Portulaca oleracea) cultivars to determine energy-efficient lighting strategies for CEA. Green and golden purslane cultivars were cultivated in CEA chambers and four experimental treatments consisting of PPFDs of 150, 200, 250, and 300 ± 10 µmol m−2s−1 were performed, representing daily light integrals (DLIs) of 8.64–17.28 mol m−2d−1 throughout a 16 h photoperiod. The results show that photoresponses to light PPFDs are cultivar-specific. The green cultivar accumulates 174% more dry weight at 300 PPFD compared to the golden cultivar, and also has a higher LUE, but a lower ETR. Dry weight accumulation, plant height, and leaf area dependence on light intensity do not highlight the economic significance of light PPFD/DLI. The derivative parameter (Δ fresh weight (%)/ΔDLI %) more efficiently explains how the percentage increase in DLI due to an increased PPFD affects the percentage of biomass gain between these PPFD treatments. For both cultivars, the relative fresh weight gain is maximal when the lighting PPFD increases from 200 to 250 µmol m−2s−1 and declines with PPFD increases from 250 to 300.This study aims to evaluate the impacts of the lighting photosynthetic photon flux density (PPFD) on the growth, photosynthesis, and antioxidant response of common purslane ( The CEA market size was valued at USD 97.42 million in 2022 and is calculated to reach USD 339.94 million by 2030, rapidly growing at a CAGR of 14.53% from 2023 to 2030 . For exa−2 per day; in a literature review, a 16 h photoperiod and 250 µmol m−2s−1 were found to be the average optimum for yield and resource use efficiency in hydroponic lettuce cultivation [−2s−1 results in an improved photosynthetic performance, raises the antioxidant capacity, and improves the nutritional value of lettuce [Brassicaceae plants [The lighting spectrum, photoperiod, and photosynthetic photon flux density (PPFD) can be optimized, maximizing the efficiency with which plants intercept and use light to produce marketable biomass, which has the potential to reduce input costs and increase yields and profitability ,5,8. Thetivation ,10. It w lettuce , rocket  lettuce , kale [1 lettuce , mizuna e plants . Howevere plants . The rese plants .Brassicacea sp., etc. [Portulaca olearacea L.) is a herbaceous annual plant with fleshy stems and thick, succulent leaves. It has received specific attention in recent years due to its exceptional nutritional content, mainly resulting from high contents of omega-3 fatty acids, polysaccharides, alkaloids, terpenoids, sterols, phenols, flavonoids, vitamins A, C, E, and B, and minerals such as potassium, calcium, and magnesium, which provide neuroprotective, antimicrobial, antidiabetic, antioxidant, anti-inflammatory, antiulcerogenic, anticancer, and anticholinesterase properties [The assortment of vegetables cultivated in CEA is relatively narrow and research is based on high-demand species, such as lettuce, basil, spinach, p., etc. . Howeverp., etc. . Wild edp., etc. ,20. Pursoperties ,21. Knowoperties . Severaloperties ,23. It wPurslane is a promising candidate for cultivation in CEA; however, it is a C4 halophyte, which undergoes CAM under stress conditions . Therefo−2s−1 PPFD exposure and a 180% higher dry weight at 300 µmol m−2s−1 PPFD compared to the golden cultivar. Dry weight percentages are similar between PPFD treatments (~4.3 for golden and 5.1 for green purslane), as dry and fresh weight accumulation  and from 200 to 250 µmol m−2s−1 (11.52 to 14.4 mol m−2 per day) resulted in a remarkable increase in plant leaf area in both cultivars. The leaf area of green purslane plants was determined to be 80 and 34% higher, respectively, while the leaf area increased by 53 and 40% in golden purslane plants. According to the two-way ANOVA results (p < 0.01) impact on the measured biometric parameters.Plant heights and leaf areas c,d also  results e, both tPortulaca olearacea growth parameters differs at lower and higher PPFD values. −2s−1 PPFD increment adds 2.88 mol m−2d−1 to the DLI compared to the previous treatment; however, the percentage of the total DLI increase declined. The DLI increases by 33, 25, and 20% due to PPFD shifts from 150 to 200, 200 to 250, and 250 to 300 µmol m−2s−1, respectively. Both cultivars represent the same trend for photon utilization efficiency for fresh weight gain. The relative fresh weight gain is the highest when the PPFD increases from 200 to 250 µmol m−2s−1 . Lower Δ fresh weight %/Δ DLI % values when the PPFD increases from 150 to 200 µmol m−2s−1 indicate that the lighting intensity is still not sufficient for the potential purslane biomass productivity. Decreased ratio values when the PPFD increases from 250 to 300 µmol m−2s−1 (2.20 for green and 0.71 for golden cultivar) indicate that at this light intensity, additional photosynthetic photons are not efficiently utilized for biomass gain and golden purslane plants possibly experience growth inhibition due to the excess light intensity. While In practical evaluations, the photosynthetic photon utilization efficiency is not fully reflected in dry and fresh weight accumulation depending on the light intensity. The impact of light intensity increases on −2s−1). However, at higher PPFDs, the second photosystem’s (PSII) capacity and electron transport rate significantly differ between both purslane cultivars. The golden cultivar . However, for the green cultivar, a further increase in the PPFD strongly (R > 0.8) correlates with DPPH, FRAP, and total phenolic compound (TPC) contents, as well as biometric indices  and FRAP (R = 0.93) values  values .−2s−1 PPFD treatments according the F1 component. According to the F2 component, for the green cultivar, 250 and 300 µmol m−2s−1 PPFD treatments significantly differ according to the chlorophyll index, while for the golden cultivar they differ according to the DPPH free radical scavenging activity, FRAP antioxidant power, and total phenolic content.The PCA score scatterplot a,c shows2 assimilation rate, and growth rate. Kim and van Iersel (2022) also proposed that the anthocyanin content in leaves may be associated with reduced growth, because light absorption by anthocyanins reduces the amount of photons available for photosynthesis [Providing an adequate lighting PPFD in CEA is expensive, and electricity costs could limit the widespread adoption of indoor plant cultivation systems on a commercial scale . Therefoynthesis . Screeniynthesis  discover−2s−1 (DLI increase from 11.52 to 14.4 moles m−2 per day) and declines with a PPFD increase from 250 to 300 µmol m−2s−1 (DLI increase from 14.4 to 17.28 mol m−2 per day). This suggests that the most efficient light energy conversion to purslane biomass occurs at 250 µmol m−2s−1. When the light saturation point is reached, only increasing the photoperiod would promote further efficient biomass gains [−2s−1 compared to continuous lighting or higher PPFDs with shorter photoperiod. In addition, higher PPFDs resulted in higher mineral and nutrient contents [Dry weight accumulation, plant height, and leaf area dependence on light intensity do not highlight the economic significance of light PPFDs/DLIs. In this study, a higher PPFD corresponded to increased DLI values, as the photoperiod was kept constant. The derivative parameter (Δ fresh weight (%)/ΔDLI %) explains how the percentage increase in DLI due to an increased PPFD affects the percentage of biomass gain between these PPFD treatments. Relative values are qualitative justification of the most efficient light intensity. The same tendency is observed for both cultivars, however, it is more clear for the golden cultivar. The relative fresh weight gain is maximal when lighting PPFD is increased from 200 to 250 µmol mss gains  investigcontents ,31.The results of this study indicate that for reasonable economic efficiency of purslane lighting in CEA, the direct evaluation of plant photoresponses to the light PPFD is not sufficient. The derivative parameter (Δ fresh weight (%)/ΔDLI %) better represents the utilization of electric energy at different PPFD levels for purslane biomass gain. However, the cultivar-specific response to the applied PPFD highlights the necessity for more specific research on efficient CEA technologies.2 level of 1000 ppm within the chamber. Artificial lighting was provided by 4-channel controllable light emitting diode (LED) units  with an equal spectral composition   were germinated in rockwool cubes  and soaked in distilled water under a lighting photosynthetic photon flux density (PPFD) of 200 µmol m−2s−1. On the 10th day after germination, seedlings were thinned out, leaving 5 plants per cube, and transferred into deep water culture (DWC) hydroponic systems. Each experimental PPFD treatment consisted of 3  DWC tanks of 40 L volume, with 12 tanks in total. Each DWC tank contained 12 mesh pots, with 7 plants in each. A commercial hydroponic nutrient solution was used (Hydro A (NPK(3-0-1), CaO 5.9%, MgO 0.4%) and Hydro B ; Plagron Ommelpad, NL), using 2.5 mL of each component per liter of water. The initial buffer capacity of the hydroponic solution was enhanced by the addition of 3 mM MES (2-(N-morpholino) ethane sulfonic acid) as a buffering agent and therefore pH values were passively maintained in the range of 6.0–6.5 throughout the cultivation period.Seeds of green and golden cultivars of common purslane  of 8.64–17.28 mol m−2d−1 throughout the 16 h photoperiod (Lighting PPFD treatments. Photosynthetic photon flux densities (PPFDs) common in controlled-environment agriculture were selected for the experimental treatments. Four experimental treatments of PPFDs of 150, 200, 250, and 300 ± 10 µmol mtoperiod , while mAnalyses were performed 35 days after sowing. All measurements in each experimental replication were performed in 3 analytical replications . Plant material was lyophilized  and ground, and dry plant material was used for the determination of antioxidant properties.Biometric measurements. Plant above-ground parts were used to determine plant height (PH) and fresh (FW) and dry  weights. The leaf area (LA) was determined using a benchtop leaf area meter . To calculate the LUE (light use efficiency), the shoot dry weight was divided by the total incident light that the plant received over the growing period. The ratio of Δ fresh plant weight (%)/Δ DLI (%) was calculated to compare the impact of light intensity increases under different PPFD treatments.−1m−2 and chlorophyll fluorescence parameters were recorded.The ETR (electron transport rate) was determined using an imaging-PAM fluorometer ) on light-acclimated (30 min) plants. Three plants from each experimental replication (9 plants per PPFD treatment) were used for measurements. PPFDs were gradually increased at fixed time intervals from 0, 2, 18, 54, 140, 285, 481, 728, and 910 to 1196 µmol s−2) and flavonol  contents and nitrogen balance indexes .Leaf optical indexes. A Dualex meter  was used to determine chlorophyll  free radical scavenging activity and ferric reduction antioxidant power (FRAP), and the total contents of phenolic compounds (TPCs) were evaluated in dry plant material. Lyophilized plant material (0.01 g) was ground with 5 mL 80% methanol, incubated for 24 h, and centrifuged , and the supernatant was used for analysis.