Sadala Simon, 1880 collected in Lowland Evergreen rainforests at the Tiputini Biodiversity Station, Yasuni Biosphere Reserve, Amazonian Ecuador. This new species corresponds to the first record of the genus from Ecuador. Females of the new species of Sadala are similar to S. punicea and S. nanay, by having the epigyne with a median septum diamond‐shaped posteriorly. The new species is easily distinguished from S. punicea and S. nanay by having relatively straight anterior lateral margins of the median septum. This study increases to 10 the number of described species of Sadala.We describe a new species of giant crab spider of the genus  Sadala is described from Ecuador. The new species was discovered at the Tiputini Biodiversity Station, a research station in the Yasuni Biosphere Reserve, Amazonian lowlands of Ecuador.The first species of spider of the genus  TBS is a research station founded in 1994 by Universidad San Francisco de Quito USFQ on a 744‐ha tract of undisturbed Lowland Evergreen Forest on the northern bank of the Tiputini, within the Yasuni Biosphere Reserve, one of the most biodiverse regions in the world , a citizen science platform by the California Academy of Science and National Geographic. Data search and extraction were conducted in July 2022. For each occurrence point, we compiled geographic data and all other associated information, and localities were reviewed and validated individually, following protocols described by Cisneros‐Heredia and Peñaherrera‐Romero , provincia de Orellana, República del Ecuador, 30 May 2022, P. Peñaherrera‐R., R. J. León‐E., A. Guerrero‐Campoverde, D. F. Cisneros‐Heredia leg.Paratypes. ZSFQ‐i8259, same data as holotype but collected at Matapalo trail on 29 May 2022; ZSFQ‐i8260 at Guacamayo trail on 02 July 2022.Etymology. The specific name is a patronym for Victor Raul Peñaherrera de la Cadena, grandfather of Pedro Peñaherrera‐R., who in life had the project of narrating the history of Ecuador in a book that could not be completed and now will form part of the natural history of Ecuador immortalized in this species.Generic placement. The new species is assigned to the genus Sadala by having chelicerae with three promarginal teeth and intermarginal denticles, 5–10 escort setae at the base of fang, short‐toothed female palpal claw, three pairs of spines on ventral tibiae I–II, and median septum of epigyne with triangular scape‐like projection . Also, S. rauli differs from S. punicea by having a MS more than 1.5 times longer than wide with elliptical CO. Figures  and 2. TDescription of holotype. Female holotype: Total length 21.62. Cephalothorax: 7.44 long, 7.36 wide. Abdomen: 11.75 long, 7.94 wide. Eyes diameters: 0.47, 0.37, 0.31, 0.35; interdistance: 0.43, 0.45, 0.80, 0.86, 0.47, 0.56. Legs: I (8.70/3.44/7.51/8.33/2.17); II (9.49/3.35/8.87/8.70/2.13); III (7.24/3.08/5.82/5.39/1.83); IV (8.24/2.90/6.39/6.96/2.14). Spination follows the generic pattern except for patellae I–II: p0‐1‐0, v0, d0, r0‐1‐0; III: p0‐2‐0, v0, d0, r0. Epigyne: EF as long as wide; MAB embedded (cleared during soft tissue digestion); TP wider than long; LL closer to each other posteriorly , while five others are known from nearby localities in the department of Iquitos, Peru, and the state of Amazonas, Brazil  ; data curation ; formal analysis ; investigation ; methodology (lead); project administration (supporting); software ; validation ; visualization ; writing – original draft ; writing – review and editing . Diego F. Cisneros‐Heredia: Conceptualization ; data curation ; formal analysis ; funding acquisition (lead); investigation ; methodology (supporting); project administration (lead); resources (lead); software ; supervision (lead); validation ; visualization ; writing – original draft ; writing – review and editing .Universidad San Francisco de Quito USFQ and Tiputini Biodiversity Station supported this work through operative funds assigned to the Institute of Tropical Biodiversity IBIOTROP and research and outreach funds assigned to Diego F. Cisneros‐Heredia.None.


Malignant pleural mesothelioma (MPM) is an aggressive cancer of the cells lining the pleural cavity with a low overall incidence. The National Cancer Database (NCDB) released in August 2022 updated data that reflect the newest trends in MPM.t‐test and independent‐samples proportions test were used for means analysis. Survival was assessed by the Kaplan–Meier method using SPSS version 28.The NCDB was queried for patients diagnosed with MPM between 2004 and 2020. Variables collected included demographics, tumor characteristics, and treatment. Student's p < 0.001). Since 2010, there has been a significant increase in patients offered treatment with 73.9% receiving some therapy (p < 0.01): 50.5% received chemotherapy, 27.6% surgery, 8.6% radiation, and 5.4% immunotherapy. The median overall survival was 10.3 months from diagnosis [95% CI: 10.2–10.5]. Risk factors associated with 30‐day mortality from surgical intervention included age , male gender , poorly differentiated grade , Stage 4 , and epithelioid histology .A total of 41,074 patients were diagnosed with mesothelioma, with a steady incidence (0.25%) between 2004 and 2017. The mean age of diagnosis was 70 (SD 13). 73.2% of the patients were males, 69% had no comorbidities, and 93.3% were white. More patients were diagnosed at Stage 1 after 2008  database proved the most updated trends in the prevalence, management, and diagnosis of malignant mesothelioma. Due to the small numbers of patients at most academic centers, the NCDB provides a robust dataset to draw upon broad data points in treatment discussions with patients. If left untreated, median survival is 4–12 months following diagnosis.Mesothelioma has three major subtypes: epithelioid, sarcomatoid, and biphasic. Surgery is the standard of care for resectable epithelioid MPM and has improved survival with a 5‐year survival of 10%–15%. Patients with sarcomatoid and biphasic histologies may not benefit from surgery.The aim of our study was to understand the most recent trends over the last two decades in incidence, histology, treatment, prognostic factors, and major differences in survival between patients with mesothelioma in the United States. Given the low incidence of this disease and insufficient data from any single academic center, we elected to utilize the NCDB in order to generate sufficient data for statistical significance. Identifying predictors of survival and factors associated with increased risk can help providers and patients in choosing their care options. This study is the first that uses the latest available data of the recently released NCDB public user files (PUF) 2020.22.1Established in 1989, the NCDB is a nationwide, facility‐based, comprehensive clinical surveillance resource oncology database that currently captures 72% of all newly diagnosed malignancies in the United States annually.Patients included in our study were those with a history of pleural mesothelioma diagnosed between 2004 and 2020. All patients were 18 years or older and underwent treatment in a center designated by the Commission on Cancer. Patients with missing data were excluded from this study. Since this was an analysis of publicly available data, a waiver was granted for institutional review board revisions.2.2The variables collected included patient demographics such as age at diagnosis, sex, race, insurance status, annual income, educational level, location in an urban versus rural setting, type of facility, and the Charlson–Deyo (CD) score. Other variables were related to tumor staging and treatment. They included histology, grade, type of treatment and staging defined according to the 6th Edition (2004–2009), 7th Edition Staging criteria (2010–2017), and 8th Edition Staging criteria (2018+). The outcomes that were analyzed included 30‐ and 90‐day mortality, and last contact or death months from diagnosis.Two‐, 5‐year, and OS was assessed by the Kaplan–Meier method and life. An event was defined as patient death and patients were censored as to have survived if they were alive at the end of the studied interval. Each treatment method was sub‐analyzed by stratifying into NCDB analytic stage group and tumor histology. The log‐rank test was used for comparisons between groups. Two‐ and 5‐year follow‐up intervals from diagnosis were used to compare median survival between subgroups.p < 0.2 on bivariate analysis were included in the final mode. Backward elimination was used to retain variables with a p < 0.05 significance value. The final model is reported as odds ratio (OR), 95% confidence interval, and p value.The log‐rank test was used for identifying predictors for 30‐, 90‐day, and OS. Clinically relevant variables and those who had a 33.1Over the 17 years evaluated, 41,074 patients were diagnosed with mesothelioma, with an average yearly 2417 new cases. The absolute number of patients newly diagnosed with mesothelioma is slightly increasing as seen in Figure p = 0.3). The ages of patients ranged from 18 to 90. Most patients were between 71 and 80 . 