Background: Changes in platelet count (PLT) are strongly associated with patient survival and may be clinically indicative of certain underlying diseases. However, there were few studies on the prognosis of patients with cancer cachexia.Objective: The purpose of this study was to investigate the relationship between PLT and 1-year survival in patients with cancer cachexia.Methods: We performed a nested case-control study of data from a multicenter clinical study of cancer. There were 252 patients with cancer cachexia whose survival time was less than or equal to 1 year and 252 patients with cancer cachexia whose survival time was more than 1 year meeting the inclusion criteria. The mortality risk and the adjusted risk were estimated by logistic regression and displayed as odds ratios (ORs) and 95% confidence intervals (95% CIs).Results: PLT was negatively correlated with 1-year overall survival (OS) of patients with cancer cachexia (increased per standard deviation (SD): OR = 1.29; 95% CI: 1.05-1.60; P = 0.018). The higher the PLT, the lower the OS of patients. When classified by dichotomy , OS of patients in the D2 group was worse . When classified by quartile , OS of patients in the Q4 group was poorer . In addition, patients with a low PLT (< 296×109/L) and either a high total bilirubin (TBIL) (≥ 17.1 µmol/L) or a smoking history had poor 1-year survival. Based on our primary cohort study, we conducted a survival analysis of 3130 patients with cancer cachexia and found that OS was better in patients with low PLT (< 296×109/L).Conclusion: PLT was negatively correlated with 1-year overall survival of patients with cancer cachexia. Cachexia is extremely common among all cancer deaths worldwide, with a prevalence of more than 50% 9/L on the fifth day following liver transplantation presented a high mortality rate and poor graft survival within one year after operation In recent years, the relationship between platelets and cancer has received extensive attention. Platelet count (PLT) is associated with prognosis in many diseases. For instance, it can be used as a predictor of death and graft loss after liver transplantation. Patients with a PLT < 70 × 10This study aims to explore the predictive function of PLT in the clinic, since in addition to its crucial role in hemostasis, platelet is increasingly recognized as an inflammatory mediator regulating the immuno-oncological system Declaration of Helsinki. In total, we identified 252 patients with cancer cachexia whose survival time was less than or equal to 1 year and matched 252 controls with a survival time of more than 1 year. We then paired the case and control groups in a 1: 1 ratio based on age (±5 years), gender, tumor type, tumor stage, and location of hospitalization. The median survival estimates along with the two-sided 95% CI of patients with survival time greater than 1 year and patients with survival time less than or equal to 1 year were as follows: 37.5 months  and 6.13 months , respectively. All pathological stages in our study were defined in accordance with the American Joint Committee on Cancer TNM staging system (8th edition) This study was a nested case-control study with data obtained from 40 clinical centers in China from 2013 to 2020. Cancer patients aged 18 years or older were enrolled and patients with incomplete PLT data were excluded  = weight (kg) / height2 (m2). Mid-upper arm circumference (MAC) and triceps skin fold (TSF) were measured at the acromion and at the midpoint of the olecranon crest of the dominant arm. The subject was placed in a supine position with the knee flexed 90 degrees. MAC was measured with a plastic metric tape, while TSF was measured with a conventional skin crease caliper. A Jamar dynamometer was employed to measure hand grip strength (HGS) of the dominant hand. Information on smoking status, alcohol consumption and tea consumption were obtained through a lifestyle questionnaire. The OS was the primary outcome in this study, which included mortality due to any cause. Evidence of death was obtained from regular follow-up of the patients.The diagnosis of cancer cachexia was based on Fearon's criteria The subjects of laboratory testing mainly included total protein, albumin, neutrophils, total bilirubin (TBIL), aspartic transaminase (AST), alanine transaminase (ALT), hemoglobin, white blood cell (WBC), lymphocyte, red blood cell (RBC), as well as PLT. All blood tests were performed after at least 9 hours of fasting and before anti-tumor treatment within the first 24-hour hospitalization. All the study outcomes were reviewed and adjudicated by an independent Endpoint Adjudication Committee, whose members were unaware of the specific assignments of study group.9/L) group and a low PLT (< 296×109/L) group. Ultimately, 3130 patients with cancer cachexia were identified based on clinical diagnoses in the medical records, and a subsequent cohort study was conducted according to the matching principle of a 1: 1 ratio.Prognostic validation was performed in the cachexia cohort based on the truncation level of the nested case-control study. We collected data on 50,000 patients with cancer from 2013 to the end of 2020, and then divided them into a high PLT  and 95% confidence intervals (95% CIs) for 1-year survival of cancer patients were constructed by modeling risk factors as continuous variables, as well as modeling dichotomous and quartile PLTs using the chi-square test. Adjusted matching variables included BMI, HGS, MAC, TBIL, WBC, RBC, TSF, chemotherapy, radiotherapy, and surgery. Correction factors were selected using stepwise regression. In addition, heterogeneity among subgroups was evaluated by a conditional logistic regression method, and the influence of preoperative treatment was excluded by sensitivity analysis, and the interaction between PLT and subgroups was examined by probability ratio. Survival analysis of the basic cohort (n = 3130) was performed by the Kaplan-Meier method and survival curves. In this study, a two-tailed 9/L ± 3.98×109/L vs. 6.45×109/L ± 3.21×109/L), neutrophil count (5.59×109/L ± 3.77×109/L vs. 4.36×109/L ± 3.90×109/L), as well as PLT (261×109/L ± 116×109/L vs. 238×109/L ± 96×109/L). However, HGS (21.85 kg ± 9.56 kg vs. 24.25 kg ± 8.50 kg), BMI(20.15 kg/m2 ± 2.90 kg/m2 vs. 21.18 kg/m2 ± 3.26 kg/m2) total protein (65.28 g/L ± 8.47 g/L vs. 67.63 g/L ± 6.86 g/L) and albumin (35.09 g/L ± 5.32 g/L vs. 38.30 g/L ± 5.92 g/L) levels, blood components including Hb (113.29 g/L ± 20.76 g/L vs. 120.38 g/L ± 17.97 g/L), lymphocyte count (1.33×109/L ± 0.76×109/L vs. 1.53×109/L ± 0.65×109/L), RBC (3.90×1012/L ± 0.72×1012/L vs. 4.16×1012/L ± 0.59×1012/L), as well as MAC (24.44 cm ± 3.29 cm vs. 25.22 cm ± 3.66 cm) and TSF (12.27 mm ± 6.83 mm vs. 14.53 mm ± 7.37 mm) were lower in the patient population with shorter survival , we found that the D2 group had poorer OS compared with the D1 group . While when patients' PLT levels were divided into quartiles , the Q4 group had a relatively higher risk  and worse 1-year OS compared with the Q1-Q3 group  (Table 9/L) were negatively correlated with patient prognosis (P < 0.05). The negative association between PLT and 1-year survival was stronger in the high TBIL group  than in the low TBIL group   than in those without a history of smoking   Figure . Similar) Figure .9/L) of the nested case-control study, we performed prognostic validation in the cachexia cohort. The cohort study (n=3130) showed that compared with patients with high PLT (≥ 296×109/L), patients with low PLT (< 296×109/L) had better OS , indicating that thrombocytosis was prevalent in patients with non-small cell cancer 9/L) could predict poor prognosis in lung cancer patients 9/L', while it decreased when the ΔPLT ≤ or ≥ 20×109/L, which is in favor of our findingsOverall, in this hospital-based retrospective nested case-control study, a higher PLT was associated with a poorer OS. The relationship between PLT and survival has been examined in several previous studies. In an analysis of 285 patients with non-small cell lung cancer who underwent consecutive therapeutic pneumonectomy, the rates of thrombocytosis were 22.41% and 3.82% in stage III + IV and stage I patients, respectively  when both PLT and TBIL were high, suggesting that those patients with higher PLT and TBIL would suffer from a higher risk of death compared to those with lower PLT and TBIL.in vivo and leads to platelet activation and aggregation. Meanwhile, smoking may activate thrombopoietin which stimulates platelet production A study showed that in stage IV colorectal cancer patients, elevated TBIL and DBIL were associated with poorer OS P < 0.00001) Platelets, and platelet related indicators, including PLT, mean platelet volume (MPV), platelet distribution width (PDW), platelet crit (PCT), and platelet-to-lymphocyte ratio (PLR), are important in the clinical observation of the prognosis of certain cancers. Using a retrospective analysis, Huang The ability of PLT in predicting the prognosis of cancer cachexia patients may be related to thromboembolism. In cancer patients, the endogenous ligand podoplanin binds to C-type lectin-like receptor 2 to induce platelet activation, promoting hematological cancer metastasis and cancer associated thrombosis Currently, this correlation was found for the first time in our study, which provided great help and convenience for the prognostic management of patients with cancer cachexia. However, this paper has several limitations in the following aspects. First, participants' PLT was evaluated only at baseline, so we could not explore the impact of dynamic changes in PLT on the survival of cancer patients. Second, our included sample size (n=252) was not sufficiently representative of all patients with cancer cachexia. Further studies need to expand the sample size to increase credibility. Third, our study subjects were of a single ethnicity, multi-ethnic studies may be conducted in the future to generalize our conclusions. Finally, this study is short of a systematic review addressing multiple platelet indices  which could be fixed out in future study design. Due to the aforementioned limitations, these findings require further verification in the future.In summary, PLT was negatively correlated with 1-year OS in patients with cancer cachexia, which was validated in the total independent population cohort. In addition, patients with a high TBIL and a smoking history had a lower 1-year survival rate. Our findings, to some extent, provide certain guidance for the prognostic management of patients with cancer cachexia.