−1 DW).A stable 126.8 µM DPPH  solution was prepared in methanol. Analyses were conducted in 96-well plates by mixing 290 µL of DPPH solution with 20 µL of plant extract in each well; the absorbance was read at 515 nm  at the 16th minute . Results3+ ion reduction to Fe2+. A fresh working solution was prepared by mixing the following solutions in a 10:1:1 volumetric ratio: 10 mM TPTZ  solution in 300 mM, pH 3.6 acetate buffer, 40 mM HCl, and 20 mM FeCl3 × 6H2O. Analyses were conducted in 96-well plates by mixing 290 µL of working solution with 20 µL of plant extract in each well and incubating it in the dark for 30 min; the absorbance was read at 593 nm  [2(SO4)3 (0.005–0.5 mM) (Iron (III) sulphate; 97% purity; Sigma-Aldrich, Burlington, MA, USA) were used to determine the calibration curve. Results were expressed as µmol of Fe2+ reduced by g−1 of dry plant weight (µmol g−1 DW).The FRAP method is based on FeGermany) . Solutiow/v) Folin–Ciocalteu reagent and 160 µL of 1 M Na2CO3 solution and incubating it in the dark for 20 min. Afterwards, absorbance was read at 765 nm  [−1 DW).Analyses of total phenolic compound (TPC) contents were conducted by mixing 20 µL of plant extract with 20 µL of 10% (Germany) . The conp ≤ 0.05. For result modeling, a correlation analysis and a multivariate principal component analysis (PCA) were performed. Data were evaluated using Microsoft® Excel® for Microsoft 365 MSO (Version 2309 Build 16.0.16827.20166) 64–bit and compatible XLStat version 2022.1.2.1279 (64–bit)  software packages.Statistical analysis. The results are presented as the average ± standard deviation of 3 experimental and 3 analytical replications, n = 9 per PPFD treatment. A one-way ANOVA, using Tukey’s HSD test, was employed to determine differences between light treatments; a two-way ANOVA was used to evaluate the significance of the impact and interaction of variables (cultivar and lighting treatment) at the confidence level of −2s−1 (DLI increases from 14.4 to 17.28 mol m−2 per day), and the most efficient light energy conversion to purslane biomass occurs at 250 µmol m−2s−1. However, the species- and even cultivar-specific responses to the applied PPFD highlight the necessity for more specific research on efficient CEA technologies.For a reasonable economic efficiency of plant lighting in CEA, the direct evaluation of plant photoresponses to light PPFDs/DLIs is not sufficient. For both green and golden purslane cultivars, increasing the light intensity linearly influences dry and fresh weight accumulation; however, the derivative parameter (Δ fresh weight (%)/ΔDLI %) shows that biomass gains, considering the lighting DLI input, decline when the PPFD increases from 250 to 300 µmol m


Scoliosis is widely prevalent among osteogenesis imperfecta (OI) patients, and is progressive with age. However, factors affecting scoliosis in OI are not well known.We retrospectively retrieved longitudinal radiographic and clinical records of consecutive OI patients seeking treatments at our hospital from 2014 to 2022, graded their pre-operative spinal conditions into four outcome groups, estimated their progression rates, and descriptively and inferentially analyzed the genetic and non-genetic factors that may affect the outcomes and progression rates.COL1A1 or COL1A2, followed by mutations in WNT1 (9.0%), IFITM5 (9.0%) and other OI risk genes. With an average age of 12.0 years (interquartile range [IQR] 6.9–16.1), 70.7% of the cohort had scoliosis (Cobb angle > 10°), including 106 (36.5%) mild (10°–25°), 40 (13.8%) moderate (25°–50°), and 59 (20.3%) severe (> 50°) scoliosis patients. Patients with either COL1A1 and COL1A2 were strongly biased toward having mild or no scoliosis, whereas patients with mutations in IFITM5, WNT1 and other recessive genes were more evenly distributed among the four outcome grades. Lower-limb discrepancy, bone mineral density (BMD) and age of first drug used were all significantly correlated with severity outcomes. Using multivariate logistic regression, we estimated that each year older adds an odds ratio of 1.13  in progression into advanced stages of scoliosis. We estimated a cohort-wide progression rate of 2.7 degrees per year (95% CI 2.4–3.0). Early-onset patients experienced fast progressions during both infantile and adolescent stages. Twenty-five of the 59 (42.8%) patients with severe scoliosis underwent spinal surgeries, enjoying an average Cobb angle reduction of 33° (IQR 23–40) postoperatively.In all, 290 OI patients met the inclusion criteria, where 221 had genetic records. Of these 221, about 2/3 had mutations in COL1A1, mutations in COL1A2 were less damaging while those on IFITM5 and other recessive genes conferred damaging effects. Progression rates were the fastest in the adolescent adult age-group.The severity and progression of scoliosis in osteogenesis imperfecta were affected by genetic factors including genotypes and mutation types, and non-genetic factors including age and BMD. As compared with The online version contains supplementary material available at 10.1186/s13023-023-02906-z. Osteogenesis imperfecta (OI) is a rare congenital musculoskeletal disorder caused by mutations in ~ 20 genes related to type I collagen synthesis, with impact on osteoblast differentiation and mineralization in bone , 2. OI pIFITM5 (c. -14C > T) [COL1A1/2 and IFITM5 mutations remain categorized according to the Sillence scheme, while those with mutations in the other 17 genes are subtyped from VI (OMIM #613982) to XXII (OMIM #619795) [Traditionally, OI was clinically grouped into four subtypes  based on the Sillence classification, with type I being mildest, followed by type IV and then type III. Type II is perinatally lethal and thus most serious . The mod14C > T) , 8. WithScoliosis, which affects physical mobilities and cardiopulmonary functions, is a form of lateral deformity of the spine (defined as Cobb angle > 10°), categorized into idiopathic (i.e. unknown causes), congenital and neuromuscular subtypes . ScoliosCOL1A1/A2, IFITM5, WNT1, SERPINF1, FKBP10, etc. Genetic inheritance (AD/AR) and mutation types  were also documented. We also retrieved information of skeletal maturity, bone density, drug history, Sillence subtypes, and conditions in the limbs. Based on the Cobb angle of the major curves, we stratified the patients into four severity grades: non-scoliotic, mild, moderate and severe [To delineate the relationship between OI scoliosis and potential risk factors, we retrieved the medical records of all consecutive OI patients from 2014 to 2022 seeking treatments at our hospital. Genetic testing results were included where possible, with affected genes covering d severe . We thenRecords of all patients diagnosed with osteogenesis imperfecta (OI) in our hospital from August 2014 to November 2022 were retrieved for the current study (n = 308). Eighteen patients without radiographs of the spine were excluded. For each of the remaining 290 patients with spine radiographs, sitting or standing Cobb angles at the thoracic, thoracic-lumbar (TL), and lumbar regions were measured. Each patient may thus have up to three curves, of which the one with the maximum Cobb angle was designated the major curve. All Cobb angles were measured by three experienced pediatric orthopedic surgeons . Each patient had one or multiple spinal radiograph follow-ups. Data from the follow-up with maximum major curve Cobb angle was used to grade scoliosis severity. Patients with Cobb angle below 10° were considered non-scoliotic, while those with preoperative Cobb angles between 10°–25°, 25°–50° and > 50° were considered mild, moderate and severe scoliosis, respectively.Drug treatment history, including the dates and types of anti-osteoporotic agents, was retrieved. Patients were contacted for confirmation of first date of drug treatment and the menarches for female patients. The BMD data were retrieved from the Discovery DXA system  at our hospital. The total hip and lumber regions (L1–L4) BMDs were used. Weight and height corresponding to each BMD measurement were also collected.The patients were clinically categorized based on the radiographic features, BMD reports and drug treatment histories according to the criteria of modified Sillence classification , where pCOL1A1, COL1A2, IFITM5, SERPINF1, CRTAP, P3H1, PPIB, SERPINH1, FKBP10, BMP1, SP7, TMEM38B, WNT1, CREB3L1, SPARC, TENT5A, MBTPS2, MESD, KDELR2) and 5 OI related genes  were included in the sequencing panel. Sequencing results from 167 out of 221 patients were previously published by our team [COL1A1 and COL1A2 were classified into variants with a qualitative impact (missense) and those with a quantitative  impact.Targeted amplicon sequencing was performed on 221 out of the 290 patients. Nineteen OI causative genes , late-onset (LOS), and ‘unknown’, based on radiographic evidence and a recommended consensus of demarcation point at the age of 10 . The EOSTo estimate the progression rates, we first excluded the 40 postoperative data-points in the 25 patients with surgical intervention on the spines, and obtained 606 preoperative Cobb angles of major curves in 290 patients. The progression rate was calculated as (angle difference)/(age difference in years) between successive data-points of the same patient. For the first data-point in each patient, this was calculated as (first angle)/(first age), which effectively assumed a constant progression rate since birth. The mid-point of the two ages associated with the two adjacent radiographs was used as the age corresponding with the progression rate estimate.t-test was used for univariate analyses. For those with multiple groups, one-way ANOVA was used. For categorical variables, Pearson’s Chi-squared test was used. For multivariate analyses with dichotomous dependent variables, logistic regression was used. The progression rates with respect to age were fitted using a local polynomial model (LOESS). In all cases, p-values were reported rounding to three digits of significance, and P < 0.05 was considered statistically significant.The statistical analyses were conducted on the R platform (version 4.0.0). For dichotomous quantitative variables, Student's 2p = 0.93) (Table p < 0.001) , among whom no significant variation was detected (p = 0.609).In all, 290 Chinese patients diagnosed with OI were enrolled in the current study, where 39 13.4%), 67 (23.1%), 162 (55.9%), and 22 (7.6%) were classified as types I, III, IV and V, respectively . Most of these patients were symptomatically mild or moderate, with only 16.5% (28/145) being classified as type III, lower than the cohort-wide percentage (23.1%). Single-point mutation on 5’-UTR of IFITM5 (c.