CCND expression, with a marked enrichment of acquired del(17p) in CCND2 over CCND1 tumors. Acquired CNAs were not influenced by high-dose melphalan or lenalidomide maintenance randomization. A branching evolution pattern was significantly associated with inferior overall survival  2.61, P = 0.0048). As an individual lesion, acquisition of gain(1q) at relapse was associated with shorter OS, independent of other risk markers or time of relapse . There is an increasing need for rational therapy sequencing in MM. Our data supports the value of repeat molecular profiling to characterize disease evolution and inform management of MM relapse.Structural chromosomal changes including copy number aberrations (CNAs) are a major feature of multiple myeloma (MM), however their evolution in context of modern biological therapy is not well characterized. To investigate acquisition of CNAs and their prognostic relevance in context of first-line therapy, we profiled tumor diagnosis–relapse pairs from 178 NCRI Myeloma XI (ISRCTN49407852) trial patients using digital multiplex ligation-dependent probe amplification. CNA profiles acquired at relapse differed substantially between MM subtypes: hyperdiploid (HRD) tumors evolved predominantly in branching pattern vs. linear pattern in t vs. stable pattern in t. CNA acquisition also differed between subtypes based on  WHSC1/MMSET/NSD2, FGFR3, CCND1, MAF, and MAFB) through juxtaposition to the immunoglobulin heavy-chain locus , 2019, 2P value of ≤0.05 was considered statistically significant.Analyses were performed in R version 3.5.2 using “dplyr”, “tidyr”, “stats”, “survival” and “survminer”, “ComplexHeatmap” and “ggpubr” packages. The association between categorical variables was examined using Fishers exact test and between continuous variables using the Wilcoxon signed-rank test. Progression-free survival (PFS) was defined as time from induction randomization to progression, according to International Myeloma Working Group criteria, or death of any cause. Overall survival (OS) was time from induction randomization to death of any cause. Cox proportional hazards (PH) regression was used to estimate univariate and multivariable hazard ratios (HRs) and 95% confidence intervals (CI). To examine the predictive value of evolution in CNA status for subsequent therapy, high-risk CNA were considered as time dependent covariates within the multivariate model. The PH assumption was tested to investigate whether time dependent covariate effects persisted irrespective of time of acquisition . MultivaP = 0.0058). As previously well-documented [Relapse was associated with the acquisition of new CNAs in 87.1% of tumors, most (73.2%) being large-scale chromosome changes. Across all patients, relapse was associated with a higher number of CNAs: median 11.5 (range 0–34) vs. median 12 (range 0–29)  , del(13q) (10%), gain or amplification of 11q (9%) and del(17p)/8%) Fig. . Althoug8%) Fig. .Fig. 1ReCDKN2C or 1p12 implicating FAM46C . In nine of 22 tumors with 1p12 (FAM46C) deletion at diagnosis, the deletion was not clonally detectable at relapse; in contrast all but one of the 8 presentation 1p32.3 (CDKN2C) deletions were detectable at relapse.In contrast to 1q gain, which generally involved the whole chromosomal arm, clonal 1p deletions tended to be sub-chromosomal  tumors. In three of 15 tumors with MYC gain at presentation, reversion to diploid status was a feature of relapse.Focal gain at relapse also affected 8q24.21, involving the CRBN (3p26.2), IKZF1 (7p12.2), IKZF3 (17q12), and IRF4 (6p25.3)—were infrequent .Sub-chromosomal CNAs involving IMiD response genes—CRBN p26.2, IKMYC at diagnosis progressed to amplification at relapse. This was also a feature with gain of 15q (4.4%) and 19p (2.3%), albeit at low frequency.We next examined for clonal emergence at relapse of sub-clonal changes at diagnosis, focusing on the most frequent drivers—gain(1q) and del(17p). We and others showed before that calling of CNAs using conservative high confidence MLPA cutoff values detects clonal infiltration equivalent to about >20% by FISH, but that calling of minor sub clones is also feasible . Thirty We and others have demonstrated that patients with double or triple hit tumors , t, t, gain(1q), or del(17p)) have an especially poor prognosis [;14, t, t, t/t14;20)), and those with HRD, sub-grouped into those with and without gain of 11, in analogy to Translocation and Cyclin D (TC) classification (Supplementary Table 0), and t;14, t, linear (22.5%), linear loss (19.1%), and stable (12.9%) Fig. . In HRD Both del13) (94.7%) and gain/amp (1q) 68.4%) were seen at high frequency in t MM at presentation, and the CNAs were seen at even higher frequencies in relapse tumors at 100% and 79.0%, respectively  8.4% wereCCND expression; CCND1 (D1), CCND2 (D2), or both (D1 + D2) , del(1p)/CDKN2C , gain(8q)/MYC , and del(17p)  considered as time dependent variables, i.e., including their acquisition at relapse, were all independently associated with shorter OS (Table Gain1q) . Median OS was 44.3 vs. 47.9 vs. 67.1 months for gain(1q) at diagnosis, new gain(1q) at relapse and normal 1q copy number at both time points, respectively (log-rank P = 0.007)  at relapse. Gain(1q) from presentation and evolution of new gain(1q) at relapse were both associated with significantly shorter OS compared to normal 1q copy number  Fig. .Fig. 5ReCDKN2C) or gain(8q/MYC) at relapse were not clearly associated with a significantly worse OS in subgroup analyses, although our power to demonstrate a relationship was naturally limited by low frequency of these CNAs. Median OS was 31.3 vs. 60.5 vs. 65.4 months for del(17p) at diagnosis, emergence at relapse and absence at both, respectively (log-rank P < 0.001)  at diagnosis, emergence at relapse or absence, respectively (log-rank P = 0.03) , 38.3 vs. 36.2 vs. 63.4 months, respectively (log-rank P = 0.07) , del(1p/P = 0.0048), with median OS being 44.6 vs. 59.6 vs. 62.0 vs. 68.1 months for branching, linear, no change and linear loss respectively (log-rank P = 0.02) , in line with its driver role in B-cell malignancies [Our analysis demonstrates that progression of MM is characterized by emergence of clones with additional large-scale chromosomal changes, commonly involving 1q. Gain or amp(1q) frequently co-evolves with other CNAs, either implicating 1q directly in their genesis as per “jumping 1q” hypothesis, or highlighting 1q as a region providing clonal advantage for genetically instable tumors . While agnancies , 29.CCND2 and CCND1 driven tumors [CCND2 driven tumors which has, to our knowledge, not been described before. These differences are features of subtypes irrespective of therapy. Our findings are in keeping with published observations for evolution of pre-therapeutic mutational patterns but extend these in context of therapy and relapse [By considering MM molecular subtype, we highlight differences in evolution trajectories, particularly between HRD and IG translocated MM, but also between t and t and/or between n tumors . Of note relapse –32. We aWe identified only a low frequency of CNAs to which IMiD response genes map. In conjuncture with published somatic SNV data, results suggest mutation of these genes per se is not the major determinant of acquired resistance to lenalidomide, or other members of this class of agent in MM , 33–35.CDKN2C) and gain(8q/MYC), our power to robustly assert clinical relevance of acquired lesions at relapse is inevitably limited by the lower frequency of these CNAs [We demonstrate, to our knowledge for the first time, in a randomized controlled trial that acquisition of gain(1q) at relapse is independently and strongly associated with inferior OS . Althougese CNAs . Clinicaese CNAs –40.It is unknown whether sub-clones residing outside of the standard iliac crest bone marrow sampling area at diagnosis are the main source of clonal evolution detected at relapse or whether these predominantly emerge through ongoing genomic instability . Multi-rAll patients received uniform trial treatment, in particular providing insight into thalidomide and lenalidomide associated CNA changes. However, patients also received low-doses of the oral alkylator cyclophosphamide during induction, which may have specific impact on CNA changes, potentially limiting generalizability of our findings.In addition, molecular information generated with a targeted tool like digitalMLPA is inherently focused and does not discriminate complex processes potentially underlying identified CNAs, such as chromotripsis or chromoplexy, which are detectable by discovery tools such as whole genome sequencing, or single nucleotide variants captured by myeloma specific CNA/sequencing panels , 45–47. Genetic re-profiling at relapse in MM is currently not widely recommended and, as a consequence, often not reimbursed , 39, 49.Our findings strongly support repeated tumor molecular analysis in MM in context of modern treatment, even in circumstances where only selected markers such as 1q can be tested, as a means of tailoring patient treatment beyond first-line therapy.Supplementary MethodsSupplementary TablesSupplementary Figures


We found that FBXO22 expression was significantly increased in epithelial ovarian cancers tissues and was closely correlated with clinical pathological factors. As a result, we found that FBXO22 promoted the growth and metastasis, as well as inhibited the autophagy flux. In addition, we identified that FBXO22 performed these functions via the MAPK/ERK pathway. Our results first reported the function of FBXO22 in epithelial ovarian cancer and the correlation between FBXO22 and autophagy, suggesting FBXO22 as a novel target of epithelial ovarian cancers assessment and treatment.E3 ubiquitin ligase F-box only protein 22 (FBXO22), which targets the key regulators of cellular activities for ubiquitylation and degradation, plays an important role in tumorigenesis and metastasis. However, the function of FBXO22 in epithelial ovarian cancers has not been reported. This study aims to explore the biological function of FBXO22 in epithelial ovarian cancers progression and metastasis and its specific regulation mechanism. Immunohistochemistry analysis of tissue microarray was performed to evaluate the expression of FBXO22 in epithelial ovarian cancers patients. The proliferative ability of epithelial ovarian cancers cells was examined by the CCK8. The metastasis ability was detected by the wound healing assay, migration and invasion assays. Western blot was used to verify the relationship between FBXO22 expression and mitogen-activated protein kinase related proteins. Autophagic flux was detected by electron microscopy, mRFP-GFP-LC3 adenovirus, lysosomal tracker and western blot. For  Ovarian cancer is the seventh most common cancer among women, and the age-standardized rates in developed and developing countries are approximately 9.4 and 5.0 per 100,000 people, respectively . DespiteEarly diagnosis increases the chance of recovery, but due to the nonspecific symptoms of the disease in the early stage, recovery is hindered. Advanced ovarian cancer is aggressive, has rapid growth and spread and chemotherapy/radiotherapy resistance, and recurs. At present, cytoreductive surgery is clinically adopted, along with chemotherapy using cisplatin and paclitaxel, but the multidrug resistance of ovarian cancer cells has severely reduced the long-term efficacy of these treatments; the 5-year survival rate is only 30% . TherefoAutophagy is a lysosome-dependent degradation pathway that widely occurs in eukaryotic cells. Under normal physiological conditions, autophagy facilitates the maintenance of cell homeostasis and promotes cell survival; however, excessive autophagy can cause cell death through a process called “autophagic cell death,” along with apoptosis and necrosis . This “dThe mitogen-activated protein kinase (MAPK) family, which includes ERK2⁄ERK1 , p38 MAPK, ERK5, and c-Jun-NH2-terminal kinases (JNK1⁄2⁄3), plays a crucial role in nearly all cell functions . DependiFBXO22 is a member of the F-box protein family . The SCFin vitro and in vivo. Furthermore, we explore the mechanism and prospected the potential and profound significance of FBXO22 in epithelial ovarian cancers assessment and treatment.In this study, we demonstrated the expression of FBXO22 in patients. We investigated the biological functions of FBXO22 A tissue microarray (TMA) containing 251 EOCs tissues and 10 matched normal tissues were constructed by Jiangsu Cancer Biotherapy Institute . The tissues, which were embedded in paraffin blocks, were collected from the Pathology Department of Affiliated Hospital of Xuzhou Medical University . All the patients underwent definitive diagnosis of epithelial ovarian cancers and then radical surgery at the above hospital. Detailed clinical information of each specimen was recorded accurately and completely. All the tissue specimens were obtained from the patient, all of whom provided informed consent, and the use of human specimens was approved by the Review Board of the Affiliated Hospital of Xuzhou Medical