C. M. Jansen). To date, no study dealing with in vitro indirect regeneration system of korarima has been reported. Thus, in this study, we developed an efficient and reproducible protocol for in vitro regeneration of korarima via callus. The procedure involved soaking seeds in 50% H2SO4 for 16 h that resulted in 92.5% germination on plant growth regulators (PGRs)-free half-strength Murashige and Skoog (MS) basal medium after a month. Shoot and rhizome induction rate of 93.75% was obtained on the MS medium containing 1.5 mg/l BAP in combination with 0.1 mg/l IBA after five weeks. Whitish yellow friable callus was obtained from rhizome culture taken from in vitro grown plantlets. The MS medium containing 2.0 mg/l 2, 4D in combination with 0.5 mg/l kinetin, resulted in 77.5% callus induction. The shoot regeneration rate of 45% was obtained from callus on the MS medium containing 2.0 mg/l TDZ in combination with 0.5 mg/l IBA. The mean shoot number of 10.83 per explant was obtained upon multiplication on the MS medium containing 1.5 mg/l BAP with a mean shoot height of 5.37 cm. The best rooting responses were obtained on half MS medium supplemented with 0.5 mg/l IAA resulting in a mean number of root of 18.59, mean root length of 9.71 cm, and mean shoot height of 7.32 cm. The plantlets showed 75% survival efficiency after acclimatization. The present regeneration protocol offers a conceivable system towards effective conservation and genetic improvement of the crop by increasing the efficiency of genetic transformation.Developing an  Aframomum corrorima (Braun) P. C. M. Jansen) (Zingiberaceae) is a threatened plant species native to Ethiopia [Korarima , Ethiopia, and stored under dry conditions at room temperature until the experiments were started. Voucher specimens were collected, pressed, and deposited in the National Herbarium of Addis Ababa University (AAU). The plant identification was performed both in the field and at the National Herbarium of AAU. Furthermore, the plant name was checked and verified from the African plant database database  and httpine.org/  for its The experiment used the MS  basal mein vitro seed germination, to overcome the hard seed coat, seeds were soaked in 50% H2SO4 solution for 16 hours before sowing following [For the initiation of aseptic culture, capsules were cut open to release seeds, dried in shade for three days, and kept in sterile plastic bags. The seeds were taken out of plastic bags using forceps and washed three times with tap water and commercial liquid detergent. The washed seeds were kept under running tap water for 10 minutes. The seeds were then transferred to the laminar flow hood, washed in 70% (v/v) ethanol for one minute, and rinsed three times in sterile distilled water. This was followed by surface sterilization with sodium hypochlorite (NaOCl) solution that contains 5% active chlorine, at 20% NaOCl (1% active chlorine) concentrations for 20 minutes of exposure time. The seeds were rinsed five times with sterile distilled water followed by culturing in glass jars containing 50 ml plant growth regulator (PGR)-free half-strength MS basal solid medium. For optimum ollowing , 25.in vitro germinated seedlings and cultured on agar (0.7% plant propagation agar)-solidified MS basal media supplemented with 0.5, 1, 1.5, 2, 2.5, and 3 mg/l BAP alone and each BAP level in combination with 0.1 and 0.2 mg/l IBA. The liquid MS medium fortified with the above six levels of BAP was also used in the experiment. Therefore, the experiment was laid out using a complete random design (CRD) with a treatment combination of six concentrations of BAP and two concentrations of IBA that provides 1 × 6 factorial combinations in both cases of solid and liquid media and 6 × 2 for solid media.For shoot induction, shoot apices (1–1.5 cm) were taken from 45 days old Subculturing was carried out twice on the same fresh medium and maintained in growth room until suitable rhizomes develop for use as explant in subsequent callus induction experiment. The percentage of shoot tips that developed rhizomatous shoots, the number of shoots per explant, the height of microshoot, and number of leaves per microshoot were recorded after six weeks.in vitro shoot tip cultures were cut into small pieces and punched using forceps, wounded and cultured on the MS medium containing 30 g/l sucrose and 2, 4-D , NAA  and 2, 4-D  in combination with KN (0.5 and 1.0 mg/l) and NAA (0.5 and 1.0 mg/l) each. Accordingly, the experiment was laid out using the CRD with 5 × 3 and 4 × 2 × 2 factorial combinations. Five pieces of rhizomes were cultured in Magenta culture vessels. The culture was transferred to the same fresh medium every four weeks and maintained in darkness for 12weeks at room temperature. The percentage of rhizomes that induced callus with their color and texture was observed and recorded after nine weeks of the initial culture.Young rhizomes (0.5–1.0 cm long) induced on After three months, the produced calli were transferred to the shoot regeneration medium containing 0.5, 1.0, 1.5, 2.0, and 3.0 mg/l BAP and TDZ alone as well as 1.0, 2.0, and 3.0 mg/l BAP and TDZ in combination with 0.5 mg/l and 1.0 mg/l IBA each. Therefore, the experiment was laid out in the CRD with 5 × 5 and 3 × 3 × 2 factorial combinations. Then, the cultures were kept for a 12-hour photoperiod and covered with loose transparent soft papers for fifteen days. The cultures were then uncovered and maintained under eight-hour light conditions at 25 ± 2°C for another 15 days after which they were transferred to 16-hour photoperiod. Regenerated shoots were then separated and transferred on the same fresh medium. The percentage of calli that regenerated shoot, the number of shoots proliferated per explant, and shoot height were recorded after nine weeks.Microshoots that responded well were transferred to a shoot multiplication medium. The shoot multiplication medium was the MS medium containing BAP, TDZ, and KN at 0.5, 1.0, 1.5, 2.0 ,and 2.5 mg/l each and 1.0, 2.0, and 3.0 mg/l BAP combined with 0.1, 0.3, and 0.5 mg/l IBA. Thus, the experiment was arranged in a 3 × 5 and 3 × 3 factorial combinations in CRD. After five weeks, the multiplied shoots were separated and subcultured on the same fresh medium for three weeks. The number of shoots proliferated per shoot bud/microshoot explant and the length of shoots were recorded after six weeks of initial culture. However, the number of shoots and length of shoots produced after initial culture as well as at two successive subcultures within three weeks were recorded, and the mean values were considered for analysis.For rooting, microcuttings (2–3 cm long) produced through regeneration were excised and cultured on the half-strength MS medium supplemented with each of IBA, IAA, and NAA at 0.25, 0.5, 0.75, 1.0, 1.25, and 1.5 mg/l concentrations using the CRD of 3 × 6 factorial combinations. The number of roots, root length, and shoot height were recorded after five weeks of culture.After five weeks, well-rooted plantlets were removed from the culture vessels, and the roots were washed carefully under slightly warm water to facilitate the removal of adhering agar from the root surface, rinsed in a fungicidal solution of 3% Kocide‐101, and transferred to a sterilized potting mix of forest soil, compost, and sand at a ratio (v/v) of 1 : 2 : 1, respectively. The plantlets were then kept under a plastic cover of high humidity (80–90%) to prevent desiccation for ten days. Starting from the seventh day, the relative humidity within the plastic cover was reduced gradually to reach about 60% at the end of the tenth day. After the tenth day, the plantlets were transferred to a 70% shade net, where they were retained for a month. Later, they were transferred to a 30% shade net and maintained for another month, prior to transfer to the field. The percentage of survived plants was recorded after two months in greenhouse.All experiments were laid in the CRD with factorial treatment combinations, having five explants per culture vessel and four replications per treatment. All the experiments were repeated two times to ensure reproducibility of the results and the average were considered for analysis. Control experiment was laid together in all cases.Means of the data from the two repetitions for each experiment were subjected to statistical analyses using SAS  statistiin vitro growth and development on the PGR-free half-strength MS solid media, germination percentage of 92.5% was obtained from one-week-old seeds soaked in 50% H2SO4 for 16 hours  effect on the rate of shoot induction, number of shoot, shoot length, and number of leaf per shoot  on the rate of callus induction  on the rate of shoot regeneration, the number of shoots produced, and the height of shoots obtained as evidenced by ANOVA  was obtained on the MS medium containing 2.0 mg/l TDZ combined with 0.5 mg/l IBA followed by 43.33% obtained on the medium fortified with 2.0 mg/l BAP combined with 0.5 mg/l IBA . After nin vitro shoot regeneration could be affected by PGR composition of the growth medium.It is well known that PGR composition influences the direction of growth and development of cultured dedifferentiated calli , 38. HenThe synergy of TDZ and IBA was found to be effective for the shoot regeneration of korarima. The two highest regeneration rates (45% and 43%) were obtained on the MS medium containing 2.0 mg/l TDZ combined with 0.5 mg/l IBA and MS medium fortified with 2.0 mg/l BAP combined with 0.5 mg/l IBA . The meaCallus tissues cultured on the PGR-free MS medium, MS medium fortified with 3.0 mg/l BAP, and MS medium fortified with 3.0 mg/l TDZ did not show shoot regeneration. This result backs the actual knowledge about the need for the interactive activity of cytokinins and auxins in promoting cell division, growth, and development in plants , 36, 37.p < 0.0001) on in vitro shoot multiplication rate and shoot height of korarima , with a mean shoot height of 5.37 cm, was obtained on the MS basal medium supplemented with 1.5 mg/l BAP Figures  followedIn vitro propagation is an advanced technique for producing a large number of genetically uniform and pathogen free plants in limited time and space [In vitro shoot multiplication implies multiplication from one culture to many cultures and then many cultures to further many cultures, which is essentially a major criterion for successful commercial micropropagation. In the present study, the use of different types and concentrations of plant growth regulators highly affected the in vitro proliferation rate of korarima and subculturing was also found to be important for obtaining the optimum shoot multiplication result.nd space , which iAn increase in the number of shoots per shoot bud culture with an increased level of BAP and KN from zero to 1.5 or 2.0 mg/l  is due tin vitro. Adult propagules respond poorly in micropropagation due to phenolic exudates, which causes blackening [The proliferation rate had shown a progressive increase from the initial culture to the first subculture and second subculture by an average of 16.66% and 25%, respectively. Subculturing by trimming the base promotes the shoot proliferation rate as it is easier to establish juvenile than adult explants ackening , 41. Addackening . In thisMost of the shoot buds cultured on the MS medium containing 1.0 mg/l BAP combined with 0.5 mg/l IBA resulted in callus formation, whereas those at 2.0 mg/l and 3.0 mg/l BAP combined with the same 0.5 mg/l IBA resulted in shoot formation. This effect might be attributed to unsuitable proportion among cytokinin and auxin for proper stimulation of shoot growth .In the present study, the MS medium containing 1.5 mg/l BAP resulted in the highest number of shoots per explant. The mean number of shoots reported by  on this Mean shoot number of 16.6 per explant has been reported by  on the Mp < 0.0001) effect on the mean root number and length as well as shoot height (All (100%) of the microcuttings cultured on the half-strength MS medium containing 0.5, 1.0, and 2.0 mg/l of the above auxins were rooted after four weeks. The ANOVA also indicated that the variations in the concentration of the auxins had a very highly significant (t height .The highest mean number of roots (18.59), mean root length (9.71 cm), and mean shoot height (7.32 cm) were recorded from the half-strength MS medium containing 0.5 mg/l IAA followed by the half-strength MS medium containing 0.75 mg/l IBA resulting in a mean root number of 16.59, mean root length of 8.16 cm, and mean shoot height of 7.6 cm . The halin vitro rooting. In the present study, all IAA, IBA, and NAA were found to be effective in promoting root formation and enhancing shoot elongation on the half-strength MS basal medium.Rooting is an important process to the success of micropropagation. Without an effective root system, plant acclimatization is difficult and the rate of plant propagation may be affected substantially . ConcentSimilar to the present result,  reportedParallel to , 23, theAfter rooting, 143 plantlets were transferred to pots (3 plantlets per pot), containing a mixture of forest soil, compost, and sand in 1 : 2 : 1 ratio. The pots were then shielded with polyethylene plastic covers and placed in the greenhouse for acclimatization to outer environmental conditions, out of which, 107, that is, 75%, were survived and established well in pots after two months .in vitro regeneration protocol for korarima was established. The protocol could provide a possible system towards effective germplasm conservation and genetic improvement of the crop, and increase the efficiency of transformation protocols employing efficient regeneration system.An