-14C > T) occurred in 20 patients, two of whom also carried pathogenic mutations in COL1A1  inheritance, including COL1A1/2 and IFITM5 variants, accounted for 75.1% (166/221) of the patients with genetic tests. Another two patients were clinically classified as type V OI without genetic testing, as they carried typical radiographic features, including hyperplastic callus, inter-osseous ossification and radial head dislocation [WNT1 ; SERPINF1 , FKBP10  and other genes  that affected only 1 ~ 2 patients. Fifteen (35.7%) of these AR patients were type III, which was over twice as high (χ2p = 0.005) as the percentage of type III in the 166 AD patients (17.5%). This is consistent with previous reports that AR patients tend to have more severe phenotypes than AD patients [Among the 221 patients with genetic testing, ~ 2/3 (145 patients) carried pathogenic mutations in genes encoding type I collagen  per patient. Over half (59.7%) of the patients had two follow-ups or more (Table –10 per pp < 0.001), with mean ages of 8.4 ± 8.7, 10.6 ± 6.4, 15 ± 7.5, and 17.9 ± 6.3 years for the non-scoliotic, mild, moderate and severe groups, respectively , curve property and gender. The severity was positively associated with age (one-way ANOVA p = 0.068). For the mild and moderate cases, the apex tended to be in the upper lumbar (L1–L2) or lower thoracic (T7–T12) regions, whereas for the severe cases, the apex was most commonly found in the T7–T8 region (Table 2p < 0.001) (Table 2p = 0.262).Interestingly, the sites of the scoliosis apex, the vertebrae corresponding to the maximum convexities, appeared borderline different among the different severity grades (on Table . Among t1) Table . On the COL1A1 or COL1A2 alone tended to have a milder form of scoliosis. In particular, among the 76 patients carrying COL1A1 mutations, 23 were non-scoliotic and 29 were mildly scoliotic, accounting for > 2/3 of COL1A1 patients. Only 11 (14.5%) and 13 (17.1%) patients in this group had moderate and severe scoliosis, respectively. Similarly, patients with mutations in COL1A2 also tended to be milder. Fifty-four of the 69 (78.3%) COL1A2 patients were non-scoliotic or mildly scoliotic, while only 8 and 7 had moderate and severe scoliosis, respectively. Patients carrying qualitative mutations (i.e. missense mutations) were known to have more severe phenotypes than those carrying quantitative ones (frameshift or stop codon) [ COL1A1 and COL1A2 is higher than quantitative mutations. In both COL1A1 (χ2p = 0.438) and COL1A2 (χ2p = 0.136), carrying qualitative or quantitative mutations did not show a significant difference in scoliosis severities (Table Patients with pathogenic variants in p codon) . In modeIFITM5 mutation, scoliosis severities were not more biased towards the non-scoliotic or mild groups. Three (15%) patients were non-scoliotic , or the patients with other AR gene mutations (χ2p = 0.95). In particular, all six patients with mutations in FKBP10 (type XI OI) had scoliosis [For the 20 patients with ic Table  and ten coliosis . Due to 2p = 0.93), suggesting the representation of the cohort and the accuracy of clinical diagnoses based on radiographic features.Interestingly, among the 13 patients without mutations in the tested genes, scoliosis severities were also not biased towards non-scoliotic or mild grades. On the other hand, the severity distribution among the 69 untested patients was highly similar to that of the 221 tested patients  . To reveDue to the relatively young ages in our cohort, 179 patients (61.7%) still had open triradiate cartilage during their last radiographs. Another 50 patients were over the age of 18 and their triradiate cartilage were considered closed with the closure age unknown. This left 61 patients whose closure age were captured by radiographs Table . Among t2p < 0.001). The percentage of patients with LLD was significantly higher in the severe group (67.8%) than the non-scoliotic (32.9%), mild (50.9%), and moderate (45.0%) groups  of the cohort had lower-limb discrepancies (LLD), and its prevalence was significantly associated with scoliosis severity . Post-hoc analysis by Tukey honest significant difference test showed that the severe group had the lowest BMD Z-scores (− 3.5 ± 1.9), as compared with the non-scoliotic (p < 0.001) and mild (p < 0.001) groups. No difference (p = 0.99) was observed between the non-scoliotic and the mild groups.In patients with AIS, BMD was known to be lower, although no correlation was found with the severity of spinal deformities . Among tp < 0.001), with the severe group having evidently higher age of first drug use (average 10.3yo) than other groups   Table .Next, we focused on OI scoliosis severity with respect to individual genetic and non-genetic factors. In reality, these factors are often interdependent. For example, skeletal maturity measures, including the Risser sign and the closure age of triradiate cartilage, are highly dependent on the patient ages of available radiographs. Sillence grades are dependent on the presence of lower-limb discrepancy and scoliosis, which in turn depends on genetics. BMD Z-scores depend on genetics and drug history. Thus, genetics , age, drug history (including drug types and ages of first use) and gender are “fundamental” independent factors, whereas Sillence grades, the presence of LLD, and skeletal maturity may be considered “intermediate” factors that are themselves the outcomes of one or multiple other fundamental factors. We asked how some of these factors may increase or decrease the chance (in terms of odds ratio) of progressing into the advanced stages scoliosis (moderate or severe) of scoliosis.p < 0.001) of developing moderate or severe scoliosis. Gender, on the other hand, was not predictive of progression  non-scoliotic or mild, and; (2) advanced stages of scoliosis (moderate or severe). As Sillence grading had taken scoliosis into consideration, we excluded Sillence grading from the model, and considered age corresponding to the maximum Cobb angles, gender, genotypes, drug history, BMD Z-scores and LLD as predictors. Regression results showed that age was the most significant predictor Table , with eaon Table .Table 5LIFITM5 and WNT1 mutations increased the OR by 3.71  and 3.37  times, respectively. Mutations in all other AR genes also significantly increased the OR by 3.16  times.Genetic variants appeared predictable for moderate/severe and severe scoliosis Table . In partp = 0.04). But we cautioned the interpretation of this result, as pamidronate is an older bisphosphonate and was used by older patients, making it a confounder of the age factor. Bone mineral density (BMD) was a strong predictor, with each one standard deviation increase resulting in 0.82  times lower OR. The lower-limb discrepancy did not appear to affect the severity grades at all , but to the 145 patients carrying mutations in these two genes only. We found that COL1A2 appeared significantly less severe than COL1A1, with an OR of 0.28  for progression into moderate or severe scoliosis .Since patients with p = 0.015) than the baseline level (represented by COL1A1), and patients with WNT1 mutations had higher progression rates on average  . We also included patients without scoliosis at the cutoff date of this study. Overall, the mean progression rate was 2.7°/year (95% CI 2.4–3.0) with a peak at ~ 12.5 years of age  underwent spinal surgeries, which represent 42.8% of the 59 patients with severe scoliosis. These patients had an average preoperative Cobb angle of 85.3° (IQR 64–96). After spinal surgery, they enjoyed an average reduction of 33° (IQR 23–40). These patients are currently being followed up. The surgical details and follow-up outcomes will be reported separately.Scoliosis is one of the most prevalent conditions among osteogenesis imperfecta patients and is well-known to be progressive with age , 12. It COL1A1 and COL1A2 genotypes were strongly biased towards having mild or no scoliosis at all, whereas patients with pathogenic variants on IFITM5, WNT1 and other recessive genes did not display such a pattern. Due to the relatively small number of cases in IFITM5 and recessive genes, it was difficult to statistically delineate their effects on the outcomes, although their fractions of moderate or severe cases were comparable.We stratified the cohort by four scoliosis outcome grades, including non-scoliotic, mild, moderate and severe, as measured by their maximum  Cobb angles of the major curves. We then performed univariate and multivariate analyses between the outcome and a set of genetic and non-genetic factors. We found that patients with COL1A2 was less damaging than COL1A1 in progressing into advanced stages of scoliosis. The mutation types, in terms of qualitative or quantitative changes, had a weak influence on the outcomes. Neither within each collagen gene nor the two genes combined did the mutation types have a significant association with the severity grades. Among the non-genetic factors, we found that skeletal maturity, lower-limb deformities, and drug history were all individually associated with severity outcomes , and was lower in COL1A1/2 patients in our study and socioeconomic reasons. Unlike in the West where 85–90% of the OI patients seeking medical treatments were affected by COL1A1/2 mutations [Overall, we noted both the outcome grades and progression rates were more severe in our cohort than in the literature. At 70.7%, the prevalence of scoliosis was considerably higher in our cohort than previously reported , 12, 16.utations , 29, ourutations . Subtypeutations . At an autations .We also studied the onset age of scoliosis in OI. Based on radiographic evidence, we stratified the scoliosis group into early-onset (EOS), late-onset (LOS) and ‘unknown’. The ‘unknown’ group represented over half of all scoliotic OI . Since eight genes were involved among these cases, the number of cases per genotype was rather small. This distribution bias may cause difficulty in estimating genotype-specific effects on OI scoliosis. We addressed this by a two-step approach, whereby the two collagen genes were first considered as a single group, before a second analysis on the 145 patients affected by these two genes only was conducted, where COL1A1 and COL1A2 were now treated as separate groups. Sillence classification was often used as an independent variable to explain the scoliosis outcomes [Although we tried to make our cohort as representative as possible by including all consecutive cases, the non-outcomes , 12, altoutcomes . To avoiLastly, it is noteworthy that the cases in the current study only represented OI patients seeking treatment at our hospital, as a result of which some milder cases not needing medical treatment were not screened. Our results thus may appear more severe than the actual situation among the broader OI community.In all, a comprehensive study of scoliosis in osteogenesis imperfecta was undertaken to identify the genetic and non-genetic factors affecting its severity and progression, and it is hoped insights from this study may be helpful in making certain clinical decisions.Additional file 1. Supplementary information.Additional file 2. Anonymized genetic testing results of the 54 patients newly reported in the current study.