73.2% of the patients diagnosed were males compared to 26.8% females and that distribution was constant across the years. Most of the patients had no comorbidities, were White, and treated at a comprehensive community cancer program or academic/research program. Although most patients did not receive palliative care, the percentage of patients who were referred to palliative care increased from 8.8% in 2004 to 13.4% in 2020 (p < 0.001)  and median 73. It did not significantly change over the past 17 years   Figure . There w) Figure .p < 0.001)  with 53.5% of tumors without a histologic subtype specified. Prior to 2010, 98% of the data did not specify the histologic subtype compared to only 26% after 2010  Figure . Histolo3.3p < 0.01). The treatment modalities were surgical, chemotherapy, radiotherapy, and immunotherapy.Since 2010, 73.9% of patients received treatment . The mean number of days patients stayed in the hospital post‐operation was 7.8 days (SD 10), with no statistically significant difference between 2004 and 2020 (p = 0.8).Thirty percent were offered surgery and 27.6% of patients underwent surgery. There were no statistically significant differences between the proportion of patients offered surgery in 2004 and 2020 (3.3.2p < 0.001). Radiation was administered with a mean of 113 (SD 113) days after diagnosis. It was administered for a mean of 60 days (SD 233).8.6% of patients underwent radiation. There was a slight but significant increase in the proportion of patients who underwent radiation in 2020 compared to 2004 from 20% to 21% (3.3.3p = 0.002). There was also a statistically significant increase in the use of multimodal therapy from 30% in 2004 to 42% in 2020 (p < 0.001). On average, it was administered 55 (SD 48) days from diagnosis.50.5% of patients received chemotherapy. Among those, 33.1% received the treatment perioperatively and the rest as definitive chemotherapy. There was a statistically significant increase in chemotherapy utilization from 56.8% in 2004 to 61.5% in 2020 (p < 0.001).Only 5.4% received immunotherapy. There has been a significant increase in its utilization from 2004 to 2020 from 0.6% to 27% .Patients who had undergone surgery had a median survival of 19.8 months [95 CI: 19.2–20.3] compared to 7.9 months [95% CI: 7.8–8.1] in those who had not undergone surgery. (p < 0.001). The 5‐year survival was 5% among those who did not undergo surgery compared to 16% among those who did (p < 0.001)   Table .4.34.3.1p < 0.001).Those who underwent chemotherapy had a median survival of 14 months [95% CI: 13.8–14.3] compared to 6.01 months [95% CI: 5.8–6.2] among those who did not (p < 0.001). When comparing stages among those who had received chemotherapy, patients with Stage 2 (p = 0.4) and Stage 3 (p = 0.2) had comparable 2‐ and 5‐ year survivals to Stage 1. However, those who were Stage 4 had a worse survival compared to all other stages (p < 0.001)  .Those who received immunotherapy had a median survival of 15.2 months [95% CI: 14.4–16] compared to 10.1 months [95% CI: 9.9–10.3] in those who had not received immunotherapy  but both were superior to sarcomatoid histology (p < 0.001). When comparing stages among those who had received surgery, patients with Stage 2 (p = 0.44) and Stage 3 (p = 0.07) had comparable 2‐ and 5‐year survivals to Stage 1. However, those who were Stage 4 had a worse survival compared to all other stages (p < 0.001) .Those who received radiotherapy had a median survival of 12.2 months [95% CI: 11.5–12.8], which was superior to those who had not received radiotherapy with a median survival of 10.2 months [95% CI: 10–10.4] (p < 0.001). When comparing stages among those who had received surgery, patients with Stage II (p = 0.2) and Stage III (p = 0.1) had comparable 2‐ and 5‐year percentage survivals to Stage I. However, those who were Stage IV had a worse percentage survival compared to all other stages (p < 0.001)  Table .4.5p < 0.001), being female , having a CD score of 1 , receiving care at a community cancer network , well‐differentiated grade , Stage 1 disease , epithelioid histology , chemotherapy , radiation , and immunotherapy .Thirty‐day mortality correlated on a univariate level with mean age at diagnosis , being male , poorly differentiated grade , Stage 4 , epithelioid histology , treatment with chemotherapy , and radiation treatment  all correlated with 30‐day survival.On the multivariate level, age at diagnosis , being male , CD score of 1 , receiving care at a community cancer center and integrated cancer network program , well‐differentiated grade , Stage 1 disease , the presence of malignant pleural effusion , chemotherapy , radiation , and immunotherapy .On the univariate level, 90‐day mortality correlated with mean age at diagnosis , being male , undifferentiated tumor grade , Stage 1 , the presence of a pleural effusion , tumor histology as epithelioid , having undergone radiation therapy , having undergone chemotherapy , and having undergone radiation therapy .On the multivariate level, age at diagnosis . Histologically, surgery is recommended only for the epithelioid subtype but may be considered for biphasic histology if the patient has early‐stage disease (“low volume disease”). This was reflected in our study with the significant increased survival of those with epithelioid subtypes who underwent surgery compared to those with other histologies. In our study, there were no significant differences between the proportion of patients offered surgery between 2004 and 2020. The surgical procedures offered are extra‐pleural pneumonectomy, extended pleurectomy decortication, pleurectomy decortication, and partial pleurectomy. Pleurectomy decortication and partial pleurectomy are debulking procedures which improve quality of life but do not necessarily prolong survival.In our study, OS for patients who underwent surgery alone was 19.8 months [95% CI: 19.5–20.3], which was superior to those who did not undergo surgery 7.9 months [95% CI: 7.8–8.1]  to determine the utility of immunotherapy. Over the past 10 years, there was an increase in patients who were offered treatment and a steady increase in survival. Traditionally, patient with a non‐epithelioid MPM have a worse prognosis and tend to be chemo‐resistant. Several studies suggest that the advent of immunotherapy can help address this subgroup as they have an increased expression of PDL‐1, the target of pembrolizumab.This is a retrospective study, and it is hard to fully characterize the selection bias in the subgroups compared. Furthermore, the NCDB allows for analysis of rare tumors because of the large network it encompasses on a national level. It is heavily reliant on accurate reporting from hospital populations, which is not generalizable and representative of the entire population. It also does not break down surgery to the actual procedure performed for the treatment of MPM but rather into biopsy, debulking resection, en bloc resection, and biopsy. As such, granular analysis of the operations performed and a comparative study cannot be done.In summary, the mainstay of management of MPM is through a multidisciplinary team and expert consensus based on stage and histologic subtype. Stage 1–3a and epithelioid histology are recommended surgical exploration with neoadjuvant or adjuvant chemotherapy. Otherwise, if the tumor is Stage 3 or 4 or unresectable, systemic therapy and/or supportive care are the current recommendation.Patrick Bou‐Samra: Writing‐original draft, conceptualization, methodology, formal analysis, data curation, project administration Austin Chang: conceptualization, methodology, data analysis, reviewing, editing Feredun Azari: conceptualization, project administration, reviewing, editing Gregory Kennedy: conceptualization, reviewing, editing Alix Segil: data curation, reviewing, editing Emily Guo: data curation, reviewing, editing Melina Marmarelis: conceptualization, reviewing, editing, supervision Corey Langer: methodology, reviewing, editing, supervision Sunil Singhal: conceptualization, data curation, original draft preparation, reviewing, editing, supervisionThere are no conflicts of interest or financial disclosures for any of the authors listed above.