Paris polyphylla with a giant genome. The lack of genomic data and tissue specificity causes the study of the biosynthetic pathway notably difficult. Here, we report an effective method for the prediction of key genes of polyphyllin biosynthesis. Full-length transcriptome from eight different organs via hybrid sequencing of next generation sequencingand third generation sequencing platforms annotated two 2,3-oxidosqualene cyclases (OSCs), 216 cytochrome P450s (CYPs), and 199 UDP glycosyltransferases (UGTs). Combining metabolic differences, gene-weighted co-expression network analysis, and phylogenetic trees, the candidate ranges of OSC, CYP, and UGT genes were further narrowed down to 2, 15, and 24, respectively. Beside the three previously characterized CYPs, we identified the OSC involved in the synthesis of cycloartenol and the UGT (PpUGT73CR1) at the C-3 position of diosgenin and pennogenin in P. polyphylla. This study provides an idea for the investigation of gene cluster deficiency biosynthesis pathways in medicinal plants.The genes in polyphyllins pathway mixed with other steroid biosynthetic genes form an extremely complex biosynthetic network in  Paris polyphylla, and the approach may be relevant to other medicinal plants.Xin Hua, Wei Song, and ZheYong Xue et al. report an effective method to predict key genes involved in polyphyllin biosynthesis in plants. Their results provide further insight into biosynthesis pathways in  P. polyphylla var. yunnanensis is a member of Liliaceae family and one of the most famous medicinal plants in China. The rhizome of this plant is an important component of the traditional Chinese medicines “Yunnan Baiyao” and “Gongxue Ning”1, which have pharmacological activities, such as hemostasis, analgesic, sedation, anti-inflammatory, and anti-tumor effects4. The main active components of this plant are steroidal saponins, also known as polyphyllins, accounting for about 80% of the total number of active compounds5. Polyphyllins have aroused great interest for their rich pharmacological activities, including anti-inflammatory, vascular protection, hypoglycemic, immunomodulatory, antiparasitic, hypocholesterolemic, antifungal, anti-parasitic, and anti-tumor effects7. However, the species is at risk of extinction due to its slow growth and excessive exploitation8. Given their complex molecular structures, polyphyllins are unlikely to be chemically synthesized for commercial usages. Therefore, metabolic engineering may be an effective method to provide a stable source of polyphyllins. The metabolic engineering strategy largely relies on the biosynthetic pathway of polyphyllins, which still has not been fully elucidated.8. However, the sources of diosgenin mainly depend on the extraction from several specific plants, such as yam (Dioscorea genus) and fenugreek (Trigonella foenum-graecum). The biosynthetic pathway of polyphyllins begins with the condensation of two molecules of isopentenyl diphosphate and one molecule of dimethylallyl diphosphate, which is then catalyzed by farnesyl diphosphate synthase (FPS) to form farnesyl diphosphate 9. Two FPP molecules are catalyzed by squalene synthase (SQS) to produce a linear C30 molecule, squalene, which is further cycled by squalene epoxidase to 2,3-oxidosqualene9. Then, 2,3-oxidosqualene is cyclized by a cycloartenol synthase (CAS) to form cycloartenol, which is then modified through a series of oxidation and reduction to form cholesterol11. Enzymes in the CYP90G family can catalyze the hydroxylation of cholesterol C-16 and C-22 with the closure of E ring. Then, 16S,22S-dihydroxycholesterol is further hydroxylated at C-26 and forms an F ring to produce diosgenin under the action of cytochrome P450s (CYPs), such as PpCYP94D10812. However, how steroidal skeleton α-hydroxylates at C-17 form pennogenin is still unknown. Subsequently, diosgenin and pennogenin are glycosylated by UDP glycosyltransferases (UGTs) to form various polyphyllins te FPP, C59. Two F15. The biosynthesis of several specified metabolites, such as the ginsenosides in Araliaceae family, lacks tissue specificity. A variety of ginsenosides are widely found in multiple tissue parts of the plant, and the complex distribution pattern of ginsenoside components hinders the prediction of the exact genes involved in biosynthetic pathway application by simple differential expression analysis16. The divergent evolution of CYP and UGT families generated numerous individual members that are phylogenetically close and can decorate diverse type of natural products. For this reason, predicting the related pathway gene solely based on phylogenetic analysis is impossible17. A previous study reported the extremely huge genome size of Parideae species (about 50 pg) . In addition, the biosynthetic genes of specific plant metabolites are scattered in different regions of the genome, further increasing the difficulty in identifying candidates precisely by the physical distance of metabolic pathway-related genes19, despite the generation and good assembly of whole-genome sequences21. Therefore, an efficient strategy needs to be developed and improved urgently to accurately predict the key genes in the complex none-clustered biosynthetic pathway of specialized plant metabolites.Although genomic and transcriptional information of numerous medicinal plants has been generated and made available to the public, the progress of candidate gene mining and whole pathway dissection of specialized plant metabolites remains slow due to the following factorsP. polyphylla var. yunnanensis. The weighted gene co-expression network analysis (WGCNA) combining the distributions of specific metabolites, different gene expressions, and phylogenetic analysis was further used to predict the key genes involved in the biosynthesis of polyphyllins. Then, candidate genes containing several assumed 2,3-oxidosqualene cyclase (OSC) genes and UGTs were functionally verified. This study may provide a strong basis for characterizing the steps of biosynthesis of polyphyllins of P. polyphylla var. yunnanensis, thus promoting the production of such important chemicals via synthetic biology.In this study, full-length transcriptome analysis using hybrid sequencing strategy based on single-molecule sequencing and paired-end mRNA sequencing was performed on eight different tissues from 22 to evaluate the integrity of the transcriptome, the results showed that complete and single-copy duplicated transcript sequences accounted for 69.92%, the fragmented ones accounted for 5.97%, and those missing accounted for 26.11%.A total of 292.69 Gb clean data for 21 sequencing libraries, including three biological replicates of rhizome, fibrous root, stem, leaf, and ripe fruit and two biological replicates of stigma, anther, and petals, were obtained by Illumina sequencing  unigenes from 39,875 transcripts. Afterward, we performed comparisons in the Swissprot, eggNOG, KOG, and Pfam databases and annotated 29,475 (73.92%), 37,673 (94.48%), 24,581 (61.65%), and 33,607 (84.28%) unigenes.P. polyphylla var. yunnanensis and other 51 species showed that different branches distinguished the classification of OSC genes. The different OSC subfamilies are distributed in terms of various activities, especially for the skeletons of catalytic products, including cycloartenol, β-amyrin, lanosterol, lupeol, α-amyrin, friedelin, dammarenediol II, and mixed products  and CYP86 (60) families have the largest number of P450 genes, followed by the CYP85 (37) and CYP72 (21) families, whereas CYP710 (4), CYP97(4), CYP74 (2), CYP711 (2) and CYP51 (1) has the smallest number of P450 genes  in different tissues of sis Fig. . Diosgenles Fig. . When thBased on the correlation coefficient, we observed that trillin clustered well with the lavenderblush3 0.82), lightblue3 (0.72), and white (0.73) modules in the co-expression network. Polyphyllin I had the highest correlation with Coral (0.72) and orangered4 (0.7) modules, and polyphyllin VII showed a high correlation with antiquewhite4 (0.81) and antiquewhite1 (0.76). Diosgenin and cholesterol were correlated with the antiquewhite4 module, and the correlation coefficients were 0.66 and 0.65, respectively. Polyphyllin II was correlated with lavenderblush3 (0.64) module, but polyphyllin VI and prosapogenin A showed no specific correlation with a certain module. The clustering results indicated that the genes in the orangered4, lavenderblush3, lightblue3, coral, antiquewhite1, white, and antiquewhite4 modules are likely to be involved in the biosynthesis of polyphyllins. Furthermore, we analyzed the upstream genes involved in the metabolic pathways of cholesterol or other phytosterols in the above seven modules. The coral, antiquewhite4, and lavenderblush3 modules contained relatively rich sterol synthesis upstream genes  , PpCYP90B27 (F01_transcript/40246), and PpCYP72A616 (F01_transcript/40246), have been reported to be involved in the biosynthesis of polyphyllins. These three genes ranked 1st, 3rd, and 6th place in the list of candidate genes, respectively. These candidate genes are very likely to be involved in the biosynthesis of polyphyllins. The genes that appeared in the candidate clades of phylogenetic tree but were not included in the candidate modules of WGCNA should not be ignored. They may also be involved in the biosynthesis of polyphyllins. All candidate OSC, CYP, and UGT genes can be found in GenBank (BankIt2522666: OL654188–OL654276).In addition, we predicted the candidate ely Fig. . Among tOSC gene in lower plants, higher plants always have multiple OSC gens in their genomes9. A total of 13 OSC-related transcriptome variants were found in the transcriptome, which may represent two non-redundant OSCs (PpOSC1 and PpOSC2). To investigate the activity of the two candidates, we cloned two genes transferred them into optimized yeast strains (SQSQ-pPOSC1 and SQSQ-pPOSC2). Cycloartenol production was evidently observed in the SQ-PPOSC1 strains, whereas it was absent in SQ-PPOSC2 strains, suggesting that PpOSC1 gene encodes CAS in P. polyphylla var. yunnanensis  of diosgenin and pennogenin were 177.13 ± 8.91 and 87.7 ± 3.27 nmol/min/mg, respectively. Michaelis constant (Km) reflected the affinity between the enzyme and substrate to a certain extent. Compared with pennogenin (Km = 73.43 ± 8.16 µM), PpUGT73CR1 had a higher affinity for diosgenin (Km = 53.69 ± 9.37 µM). The enzymatic catalytic constant (Kcat) of PpUGT73CR1 for diosgenin and pennogenin were 0.24 and 0.12 s−1, respectively, and the calculated conversion efficiencies (Kcat/Km) were 4.47 (diosgenin) and 1.62 (pennogenin) mM−1 