3β as compared with the blank control (P < 0.01) in the levels of β-catenin, c-Myc, cyclin D1, and GSK-3β (P < 0.01) in the LPS–MEL groups.MEL (0.5 and 5 μM) treatment alone did not influence ( control . LPS stid GSK-3β . CompareAs shown in P < 0.01) at 10 min, whereas the of p-AKT/AKT ratio increased (P < 0.05) at 5 and 10 min and decreased at 15 min. Based on these results, the time point of 10 min was selected for subsequent experiments.The influence of MEL on the PI3K/AKT pathway was detected using western blot and is depicted in P > 0.05) on the PI3K/AKT pathway (P < 0.01) in the LPS group. Compared with the LPS group, the phosphorylation levels of PI3K and AKT decreased (P < 0.01) in the LPS–MEL groups  showed no effect ( pathway . CompareL groups .PTGS1 and PTGS2 genes, respectively. In this study, MEL reduced the PTGS2 but not PTGS1 transcription in BEEC stimulated with LPS, implying its preferential inhibitory effect on COX-2 over COX-1. MEL inhibited PI3K/AKT and Wnt/β-catenin pathways and the downstream c-Myc, cyclin D1, and VEGF, resulting in the cell cycle block and the downregulation of cell proliferation in LPS-stimulated BEEC. This was similar to the report that the cell survival was inhibited by MEL treatment in LPS-challenged bovine mammary epithelial cells (The COX-1 and COX-2 were encoded by al cells . These rThe results showed that 0.5 and 5 μM MEL with or without 10 μg/ml LPS treatment had no effect on BEEC viability. By observing the cell scratch test, MEL alone had no effect on cell migration. This was consistent with the results of Goncalves et al. , showingPTGS2 transcription in BEEC, and many studies supported this result (TLR4 gene expression. However, MEL did not attenuate the LPS-induced TLR4 gene expression, which may be explained by its effect on the downstream NF-κB pathway (VEGFA, CCN2, IGF1R, TGFB1, and TGFB3 in LPS-stimulated BEEC to varying degrees. VEGF and CTGF were encoded by VEGFA and CCN2 genes, respectively. Quintana et al. found that MEL inhibited VEGF expression in ovarian hyperstimulation syndrome (VEGFA, CCN2, IGF1R, TGFB1, and TGFB3 and inhibited the proliferation and growth of BEEC.COX-2 is a pathological inducer, which catalyzes the synthesis of PG and participates in inflammatory reaction. Our results showed that MEL inhibited LPS-induced s result , 32. It s result . The recs result . Our res pathway . Growth  pathway , 35, 36.syndrome . Other ssyndrome . Tolfenasyndrome . These rTGFB1, suggesting another potential target of MEL in inhibiting the Wnt/β-catenin pathway.Wnt/β-catenin is involved in wound healing, uterine development, embryo implantation, and uterine involution , 41. CheIGF1R in the LPS–MEL groups may further inhibit the activation of the PI3K/AKT pathway. Further studies are required to clarify the mechanism.The PI3K/AKT pathway regulates various cellular processes such as cell proliferation and differentiation , 51. AKTVEGFA, CCN2, IGF1R, TGFB1, and TGFB3 in LPS-challenged BEEC. This inhibitory effect of MEL was possibly mediated by Wnt/β-catenin and PI3K/AKT pathways.In conclusion, MEL reduced the cell proliferation, blocked the cell cycle progression, and inhibited the gene expressions of The original contributions presented in the study are included in the article/LC: conceptualization and methodology. YQ: data curation, writing—original draft preparation, and software. HC: visualization, software, and validation. JD and JuL: investigation. HW and CQ: supervision. JiL: supervision, writing—reviewing, and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.