Streptococcus agalactiae , group B Streptococcus (GBS), a major cause of infection in a wide variety of diseases, have been compared in different human and animal sources. We aimed to compare the bacterial proteome and metabolome profiles of human and animal S. agalactiae strains to delineate biological interactions relevant to infection. With the innovative advancement in mass spectrometry, a comparative result between both strains provided a solid impression of different responses to the host. For instance, stress-related proteins (Asp23/Gls24 family envelope stress response protein and heat shock protein 70), which play a role in the survival of GBS under extreme environmental conditions or during treatment, are highly expressed in human and animal strains. One human strain contains ꞵ-lactamase (serine hydrolase) and biofilm regulatory protein (lytR), which are important virulence regulators and potential targets for the design of novel antimicrobials. Another human strain contains the aminoglycosides-resistance bifunctional AAC/APH (A0A0U2QMQ5) protein, which confers resistance to almost all clinically used aminoglycosides. Fifteen different metabolites were annotated between the two groups. L-aspartic acid, ureidopropionic acid, adenosine monophosphate, L-tryptophan, and guanosine monophosphate were annotated at higher levels in human strains. Butyric acid, fumaric acid, isoleucine, leucine, and hippuric acid have been found in both human and animal strains. Certain metabolites were uniquely expressed in animal strains, with fold changes greater than 2. For example, putrescine modulates biofilm formation. Overall, this study provides biological insights into the substantial possible bacterial response reflected in its macromolecular production, either at the proteomic or metabolomic level. Streptococcus agalactiae , a group B Streptococcus (GBS), is a gram-positive bacterium that is considered an important pathogen regularly associated with mastitis in bovine and neonatal meningitis in humans. In humans, it is a dominant contributor to newborn infectious diseases in several regions, and a high number of females carry it asymptomatically. GBS is the leading cause of newborn sepsis and meningitis, with substantial mortality and morbidity, particularly in cases of extensive neonatal infections. It is also a significant pathogen in the elderly and in people with associated illnesses that impede immune defense systems against pathogenic microorganisms. Vertical GBS transmission to infants during accouchement is responsible for certain occurrences of neonatal GBS infections. Colonization occurs in approximately 40% of pregnant women. Approximately 5% of GBS-infected newborns die, while those who escape typically suffer from serious neurological consequences, such as mental retardation and sight or hearing impairment1.S. agalactiae is considered one of the most frequent mastitis-causing bacteria in dairy cattle, causing significant reductions in the quantity and quality of milk and representing a huge economic problem for the dairy industry, particularly because this bacterium is extremely contagious within a farm and significantly affect milk productivity3. GBS invades the mammary glands of dairy cattle through the skin and teats4. Mastitis caused by GBS is a chronic illness with few acute epidemics and no notable associated symptoms, but it affects milk output5.In addition, S. agalactiae in humans and cattle raises the prospect of interspecies transmission. This concern is especially significant in view of the re-emergence of S. agalactiae in adults and animals. Numerous S. agalactiae comparisons between humans and cattle have been reported, and most studies have indicated that isolates from these species constitute substantially separate groups in terms of their core and accessory genomes8.The presence of 9. This aids in a deeper understanding of infection and the advancement of antimicrobial strategies to fight infection.There is barely any information at the level of the functional proteome presented in GBS and its physiological status in both human and bovine GBS. Proteomics reveals valuable information about the virulence of the pathogen and is a very useful procedure for understanding how bacteria respond to external environmental factors. It is a powerful method to investigate bacterial response profiling from system-level studies and can enhance our knowledge of bacterial adaptation, antibiotic resistance mechanisms, and tolerance development10. The availability of databases on bacterial gene regulation and metabolic pathways allows for the investigation of prevalent bacterial systems, such as strain identification and differentiation, drug mechanisms of action, and metabolic changes in response to antimicrobial treatment11.On the other side, metabolomics is a comprehensive examination of the metabolites found within the living systems. The approach has been widely used to help better understand the complex bacterial cellular metabolic states and changes as one of the important "omics" tools. It has also provided important information for elucidating the disease pathophysiology and identifying novel biomarkersS. agalactiae strains and their related metabolites. In this study, we used different techniques to compare the proteomic and metabolomic signatures of human and animal S. agalactiae strains.To the best of our knowledge, no published data have investigated the link between the proteomic profile of clinical human and animal All the GBS strains 100%) were resistant to penicillin, amoxicillin, clindamycin, ceftriaxone, cephalexin, cloxicillin, cefepime, tetracycline, erythromycin, and streptomycin followed by ciprofloxacin (69.6%), amoxicillin-clavulanic acid (60.9%), and cefoperazone (43.5%). However, high susceptibility was observed for impenem (100%) followed by sulfamethaxole-trimethoprime (69.6%). All isolates were multidrug-resistant (MDR) with multiple antibiotic resistant index (MAR) ranged from 0.66 to 0.93  were analyzed. Quality validation showed that distribution of mass errors was lower than 0.02 Da indicating a good quality of sample preparation. A total of 2527 S. agalactiae proteins were identified with 99% confidence at the peptide and protein levels  broth sub-culture of Of the annotated components, 1659/2527 have an assigned molecular function, 1082/2527 are involved in cellular compartment, and 1080/2527 are assigned known biological processes (Supplementary Table www.ebi.ac.uk/pride) via ProteomeXchange with identifier PXD035678.The identified proteins are available in the PRIDE database  were annotated to a specific function using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases.GO annotation indicated that the DAPs of human and animal strains could be classified into 18 and 16 categories, respectively, which included many important biological and cellular processes , cell wall/membrane/envelop biogenesis (13.38%), unknown function (11.04%), translation (9.69%), and replication (8.36%). The other protein functions were carbohydrate transport and metabolism, energy production and conversion, post-translational modification, inorganic ion transport and metabolism, nucleotide transport and metabolism, cell cycle control, signal transduction mechanisms, coenzyme transport and metabolism, transcription, defence mechanisms, intracellular trafficking, lipid transport and metabolism, and secondary metabolites biosynthesis.S. agalactiae strains were involved in microbial metabolism, genetic information processing, and defense mechanism , carbohydrate transport and metabolism (16.51%), amino acid transport and metabolism (9.91%), signal transduction mechanisms 8.49%), post-translational modification (8.02%), and defense mechanisms (6.13%). KEGG orthology (KO) annotation for these proteins revealed that the preponderant reported KO for unique proteins of both human and animals %, carboh9%, post-S. agalactiae strains was clearly observed  and P value between average biological replicates of the two groups , Glycolysis/Gluconeogenesis, and RNA polymerase Table . These pS. agalactiae for both human and animal strains was used for metabolites extraction. The metabolome of 23 strains  were analyzed. A total of 325 measurable and reproducible metabolite signals were obtained from both human and animal strains  indicated significant increases in the relative concentration of 15 different metabolites/small molecules between the two groups. The different metabolites included L-aspartic acid, glycine, L-tryptophan, succinic acid, dopamine, sarcosine, ureidopropionic acid, indole, guanosine monophosphate, putrescine, 2-phenylpropionate, adenosine monophosphate, and D-alpha-aminobutyric acid.As presented in Fig. Human strains contained higher levels of L-aspartic acid, ureidopropionic acid, adenosine monophosphate, L-tryptophan, and guanosine monophosphate than did animal strains.To better evaluate how multiple pathways differed between the human and the animal strains, a KEGG functional enrichment analysis of the pathways related to the different metabolites was conducted Table .Table 3DPathway topology analysis showed 29 shared metabolic pathways between the two groups, mainly metabolic pathway, phenylalanine metabolism, ABC transporters, alanine, aspartate and glutamate metabolism, pyrimidine metabolism, butanoate metabolism, glycine, serine and threonine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, arginine and proline metabolism, purine metabolism, two-component system, aminoacyl-tRNA biosynthesis, D-glutamine and D-glutamate metabolism, and citrate cycle (TCA cycle)  included L-histidine, L-arginine, guanosine diphosphate, isopentenyl pyrophosphate, malonic semialdehyde, alpha-linolenic acid, guanosine, 2-phenylbutyric acid, pargyline, 1-pyrroline, O-phosphoethanolamine, phosphorylcholine, 4-hydroxyphenylpyruvic acid, amphetamine, 2,6-dimethylpyridine, and PE (16:1(9Z)/16:1(9Z)). Whereas, 12 metabolites were uniquely annotated in animal group including 3-methyl pyruvic acid, galactonic acid, indole-3-propionic acid, ꞵ-hydroxy butyric acid, indoleacetic acid, phenylacetic acid, rivastigmine, deoxyuridine, oxalic acid, phenylglyoxal, 3-(Methylthio)-1-propene, and niacinamide.As revealed in Fig. S. agalactiae is an important host-generalist opportunistic pathogen causing disease in humans and cattle14. It is a leading cause of meningitis and sepsis in newborns and a causative agent of invasive infections in adults, including