Blumea balsamifera (L.) DC., a perennial herb in the Asteraceae family native to China and Southeast Asia, has a notable history of medicinal use due to its pharmacological properties. Using UPLC–Q–Orbitrap HRMS techniques, we systematically investigated the chemical constituents of this plant. A total of 31 constituents were identified, of which 14 were flavonoid compounds. Significantly, 18 of these compounds were identified in B. balsamifera for the first time. Furthermore, the mass spectrometry fragmentation patterns of significant chemical constituents identified in B. balsamifera were analyzed, providing important insights into their structural characteristics. The in vitro antioxidative potential of the methanol extract of B. balsamifera was assessed using DPPH and ABTS free-radical-scavenging assays, total antioxidative capacity, and reducing power. The antioxidative activity exhibited a direct correlation with the mass concentration of the extract, with IC50 values of 105.1 ± 0.503 μg/mL and 12.49 ± 0.341 μg/mL for DPPH and ABTS, respectively. For total antioxidant capacity, the absorbance was 0.454 ± 0.009 at 400 μg/mL. In addition, the reducing power was 1.099 ± 0.03 at 2000 μg/mL. This study affirms that UPLC–Q–Orbitrap HRMS can effectively discern the chemical constituents in B. balsamifera, primarily its flavonoid compounds, and substantiates its antioxidative properties. This underscores its potential utility as a natural antioxidant in the food, pharmaceutical, and cosmetics sectors. This research provides a valuable theoretical basis and reference value for the comprehensive development and utilization of B. balsamifera and expands our understanding of this medicinally valuable plant. Oxidative stress, a pathological condition induced by an imbalance between reactive oxygen species’ (ROS) production and the biological system’s ability to detoxify these reactive intermediates, contributes to several chronic diseases such as cancer, diabetes, cardiovascular diseases, neurodegenerative disorders, and aging. Antioxidants have the potential to quell this imbalance by neutralizing ROS, thereby preventing or delaying cellular damage . Plant–dBlumea balsamifera (L.) DC., a herbaceous plant from the Asteraceae family, is extensively distributed in tropical and subtropical regions, including countries such as China, Thailand, Vietnam, the Philippines, and Malaysia −, with its molecular formula deduced as C27H30O16 using Xcalibar 4.0 software. Upon induced collision dissociation, it loses to form three kinds of fragment ions. Firstly, it loses a sugar moiety C12H21O9 to form a fragment m/z: 300.0281. Secondly, it undergoes a cleavage from the sugar moiety, losing C10H18O8 to form a fragment m/z: 343.0423. Thirdly, it loses a sugar moiety C12H20O9 to form a fragment ion m/z: 301.0359, which, through five types of cleavage pathways, yields different fragments. These pathways include: losing a molecule C7H3O2 at the C–ring’s C–2 site to produce fragment m/z: 178.9989; losing a water molecule on the A–ring to yield fragment m/z: 283.0242, which continues to cleave a molecule of CO from the C–ring to yield fragment m/z: 255.0304 and, through rearrangement, further loses a molecule of CO from the C–ring to yield fragment m/z: 227.0358; undergoing a RDA cleavage on the C–ring, losing C8H6O3, to yield fragment m/z: 151.0037; losing a molecule of CH2O on the C–ring to yield fragment m/z: 271.0254, which continues to lose a molecule of CO on the C–ring to yield fragment m/z: 243.0307; and cleaving a CO molecule at the carbonyl on the C–ring to yield fragment m/z: 273.0405, which continues to cleave through three pathways, losing C7H4O4, CO2, and C8H5O4, respectively, to yield fragments m/z: 121.0300, m/z: 229.9944, and m/z: 108.0217. By comparison with the corresponding literature references −. Using Xcalibar 4.0 software, the molecular formula is deduced to be C21H22O10. Upon collision-induced dissociation, it loses C3H6O3 and C4H8O4 to yield secondary fragment ions m/z: 343.0833 and m/z: 313.0727, which are characteristic fragment ions of hexose flavonoid glycosides. The m/z: 313.0727 ion loses CO from the sugar moiety residue to yield fragment ion m/z: 285.0797, with four cleavage pathways. Firstly, it loses a methyl group from the C–ring to form fragment m/z: 271.0619, which continues to cleave in three pathways: losing the B–ring to yield fragment m/z: 177.0199, which further loses a carbonyl group from the A–ring to yield fragment m/z: 149.0246; cleaving C3O2 from the A–ring to yield fragment m/z: 203.0870; and losing C2H2O from the C–ring to yield fragment m/z: 229.0054, which further loses a molecule of CO from the B–ring to yield fragment m/z: 201.0718. Secondly, it loses the A–ring and C–ring from the connection between the A and B rings to yield fragment m/z: 93.0347. Thirdly, a RDA cleavage occurs on the C–ring, cleaving C8H8O from C–1 and C–3 to yield fragment m/z: 165.0196, which continues to lose a methyl group from the A–ring to yield fragment m/z: 151.0039. Fourthly, cleaving C8H6O4 from the C–ring yields fragment m/z: 119.0506. The pseudomolecular ion peak m/z: 433.1154 loses a water molecule from the B–ring to form fragment m/z: 415.1044. By comparison with the corresponding literature references − in the negative ion mode. The molecular formula C16H18O9 was discerned utilizing the Xcalibur 4.0 software. Upon collision-induced dissociation, the molecule generated a variety of fragment ions, as described below: fragmentation through ester bond breakage resulted in a fragment at m/z: 191.0561, which, upon further loss of a water molecule, yielded a fragment at m/z: 173.0456. Another fragmentation process involving ester bond cleavage produced a fragment at m/z: 179.0353, which, after the loss of a water molecule, formed a fragment at m/z: 161.0243. Cleavage to the left of the carbonyl group, resulting in the loss of C7H12O6, generated a fragment at m/z: 161.0243. The loss of C8H12O7 led to the formation of a fragment at m/z: 133.0295. Cleavage at the alkyl group of the phenyl ring, which resulted in the loss of C10H12O7, generated a fragment at m/z: 109.0308. Lastly, cleavage to the left of the carbon–carbon double bond, accompanied by the loss of C8H10O7, produced a fragment at m/z: 135.0454. Drawing upon mass spectral data analysis, in comparison with the relevant literature references + in the positive ion mode. Its molecular formula, C27H28N2O4, was determined utilizing Xcalibur 4.0 software. Following collision-induced dissociation, it generated five distinct types of fragment ions as described below: the loss of C16H12NO2 yielded a fragment at m/z: 194.1179, which further underwent the loss of C2H4O2, resulting in a fragment at m/z: 134.0968. The loss of C12H14NO3 led to the formation of a fragment at m/z: 224.1074. The loss of C11H14NO2 produced a fragment at m/z: 252.1023. This fragment, upon further loss of C9H9NO, generated a fragment ion at m/z: 105.033, which was the most stable fragment ion peak under these conditions. The loss of C24H18NO2 resulted in a fragment at m/z: 88.5351. The loss of C16H14N2O2 yielded a fragment at m/z: 177.0927, which further lost C2O2 and C2H3O to yield fragments at m/z: 117.0702 and m/z: 134.0968, respectively. The loss of C16H14NO2 and C2H3O2 led to the formation of fragments at m/z: 194.1179 and m/z: 134.0968, respectively. Based on mass spectral data analysis and comparison with the related literature reference −. Its molecular formula, C19H18O3, was ascertained using the Xcalibur 4.0 software. Subsequent to collision-induced dissociation, it gave rise to three distinct types of fragment ions, as detailed below: the loss of a molecule of H2O resulted in a fragment at m/z: 277.2161. Further losses of CO and C3H6 yielded fragments at m/z: 249.1274 and m/z: 235.1692, respectively. Dimethyl cleavage involving the loss of a CH3 molecule produced a fragment at m/z: 280.1093. A