Chitiniphilus and Chitinolyticbacter were enriched significantly. A novel LPMO (M2822) was found in the metagenome of the enriched microbiota. Phylogenetic analysis showed that M2822 had a unique phylogenetic position in auxiliary activity (AA) 10 family. The analysis of enzymatic hydrolysate showed that M2822 had chitin activity. When M2822 synergized with commercial chitinase to degrade chitin, the yield of N-acetyl glycosamine was 83.6% higher than chitinase alone. The optimum temperature and pH for M2822 activity were 35°C and 6.0. The synergistic action of M2822 and chitin-degrading enzymes secreted by Chitiniphilus sp. LZ32 could efficiently hydrolyze shrimp shell powder. After 12 h of enzymatic hydrolysis, chitin oligosaccharides (COS) yield reached 4,724 μg/mL. To our knowledge, this work is the first study to mine chitin activity LPMO in the metagenome of enriched microbiota. The obtained M2822 showed application prospects in the efficient production of COS.Lytic polysaccharide monooxygenases (LPMO) are expected to change the current status of chitin resource utilization. This study reports that targeted enrichment of the microbiota was performed with chitin by the selective gradient culture technique, and a novel LPMO (M2822) was identified from the enrichment microbiota metagenome. First, soil samples were screened based on soil bacterial species and chitinase biodiversity. Then gradient enrichment culture with different chitin concentrations was carried out. The efficiency of chitin powder degradation was increased by 10.67 times through enrichment, and chitin degradation species  Chitin is the second-most abundant biosynthetic bio-polysaccharide after cellulose and is found in large quantities in shrimp and crab seafood waste . Most of2O2 production from LPMOs  of the helper activity (AA) family. Each family has a different biological origin and substrate specificity . LPMOs pStreptomyces griseus show the catalytic facilitation of several chitinases  were used in the sub-cloning and expression of the LPMO gene from the metagenome, respectively. The pET-22b(+) plasmids were used as the expression vector of the LPMO gene. The chitin oligosaccharides (polymerization degrees 2 to 6) were acquired from Qingdao HEHAI Biotech Co., Ltd. .The 2,6-Dimethoxyphenol , chitinase and chitin powder (CP) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. . The shrimp were purchased from a local seafood market to obtain shrimp shells, washed thoroughly with tap water to remove residual shrimp meat, and dried in a vacuum oven at 60°C for 24 h. The dried material was pulverized in a disintegrator and then passed through a 40 mesh sieve to produce shrimp shell powder (SSP). Preparation of β-chitin according to the 2HPO4, 0.3 g/L KH2PO4 and 0.5 g/L MgSO4, and chitin. The gradient acclimation was carried out with chitin content of 1 g/L, 2 g/L, and 4 g/L.Soil samples were collected in Liaocheng, Shandong, China . 1 g soil sample was inoculated into a 250 mL flask containing 50 mL enrichment medium and incubated at 30°C and 180 rpm. The culture was finished when the CP in the fermentation broth was degraded to be invisible to the naked eye. The next batch of enrichment culture was carried out with 15% inoculation until the degradation of CP could be completed within 3 days. The enrichment medium comprised of 2 g/L peptone, 1 g/L glucose, 0.7 g/L KThe total DNA of soil samples was extracted by E.Z.N.A. soil DNA kit . After quality inspection and quantification, the V3-V4 region of 16S rRNA was amplified by PCR using 338F (5´-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5´-GGACTACHVGGGTWTCTAAT-3′). The conserved region of chitinase was amplified by combining primers ChiF (5´-CGTGGACATCGACTGGGARTWYCC-3′) and ChiR (5´-CCCAGGCGCCGTAGARRTCRTARSWCA-3′). PCR products were detected, quantified and sequenced by the Miseq PE300 platform of Illumina Company. The fastp software was used for quality control of original sequencing sequences, and Flash software was used for splicing. Using Usearch software,The entire DNA of the enrichment bacterial community was extracted using the E.Z.N.A. soil DNA kit. After concentration, purity and integrity testing, ultrasound treatment was performed, and a 450 bp insertion library was constructed. The fastp software is used to filter the original data, megahit is used to mix and splice multiple samples, bowite2 is used to extract unmapped reads, and SPAdes is used to splice again to get more complete contigs assembly results. Prodigal was used to predict the ORF of splicing results, and CD-HIT software was used to obtain non-redundant gene sets. Phylogenetic trees were drawn with GraPhlAn . Use theInterProScan was used to search and analyze conserved domains and feature sequences. Signal peptide analysis was performed with signal P5.0 server. The map of the protein domain was drawn by IBS 1.0.3 software. Phylogenetic analysis was performed using MEGA7 software and optimized and annotated on the iTOL web server. ClustalX software was used for sequence alignment between full-length M2822 and five characteristic LPMO10s. Sequence similarity and secondary structure analyses are carried out using ESPRIPT 3.0 network server. Modelling structural homology in dense modelling mode using a Phyre2 server . The modE. coli BL21 (DE3) to construct M2822 expression strain E. coli BL21-M2822. It was inoculated into a new LB medium containing ampicillin (50 μ g/ml) and cultured at 37°C at 200 rpm until the OD600 nm value was 0.6. The culture was cooled to 15°C and left standing for 30 min. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to the final concentration of 0.5 mmol/l, cultured at 15°C for 8 h, centrifuged at 8000 rpm for 10 min to collect the cells. Extraction of M2822 protein from the periplasm of E. coli BL21-M2822 using an osmotic shock method . The PCR fragment was inserted between pelB and His pET-22b(+) vector tag to construct recombinant plasmid pET-22b-m2822. The recombinant plasmid was transformed into k method . The solThe chitin degradation activity of M2822 was carried out according to our previous study and made minor modifications . The reaMicrocrystalline cellulose (MCC), powdered α-chitin, powdered β-chitin and colloidal α-chitin (CC) were added into M2822 solution at a concentration of 10 g/L, respectively, incubated at 25 ° C and 200 rpm for 30 min, then centrifuged at 6000 rpm for 10 min to collect the supernatant. The protein content of the solution before substrate addition and the supernatant after incubation was examined using the BCA protein assay kit. The binding ability of M2822 to the insoluble substrate was evaluated by the decrease of M2822 in the supernatant.2O2 and 0.143 mg/mL M2822. The samples were incubated at 30°C under 300 rpm shaking for 12 h, and samples were taken to detect at selected time points. The content of N-acetyl glucosamine (GlcNAc) produced by enzymatic hydrolysis was quantitatively detected according to the HPLC detection method of A synergistic reaction system of M2822 and commercial chitinase was established in Britton-Robinson buffer (pH 6.8) using powder α-chitin as substrate. The reaction system contained 10 mg/ml chitin, 1 mg/mL chitinase, 100 μ mol/L H2O2 as substrate according to the method of 2O2 substrates in the reaction system. The additional amounts of 2, 6-DMP and H2O2 in the reaction system were determined by optimization. The 200 μL reaction system contains 2.5 μM M2822, 100 μm H2O2 and different concentrations of 2, 6-DMP (1–50 mmol/L). After incubation at 30°C for 15 min, the M2822 was added to the enzyme-labelled plate, and then the reaction was carried out for 300 s at 30°C. The absorbance value increment of the reaction product coerulignone at 469nm was measured. The amount of enzyme needed to produce 1 μM coerulignone in 1 min was defined as one enzyme activity unit (U). Under the condition of 5 mmol/L 2, 6-DMP, the optimal conditions were obtained by changing the concentration of H2O2 (10–500 μ mol/L).The activity of M2822 was determined with 2, 6-dimethylphenol  and H2O2 as substrates in Britton-Robinson buffer at pH 6.8 in the temperature range of 25–50°C. The highest enzyme activity determined is defined as relative 100%. To evaluate the thermal stability of M2822, it was treated at different temperatures  for 8 h, and samples were taken at selected time points to detect the residual activity of M2822. The initial activity of m2822 was 100% in each condition. According to In order to investigate the temperature effect on M2822 activity, the enzyme reaction was performed with 5 mM 2,6-DMP and 0.1 mM H2O2 were determined under the optimum conditions. 2.5 μM M2822, 5 mmol/L 2, 6-DMP and different concentrations of H2O2 (5–500 μmol/L) were incubated at 35°C for 300 s in the Britton-Robinson reaction system at pH 6.0.The kinetic parameters of M2822 for HC. sp. LZ32 (ChiMix32) was prepared according to the previous method  and glycoside hydrolase family 18 chitinase degenerate primers (ChiF and ChiR) . Three sChromobacteriaceae, Family Neisseriaceae, Genus Chitiniphilus, and Genus Chitinolyticbacter accounted for 99.49, 96.35, 90.40, 61.10, 29.07, 49.70, and 28.61%, respectively.The macro genome of LNM32 generated 1.597 Gb of original sequence data. After data quality control, clean reads splicing and assembly, and gene prediction, a non-redundant gene set was constructed. The number of genes was 3,643, and the N50 value was 1,203 bp. The abundance of each gene was calculated and counted. The high-throughput sequencing of the LNM32 metagenome showed that all species in LNM32 were bacteria, of which phylum Proteobacteria, Class Betaproteobacteria, Order Neisseriales, Family Chromobacteriaceae and Family Neisseriaceae is 61.10 and 29.07%, respectively  and C. shinanonensis DSM 23277 (GCA_000374805. 