College.2O2 for 30 min. Antigen retrieval was performed by heating the TMA slides immersed in a retrieval solution  at 95°C for 30 min in a pressure boiler. After 1 h of blocking with 5% normal goat serum, the sections were incubated with polyclonal rabbit anti-FBXO22 antibody  overnight at 4 °C. The slides were immersed in a response enhancer for 20 min and biotin-labeled secondary antibody  for 20 min at room temperature and then with avidin–peroxidase reagent and 3,3′-diaminobenzidine  substrate. After hematoxylin counterstaining and dehydration, the sections were sealed with cover slips.TMA immunohistochemistry was implemented according to the streptavidin–peroxidase (Sp) method. A standard PV-9001 kit  was used. Before immunostaining, TMA slides were dewaxed at 60°C for 2 h, then deparaffinized with xylene and hydrated with graded ethanol and distilled water. Endogenous peroxidases were inhibited with 3% HTwo pathologists separately examined the TMAs under blinded experimental conditions. The staining scores of FBXO22 were evaluated according to the intensity and percentage of cells with positive staining. The staining intensity of FBXO22 was scored 0, 1, 2, or 3 ; the percentage of the FBXO22- positive stained cells was graded as 0 (<5%), 1 (5–25%), 2 (25–50%), 3 (50–75%), or 4 (75–100%). The immunoreactive score (IRS) of each section was calculated by multiplying the scores of staining intensity and the percentage of positive cells. On the basis of the IRS, staining patterns were divided into two classes: negative (IRS: 0–6) and positive (IRS: 7–12) expression.2.Human epithelial ovarian cancers cell lines  and human ovarian epithelium cells (IOSE) were obtained from the Type Culture Collection of the Chinese Academy of Sciences . The OVCAR3 cells were cultured in an RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS) and 0.1 mg/ml insulin. The HO8910, A2780, and IOSE cell lines were cultured in an RPMI 1640 medium supplemented with 10% FBS. Then, 100 U/ml streptomycin/penicillin was added to the RPMI 1640 medium. All the cells were cultured in an incubator at 37°C with 5% COCells were seeded on coverslips, fixed in 4% paraformaldehyde at room temperature for 15 min, rinsed in PBS for three times, permeabilized in 0.3% Triton X-100 for 15 min, rinsed in PBS for three times, and blocked in 2% bovine serum albumin at room temperature for 1 h. Then, coverslips were incubated with primary antibodies 1:100 diluted in staining buffer  overnight at 4°C in a humid chamber. After washing three times, secondary antibodies  were applied in a 1:200 dilution in staining buffer for 1 h at 37°C in a humid chamber in the dark. After washing, coverslips were mounted with Vectorshield with 4′,6-diamidino-2-phenylindole . Fluorescent signals were captured with a confocal laser scanning microscope .NC: sense, 5′-UUCUCCGAACGUGUCACGUTT-3′;antisense, 5′-ACGUGACACGUUCGGAGATT-3′.siFBXO22#1: sense, 5′-GGUGGGAGCCAGUAAUUAUTT-3′;antisense, 5′-AUAAUUACUGGCUCCCACCTT-3′.siFBXO22#2: sense, 5′-GUUCGCAUCUUACCACAUATT-3′;antisense, 5′-UAUGUGGUAAGAUGCGAACTT-3′.siFBXO22#3: sense, 5′-GCACCUUCGUGUUGAGUAATT-3′;antisense, 5′-UUACUCAACACGAAGGUGCTT-3′.siATG5: sense, 5′-CAGUUUGGCACAAUCAAUATT -3′;antisense, 5′-UAUUGAUUGUGCCAAACUGTT-3′.Small interfering RNA (siRNA) specific for FBXO2 , autophagy related 5 , and nonspecific control (NC) were purchased from Gene-Pharma  and transfected with siLentFect lipid reagent  according to the manufacturer’s protocol when the epithelial ovarian cancers cells grew to 30–40% confluency. Approximately 6 h after transfection, the medium containing transfection reagents was replaced with a fresh medium. The siRNA sequences were as follows:Overexpression plasmid (HA-FBXO22) and nonspecific control (pcDNA3.1-Ctrl) were purchased from You Bio  and transfected with Lipo2000  according to the manufacturer’s protocol when the epithelial ovarian cancers cells grew to 80–90% confluency. Approximately 6 h after transfection, the medium containing transfection reagents was replaced with a fresh medium.shFBXO22-LV3, Ctrl-LV3, Flag-FBXO22-LV5, and Ctrl-LV5 lentivirus (Gene-Pharma) were used for the stable suppression and overexpression of FBXO22. After 48 h of infection on HO8910 cells with the lentivirus, stable cells were extracted using 2 μg/ml puromycin for 14 days.This procedure was completed using the following primary antibodies raised against: FBXO22, GAPDH, P62, BECN1 and ATG5 (Proteintech Group); MMP-2, ERK, p-ERK, P38, p-P38, p-90RSK, RAC1 and BCL-2 , and LC3 I/II (Abcam). The secondary antibodies were goat anti-rabbit and goat anti-mouse corresponding HRP . The protein bands were determined using a Tanon 5200 automated chemiluminescent imaging analysis system with ECL reagents .3 cells were seeded in a 96-well culture plate and then incubated at 37°C in a humidified atmosphere containing 5% CO2 for 24 h. Then, 10 μl of CCK-8 solution was added 24, 48, 72, and 96 h after plating, and the cells were incubated at 37°C for another 1 h. Absorbance was determined at 450 nm.After knock down or overexpression of FBXO22 in HO8910 and OVCAR3 cells, cell proliferation was monitored by the cell counting kit-8 . After knockdown or overexpression of FBXO22 in HO8910 and OVCAR3 cells, 5 × 10In the wound healing assays, cells were planted in six-well plates and cultured to 80% confluence. Then, a sterile 10 μl pipette tip was used in making artificial scratches in each well. Suspended cells were washed away with PBS, then the cells were cultured in a medium with 1% FBS. Cell migration distance was photographed at 0 and 24 h under an inverted light microscope.5 cells were seeded into the upper chamber for the migration assay kit. In the same chamber, 4 × 105 cells were seeded into a medium without serum for the invasion assay. The lower chamber was filled with RPMI 1640 supplemented with 20% FBS. After 24 and 48 h of incubation at 37°C in 5% CO2, the cells were fixed in the membrane with methanol (90%) and then stained with crystal violet. The cells in the upper chamber were removed gently. Afterward, the traversed cells were dried and counted.Assays on cell migration and invasion were performed using Transwell filter inserts (8.0 μm pore size with polycarbonate membranes) precoated with Matrigel  and those that were not. The cells underwent serum starvation overnight, and 2 × 107) were pre-fixed in 2.5% glutaraldehyde and 1% osmium tetroxide and then incubated with 1% OsO4 for 3 h at 4°C, dehydrated in a graded series of ethanol, and flat-embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate and observed under a transmission electron microscope (Tecnai G2 Spirit Twin/*Tecnai G2 Spirit Twin).shFBXO22–LV3, Ctrl–LV3, Flag–FBXO22–LV5, and Ctrl–LV5 cells  were infected for 24 h. Fluorescent signals were captured with a confocal laser scanning microscope . The number of autolysosomes and autophagosomes was determined by counting the red puncta or yellow puncta, respectively. Thirty randomly selected cells per experimental group were analyzed.2 for 1 h. Fluorescent signals were captured with a confocal laser scanning microscope .A lysosomal tracker  was diluted with HBSS  and then incubated at 37 °C under a humidified atmosphere containing 5% CO6. Five weeks after injection, the mice were sacrificed, and their subcutaneous tumors were excised and fixed in 10% buffered formalin for the detection of FBXO22 and Ki67expression levels through IHC analysis.Eight six-week-old BALB/c nude mice were used. Stable HO8910 cell lines constructed with Ctrl-LV3 and shFBXO22-LV3 were injected into the left and right backs successively with the number of 5 × 106) respectively. Fourty days after injection, the mice were sacrificed, and their organs including colon, lung, kidney, liver, spleen and heart were excised and made into paraffin sections for analysis of metastasis by HE staining.Sixteen six-week-old BALB/c nude mice were divided into two groups and injected i.p., with Ctrl-LV3 and shFBXO22-LV3 cells . Differences between two groups were analyzed with Student’s t test, whereas differences among more than two groups were evaluated by one-way ANOVA for independent samples or ANOVA for repeated measurements followed by Tukey post-hoc test. In all cases, To evaluate whether the FBXO22 protein expression is related to EOCs, we performed immunohistochemistry experiments across an entire TMA containing 251 EOCs tissues and 10 matched normal tissues. The resulting FBXO22 staining intensity was quantified using IRS. Samples with IRS in the range of 0–6 or 7–12 were classified as negative or positive FBXO22 expression, respectively . The stap < 0.001) and pathological grades (p = 0.015) (p = 0.662). Analysis of the epithelial ovarian cancer patients in TCGA shown that there was no significant difference in survival (p = 0.203). These results shown that EOCs patients have higher expression of FBXO22 and the expression of FBXO22 is associated with clinical pathological factors.Moreover, the FBXO22 expression was positively correlated with clinical FIGO stages (= 0.015) ; Table 1Consistently, increased FBXO22 expression was also detected in three EOC cell lines  as compared with normal ovarian epithelial cell line IOSE . MoreoveTo investigate the biological function of FBXO22 in epithelial ovarian cancers cell proliferation, we transfected HO8910 and OVCAR3 cells with negative control siRNA (NC) and siRNAs targeting FBXO22  successively . Then, Cin vivo. The cells were subcutaneously injected into BALB/c nude mice. The mice were sacrificed 5 weeks after the implementation, and the tumors were exposed  and shFBXO22-LV3 (sh-FBXO22) stable cell lines to verify the function of FBXO22 in the regulation of epithelial ovarian cancers cell  exposed . Differe exposed . The res cancers . The resThe effect of FBXO22 on migration and invasion of EOCs were also explored. Knockdown of FBXO22 significantly inhibited the migration ability of HO8910 and OVCAR3 cells when compared with control cells , as evid6 HO8910 NC/sh-FBXO22 cells were injected to 7-week-old nude mice, and the peritoneal metastasis status was analyzed 8 weeks later. Compared with the NC group, sh-FBXO22 cells formed fewer metastatic foci in colon, lung, kidney, liver and spleen , as important enzymes that degrade the extracellular matrix, play an important role in mediating tumor angiogenesis, metastasis, and invasion. MMP-2 is an important MMP . We deteMAPKs constitute a group of serine threonine protein kinases that can be activated by a series of extracellular stimulus signals and mediate signal transduction from the cell membrane to the nucleus. They regulate many physiological activities, such as inflammation, apoptosis, carcinogenesis, invasion, and metastasis, particularly those of tumor cells. ERK1/2 and P38 are the two important members of MAPK . We deteSignificantly, TRAF6 and BCL-2, which were proved correlated with FBXO22 in former study, reported to regulate autophagy . Thus, wThen, stable cell lines were established . AutophaChloroquine (CQ), an inhibitor of LC3B degradation in autolysosomes, was used to verify autophagy flux. Autophagy flux increased after FBXO22 knockout but decreased after FBXO22 overexpression, and was negatively correlated with p-ERK  lane 7,8To validate the essential functions of p-ERK, ERK1/2 inhibitor 1, a potent orally bioavailable ERK1/2 inhibitor, was used. The results shown that the level of p-ERK was decreased by the ERK inhibitor, which enhanced autophagy flux in HO8910 cells with FBXO22 knockdown or overexpression . MoreoveIn conclusion, FBXO22 inhibits the autophagy of EOCs, and the inhibition function is p-ERK dependent.in vivo and in vitro, showing that FBXO22 is a potential target for the clinical diagnosis and treatment of epithelial ovarian cancers.There is tremendous amount of research and focus on EOC as it presents in advanced stages and is the most fatal of the gynecologic malignancies. Since EOC does not have symptoms specific to cancer, there are no early screening and detection modalities . Thus, aE3 ligase is the core component of the ubiquitination cascade. It controls the specificity of a substrate and directly binds to the substrate. Hundreds of E3 ubiquitin ligases are found in humans . Among t+/− mice  shown that spontaneous tumors occur in BECN1+/− mice .Some core proteins engage in crosstalk between apoptosis and autophagy, including BCL-2 family members. Anti-apoptotic BCL-2 members inhibit autophagy by binding to BECN1, which contains a functional BH3 domain that inserts into the hydrophobic groove of anti-apoptotic BCL-2 members to regulate autophagy . Post-trEpithelial ovarian cancer is recognized as a heterogeneous disease and is classified according to histologic subtype: high-grade serous, low-grade serous, clear cell, endometrioid, and mucinous . The mitIn summary, our study is the first to report that FBXO22 promotes growth and metastasis and inhibits autophagy in epithelial ovarian cancers, and these functions depend on the MAPK/ERK pathway. These findings suggest FBXO22 as a novel target of epithelial ovarian cancers assessment and treatment.