subsequent loss of H2O gave rise to a fragment at m/z: 262.0860. Consecutive losses of a CO molecule resulted in fragments at m/z: 235.1692 and m/z: 206.1085, respectively. The loss of a molecule of CHO led to the formation of a fragment at m/z: 266.0934. A subsequent loss of CH3 generated a fragment at m/z: 252.0796. Drawing upon the analysis of mass spectral data and comparisons with the related literature references [The compound tanshinone IIA, operating in the positive ion mode, presented a quasi-molecular ion peak at ferences , the comB. balsamifera, alongside an ascorbic acid control, is depicted in The DPPH radical scavenging capacity of the methanol extract of 50) of the sample was calculated. A smaller IC50 value indicated stronger antioxidant activity. The IC50 values for ascorbic acid and the methanol extract in scavenging DPPH radicals were as follows: ascorbic acid (0.98 ± 0.073 μg/mL) < methanol extract (105.1 ± 0.503 μg/mL), as shown in The value of the half-inhibitory concentration  < methanol extract (12.49 ± 0.341 μg/mL), as indicated in The ICB. balsamifera methanol extract and ascorbic acid control are shown in The total antioxidant capacity of B. balsamifera methanol extract and ascorbic acid control are shown in The reducing power of B. balsamifera, a plant with significant ethnobotanical relevance. An advanced UPLC–Q–Orbitrap HRMS technique was used to identify 31 compounds in the plant, including 18 compounds that have not been reported before. These compounds belong to various categories such as flavonoids, organic acids, quinones, and alkaloids. The identification of these compounds provides a chemical basis for understanding the medicinal value and mechanism of action of B. balsamifera. There are still many unknown peaks in the mass spectrum that have not been identified due to the lack of reference mass spectrometry data and substances for the corresponding compounds. Additional analysis is required to clarify these unknown compounds. Flavonoids formed the most abundant group among the identified compounds, with hemiphloin, taxifolin, rutin, and quercetin standing out in terms of their quantity. These flavonoids are well-documented for their potent antioxidant capabilities, which can be linked to the significant antioxidant activity observed in B. balsamifera. The detailed mass fragmentation patterns of these representative compounds could serve as a valuable reference for future phytochemical studies on this plant.This study analyzed the phytochemical composition and antioxidant potential of B. balsamifera has garnered significant interest in recent years in the quest for natural sources of antioxidants. Studies evaluating the antioxidant activity of this plant have primarily focused on its leaves. For instance, Ginting et al. investigated the antioxidant activity of n-hexane extract and its fractions from B. balsamifera leaves using a DPPH assay. However, the results showed that the antioxidant activity of the extracts was insignificant [B. balsamifera leaf extracts from different growth cycles using DPPH, ABTS, and FRAP assays. They found that the immature leaves of B. balsamifera were rich in quercetin and phenols and exhibited significant antioxidant activity [B. balsamifera picked at different times. They reported that the essential oils of B. balsamifera collected from October to December had higher antioxidant activity [B. balsamifera was assessed by evaluating its DPPH and ABTS free radical scavenging abilities, total antioxidative capacity, and reducing power. The results indicated that the methanol extract of the plant has considerable radical scavenging capabilities, thereby aligning with its traditional use as a natural antioxidant. There was a positive correlation observed between the antioxidant activities and the mass concentration of the methanol extract, as well as the polyphenol content, with a particular emphasis on flavonoids. Flavonoids are recognized for their strong antioxidant properties, thus contributing to the observed relationship. Although the potency was not as high as ascorbic acid, a benchmark antioxidant, it was noteworthy given the natural origin of the plant extract. This study contributes significantly to the existing body of knowledge about B. balsamifera, shedding light on its chemical diversity and antioxidant potential. These findings suggest that this plant could be a promising source of bioactive compounds for further pharmacological studies and possible drug development. Furthermore, the identification of flavonoids and other compounds could pave the way for the development of new antioxidant agents.nificant . Jirakitactivity . Wang etactivity . In thisB. balsamifera methanol extract, which may not reflect the full spectrum of its bioactive components and pharmacological effects. However, the exploration of B. balsamifera’s therapeutic potential should not stop here. There are avenues for further investigations, such as in vivo antioxidant tests, and evaluations of other possible bioactivities. Additionally, the interactions between the identified compounds and their collective impact on the overall antioxidant activity of B. balsamifera could be an interesting focus for future research. This will not only provide a more holistic understanding of the plant’s therapeutic value but could also contribute to the optimization of extraction and formulation processes for therapeutic applications. Future research should also conduct phylogenetic and chemotaxonomic studies to clarify the relationship between B. balsamifera and other species in the genus Blumea or the family Asteraceae.The study only focused on the chemical constituents and antioxidant activities of B. balsamifera (L.) DC. was collected in July 2022 from the Yulin medicinal plant market in Guangxi, China. The plant material was authenticated by Associate Researcher Zheng Xilong from the School of Chinese Medicine Resources, Guangdong Pharmaceutical University. The dried aerial parts (moisture content not more than 10%) were ground using a DFY–300C swing pulverizer . The dry coarse powder of B. balsamifera was passed through a 50 mesh sieve (with a particle size ranging from 180 μm to 270 μm), and 10 g powder was accurately weighed and placed in a 50 mL flask. Methanol was added at a solid-to-liquid ratio of 1:50, and the mixture was extracted by ultrasound at a power of 300 W for 1 time, a temperature of 60 °C and time of 30 min, followed by centrifugation at 1500× g for 15 min. A volume of 200 μL of supernatant was taken and diluted to 1 mL with methanol. Then, it was filtered through a 0.22 μm membrane and stored at 4 °C before UPLC-Q-Orbitrap HRMS analysis. The remaining methanol extract was evaporated to obtain the crude methanol extract of B. balsamifera (BBME), which was stored at 4 °C for later use.Analytical grade methanol , chromatography-grade acetonitrile (obtained from Honeywell Trading (Shanghai) Co., Ltd., Shanghai, China), distilled water , rutin, and quercetin , isoquercitrin, 1,3–dicaffeoylquinic acid, and chlorogenic acid . The purity of all reference compounds exceeded 98%, including ascorbic acid  and 1,1–diphenyl–2–trinitrophenylhydrazine, 2,2–diazo–bis (3–ethylbenzothiazole–6–sulfonic acid) .18 column  with a flow rate of 0.3 mL·min–1 and a column temperature of 35 °C. The injection volume was 2.00 μL. The mobile phase consisted of acetonitrile (A) and 0.1% formic acid aqueous solution (B), using a gradient elution program . The mass spectrometer was operated in positive and negative ion-switching modes with the following conditions: heated electrospray ionization (HESI) source; spray voltage, 3.5 kV; capillary temperature, 325 °C; auxiliary gas temperature, 300 °C; mass scan range (m/z), 100~1500; sheath gas, auxiliary gas, and sweep gas were all nitrogen with flow rates of 50 L·min–1, 8 L·min–1, and 1 L·min–1, respectively, and; resolution, 60,000. The B. balsamifera sample was