1) were analyzed by dbCAN. The results showed that both strains had only one AA10 family LPMO. Therefore, AA10 family LPMO may play an essential role in the degradation of chitin by LNM32. M2822 was selected for further study.Species taxonomic hierarchy annotation results showed that all species in LNM32 were bacterial, among which phylum Proteobacteria, Class Betaproteobacteria, and Order Neisseriales account for 99.49, 96.35, and 90.40% of their taxonomic hierarchy, respectively . Under Oectively . At the ectively . The numectively . Some stectively . The CAZectively . Furtherectively . Given tC. shinanonensis (BDB32680.1), and also showed 56.72% identity (99% query cover) to a LPMO from C. piscinae (WP_194115059.1). These LPMOs have not been further studied. A phylogenetic analysis of M2822 and previously reported typical AA10s , and chitin-binding domain of chitinase C (amino acids 377–425), and chitin-binding domain of Chi A1-like proteins (amino acids 476–522). Based on the function of each conserved domain, M2822 was predicted to be an LPMO with chitin activity in the AA10 family. The Phyre2 server  was transformed and induced to be expressed by IPTG. The M2822 protein (54 kDa) was obtained by osmotic impingement method  2 to 7 aldehyde acid. Similar results were reported in other studies on LPMOs (The nucleotides of the M2822 gene were inserted into the pET-22b(+) vector , and E. t method , and purt method . Enzymatose acid . For exaon LPMOs . TherefoPurified M2822 was incubated with excess microcrystalline cellulose (MCC), α-CP, β-CP, and colloidal α-chitin (CC), and the content of free protein after the binding was detected to investigate the ability of M2822 to bind to insoluble substrates. As shown in The synergistic effect of M2822 on chitinase in the process of chitin degradation was verified by detecting the amount of GlcNAc produced in the reaction system. Synergistic experiments showed that the degradation of CP by chitinase was significantly improved in the presence of M2822 . When M22O2 concentration was increased under the condition of 5 mM 2,6-DMP, the specific activity that M2822 could detect will also be increased. When the H2O2 concentration was 200 μM, the specific activity detected was only slightly higher than 100 μM  were prepared by combining M2822 with chitin-degrading enzymes. Considering the high cost of commercial chitin-degrading enzymes, the enzyme used to prepare CDEC is ChiMix32, which is a crude enzyme secreted by f 2 to 6 . Three sf 2 to 6 . Comparef 2 to 6 . AlthougE. coli, and its enzymatic properties were characterized. Enzyme cocktails composed of M2822 and chitin-degrading enzymes could degrade SSP into the COS of PD2-6. M2822 has significant application potential in the biorefinery of chitin-rich biomass.This study obtained an enrichment microbiota with a high degradation performance of powdered chitin through the gradient enrichment of soil samples. A novel AA10 family LPMO M2822 was successfully captured. M2822 could cooperate with chitinase and significantly improve the degradation efficiency of powdered chitin. M2822 was expressed in The original contributions presented in the study are included in the article/YZ: investigation, project administration, resources, and writing – review and editing. DP: investigation, data curation, and writing – review and editing. PX: investigation, formal analysis, and writing-review and editing. QX: investigation, and writing – review and editing. FG: formal analysis, and writing – review and editing. XZ: investigation, writing – review and editing, supervision, investigation, formal analysis, and writing – original draft. HX: supervision and investigation. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (No. 32100065), the Natural Science Foundation of Shandong Province of China (No. ZR2018QC002), and the Doctoral Research Startup Foundation of Liaocheng University (No. 318052041).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.


Early-onset Parkinson’s disease (EOPD) labels those cases with onset earlier than fifty. Although peculiarities emerged either in clinical or pathological features, EOPD is managed alike typical, late-onset PD. A customized approach would be, instead, better appropriate. Accordingly, a deeper characterization of the clinical course, with an estimation of the disease progression rate, the therapy flow, and the main motor and non-motor complications occurrence, is needed.A longitudinal cohort of 193 EOPD patients (selected on a single-centre population of 2000 PD cases) was retrospectively analysed, providing descriptive statics on a series of clinical parameters  and modelling the trajectories from diagnosis to 10 years later of both Hoehn and Yahr (H&Y) stage and levodopa equivalent daily dose (LEDD).EOPD had a prevalence of 9.7%, including few monogenic cases. It mostly appeared as a motor syndrome, with asymmetric, rigid-akinetic presentation. H&Y linearly progressed with an increment of 0.92 points/10 years; LEDD flow had a non-linear trend, increasing of 526.90 mg/day in 0–5 years, and 166.83 mg/day in 5–10 years. Motor fluctuations started 6.5 ± 3.2 years from onset, affecting up to 80% of the cohort. Neuropsychiatric troubles interested the 50%, sexual complaints the 12%. Gender-specific motor disturbances emerged.We shaped EOPD course, modelling a “brain-first” PD subtype, slowly progressive, with non-linear dopaminergic requirement. Major burden mostly resulted from motor fluctuations, neuropsychiatric complications, sexual and marital complaints, with a considerable gender-effect. Parkinson disease (PD) is a disabling, neurodegenerative disorder due to the loss of dopaminergic nigral cells and the brain accumulation of alpha-synuclein containing Lewy bodies. PD commonly occurs in elderly, and, more rarely, in younger subjects (before the fifty), as early-onset PD (EOPD) , 2.EOPD may include either monogenic forms or, mostly, sporadic cases. If EOPD differs from typical late-onset PD (LOPD) is still matter of debate. There are data suggesting distinctive features for these two conditions, regarding both the pathogenesis and the clinical course , 4. EvenThis study aims to shape EOPD course, from onset to later disease phases, employing real-world data from a single longitudinal cohort, quantifying epidemiological and clinical features, progression of motor disturbances, changes in therapeutic regimen and main complications overall. Establishing such trajectories would be crucial either to improve understanding and management of EOPD patients or construct future observations.We performed a retrospective longitudinal study following recent standardized guidelines . MedicalFor each EOPD patient, we collected AOO, gender, family history of PD (presence of at least one affected relative up to second degree), motor phenotype at onset  diagnosed according the UK Brain Bank Criteria [To allow comparative analysis, a control group of Criteria  or the 2Criteria  was alsoCriteria . For eacMedical charts reviewed for the study were all filled in by movement disorders specialists. Motor and non-motor conditions were diagnosed following current criteria. In particular, for neuropsychiatric disturbances, the DSM-V definitions were used.The study was conducted in agreement with principles of Helsinki declarations. Local ethical committee approved the study.t test. Correlations analysis was performed by Pearson’s test. Statistical significance was set at p < 0.05. Analysis was conducted by means IBM-SPSS 26.Descriptive statics  was run on both qualitative and quantitative variables. Frequencies among the groups were compared by Chi square test. Kolmogorov–Smirnov test showed that quantitative variables had a normal distribution. The rate of disease progression, in terms of H&Y score variation over 10 years, was estimated by linear regression. The flow of therapy was also outlined, by using the LEDD variation over 10 years into a quadratic regression analysis, as it better fits compared to a simple linear model. The slope of the regression models was then compared using the We identified 193 EOPD patients on a whole PD population of 2000 cases . Age at onset (mean ± st.dev), gender distribution, frequency of main comorbidities and motor features at onset have been summarized in Table p < 001). In LOPD patients, male to female ratio was 1.6:1 , with no significant difference compared to the ratio in EOPD (1.2:1). Mean age and disease duration of the LOPD group were 80.2 ± 3.9 years and 10.0 ± 3.1 years respectively. Regarding comorbidities, diabetes  and blood hypertension  were more prevalent in LOPD than EOPD . Further details in Fig. In the LOPD group, AAO was 70.2 ± 5.0 years (range 54–87), significantly different from the one of EOPD , tremor dominant phenotype , mixed phenotype . Presenting motor signs have been reported in Table According to standard subtypes classification of Rajput et al. , 178 outT0 = 1.51 ± 0.52; H&YT5 = 1.92 ± 0.32; H&YT10 = 2.43 ± 0.45). Motor progression in terms of H&Y score change over 10 years has been