s−1. Thus, the catalytic efficiency of PpUGT73CR1 for diosgenin was higher than that of pennogenin.To study the promiscuity of UGT to diverse substrates, we evaluated the catalytic capability of PpUGT73CR1 on diosgenin and pennogenin. Subsequently, the enzymatic kinetics of PpUGT73CR1 catalyzing different substrates were studied  all come from the coral module. These results showed the accuracy and reliability of this prediction method.We constructed a co-expression network of metabolites with gene expression levels in different tissue of Solanum aculeatissimum shows the glycosylation activity of diosgenin, nuatigenin, tigogenin, and other glycosyltransferases31. In this study, c-3 glycosyltransferases of diosgenin and pennogenin were found in P. polyphylla var. yunnanensis. According to the study of substrate promiscuity and enzyme kinetics, PpUGT73CR1 had a better affinity and catalytic capability with diosgenin compared with pennogenin.Several studies focused on glycosylation modification of steroidal sapogenins. A C-3 glycosyltransferase SAGT4a in 33, and several P450s. Other research reported the glycosylation modification of diosgenin. Therefore, the key genes involved in the biosynthesis pathway of polyphyllin can be possibly predicted by using our prediction method combining the evolutionary tree, co-expression network, and gene expression quantity. This method is also generally applicable to the prediction of key genes in plants lacking genome data.At present, the analysis of steroidal sapogenin biosynthetic pathways is progressing slowly and remains in the exploratory stage. Research has focused on the cloning and regulation of functional genes upstream of terpenoid biosynthetic pathways, such as HMGR, FPS, SS, CAS, etc.Paris polyphylla var. yunnanensis, and the gene expression and WGCNA method were used to predict the OSCs, CYPs, and UGTs involved in the biosynthesis of sapogenin. Among the predicted candidate genes, we identified an OSC gene (PpOSC1) and a diosgenin/pennogenin C-3 UGT gene (PpUGT73CR1). This study improves our understanding of the biosynthetic pathways of polyphyllins, providing a basis for further elucidation of the pharmacologically active triterpene/sapogenin biosynthesis and an efficient strategy to study the complex pathway of other specialized plant metabolites.Polyphyllin has a variety of pharmacological activities, but the analysis of the biosynthetic pathway of polyphyllin is incomplete. We performed splicing and full-length transcriptome sequencing of rhizomes, fibrous roots, stems, leaves, ripe fruits, stigma, petals, and pistil tissues of P. polyphylla var. yunnanensis were collected from Dali, Yunnan, China. The rhizomes, fibrous roots, stems, leaves, ripe fruits, stigma, petals, and pistil were harvested in 5-year-old healthy plants , in accordance with the manufacturer’s protocol. Three biological replicates of rhizome, fibrous root, stem, leaf, and ripe fruit were determined, and two biological replicates of stigma, anther, and petal were tested due to difficulty in sample collection and insufficient RNA quality. RNA quality was examined using Agilent 2100 . The cDNA library was constructed and sequenced by Biomarker Technologies Corporation . A single tissue was sequenced using an Illumina Hiseq 2000 platform, and full-length transcriptome sequencing for the mixture of different tissues was performed using PacBio Sequel platform. The PacBio long reads were filtered, and redundant sequences were removed using CD-HIT-EST program. In full-length transcriptome data, sequences with polymerase read <50 bp and sequence accuracy <0.9 were filtered out. Then, the clean reads from Illumina sequencing were mapped into the non-redundant long-reads to calculate the fragments per kilobase per million (FPKM) values using DESeq2. Specifically, FPKM = cDNA fragments/mapped fragments (millions) × transcript length (kb), where cDNA fragments represent the number of fragments aligned to a transcript, mapped fragments (millions) is the total number of fragments aligned to the transcript; transcript length (kb) denotes the transcript length. The filtered full-length transcripts were functionally annotated using non-redundant (nr), SWISSPROT, Gene Ontology, Clusters of Orthologous Genes, EuKaryotic Orthologous Groups (KOG), PFAM, and Kyoto Encyclopedia of Genes and Genomes databases, respectively.27. A bootstrap resampling analysis with 1000 replicates was performed to evaluate the topology of phylogeny.Transcripts belonging to OSCs, CYPs, and UGTs were identified using BLAST software. The transcripts with a length under 1000 bp were removed. Supplementary Data g for 5 min. The supernatant was transferred to a new centrifuge tube and added with 300 µL n-hexane for extraction. After extraction, the n-hexane layer was sucked and removed, and the process above was repeated. SpinVac was applied for solvent removal in the samples. The samples were redissolved with 500 ml distilled water. Then, extraction was performed twice with 500 mL n-butanol. Nitrogen was used to dry the organic phase of the sample and redissolved sample with the mobile phase during measurement.The tissues frozen at −80 °C were lyophilized in a freeze dryer. Then, 20 mg dry materials from different tissues were weighted and placed in a 2 mL centrifuge tube. A total of 1 mL 80% methanol containing 20 µg internal standard was added to the sample . The samples were further extracted at 1400 rpm for 2 h and centrifuged at 10,000 × 2, 1.7 µm) at 40 °C. The 0.1% formic acid water was used as mobile phase A, and 0.1% formic acid in acetonitrile was used as mobile phase B. The gradient was 0–1 min, 5–52% B; 1–6 min, 52–56% B; 6–7 min, 56–95% B; 7–7.5 min, 95–95% B; 7.5–9 min, 95–5% B; 9–10 min, 5–5% B. The flow rate was 0.25 mL min−1, and the injection volume was 5 µL. The electrospray ionization source interface operated in negative ionization mode was used in this study. The ion spray voltage was set at −4500 V. Supplementary Table m/z, and the solvent delay was 10 min. The GC conditions were as follows. The sample (1 µL) was injected in split mode (10:1) at 250 °C under a He flow rate of 1.2 mL min−1, and the temperature cycle involved the initial injection temperature of 170 °C for 2 min, 170–290 °C for 6 °C per minute, holding for 4 min after the temperature reached 290 °C, and raising the temperature from 290 to 340 °C at 25 °C per minute.The analysis was performed on a Waters ACQUITY ultra-performance liquid chromatography (LC) system coupled with an AB Sciex 5500 Qtrap mass spectrometer . Chromatographic separation was achieved on an ACQUITY BEH C18 column . Based on the TOM, we used the average-linkage hierarchical clustering method to cluster genes, following the standard of the hybrid dynamic shearing tree and set the minimum number of genes for each gene network module to 30. The power β was selected based on the scale-free topology criterion. The modules were detected as branches of the dendrogram using the dynamic tree-cut, and a cut-off height of 0.25 was used to merge the branches to the final modules.Gene co-expression networks were constructed using the WGCNA approach with R packages (version 3.2.2)Finally, the gene visual network was described by using a heatmap. The heatmap depicts the TOM among all genes in the analysis. Light color represents a low overlap, and the progressively darker red color represents higher overlap. Blocks of darker colors along the diagonal are the modules, and a very strong association existed between the genes that are contained within these red modules. These red modules were the focus of our genetic prediction.Nicotiana benthamiana. Supplementary Table PpOSC1 and PpOSC2 were cloned from P. polyphylla var. yunnanensis and transferred into pδHis plasmid. The plasmid was transformed into yeast strain BY-SQ1 using the standard lithium acetate approach. The yeast strains SQ-PpOSC1 and SQ-PpOSC2 were precultured in 5 mL synthetic defined medium with glucose as carbon source and uracil and histidine omitted (SD-URA-HIS) at 30 °C and 220 rpm for 24 h. Precultures were inoculated at an initial optical density (OD)600 of 0.05 in 50 mL SD-URA-HIS in 250 mL flasks and grown under the same condition for 72 h. The cells were harvested, resuspended in 2 mL 10% KOH (w/v) and 90% ethanol (v/v), heated for 2 h at 75 °C, and cooled and extracted once with 0.5 mL ethyl acetate. After centrifugation, the ethyl acetate phase was collected and dried by centrifugal vacuum evaporator. Derivatization of the dried products was conducted with 1-(trimethylsilyl)imidazole-pyridine mixture at 70 °C for 30 min to prepare the sample for analysis.The function of OSC gene was verified by yeast strain and Nicotiana benthamiana, the coding regions of candidate OSC genes were cloned from P. polyphylla var. yunnanensis into the pEAQ-HT-DEST1 vector. After sequence verification, pEAQ-HT-DEST1 vectors carrying OSC genes were separately transferred into Agrobacterium tumefaciens strain GV3101 and cultured overnight at 28 °C and 220 rpm. Then, 1 mL culture was used to inoculate 10 mL Luria–Bertani (LB) medium containing 50 µg/mL kanamycin, 25 µg/mL rifampicin, and 25 µg/mL gentamicin for overnight growth. The following day, the cultures were centrifuged , and cells were resuspended in infiltration buffer (10 mM MES (C6H13NO4S), pH 5.6, 10 mM MgCl2, and 100 µM acetosyringone) to a final OD600 of 0.4. The leaves of 6-week-old N. benthamiana were infiltrated with A. tumefaciens solution as follows. A 5 mL needle-free syringe was used to gently push the bacterial mixture into the abaxial surface until the entire leaf was filled with agrobacterium. The infiltrated leaves were cultured at 22 °C, exposed to light for 10 h a day, and harvested at 6th day after infiltration. For metabolite extraction, leaf disks in diameter 1 cm were prepared from Agrobacterium-infiltrated N. benthamiana and dried with a vacuum freeze-dryer. Then, the leaves were ground into powder, and 10 mg powder was weighed and placed a 2 mL tube for use. Then, 2 mL lysate was added to each sample and heated in the water bath (75 °C for 1 h). After the samples were completely dried, 300 µL ethyl acetate and 500 µL water were added and mixed with vortex shock and centrifuged for 10 min to facilitate separation. Next, 100 µL was removed from the upper layer (ethyl acetate layer) and transferred to a special glass tube. The liquid was blow-dried with nitrogen and added with 50 µL 1-(trimethylsilyl) imidazole-pyridine mixture. After vortex-mixing–heating at 70 °C for 30 min, the mixture was analyzed with GC–MS same as cholesterol analysis above.In the transient expression system of P. polyphylla var. yunnanensis was extracted and reverse transcribed to obtain cDNA. The candidate polymerase chain reaction (PCR) primers for UGTs were designed based on the transcriptome sequence. The PCR procedure was as follows: 95 °C for 3 min; 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 90 s in 33 cycles; 72 °C for 5 min. The primers of UGT genes are shown in Supplementary Data E. coli DH5α.The total RNA from the 600 of = 0.6. A total of 0.2 mM isopropyl β-d-1-thiogalactopyranoside was added to the culture medium, induced at 16 °C for 16 h, and centrifuged at 4 °C at 5000 rpm to collect the bacteria. The bacterial cells were suspended in 10 mM phosphate buffer (pH 7.4), and the cells were disrupted by ultrasound in an ice bath. Then, the cells were centrifuged at 12,000 × g at 4 °C for 20 min. The bacterial supernatant was purified using glutathione beads  and concentrated using Millipore ultrafiltration tubes . Pierce BCA Protein Assay Kit  was used to quantify the target protein.The plasmid with correct sequencing was transformed into Rosetta-gami B (DE3) pLysS and inoculated into LB liquid medium containing ampicillin (100 mg/L) and then cultured at 37 °C at 180 rpm until the OD2, 5 mM glucose donor (UDP-glucose), 1 mM glucose receptor (diosgenin/pennogenin), and purified enzyme of PpUGT73CR1 in a final volume of 100 µL. After overnight incubation at 37 °C, an equal volume of ice methanol was added to stop the reaction. The product was concentrated and dried, dissolved in 100 µL chromatographic methanol, and centrifuged at 12,000 rpm for 10 min, and the supernatant was obtained for testing. The reaction products were identified by high-performance LC (HPLC) and LC time-of-flight mass spectrometry (LC-TOF-MS), and the Thermo Hypersil GOLD C18 column  was used for HPLC detection. The mobile phases were water (A) and acetonitrile (B). The elution gradient was as follows: 0–6 min, 20–30% B; 6–15 min, 30–60% B; 15–21 min, 60–100% B; 21–30 min, 100% B, 30–35 min, 100–20% B. The flow rate was 1 mL/min, the column temperature was 30 °C, the injection volume was 10 µL, and the detection wavelength was 210 nm. LC-TOF-MS was performed using the AB Sciex Tripletof 6600  in a positive ionization mode.The enzymatic reaction system consisted of 50 mM Tris (pH 8.0), 1 mM MgClFor the kinetic analysis of UGT73CR1, the reaction mixture contained 50 mM Tris−HCl (pH 8.0), 5 mM UDP-glucose, acceptor substrate (20–400 µM diosgenin and pennogenin), and 1 µg purified UGT73CR1 in a final volume of 100 µL. The reaction was incubated at 37 °C for 30 min. HPLC analysis was used to quantify the target product in each reaction. The Michaelis–Menten parameters were calculated by kinetic model using Prism 7 . All data are presented as means ± standard deviation of three independent experiments.All data calculations in this research are from more than three independent experiments. The WGCNA analysis and heatmap drawing of CYPs and UGTs were generated with R packages (version 3.2.2) and the concentration data of polyphyllins were processed by Graphpad 7.0 and presented with mean ± standard deviation.Further information on research design is available in the Transparent Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary


In 1948, Claude Elwood Shannon  then buip-th Rényi entropy [p-th Rényi entropy power is given byThe  entropy ,4 of a pined asHp(f):=1iven byNp(f):=eentropy power inequality (EPI) is one of the most important information inequalities [Shannon’s ualities , which hualities ,11,12,13ualities .n-dimensional random vector introduced by Costa [probability density of Let by Costa ,15,16,17Costa’s differential entropy is defined to beShannon entropy power t; that is, Related to EPI, Costa  proved tgiven in ,19,20,21concavity of entropy power is a property which is not restricted to the Shannon entropy power . Precisely, we give a propositional logic formula In this paper, we give a generalization for the -Toscani  and contgiven in  consist The formula given in ,16,17,23mination . In thisThe rest of this paper is organized as follows. In In this section, we present a procedure to prove the CREP. To make the paper concise, we only give those steps that are needed in this paper.t andLet u to denote u with respect to the differential operators v has order degree, order, and total order are defined to be Let kth-order differentially homogenous polynomial or simply a kth-order differential form, if all its monomials have degree k and total order k. Let k and total order k. Then, the set of kth-order differential forms is an lexicographic order for the monomials defined in [A polynomial in fined in ,16,17.CREP. The property In this section, we give the procedure to prove the y proved . We focuCREP into the proof of an integral inequality, as shown by the following lemma, the proof of which is given in In step 1, we reduce the proof of Lemma 1.The proof of under the condition u of R is called an equational or inequality constraint ifIn step 2, we compute the constraints, which are relations satisfied by the probability density E2,n in  is a fouThe method to compute the constraints is given in S is a sum of squares (SOS). Details of this step and the formula In step 3, we find a propositional formula To summarize the proof procedure, we haveTheorem 1.The CREP is true if Proof. S is an SOS and hence By Lemma 1, we have the following proof for CREP:second-order equational constraint is a fourth-order differential form in In this section, we show how to find the second-order equational constraints. A Property 1.Let withWhen ows from . While Using Property 1, we can compute 28 second-order equational constraints using the method given in ,16,17:, w, w(22)Remark 1.In , the sterding to , and , we, we25), fined in . Then, tIn this section, we prove a generalized CREP using the procedure given in Theorem 2.Let  solving  and sati solving . Then, wfined in  satisfieunder the condition The proof of the above theorem consists of three steps, which are given in the following three subsections.We first give an inequality constraint. Denote From  and 29)29), in oProblem 1.Find a formula under the conditions given in .Since From , in ordeProblem 2.Find a formula a and b are fixed and are treated as symbols.In this section, we simplify C2,n in . Note thOur goal is to reduce fined in .quadratic monomial. Write monomials in We simplify the constraints in  as folloWe now simplify In order for From , in ordeProblem 3.Find a propositional formula In principle, Problem 3 can be solved with the quantifier elimination . In thisBy the knowledge of linear algebra, fined in . FurtherLemma 5.We haveWe present an explicit formula for Φ in . First, ⌀. Define i-th row and the j-th column in We define Φ in  using TaFor example, uch that  is true uch that , and theWe now give the main result of the paper, which implies Theorem 2. The proof for the theorem can be found in Theorem 3.The sufficient and necessary condition for Problem 3— that is,  must be where fined in  and ∨ meWe show that our result includes the result proved in  and moreIn , the CREroved in  is a speWe can also prove the result in  directly)2≤0 in , we obtaWe can use the SDP code in . By , μ4≤μ≤μCase 1.2: We now eliminate μ6). By , μ7≤μ≤μCase 1.3: Case 1.3.1: We now eliminate Case 1.3.2: Case 1.3.2.1: If We now eliminate Case 1.3.2.2: If We now eliminate Case 1.4: Case 1.4.1: Similar to Case 1.3.2.1, We now eliminate Case 1.4.2: Similar to Case 1.3.2.2, We now eliminate Case 1.5: Case 1.5.1: Similar to Case 1.3.2.1, we have We now eliminate Case 1.5.2: Similar to Case 1.3.2.2, we have We now eliminate Case 1.6: We now eliminate Case 1.7: Case 1.7.1: If we select We now eliminate Case 1.7.2: If we select p satisfies Case 2: 1. From , we haveand from , Φ(n,p,Case 2.1: We now eliminate Case 2.2: Case 2.3: Case 2.4: Case 3: Case 3.1: If Case 3.2: If p and This paper is an extension of the work ,16,17 totic form  whose comination . Some inmination ,27 can hBased on the above method, we give a sufficient condition -Toscani . By TheoFor future research, it is interesting to see whether the three conjectures about Costa’s differential entropy studied in  can be g


The effects of postoperative intervertebral height (IH) changes on the clinical and radiological outcomes after anterior cervical decompression and fusion (ACDF) surgery using a zero-profile device remain unclear.We retrospectively reviewed patients who had undergone ACDF using a zero-profile device from March 2012 to February 2016 at our institution. Based on the postoperative IH variation, the patients were divided into group A with postoperative IH 0 to 2 mm, group B with postoperative IH 2 to 4 mm, and group C with postoperative IH greater than 4 mm. Clinical efficacy was evaluated using JOA, VAS, and NDI scores in the groups. Imaging parameters including the IH, cervical lordosis, fusion rate, intervertebral foramen (IVF) diameter and complications such as subsidence, dysphagia, and ASD were also compared across the three groups.The average IH increased significantly from 6.72 mm preoperatively to 10.46 mm 1 week after surgery, and then gradually decreased to 7.48 mm at the final follow-up. The fusion rate was 61.90% in group A, 63.23% in group B, 53.57% in group C at 3 months, 73.81% in group A, 79.41% in group B, 67.86% in group C at 6 months, 90.48% in group A, 95.59% in group B, 92.86% in group C 1 year after surgery, and at the last follow-up, the fusion rate of three groups was all 100%. The IVF diameter was 6.52 ± 1.80 mm in group A, 9.55 ± 2.36 mm in group B, and 9.34 ± 1.62 mm in group C. ASD at the superior and inferior levels affected 11.90 and 16.67% patients in group A, 5.88 and 7.38% in group B, and 14.28 and 10.71% in group C. Regarding the 3 groups, the subsidence rates were 7.14, 4.41, and 14.29%, respectively.No clear correlation was found between IH changes and clinical efficacy within a year of surgery. However, the IH may affect various complications after ACDF. If postoperative IH changes are maintained at 2 to 4 mm after a year, a satisfactory imaging parameters and relatively low complications may be achieved after ACDF surgery using a zero-profile device. Cervical disc degenerative disease (CDDD) is a spinal disorder that commonly affects middle-aged and older adults and may cause neck discomfort, radiating upper extremity pain, and neurologic abnormalities. Surgery is recommended if the patient does not respond to conservative treatment. Anterior cervical decompression and fusion (ACDF) using a traditional plate-cage construct (PCC) system is the main spinal surgery approach to treat symptomatic cervical disc disease . HoweverSatisfactory reconstruction and consistent maintenance of the intervertebral height (IH) influence the cervical surgery outcome. IH distraction is associated with neck pain, the occurrence of ASD and neural functional recovery after cervical surgery . HoweverHere, we examined IH changes following zero-profile ACDF and analysed the relationship between IH and clinical efficacy and imaging parameters.Ethical approval for this study was granted by our institutional ethics committee. All the participants provided informed consent for analysis of their clinical data.The data on single-level CDDD were collected retrospectively from March 2012 to January 2016 at our institution. The primary inclusion criteria were patients aged 18–65 years, symptomatic cervical disc degenerative diseases (CDDDs) with spondylotic radiculopathy or myelopathy at 1 level from C3 to C7 that correlate with imaging findings, patients showing a poor effect on conservative treatment or unclear improvement after at least 3 months with a worsening condition. Patients were excluded if they had received cervical disc arthroplasty (CDA), were treated with hybrid surgery (CDA incorporated with fusion), had undergone ACDF using another implant, or had been treated with multilevel surgery. Patients with infections, osteoporosis, spinal fractures, spinal deformity, allergy to the device material, ankylosing spondylitis, rheumatoid arthritis, and previous cervical spine surgery were also excluded.All anterior fusions were performed using the Smith-Robinson technique and a right-sided approach by the same surgeon. After confirmation and exposure of the appropriate vertebral levels, a Caspar distracter was used, and disc material was removed. The endplate cartilage was scraped using a curette or high-speed electric drill to prepare for bone grafting. The posterior longitudinal ligament, osteophytes, and other compressive elements were removed to ensure adequate dural and neural decompression. After measuring the intervertebral height and width, the appropriate Zero-P implant filled with b-tricalcium phosphate was inserted with an implant holder/aiming device.The patients wore a soft collar for 3 months after surgery, began functional exercise a day after the operation and were given a home exercise regimen at discharge.The clinical outcomes were evaluated using the visual analog scale (VAS) arm and neck score, Japanese Orthopedic Association (JOA) score, and neck disability index (NDI). The VAS evaluated neck and arm pain, the JOA score evaluated myelopathy status, and the NDI assessed neck function. These clinical outcomes were measured preoperatively and at 3 months, 6 months, and 12 months and at the last postoperative follow-up.Independent radiologists performed radiographic imaging using standing lateral, flexion and extension radiographs. Radiologic measurements included the IH, cervical curvature, the functional spine unit (FSU), the intervertebral