analyzed under the chromatographic and mass spectrometric conditions described above, and the total ion and MS2 chromatograms . Chromatographic separation was carried out on a Hypersil GOLD Catograms  in both –6), and the isotopic abundance ratios. The possible chemical constituents were identified by comparing the B. balsamifera chemical composition database information , reference substances, database matching , and fragmentation patterns, and confirmed through literature comparison.The molecular formulas of the compounds corresponding to the chromatographic peaks were determined using the high-resolution exact mass values calculated by Xcalibur 4.1 software, the deviation between the measured and theoretical relative molecular masses . The IC50 values of the samples were calculated by regressing the sample concentrations with the clearance rates.The method is rapid and simple, and has been widely used to determine the antioxidant activity of biological samples, pure compounds and extracts in vitro ,59. The •+) was diluted with ethanol to give an absorbance of 0.70 ± 0.02 at 734 nm. An amount of 2 mL of the BBME solutions of different concentrations  was mixed with 2 mL of ABTS•+ solution and the mixture was left to stand in darkness for 30 min. Absorbance was measured at 734 nm. Ascorbic acid was used as a positive control. The percentage inhibition of ABTS•+ radical was calculated using the following equation:1 represents the absorbance of the sample, A2 represents the absorbance of the sample and ethanol without ABTS, and A0 represents the absorbance of the control (the methanol in replace of the sample). The IC50 values of the samples were calculated by regressing the sample concentrations with the clearance rates.The 2,2′–azino–bis–3–ethylbenzthiazoline–6–sulphonic acid (ABTS) is a protonated radical. The method measures the antioxidant-scavenging ability of the ABTS radical generated by potassium persulfate in the aqueous phase. It has a characteristic maximum absorbance value at 734 nm and decreases with the scavenging of protonated radicals . The antThe total antioxidant capacity of the BBME was measured using the phosphomolybdenum method . The metThe reducing power of an active substance can be an important indicator of its potential antioxidant activity. The absorbance measured at 700 nm was used to demonstrate the reducing power. An increase in the absorbance of the measured sample indicates an increase in the reducing power of the sample tested . The totp < 0.05).All tests were conducted in triplicate. Data were presented as mean ± SD. Statistical analysis was performed using the SPSS software . The data were subjected to one-way analysis of variance (ANOVA), and the significant differences were evaluated using Duncan’s test (


The aim of this study was to investigate the physiological impact of airway pressure release ventilation (APRV) on patients with early moderate-to-severe acute respiratory distress syndrome (ARDS) by electrical impedance tomography (EIT).In this single-center prospective physiological study, adult patients with early moderate-to-severe ARDS mechanically ventilated with APRV were assessed by EIT shortly after APRV (T0), and 6 h (T1), 12 h (T2), and 24 h (T3) after APRV initiation. Regional ventilation and perfusion distribution, dead space (%), shunt (%), and ventilation/perfusion matching (%) based on EIT measurement at different time points were compared. Additionally, clinical variables related to respiratory and hemodynamic condition were analyzed.p < 0.001]. The other is the center of ventilation, which gradually shifted towards the dorsal region . The dorsal ventilation/perfusion matching increased significantly from T0 to T3 . Better dorsal ventilation (%) was significantly correlated with higher PaO2/FiO2  and lower PaCO2 .Twelve patients were included in the study. After APRV, lung ventilation and perfusion were significantly redistributed to dorsal region. One indicator of ventilation distribution heterogeneity is the global inhomogeneity index, which decreased gradually [0.61 (0.55–0.62) to 0.50 (0.42–0.53), APRV optimizes the distribution of ventilation and perfusion, reducing lung heterogeneity, which potentially reduces the risk of ventilator-induced lung injury.The online version contains supplementary material available at 10.1186/s13054-023-04469-8. Airway pressure release ventilation (APRV) is a highly effective strategy improving lung recruitment and oxygenation in clinical studies, but its effects on lung injury and mortality is debatable , 2. AnimElectrical impedance tomography (EIT), a noninvasive, bedside, and radiation-free technique, has been proposed as a valid method monitoring lung ventilation and perfusion . To uncoWe conducted a prospective, observational study in the general ICU of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, from March 2022 to June 2022. The study was approved by the Institutional Research and Ethics Committee of Union Hospital (NO. 2022–0048). Written informed consents were obtained from the patients’ legal representatives. The study was registered before enrollment at chictr.org.cn (ChiCTR2200057638).2/ FiO2 ≤ 200 mmHg with positive end-expiratory pressure ≥ 5 cmH2O according to the Berlin definition , and CoV gradually changed towards dorsal regions . Patients trend to have an increased global V/Q matching (%) (63.41 ± 13.47 to 68.58 ± 11.70%) and decreased shunt-EIT (%) (13.80 ± 7.53 to 8.39 ± 5.18%) from T0 to T3, but not significantly; however, the dorsal V/Q matching (%) increased significantly   and a decrease in global functional shunt , whereas patients with severe ARDS had no improvement . Additionally, PaO2/FiO2 increased from 109.5 ± 36.44 to 222.2 ± 58.46 mmHg significantly (p < 0.001), and PaCO2 decreased from 44.27 ± 8.58 to 36.78 ± 4.20 mmHg significantly (p = 0.032). Better dorsal ventilation (%) was significantly correlated with higher PaO2/FiO2  and lower PaCO2   to 37.95 (36.00–44.25) mL/cmHTo the best of our knowledge, this is the first study using EIT to assess the physiological effects of APRV on lung ventilation and perfusion distribution, and V/Q matching in patients with early moderate-to-severe ARDS.This study showed that dorsal region recruited after 24 h of APRV; moreover, the lung ventilation became more homogenous according to the results of GI index and CoV, suggesting that APRV has the potential to reduce the risk of VILI. Interestingly, we observed that ventilation redistributed prior to perfusion redistribution, implying that optimized ventilation facilitated in perfusion improvement. Dorsal alveolar recruitment reduced pulmonary vascular resistance, facilitating significant improvement of dorsal perfusion and V/Q matching. On the contrary, if lung inflation caused overexpansion rather than alveolar recruitment, an increase in pulmonary vascular resistance in hyperinflated regions would also direct perfusion toward nonaerated dorsal regions, ultimately leading to increased functional shunt and dead space .The number of unmatched regions is associated with ARDS severity and the risk of VILI, as well as being an independent predictor of mortality . Our stuThere are several limitations in our study. First, the sample size was limited. Second, we didn’t focus on the effects of spontaneous breathing on APRV. The effects of APRV with or without spontaneous breathing on ARDS patients are still being debated . Third, APRV is a strategy based on pathophysiology that provides lung recruitment, stabilization, and homogeneity, potentially protecting injured lungs in patients with early moderate-to-severe ARDS.Additional file 1. Methods. Table S1. Exclusion criteria. Table S2. Patients’ main characteristics. Table S3. Subgroup analysis of ventilation and perfusion distribution evolution at T0, T1, T2, and T3. Fig. S1. Ventilation and perfusion measured by EIT in a representative patient at different time points.