modelled by linear regression, estimating a R2 coefficient of 0.29 (p < 0.0001) .For 47 patients, longitudinal H&Y scores at diagnosis (T0), 5 (T5) and 10 years (T10) later were available  were suffering with RBD and 56 (29.0%) with constipation, mostly in association. Along the entire disease course (from onset to last visit), 50.8% of patients developed neuropsychiatric symptoms, including depression, anxiety, apathy and psychosis. Three patients (1.6%) attempted suicide. Twenty-one patients (10.9%) had cognitive impairment and 16 patients (8.3%) visual hallucinations.p < 0.0001 and p < 0.0001, respectively). Occurrence of neuropsychiatric manifestations did not differ between the two groups (56.5% in LOPD vs 50.8% in EOPD).In LOPD group, cognitive impairment occurred in 84 patients (49%) and visual hallucinations in 54 (31.4%), a prevalence significantly higher than in EOPD , monoamine oxidase (MAO) B inhibitors  or a combination of them . Levodopa was the first choice in 19 cases (13.5%), in monotherapy (n = 17), associated to DA (n = 1) or to MAO B inhibitor (n = 1). In general, levodopa was later introduced (4.5 ± 3.8 years from disturbances onset or 2.9 ± 3.3 years from the diagnosis), being finally used in 148/171 patients .For 141/193 EOPD patients, data on initiation therapy were available. Patients mostly received dopamine agonists (DA) . The flow of therapy in terms of LEDD value changes over 10 years has been modelled by quadratic regression, estimating a R2 coefficient of 0.701 (p < 0.0001) . Mean difference between T10 and T5 LEDD was not statistically significant (p = 0.11).For 60 patients, longitudinal LEDD values at diagnosis (T0), 5 (T5) and 10 years (T10) later were available .In LOPD patients with 10.0 ± 3.8 years of disease duration, LEDD was 833.86 ± 333.34 mg/day, which was significantly higher than LEDDp = 0.003), but no gender differences resulted when considering the time interval from disease onset .Motor fluctuations affected 77.7% of 148 EOPD patients under levodopa, having been appeared 6.5 ± 3.2 years from the disease onset and 1.8 ± 1.7 years from levodopa initiation. LIDs affected 60.8% of 148 levodopa users, 6.7 ± 3.2 years after disease onset and 2.6 ± 2.4 after levodopa initiation. LIDs occurred closer to levodopa initiation in females than males .In LOPD group, 180 patients were treated with levodopa. LIDs occurred in 88 of them (48.9%), a prevalence lower than in EOPD (Impulse control disorder (ICD) occurred in 72 EOPD patients, mostly gambling Figs. .Fig. 2Ven = 13, 76.5%), while 23.5% received two DATs.Advanced therapies  were proposed to 26 out 148 EOPD patients under levodopa (15.2%). Nine refused. The remaining 17 mostly underwent STN-DBS , impotence , reduced sexual desire  or anorgasmia . Four patients had both hypersexuality and impotence. One patient attempted unsuccessfully pregnancy. Three out 86 female patients (3.41%) complained with worsening of motor symptoms during menses.Along the disease course, 4/171 EOPD patients divorced , while two became widowed. Five patients (2.9%) developed a delirium of jealousy . Sexual difficulties were reported by 24/193 patients (12.4%), namely hypersexuality (PRKN gene (parkin), six (33%) heterozygous for pathogenic variants in LRKK2 gene (leucine rich repeat kinase 2), five (28%) heterozygous for pathogenic variants in GBA gene (acid beta glucocerebrosidase) and one (6%) homozygous for pathogenic variants in PINK1 gene (PTEN induced putative kinase 1). Five patients carried variants of unknown significance (VUS).In EOPD patients, a genetic origin was ascertained in 9.3% of cases (18/193 patients). Six patients (33%) were homozygous for pathogenic variants in This study analysed main features of a large, single-centre, longitudinal cohort, to shape EOPD and disclose those elements that might drive a customized approach for such a burdening condition, affecting people in the prime of its life. EOPD is felt as an emerging problem, because of the worldwide increase of frequency . In our The 9.3% of our EOPD cases were pure inherited forms (monogenic PD), in agreement with existing literature . NeverthPhenotype at onset of our EOPD patients mostly matched the rigid-akinetic motor subtype (50%), being the tremor-dominant and the mixed one little rarer, as noticed before . PD motoEOPD patients were diagnosed 1.52 ± 1.64 years after symptoms onset, and mostly received therapy with DAs or MAO B inhibitors . DAs, indeed, represent the most preferred starting treatment and, in general, the most frequently prescribed drugs in EOPD patients , althougOur EOPD patients have been longitudinally followed-up in average for 2.75 ± 4.1 years, with the first visit 5 ± 5.07 years after onset and the last one 9.3 ± 5.8. Prospective observation allowed us tracking the evolution of motor disturbances, the flow of dopaminergic therapy and the occurrence of a series of non-motor disturbances or disease-related troubles. Precisely, we could estimate on a subgroup of patients the rate of H&Y score progression from diagnosis to 10 years later, modelling a linear growth. We measured that H&Y score increased of 0.41 points in the first 5 years and of 0.51 points in the successive 5 years . Previous studies, even in naturalistic, real-life contexts, showed that PD patients with typical LOPD basically gain one point of H&Y score every 2 years, with a faster progression in those patients with later or more severe disease onset . EOPD paT5–T0 increase = 526.90 mg/day), followed by a substantially slower increase at later phases of the disease (LEDDT10−T5 increase = 166.83 mg/day), predicting an overall LEDD of 693.73 mg/day at 10 years from onset. Our LOPD patients had significantly higher LEDD at about 10 years of disease duration, but we could not track the trend over time because of the missing follow-up.The flow of dopaminergic therapy, in terms of LEDD value variation, exhibited a non-linear trend (modelled by quadratic regression) in EOPD patients. We observed an early, rapid increase of the dopaminergic dosage  or, rather, to the occurrence of motor fluctuations, and LID especially. In fact, motor fluctuations overall, and LID specifically, affected up to 80% and 60% of EOPD patients respectively (a percentage significantly higher than LOPD), appearing about 6 years after disease onset or a couple of years from levodopa initiation. The faster LEDD increase might contribute to fluctuations and LID onset of EOPD patients , althougNeuropsychiatric symptoms  affected up to 50% of patients along the entire disease course, while three patients (1.6%) attempted suicide. EOPD patients are very prone to depression and anxiety  and may EOPD may also burden on married life and sexuality, although the real prevalence of these problems could be often underestimated, because of embarrassment or discretion in referring to physicians . We founDifferently from LOPD, EOPD involves fertile age, complicating menses, pregnancy, birthing and breastfeeding. In our cohort, three female patients complained with worsening of motor disturbances during menses, which is in line with some previous reports , althougThis study has several limitations, including the retrospective design and the related bias, some lacking data, the poor assessment of non-motor disturbances and the absence of a control group. We also merged both sporadic and inherited cases, which indeed may exhibit some distinctive trait. Nevertheless, we provided a series of real-world data from a longitudinal cohort supporting a separate approach to EOPD, as a clinical entity distinct from classical LOPD.This study provided several useful real-world data regarding EOPD. First, we found that the prevalence of EOPD can be higher than expected from previous epidemiological predictions. Most of the cases were sporadic, although genetic test has not been performed systematically to the entire population. Phenotype at onset of EOPD basically consisted of an asymmetric rigid-akinetic syndrome with a low burden of constipation and RBD, which indeed seems to suggest a central origin of neuropathology (“brain-first” PD subtype). Then, we modelled either the disease progression, showing a slow, linear evolution (0.9 H&Y score points per decade), or the flow of the dopaminergic therapy, outlining a non-linear progression with a faster LEDD increase in the first years of the disease (526.90 mg/day) and a milder increase later (166.83 mg/day), probably reflecting motoric needs and complications. Therefore, we drew a clinical course typically characterized by motor fluctuations, neuropsychiatric disturbances, ICD and other sexual or marital troubles. Finally, we provided some comparisons with LOPD patients, showing peculiar differences regarding the therapy, the complications and the comorbidities.All these data might be relevant to develop a customized approach to EOPD. In particular, we provided meaningful information for possible trial or preventive strategies design, including the progression rate of motor impairment and therapy flow, as well as the potential “brain-first” origin of EOPD. Then, we identified several critical hints to drive therapeutic choices, such as prevalence of motor and non-motor complications and the need for adequate psychological support for social and familial issues. On the other hand, further themes deserving appropriate investigations emerged, such as those related to pathogenesis or gender differences.