foramen (IVF) diameter, adjacent segment degeneration (ASD), the fusion rate and implant subsidence. The IH was calculated using the formula a-(b + c), where a is the distance from the midpoint of the upper endplate of the cephalad vertebral body to the lower endplate of the caudal vertebral body, b is the distance from the midpoint of the upper endplate of the cephalad vertebral body to the midpoint of the lower endplate, and c is the distance from the midpoint of the upper endplate to the midpoint of the lower endplate Fig. . CervicaRadiographic assessments were performed twice by two independent surgeons, and the mean values were used for statistical analysis. The results are presented as means ± standard deviation. Student’s t-test or the Mann–Whitney U test was used to compare continuous data between two groups, depending on whether the data were normally distributed. Chi-squared test or Fisher’s exact test was used to compare categorical data between the groups. Odds ratios with 95% confidence intervals were calculated for differences between the groups. P = < 0.05 was considered statistically significant. SPSS version 22.0 (IBM Corp.) was used for statistical analyses.One hundred forty-eight patients were enrolled in the study; among them, ten patients were excluded because of incomplete postoperative imaging data and did not attend the final follow-up. Thus, 138 consecutive patients  qualified for further analyses. The mean age was 48.03 ± 9.51 years, with a mean follow-up of 22.83 ± 6.272 months (range: 18–36 months). Twelve patients were at the C3–4 level, 34 at the C4–5 level, 71 at C5–6, and 21 at C6–7.p = < 0.05). The average IH was 7.48 mm at the final follow-up (p = > 0.05) . The fusion rate was 61.90% in group A, 63.23% in group B, 53.57% in group C at 3 months, 73.81% in group A, 79.41% in group B, 67.86% in group C at 6 months, 90.48% in group A, 95.59% in group B, 92.86% in group C 1 year after surgery, and at the last follow-up, the fusion rate of three groups was all 100%. The fusion rate of patients in group C was significantly lower than that in the other two groups in the first 6 months (p = < 0.05). The mean IVF diameter was 6.52 ± 1.80 mm (variation = 2.58 ± 0.51 mm) in group A, 9.55 ± 2.36 mm (variation = 0.87 ± 1.21 mm) in group B, and 9.34 ± 1.62 mm (variation = 1.47 ± 1.30 mm) in group C. The IVF diameter in group A was significantly different than that in the other two groups, while the variation differed significantly between groups A and B 1 year post-surgery . The incidence of implant subsidence was 7.14% in group A, 4.41% in group B, and 14.29% in group C. The rates in group B were significantly different from those in group C for implant subsidence. The incidence rates of dysphagia and local kyphosis did not differ significantly among the groups  of group A patients, 5.88% (4/68) of group B patients, and 14.28% (4/28) of group C patients. The ASD at the inferior level affected 16.67% (7/42) of group A patients, 7.35% (5/68) of group B patients, and 10.71% (3/28) of group C patients. Group B rates were significantly lower than those of the other groups  was effectively improved in all patients. The average IH rose significantly from 6.72 mm preoperatively to 10.46 mm 1 week before being progressively reduced to 9.58 mm at 3 months, 8.73 mm at 6 months, 7.58 mm at 1 year, and 7.50 mm at the last follow-up. Thus, the IH changed subtly 1 year after ACDF  [Achieving complete nerve root decompression in cervical spondylotic radiculopathy patients with intervertebral foramen stenosis is challenging. Frequently, no remission for radicular pain or reoccurrence is observed after temporary relief in such patients. Although the pathogenesis of cervical radiculopathy is not completely understood, stenosis of the intervertebral foramina is considered among its main mechanistic underpinnings . AdditioAn in vitro biomechanical study used a calibrated distractor and a subminiature load cell on 17 cadaveric cervical specimens to investigate the effects of IH and distractive forces on a cervical spine model. In that study, the longer was the intervertebral space distance, the greater was the compressive load produced between the implant and vertebral body end plate, which may decrease the fusion rate . Here, tProsthesis subsidence affects 7–25% of patients undergoing ACDF surgery using the Zero-P device –19. Our We found that IH is strongly associated with the occurrence of ASD, consistent with a prior study . In thatOur study has the following limitations. Because it was a retrospective study, patient selection bias was unavoidable. Thus, randomized controlled studies are needed to validate our findings. Although our measuring method was conducted according to previous studies, we acknowledge that potentially inherent radiographic imaging error may be a major limitation. Additionally, we did not evaluate effective methods for maintaining the postoperative IH. Given that our study mainly investigated the effects of postoperative IH changes on clinical and radiographic outcomes, further studies must focus on effective interventions for maintaining the postoperative IH at 2 to 4 mm.We found that ACDF surgery using a zero-profile device could not sustain the IH attained 1 week after surgery. No clear correlation was found between IH changes and clinical efficacy within a year of surgery. However, the IH may affect various complications within a year after ACDF. If postoperative IH changes can be maintained at 2 to 4 mm after a year, a satisfactory fusion rate and IVF diameter and a relatively low implant subsidence and ASD may be achieved after ACDF surgery using a zero-profile device.


Staphylococcus aureus as a maximum 20 mm zone of inhibition was observed. The data presented in the present study proves that such composites are an efficient antibacterial agent and suitable for therapy for severe infections.In the present work, multiwalled carbon nanotube (MWCNT)/zinc oxide (ZnO) nanoparticles at pH 6.0, 7.0, 8.0, and 10.0 were prepared through the coprecipitation method to improve the antibacterial activity. Morphological, structural, and optical analysis, such as Fourier transform infrared spectroscopy, X-ray diffraction, and field emission scanning electron microscopy, was used to investigate the formation of composites. Analysis revealed that there were variations in morphology from agglomerated structures to rod-like then flower-like structures as pH varied from 6.0 to 10.0. The MWCNT/ZnO composite enhanced the antibacterial activity especially for  The infectious disease affects human health and is caused by pathogenic microbes. For the treatment of these diseases, the first choice is antibiotics. However, high mutation and morphological changes lead to microbial resistance. To overcome this microbial resistance, a new nanoparticle-based strategy has gained more attention in recent times. Piktel et al. studied the antibacterial effect of various shape gold nanoparticles against different microbes . Their s2O3) 2- immediately starts to take place  which identifies the elements in the sample. Figures , and 77° [The crystalline phase of prepared ZnO NPs at different pH 6.0, 7.0, 8.0, and 10.0 and their composites were analyzed with XRD. The XRD pattern of MWCNT and FMWCNT is shown in  and 77° . The XRD+ ions and a low concentration of OH− ions in the solution. However, at pH 7.0, ZnO peaks are obtained but peaks are broader and less intense. However, ZnO NPs withpH ≥ 8showed the crystalline nature with all peaks (100), (002), (101), (102), (110), (103), (200), (112), (201), and (202) which are found to be at position 31.67°, 34.31°, 36.14°, 47.40°, 56.52°, 62.73°, 66.28°, 67.91°, 69.03°, and 77.03°, respectively [a = b = 3.25 Å and c = 5.20 Å) for ZnO NPs were measured for the peaks that confirm the hexagonal wurtzite structure as confirmed by the JCPDS 36-1451 data. ForpH ≥ 7, the ZnO peaks were observed which shows that the precursors were completely decomposed and no other products were formed. Additionally, higher intensity and narrower width peaks confirmed that the obtained ZnO NPs have good crystallinity for pH ≥ 8.ectively . It can −1. The FTIR spectrum of MWCNT, FMWCNT, ZnO NPs, and MWCNT/ZnO composites synthesized at pH 6.0, 7.0, 8.0, and 10.0 is shown in Figures −1 and 1714 cm−1 were attributed to the carboxylic group bands C-O and C=O, respectively. The peak at 3449 cm−1 confirmed the presence of hydroxyl group –OH, whereas in FMWCNTS, carboxylic groups formed on the surface of MWCNT by acid treatment are shown at 1800 cm−1 and stretching at about 3466 cm−1 [−1 showed the presence of the –OH group. From the figure, it can be observed that the position of –OH peak changes from pH 6.0 to a broad peak at pH 10.0. This moment of –OH peak plays an important role in the formation of ZnO NPs, which can be seen in FESEM images, while the C-O-C peak is appearing between 1025 and 943 cm−1 and the peak at 2360 cm−1 showed the presence of C=O. The peaks of C=O and C-O-C are not much affected on the synthesis of ZnO NPs as the bands are not much different with pH change. The ZnO absorption band near 529 cm−1 was observed in the spectrum of ZnO NPs. From the graph, it can be observed that all peaks of ZnO were shifted due to changes in the morphology of ZnO NPs. In a similar manner, −1 corresponded to the –OH vibrational mode which appears due to the possible water and acid used during the purification process identified from the MWCNT sample [−1 is related to the C-H and C=C band characteristics of carbon nanotubes [−1 and 977 cm−1 is related to the characteristics for ZnO and Zn-C for MWCNTs/ZnO, providing the MWCNT decoration with ZnO NPs [FTIR was performed to analyze the presence of functional groups and the quality of synthesized materials. The spectrum was plotted in the range of 500–4000 cm466 cm−1 . SimilarT sample . The peaanotubes . The pea ZnO NPs .TGA is a useful method of thermal analysis based on the measurement of mass change related to the temperature for the quantitative determination of the thermal degradation. The thermal properties of ZnO NPs were determined by TGA/DSC (Mettler Toledo TGA/DSC 1). The ZnO NPs  were heated up to 800°C at 10°C/min. The results obtained are shown in Staphylococcus aureus (S.A), Escherichia coli (E. coli), Klebsiella pneumonia (K.P), and Pseudomonas aeruginosa (Pseudo) as shown in Figures S.A bacteria were more sensitive compared to E. coli, K.P, and Pseudo for MWCNT/ZnO composites. The observed value of the zone of inhibition is shown in S.A. These results suggest that the efficiency is limited against gram-negative bacteria. This can be related to the particularities of their cellular wall (single-layered structure) and eukaryote organization.Bacterial resistance is based on its defensive barriers which are the cell wall and cell membrane. Nanoparticles (NPs) use different pathways of cell membranes to adsorb in gram-positive and gram-negative bacteria . For antS.A, E. coli, K.P, and Pseudo were 0.25%, 0.5%, 0.5%, and 1%, respectively. This showed that MWCNT/ZnO NPs synthesized at pH 10.0 are most effective against S.A, where they successfully inhibit the growth of bacteria at the MIC as low as 0.25 mgml−1.Quantitative tests suggested that MWCNTs/ZnO NPs synthesized at pH 10.0 have more pronounced antibacterial effects compared to other composite . The valIn the present work, novel composite MWCNT/ZnO NPs synthesized at different pH 6.0, 7.0, 8.0, and 10.0 are reported to improve the antibacterial activity. The formation of MWCNT/ZnO composites was confirmed by morphological, structural, and FTIR analysis. The variation of pH showed different morphologies from agglomerated structures to rod-like than flower-like structures. The in vitro results of MWCNT/ZnO composites show significant antibacterial activity against S.A, E. coli, K.P, and Pseudo strains. The higher antibacterial activity was observed for S.A with the largest diameter of zone of inhibition (20 mm). These results suggest that MWCNT/ZnO has potential application as an antibacterial material for simultaneous concentrating and inactivating pathogenic bacteria.