meningitis, endocarditis, osteoarticular, and soft tissue infections15. GBS bovine mastitis is a major veterinary and economic issue for the dairy industry worldwide because of the treatment costs, reduced milk production, milk that must be discarded, and culling and replacement16. S. agalactiae is recognized as an emerging pathogen in adult humans throughout the world and as a reemerging pathogen in dairy cattle17. Recent studies have demonstrated the importance of bioinformatics techniques and algorithms for investigating pathogenesis-related proteins and identifying druggable targets and innovative treatments19. To our knowledge, no published study has analyzed the relationship between the proteomic profiles of clinical human and animal MDR S. agalactiae strains and their associated metabolites. This is the first study to generate information on the proteomic and metabolomic profiles of human versus animal GBS strains. To identify proteomic biomarkers that could better characterize the pathogenesis, we studied 23 S. agalactiae strains of human and animal origin. Catabolic ornithine carbamoyltransferase was the only shared protein identified by Swiss-prot between human and animal strains which is involved in arginine metabolic process. Out of the 29 differentially significant shared proteins between human and animal strains  that are highly expressed in human and animal strains may explain their role in the survival of GBS under extreme environmental conditions or during treatment. This finding is confirmed by previous studies showing that the stress-related proteins identified in Lactiplantibacillus plantarum 9010 help its survival under harsh gastrointestinal environmental conditions26. These proteins may contribute to antibiotic resistance associated with virulence28.Enolase is a shared protein between human and animal GBS. Enolase is a moonlighting protein with multiple functions in the cell, including glycolysis, tRNA confirmation, and a cell surface plasminogen receptor that can be activated to plasmin, promoting the spread of bacteria, penetration of endothelial cells, and organ invasionS. aureus isolates22. Moreover, the late competence protein ComGE (A0A1D0 CSP9), was found in one animal isolate (code No. 40). This may be due to the use of antibiotics and hydrogen peroxide in the farm. This assumption is further supported by the findings of Chanda et al.29 that the promoter of S. aureus cspC is strongly induced in response to exposure to antibiotics and hydrogen peroxide. In addition, cspC was present at a significantly higher level in methicillin-resistant S. aureus than in methicillin-sensitive S. aureus28. Mobile genetic elements, in particularly lysogenic prophages, play an important role in the evolution of GBS, its adaptation to host species, its high virulence, and its ability to cause invasive infections in humans30. Also, acquisition of prophages is associated with host adaptation of the cattle lineage31.Furthermore, it is evident that several proteins not only distinguish human and animal strains but are also unique to certain isolates. For instance, the protein alkyl hydroperoxide reductase subunit C  was found in one isolate (code No. 75) from cow mastitis  contained ꞵ-lactamase (serine hydrolase). This strain contains also a biofilm regulatory protein (lytR) that has been described as an important virulence regulator and a potential target for the design of novel antimicrobials34. Another human strain contains aminoglycosides-resistance bifunctional AAC/APH (A0A0U2QMQ5) protein that confers resistance to almost all clinically used aminoglycosides35.Antibiotic resistance is a global public health problem and a development threat. As bacteria develop resistance via the selection of existing mutations and acquisition of new genes at a rate that far outpaces our capacity to generate novel antimicrobials, our current therapeutic arsenal is becoming increasingly constrainedS. pneumoniae is responsible for fluoroquinolone resistance36. In this study, an ABC-type multidrug transport system with a permease component  was identified in two animal strains (codes 75 and 172) and one human strain (code 24).ATP-binding cassette (ABC) transporters are one of the largest families of membrane proteins that include a number of therapeutically significant members that expel drugs. An ABC transporter from tetA37 was found in one animal strain (code 105).Moreover, a TetR family transcriptional regulator (TetR/AcrR family) involved in regulating the expression of the tetracycline resistance determinant encoded by 38 discussed the multifaceted functions of FcαRI and IgA during homeostasis, infection, and chronic inflammation and their potential as novel therapeutic targets. It was reported that many clinical isolates of S. pyogenes and a majority (70%) of type lb human GBS expressed IgA Fc-receptors (ꞵ antigen)39. Here, the IgA FC receptor was present in only one animal isolate (code No. 91).Recently, van Gool and Egmond41. Hence, bacterial kinases have received considerable attention as prospective targets for the discovery of novel antimicrobial agents40. Interestingly, animal strains uniquely contained histidine kinase  and HPr kinase/phosphorylase (HPrK/P) proteins (isolates code no. 20 and 46), whereas penicillin-binding proteins 1 B and 2 B were unique in human strains  and 16S rRNA methyltransferases (n = 13) that could provide a novel drug target for stopping the emergence of MDR bacteria as they simultaneously inhibit translation and eliminate the ability to methylate the substrates. The key finding of our study is the unique detection of 16S rRNA m5C967 methyltransferase , a TrmH family tRNA/rRNA methyltransferase YacO, cytosine-specific methyltransferase, 23S rRNA (guanosine (2251)-2′-O)-methyltransferase RlmB, rRNA methyltransferase, and methyltransferase (yhdJ_1) in human GBS. Animal GBS contains ribosomal RNA small subunit methyltransferase I (rsmI), putative tRNA (cytidine (34)-2′-O)-methyltransferase, cytosine-specific methyltransferase, and 5-methyltetrahydropteroyltriglutamate—homocysteine methyltransferase (met E).Kinases are crucial for bacterial growth, biofilm formation, and antibiotic resistance. Recent studies have reported the important role of penicillin-binding protein and serine/threonine kinase-associated (PASTA) kinases in regulating resistance to β-lactam antibiotics44. It is a useful tool for detecting alterations in metabolites resulting from mastitis and antibiotic treatment46. Among the annotated metabolites in human and animal GBS, uridine 5′-diphosphate, hypoxanthine, and guanine were prevalent and upregulated in human samples compared to animal samples, whereas phenylpyruvic acid was downregulated  and were identified as S. agalactiae by CAMP positive reaction, β-hemolysis on blood agar, and positive sodium hippurate hydrolysis test66. Further confirmation by PCR was performed using two sets of primers targeting the cfb gene which encodes the CAMP factor and the scpB gene that encodes C5a peptidase67. Reference strain: S. agalactiae (ATCC 12386) was involved in our study.Sixteen and seven These strains were stored at − 80 °C in 80% double strength broth and glycerol until use. The strains were cultured before use on tryptic soya agar plates then sub-cultured on brain heart infusion broth .S. agalactiae strains involved in our study were tested against 15 antimicrobial discs of 7 groups using the Kirby-Bauer disk diffusion assay following the Clinical and Laboratory Standards Institute guidelines and interpretative criteria68. The tested antimicrobial discs  were penicillin (10 U), a, amoxicillin (25 µg), cloxacillin (1 µg), amoxicillin-clavulanic acid (20 + 10 µg), cephalexin (30 µg), cephoperazone (75 µg), ceftriaxone (30 µg), cefepime (30 µg), impenem (10 µg), ciprofloxacin (5 µg), tetracycline (30 µg), erythromycin (15 µg), trimethoprime -sulphamethaxazole (23.75 + 1.25 µg), streptomycin (10 µg), and clindamycin (2 µg).The antimicrobial susceptibilities of S. agalactiae isolates from human and animal sources; respectively, were subjected to total protein extraction. Proteins were precipitated from the collected media using four times chilled acetone. After incubation at − 80 °C for 30 min and at − 20 °C overnight, samples were centrifuged at 10,000 RPM for 30 min. Protein extract of cells was obtained by adding 50 µl lysis solution  with Complete ultra-proteases . Samples were incubated at 37 °C for 1 h with occasional vortex, then centrifuged at 12,000 rpm for 20 min. Protein assays of the extracts were performed using Bicinchoninic acid (BCA) assay  at Å562 nm prior to digestion.Brain heart infusion broth sub-cultures of 16 and 7 69. Peptides were assayed using BCA method  at Å562 nm prior to injection to be 1.5ug/10 µl.Thirty µg of cell protein lysate from each sample were subjected to in solution digestion. In brief, protein pellets were re-suspended in 8 M urea lysis solution and reduced with 5 mM tris (2-carboxyethyl) phosphine (TCEP) for 30 min. Alkylation of Cysteine residues was performed using 10 mM iodoacetamide for 30 min in dark area. Samples were diluted to final concentration 2 M urea with 100 mM Tris-HCl, pH 8.5 prior to digestion with trypsin. For endopeptidase digestion, modified porcine trypsin  was added at 40:1 (protein: protease mass ratio) and incubated overnight in a thermo-shaker at 600 rpm at 37 °C. Digested peptide solution was acidified using 90% formic acid to a final pH of 2.0. The resultant peptide mixture was cleaned up using stage tip as discussed earlierNano-LC MS/MS analysis was performed using TripleTOF 5600 +  interfaced at the front end with Eksigent nanoLC 400 autosampler with Ekspert nanoLC 425 pump. Peptides were trapped on CHROMXP C18CL 5 µm (10 × 0.5 mm)  on trap and elute mode. MS and MS/MS ranges were 400–1250 m/z and 170–1500 m/z, respectively. A design of a 120-min liner gradient 3–80% solution  was performed. The 40 most intense ions were sequentially selected under data dependent acquisition (DDA) mode with a charge state 2–5. For each cycle, survey full scan MS and MS/MS spectra were acquired at resolution of 35.000 and 15.000, respectively. For high precision, external calibration was programmed and run during sample batches to correct for possible TOF deviation.70. Peptides were identified from MS/MS spectra, then the Pro Group™ Algorithm was used to assemble peptide identifications into a list of protein identifications. S. agalactiae database  was used. Analysis was searched with