Here we use a soil–vegetation gradient in an Australian sub-alpine ecosystem to examine links between composition of soil microbial communities, and the fluxes of greenhouse gases they regulate. For each soil/vegetation type , we measured carbon dioxide (CO2) and CH4 fluxes and their production/consumption at 5 cm intervals to a depth of 30 cm. All soils were sources of CO2, ranging from 49 to 93 mg CO2 m−2 h−1. Forest soils were strong net sinks for CH4, at rates of up to −413 µg CH4 m−2 h−1. Grassland soils varied, with some soils acting as sources and some as sinks, but overall averaged −97 µg CH4 m−2 h−1. Bog soils were net sources of CH4 (+340 µg CH4 m−2 h−1). Methanotrophs were dominated by USCα in forest and grassland soils, and Candidatus Methylomirabilis in the bog soils. Methylocystis were also detected at relatively low abundance in all soils. Our study suggests that there is a disproportionately large contribution of these ecosystems to the global soil CH4 sink, which highlights our dependence on soil ecosystem services in remote locations driven by unique populations of soil microbes. It is paramount to explore and understand these remote, hard-to-reach ecosystems to better understand biogeochemical cycles that underpin global sustainability.Soil-to-atmosphere methane (CH In some cases, soils can switch from being a net source to a net sink for CH4 in a matter of minutes to hours [Counteracting biogeochemical processes that consume or produce greenhouse gases (GHGs) regulates whether soils act as net sources or sinks. The magnitude and spatial distributions of these competing processes—whether across landscapes, with soil depth ,2,3, or sumption . On the to hours ,9,10. Sito hours ,12,13.2, and is responsible for ~17% of anthropogenic warming [4 are controlled by abundance and composition of specific microbial communities [4 to microbial biomass and CO2 is, for example, restricted to distinct groups of specialized methanotrophic microorganisms. Aerobic methanotrophs belonging to the α-proteobacteria and γ-proteobacteria groups have been studied for decades and detected across a wide range of habitats [Candidatus Methylomirabilis oxyfera, from the NC10 phylum [4-consuming bacteria whose activity has been known for decades but had evaded efforts to be isolated or characterized until recently [4 oxidation is catalyzed by methane monooxygenase (MMO) enzymes. The pmoA gene encoding a subunit of the membrane-bound MMO (pMMO) enzyme is present in most aerobic methanotrophs and is frequently used as a genetic marker.Methane is a potent greenhouse gas, 34 times as potent as CO warming . Soil somunities ,16, but munities ,19. Oxidhabitats . Groundbhabitats  and intr0 phylum . In addirecently ,24,25. T4 either from CO2 and H2 (hydrogenotrophic methanogenesis), acetate (acetoclastic methanogenesis), formate, or methylated one-carbon (C1) compounds [mcrA gene, which is used as a genetic marker for methanogens in the environment. A few recent studies have shown that even water-logged soil profiles tend to have an aerobic upper layer that, via its capacity for CH4 oxidation, helps mitigate CH4 release to the atmosphere from lower soil depths. In some cases, this layer can remove 80–97% of CH4 produced at lower depths [Methanogenesis in soil typically requires anaerobic and low redox conditions, with depletion of strong electronegative terminal electron acceptors, e.g., nitrate and iron. Methanogens belong to the Euryarchaeota phylum, and produce CHompounds . All knor depths ,28,29. I2 and CH4 fluxes. These soils are of particular interest given their previously observed, rapid rates of CH4 oxidation in forest and grassland soils (unpublished data) and likely biogeochemical sensitivity to climate change. This endemic, mosaic ecosystem is particularly prone to wildfires in summer, while being snow covered in winter [4 sink strength of these soils. Our objective was to determine the role of soil microbial communities in regulating the production/consumption of CH4 across this unique soil–vegetation gradient.Local soil and vegetation gradients provide an opportunity to explore mechanisms that regulate soil GHG fluxes ,31,32. Sn winter ,34. ClimOur study sites were located in an area known locally as the Snowy Plains, within the Snowy Mountains region of southern New South Wales. The Snowy Plains form part of the Australian Alps montane grasslands and lie adjacent to the Kosciuszko National Park . Grasslands are dominated mostly by Poa hiemata and Poa costiniana. Forests are dominated by Snow Gums , with the N-fixing shrub Bossiaea foliosa, and some Poa spp. as understory and ground layers.The area is a mosaic ecosystem containing a mixture of bog, grassland, and forest and has been well described elsewhere . Alpine 2 and CH4) sampling (4 production and uptake peaked (based on prior gas measurements).We used four topographic transects  to guide our soil and GHG . At each depth, a subsample (~3–5 g of soil) was immediately placed into a 2 mL Eppendorf tube, then placed in liquid nitrogen and stored at −80 °C until DNA was extracted. The remainder of the soil core was placed in a protective PVC sleeve, and then stored in an iced cooler until reaching the laboratory.Immediately after completing in situ gas sampling, the chambers were removed, and a 5-cm-diameter, 30-cm-deep soil core was taken directly in the center of the collar. These cores were used for determining ancillary soil properties, microbial community analyses, and to measure net CO2 and CH4 production and consumption occurring at each depth . Dissolved organic C and N were analyzed on the same extracts on a Shimadzu TOC-N Analyzer . Electrical conductivity and pH were measured on SevenMulti probe  with a 1:1 (w:w) ratio with de-ionized water. Heavy elements were analyzed by X-ray fluorescence using a Niton XL3t Ultra Analyzer .After incubation, soils from the six 5-cm subsections were sieved (2 mm) and rocks and roots were removed and weighed. A subsample of each soil was ground for total C and N analysis on a TruSpec Elemental Analyzer . Soil texture was analyzed by using the hydrometer method . Soil in® Soil kit , which disrupts microbial cells by bead beating (30 s at 5.5 m s−1). DNA purity and quantity were determined using a NanoDrop® Spectrophotometer ND-1000 . DNA at a concentration of 10 ng μL−1 was stored at −20 °C for further molecular analysis. The abundances of bacterial and archaeal 16S rRNA genes as well as pmoA genes were quantified using an iCycler Instrument . For all assays, standards containing known numbers of DNA copies of the target gene were used. qPCR conditions for archaeal and bacterial 16S rRNA genes were based on dual-labeled probes. For bacterial 16S rRNA genes, primers Bac338F and Bac805R, and probe Bac516P were used [2  and 12.5 µL of JumpStart Ready Mix (Sigma-Aldrich) in a final volume of 25 µL. The volume of 1 µL of UltraPure non-acetylated BSA  was added to archaeal 16S rRNA gene reactions. The program used for both assays included an initial denaturation of 94 °C for 5 min, followed by 35 cycles of 95 °C for 30 s and 62 °C for 60 s extension and signal reading [pmoA genes used a SYBR Green method with primers A189F/mb661R [2 (Sigma-Aldrich), 0.25 µL of FITC , 12.5 µL of SYBR Green JumpStart Taq Ready Mix (Sigma-Aldrich) and 0.6 µL of UltraPure non-acetylated BSA  in a final volume of 25 µL. The qPCR program included an initial denaturation of 94 °C for 6 min, followed by 45 cycles of 94 °C for 25 s, 65.5 °C for 20 s, 72 °C for 35 s, plus 72 °C for 10 s for the plate read. A final melting curve was included as follows: 100 cycles of 75–94.8 °C for 6 s, +0.2 °C cycle−1 [pmoA genes were obtained, all with R2 values > 0.99. Technical duplicates were performed for each of the samples.DNA was extracted using the NucleoSpinere used . For arcere used . Conditi reading . The qPCF/mb661R . Each qP cycle−1 . PurifiepmoA genes. PCR primers 515F and 806R targeting the V4 region of the 16S rRNA gene (approximately 250 bp) were used [pmoA genes was performed via a semi-nested PCR approach using the primers A189F/A682R for the first round PCR [MiSeq Illumina sequencing was performed for total 16S rRNA and ere used  with an ere used . The ampound PCR  as folloound PCR . Aliquotound PCR . IndividpmoA gene, one-end run was performed, and the forward adaptor was trimmed using cutadapt. Downstream processing was performed with UPARSE [pmoA genes were classified using the same method, but using the pmoA database [For 16S rRNA genes, quality filtering and trimming of forward and reverse adaptors from the sequences were performed with cutadapt . Forwardh UPARSE  and UCHIh UPARSE  followinh UPARSE . For 16Sh UPARSE . The pmodatabase . SequencA USCα 16S rRNA gene sequence has recently been identified , which e2 and gene abundance data). All univariate statistics were conducted with R software version 3.0.1 [2 value and lowest p-value. Comparisons of variance among soil/vegetation types  were completed using 1-way ANOVA (α = 0.05) with transect considered random, and depth not included in interactions since depth effects are not independent. Post hoc tests were completed using emmeans, adjusted using Tukey’s test for multiple comparisons. We used the vegan package [decostand function for ordination of Hellinger distances. Influence of environmental variables on the total diversity of 16S rRNA and pmoA genes was analyzed by the envfit function . Heatmaps were constructed with the gplots package [prcomp function. The OTUs explaining most of the differences between samples were defined as the ten OTUs contributing the largest absolute loadings in the first and second dimensions of the PCA, obtained from the rotation output file [hclust function. The heatmap was constructed using the heatmap.2 function in the ggplot package [Greenhouse gas fluxes (both field and incubation) were calculated using linear regression, or as the change in GHG concentrations over the time. Data were screened for normality and heterogeneity of variances, and when not conforming, log transformed  for staton 3.0.1 . We used package  for mult package . Principput file . For hie package .4 production across the Australian Alps based on a number of geographic information system (GIS) datasets. The datasets included elevation and aspect (30 m pixel) as well as daily estimates of maximum and minimum air temperature [4 fluxes were determined in the field, the location of 12 sites was used to extract data from each of the raster datasets using ArcGIS . These data were then used to develop a linear regression model using R (v 4.0.2). The final linear regression model  included the main factors  as well as soil moisture and maximum air temperature and their interactions. The spatial extent of each ecosystem across the Australian Alps bioregion [4 fluxes for each of the ecosystems measured across the Australian Alps based on our linear regression model and data for soil moisture and temperature.We developed a model to estimate annual CHperature ,54 and sioregion  was baseioregion  and mappioregion ). These 2O g dry soil−1 for bog soils  across the soils ranged from 2.5 to 17.4 mg CO2 m−2 h−1, in spring and summer, respectively (2 production (measured in the laboratory) decreased with depth. CO2 production thus dominated by emissions from the top 0–5 cm of soil that ranged from 72 to 2357 µg CO2 g−1 d−1. Contrastingly, at 25–30 cm depth, CO2 production ranged from 4 to 197 µg CO2 g−1 d−1. Forest and bog soils showed significantly greater CO2 production in summer than the grassland soil  at 5–10 cm depth (p = 0.003), and moderate CH4 production at 25–30 depth ranging from 87 to 704 µg CH4 g−1 d−1  offset CO2 emissions by 54 to 56% for grassland and forest soils, respectively .Across all soils, Illumina sequencing resulted in 440,504 archaeal, 24,293,004 bacterial, and 2,258,405 ch other . In the ch other . Contrasch other . More sppmoA genes. Both 16S rRNA and pmoA gene sequence data revealed that the forest and grassland soils were dominated by USCα .Non-metric multidimensional scaling (NMDS), similar to the clustering observed in og soils . Grasslaog soils . BacteripmoA gene profiles were relatively tightly clustered except for one sample at 10–15 cm depth, whereas they varied more across depths in the grassland and bog soils.  