Aseptic loosening is one of the major reasons for late revision in total knee arthroplasty (TKA). The risk of aseptic loosening can be detected using radiostereometric analysis (RSA), whereby micromovements (migration) can be measured, and thus RSA is recommended in the phased introduction of orthopedic implants. Decrease in bone mineral density (BMD), as measured by dual-energy x ray absorptiometry (DXA), is related to the breaking strength of the bone, which is measured concurrently by RSA. The aim of the study was to evaluate bone remodeling and implant migration with cemented asymmetrical tibial and uncemented femoral components after TKA with a follow up period of 2 years.This was a prospective longitudinal cohort study of 29 patients , received a hybrid Persona® TKA  consisting of a cemented tibial, an all-polyethylene patella, and uncemented trabecular metal femoral components. Follow up: preoperative, 1 week, and 3, 6, 12 and 24 months after surgery, and double examinations for RSA and DXA were performed at 12 months. RSA results were presented as maximal total point of motion (MTPM) and segmental motion (translation and rotation), and DXA results were presented as changes in BMD in different regions of interest (ROI).MTPM at 3, 6, 12, and 24 months was 0.65 mm, 0.84 mm, 0.92 mm, and 0.96 mm for the femoral component and 0.54 mm, 0.60 mm, 0.64 mm, and 0.68 mm, respectively, for the tibial component. The highest MTPM occurred within the first 3 months. Afterwards most of the curves flattened and stabilized. Between 12 and 24 months after surgery, 16% of femoral components had migrated by more than 0.10 mm and 15% of tibial components had migrated by more than 0.2 mm. Percentage change in BMD in each ROI for distal femur was as follows: ROI I 26.7%, ROI II 9.2% and ROI III 3.3%. BMD and at the proximal tibia: ROI I 8.2%, ROI II 8.6% and ROI III 7.0% after 2 years compared with 1 week postoperative results. There was no significant correlation between maximal percentwise change in BMD and MTPM after 2 years.Migration patterns and changes in BMD related to femoral components after TKA in our study correspond well with previous studies; we observed marginally greater migration with the tibial component.The online version contains supplementary material available at 10.1186/s43019-021-00111-5. Total knee arthroplasty (TKA) is, in general, a very successful treatment for patients with symptomatic osteoarthritis (OA), and register studies indicate implant survival of more than 90% after 10 years . One of The risk of aseptic loosening can be detected by radiostereometric analysis (RSA), whereby micromovements, described as migration, can be measured, and thus RSA is recommended as a standard in the phased introduction of new orthopedic implants  with 2-yDual-energy x ray absorptiometry (DXA) can be used to measure changes in bone mineral density (BMD) after TKA , 12; a sThe aim of this study was to evaluate implant migration using MBRSA and bone remodeling using DXA, and to assess correlation between implant migration and bone remodeling in patients with cemented asymmetrical tibial and uncemented femoral TKA components over a follow up period of 2 years.We performed a prospective longitudinal cohort study of patients Fig.  demogra who undeWe included 31 patients in the study; 29 patients were available for follow up as 1 patient declined to participate in the study after surgery, 1 patient had a change of tibial insert to an ultra-congruent (UC) during initial surgery, and 1 patient did not attend to the preoperative appointment   were placed in both the proximal tibia, the polyethylene insert and the distal femur, using an inserter that positions and inserts markers in bone one at a time . The same assistant positioned the beads in each procedure to minimize variation and we aimed for the widest possible non-linear spread between the beads. The tantalum markers placed in the polyethylene insert were not used for the analyses in this study because MBRSA was used to evaluate migration and segmental motion.RSA performed 1 week after surgery  was used as the baseline for RSA measurements and follow up examinations were performed at 3, 6, 12, and 24 months after surgery. RSA was performed with the patient in a standardized supine position, with the knee placed in a biplane plexiglass calibration cage . Two moveable ceiling-fixed x ray tubes  were positioned at a 90° angle to each other, one positioned for the anterior-posterior projection and the other for the medial-lateral projection. Both tubes were placed 100 cm from the x ray detectors in moveable cassettes, and intensity was set at 50 kV and 25 mA seconds (mAs). The radiographic images were stored in digital imaging and communication in medicine (DICOM) format with a resolution of 10 pixels per millimeter, in the picture archiving and communication system (PACS). All examinations were performed by the same two researchers.RSA analysis Fig.  was perfThe distribution of tantalum markers is expressed by the condition number (CN), whereas mean error (ME) is an expression of the stability of the tantalum markers; both CN and ME are calculated by the analysis software. We were aiming for a low CN, which indicates a non-linear distribution with wide dissemination of the markers. The upper limits for CN and ME were set at 150 and 0.35 mm, respectively, according to guidelines . Migratin = 22) were obtained at the 12-month follow up. Patients were requested to stand up between each examination and were positioned again after 5 min in the aforementioned supine position and additional RSA radiographic images were obtained. We evaluated the measurement precision for RSA. Precision was defined as the standard deviation of the difference (SDdiff) and precision error was expressed as 1.96 x SDdiff [Double RSA radiographic images , and after 3, 6, 12, and 24 months. The distal femur in the affected limb was scanned in the sagittal plane, with the patient positioned in the lateral decubitus position, with the affected knee placed nearest to the examination table and in slight flexion, to obtain a true lateral projection. The proximal tibia on the affected limb was scanned in the anterior-posterior plane, with the patient placed in the supine position with the knee fully extended and the lower limb slightly rotated inward, to avoid superimposition of the fibula and tibia.DXA was performed by two experienced technicians using a Norland XR-46 bone densitometer . The proximal tibia and distal femur were scanned using customized software for research with a pixel size of 0.5 × 0.5 mm and a speed of 45 mm/sec.Both femoral and tibial DXA scans were analyzed by creating three regions of interest (ROI) on the computerized scan plots Fig. A and B fn = 16) were obtained at the 12-month follow up. Patients were requested to stand up between each examination and were positioned again after 5 min using the aforementioned positioning and then rescanned. The precision error of the BMD measurements in the various ROI of the proximal tibia and the distal femur was calculated from the double measurements and expressed as the mean coefficient of variation (CV) (CV = (standard deviation (SD)/mean) × 100%).Double DXA scans (The Knee Society score (KSS) and the Oxford knee score (OKS) were calculated preoperatively and postoperatively after 1 and 2 years. The KSS is a physician-completed score and consists of a clinical and a functional score. Clinical scores include pain, extension lag, total range of flexion, alignment, stability , and if present, flexion contracture. Functional scores include quality of walking, whether walking aids are used, and the ability to use stairs. A KSS score below 60 is considered poor, 60–69 fair, 70–79 good, and 80–100 excellent .The OKS is a patient-reported score and consists of 12 items to assess function during the past 4 weeks, where a score of 0 (minimum) may indicate severe OA and 48 (maximum) may indicate satisfactory function .t test for paired data was used to compare time-related change (0–24 months) in BMD, and percentage time-related mean change in BMD was presented with 95CI. The OKS and KSS were expressed as the mean with 95CI and preoperative and 2-year followup values were compared using the paired t test.Data on MBRSA translation (millimeters) and rotation (degrees) were expressed as mean values with 95% confidence intervals (95CI). As recommended by Valstar et al. , all traThe size of our study population size corresponds well with the number of required participants as determined from previous sample size calculations for RSA and DXA studies when comparing two different implants. RSA has high accuracy and therefore a small number of participants can be studied .p <0.05 and confidence intervals were reported at 95%.Mean annual migration of 0.09–0.10 mm for femoral components is comparable with a good long-term outcome , 27. AccApproval from the local Ethical Committee (case no. H-16035883) and Danish Data Protection Agency  was obtained. All patients were informed about the study orally and in writing by the principal investigator and informed consent was obtained prior to inclusion, in accordance with the Helsinki Declaration.The precision error for measurement of MTPM from 22 femoral double examinations was 0.19 mm. Precision error for the segmental motion was 0.20°, 0.25°, and 0.24° for X, Y, and Z rotations, respectively, and precision error for the corresponding translational segmental motion was 0.16 mm, 0.07 mm, and 0.18 mm, respectively.The greatest increase in mean MTPM (0.65 mm) occurred within the first 3 months. Afterwards, the curve flattened and stabilized, and the mean MTPM after 24 months was 0.96 mm  after 6 months, 0.92 mm (range 0.17–4.93 mm) after 12 months and 0.96 mm (range 0.2–5.36 mm) after 24 months. Implant migration > 0.10 mm was observed between 12 and 24 months in 16% of patients (4 out of 25 patients).A spaghetti plot demonstrates the individual MTPM Fig. . PatientThe highest mean rotational and translational segmental motion was found around the Y axes Fig. ; mean roThe mean CN was 58.4 (range 20.5–97.0) and mean ME was 0.16 (range 0.03–0.43). All CN values were acceptable, whereas one ME value (0.43) was above the maximum value of 0.35 as recommended by guidelines .The precision error for measurement of MTPM from evaluation of the 22 double tibial examinations was 0.33 mm, 0.20°, 0.63°, and 0.21° for rotational segment motion, X, Y, and Z rotations, respectively, and 0.14 mm, 0.09 mm, and 0.19 mm, respectively, for the corresponding translational segment motion. The greatest increase in mean MTPM (0.54 mm) was seen after 3 months of follow up and then the curve considerably flattened as an expression of stabilization of the tibial component, with mean MTPM of 0.61 mm range 0.17–1.99 mm) after 6 months, 0.65 mm (range 0.13–2.82 mm) after 12 months, and 0.69 mm (range 0.12–3.2 mm) after 24 months  and mean ME was 0.17 (range 0.06–0.4). All CN values were acceptable and one ME value (0.4) was above the maximum value of 0.35 as recommended by guidelines .The precision error expressed as the CV for measurement of BMD at each ROI was calculated from 16 double examinations. The CV for the distal femur was 1.4% (95CI 0.89–1.9), 1.3% (95CI 0.43-2.11), and 0.9% (95CI 0.5–1.4) for ROI I, ROI II, and ROI III, respectively. The corresponding results for the proximal tibia were 1.3% (95CI 0.69–1.95), 1.8% (95CI 0.86–2.68), and 2.1% (95CI 0.9–3.25), respectively.At both the distal femur and the proximal tibia and at all ROI, there was a statistically significant decrease in BMD at 2 years compared with the immediate postoperative measurement Fig. .Fig. 10The greatest mean BMD decrease at the distal femur was at ROI I (anterior) with 26.7% decrease (95CI 17.3–36.1%) after 2 years, while the decrease at ROI II (posterior) and at ROI III  was 9.2% (95CI -3–21.5%) and 3.3% (95CI -5.55–12.1%) respectively. A decrease in BMD after 24 months was also observed in the proximal tibia and it was almost the same at all three ROI with 9.5% (95CI 4.7–14.3%) at ROI I , 9.6% (95CI 2.5–16.7%) at ROI II , and 7.2% (95CI 0.6–13.8%) at ROI III , respectively. There was no significant correlation between MTPM and BMD after 2 years Fig. .Fig. 11n = 29) showed a significant increase (p <0.001) from a score of 25 (range 13–38) preoperatively to 44 (range 35–48) at the 2-year follow up. The KSS for function increased from 54 (range 10–100) preoperatively to 94 (50–100) at 2 years (p <0.001), and the corresponding KSS clinical score increased from 38 (range 10–79) to 87 (range 60–90) (p <0.001).The 2-year clinical outcome determined by the OKS  with migration > 0.10 mm at 12–24 months.Revisions related to femoral components, regardless of fixation, are rare , 27. ThiGao et al.  identifiThe findings on femoral components in our study correspond well with previous studies , 30–32. One possibility for further studies could be to examine the migration pattern of femoral components in patients who underwent revision due to aseptic loosening, to identify any pattern.For the tibial components as with the femoral component, the greatest increase in mean MTPM (0.54 mm) was seen after 3 months of follow up. Mean MTPM was 0.61 mm after 6 months, 0.65 mm after 12 months, and 0.69 mm after 24 months. Between 12 and 24 months after surgery, 14.8% of patients (4 out of 27 patients) had migration > 0.2 mm.Pijls et al.  identifiFrom Leande et al.  interpreMany RSA studies have been effectuated using a different type of fixation and prosthesis design for the tibial component. If we compare our results with previous studies using cemented fixations , 32–34, Furthermore, Ryd et al.  state thA decrease in BMD of 26.7% was observed in ROI I at the distal femur after 24 months, and the respective decrease in ROI II and ROI III was 9.2% and 3.3%, respectively. The corresponding decrease in BMD at the proximal tibia in ROI I, II, and III was around 9%. The decrease in BMD at the distal femur and proximal tibia after TKA is a known consequence of postoperative adaptive bone remodeling –39. BMD Quantitative studies have been performed on periprosthetic bone remodeling at the distal femur after primary TKA, but in general, the greatest bone loss is seen in the anterior part of the bone where the decrease in BMD typically reaches 23.6–36.0% after 2 years with uncemented femoral components , 35, 36.The greatest decrease in BMD at the proximal tibia is often in ROI I  and previous studies have identified a decrease with cemented tibial components between 4.4% after 1 year  and up tTo our knowledge, there are no studies to indicate the range of decrease in BMD associated with periprosthetic fracture; one of the reasons for this could be that periprosthetic fracture is not only associated with a decrease in BMD but also has a multifactorial genesis. Decrease in BMD in the present study was caused by local adaptive bone remodeling.This study has no randomization between a current standard prosthesis and the new implant, which would be the preferred way to test a new implant; with the patients blinded to the type of prosthesis, the clinical outcome could be determined more accurately. Results from 29 patients for one type of prosthesis are acceptable for studying implant migration and adaptive bone remodeling after TKA, but to interpret functional results more patients are needed.Migration patterns for femoral component and changes in BMD in our study correspond well with findings in previous studies, and we observed marginally higher migration with the tibial component. There was no significant correlation between MTPM and BMD. Those components at risk need further evaluation with 5-year postoperative follow up.Additional file 1.