Bias Correction using and biological modifications as ID focus. To ensure high-quality results, the false discovery rate (FDR) was maintained at 1% of the protein level. All figures are generated using the ggplot 2 package for the statistical programming language R71.Raw LC/MS/MS data in Wiff format were searched using ProteinPilot™ SoftwareS. agalactiae isolates from human and animal sources; respectively, were subjected to total metabolites extraction. Both intra- and extra-cellular metabolites were used by vertexing the bacterial pellets in the BHI broth. One mL of each broth culture was thawed on ice. A pre-cooled methanol 80% was added to each sample72. A 100 µl of methanol containing 10 ng/mL internal standards (Olopatadine and Atorvastatin) was added to each sample then agitation for 2 min followed by thawing and freezing for two cycles, 15 min each. The mixture was vortexed and whirled for at least 2 min. Samples were ultra-sonicated for 15 min at a temperature less than 25 °C and were centrifuged at 12,000 rpm at 4 °C for 10 min. The supernatant was transferred to a clean tube. And each sample was further dried using a speed vac at 30 °C and reconstituted in a solvent consisting of water: methanol: acetonitrile 2:1:1, respectively. The extracted samples were then subjected to LC–MS/MS analysis. A pooled quality control (QC) was obtained by mixing 10 µl of each sample.Brain heart infusion broth (BHI) sub-cultures of 16 and 7 +) and negative-ion (ESI–) modes. IDA acquisition method consisted of a single TOF scan from 50 to 1100 Da followed by product ion TOF scan from 50 to 1000. The mobile phase solutions consisted of solution (A); 5 mM ammonium formate in 1% methanol (pH 3.0) for positive mode, solution (B); acetonitrile, and solution (C); 5 mM ammonium formate in 1% methanol (pH 8.0) for negative mode. Gradient elution was sustained at 0% B for 1.0 min, 0% to 90% B in 20 min, 90% for 4.0 min, 90% B to 0% B in 1.0 min, and finally re-equilibrating with 0% B for 3.0 min. The order for samples was random, with an injection of QC sample every ten injections. Mass calibration was automatically performed every 2.5 h analysis by an automated calibration delivery system using APCI calibration solution (AB SCIEX). Blank sample used to check solvents quality and monitor carryover. Data collections were created using Analyst TF (v 1.7.1).Chromatographic separation was carried out using a SCIEX EXION LC™ AC UHPLC system equipped with Acquity XSelect HSS T3 analytical column 2.1 × 150 mm, 2.5 µm ID . Mass spectrometric analysis was performed using Triple TOF™ 5600 + system  equipped with a Duo-Spray source operated using the positive-ion  to validate the MS1 and MS2 of the significant molecules.MS-DIAL platform was used for small molecules identification74, Log transformed and scaled. All process was achieved using R environment. Differential expression analysis was performed using Mann Whitney test with FDR 1%75. For metabolomics, peak intensities were parsed for further analysis. Filtration process was applied with a cutoff 50% group and FDR < 0.1 (10%). Data were normalized using PQN and scaled then subjected to statistical analysis. For metabolomics, T test was used. Heatmap analysis was plotted based on NSAF abundance after applying the abovementioned parameters with p value 0.05 and FDR 1%. Dendrogram associated with heatmap was generated using the Euclidean distance calculation. KEGG pathway maps were created using the KEGG database76.Protein abundance represented by NSAF was subjected to filtration process based on its appearance within the replicates where proteins were omitted if it did not show within 20% group. Data were normalized using Probabilistic Quotient Normalization (PQN)This study was approved by Suez Canal University ethical board (No. 202103RH2). All experiments were performed in accordance with relevant guidelines and regulations. An informed consent was obtained from all subjects in this study.Supplementary Legends.Supplementary Figure 1.Supplementary Figure 2.Supplementary Figures.Supplementary Figure 3.Supplementary Figure 4.Supplementary Figure 5.Supplementary Figure 6.Supplementary Figure 7.Supplementary Figure 8.Supplementary Table 1.Supplementary Table 2.Supplementary Table 3.Supplementary Table 4.Supplementary Table 5.Supplementary Table 6.Supplementary Table 7.


For high-temperature behavior, the maximum superplasticity or ductility of 416% was demonstrated in this fine-grained alloy with an average grain size of 10 μm at 573 K and 1.67 × 10−3 s−1. A power-law constitutive equation was established. The stress exponent was 2.29 (≈2) (strain rate sensitivity 0.44), and the deformation activation energy was 162.02 kJ/mol. This evidence confirmed that the dominant deformation mechanism at elevated temperatures is grain boundary and interphase boundary sliding controlled by lattice diffusion.Magnesium-lithium alloy is the lightest alloy to date. To explore its room temperature strength and high-temperature ductility, a plate of a new fine-grained Mg-9.13Li-3.74Al-0.31Sr-0.11Y alloy was fabricated by asymmetric rolling, and the rolled plate was subjected to friction stir processing (FSP). The microstructure and mechanical properties at room and elevated temperatures were investigated by optical microscopy, X-ray diffraction (XRD), energy dispersive spectroscopy (EDS), and tensile tester. Grain refinement with an average grain size in the α-Mg phase of 1.65 μm and an average grain size in the β-Li phase of 4.24 μm was achieved in the water-cooled FSP alloy. For room temperature behavior, the ultimate tensile strength of 208 ± 4 MPa, yield strength of 193 ± 2 MPa, and elongation of 48.2% were obtained in the water-cooled FSP alloy. XRD and EDS analyses revealed that the present alloy consists of α-Mg and β-Li phases, Al Magnesium-lithium (Mg-Li) alloys have found certain applications in the automobile industry, 3C  electronics, weapons, and spaceflight industries owing to their extremely low density, excellent specific stiffness, good specific strength, good electromagnetic shielding properties, and damping properties. Due to the demand of aviation and spaceflight industries, studies on the room temperature microstructure, mechanical properties, and strengthening have been reported in some Mg-Li alloys ,2,3,4. MSevere plastic deformation has been extensively studied over past decades ,17. In MIn this work, our aims include several aspects. Firstly, a novel Mg-9Li-4Al-0.5Sr-0.5Y alloy is fabricated by asymmetric rolling + FSP. Secondly, the microstructures and mechanical properties at room and elevated temperatures are studied. Thirdly, the room temperature strengthening mechanism and high-temperature superplasticity mechanism are expounded.The melting and casting of the Mg-Li-Al-Sr-Y alloy were performed using Jackson’s approach. The flux (LiCl + LiF) coverage and argon atmosphere were adopted to prevent lithium vaporization. The analyzed chemical composition was Mg-9.13Li-3.74Al-0.31Sr-0.11Y in wt.% . The ingot was homogenized at 523 K for 20 h. After milling the ingot surface, the milled ingot was asymmetrically rolled at 523 K to 6 mm thick with a reduction of 75%. 75% thickness reduction was applied in six passes. Then the rolled plates were subjected to friction stir processing. The schematic diagram of the FSP is shown in −2–1.67 × 10−4 s−1. After tension, the tensile samples were quenched into water to preserve the high-temperature microstructures. Dog bone samples were spark discharge machined from the FSPed plate perpendicular to the forward direction of FSP. The sample dimensions were 3 mm (gauge length) × 2 mm (gauge width) × 2 mm (gauge thickness). The tensile test velocity at room temperature was 0.3 mm/min. The number of testing samples was three. After being held at the designated temperature for 15 min, high-temperature tensile tests were performed for water-FSP samples in a SANS-CMT5105 universal tensile tester in the temperature range of 473–623 K over the strain rates of 1.67 × 10The samples for optical microstructure observation were ground, polished, and etched as per the conventional metallographic method. The etching solution was 4% HCl and 96% ethanol. The optical examination was carried out on an Olympus DSX500 optical microscope. Image-Pro-Plus (IPP) software was used to measure the grain size and phase volume fraction. The samples for X-ray diffraction (XRD) detection were ground and polished to obtain a flat surface. The scanning angle was 10–90°. The scanning velocity was 6°/min. The voltage was 40 kV. The current was 40 mA. The XRD test was carried out on the X’Pert Pro X-ray diffractometer. Jade 6.5 software was used to analyze the phase composition.The scanning electron microscopy (SEM) samples were taken from the XRD samples. SEM observations and energy dispersive spectroscopy (EDS) analysis were conducted on an SSX-550 scanning electron microscope.2Y, Al4Sr, MgLi2Al, and AlLi intermetallic compounds. The α-Mg and β-Li phases achieve solid solution strengthening during processing. Al2Y and Al4Sr compounds possess high melting points and can realize the second phase strengthening during processing. MgLi2Al is a metastable strengthening compound during thermal processing and can be transformed into a soft AlLi compound phase at room temperature. As shown in 2Y and Al4Sr compounds. As shown in 2Y and Al4Sr compounds. −3 s−1, as shown in −4 s−1 exhibits strain hardening. As shown in −2 s−1 exhibits strain softening after the peak stress. This issue will be discussed in A is a dimensionless constant, G is the shear modulus, a function of temperature, b is the magnitude of Burgers vector of dislocation, b (Mg) = 3.21 × 10−10 m [k is Boltzmann’s constant, k = 1.38 × 10−23 J/K, T is the absolute temperature, d is the grain size, p is the grain size exponent, σ is the applied stress, n is the stress exponent , D0 is the frequency factor for diffusion, Q is the deformation activation energy, and R is the universal gas constant. Here, the n-value, p-Value, and Q-value will be determined. The shear modulus of G for pure Mg is given by [To deepen understanding of the deformation mechanism at elevated temperatures, a power-law constitutive equation is established and is suitable for application in superplastic forming process control. Power-law constitutive equation at elevated temperature is generally given by ,36:(1)ε˙ 10−10 m , k is Bon1/ relation in this alloy to determine the threshold stress and stress exponent. At the