The toxic effect of doxorubicin on the heart limits its clinical usage in cancer therapy. This work intended to investigate, for the first time, the efficacy of rifampicin administration against doxorubicin-induction of cardiotoxicity in mice. Forty adult male albino mice were distributed into four sets: Control, Doxorubicin, Doxorubicin + Rifampicin 0.107, and Doxorubicin + Rifampicin 0.214, with n = 10 for each. Heart histopathology and biochemical assays for heart function tests , oxidative stress [malondialdehyde (MDA) and superoxide dismutase (SOD)], and minerals  were done.p < 0.001, as well as increases in cTnI and ANP levels, p < 0.001, increased oxidative stress , high minerals , with reduced VEGF concentration, p < 0.001, and low antioxidant  were observed in the Doxorubicin group compared to control. Co-treatment with rifampicin significantly (p < 0.001) reduced the increased oxidative stress, high Na and K, increased LDH, CK, AST, cTnI, and ANP, and elevated the low SOD toward the normal ranges. Our histological data supported our biochemical data; rifampicin dose 0.214 mg/kg showed better improvements than dose 0107.Doxorubicin-induced cardiotoxicity using a total dose of 15 mg/kg was confirmed histologically. Cardiomyocytes showed congestion, necrosis, edema, and inflammatory cell infiltration. Biochemically, elevations in LDH, CK, and AST activities, Our results demonstrated that rifampicin could help protect the body against doxorubicin-induced cardiotoxicity through its antioxidative effect. Cardiovascular diseases are the chief cause of death globally; they were responsible for 32% of all deaths in the globe in 2019. Approximately 75% of deaths were in low- and middle-income nations . Many reStreptococcus peucetius var. caesius. It is commercially known as Adriamycin, Doxil, Rubex, widely utilized as a chemotherapeutic to treat various types of solid tumors. However, the doxorubicin drug's severe side effects, such as cardiotoxicity, hepatotoxicity, nephrotoxicity, and neurotoxicity, limit its clinical use  and Rifactine (300 mg rifampicin)  were used. Meanwhile, nitro blue tetrazolium (≥ 90%), nicotinamide adenine dinucleotide reduced form , sodium pyrophosphate tetrabasic (≥ 95%), phenazine methosulfate (≥ 90%), trichloroacetic acid (≥ 99%), and thiobarbituric acid (≥ 98%) were obtained from Sigma-Aldrich, Saint Louis, MO, United States. Perchloric acid (70%) was bought from LOBA CHEMIE PVT.LTD, Mumbai, Maharashtra, India. Absolute ethanol (99.9%) was acquired from Alfa Chemical Group, Hadayek El-Kobba, Cairo, Egypt. In addition, potassium dihydrogen orthophosphate (98–100%) and sodium chloride saline solution (0.9%) were purchased from ELNASR PHARMACEUTICAL CHEMICALS, Qalyubia, Egypt. Di-potassium hydrogen phosphate (99%) was bought from HiMedia Laboratories C40, Maharashtra, India. All other solvents and chemicals obtained for the experiments were of the highest available purity and were used as received.Forty adult male Swiss albino mice (body weight of 20–25 g) obtained from Theodor Bilharz Research Institute, Giza, Egypt, were used. Mice were housed at a temperature of 23 ± 2 °C, appropriate humidity, and 12:12 h light/dark cycle following the standards summed up in the “Guide for the Care and Use of Laboratory Animals” designed by the National Academy of Science and published by the National Institute of Health. Mice were accommodated for one week prior using in the experiment. They were kept in clean polypropylene cages and fed with standard mice pellet diet and water ad libitum.To induce cardiotoxicity in mice, a cumulative dose of 15 mg doxorubicin/kg was used , 17 as fBecause rifampicin is an already-approved drug used in clinics to treat patients with doses up to 300–600 mg/day (5–10 mg/kg). Moreover, because drug adverse events are dose-related and for safety purposes, two rifampicin doses equal to 0.107  and 0.21Practically, rifampicin powder was first dissolved as a stock solution of 1 mM concentration in absolute ethanol, and the volume corresponding to 0.107 mg/kg or 0.214 mg/kg was diluted in saline  just before injection.Control (negative control): normal healthy miceDoxorubicin : doxorubicin 5 mg/kg (three doses); i.p. on the 1st, 5th, and 9th dayDoxorubicin + Rifampicin 0.107 (first treated cardiotoxicity group with rifampicin): doxorubicin 5 mg/kg (three doses); i.p. on the 1st, 5th, and 9th day + rifampicin 0.107 mg/kg/day; i.p., for 14 daysDoxorubicin + Rifampicin 0.214 (the second treated cardiotoxicity group with rifampicin): doxorubicin 5  mg/kg (three doses); i.p. on the 1st, 5th, and 9th day + rifampicin 0.214 mg/kg/day; i.p., for 14 days.Forty mice were divided into four groups, each containing ten mice, and treated as follows Table :Control After the last drug injection on the 14th day, the mice fasted for eight hours before being euthanized by cervical dislocation while under general anesthesia. The sacrifice was carried out following the Guide for the Care and Use of Laboratory Animals and in compliance with the ARRIVE guidelines, also approved by our Faculty. Blood samples, just before euthanasia, were collected via cardiac puncture under the effect of combined ketamine (75 mg/kg)/xylazine (10 mg/kg) anesthesia. After clotting, blood was centrifuged. The obtained serum was stored at − 20 °C till use. Rapidly, the heart was separated, washed, dried on filter papers, and weighed. A part of the tissue was kept in 10% buffered formalin for processing for histopathological examination. A 10% W/V tissue homogenate in ice-cold PBS was prepared, centrifuged, and the clear supernatant was stored at − 20 °C till use.Estimations of phosphorus, sodium, potassium, calcium, CK, LDH, and AST in serum, besides assays of cTn I, ANP, and VEGF levels in tissue homogenate, were performed following kit manufacturer instructions.+. The rate of NADH/NAD+ conversion was followed at 340 nm, and it was proportional to LDH activity [The colorimetric kit for LDH assay was purchased from Chema Diagnostica, Italy. Using LDH, pyruvate is converted into L-lactate and NADH into NADactivity .Mouse ANP and Rat VEGF-A  immunoassay kits that use the Sandwich-ELISA principle  were bouColorimetrically, CK and AST activities were measured using the N-acetyl-cystein-activated CK and AST kits obtained from Biomed Diagnostics, Germany. The reaction of amino group transfer from aspartate to α-ketoglutarate, producing glutamate and oxaloacetate, was catalyzed by AST. AST activity was proportional to the amount of the formed oxaloacetate, which was measured by reacting with 2,4-dinitrophenylhydrazine in an alkaline solution at 505 nm . For CK,The Finecare TM cTn I Quantitative kit based on fluorescence immunoassay technology  was intended to assess cTn I. By adding the specimen to the sample well of the test cartridge, the fluorescence-labeled detector cTn I antibody bound to the cTn I antigen in the sample. When the sample mixture migrated on the nitrocellulose matrix of the test strip via capillary action, the complexes of the detector antibody and cTn I were caught by the cTn I antibody that had been immobilized on the test strip. Consequently, the more cTn I antigen is in the sample, the more complexes were accumulated on the strip. The signal intensity of fluorescence of the detector antibody reflected the amount of cTn I captured, and the Finecare TM FIA system showed cTn I concentrations in the sample .2+ ions were reacted in an alkaline medium with methyl thymol blue, producing a blue color that was measured at 585 nm [+ ions with magnesium acetate were converted into sodium magnesium uranyl acetate, and the residual uranyl acetate was reacted with potassium ferrocyanide to form a colored complex that was measured at 545 nm [Kits for colorimetric determination of calcium, phosphorus, sodium, and potassium were obtained from Biodiagnostics (diagnostic and research reagents), Giza, Egypt. In short, Cat 585 nm . For phot 585 nm . About st 545 nm . While pt 545 nm .SOD activity in tissue was assayed by the method of Dechatelet et al.  dependinRoutinely, heart tissues were fixed in 10% buffered formalin. After dehydration by passing in an ascending series of alcohol and clearance with xylene, fixed tissue was immersed in paraffin wax, sliced into 5–6 μm sections in a rotary microtome, then stained with the nuclear stain hematoxylin and the counterstain eosin (H&E). Under light microscopy, a pathologist unaware of the mice treatment examined and evaluated the stained sections. At least 3-sections were examined by the pathologist per one tissue sample. The magnification X 200 was used. The assessed variables include the evaluation of the parameters of myocardial necrosis , inflammation, and fibrosis.P < 0.05 was assumed significant.For comparison between the means of more than two groups, the statistical analysis by the one-way analysis of variance (ANOVA) for parameters with Gaussian distribution was done using the Instat software, version 3.10 , followed by Tukey-Karmer multiple-comparisons test. For data with non-Gaussian distribution Kruskal–Wallis test was employed, followed by Dunnett’s test for multiple comparisons. Data was presented as mean ± standard deviation (S.D). p < 0.001) resulted in approximately a 60% reduction in HW and reduced HW/BW ratio to 64% of the Control group value; as the mean values of HW and HW/BW ratio were of 0.122 g and 0.438% in the Control group and 0.0489 and 0.28% in the Doxorubicin group. In the concomitant treatment of rifampicin with doxorubicin, with rising the rifampicin dose mean levels of HW and HW/BW were significantly (p < 0.001) increased to reach 0.061 and 0.067 g, and 0.3 and 0.34% with the 0.107 mg/kg dose and the 0.214 mg/kg dose, respectively compared to doxorubicin alone. However, by comparing the 0.107 and 0.214 mg/kg doses together, the differences in HW or HW/BW ratio were non-significant.In Fig. p < 0.001). Compared with the Doxorubicin group, in Rifampicin-treated groups (Doxorubicin + Rifampicin 0.107 and Doxorubicin + Rifampicin 0.214), the mean levels of these enzymes were significantly (p < 0.001) decreased, to 217.45, 41.3, and 52.71 U/L, respectively in Doxorubicin + Rifampicin 0.107 and to 143.62, 37.8, 41.08 U/L, respectively in Doxorubicin + Rifampicin 0.214, toward normalization with increasing the dose of rifampicin. Statistically, when Rifampicin-treated groups were compared together, differences were significant (p < 0.001) in the levels of CK and AST but non-significant in the LDH level.As illustrated in Fig. p < 0.001). In Doxorubicin + Rifampicin 0.107 and Doxorubicin + Rifampicin 0.214 groups, there were decreases towards normal  compared with the Doxorubicin group in a dose-dependent manner (p < 0.001). When the two rifampicin groups were compared with each other, there was a significant difference (p < 0.001) in cTn I level but not in the ANP level (p > 0.05). On the contrary, compared to the Control group, the VEGF mean level displayed a 36% decrease in the Doxorubicin group  and re-increased to 658.64 and 720.58 pg/mL depending on the dose significantly (p < 0.001) by rifampicin treatment. Moreover, there was a significant difference (p < 0.05) in VEGF level in the Doxorubicin + Rifampicin 0.107 group compared to the Doxorubicin + Rifampicin 0.214 group , and the ANP mean level showed an elevation to 92.74 pg/mL (243%) in the Doxorubicin group compared to those in the Control group  elevated by doxorubicin to 218%, 104%, 115%, and 149%, respectively. They displayed mean levels of 66.39 mmol/L, 9.44 mg/dL, 6.76 mg/dL, and 4.68 mmol/L in the Control group, and 144.67 mmol/L, 9.79 mg/dL, 7.75 mg/dL, and 6.99 mmol/L, respectively in the Doxorubicin group. Rifampicin treatment lowered these levels depending on the dose in Doxorubicin + Rifampicin 0.107 and Doxorubicin + Rifampicin 0.214 groups to levels closer to these in the Control group. However, in the comparison between the Rifampicin-treated groups, all the differences were not significant (p > 0.05).As revealed in Fig. p < 0.001) the MDA mean level to 223%, from 0.048 to 0.107 nmol/g ptn, and decreased (p < 0.001) SOD by 51% (from 43.66 to 22.37% inhibition) compared to control. The Doxorubicin + Rifampicin 0.107 and 0.214 groups showed decreases in MDA to 0.06 and 0.056 nmol/g ptn, by 56% and 52%, respectively (p < 0.001), alongside elevations in SOD to 37.24 and 36.12% inhibition, by 166% and 161.5%, respectively (p < 0.001) compared to the Doxorubicin group. Differences between the Doxorubicin + Rifampicin groups with each other in MDA or SOD levels were statistically non-significant  improved levels of the above biomarkers toward normal concomitant to (2) architectural improvement in the form of moderate and mild affection of the cardiac muscle shown histopathologically with rifampicin dose of 0.107 mg/kg and 0.214 mg/kg, respectively.A limitation of our work is that due to financial causes, we could not study the effect of a longer duration of rifampicin co-treatment on tissue recovery. Also, we could not assess anti-inflammatory and anti-apoptotic mechanisms. Another limitation is that we could not carry out Echocardiography to assess cardiac function.In conclusion, our data demonstrate that rifampicin as antioxidant can help the host body face the manifestations of doxorubicin-induced cardiotoxicity; it achieved success in alleviating oxidative stress, amelioration and retrieving the heart structure and function back close to its healthy state. Therefore, rifampicin co-treatment can protect against doxorubicin-induced cardiotoxicity. Finally, our current results recommend future using of rifampicin in combination with doxorubicin in the course of treating cancer to protect against doxorubicin-induced cardiotoxicity after more validation via future research.