The changes in gene expression levels of citrate synthase (CS), cytochrome oxidase (COX19), pyruvate dehydrogenase (Pdh1), and ATP synthase (ATP6), which is involved in mitochondrial respiration, were studied in root and cotyledon tissues. Significant increases were recorded in the gene expression levels of all studied enzymes. In addition, progesterone applications stimulated activities of malate synthase (MS), isocitrate lyase (ICL), and alpha-amylase, which are important enzymes of the germination step. The changes in gene expression levels of mas1 and icl1 were found parallel to the rise in these enzymes’ activities. It was determined similar increases in root and coleoptile lengths and total soluble protein and total carbohydrate contents. The most remarkable changes were detected in 10−8 mol·L−1 progesterone-treated seedlings. These results clearly indicate that progesterone stimulates mitochondrial respiration by inducing biochemical and molecular parameters and thus accelerates seed germination thanks to the activation of other pathways related to mitochondrial respiration.This research aimed to investigate the effects of progesterone, a mammalian steroid sex hormone, on the mitochondrial respiration in germinating maize seeds. For this purpose, maize seeds were divided into four different groups (control, 10 Mammalian sex hormones (MSHs) are a member of steroids and are naturally present in plants . After tIn the present study, we aimed to enlighten based on physiological, biochemical, and molecular features the possible stimulating effect of progesterone on seed germination and early growth parameters by considering the importance of mitochondrial respiration in the stage of seed germination. This is the first detailed investigation-exhibiting role of progesterone on mitochondrial respiration in the stage of seed germination and early plant growth.−6, 10−8, and 10−10 mol·L−1, progesterone supplementations markedly increased the root length by 38.79, 63.96, and 51.17%, respectively, as compared to the control group. The values of the root length were recorded as 10.42, 12.31, and 11.35 cm in progesterone-treated seeds. In 10−6, 10−8, and 10−10 mol·L−1, progesterone supplementations significantly enhanced the coleoptile length by 9.37, 27.89, and 15.86%, respectively, as compared to the control group. The values of the coleoptile length were recorded as 6.91, 8.08, and 7.32 cm in progesterone-treated seeds  exhibited significant increases in endosperms of maize seedlings compared to the control group of all of the enzymes studied.The activities of amylase, isocitrate lyase, and malate synthase were presented in −6, 10−8, and 10−10 mol·L−1 progesterone in comparison to the control, respectively. The values of the amylase activity were recorded as 73.19, 82.40, 93.43, and 84.11 U mg protein−1 FW in the control, and 10−6, 10−8, and 10−10 mol·L−1 progesterone applications, respectively.The amylase activity increased by 12.58, 27.65, and 14.92% in 10−6, 10−8, and 10−10 mol·L−1 progesterone applications, respectively. The values of the ICL activity were recorded as 1.53, 2.06, 2.56, and 2.00 U mg protein−1 FW in the control, and 10−6, 10−8, and 10−10 mol·L−1 progesterone applications, respectively.As seen in −6, 10−8, and 10−10 mol·L−1 progesterone compared with the control group, respectively. The values of the MS activity were recorded as 7.17, 10.31, 13.66, and 10.74 U mg protein−1 FW in the control, and 10−6, 10−8, and 10−10 mol·L−1 progesterone applications, respectively.Malate synthase activity caused remarkable increases by 43.70, 90.44, and 49.80% in 10icl1 and mas1 gene levels increased under the 10−6, 10−8, and 10−10 mol·L−1 progesterone supplementation compared to the control  and malate synthase (MS) have major effects in the mobilization of storage lipids during germination ,28. Thes control . There i control . The obtCS), cytochrome oxidase (COX19), pyruvate dehydrogenase (Pdh1), and ATP synthase (ATP6) were also analyzed to enlighten the effect of progesterone on the mitochondrial respiration pathway. The expression levels of genes encoding CS and COX19 were presented in Pdh1 compared to control in both root and coleoptile tissues of maize  were used for this experiment. Firstly, all seeds were subjected to surface sterilization by using 96% ethanol for a short time, and then 5% NaClO for 10 min and washed with ultra-distilled water several times. After sterilization, the seeds were divided into four different groups; control group, 10−6 mol·L−1 PRG, 10−8 mol·L−1 PRG, and 10−10 mol·L−1 PRG. The optimum concentrations obtained in the present study were determined according to the previous studies in the literature. The concentration range was chosen from 10−4 to 10−12 mol·L−1 progesterone. After the pre-experiments, this range was determined as 10−6, 10−8, and 10−10 mol·L−1 progesterone. To prepare the stock solution (10−4 mol·L−1), progesterone was weighed and dissolved first in a small volume of methanol (2 or 3 mL for 1 L solutions), and the volume of solution was completed with sterile ultra-distilled water. Then the prepared stock solution was diluted in sterile ultra-distilled water in order to obtain 10−6, 10−8, and 10−10 mol·L−1 concentrations. The sterilized seeds were shared equally among all groups and were imbibed in their own solutions. After the imbibition period, ten seeds per dish were placed in Whatman No. 1 filter paper-lined glass Petri dishes. Each treatment group was supplied with its own progesterone solutions, while control seeds were supplied with sterile ultra-distilled water containing methanol (2 or 3 mL for 1 L). All groups were incubated in a germination cabinet, which was set at 24.5 ± 0.5 °C, and under dark conditions for 4 d. On the 4th day, all seedlings were harvested, and endosperm, coleoptile, and roots of seedlings were separated and stored at −86 °C until the determination of changes in physiological, biochemical, and molecular parameters.Maize seeds , and samples were centrifuged at 15,000 rpm at 4 °C for 15 min. After the centrifugation period, 5 μL supernatant was transferred to wells of the plate, and BCA reagent (bicinchoninic acid + CuSO4) was added into the samples. The plate was incubated at 65 °C for 15 min. The changes of absorbance were recorded at 562 nm, and protein values were determined by utilizing the standard chart.The amount of total soluble protein was estimated according to Smith et al.  by using2CO3 powder was added into samples until the reaction stopped. An amount of 45 mL of distilled water was added on top of the samples, and they were centrifuged at 15,000 rpm for 15 min. After the centrifugation, 1 mL of supernatant was taken, and an anthrone solution was added. After cooling, the absorbance changes were recorded at 630 nm. The standard chart was used to determine the total carbohydrate content.The total carbohydrate content was determined according to the method described by Dische  with a sg for 20 min. After the centrifugation, 500 μL of supernatant was mixed with 500 μL 40 mM EDTA and was added into 1 mL of reaction buffer including 200 μmol CaCl2, 150 mg of soluble starch, and 600 mg of KH2PO4 dissolved in 100 mL of distilled water. The samples were incubated at 30 °C for 5 min. Then, the reaction was stopped by adding 1 mL of iodine reagent (3 mg of KI and 0.3 mg of I were dissolved in 0.05 N HCl). Thirteen milliliters of distilled water were added to the samples, and absorbance changes were recorded at 620 nm. One unit of amylase activity was identified as the quantity of enzyme that caused the changes in the absorbance of 0.1 [Amylase activity was determined according to the procedure defined by Juliano and Varner . The ende of 0.1 .g for 10 min. After centrifugation, the pellet was taken and was homogenized 6 mL of extraction buffer. All samples were homogenized at 23,000 rpm for 5 h.For the extraction of isocitrate lyase and malate synthase activity, 10 g of endosperm tissue was extracted in extraction buffer containing 400 mM sucrose, 165 mM N-trishydroxymethy glycine (pH 7.5), and 10 mM dithiothreitol. The samples were centrifuged at 270× −1 M−1). About 15 μg of supernatant was mixed with 1.2 mL of reaction buffer, including 87 mM phosphate buffer (pH 6.9), 4.6 mM dithiothreitol, 8.7 mM MgCl2, 13 mM isocitrate, and 10 mM phenylhydrazine. The reaction was started by adding the isocitrate to the mixture, and the absorbance values were determined at 324 nm.Isocitrate lyase activity was conducted by the method of Dixon and Kornberg . Enzyme 2, 18 mM acetyl coenzyme-A, and 20 mM sodium glyoxylate. The values of absorbance were recorded at 412 nm.Malate synthase activity was determined according to Hock and Beevers . About 2icl1, mas1, CS, COX19, Pdh1, and ATP6 mRNA accumulation were given as a heat map with dendrograms indicating co-regulated genes. The primer series of the genes were presented in The isolation of total RNA and quantitative real-time PCR reactions were performed according to the producer’s analysis protocol. All tissue samples were conducted in a Qiacube device by using RNeasy plant mini kit  for RNA isolation. The RNA’s purity of samples was measured at the Qiaexpert. cDNA synthesis was conducted by producer’s procedures by using Nanoscript 2 RT kit (Primer Design). Specific gene primers were provided from Qiagen Company. The relative gene expression analysis was conducted by Qiagen-Geneglobe . β-actinp < 0.05 (Duncan’s multiple range method). The statistical analyses were conducted using SPSS 20.0, and the standard errors were shown in all tables and figures.The assay was randomly conducted with five biological replications and three parallel. To compare significant differences between the samples was used the analysis of variance (ANOVA). Statistical significance was defined as