true strain of 0.2, the values of threshold stress n1/ relation. The stress exponents are taken as 2, 3, 4, and 5. When nn = 3, 4, and 5 are excluded. The determination coefficient, R2, of 0.9888 with high correlation accuracy when n = 2. Thus, the true stress exponent is determined to be 2. Threshold stress According to Equation (1), the deformation activation energy is given by:−3 s−1 is more than 400% [p-value should be determined to check whether p = 2 for lattice diffusion or p = 3 for grain boundary diffusion. The effective diffusion coefficient is given by [x = 1 × 10−2 for superplasticity, gbf = w is the grain boundary width, w = 2b. As per our previous model [The deformation mechanism in the present alloy is grain boundary sliding because the maximum elongation of 416% at 573 K and 1.67 × 10han 400% . Thus, tThe grain sizes are shown in p = 2 as per reference [Q = 147.83~178.14 kJ/mol, as shown in Q = 162.02 kJ/mol. When eference . SubstitD0 = 1.0 × 10−4 m2·s−1 [The diffusion coefficient is given by 4 m2·s−1 . SubstitR2, is 0.90. Thus, the power-law constitutive equation is obtained as the following: Microstructure refinement has a direct influence on mechanical properties at room temperature. As shown in 2Y, Al4Sr, MgLi2Al, and AlLi intermetallic compounds, hinder the dislocation motion and cause Orowan strengthening during the processing process. Thus, solid solution strengthening, dislocation strengthening, grain boundary strengthening, and Orowan strengthening contribute to the yield strength and improve the load-bearing capability of the present alloy. On the other hand, various strengthening mechanisms improve yield strength in the LASY9400 alloy. In the as-cast state, the interaction between solvent Mg and solutes like Al, Sr, and Y leads to lattice distortion and increases the resistance to dislocation motion in the α-Mg and β-Li solid solution. Thus, solid solution strengthening occurs. Asymmetric rolling exerts shear strain on the alloy, increases the dislocation density, enhances the flow stress, and results in strain hardening or dislocation strengthening. Asymmetric rolling and FSP lead to pronounced grain refinement, increase the number of grain boundaries, increase the resistance of grain boundary to dislocation motion, and result in Hall-Petch strengthening or grain boundary strengthening. Finally, the second phases, such as AlCooling modes of FSP have a certain influence on mechanical properties. Different from available reports on the pronounced effect of liquid nitrogen FSP on WE54 Mg alloy ,44, the −3 s−1 in this alloy. Secondly, the phase proportion of α-Mg to β-Li in B = D/kT, where B is the atomic mobility, D is the diffusion coefficient, k is Boltzmann’s constant, and T is the absolute temperature, the migration velocity of grain coarsening is directly proportional to the atomic mobility. Thus, the grain coarsening velocity is directly proportional to the diffusion coefficient. This indicates the accelerated atomic diffusion at 573 K, which is the atomic diffusion cause of grain coarsening at 573 K in this alloy. The microstructures, such as grain size and shape, phase proportion, solutes, and grain coarsening, have a direct influence on the ductility at elevated temperatures. Firstly, the grains are equiaxed and fine below 10 μm. This fine-grained microstructure provides a prerequisite for inducing more than 400% elongation. Also, fine and equiaxed grains contain a large number of high-angle grain boundaries, which facilitate grain boundary sliding. Microscopic grain boundary sliding gives large macroscopic strain. Thus, the maximum elongation of 416% is demonstrated at 573 K and 1.67 × 10−4 s−1 and the peculiar softening curve at 573 K and 1.67 × 10−2 s−1 are analyzed. The peculiar hardening curve at 523 K and 1.67 × 10−4 s−1 is because at a lower strain rate, dynamic grain coarsening occurs, dislocation density increases, and flow stress increases. The peculiar softening curve at 573 K and 1.67 × 10−2 s−1 is because at 573 K and higher strain rate, thermal activation accelerates, atomic diffusion enhances, and stress is relaxed. As a result, the flow stress decreases.As shown in 2Y, Al4Sr, MgLi2Al, and AlLi intermetallic compounds and grain coarsening at 573 and 623 K increase the difficulty of alloy deformation and increase the resistance to the dislocation activity as an accommodation mechanism of boundary sliding. Thus, the dominant deformation mechanism in this alloy at elevated temperatures is grain boundary and interphase boundary sliding controlled by lattice diffusion. As shown in (1)2Y, Al4Sr, MgLi2Al, and AlLi intermetallic compounds.A new fine-grained LASY9400 alloy has been fabricated by asymmetric rolling and friction stir processing. Grain refinement with an average grain size in the α-Mg phase of 1.65 μm and an average grain size in the β-Li phase of 4.24 μm has been achieved in the water-cooled FSP alloy. For room-temperature performance, the ultimate tensile strength of 208 ± 4 MPa, yield strength of 193 ± 2 MPa, and elongation of 48.2% were obtained in the water-cooled FSP alloy. XRD and EDS analyses revealed that the present alloy consists of α-Mg and β-Li phases, and Al(2)−3 s−1.For high-temperature performance, the maximum superplasticity or ductility of 416% was demonstrated in this fine-grained alloy with an average grain size of 10 μm at 573 K and 1.67 × 10(3)A power-law constitutive equation was established. The stress exponent was 2.29 (≈2) (strain rate sensitivity 0.44), and the deformation activation energy was 162.02 kJ/mol. This evidence confirmed that the dominant deformation mechanism at elevated temperatures is grain boundary and interphase boundary sliding controlled by lattice diffusion.(4)Statement of novelty and outstanding achievements: In this report, a novel fine-grained LASY9400 alloy has been fabricated by our innovative asymmetric rolling and friction stir processing technique. Reasonable room-temperature properties and high-temperature superplasticity were obtained. The effect of cooling media on FSP microstructure and properties is rarely reported in the Mg-Li alloy system. The diffusion mechanism of grain coarsening has been quantified and elucidated. The striking achievement is to use our formulae of diffusivity and activation energy established in a simple system binary Mg-Li alloy to this complex multicomponent alloy. Another striking achievement is that the dislocation climb is revealed via a normalized plot in this multicomponent alloy at elevated temperatures. The processing, properties, and established constitutive equation can be applied to the engineering process and superplastic forming control process in the manufacturing field.


Gastric cancer remains a global health threat, particularly in Asian countries. Current treatment methods include surgery, chemotherapy, and radiation therapy. However, they all have limitations, such as adverse side effects, tumor resistance, and patient tolerance. Hyperthermia therapy uses heat to selectively target and destroy cancer cells, but it has limited efficacy when used alone. Linderae Radix (LR), a natural compound with thermogenic effects, has the potential to enhance the therapeutic efficacy of hyperthermia treatment. In this study, we investigated the synergistic anticancer effects of cotreatment with LR and 43 °C hyperthermia in AGS gastric cancer cells. The cotreatment inhibited AGS cell proliferation, induced apoptosis, caused cell cycle arrest, suppressed heat-induced heat shock responses, increased reactive oxygen species (ROS) generation, and promoted mitogen-activated protein kinase phosphorylation. N-acetylcysteine pretreatment abolished the apoptotic effect of LR and hyperthermia cotreatment, indicating the crucial role of ROS in mediating the observed anticancer effects. These findings highlight the potential of LR as an adjuvant to hyperthermia therapy for gastric cancer. Further research is needed to validate these findings in vivo, explore the underlying molecular pathways, and optimize treatment protocols for the development of novel and effective therapeutic strategies for patients with gastric cancer. Gastric cancer poses a formidable global health challenge, especially in Asian countries, where its incidence and mortality rates are strikingly high ,2,3. TraCancer hyperthermia therapy, a technique that utilizes heat to selectively target and eliminate cancer cells, has emerged as a potential solution to overcome the limitations of current approaches of gastric cancer treatment ,7,8,9. HLinderae Radix (LR), a natural herb steeped in traditional medicinal history, is renowned for its thermogenic properties and has been harnessed to treat various gastrointestinal ailments, such as indigestion, abdominal discomfort, and bloating ,14,15. RBased on this hypothesis, our study was designed to investigate the synergistic effects of LR and hyperthermia treatment. We aimed to explore the possibility of boosting the anticancer capabilities of both treatment modalities while minimizing the activation of heat shock responses that could promote cell survival and tumor resistance. Furthermore, we sought to elucidate the molecular mechanisms underlying these observed effects to understand the potential benefits of this treatment.Lindera strichnifolia  was ground and homogenized using a homogenizer. After soaking for 24 h at room temperature in 70% EtOH, the extract was produced. The resulting extract was filtered (pore size: 5 μm), concentrated under reduced pressure, and lyophilized to obtain a sample. Samples at concentrations of 80, 100, and 120 μg/mL were prepared using dimethyl sulfoxide (DMSO) . The solutions were stored at 4 °C until further use.Chromatographic analysis was performed using ultraperformance liquid chromatography-electrospray ionization/quadrupole time-of-flight high-definition mass spectrometry/mass spectrometry (UPLC-ESI-QTOF-MS/MS) to identify the chemical components of the ethanolic extract. The extract was shaken in 50% methanol using a vortex mixer for 30 s and sonicated for 10 min. The supernatants were filtered through a 0.2 μm hydrophilic polytetrafluoroethylene syringe filter . The filtrate was diluted to 20 mg/mL and transferred to an LC vial prior to use. The LC–MS system consisted of a Thermo Scientific Vanquish UHPLC system  with an ACQUTY UPLC HSS T3 column  and a Triple TOF5600+ mass spectrometer system .The QTOF MS system was equipped with an electrospray ionization (ESI) source in positive and