Previous studies have found that the triglyceride glucose index (TyG index) trajectories are associated with cardiovascular diseases. However, the association between the patterns of TyG index trajectories and risk for hypertension has not been investigated. In a longitudinal general population, we aimed to identify distinct TyG index trajectories over 12 years and describe their association with incidence of hypertension.Of the 15,056 adults retrospectively recruited from the Physical Examination Center of the Second Affiliated Hospital of Dalian Medical University in northeast of China from 2011 to 2022. TyG index was calculated as ln (fasting TG [mg/dL] × FPG [mg/dL]/2) and the TyG index trajectories were developed using group-based trajectory modelling. Cox regression analysis was accomplished to assess the association between TyG index and incidence of hypertension.The median age of the population was 38 years, and 7352 (48.83%) of the participants were men. Three distinct TyG index trajectories were identified: “low increasing” (N = 7241), “moderate increasing” (N = 6448), and “high stable” (N = 1367). Using “low increasing” trajectory as a reference, “moderate increasing” and “high stable” trajectory were associated with increased risk of hypertension . After adjusting for baseline sex, age, diabetes, smoking, systolic blood pressure, diastolic blood pressure, BMI, cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, blood glucose, triglyceride, urea, uric acid, and glomerular filtration rate, the HR were slightly attenuate in “moderate increasing” and “high stable” trajectories to 1.38 (95% CI 1.23–1.54) and 1.69 (95% CI 1.40–2.02) respectively. Meanwhile, similar results were observed in multiple sensitivity analyses. The HR of the “moderate increasing” and “high stable” trajectory groups were 2.63 (95% CI 2.30–3.00) and 4.66 (95% CI 3.66–5.93) in female, and 1.66 (95% CI 1.48–1.86) and 2.33 (95% CI 2.04–2.66) in male.Elevated TyG index at baseline and long-term TyG index trajectories were associated with the risk of hypertension. Early identification of increasing TyG index could provide insights for preventing hypertension later in life.The online version contains supplementary material available at 10.1186/s12933-023-02087-w. Hypertension is a momentous cause of cardiovascular disease, especially in developing countries –3. MeanwRecent studies have demonstrated that the triglyceride-glucose (TyG) index is correlated with incident hypertension –15 and aConsequently, we employed large-scale retrospective data recruited from 2011 to 2022 to identify various trajectories of the TyG index by group-based trajectory modeling (GBTM), and to estimate the correlation between TyG index trajectories and the prevalence of hypertension in the general populace.–92 years, from the health examination database from 2011 to 2022. All available data at every visit of all participants, including age, sex, height, weight, BMI, smoking, alcohol consumption status, medical history, and family history, were taken into account. For the purposes of this study, we excluded participants using the following criteria: Firstly, participants with fewer than 3 physical examination records  were excluded. Secondly, we eliminated individuals who had been diagnosed with hypertension (n = 5818), a prior record of cardiovascular disease (n = 48), a previous history of both benign and malignant tumors  (n = 15), or missing the history of disease (n = 898) at baseline. Thirdly, we also excluded those with less than 3 TyG index measurements (n = 1327). Therefore, the remaining 15,056 participants with 59,999 measures were included for further analysis /height (m) one day . A drink100 ml/d . Systoli100 ml/d , 20. Stu100 ml/d . Diabete100 ml/d . Hyperte100 ml/d .Statistical analyses were performed using R (version 4.2.2), Stata (version 16), and GraphPad Prism 9 software. Continuous variables with normal distributions were described as mean ± standard deviation (SD), while skewed distributions were presented using medians with interquartile ranges. Normal distribution data was tested using Kolmogorov Smirnov test, and the Mann–Whitney U test or Kruskal–Wallis test was used for skewed distributions. Categorical variables were described as percentages and compared using chi-square test. A two-sided P value of < 0.05 was recognized as statistically significant.Group-Based trajectory modeling (GBTM) was used to identify TyG trajectory subgroups that shared similar growth patterns of the TyG index over time , 28. TheCox proportional hazards model was calculated to estimate the association between different TyG index trajectories and hypertension risk, presented as hazard ratio (HR) and 95% confidence intervals (95%CI). Two multivariate models were conducted to adjusted for potential confounding elements: Model 1 was adjusted for age and sex. Model 2 was further adjusted for baseline diabetes, smoking, SBP, DBP, BMI, TC, HDL, LDL, TG, FBG, urea, UA and eGFR. We further used the restricted cubic spline regression model, with 5 knots, to more thoroughly assess the dose–response relationship between the TyG index and new-onset hypertension. Additionally, the Kaplan–Meier method was used to estimate the incidence curves of hypertension over the total follow-up period.To further confirm the robustness of our results, we conducted 4 sensitivity analyses. Firstly, to exclude the influence of medications, we excluded participants who used any antidiabetic or lipid-lowering medications to assess whether the results were affected. Secondly, to exclude the potential bias of missing data, we completed the missing data with multiple imputations. Thus, we created five imputed data sets, selecting the most appropriate dataset to input the missing variables. Additionally, all variables missing at baseline and blood pressure missing during follow-up were presented in Additional file Furthermore, to gain a more comprehensive understanding of the potential relationship between TyG index trajectories and the risk of hypertension, we also conducted subgroup analyses based on different gender, age, and BMI levels.–47) years, and 7352 (48.83%) were male. The median TyG index was 8.44 at baseline. On average, all participants had a mean of 3.99 individual physical examination records and a mean 4.13 years of follow up. During follow-up, 3017 participants who were identified as having hypertension. The baseline characteristics were described according to the quartile range of the TyG index in Additional file In total, 15,056 eligible individuals were included, the median age was 38 (30–43.00) years in the “low increasing” trajectory to 45.00 (36.00–52.00) years of the “high stable” trajectory. The levels of TyG, BMI, FPG, TC, TG, LDL, diabetes, smoking, drinking, and family history of diabetes were higher in the “high stable” trajectory compared to other two trajectories, and HDL was lower in the “high stable” trajectory. However, the family of hypertension was similar in three trajectories. Notably, participants in the “high stable” trajectory had higher SBP and DBP levels than their counterparts in the “low increasing” and “moderate increasing” trajectory.We identified three distinct TyG index trajectories among the 15,056 participants: “low increasing” , “moderate increasing” , and “high stable”  , 26.21% , and 39.28% (n = 537) in the “low increasing”, “moderate increasing”, and “high stable” trajectory groups, respectively (Table –2.67) and 3.88 (3.48–4.33), respectively. These associations were moderately attenuated when adjusted for age and sex, the HR and 95% CI of “moderate increasing” and “high stable” trajectories were 1.77 (1.62–1.93) and 2.36 (2.10–2.65). Further adjustment for potential confounders of the TyG index and hypertension showed that the HR (95% CI) of “moderate increasing” and “high stable” trajectories were 1.38 (1.23–1.54) and 1.69 (1.40–2.02), respectively. The positive association remained statistically significant.Table Furthermore, the Kaplan–Meier curve showed that the slope of the tangent line at the same time in the “moderate increasing” and “high stable” trajectory groups is greater compared to the “low increasing” trajectory group. This indicates that means the cumulative incidence of hypertension increased with the TyG index trajectory group Fig. .Fig. 3KaFour sensitivity analyses were performed, and the results were consistent Table . First, 2) status at baseline. For males, the incidence of hypertension was 28.10% throughout the follow-up period, while for females, it was lower at 12.34%. For males, the risk of outcome events in the “moderate increasing” and “high stable” trajectory groups were 1.66 (1.48–1.86) and 2.33 (2.04–2.66),respectively. On the other hand, for females, a higher risk was observed in the “moderate increasing”  