A healthy blood–brain barrier (BBB) shields the brain from high concentrations of blood glutamate, which can cause neurotoxicity and neurodegeneration. It is believed that traumatic brain injury (TBI) causes long-term BBB disruption, subsequently increasing brain glutamate in the blood, in addition to increased glutamate resulting from the neuronal injury. Here, we investigate the relationship between blood and brain glutamate levels in the context of BBB permeability. Rats exposed to BBB disruption through an osmotic model or TBI and treated with intravenous glutamate or saline were compared to control rats with an intact BBB treated with intravenous glutamate or saline. After BBB disruption and glutamate administration, the concentrations of glutamate in the cerebrospinal fluid and blood and brain tissue were analyzed. The results showed a strong correlation between the brain and blood glutamate concentrations in the groups with BBB disruption. We conclude that a healthy BBB protects the brain from high levels of blood glutamate, and the permeability of the BBB is a vital component in regulating levels of glutamate in the brain. These findings bring a new approach to treating the consequences of TBI and other diseases where long-term disruption of the BBB is the central mechanism of their development. Traumatic brain injury (TBI) is associated with neurologic and psychiatric effects, including depression, anxiety, and aggression ,2,3,4,5.Glutamate is a free amino acid in the brain , with a Disruption of the BBB has a crucial role to play in producing higher levels of brain glutamate in the CSF and ECF. The peak of the destruction of the BBB after brain insult occurs 5–6 h after the injury . StudiesWe have recently hypothesized that the integrity of the BBB is partially responsible for limiting pathologically increased levels of ECF and CSF glutamate in the post-TBI phase of neuronal death and establishes the maximal level of glutamate in the brain after TBI . In addiDespite the wide spectrum of sensitivity of nervous tissue to high concentrations of glutamate, data suggest that even a slight increase of 10% of extracellular brain glutamate (glutamate neurotoxicity) over a long period of time can activate neurodegenerative cascades ,17. TherGlutamate neurotoxicity involves excessive glutamate that induces neuronal degeneration and dysfunction, causing neurotoxicity ,24,25. IFollowing acute brain injury, such as ischemic strokes, cerebral ischemia, TBI, hypoglycemia, and epilepsy, increased extracellular glutamate can produce excitotoxicity due to the overaction of ionotropic glutamate receptors ,30,31,32The main purpose of this manuscript is to investigate the role of BBB disruption as a factor that contributes to excess glutamate through a rat model of TBI and model of BBB disruption. We, thus, outline the proposed pathways of the process and supply evidence to demonstrate this association. We propose that this relationship provides a useful tool to understanding the mechanisms for the possible treatment of TBI and its sequelae.p < 0.01, r = 0.749). The data were measured as a count and expressed as the median and 25–75 percentile range (Mann–Whitney U test). No significant difference was observed between the males and females.There was no evidence of neurological deficit in the control group. The NSS at 5 h was significantly greater in TBI rats compared to the control group (10(7–13) vs. 0(0–0), U = 0, We employed a 3-way ANOVA to determine the main effects of the model of BBB disruption , treatment , and sex  on outcomes such as blood glutamate concentration, CSF glutamate concentration, and Evans blue extravasation index.3,199 = 270, p < 0.01, pη2 = 0.818). A Bonferroni post-hoc analysis showed a significantly higher Evans blue extravasation index , TBI at 24 h , and HOBBB  compared to the control rats (−0.055 × 10−7 g ± 0.187 × 10−7 g). Our analysis found no significant effects of sex and treatment, and no influence on BBB disruption . Since no sex or treatment differences were found in the Evans blue extravasation index, an initial t-test for the study groups was performed and confirmed that sex and treatment did not affect the Evans blue extravasation index. The data are measurements in grams of extravasated Evans blue dye per gram of brain tissue and expressed as mean ± SD . Post-hoc analysis with a Bonferroni test denoted a significantly higher level of blood glutamate for rats receiving glutamate 0.423 g/kg , glutamate 0.845 g/kg  and glutamate 1.69 g/kg  compared to the placebo (224 µM/L ± 26 µM/L). No effects of sex and severity of BBB disruption or interaction between sex and severity of BBB disruption on blood glutamate levels were found . Since no difference in sex or severity of BBB disruption was found in blood glutamate levels, an initial t-test for the study groups confirmed the result that sex and severity of BBB disruption did not demonstrate a significant difference in blood glutamate levels. The data are measurements in µM/L and expressed as a mean ± SD  and BBB disruption . Using a Bonferroni test for post-hoc analysis, we observed significantly higher levels of CSF glutamate in several groups, including those receiving glutamate doses of 0.423 g/kg , 0.845 g/kg , and 1.69 g/kg , as well as those undergoing BBB opening procedures with TBI at 6 h , TBI at 24 h , and HOBBB , compared to the control group (4 µM/L ± 2.8 µM/L). Sex or interaction between sex and severity of BBB disruption or treatment regarding CSF glutamate levels were not found to have a significant effect . The data are measurements in µM/L and expressed as mean ± SD . Tukey’s post-hoc analysis indicated that the control group (−0.055 × 10−7 g ± 0.187 × 10−7 g) had significantly lower Evans blue extravasation index than the TBI-based BBB opening groups with TBI at 6 h , TBI at 6 h + glutamate 1.69 g/kg , TBI 24 h  and TBI at 24 h + glutamate 1.69 g/kg . Tukey’s post-hoc analysis revealed that the control group (−0.055 × 10−7 g ± 0.187 × 10−7 g) had a significantly lower Evans blue extravasation index than the HOBBB groups given mannitol , mannitol + glutamate 0.423 g/kg , mannitol + glutamate 0.845 g/kg  and mannitol + glutamate 1.69 g/kg . The glutamate 1.69 g/kg (0.06 × 10−7 g ± 0.13 × 10−7 g) group was not different from the control group . According to Tukey’s post-hoc analysis, the control group (0.039 × 10−7 g ± 0.175 × 10−7 g) had a significantly lower Evans blue extravasation index than the TBI-based BBB opening groups with TBI at 6 h , TBI at 6 h + glutamate 1.69 g/kg , TBI at 24 h  and TBI at 24 h + glutamate 1.69 g/kg  . According to Tukey’s post-hoc analysis, the control group (0.039 × 10−7 g ± 0.175 × 10−7 g) had a significantly lower Evans blue extravasation index than the HOBBB groups given mannitol , mannitol + glutamate 0.423 g/kg , mannitol + glutamate 0.845 g/kg , and mannitol + glutamate 1.69 g/kg . The glutamate 1.69 g/kg (0.059 × 10−7 g ± 0.1 × 10−7 g) group was not different from the control group  see . A one-wroup see .4,95 = 451, p < 0.01, η2 = 0.95). Tukey’s post-hoc analysis showed a significantly higher level of blood glutamate for the treatment of TBI at 6 h + glutamate 1.69 g/kg  and TBI at 24 h + glutamate 1.69 g/kg  compared to the control rats (224 µM/L ± 26 µM/L). The data are measurements in µM/L and expressed as mean ± SD . Tukey’s post-hoc analysis showed a significant higher level of blood glutamate for the treatment of mannitol + glutamate 0.423 g/kg , mannitol + glutamate 0.845 g/kg , mannitol + glutamate 1.69 g/kg  and glutamate 1.69 g/kg  compared to the control rats (224 µM/L ± 26 µM/L). The data are measurements in µM/L and expressed as mean ± SD (see A one-way ANOVA indicated a significant difference in the blood glutamate level between the TBI-based BBB opening study groups (F± SD see e. A one-± SD see f.p < 0.01), TBI at 6 h  = 9.29, p < 0.01), TBI at 24 h  = 8.47, p < 0.01) and TBI at 24 h + glutamate 1.69 g/kg  = 15.4, p < 0.01) groups compared to the control rats (4.1 µM/L ± 2.8 µM/L). The data are measurements in µM/L and expressed as mean ± SD  = 15.7, ± SD see g. 5,114 = 290, p < 0.01, η2 = 0.927) and a subsequent Tukey post-hoc analysis showed a significant higher level of CSF glutamate for mannitol , mannitol + glutamate 0.423 g/kg , mannitol + glutamate 0.845 g/kg  and mannitol + glutamate 1.69 g/kg  compared to the control rats (4 µM/L ± 2.8 µM/L). The glutamate 1.69 g/kg (3.7 µM/L ± 2.8 µM/L) group was not different from the control group. The data are measurements in µM/L and expressed as mean ± SD  = 0.873, p < 0.01, see p(40) = 0.966, p < 0.01, see p(80) = 0.917, p < 0.01, see p(60) = 0.927, p < 0.01, see s(4) = 0.965, p < 0.01, see Correlations were determined between CSF glutamate, blood glutamate, and the degree of BBB permeability see a–e. A stIn this study, we examined the role of BBB disruption as a key factor that contributes to the excess of glutamate in the CSF. We specifically studied the effect of different concentrations of blood glutamate on several models of BBB disruption, including both (1) an isolated model of BBB disruption known as HOBBB, and (2) traditional TBI (at 6 h and 24 h), which has multiple pathological processes, and compared the study rats to the control rats. The primary conclusion of this study is that while a healthy BBB