negative ion modes and used to complete the high-resolution experiment. The elution program for UHPLC separation, which employed 0.1% formic acid in water as eluent A and 0.1% formic acid in acetonitrile as eluent B, was as follows: 0–1 min, 5% B; 1–4 min, 5–15% B; 4–1 1 min, 15–35% B; 11–17 min, 35–50% B; 17–21 min, 50% B; 21–25 min, 50–100% B; and 25–29 min, 100% B and equilibration with 5% B for 4 min at a flow rate of 0.4 mL/min. The column temperature was 40 °C, and the autosampler was maintained at 4 °C. The injection volume of each sample solution was 2 μL. Data acquisition and processing for qualitative analysis were conducted using Analyst TF 1.7, PeakVeiw2.2, and MasterView . The MS/MS data for qualitative analysis were processed using PeakView and MasterView to screen for probable metabolites based on accurate mass and isotope distributions.2 .The AGS stomach cancer cell line was provided by the Korean Cell Line Bank . The cells were cultured in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum  and 1% penicillin–streptomycin  in an incubator at 37 °C with humidified air containing 5% COAGS cells were seeded in 6-well plates (0.3 × 10⁶ cells) and suspended in 3 mL of medium. The temperature environment was applied by incubating the cells for 30 min in a water bath set to certain temperatures (37 °C or 43 °C). LR was treated to AGS cells an hour prior to temperature control.2 and then soaked in a water bath with a temperature controlled at 37 °C or 43 °C for 30 min. After 48 h, each well received 20 μL of MTT (2 mg/mL in PBS)  and then was incubated for an additional 2 h. After removing the growth media, the cells were lysed in 100 μL of DMSO. Absorbance was measured at 570 nm using an automated spectrophotometric plate reader. The relative cell viability percentages were normalized to those of untreated controls. The synergistic effects of the medication and HT combination were calculated using Compusyn software (ver.1.0).The MTT assay was used to assess cell proliferation after temperature exposure to LR. AGS cells (1 × 10⁴ cells/100 μL) were seeded in 96-well plates and allowed to attach overnight. Each group contained three wells, with the untreated group acting as a control. LR was added to the plates in three concentrations  after the cells had been fixed. The plates were incubated for an hour at 37 °C in a humid atmosphere with 5% COThe vitality of the cells was assessed using a hemocytometer after trypan blue  staining . In brief, AGS cells (0.3 × 10⁶) were sown in 6-well plates and then treated to 1 h of LR therapy and heat (30 min). After 24 h of post-treatment incubation, the cells were collected, diluted 1:4 with PBS, stained, and then counted.Cell proliferation was measured using a morphological assay. AGS cells were seeded in a 6-well plate at a density of 0.3 × 10⁶ cells per well. The cells were treated with 120 μg/mL LR for 1 h after adhering to the plates and then incubated at 37 °C or 43 °C for 30 min. The cells were examined and photographed under a microscope  after 24 h.Cells were plated in a 6-well plate at a density of 0.3 × 10⁶ cells per well and then kept at 37 °C. Once the cells reached confluence, a thin scratch was created in each well using a yellow pipette tip. Images were captured at 0 h using a microscope  (0 h). After 24 h, the cells were rinsed with PBS and photographed under a light microscope (24 h).A total of 400 cells were seeded into each well of a 6-well plate and then incubated overnight. The cells were incubated for 30 min at 37 °C or 43 °C and then treated with 120 μg/mL LR for an hour. After a week, the cells were stained with crystal violet solution  for 10 min at room temperature and then washed with PBS. Colonies were examined under a microscope .Protein was extracted from AGS cells after indicated treatments. After calculating protein concentration, equal amounts of the SDS-PAGE-separated lysates were transferred onto polyvinylidene difluoride (PVDF) membranes, which were subsequently blocked at room temperature with 1 × TBS containing 0.1% Tween 20 and 5% skim milk. The membranes were incubated at 4 °C overnight with the following primary antibodies: anti-caspase-3, anti-caspase-8, anti-caspase-9, anti-survivin, anti-HSP27, anti-HSP70, anti-HSP90, anti-p-ERK (Thr202/Tyr204), anti-ERK, anti-p-p38 (Thr180/Tyr182), anti-p38, anti-p-JNK (Thr183/Tyr185), anti-JNK , anti-β-actin, anti- Bcl-xL, anti-Bcl-2, anti-cyclin B1, anti-cyclin D1, anti-MMP9, anti-MMP2, anti-VEGF , anti-HSF1, anti-pHSF1 , and anti-cleaved caspase . The membranes were washed three times before exposure to diluted anti-rabbit or anti-mouse IgG secondary antibodies  for an hour at room temperature. The blots were washed thrice with 1 × TBS-T buffer for 10 min between each stage. The membranes were identified using enhanced chemiluminescence .Apoptosis was examined by flow cytometry using the annexin V-FITC detection kit . Briefly, AGS cells (0.3 × 10⁶ cells/well in a 6-well plate) were subjected to LR and HT for 24 h. The cells were collected and stained with annexin V-FITC in 1× cold binding buffer under a light-blocking cover for 15 min at room temperature. Propidium iodide (PI) staining was performed using 1× cold binding buffer after removing the supernatant.AGS cells (0.3 × 10⁶ cells/well in 6-well plates) were cotreated for 24 h. To measure the cell cycle phase, cells were collected, frozen in 70% ice-cold EtOH for 24 h incubation, washed in 1× cold PBS, and then resuspended in PBS supplemented with 1 mg/mL PI and 10 mg/mL RNase A in a dark environment for 10 min. The cell cycle was analyzed using a flow cytometer.TM D399). A 6-well plate containing AGS cells (0.3 × 10⁶ cells per well) was subjected to cotreatment for 4 h. After collecting the cells, 10 μM reagent was added and then incubated at 37 °C for 40 min without light for reaction. ROS levels were measured using flow cytometry.A ROS experiment was carried out using 2′,7′-dichlorofluorescin diacetate , boldine (5), aesculitannin B (6), norboldine (10), linderalactone (12), lindenanolide E (15), and lindenanolide 16). These compounds are reported as the major components of LR  [16,17,1. These cThe effect of simultaneous treatment with LR and hyperthermia at 37 °C and 43 °C was investigated using MTT assays. When LR was administered at the same dose (120 µg/mL), the combination of LR and 43 °C significantly reduced AGS cell viability to a greater extent than the combination of LR and 37 °C A. The deMorphological observations revealed that the cotreatment led to distinct changes in cell shape. These alterations result from the inhibitory effect of the cotreatment on cell growth D. The moTo elucidate the mechanisms underlying the synergistic effects of LR and hyperthermia, we investigated the expression levels of various factors associated with apoptosis, cell proliferation, metastasis, and angiogenesis. Our results show that treatment with LR at 43 °C dose-dependently increased the expression of activated forms of caspase 3, a key marker of programmed cell death ,20, wherThe rate of annexin V-related apoptosis in AGS cells was higher after cotreatment with LR and hyperthermia than after treatment with 43 °C hyperthermia alone or cotreatment with LR and normothermia A. A doseHSPs are molecular chaperones that maintain protein stability, facilitate transport, and transmit signals within cells ,27,28. HHSPs regulate the intracellular levels of reactive ROS; thus, a decreased expression of heat shock proteins can increase ROS levels in cancer cells . AccordiGastric cancer remains a significant global health threat, particularly in Asian countries, where the prevalence and mortality rates are alarmingly high ,37. VariOne approach to overcome these limitations is the use of hyperthermia therapy . Cancer LR is a natural herb used in traditional medicine owing to its thermogenic effects; it has been used in the treatment of various gastrointestinal disorders, such as indigestion, abdominal pain, and bloating ,45,46,47First, we found through MTT assays, morphological observations, crystal violet staining, and cell migration assays that cotreatment with LR and 43 °C hyperthermia synergistically inhibited AGS cell proliferation. These results suggest that the combination of LR and hyperthermia has a potent anticancer effect against AGS cells .The mechanisms underlying the synergistic effect induced by cotreatment with LR and hyperthermia were investigated by examining the expression of various factors associated with apoptosis, proliferation, metastasis, and angiogenesis. Apoptosis is the main mechanism of programmed cell death ,49 and iCells can also be exposed to various environmental stressors, such as cellular development or disease, temperature change, or mechanical stress . Cells fROS, a group of highly bioactive molecules, act as key signaling regulators in several cellular functions ,55. AlthOur study demonstrates that LR enhances the sensitivity of gastric cancer cells to hyperthermia treatment. This effect can be attributed to the following bioactive compounds in LR: norisoboldine (O3), boldine (O5), aesculitannin B (O6), norboldine (O10), linderalactone (O12), lindenanolide E (O15), and lindenanolide (O16). These compounds exhibit various biological activities that contribute to their synergistic effects. Norisoboldine (O3) and boldine (O5) exert anticancer activities by modulating the expression of genes related to cell cycle progression, apoptosis, and metastasis ,66,67,68Despite these promising results, this study has several limitations, mainly due to its in vitro nature. Further research is required to confirm these findings in animal models and human clinical trials. A number of attempts have been made to demonstrate the effect of hyperthermia in vivo. One of the most easily integrated and convenient methods involves using magnetic nanoparticles . HoweverIn conclusion, our study provides evidence that cotreatment with LR and hyperthermia exerts a synergistic anticancer effect on AGS cells by inducing apoptosis, promoting cell cycle arrest, and suppressing heat-induced heat shock responses. Our research endeavors to lay the groundwork for the development of groundbreaking and more efficient therapeutic strategies for patients with gastric cancer, ultimately enhancing their quality of life and survival rates. Future investigations should focus on validating these results in vivo, unraveling the molecular pathways underlying the observed effects, and refining treatment protocols.