showed distinct microbial community composition and abundance with depth. Bog soils tended to have the greatest abundance of archaeal 16S rRNA and oA genes A,C. At moA genes . Both ofoA genes . AvailaboA genes , indicat4 at shallow depths  [4 release from bog soils is mostly expected and was evidenced here  . Fast raced here E, and suced here . Previou Kingdom . Therefos of CH4 ,66,67,684 oxidation in well-drained habitats lacking substantial endogenous methanogenesis [4 consumption and relatively high USCα abundance at the 0–5 cm depth during the summer when soil-to-atmosphere CH4 fluxes were greatest  to the total GWP of CO2 emissions. All soils consumed CH4 at 0–5 cm depth. This is consistent with other studies showing fast uptake of CH4 in surface soils where O2 is more available [4 oxidation profiles with depth to those recorded here [4 consumption depends on texture, organic matter and moisture content, as well as availability of labile C and inorganic N [Forest and grassland sinks for CHvailable ,97,98. Eded here ,100,101.rganic N ,89,99.4 sinks (4 uptake have recently been reported in several long-term studies of forest soils [4 oxidation in alpine and sub-alpine regions are less obvious and less easy to value but are critical buffers against anthropogenic climate change.The native ecosystems studied here, with arguably disproportionate importance as CH4 sinks , are amost soils . More frst soils ,104, witst soils , and agr


CD complexation promoted new degradationpathways of the drug. In addition to three already known PZQ degradants,seven new degradation products were identified  and theirstructures were proposed based on HRMS/MS data. Solid complexes wereprepared by mechanochemical activation, a solvent-free and ecologicallyacceptable method.Praziquantel (PZQ) is a biopharmaceuticalclassification system(BCS) class II anthelmintic drug characterized by poor solubilityand a bitter taste, both of which can be addressed by inclusion complexationwith cyclodextrins (CD). In this work, a comprehensive investigationof praziquantel/cyclodextrin (PZQ/CD) complexes was conducted by meansof UV–vis spectroscopy, spectrofluorimetry, NMR spectroscopy,liquid chromatography-high-resolution mass spectrometry (LC-HRMS/MS),and molecular modeling. Phase solubility studies revealed that amongfour CDs tested, the randomly methylated β-CD (RMβCD)and the sulfobutylether sodium salt β-CD (SBEβCD) resultedin the highest increase in PZQ solubility (approximately 16-fold).The formation of 1:1 inclusion complexes was confirmed by HRMS, NMR,and molecular modeling. Both cyclohexane and the central pyrazinoring, as well as an aromatic part of PZQ are included in the CD centralcavity through several different binding modes, which exist simultaneously.Furthermore, the influence of CDs on PZQ stability was investigatedin solution (HCl, NaOH, H Therefore, the CD-based technology appearsas a promising approach to develop novel and more efficient formulationsfor PZQ. Nevertheless, in our previous study, which aimed to developnew, solvent-free, and ecologically more acceptable methods for thepreparation of PZQ/CD complexes in the solid state, we noticed anunfavorable effect of the inclusion complex formation on the chemicalstability of the drug.12 This promptedus to thoroughly investigate the effect of CDs on the chemical stabilityof PZQ, as this issue has not been systematically investigated sofar.The main drawbacks of PZQ include its poor watersolubility, relatedto the high dose required, as well as intensive bitter and metallictaste, which is accompanied with poor patient compliance, especiallyin the pediatric population.Ks), as datafrom the literature are rather inconsistent. For example, the valueof Ks for PZQ complexes with β-CDvaries in the range of 275–531 M–1.13 Thereafter, the complex stoichiometrywas determined via liquid chromatography quadrupole time-of-flightmass spectrometry (LC-QTOF). NMR spectroscopy and molecular modelingwere used to confirm the actual complex formation and gain insightinto the inclusion mechanism. The second part of this investigationincluded solution and solid-state stability studies, focused to determinethe effect of selected CDs on the stability of the PZQ, as well ascharacterization of observed degradation products. The results obtainedin this investigation will enable a critical evaluation of the applicabilityof CDs in the development of novel PZQ formulations with improvedfunctionality.In the first part of this investigation, several phasesolubilitystudies with PZQ and various CDs were performed to determine the exactstability constant values  Praziquantel (PZQ)was supplied by Genera d.d. (Croatia). Cyclodextrins used in thisstudy included β-cyclodextrin (β-CD), 2-hydroxypropyl-β-cyclodextrin, randomlymethylated β-cyclodextrin , and sulfobutylethersodium salt β-cyclodextrin , all obtainedfrom CycloLab (Hungary). 1,2-Deshydro praziquantel (one of the knownPZQ impurities) was obtained from Toronto Research Chemicals. Complexesin the solid state were prepared by grinding and analyzed as describedpreviously.Hydrochloric acid, min. 36.5% p.a., and ammoniumbicarbonate, min. 99.5% p.a., were obtained from Sigma-Aldrich (Germany).Sodium hydroxide, pellets 2–5 mm p.a., ammonia solution, min.25% p.a., and hydrogen peroxide, min. 30% p.a., were obtained fromKemika (Croatia). Acetonitrile and methanol, LC-MS grade, as wellas acetonitrile, methanol, and isopropanol, LC grade, were obtainedfrom Merck (Germany). Ultrapure water was obtained from a Milli-QAdvantage A10 purification system (Merck). All solvents used for NMRanalysis were purchased from Eurisotop.2.2l = 1 cm). The absorbance of PZQ was measuredat 267 nm in a 1:4 methanol–water solution.UV–vis spectrawere recorded on a Specord 200 spectrophotometer  over the range of 190–400 nm, using a standard quartzcell (l = 1 cm). The excitation and emission slit widths were5 nm. The fluorescence intensity of PZQ was measured at 286 nm withthe excitation wavelength set at 260 nm in a 1:4 methanol/water solution.Excitationand emission spectra were recorded on a PerkinElmer LS55 spectrofluorimeterover the range of 200–400 nm using a standard quartz cell  column and mobile phases consistingof acetonitrile and ultrapure water in a ratio of 45:55 (v/v). Theflow rate was set to 1 mL minm/z 100 to 3200.The capillary potential was 3000 V, the fragmentor voltage was 50V, the drying gas flow was 15 L min–1, and the temperaturewas 200 °C. The sheath gas flow was 11 L min–1, and the temperature was 250 °C. Nitrogen was used as a dryingand sheath gas.High-resolutionmass spectra were acquired on an Agilent 6550 SeriesAccurate-Mass Quadrupole Time-of-Flight (Q-TOF). Solutions were introduceddirectly via an Agilent 1290 Infinity II UHPLC. The electrospray ionisationmass spectrometry (ESI-MS) analysis was performed in a positive-ionmode, ranging from Accelerated solid stress testing was performedfor 3 months ina Binder Constant climate chamber, model KBF 720 at 40 °C and70% RH (relative humidity).–2 for 1 h and 700 W m–2.Photostability studies were conductedfor 8 h using an ATLAS CPS+Suntest box with a cooling aggregate, equipped with an ID65-filteredxenon lamp at 300 W m4HCO3) pH 10(solvent A) and acetonitrile (solvent B). The flow rate was set to0.5 mL min–1, and the column temperature was setto 40 °C. The volume of injection was 10 μL. The linearityof the method was determined by injecting a series of diluted stocksolutions of PZQ and PZQ-CD complexes at eight different concentrationsin the range of 0.1–2.5 mg mL–1 (detailsare shown in Supporting Information (SI) Table S6). The ESI-MS spectra were obtained in a positive-ion moderanging from m/z 50 to 1000. Thesource temperature was 140 °C, the desolvation temperature was400 °C, the cone gas flow was 50 L h–1, andthe desolvation gas flow was 750 L h–1.Chromatographic measurements for stabilitystudies were carriedout on a Waters Acquity liquid chromatography system with a UPLC diode-arraydetector (DAD) coupled to an Acquity SQ mass spectrometer. Chromatographicseparation was achieved on a Waters Acquity BEH C18  column in gradient mode using a mobile phase consistingof 10 mM ammonium bicarbonate . Thereafter, samples were shakenat ambient temperature for 48 h, filtered using 0.45 μm ChromafilXtra H-PTFE filters , and diluted accordingly.The PZQ concentration in samples was determined by UV–vis spectrophotometry,fluorescence spectroscopy, and HPLC. Under the experimental conditionsused, no interference of CDs on PZQ quantification was confirmed duringmethod development. The apparent stability constant (K1:1) and complexation efficacy (CE) were calculated fromthe solubility diagram using the following formulasS0 is theintrinsic solubility of PZQ. The experiments were repeated three times.Solubilitymeasurements were carried out according to Higuchi and Connors.2.4–1. Samples for HRMS analysis were prepared by diluting stock solutionsin solvent, consisting of methanol and water in a ratio of 50:50 (v/v).The final concentration was 4.4 × 10–5 molL–1. Samples of mixtures of CD and PZQ were preparedby mixing solutions in an equimolar ratio.Stock solutions of cyclodextrins and PZQ wereprepared by weighing analytes and diluting them with water and methanol,respectively, to achieve a final concentration of 1 mg mL2.5d6 and transferredto a 5 mm NMR tube. The final concentration in DMSO-d6 was 16 mM.PZQ (5 mg), β-CD(18.5 mg), and PZQ/β-CD complex (1:1) (23.5 mg) were dissolvedin 600 μL of DMSO-2O to achieve a concentration of 2.13 mM. Due to the precipitation ofPZQ, it was further diluted until fully dissolved to a final concentrationof 1.07 mM. To match this concentration, β-CD was also diluted.PZQ/β-CD complex (1.5 mg) was dissolved in 1.24 mL of D2O to a final concentration of 1.07 mM.PZQ (1.0 mg) and β-CD (2.6 mg)were each dissolved in D2O givinga final concentration of 3.52 mM for PZQ and 14.08 mM for β-CD.In an attemptto increase the concentration of PZQ in the solution,17.09 mg of the solid mixture containing PZQ and β-CD, in amolar ratio of 1:4, was dissolved in 1 mL of DAll four solutions (600 mL) were transferred to 5 mm NMR tubes.1H, 13C, COSY, NOESY, ROESY, DOSY,HSQCe, and HMBC). Unless otherwise stated, the spectra were acquiredat 25 °C using standard Bruker pulse sequences. NOESY spectrawere obtained with a mixing time of 500 ms. The durations of the ROESYspinlock were 200 ms (water suppression experiments) and 300 ms (recreationof the literature experiment). The data were processed using TopSpinBruker software package.The complete NMR analysis was made on the basis of one- and two-dimensionalNMR spectra  with bipolar gradient pulsesfor diffusion and three spoil gradients.d6), 32 scans (β-CD in D2O), and 256 scans(PZQ and PZQ/β-CD complex in D2O) for each gradientstep, and the linear gradient was chosen in the range from 2 to 95%.The diffusion times (Δ) were 93.6 ms (DMSO-d6) and 95.4 ms (D2O), while the gradient durations(δ) were set to 4.6 ms (DMSO-d6)and 3 ms (D2O). The spectra were processed using DynamicsCenter 2.6.3 software. The fitted function wasI0 is the signal integral, and variable (x) is the gradient strength.The spectra were acquired with 16 scans  of the docking grid was used. Visualizationwas performed with Chimera.20The 3D model of PZQwas generated using Chem3D, while 3D coordinates of β-CD weretaken from the PDB structure (PDB code: 3CGT). Docking preparation was performed withAutoDock Tools using default settings.2.7–1. For each