effectively protects the brain from elevated levels of blood glutamate, a compromised BBB contributes to a pathological excess of glutamate in the CSF, which can account for long-lasting effects of the TBI. As expected, the CSF glutamate levels in the group of rats treated with glutamate at a concentration of 1.69/kg, but that were not subjected to induction of BBB destruction, were not significantly different from the levels of CSF glutamate in the control group that did not receive glutamate. This suggests that a healthy BBB effectively protects the brain from high levels of glutamate in the blood. However, the levels of cerebrospinal fluid glutamate in all models of BBB destruction, both with mannitol and with two models of TBI (at 6 h and 24 h), were increased in all experimental groups compared with the control rats. In addition, a strong correlation was documented between (1) the levels of CSF glutamate and blood glutamate and (2) the level of CSF glutamate and the degree of BBB destruction.After additionally studying the groups with different concentrations of blood glutamate , which had the same degree of BBB destruction, we concluded that it is the degree of BBB permeability that regulates the levels of brain glutamate in healthy as well as in injured brains. It was determined that the level of glutamate in the CSF in the mannitol groups with TBI at 6 h, and TBI at 24 h did not exceed 43 µM/L. This finding confirms our hypothesis  that a lTo explore the role of BBB disruption as a key factor that contributes to excess CSF glutamate, in this study we used the following two different rat models, in addition to control groups, to study BBB permeability: (1) a model of HOBBB disruption, and (2) TBI-induced BBB disruption. The TBI model was studied at two time points that differed in the severity of BBB destruction. This effect is known as biphasic BBB destruction, the dynamics of which have been studied and described previously . The valAs expected in the TBI model, the more severe BBB permeability was recorded at 6 h compared to 24 h. Both TBI models, at 6 h and 24 h, showed a higher degree of BBB destruction compared with the HOBBB model. BBB permeability was measured separately in two hemispheres in all the experimental groups. We recorded a significant increase in BBB permeability in the injured hemisphere in the TBI model, and in the hemisphere on the side of the mannitol injection in the HOBBB disruption model compared to the contralateral hemisphere. This has been previously observed in the literature ,35. We aAs expected, we did not observe any differences in the blood glutamate concentrations between the groups of rats administered mannitol + glutamate 1.69 g/kg, TBI at 6 h + glutamate 1.69 g/kg, or TBI at 24 h + glutamate 1.69 g/kg, compared to the group with glutamate 1.69 g/kg. There were also no differences in the concentration of glutamate in the blood of rats in the TBI and mannitol groups that did not receive glutamate , compared with the control group. Multivariate statistical analysis confirmed no influence between BBB degradation and blood glutamate levels (see Results). All groups in this study showed a very strong correlation between the concentration of glutamate in the blood and the amount of intravenously administered glutamate r = 0.965.We conclude that a healthy BBB effectively protects the brain from high levels of blood glutamate. In addition, after BBB disruption, the concentration of CSF glutamate is strongly correlated with the concentration of blood glutamate and relies on the degree of disruption of the BBB; the permeability of the BBB is a central component in regulating the levels of glutamate in the brain. The experiments were sanctioned by the Animal Care Committee of Ben-Gurion University of the Negev . This experiment used 100 male and 100 female  Sprague-Dawley rats , all weighing between 300 and 350 g. Purina chow  and water were made available ad libitum. Rats were situated in environmentally controlled rooms at a temperature of 22 °C, with a 12 h light/12 h dark cycle. All the tests took place between 8 am and 6 pm.Evans Blue (E2129) and glutamate (G1251) were purchased from Merck & Co. Inc. . In addition, 25% mannitol was purchased from Hospira, Inc. .All rats were randomly separated into 10 groups of 20 rats each  to undergo hyperosmolar opening of the BBB (HOBBB), TBI, or sham operation, with a variety of treatment methods (see below for HOBBB and TBI protocols). The effects of BBB breakdown on high concentrations of cerebral glutamate were studied in rats using the following 10 treatment groups: (1) HOBBB (induced by administration of mannitol 25% 2.4 cc into intra-carotid artery); (2) HOBBB + intravenous (IV) glutamate 1.69 g/kg (85); (3) HOBBB + IV glutamate 0.845 g/kg; (4) HOBBB + IV glutamate 0.423 g/kg; (5) TBI-6 h; (6) TBI after 6 h + IV glutamate 1.69 g/kg; (7) TBI at 24 h; (8) TBI at 24 h + IV glutamate 1.69 g/kg; (9) IV glutamate 1.69 g/kg; (10) sham-operated rats. Immediately after HOBBB induction and 5 h and 23 h after TBI, the rats were intravenously administered 2% Evans blue at 4 mL/kg. One hour later, blood and CSF were collected to determine the concentration of glutamate. Rats were euthanized, and the brains were collected to assess BBB breakdown. NSS was assessed in the TBI and control groups 5 h after the injury.The rats were anesthetized with 5% inhaled isoflurane and maintained at 1.5–2.5%, with 50% medical air and 50% oxygen, a tail vein catheter was placed, and a catheter was inserted into the internal carotid artery (ICA) . MannitoTBI was performed as previously described ,38. RatsBlood glutamate levels were analyzed by collecting blood from the tail vein using a 24-gauge Neoflon  catheter, which was subsequently removed from the vein .g for 10 min at 4 °C. The pellet was discarded and the supernatant was collected, adjusted to pH 7.2 with 2 M K2CO3, and stored at −80 °C for later analysis. The concentration of glutamate was measured utilizing the fluorometric method [2SO4 and contained 11.25 U of glutamate dehydrogenase in 10 mM nicotinamide adenine dinucleotide. After an incubation period of 30 to 45 min at room temperature, the fluorescence was measured at 460 nm with excitation at 350 nm. A standard curve was ascertained with glutamate concentrations ranging from 0 to 6 mM. Measurements were performed at least in duplicates [Whole blood  was deproteinized by mixing an equal volume of ice cold 1 M perchloric acid and then centrifuging at 10,000× c method . A 60 μLplicates .Rats were anesthetized with 5% inhaled isoflurane and maintained at 1.5–2.5%, with 50% medical air and 50% oxygen, the cisterna magna was cannulated, as previously described , and 0.1g for 10 min at 4 °C. The supernatant was obtained, adjusted to pH 7.2 with 12.5 μL of 2 M K2CO3 and stored at −80 °C for later analysis [Perchloric acid (25 μL) of 0.3 M was added to freshly obtained CSF (110 μL), and then centrifuged at 10,000× analysis ,43. Analg for 20 min and the supernatant was diluted 1:3 with 96% ethanol. A fluorescence detector was employed at a 620 nm excitation wavelength (bandwidth 10 nm) and 680 nm emission wavelength (bandwidth 10 nm) [The samples of brain slices were weighed and homogenized in trichloroacetic acid, using the formula of 1 g of brain tissue in 4 mL of 50% trichloroacetic acid. It was then centrifuged at 10,000× h 10 nm) ,44. Rats were assessed for their neurological status as previously described ,45 usingp < 0.05.Statistical evaluation was carried out using the SPSS-22 package . A Kolmogorov–Smirnov test determined the correct test for comparisons between the different parameters. The data were analyzed with 3-way ANOVA for the effects of sex, BBB disruption, and treatment. Post-hoc Bonferroni tests or planned pair comparisons were performed when the interactions were significant. We ascertained the significance of the comparisons between the groups with the one-way ANOVA with a subsequent Tukey’s post-hoc test or Student’s t-tests. Using Pearson’s test (for parametric data) and Spearman’s test (for non-parametric data), we calculated the correlation between CSF and blood glutamate levels and BBB disruption. The NSS were compared by the Mann–Whitney U test. Normally distributed data and continuous variables were presented as mean ± SD. Non-parametric data were presented as median ± interquartile range. The results were considered statistically significant when Through our multi-modal approach to inducing and analyzing BBB disruption, this study explored the effects of BBB breakdown on glutamate levels in the brain and blood. We found that a healthy BBB is a critical factor in protecting the brain from excess levels of blood glutamate. Additionally, we observed that after BBB disruption, CSF glutamate concentration is linked to blood glutamate concentration, which are dependent on the severity of BBB disruption. The permeability of the BBB provides a pathway to modulating glutamate levels in the brain.We hope that these observations will contribute to new approaches in the treatment of the consequences of TBI and other diseases where long-term disruption of the BBB is the central mechanism of their development. Chronic BBB disruption is a novel idea, and we propose that it has significant impact on the consequences of brain injury.