and “high stable” trajectory groups .Considering the potential effects of sex, age, and BMI, Fig. –2.68 and HR = 4.01; 95%CI 3.57–4.49). However, there was no statistically significant difference in the “moderate increasing” and “high stable” trajectories when compared with the “low increasing” trajectory, in the participants older than 60 years.In addition, participants younger than 60 (50.00%) showed significantly increased ratio of developing hypertension compared to those older than 60 (18.70%). Meanwhile, for participants younger than 60, a significant high risk was observed in the “moderate increasing” and “high stable” trajectories . However, participants with BMI < 24 had increasing risk of hypertension in “moderate increasing”  and “high stable”  trajectories, respectively. While less pronounced, for participants with BMI ≥ 24, the HR and 95% CI of hypertension in “moderate increasing” and “high stable” trajectories were 1.64 (1.45–1.86) and 2.30 (1.86–2.67), respectively.Notably, the prevalence of hypertension in participants with BMI ≥ 24 was twice higher than that in participants with BMI < 24 , stage 2 , isolated systolic hypertension , isolated diastolic hypertension , and systolic diastolic hypertension , respectively[Previous studies have confirmed the TyG index was associated with hypertension , 29, 30. 1.66, 1.–2.11, ispectively. Cong etpectively.Various studies have proved that TyG index trajectories are closely correlated with cardiovascular events. A study of 2480 individuals, enrolled from Hanzhong Adolescent Hypertension Cohort study, found that the highest TyG index trajectory carried the greatest odds of increased arterial stiffness, with a fully adjusted odds ratio (OR) of 2.76  . AnotherWe also observed the strength of the association of TyG index trajectory and hypertension risk in the sex-stratified analysis. Our study observed that females had significant higher risk of hypertension in the “moderate increasing” and “high stable” TyG index trajectories than males. Another study based on middle-aged and older Chinese, using China Health and Retirement Longitudinal Study (CHARLS) data, also found that higher TyG index was significantly associated with stage 2 hypertension in females, but not in males . HoweverThis study has several advantages. Our research is a longitudinal study based on an abundant sample of the physical examination population, they underwent regular health examination for several years, which provides a unique opportunity to examine the longitudinal TyG index trajectory patterns during follow-up over 12 years. In addition, the GBTM approach analysis distinguishes the long-term TyG index trajectory patterns, which could provide reliable and qualitative groups that individuals exhibited specific and similar TyG index changes over time. This grouping enabled robust classification and had more statistical power.However, this study also has several limitations. First, our study enrolled individuals from the general population in northern China. Therefore, the identified trajectories could limit generalizability. Secondly, although several confounding factors we know that may have affected hypertension were enrolled in the model for adjustment, there are still unmeasured confounding components that may have influenced the results. Third, as we are retrospective cohort study, there exists an inevitable recall bias and missing data despite the fact that we had carefully adjusted the well-known and suspected risk factors.Our findings suggest that not only the baseline TyG index but also the high trajectories of TyG index growth were associated with the development of hypertension. Long-term monitoring of dynamic changes of TyG index may identify individuals at high risk of hypertension. Prevention programs that target TyG index may provide more effective for primary and secondary prevention of hypertension.Additional file 1:Table S1. Average probabilities of group assignment and Bay Information Criterion (BIC) statistics of model fits. Table S2. Baseline characteristics of participants according to quartiles of TyG index. Table S3. Baseline characteristics of participants included in the study compared to those who excluded for hypertension at baseline. Figure S1. Summary of missing data. Figure S2. Changes in standard mean differences (SMD) between different variables before and after propensity score matching (PSM). Figure S3. Incident rates of hypertension in every year. Figure S4. Comparison between groups for different sensitivity analysis. Figure S5. The restricted cubic spline curves for TyG index with risk of incident hypertension.


Lonicera caerulea L.) is an emerging fruit crop; however, determining its proper harvest time in commercial cultivation remains challenging due to its rapid fruit development characteristics. In this study, we investigated 17 agronomic traits of three blue honeysuckle cultivars harvested on 5 successive dates within their respective harvest windows. ‘Lanjingling’, ‘Wulan’, and ‘Berel’ showed solid–acid ratios (SS:TA) ranging from 10.00 to 16.01, 8.13 to 10.23, and 5.77 to 7.11, respectively; anthocyanin contents ranged from 233.85 to 276.83 mg/100 g, 236.38 to 312.23 mg/100 g, and 235.71 to 334.98 mg/100 g, respectively; vitamin C contents ranged from 88.43 to 99.68 mg/100 g, 108.13 to 191.23 mg/100 g, and 89.71 to 120.40 mg/100 g, respectively; phenolic contents ranged from 25.22 to 37.59 mg/g, 25.40 to 36.52 mg/g, and 37.66 to 50.00 mg/g, respectively. The results revealed the SS:TA value consistently increased with delayed harvesting and were significantly negatively correlated with fruit firmness, total acidity, shelf life, and respiration intensity, suggesting it is an ideal maturity indicator for blue honeysuckle berries. The factor analysis suggests that the suitable harvest date for ‘Lanjingling’ could be either 47 days after flowering (DAF) with an SS:TA value of approximately 10.0, characterized by high firmness, extended shelf life, and elevated levels of anthocyanins and phenolics; or 67 DAF (SS:TA ≈ 16.0), characterized by high vitamin C content and sweetness, and larger size and weight. For ‘Wulan’, it suggests the suitable harvest date is either 54 DAF (SS:TA ≈ 9.0), yielding fruit with high levels of anthocyanins and vitamin C; or 62 DAF (SS:TA > 10.0), yielding fruit with high sweetness and large size and weight. For ‘Berel’, it is suggested to be either 52 DAF (SS:TA ≈ 6.5), resulting in fruit with high levels of anthocyanins and vitamin C; or 62 DAF (SS:TA > 7.0), resulting in balanced levels of the fruit quality traits.Blue honeysuckle ( Lonicera caerulea L.) is a perennial deciduous shrub belonging to the Caprifoliaceae family. The fruit of the blue honeysuckle is a small berry, ranging in color from azure-blue to black-blue, with a sour to sweet taste and commonly known as ‘honeyberry’, ‘haskap’ or ‘honeysuckle berry’. Traditionally, blue honeysuckle berries have been wild-harvested in Russia, China, Korea, and Japan as an ethnomedicine [Blue honeysuckle , relatively short fruit development period (6–9 weeks depending on genotype), rapid fruit ripening, and auto-abscission, as well as its non-climacteric fruit type ,26,27. SThis study aims to determine the proper harvest time for the three main blue honeysuckle cultivars  in China by characterizing and analyzing the dynamic changes in fruit quality traits during their respective harvest windows. To our knowledge, this research represents the pioneering effort toward evaluating the harvesting times for blue honeysuckle berries worldwide. The findings would not only provide a practical approach for large-scale harvesting in commercial cultivation of blue honeysuckle in China but also serve as a valuable reference for its potential global industry expansion.https://planthardiness.ars.usda.gov/ (accessed on 1 January 2023)). The meteorological data during the harvest season at the experimental location are presented in ‘Lanjingling’  and ‘Wulan’ (CNPVP-20200390) were bred by Northeast Agricultural University (NEAU), while ‘Berel’  is a Russian hybrid cultivar that was introduced to China in 2001 . The expDuring the 2022 harvest season, the first harvest date for each cultivar was determined to be the date when 95% of the fruit had turned blue. Subsequently, based on the empirical harvest windows for the three cultivars at the experimental location , which w2 × h’ based on its cylindrical fruit shape, while for ‘Wulan’ and ‘Berel’ with oval-shaped fruits, the calculation was performed using the formula ‘4/3 π × (w/2)2 × h’. The firmness was quantified using a digital fruit durometer  equipped with a 3.5 mm diameter plunger, adhering to the manufacturer’s specifications for one punch per fruit. The fruit color was quantified using a spectrocolorimeter  with three