condition tested,300 μL of the stock solution was diluted with selected mediato achieve a final concentration of 0.6 mg mL–1.All experiments were performed at 85 °C to achieve an acceptablepercentage of degradation. Samples were collected at 0 min and 24h.Hydrolytic degradationwas carried out under acid and base conditions using hydrochloricacid (0.1 and 1 M) and sodium hydroxide (0.1 and 1 M), respectively.Hydrogen peroxide (0.3 and 3%) was used for oxidative degradation.Stock solutions of CD complexes and PZQ were prepared by weighingapproximately 2 mg of PZQ and CD complexes, followed by dilution witheither water (CD complexes) or acetonitrile (PZQ) to achieve a finalconcentration of 2 mg mL2.8–1. Priorto LC-DAD analysis, the samples were filtered through a 0.2 μmPTFE filter mounted onto a syringe. At each time point, the T0 sample was prepared prior to analysis. Foreach degradant, the relative retention time was calculated (RRT =RT(degradation product)/RT(PZQ)).PZQ and its inclusion complexes with CDs were added into a glassscrew cap vial (approximately 5 mg), weighed in quadruplicate, andplaced in a stability chamber at 40 °C and 75% RH (relative humidity)for 3 months. The samples were collected at 2 weeks and 1, 2, and3 months and prepared for analysis by adding an appropriate volumeof selected media (water (CD complexes) or acetonitrile (PZQ)) toachieve a final concentration of 2 mg mL2.9–2, 1 h; 700 W m–2, 8 h). For each time point  and T1 after 1 and 8 h), the samples were preparedby adding an appropriate volume of selected solvent (water for CDcomplexes or acetonitrile for PZQ) to achieve a concentration of 2mg mL–1. For LC-DAD analysis, the samples were filteredthrough a 0.2 μm PTFE filter mounted onto a syringe.PZQ and itsinclusion complexes with CDs were added into a glass screw cap vial(approximately 5 mg). Each sample was weighed in duplicate, and oneof them was prepared as a control sample by wrapping the vial withaluminum foil. Both samples (test and control) were simultaneouslyexposed to the same stress conditions  and water CD complexes)to obtain concentrations of 10 and 100 μg mL33.13.1.1Table S1). Basedon Higuchi and Connors,15 all observed phasediagrams were classified as AL type . The stability constants determinedfor 1:1 complexes correlate fairly well, although some discrepanciesbetween UV–vis and other methods were observed. Based on theseresults, fluorescence spectroscopy can be suggested as the optimalmethod due to its low cost, high sensitivity, and overall ease ofuse.Solubilitystudies of PZQ with selected CDs were investigated in water usingliquid chromatography, UV–vis absorption spectroscopy, andfluorescence spectroscopy. The total concentration of PZQ was determinedusing validated methods  differs from the one determined by Mourãoet al.13 (275.56 M–1).Moreover, our results are not in agreement with research done by Münsteret al.,6 where the stability constant forthe binary system with SBEβCD of 365.11 M–1 was reported. These discrepancies in stability constant values couldbe explained by differences in analytical techniques used, as wellas differences in experimental conditions, such as temperature, usedduring the study. However, the results reported in the same study6 for the binary system with HPβCD (228.71M–1) are in relatively good agreement with our data.The results obtained herein differ to some extentin comparisonto previously reported data, which showed a significant level of discrepancy.For example, the stability constant for the binary system with β-CDdetermined by Becket et al.23 Solutions of PZQ with selectedCDs were analyzed by HRMS. Signals corresponding to 1:1 PZQ/CD complexeswere observed indicating the formation of inclusion complexes , possibly due to the solvationof PZQ with molecules of DMSO making the inclusion of the drug intothe β-CD cavity unfavorable. Despite the challenges of low PZQsolubility, switching the study to D2O enabled investigationof drug–CD interactions in a medium resembling the biologicalsystem.PZQ andβ-CD complex formation has been long established;3.1.2.1Tables S3, S4, and As a first step, we fully assigned proton andcarbon chemical shifts of PZQ, β-CD, and PZQ/β-CD complex was noticed only for protonsH3 (free 3.892 ppm → complex 3.861 ppm) and H5 (free 3.787ppm → complex 3.760 ppm), positioned on the inner side of thecyclodextrin central cavity  of β-CD (l cavity 1c. This S4) proved to be morechallenging, not only due to low solubility butalso, as previously seen in DMSO-d6 , as a result of PZQ undergoing a chemical exchange between twoisomers (Table S4). The carbon chemical shift of C12 in syn rotamer was 45.9 ppm .To circumvent all of these challenges, chemical shiftsof PZQ wereextracted using a combination of proton and  spectra 2. Althou1H–13CHSQCe spectrum due to line broadening .Unfortunately, we were unable to observe these interactions with ourPZQ/β-CD 1:1 complex both in the water suppression ROESY andROESY spectrum recorded using the same experimental parameters asde Jesus et al. .As we strongly suspected that the main cause of signal absence wasthe low concentration, and in line with results obtained by phasesolubility studies , we prepared the solid mixture of PZQ/β-CD in a 1:4 molarratio to achieve a much higher PZQ concentration in D2O.At a PZQ concentration of 3.52 mM, roughly 3 times higher than incomplex at c(β-CD) = 1.07 mM, the resultingROESY spectrum  using the equation332O were calculated to be ca. 30% (at 1.07 mM β-CD)and ca. 90% (at 14.08 mM β-CD). A literature search revealedonly one previously reported DOSY experiment,11 performed on a 1:1 PZQ/β-CD complex reporting 37% of complexation.The results in p) can be used to determine association constant (Ks) in the following equation31CCD,tot and CS,tot are total concentrationsof β-CDand PZQ, respectively. Association constants were calculated to be587 and 854 M–1, at 1.07 and 14.08 mM β-CD,respectively. The former value is in good agreement with the associationconstant obtained in the phase solubility study ones with the PZQ aromatic part embedded within the β-cyclodextrincavity (Table S5) are not sufficiently differentto enable identification of the dominant binding mode. Besides vander Waals interactions, hydrogen bonds between β-CD and carbonylgroup of central pyrazino ring of PZQ can also be formed.Aiming toexplain the discrepancy between the previously postulated mode ofinclusion described in the literaturencavity 5aand (incavity 5b. Relat(a)Diffusionspectroscopy (DOSY) detectsthe difference in molecules according to their diffusion rates. Althoughfree PZQ and the complex exhibit vastly different diffusion rates,complexes with different binding modes, all simultaneously presentin solution, display an average diffusion rate due to the fast exchangebetween the different binding modes. Similarly, in proton and carbonchemical shifts of β-CD, the effect of perturbation is cumulativeand it only testifies about the drug occupying the cavity.(b)On the other hand, theROESY spectrumwould show a different set of intermolecular interactions for eachmode of binding. Considering the low solubility of PZQ at 1.07 mMβ-CD, the presence of two different isomers in exchange, andseveral different modes of binding, the concentration of each typeof complex becomes so small that it falls below the detection limitof the NMR method, thereby explaining the absence of intramolecularinteractions in ROESY spectra under such conditions. If the solutionconcentration and percentage of complexation are increased (by higherβ-CD concentration), the threshold is overcome and the interactionsbecome visible.(c)25 our NMRresults showed that PZQ cyclohexane and central pyrazino rings arealso in close contact with β-CD. This corresponds perfectlyto several different binding modes all simultaneously present in solution,as identified by molecular modeling.Finally,in addition to the previouslyreported aromatic part of PZQ,These molecular modeling results completely explained ourNMR experimentalresults:34 one can assume a similar structure of other complexes of PZQ withCDs examined.Since the CD cavity size is recognized as the critical parameterdetermining the drug–CD binding mode,3.212 suggested thatCD complexation of PZQ may compromise its chemical stability. Therefore,in the second part of the study, the effect of CDs on PZQ stabilityat different conditions was examined, and the structures of formeddegradation products were proposed.Our previous study3.2.12O2), analyzed via UPLC-DAD-MS, revealed both known andnovel degradation products, not yet described in the literature. Knowndegradation products ,35m/z 331 for NaOH, and m/z 329 and 345 for H2O238 (Table S7)) were observedin PZQ and PZQ/CD complexes. Under oxidative stress conditions, someunknown degradation products were also observed: m/z 148 and 217, both for PZQ and PZQ/CD complexes,and m/z 215 for PZQ/CD complexesand are described in detail in the following section.Samples collectedduring degradation stress conditions and PZQ/CD complexes under accelerated solid stress conditions forup to 3 months at 40 °C and 75% RH, several degradation productswere detected , as well as proposed structures fordegradation products, are summarized in A similar set of degradation products was detected for all threePZQ/CD complexes. However, analogue peaks corresponding to some ofthem were completely absent in pure PZQ chromatograms, suggestingthat CD complexation promotes new degradation pathways, as most pronouncedin the PZQ/HPβCD complex. LC-HRMS and MS/MS analyses were usedto annotate the molecular formula and to provide structural informationof degradation products. Fragments obtained from MS/MS studies (m/z 345 (IV), 273 (VII), 329 (VIII), and 311 (X),detected at relative retention times (RRT) 0.77, 0.90, 1.10, and 1.17,respectively.39 The degradation product at RRT 0.77 (m/z 345) corresponding to dihydroxypraziquantel was detectedin all three PZQ/CD complexes. The MS spectrum displayed fragment m/z 327, while fragment m/z 309 was absent. The only dihydroxypraziquantelwith such fragmentation pattern reported so far is 7,8-dihydroxypraziquantel.36 Due to the specific position of two hydroxygroups in the molecule, upon elimination of the first molecule ofwater (m/z 345 → 327), theresulting enol tautomerizes into a more stable keto form preventingthe loss of the second water molecule. This was further confirmedby the presence of fragment m/z 217,which corresponds to the loss of one water molecule and a cyclohexylketene.Among the degradation products observed,several were previouslyreported in the literature with fragment ions at m/z 273), potentially the result of amide bond hydrolysis,was also detected in all three complexes. Fragment ions observed inMS/MS spectra are in agreement with previously published results39 with m/z 256formed due to oxygen loss and m/z 163 formed due to the loss of cyclohexylketene following the cleavageof the amide bond.The degradation product detected at RRT 0.90 (m/z 329), which was onlydetected for the PZQ/HPβCD complex, based on observed fragmentsand previously reported data.38 The fragment ion m/z 311 would correspond to the loss of water; however,it was not detected in MS/MS spectra possibly due to the high fragmentationof the parent ion. The presence of fragment ions m/z 219, 201, and 83 suggests oxidation in the hexahydropyrazinoisoquinolinepart of PZQ, instead of the cyclohexyl ring. As previously reported,36 position 7, where C–H bonds are benzylicand thereby most likely a preferred point of oxidation, is the mostreactive site in PZQ.A monohydroxylated structure was suggestedfor degradation productRRT 1.10 (m/z 311) was detected in all formulationsand during mostsample collection points up to 3 months. Previously, m/z 311 was proposed to be the product of dehydrogenationof the hexahydropyrazinoisoquinoline moiety with the exact positionof the double bond undetermined.40 However, asfragment m/z 311 could also belongto a known impurity of PZQ, 1,2-deshydro praziquantel , this was later confirmed in retentiontime with UPLC-DAD via the commercially available standard.The degradation product at RRT 1.17 (m/z 148 and m/z 215), RRT 0.60 (m/z 217), RRT 0.86 (m/z 301), RRT 0.90 (m/z 273(previously published) and m/z 378),RRT 1.14 (m/z 327), and RRT 1.23(m/z 343).In addition to known degradants, the exact mass and structure wereproposed for seven new degradation products, based on HRMS, whichhave not been previously published in the literature, and are summarizedin m/z 197 and 199) and loss of CO from the parent ion (m/z 215 → 187 and m/z 217 → 189). All fragments in MS/MS spectraof degradation product m/z 215 suggestone additional double bond, compared with fragment ions from degradationproduct m/z 217. However, the datawere insufficient to accurately define the position of the doublebond. In addition, a second degradation product was observed at RRT0.57 , for which the proposed molecular formulais C9H9NO with fragment ions corresponding tothe loss of one water molecule (148 → 130), likely at position7 as the preferred point of oxidation.The degradant atRRT 0.57 was detected in all three PZQ/CD complexes,whereas the RRT 0.60 degradant was detected only in PZQ/HPβCDand PZQ/SBEβCD. Both degradation products give fragment ionsarising from loss of one water molecule  was only detected using UHPLC-QTOF-MS due to lower sensitivity.MS/MS spectra were similar to degradation product m/z 273 (RRT 0.90). Fragmentation of the parent ion(m/z 301) showed an eliminationof 28, assigned to CO, and formation of fragment ion m/z 273. Fragment ion m/z 256 isformed due to oxygen loss. Cleavage of the amide bond gives fragmention m/z 191. In MS/MS spectra ofdegradation product m/z 273, cleavingamide bond gives fragment ion m/z 163. The difference between these two fragment ions correspondsto the molecular weight of a CO group. Melo et al. proposed a degradationmechanism of PZQ and formation of fragment ions m/z 174 and 146.36 Followingcleavage of the bond on the opposite side of the nitrogen atom inamide bond, fragment ion m/z 174is formed. Fragment ion m/z 146corresponds to the loss of the CO group (m/z 174 → 146), suggesting that the position of a COgroup could be at the nitrogen atom in the pyrazine ring.The degradation productat RRT 0.86  suggest a ketone on the hexahydropyrazinoisoquinoline partof PZQ. As previously described, position 7 is most likely to be thepreferred point of oxidation.TheRRT 1.14 degradant was only detected in the PZQ/HPβCDcomplex, with an accurate mass of [M + H]m/z 343 wasdetected at RRT 1.23 . This largemolecule could be explained by a known impurity of PZQ . Accurate mass and fragment ionsin MS/MS spectra correspond to the oxidation of impurity and the formationof ketone. Fragment ion m/z 257is assigned to the cleavage of the amide bond. Following cleavageof the bond on the opposite side of the CO group, fragment ion m/z 229 is formed. Fragment ion m/z 201 is assigned to the cleavage betweennitrogen from the piperazine ring and carbon atom and loss of onewater molecule. A small amount of impurity MW 363 was found in neatPZQ  (The two degradation products found at RRT 0.90 included previouslydiscussed RT 0.95) 5, provid3.2.3–2 for 1 h, as well as under conditions at 700 W m–2 for 8 h, except for PZQ/SBEβCD with a decayof about 35% ,complexes of PZQ and β-CD can be detected,with a calculated percentage of complexation of ca. 30 and 90% atβ-CD concentrations of 1.07 and 14.08 mM, respectively. Chemicalshift perturbations and analysis of ROESY NMR spectra confirmed thatthe cyclohexane and central pyrazino ring, as well as the previouslysuggested25 aromatic part of PZQ, are inclose contact with β-CD through several different binding modes.These findings were confirmed by molecular modeling.NMR studieshave confirmed that under given conditions (DAcceleratedstress testing of solid PZQ/CD complexes for up to3 months displayed a decrease in PZQ stability in the presence ofCDs. PZQ/HPβCD showed the highest level of degradation after3 months (38%), while PZQ/RMβCD and PZQ/SBEβCD remainedrelatively stable with only minor degradation. A similar set of degradationproducts was detected across all three PZQ/CD complexes, some of whichwere previously reported in the literature. In addition, seven newdegradation products were detected and the corresponding structureswere proposed based on HRMS and MS/MS data, indicating that CD complexationpromoted new degradation pathways of the drug.Data from acceleratedsolid stress testing should be consideredwhen storing PZQ cyclodextrin formulations, avoiding high temperaturesand high humidity.


The sensitivity, specificity, and area under the curve were 98.41%, 100%, and 0.995, respectively, for the training set and 93.33%, 91.67%, and 0.983, respectively, for the test set. We further verified the significant upregulation of AC010082.1 (p < 0.01) and AC011443.1 (p < 0.05) in patients with CHD using RT-qPCR in the validation cohort. Our results suggest that lncRNA AC010082.1 and AC011443.1 are potential biomarkers of CHD. Their pathological mechanism in CHD requires further validation.Coronary heart disease (CHD) is a global health concern with high morbidity and mortality rates. This study aimed to identify the possible long non-coding RNA (lncRNA) biomarkers of CHD. The lncRNA- and mRNA-related data of patients with CHD were downloaded from the Gene Expression Omnibus database (GSE113079). The limma package was used to identify differentially expressed lncRNAs and mRNAs . Then, miRcode, TargetScan, miRDB, and miRTarBase databases were used to form the competing endogenous RNA (ceRNA) network. Furthermore, SPSS Modeler 18.0 was used to construct a logistic stepwise regression prediction model for CHD diagnosis based on DElncRNAs. Of the microarray data, 70% was used as a training set and 30% as a test set. Moreover, a validation cohort including 30 patients with CHD and 30 healthy controls was used to verify the hub lncRNA expression through real-time reverse transcription-quantitative PCR (RT-qPCR). A total of 185 DElncRNAs (114 upregulated and 71 downregulated) and 382 DEmRNAs (162 upregulated and 220 downregulated) between CHD and healthy controls were identified from the microarray data. Furthermore, through bioinformatics prediction, a 38 lncRNA-21miRNA-40 mRNA ceRNA network was constructed. Next, by constructing a logistic stepwise regression prediction model for 38 DElncRNAs, we screened two hub lncRNAs AC010082.1 and AC011443.1 ( Globally, coronary heart disease (CHD) is a major disease with high morbidity and mortality, which seriously threatens human health , killingEvidence in recent years has showed that long non-coding RNAs (lncRNAs) have a key role in the gene regulation and widely concerned . LncRNAsRecently, the proposed competing endogenous RNAs (ceRNAs) hypothesis has provided the possibility for further study of molecular mechanisms underlying several diseased conditions . CeRNAs https://www.ncbi.nlm.nih.gov/geo) with accession number GSE113079. This dataset included 93 patients with CHD and 48 healthy controls using the GPL20115 platform (Agilent-067406 Human CBC lncRNA + mRNA microarray V4.0). LncRNA and mRNA expression data from the GPL20115 platform were obtained by reannotating microarray probes according to the probe set sequences and the annotations of protein-coding genes and lncRNA records in GENCODE . The BLASTn (https://ftp.ncbi.nlm.nih.gov/blast/executables/LATEST/) was used to align the probe sequences with those of non-coding and coding transcript sequences from GENCODE. The transformed data  were considered as lncRNAs.The gene expression profile of human CHD was obtained from the GEO  adjusted using the Benjamini–Hochberg method were used to analyze the DElncRNAs and DEmRNAs | > 1 were considered as the cut-off criteria.The identification of DElncRNAs and DEmRNAs between patients with CHD and healthy controls were performed by using the “limma” package of R software (version 4.0.1) . An inde DEmRNAs . p < 0.0The prediction of lncRNAs to target miRNAs was conducted using the miRcode database . DEmRNAshttp://metascape.org) to analyze the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment of the DEmRNAs . SPSS Modeler 18.0  was used to construct a logistic stepwise regression prediction model for CHD diagnosis based on DElncRNAs. Further, the sensitivity and specificity of the model were calculated using SPSS Modeler. The receiver operating characteristic (ROC) curve was used to evaluate the effectiveness of the classification model, and the area under the curve (AUC) value was calculated from the ROC curve. ROC curves were calculated using the R package “pROC.” Principal component analysis (PCA) using the R package “ggplot2” was performed on the screened hub lncRNAs, and their expression levels in patients with CHD and healthy controls were analyzed. The selection process for screening hub lncRNAs is shown in The 141 samples from the GSE113079 dataset were randomly divided into training  and test  sets, and they maintained the similar ratio for patients with CHD and healthy controls  and 30 healthy controls  were recruited in Guang’anmen Hospital, Beijing, China, as the validation cohort. This study complied with the Declaration of Helsinki and was approved by the Ethics Committee of Guang’anmen Hospital, China Academy of Chinese Medical Sciences (no. 2019–224-KY). All subjects were aged between 42 and 77 years. CHD diagnosis was based on the European Society of Cardiology criteria, which specifies at least one vessel lesion  using coronary angiography . HealthyPeripheral blood samples (4–8 ml) were collected in the early morning from patients with CHD and healthy controls using EDTA vacuum anticoagulant blood vessels. Peripheral blood mononuclear cells (PBMCs) were isolated using the Red Blood Cell Lysis kit  and total RNA was extracted from PBMCs using TRIzol reagent  according to the manufacturers’ instructions. RNA quality and quantity were evaluated using a NanoDrop 2000 spectrophotometer . LncRNA reverse transcription was performed using the lncRcute lncRNA First-Strand cDNA Kit (TIANGEN) from 1 µg total RNA according to the manufacturer’s instructions.ACTB was used as an internal reference. The cycling PCR conditions were: 95°C for 3 min; 40 cycles of 95°C for 5 s, 55°C for 10 s, and 72°C for 15 s; 95°C for 15 s, 65°C for 15 s, and 95°C for 15 s. The primer sequences are as follows: AC010082.1 forward primer, 5′-TTTGGTCTAGGCGCTAGGAAT-3′; AC010082.1 reverse primer, 5′-CTTTTCCCCTTACCCTGCTTT-3′; AC011443.1 forward primer, 5′-TGTTCCAAGGTCAACCAAAAA-3′; AC011443.1 reverse primer, 5′-CCAAGGTGGTCAAATCTGTGT-3′; ß-actin forward primer, 5′-GAGACCTTCAACACCCCAGCC-3′; ß-actin reverse primer, 5′-AATGTCACGCACGATTTCCC-3′. Relative expression data were analyzed using the 2-∆∆CT method (Reverse transcription-quantitative PCR (RT-qPCR) was performed using the lncRcute lncRNA qPCR kit (TIANGEN) to detect relative expression using the standard protocols on the ABI7900HT Fast Real-Time PCR system . T method . R 4.0.1p < 0.05, FDR <0.05, and |log2(FC)| > 1 as screening criteria, 185 DElncRNAs (114 upregulated and 71 downregulated) and 382 DEmRNAs (162 upregulated and 220 downregulated) between CHD and healthy controls were identified. The volcano maps of DElncRNAs and DEmRNAs are shown in In all, 16,939 mRNAs and 11,874 lncRNAs were derived from the microarray data by conducting the GENCODE probe reannotation. Using We used the miRcode database to predict miRNA-targeted lncRNAs. The constructed ceRNA network only included interactions between the DEmiRNAs and DElncRNAs; thus, 1794 interactions between 47 DElncRNAs  and 274 DEmiRNAs  were identified. We mapped the abovementioned 274 DEmiRNAs against TargetScan, miRDB, and miRTarBase to search for target