technical replicates. The measurements were performed in the CIELAB color space system (L*a*b* ) employing a 10° observer and D65 illuminant. The respiration intensity was quantified using a specialized instrument designed specifically for measuring the respiration of fruits and vegetables ; in each experimental replication, a total of 6 fruits were placed into a breathing chamber with a volume of 0.10 L; the measurement was conducted three times at intervals of 30 min. The shelf life was investigated under controlled environments: 4 °C with 90% air humidity (referred to as ‘low-temperature shelf’) and 25 °C with shade (‘referred to as room temperature shelf’); 100 intact fruits for each replication were individually stored in air-permeable plastic containers without any stacking; the fruits were examined every 24 h to determine the count of sound fruits ; the shelf life (days) was recorded until the percentage of sound fruit dropped below 15%.For each replicate, 30 sound fruits were randomly selected. The fruit weight was measured using a high-precision electronic balance  with a sensitivity of 0.001 g. The vertical and transverse diameters (h and w) were measured using a digital vernier caliper  with a sensitivity of 0.01 mm. The fruit volume for ‘Lanjingling’ was calculated using the formula ‘π × (w/2)The fruits were homogenized using a high-speed tissue homogenizer  with 100 fruits per replication, and the resulting homogenate was subsequently filtered to obtain raw juice for the following measurements. The soluble solids (%) and acidity (%) were determined using a handheld refractometer  following the manufacturer’s specifications. The total anthocyanins were quantified using the pH differential method  and exprp < 0.05 was considered statistically significant), one-way ANOVA was performed using IBM SPSS Statistics for Windows (Version 27.0) [https://www.chiplot.online/ (accessed on 15 October 2023)). The principal component bi-plot was constructed using OriginPro 2021 [The raw data obtained from instruments and written records were manually imported into Microsoft Excel 2021 for conducting descriptive statistics . To asseon 27.0)  with posPro 2021  based on3, 2.06–2.42 cm3, and 2.21–2.25 cm3, and fruit weight range of 1.17–1.19 g, 1.02–1.07 g, and 1.12–1.15 g. The later harvested fruits generally exhibit slightly larger sizes and heavier weights compared to the earlier ones. Statistically significant differences were only observed in ‘Wulan’, respectively between 46DAF and 58DAF, and between 46DAF and 62DAF. Regarding fruit color, the three cultivars exhibited dark grey to dark blue hues in the L*a*b* coordinate system throughout their harvest windows. The L* values of ‘Lanjingling’, ‘Wulan’, and ‘Berel’ ranged from 33.99 to 39.45, 28.77 to 37.24, and 28.26 to 34.58, respectively; their a* values ranged from −1.48 to −1.36, −1.84 to −1.01, and from −1.69 to −0.91, respectively; their b* values ranged from −5.21 to −6.77, −4.23 to −7.24, and from −3.65 to −5.24, respectively. In addition, for all three cultivars, the absolute values of L* and b* reached their peaks at the third or fourth dates and slightly decreased thereafter, indicating a gradual lightening (L*) and bluing (b*) of fruit color as harvesting was delayed. The a* value exhibited irregular changes from the first to the last harvest date.To assess the changes in fruit appearance of the three cultivars during their respective harvest windows, we investigated fruit size, weight, and color . ‘LanjinL* and b* observed at the final harvest date can be attributed to both the degradation of cuticular wax and the dynamic synthesis of pigment metabolites, which are also likely influenced by factors such as wind strength, light intensity, air humidity, temperature, and internal metabolism. Thus, considering the rapid fruit ripening of blue honeysuckle, color indicators may not provide a reliable means for monitoring the ripeness and determining the suitable harvest time.The appearance properties of fruits play crucial roles in assessing consumer preference and acceptance of blue honeysuckle berries . In thisTo assess the tolerance to transportation and storage of the three cultivars harvested at the five different dates, we investigated fruit firmness (Ff), respiration intensity (Ri), and shelf life under 25 °C (25 °C-Sl) and 4 °C (4 °C-Sl) . OverallFruit firmness is a pivotal agronomic trait for small berry crops, influenced by the species’ maturation physiology, and significantly impacts fruit’s shelf life and transportation capacity . The simThe primary fruit tastes were evaluated by analyzing the soluble solids (SS), titratable acidity (TA), and SS:TA ratio . ThroughThe increasing trends of SS and the decreasing trends of TA along with the delay in harvesting have been widely reported in most fruit crops ,46,47. ITo assess the variations in functional constituents of the three cultivars throughout the harvesting windows, we measured the levels of total anthocyanins, phenolics, and vitamin C . The totThe bioactive metabolites present in blue honeysuckle berries, primarily anthocyanins, vitamin C, and phenolics, not only serve as essential constituents for plants but also represent the most widely recognized functional components by fruit consumers. Consequently, these compounds play a crucial role in determining the market value of blue honeysuckle berries for applications such as natural pigment production or nutritional supplementation ,49,50. TL* (r = 0.57) and vitamin C (0.59) while showing a significant negative correlation with Ff (r = −0.88), Ri (−0.81), 25 °C-SL (−0.74), 4 °C-Sl (−0.78), and TA (−0.95), respectively. Furthermore, vitamin C showed a significantly negative correlation with Ri (−0.57), 4 °C-Sl (−0.53), SS (−0.52), and TA (−0.53), respectively. However, no significant correlation was found between phenolics or anthocyanins and any traits. For ‘Wulan’ (L* (0.80) while displaying a negative correlation with a* and b* (−0.71 and −0.77), respectively. However, no significant correlation was observed between vitamin C and any other traits. For ‘Berel’ (L* (0.56) while exhibiting a negative correlation with a* (−0.63), b* (−0.64), Ff (−0.80), Ri (−0.77), 25 °C-Sl (−0.72), 4 °C-SL (−0.72), TA (−0.52) and phenolics (−0.67) respectively. The phenolic content showed a significant positive correlation with a* (0.83), b* (0.59), Ff (0.61), Ri (0.61), 25 °C-Sl (0.65), 4 °C-SL (0.77), respectively; while exhibited a significant negative correlation with L* (−0.75), vitamin C (−0.56) and SS:TA. A significant positive correlation was found between the anthocyanins and vitamin C (0.73). The vitamin C showed a negative correlation with 4°C-SL (−0.56) and phenolics (−0.56).To comprehensively understand the interrelationship between the fruit quality traits, correlation analysis was conducted on the 17 fruit quality traits of each cultivar . General ‘Wulan’ , SS:TA e ‘Berel’ , SS:TA sTo gain a deeper understanding of the interrelationship between the key quality traits and harvesting dates for each cultivar, principal component analysis (PCA) was performed based on 11 traits . Taking eigenvalue greater than 1.0 into account, three, three, and four principal components (PC) were extracted in the ‘Lanjingling’, ‘Wulan’, and ‘Berel’, respectively , and thuThe SS:TA, fruit firmness, shelf life, and health-promoting phytochemicals are crucial fruit quality traits for blue honeysuckle berries, which majorly determine the industry expansion for berry crops ,54. The To provide practical and direct information on blue honeysuckle harvesting for producers and researchers, we condensed the 17 fruit quality traits into nine key commercial indicators . Additionally, factor analysis was employed to comprehensively evaluate the harvest dates for each cultivar. The KMO statistics for the datasets of ‘Lanjingling’, ‘Wulan’, and ‘Berel’ were 0.633, 6.682, and 0.668, respectively . The BarBased on the factor analysis results and in combination with the investigation of fruit traits , our finIn order to determine the appropriate harvest dates for three major cultivars of blue honeysuckle in China, this study investigated 17 fruit traits in their respective harvest windows. Overall, with the delay in harvesting, there were increasing trends in SS:TA and decreasing trends in firmness and shelf life. Significant negative correlations were found between SS:TA and fruit firmness and respiration intensity, as well as shelf life, indicating that SS:TA serves as an ideal indicator for monitoring the maturity of blue honeysuckle berries. Comprehensively, the most suitable harvest dates for ‘Lanjingling’ were 47DAF (SS:TA ≈ 10.0) and 67DAF (SS:TA ≈ 16.0), for ‘Wulan’ were 54DAF (SS:TA ≈ 9) and 62DAF (SS:TA > 10.0), and for ‘Berel’ were 52DAF (SS:TA ≈ 6.5) and 62DAF (SS:TA > 7.0).