DEmRNAs. A total of 1912 DEmRNAs that interacted with 40 of the 274 DEmiRNAs in all three datasets were selected. After intersecting the predicted 1912 DEmRNAs and previous 382 DEmRNAs, a 38 lncRNA-21 miRNA-40 mRNA ceRNA network was constructed , 5.To determine the biological functions and pathways of the 40 DEmRNAs in the constructed ceRNA network, we used the Metascape database to analyze the GO functional enrichment and KEGG pathway enrichment . The topp < 0.05) as lncRNAs were statistically significant for model construction among the 38 independent variables. The prediction model formula is:By setting 38 DElncRNAs as independent variables, we constructed a logistic stepwise regression prediction model for CHD. The analysis reveals that AC010082.1 and AC011443.1 (Logit(P) = −13.718 + AC010082.1*8.099 + AC011443.1*3.825.p < 0.05), and that AC010082.1 and AC011443.1 have a significant positive correlation  and AC011443.1 (p = 0.02 < 0.05) were significantly higher in the CHD group than in the healthy control group, and the AC010082.1 and AC011443.1 expression levels were significantly positively correlated (r = 0.5, p < 0.01, RT-qPCR was used to verify the relative expression of AC010082.1 and AC011443.1 in the validation cohort. The results show that AC010082.1 (Despite considerable advances in modern medicine, CHD diagnosis remains difficult, particularly in the early stages. LncRNA is an important regulator of gene expression, and one of its regulatory mechanisms is ceRNA. MiRNAs can bind to mRNA through MREs, resulting in mRNA degradation or translation inhibition. When lncRNAs and mRNAs have the same MREs, lncRNAs can compete with mRNA for binding to miRNA to prevent mRNA degradation and achieve indirect regulation of mRNA expression level . The ceRβ signaling pathway, TNF signaling pathway, and leukocyte transendothelial migration. These signaling pathways have been previously shown to be closely related to the pathological mechanisms of CHD (β signaling pathway can regulate the proliferation and migration of vascular smooth muscle and endothelial cells, and affect the stability of plaque, which plays an important role in CHD pathogenesis (In this study, a ceRNA network consisting of 38 lncRNAs, 21 miRNAs, and 40 mRNAs was constructed using bioinformatics analysis. KEGG function enrichment in this network was mainly concentrated in the TGF-s of CHD . The TGFogenesis . The TNFogenesis . Leukocyogenesis . These DHowever, further lncRNA analysis using the logistic stepwise regression prediction model revealed that AC010082.1 and AC011443.1 might be important biomarkers of CHD, which was confirmed using RT-qPCR. AC010082.1 is an antisense molecule with a length of 546 bp and is located on chr7:18,429,062–18,430,738, whereas AC011443.1 is also an antisense molecule with a length of 365 bp and is located on chr19:39,134,882–39,136,463. Unfortunately, no relevant studies have been conducted on these two lncRNAs, and their direct mechanism of action remains unclear. Through bioinformatics analysis, it was found that these two DElncRNAs affect the expression of 25 mRNAs by regulating eight miRNAs. The hsa-miR-17-5p, hsa-miR-20b-5p and hsa-miR-27a-3p were jointly regulated by these two DElncRNAs, which may contain a common MRE and have a regulatory relationship through ceRNA mechanism. We found that AC010082.1 and AC011443.1 were overexpressed in patients with CHD and showed a significant positive correlation. The mechanism may be that when lncRNA AC010082.1 is highly expressed in patients with CHD, it will bind more miRNAs, so as to release the inhibition of miRNA on the other lncRNA AC011443.1, thus leading to its high expression . AC01008κB signaling pathway by binding hsa-miR-17-5p, and then affect proliferation and inflammatory response in atherosclerosis patients, which provide a potential target for treating AS (κ B pathway (ABCA1 and ABCG1 gene expression (Interestingly, we found that hsa-miR-17-5p, hsa-miR-20b-5p and hsa-miR-27a-3p co-regulated by these two DElncRNAs have important biological roles in CHD. The expression of hsa-miR-17-5p is down-regulation in patients with CHD, which could be as a biomarker of CHD  and a goating AS . Further pathway . And cir pathway . Reporte pathway . Meanwhipression .Besides, these two DElncRNAs affected TGFBR3 expression by co-regulating the miR-27a-3p sponge via bioinformatics prediction. TGFBR3 expression is significantly upregulated in patients after myocardial infarction and may serve as a therapeutic target and biomarker for myocardial damage by activating p38 MAPK to induce cardiomyocyte apoptosis . Moreovein vitro and in vivo experiments to further explore the functions of AC010082.1 and AC011443.1, and the related pathological mechanisms, are warranted.The present study had some limitations. First, we only verified the possibility of using AC010082.1 and AC011443.1 as biomarkers through RT-qPCR, and the clinical samples were obtained from only one research center. In future, larger sample sizes and data from multiple centers countrywide are required to verify our findings. In addition, In summary, our study identified AC010082.1 and AC011443.1 as possible biomarkers of CHD through bioinformatics analysis and RT-qPCR. The ceRNA network associated with these lncRNAs possibly plays a key role in the pathological process of CHD.


Mycobacterium tuberculosis results in variable degrees of lung pathology during active TB (ATB) with central involvement of neutrophils. Little is known about neutrophil-derived mediators and their role in disease severity at baseline and recovery upon TB treatment initiation.The inflammatory response to 107 adults with confirmed pulmonary TB were categorised based on lung pathology at baseline and following successful therapy using chest X-ray scores  and GeneXpert bacterial load . Plasma, sputum, and antigen-stimulated levels of MMP1, MMP3, MMP8, MMP9, MPO, S100A8/9, IL8, IL10, IL12/23(p40), GM-CSF, IFNγ, and TNF were analysed using multiplex cytokine arrays.At baseline, neutrophil counts correlated with plasma levels of MMP8 , S100A8  and GM-CSF . Levels of MMP8 (p = 3.00E−03), MMP1 (p = 1.40E−02), S100A8 (p = 1.80E−02) and IL12/23(p40) (p = 1.00E−02) were associated with severe lung damage, while sputum MPO levels were directly linked to lung damage (p = 1.80E−03), Mtb load (p = 2.10E−02) and lung recovery (p = 2.40E−02). Six months of TB therapy significantly decreased levels of major neutrophil-derived pro-inflammatory mediators: MMP1 (p = 4.90E−12 and p = 2.20E−07), MMP8 (p = 3.40E−14 and p = 1.30E−05) and MMP9 (p = 1.60E−04 and p = 1.50E−03) in plasma and sputum, respectively. Interestingly, following H37Rv whole cell lysate stimulation, S100A8 (p = 2.80E−02), MMP9 (p = 3.60E−02) and MPO (p = 9.10E−03) levels at month 6 were significantly higher compared to baseline. Sputum MMP1 (p = 1.50E−03), MMP3 (p = 7.58E−04), MMP9 (p = 2.60E−02) and TNF (p = 3.80E−02) levels were lower at month 6 compared to baseline in patients with good lung recovery.In this study, patients with severe lung pathology at baseline and persistent lung damage after treatment were associated with higher plasma and sputum levels of major pro-inflammatory neutrophil-derived mediators. Interestingly, low sputum MPO levels were associated with severe lung damage, higher Mtb burden and low recovery. Our data suggest that therapeutic agents which target these mediators should be considered for future studies on biomarkers and host-directed therapeutic approaches against TB-related lung pathology and/or lung recovery. Mycobacterium tuberculosis (Mtb) lead to more severe lung damage prior to treatment initiation (Tuberculosis (TB) caused 1.2 million deaths from HIV-negative individuals in 2019 . It has itiation  and thusMycoplasma pneumoniae infection in mice may be a result of oxidant–antioxidant imbalances which can be reduced by immunosuppression  based lung pathology scores and bacterial load before and after TB therapy. We address gaps in our understanding of TB pathogenesis by monitoring the impact of neutrophil-derived mediators on the severity of TB-induced lung pathology to inform future experiments in controlled animal models investigating TB HDTs.Ethical approval was obtained from the Medical Research Council/Gambia government joint ethics committee (SCC1523). All study participants provided written informed consent prior to collection of samples.Adult, TB patients with positive GeneXpert  results were recruited from the TB clinic at the MRC Unit The Gambia at LSHTM between April 2018 and October 2019 as part of a parent study, TB Sequel . This stMed) of all participants in our cohort was determined, 65 [29–80]. Lung damage severity (pre- and post-treatment) groupings were defined as follows: “mild pathology” (RS < RSMed) and “severe pathology” (RS ≥ RSMed).Chest radiographs were analysed based on the Ralph score (RS) . Briefly® machine  was used to determine cycle threshold (Ct) values at baseline. The lowest Ct value generated among the five rpoB probes of Xpert MTB/RIF (or of the four rpoB probes in the nested-PCR stage for GeneXpert Ultra) was taken as a measure of the Mtb cell number (Med) of all participants was computed, 17.4 [17.1–18.4]. Patients were grouped into: “high Mtb load” (Ct < CtMed) and “low Mtb load” (Ct > CtMed) groups.The GeneXpertl number . The medMed) were computed, 6.5 [1.6–14]. Patients were grouped into: “Good” (ΔRS ≥ ΔRSMed) and “Poor” (ΔRS < ΔRSMed) lung recovery groups. Nine participants had a ΔRS equal to the ΔRSMed.For each patient, RS changes (ΔRS) from BL to 6M (ΔRS = RS at BL/RS at 6M) and median change [IQR] in RS of the entire cohort  for 15 min and centrifuged. The supernatant was harvested and stored at −80°C until use.2, plates were spun  and 200–250 μl of supernatant was harvested from each well into 0.5 ml Sarstedt tubes prior to storage at −80°C for multiplex cytokine assays.Plasma was obtained from blood vials by centrifugation at 2,500 rpm and stored at −80°C prior to use. Approximately 500 µl of whole blood was stimulated with either ESAT-6/CFP-10 peptide pool , purified protein derivative , H37Rv whole cell lysate  or phorbol 12-myristate 13-acetate  along with co-stimulatory antibodies ; or unstimulated/cultured with medium alone . Following overnight incubation at 37°C, 5% COex vivo sputum) and controls were added per well. Plates were then incubated at room temperature (RT), with shaking  followed by three washes in wash buffer. Detection antibodies were diluted in detection antibody diluent as recommended and 50 μl added to each well followed by another 1 h incubation period. Following three washes, 100× streptavidin-PE was diluted in wash buffer (one in 100) and 50 μl added to each well. Plates were then incubated for 30 min and washed three times. Approximately 100 μl of assay buffer were then added to each well, plates were shaken for 2 min and read using Bioplex 200 plate reader with Bio-Plex Manager Software .Multiplex cytokine arrays were performed using a customised 13-plex inflammatory marker panel  according to the manufacturer’s instructions. The 13 analytes measured were GM-CSF, IL8/CXCL8, IL12/23(p40), MMP3, MMP9, S100A8, S100A9, TNF, IFNγ, IL10, MMP1, MMP8, and MPO. The minimum levels of detection for these were: 11.52, 2.96, 383.13, 78.48, 128.31, 74.86, 8.44, 42.35, 3.70, 40.95, 241.11, 113.00, and 26.87 pg/ml, respectively. Briefly, lyophilised standards were reconstituted and serial dilutions performed. Coupled beads were diluted in assay buffer and 50 μl were added to each well of the assay plate. Approximately 50 μl of diluted standards, blanks, samples .The two measures of ATB severity that we used were sputum GeneXpert Ct values and CXR Ralph scores. There was a weak negative correlation between patient Ct values and Ralph scores at baseline  . We alsoNeutrophil levels are associated with higher risk of lung damage  