Pasteurella multocida (P. multocida) was evaluated by establishing a piglet tissue cage infection model. Concentration of tilmicosin and bacterial numbers of P. multocida in the tissue-cage fluid were monitered. After the population of P. multocida was equal to or greater than 107 CFU/mL in a tissue cage, piglets received an oral administration of tilmicosin at a dose of 30, 40, 50, and 60 mg/kg b.w., once daily for 3 days, respectively. Bacteria were counted every 24 h after drug administration and at 48 and 72 h after the last administration. A sigmoidal Emax model was used to fit the relationship between PK/PD parameters and the antibacterial effect. AUC24h/MIC was the best PK/PD index that correlated with effectiveness of tilmicosin against P. multocida. The magnitude of AUC24h/MIC required for continuous 1/3-log, 1/2-log, and 3/4-log reductions were 19.65 h, 23.86 h, and 35.77 h, respectively, during each 24 h treatment period. In this study, when the dosage was >50 mg/kg, the AUC24h/MIC was still >35.77 h in the period of 24–48 h after the last administration due to the slow elimination, that is, tilmicosin exhibited a potent antibacterial effect against P. multocida after three successive daily administrations. The data provide meaningful guidance to optimize regimens of tilmicosin to treat respiratory tract infections caused by P. multocida.Tilmicosin is a semi-synthetic macrolide for veterinary use with strong antibacterial effect on respiratory bacteria. In this study, the pharmacokinetic/pharmacodynamic (PK/PD) integration of tilmicosin against  The infection was often accompanied by the risk of other bacteria, causing porcine respiratory disease complex (PRDC). Multiple serotypes and complex typing make it difficult to prevent and control by vaccine . Futhermore, the higher concentration in lungs relative to plasma makes it a valuable treatment option for respiratory infections. According to Zhang et al.  and elimination half-life (T1/2kel) of tilmicosin in healthy pigs were 2.27 h and 43.53 h after oral administration, respectively, and 1.64 h and 20.69 h in another study . Timicosin phosphate with a purity of 80.4% (Tilmicosin) was provided by Guangdong Dahuanong Animal Health Products . Pentobarbital sodium was purchased from Xiangbo Biotechnology Co., Ltd. Procainamide hydrochloride was supplied by Shangdong Fangming pharmaceutical Co., Ltd. Tryptic soy broth (TSB) and tryptic soy agar (TSA) were purchased from Guangdong Huankai Microbial Technology . Newborn bovine serum was supplied by Guangzhou Ruite Biotechnology .Strain C44-15 (serovar D:7) of 2.2.The study used 10 healthy crossbred piglets (Duroc × Landrace × Yorkshire) weighing 25–30 kg acquired from the Guangzhou Fine Breed Swine Farm. The piglets were given antibiotic-free feed and water over the course of the experiment. All the experimental procedures involving the animals were approved by the Committee on the Ethics of Animals of South China Agricultural University .The specifications and surgical implantation procedures for the tissue cages (TCs) matched those described previously . The TCsP. multocida suspension containing approximately 1.5 × 108 CFU/mL. Following 48 h of incubation, 0.5 mL of TCF was extracted from each tissue cage to quantify the bacteria. TCs with bacterial counts around 107 CFU/mL were selected for use in the subsequent experiment.The sterile TCs were inoculated with 1 mL of a 2.3.P. multocida was ascertained using a microdilution technique following the guidelines of the Clinical and Laboratory Standards Institute . The MIC value was derived from TSB supplemented with 5% calf serum and TCF cultures.The MIC of tilmicosin against 2.4.Ten piglets were randomly divided into four treatment groups (two piglets and four tissues cages each group) and one control group (infected but not treatment). One male and one female pig were contained in each group. Each treatment group was administered orally with tilmicosin at the dosages of 30, 40, 50, and 60 mg/kg respectively, once a day for 3 days. The control group was treated with the same amount of normal saline.g for 10 min to remove particulate matter and were kept frozen at −20°C until analyzed.For pharmacokinetics, around 0.5 mL of TCF was obtained at 1, 3, 6, 9, 12, and 24 h following each administration, and additional samples were collected at 48 and 72 h after the final dose. The samples underwent centrifugation at 3000 × For pharmacodynamics, about 0.1 mL of TCF was collected for bacterial counting at 24 h after each administration and at 48 and 72 h after the last administration. The suspension was serially diluted 10-fold in saline, with 100 μL used initially. Subsequently, 20 μL of the dilutions were dispersed onto agar plates and incubated for 12 h to enumerate the bacterial population.2.5.18 column . The mobile phase consisted of solution A  and solution B (acetonitrile) at a flow rate of 0.25 mL/min. The elution gradient was: 0–1.5 min, 10% B; 1.5–6 min, 95% B; 6–6.5 min, 5% B; 6.5–12.5 min, 5% B. The injection volume was 5 μL.The tilmicosin concentration was measured by high-performance liquid chromatography-tandem masss pectrometry (HPLC-MS/MS) using a system from Agilent Technologies . Separation was achieved on a Cg for 10 min, the clear supernatant was filtered through a 0.22-μm nylon syringe filter (JinTeng Experiment Equipment Company) and then injected into an auto sampler vial. A calibration curve was established with tilmicosin concentrations (0.005–0.5 μg/mL). PK parameters were calculated using WinNonlin software version 6.1 .The samples were allowed to thaw to ambient temperature prior to analysis. Aliquots of 200 μL TCF were added to a 1.5 mL micro centrifuge tube and then equal volume acetonitrile was added to precipitate protein. After vortexing for 2 min, and centrifuging at 12000 × 2.6.24h/MIC , Cmax/MIC , and %T > MIC . The relationship between the PK/PD indices and efficacy were evaluated using the sigmoidal maximum effect (Emax) PD model. The formula was as follows:The PK/PD parameters were calculated based on the individual pharmacokinetics of each treated infected TCs: AUC10CFU/mL) over a 24 h dosing interval; E0 denotes the change of bacterial count in the control sample; Emax is the maximum antibacterial effect during the dosing interval; Ce signifies the value of a PK/PD index ; EC50 represents the PK/PD index value producing 50% of the maximal effect; and N is the Hill coefficient defining the slope of the effect curve.Where E represents the antibacterial effect, defined as the change in bacterial count (log3.3.1.P. multocida (D:7) in both TCF and TSB broth were 0.25 μg/mL.The MIC values of tilmicosin against 3.2.24h, Cmax, and %T > MIC are presented in 24h ranged from 3.57 ± 0.20 to 13.65 ± 0.38 μg h/mL. Cmax values were obtained directly from the concentration-time curves, spanning 0.20 ± 0.03 to 0.63 ± 0.03 μg/mL. The %T > MIC values covered a range of 0 to 100%. Specifically, the AUC values from 24–48 h after the final dose at 30, 40, 50 and 60 mg/kg were 6.31 ± 0.51, 8.35 ± 0.36, 11.69 ± 0.33, and 12.87 ± 0.22 μg h/mL, respectively.The concentration-time profiles of tilmicosin in TCF following multiple dosing are depicted in 3.3.P. multocida in the piglet tissue-cage infection model across varying doses. Bacterial counts for the untreated control group remained around 107 CFU/mL. Following tilmicosin administration at 30, 40, 50, and 60 mg/kg for 3 days, the total reductions in bacteria (log10 CFU/mL) were 1.48 ± 0.13, 2.82 ± 0.10, 3.39 ± 0.11, and 3.52 ± 0.15, respectively. Tilmicosin exhibited bactericidal activity at doses of 50 and 60 mg/kg after multiple administrations.The time-kill curves depicted in 3.4.24h/MIC, Cmax/MIC, and %T > MIC) and antibacterial activity of tilmicosin against P. multocida in TCF derived from Sigmoid Emax model was shown in 2 of AUC24h/MIC, Cmax/MIC, and %T > MIC with an antibacterial effect was 0.92, 0.90, and 0.83, respectively. The obtained parameters of E0, Emax, EC50, and the Hill coefficient are listed in 24h/MIC values for a 1/3 log10CFU/mL, 1/2 log10CFU/mL, and 3/4 log10CFU/mL reduction were 19.64 h, 33.45 h, and 35.64 h, respectively, during the 24 h administration of tilmicosin. 24h/MIC, Cmax/MIC, and %T > MIC) and the antibacterial activity of tilmicosin against P. multocida in tissue cage fluid, as modeled using the Sigmoid Emax model. The coefficients of determination (R2) for AUC24h/MIC, Cmax/MIC, and %T > MIC with antibacterial effect were 0.92, 0.90, and 0.83, respectively. The derived E0, Emax, EC50, and Hill coefficient parameters are tabulated in 24h/MIC values required for a 1/3 log10CFU/mL, 1/2 log10CFU/mL, and 3/4 log10CFU/mL bacterial reduction over 24 h were 19.64 h, 33.45 h, and 35.64 h, respectively.The correlation between three PK/PD parameters  in both TCF and TSB were 0.25 μg/mL, which was also used for in vivo PK/PD analysis. In this study, the data derived from tissue fluid in tissue cage model can better integrate pharmacokinetics and pharmacodynamics.There may be differences in the antibacterial effect in artificial culture medium or in serum or tissue fluid. The MICs of tiamulin against several strains of 45 μg/mL . For cefue fluid . The MICActinobacillus pleuropneumoniae and P. multocida , which may be due to the slow elimination of tilmicosin in pigs. This is also an important reason why it is inappropriate to use% T > MIC to guide the administration of tilmicosin.It is sometimes confusing to select the best PK/PD parameters to evaluate the efficacy of drugs based on the antibacterial characteristics (time-dependent or concentration-dependent), because the characteristics of the drug itself have a lot to do with it. The classification of drugs is not the only reference for selecting parameters to describe antibacterial effect. For drugs with long elimination half-life, PK/PD parameter AUC AUC/MIC . The PK/alf-life , 34. In parasuis . The eff regimen . In the 24h/MIC and bacterial change was fitted by Sigmoid model, and the magnitude of AUC24h/MIC required for a 3/4 log10CFU/mL of bacterial killing during 24 h was 35.64 h. Bactericidal activity was defined as >3-log10 reduction in CFU/mL. In this study, tilmicosin was administered once a day for 3 days. When the dosage was >50 mg/kg, the AUC24h/MIC was still >35.64 h in the period of 24–48 h after the last administration due to the slow elimination of tilmicosin, that is, the number of bacteria could be reduced by 3 log10CFU/mL within 96 h after administration and bactericidal effects can be achieved. In this study, the counts of bacteria under 50 and 60 mg/kg were no significantly difference  and hollow fiber infection model (HFIM) showed the power in flexibility and prediction. For instance, the concentrations under different doses of tilmicosin in different tissues even in pigs with different ages can be predicted by applying a PBPK model. In a HFIM, except the total counts of bacteria, the counts of resistant subpopulation and non-inherited resistant subpopulation (persistent cells and biofilms) can be detected. In future studies, PBPK model and HFIM model should be considered.Although some useful 5.P. multocida strain D:7 was studied by establishing a tissue cage infection model in pigs. The results demonstrated that the AUC24h/MIC (R2 = 0.92) was the optimal PK/PD index for describing antibacterial activity. Further analysis indicated that an AUC24h/MIC value of 35.64 h was necessary for tilmicosin to achieve a 3/4 log10CFU/mL reduction in TCF. The bactericidal efficacy of tilmicosin against P. multocida was attained at doses of 50 mg/kg or higher, administered once daily for 3 days. These findings will facilitate guidance and optimization of clinical dosing regimens for treatment of P. multocida infections in piglets.The current study investigated the PK/PD integration of tilmicosin against The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The animal study was approved by Committee on the Ethics of Animals of South China Agricultural University. The study was conducted in accordance with the local legislation and institutional requirements.YC: Writing – original draft, Data curation, Software. XJ: Validation, Writing – original draft. SZ: Validation, Writing – original draft. WW: Writing – review & editing. HZ: Writing – original draft. HD: Conceptualization, Funding acquisition, Project administration, Writing – original draft, Writing – review & editing.This work was supported by the National Natural Science Foundation of China (grant no. 31972733).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.