and deatAt baseline, plasma concentrations of all inflammatory mediators, excluding MPO and MMP9, correlated with absolute neutrophil counts Table 2.For patients with mild lung damage at baseline, correlations were weaker and only significant for MMP8, S100A8, and S100A9 Table 3.Plasma concentrations of MMP8, MMP1, S100A8, IL12/23(p40), IFNγ, IL8, and TNF were significantly elevated in patients with severe lung damage at baseline compared to those with mild damage , and IFNγ  even after adjusting for sex  levels were higher in patients with severe lung damage at baseline  and plasma concentrations of MMP3, IL8, IL10, IL12/23(p40), GM-CSF, and TNF  were higher in males compared to females , TNF (p = 2.10E−05), IFNγ (ns), S100A8 (ns), and MPO (ns) were higher at month 6 compared to baseline  severe lung damage (Patients with good lung recovery had higher baseline sputum MPO (p = 4.70E−02) and IL10 (p = 2.70E−02) levels compared to patients with poor recovery  from baseline to month 6 in patients with good lung recovery but not in those with poor recovery (vs mild lung damage or high vs low Mtb loads at any time point (not shown).In whole blood stimulated supernatants, with the exception of IFNγ , IL12/23(p40)  and S100A8  concentrations which were higher in mild compared to severe lung damage; MMP1  which was higher in high compared to low Mtb load and; S100A8  which was higher in low compared to high Mtb load, there were no other significant differences in inflammatory mediator levels of whole blood stimulated samples  from patients in severe ). Additionally, sputum levels of MMP9 declined significantly from baseline to month 6 in patients with low Mtb load (p = 3.20E−03) but not in patients with high Mtb load  at month 6 , for which neutrophils are not necessarily the major sources.via an MPO-dependent process  and endothelial cells have been shown to cooperate in detection and capture of pathogens in lung capillaries .We understand that functional analysis of neutrophils is technically difficult considering that they are short-lived, easily activated by laboratory processing methods and cannot be cryopreserved. Nevertheless, we support future investigation of mechanistical pathways that promote the secretion of these mediators or increased production of neutrophil subsets that produce them to achieve desirable outcomes during ATB. We could not confirm the link between MPO and bacterial burden using Mtb killing assays Variability in immune responses between genders have been linked to: specific immune cell types, age, levels of sex hormones, environmental factors  and disease states . In accoMoreover, patients showing good lung recovery had higher sputum MPO concentrations at baseline. In contrast, MMP8 and sputum TNF levels were positively associated with poor recovery after adjusting for sex. These suggest that MPO, MMP8, and TNF play a considerable role in determining the degree of recovery from severe TB-related lung damage after treatment. It also supports future investigation of these mediators as proxies for predicting lung recovery following injury.As treatment progresses, sputum and plasma concentrations of MMP1, 8, 9 and plasma levels of S100A8/9 and MMP3 decrease rapidly, suggesting that the neutrophil-related inflammatory response and matrix-degrading activity are not only fuelled by MMPs , 33, 67), 6733, 6Post-therapy, we observed high plasma S100A8 levels in severe compared to mild lung damage group, meanwhile plasma S100A8 and sputum MMP9 were significantly higher in patients with initially high Mtb load compared to the initially low Mtb load group. S100A9 and MMP9 are neutrophil-derived mediators, suggesting that severe lung damage at presentation may contribute to heightened residual neutrophil activity after treatment. Also, post-treatment levels of sputum MMP1, MMP9, and IL8 were higher in patients with poor lung recovery compared to those with good lung recovery. This suggests that unresolved lung damage is linked to continuous neutrophil activity and persistent leucocyte infiltration in the lungs post-treatment. While, previous studies suggested that for patients with severe lung damage, recovery may only begin many months after the end of standard ATT , 69, a pvia T-cell exhaustion which leads to either higher levels of immune-inhibitory molecules like PD-1 (For whole blood stimulated supernatants (notably with H37Rv whole cell lysate), the increased levels of GM-CSF , TNF , IFNγ, S100A8, MPO and MMP9 after treatment compared to baseline hint at an enhanced sensitivity of immune cells to pathogen stimulation. Previous studies have reported lower cytokine production by T-cells pre-treatment, suggesting that continual pathogen stimulation results in T-cell exhaustion which is then restored after treatment  at presentation. We hereby highlight the relationship between neutrophil-derived inflammatory mediator levels and radiological disease severity irrespective of Mtb burden. We also report that S100A8/9 and other neutrophilic mediators like MMP9 and IL8 may be responsible for unresolving lung damage in patients with poor lung recovery. Finally, we recommend targeting S100A8/9, MMP8, and MPO for developing host-directed therapies against TB-induced lung pathology and to promote recovery.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Medical Research Council/Gambia government joint ethics committee (SCC1523). The patients/participants provided their written informed consent to participate in this study.CN: Conceptualisation, Data curation, Formal analysis, Investigation, Methodology and writing of manuscript. OO: Patient recruitment and follow-up, clinical data, and review of manuscript. SD: Data Management. JM: Assisted with wet-lab experiments. IS: Assisted with wet-lab experiments. SC: Funding acquisition and review of manuscript. AB: Data curation and Formal analysis. AR: Conceptualisation, Funding acquisition, data analysis, and review of manuscript. CG: Supervision, Methodology, and manuscript review. JS: Supervision, Conceptualisation, Data curation, Methodology, Funding acquisition, and review of manuscript. All authors contributed to the article and approved the submitted version.This work was supported by a PhD student stipend from TB Sequel (grant number 66.3010.7-002.00) funded by the German Ministry for Education and Research (BMBF).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Given this effect of purinergic receptors on the viability of dopaminergic neurons model cells and that Ras GTPases control Erk1/2-regulated mitogen-activated cell proliferation and survival, we have investigated the role of the small GTPases of the Ras superfamily, together with their regulatory and effector molecules as the potential molecular intermediates in the P2X7R-regulated cell death of SN4741 dopaminergic neurons model cells. Here, we demonstrate that the neuronal response to purinergic stimulation involves the Calmodulin/RasGRF1 activation of the small GTPase Ras and Erk1/2. We also demonstrate that tyrosine phosphatase PTPRβ and other tyrosine phosphatases regulate the small GTPase activation pathway and neuronal viability. Our work expands the knowledge on the intracellular responses of dopaminergic cells by identifying new participating molecules and signaling pathways. In this sense, the study of the molecular circuitry of these neurons is key to understanding the functional effects of ATP, as well as considering the importance of these cells in Parkinson’s Disease.ATP, one of the signaling molecules most commonly secreted in the nervous system and capable of stimulating multiple pathways, binds to the ionotropic purinergic receptors, in particular, the P2X Anti-RAP1 sc:65 antibody (Dil. 1:1000) was obtained from Santa Cruz Biotechnology, Inc. ; rabbit monoclonal anti-Erk1/2 (Dil. 1:1000) and anti-phospho-Erk1/2 (Dil. 1:1000) were obtained from Cell Signaling Technology . Monoclonal Anti-β-Actin antibody produced in mouse (Dil. 1:5000) were obtained from Cell Signaling Technology . The Clarity enhanced chemiluminescence (ECL) reagent was obtained from BioRad Laboratories Inc .2 atmosphere and a 95% relative humidity, as indicated by Son et al. [6 cells/200 µL of complete medium previously chilled on ice. For each experimental point, 15 ng of esiRNA  were added to a previously cooled electroporation cuvette and then 200 µL of the cell suspension were added. Electroporation was performed in a Gene Pulser Xcell Electroporator (Bio-Rad) at 260 V and 950 µF [SN4741 cells were cultured in DMEM , supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), 100 units/mL of penicillin, and 100 ng/mL of streptomycin (Sigma-Aldrich) at 33 °C in a 5% COn et al. . For tra2 atmosphere and a 95% relative humidity. A low passage number (0 to 20) of cell lines were maintained in 100-mm plates for adherent cells . For plasmid and esiRNA transfections, HEK293 cells were seeded at 300.000 cells/well. The transfection was performed using XtremeGENE HD Roche  transfection reagent following manufacturer′s recommendations. To silence endogenous expression of human hPTPRβ to inhibit endogenous microRNA activity, cells were transfected with commercial esiRNAs following manufacturer’s recommendations. MISSION® esiRNA targeting EGFP (reference EHUEGFP) (EGFP was used as a negative transfection control because it was lacking in our cellular system) was used, as well as MISSION® esiRNA targeting human hPTPRβ (reference EHU158501)Human embryonic kidney (HEK) 293 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 1% Penicilin/Streptomicin, and 1% Sodium pyruvate at 37 °C in a 5% CORas and Rap1 pull-down assay was performed using a GST fusion protein containing the RAP1-binding domain of Ral guanine nucleotide dissociation stimulator  . Rac1 pu2, 1 mM PMSF, 10 μg/mL aprotinin, 10 μg/mL leupeptin) [Cells were transfected with the indicated constructs or previously treated with specific inhibitors and, finally, lysed in 500 μL of lysis buffer  . Cell ly3 cells/well in a 96-well plate. Then, cells were previously treated (+), or not (−), with A-740003, GF109203X hydrochloride, or sodium orthovanadate. The next day, cells were treated, which was stopped by removing the medium and washing cells with cold 1X PBS. One percent FBS medium was added to avoid cellular proliferation during the test. Cell viability was measured with CellTiter 96 Non-Radioactive Cell Proliferation Assay (MTT) following the manufacturer’s instructions : Briefly, 10 µL MTT reagent (MTT labeling reagent) was added to the fresh medium in each well. The plate was incubated at 33 °C in 5% CO2 for 4 h, and the reaction was stopped by adding 100 µL of kit solubilization solution to each well overnight at 33 °C, and 5% CO2. The absorbance was measured in a microplate reader ) at the wavelength of 570 nm. The values obtained were transformed into percentages, setting the value of 100% viability to the net absorbance of control cells. The rest of the values corresponding to different treatments are presented as percentages over the control.SN4741 cells were seeded in triplicate at 30 × 10To verify the MTT assay, we counted with a Neubauer counting chamber with cover glasses (dimensions are 24 mm L × 26 mm W × 0.4 mm H) (Blau brand). Cells were counted under the microscope. Sample preparation was done by an experienced senior laboratory personnel researcher. In order to reduce the errors caused by subjective central tendency. The samples were diluted with Trypan blue solution (Sigma-Aldrich) in a normal phosphate buffer saline at 1:5 ratio.p < 0.05, ** p < 0.01, *** p < 0.001).All data are presented as mean ± S.E.M. Statistical analysis was performed using GraphPad Prism statistical software ). Comparisons between multiple experimental groups were made using one-way analysis of variance (ANOVA), followed by Bonferroni’s post hoc test. To determine the significance between data means, (*