Vigna unguiculata is an important legume crop worldwide. The subsp. sesquipedalis and unguiculata are the two major types grown; the former is mainly grown in Asia to produce fresh pods, while the latter is mainly grown in Africa to produce seeds. Here, a chromosome‐scale genome for subsp. sesquipedalis was generated by combining high‐fidelity (HiFi) long‐read sequencing with high‐throughput chromosome conformation capture (Hi‐C) technology. The genome size for all contigs and N50 were 594 and 18.5 Mb, respectively. The Hi‐C interaction map helped cluster 91% of the contigs into 11 chromosomes. Genome comparisons between subsp. sesquipedalis and unguiculata revealed extensive genomic variations, and some variations resulted in gene loss. A germplasm panel with 315 accessions of V. unguiculata was resequenced, and a genomic variation map was constructed. Population structure and phylogenetic analyses suggested that subsp. sesquipedalis originated from subsp. unguiculata. Highly differentiated genomic regions were also identified, and a number of genes functionally enriched in adaptations were located in these regions. Two traits, pod length (PL) and pod width (PW), were observed for this germplasm, and genome‐wide association analysis of these traits was performed. The quantitative trait loci (QTLs) for these two traits were identified, and their candidate genes were uncovered. Interestingly, genomic regions of PL QTLs also showed strong signals of artificial selection. Taken together, the results of this study provide novel insights into the population differentiation and genetic basis of key agricultural traits in V. unguiculata. Vigna unguiculata [L.] Walp. (2n = 2x = 22), a member of the Fabaceae family, is an annual tropical or subtropical herb grown for human food and animal feed as a green manure, hay, or silage . The Latin name Unguiculata refers to the small stalks of their petals, meaning “with a small claw” or “with a small nail”. Its common name, “cowpea”, is based on its use as fodder for cows  is mainly grown in Asia, especially in China, for the production of fresh pods. According to the molecular evidence, it is easy to predict that subsp. sesquipedalis originated from subsp. unguiculata  Hepper var. mungo], a long‐read‐based draft genome sequence was assembled with a cumulative size of ∼454.4 Mb and an N50 size of 42.88 Mb   genome of 475.64 Mb containing 26 736 protein‐coding genes with an N50 of 18.26 Mb was obtained , a 519.4 Mb sequence scaffold was generated with an N50 of 16.4 Mb  was used as plant material for genome sequencing and assembly. Previously, V. unguiculata subsp. sesquipedalis var. Xiabao II  was used for de novo genome assembly with short‐read sequencing  of a large panel of germplasms has become feasible  long‐read sequencing with a Hi‐C interaction map. Genome comparison between the subsp. sesquipedalis and unguiculata revealed extensive genomic variations, and these variations resulted in gene loss. A V. unguiculata germplasm panel with 315 accessions  was resequenced, and a genomic variation map was constructed. With further investigation of this genomic variation map, population differentiation and selection signals in genome were identified. Two traits, pod length (PL) and pod width (PW), were observed for these 278 accessions, and a GWAS was performed for both traits. The genetic basis of the two traits was uncovered, and some candidate genes for the regulation of variations in the two traits were also predicted. We provide novel insights into the population differentiation and genetic basis of key agricultural traits in V. unguiculata.Here, a high‐quality chromosome‐scale genome for V. unguiculata subsp. sesquipedalis shows long pods (above 60 cm), white flowers and variegated seeds  of s Figure . To asses Figure . The genV. unguiculata germplasm panel, in which most lines are V. unguiculata subsp. sesquipedalis, were mapped onto the A147 genome, and most of these data showed a >98% mapping rate. Second, a total of 10 samples with approximately 75 Gb of RNA‐Seq data downloaded from SRA under project ID PRJNA763044 were also mapped onto the A147 genome by using a standardized RNA‐Seq analysis pipeline, and the average mapping rate was 95.64 ± 1.4%  reads of our 4% Table . Both ty4% Table . Fourth,ab initio, homology‐based, and transcriptome‐based gene prediction for A147 was also performed. In total, 27 439 genes encoding 41 725 proteins were predicted with an average length of 5621.4 and 1468.7 bp for genes and coding DNA sequences (CDSs)  Table .V. unguiculata subsp. unguiculata line IT97K was also assembled and released  genes were lost in the genome of A147 because of SV deletions. A comprehensive genome‐wide identification of R genes in these two genomes was performed. There were 161, 48 and 255 genes in the three types of R genes with NB‐ARC, TIR and LRR1 domains in the A147 genome  is illustrated in Figure R genes were lost. Taken together, our data suggested that SVs between A147 and IT97K resulted in extensive variations in gene contents and that these variations might play major roles in the differentiation between the two subspecies in V. unguiculata. Additionally, although we could not exclude some variations identified between A147 and IT97K that were attributed to different strategies for genome assembly and annotation, most of these variations would be reliable because both genome assemblies are of high quality.Structural variations (SVs) are >50 bp genomic variations among different genomes and include deletions, insertions, duplications, contractions, inversions and translocations. With comparisons of A147 with IT97K genomes, there were 7327 SVs, including 2837, 2871, 385, 243, 95 and 516 for deletions, insertions, duplications, contractions, inversions and translocations Table , respecte Figure , respecte Figure , respects Tables  and S9. Vitis vinifera, Populus trichocarpa, Glycine max, V. angularis, V. radiata, Phaseolus vulgaris, Arabidopsis thaliana, IT97K and A147, suggested that the divergence between IT97K and A147 occurred recently  reads were mapped onto the high‐quality chromosome‐scale genome of A147, and genomic variants, including SNPs and InDels, were identified. After quality filtering, a total of 17 831 792 genomic variants were identified for these 315 V. unguiculata accessions. The top three locations for these 17 831 792 genomic variants were intergenic 13 747 834 (53.939%), upstream of genes 4 273 944 (16.679%) and downstream of gene 3 923 595 (15.394%)  Table . A total%) Table .V. unguiculata accessions was predicted by using admixture software  for all 315 accessions was approximately 300 kb. The nucleotide diversity (Pi) for subsp. unguiculata and sesquipedalis were 0.276 ± 0.158 and 0.118 ± 0.157, respectively. These data revealed that the nucleotide diversity is much higher in subsp. unguiculata than in sesquipedalis, which is also consistent with the LD decay analysis.The phylogenetic analysis also revealed that all accessions of subsp. d Figure . Pop6‐Pod Figure . Clearlys Figure . The oveV. unguiculata germplasm panel. Population structure analysis supported that a large number of subpopulations were in this germplasm panel, especially in subsp. sesquipedalis. The data also supported that subsp. unguiculata diverged first and subsp. sesquipedalis originated from subsp. unguiculata. The nucleotide diversity is much higher in subsp. unguiculata than in sesquipedalis, and this phenomenon might result in LD decay faster than that in subsp. unguiculata.Collectively, the above analyses showed that there were a large number of genomic variants in the unguiculata is mainly distributed in Africa, while subsp. sesquipedalis is mainly planted in East Asia. Distinct growth environments in these two areas might shape different genomic characteristics for these two subspecies. To prove this prediction, two parameters, the sliding window of Fst and the cross‐population composite likelihood ratio (XP‐CLR) on 11 chromosomes for the two subpopulations, were calculated. The plots for these two parameters are illustrated in Figure Fst and XP‐CLR, respectively. When gaining insight into the functions of genes located in peak regions on each chromosome, a number of genes clearly showed functions related to biotic and abiotic resistance. For example, R genes, pathogen resistance (PR) genes and heat‐ and drought‐related genes could be found on the peaks of both XP‐CLR and Fst  revealed 47 genomic variants that formed two haplotypes (Hap1 and Hap2) and three genotypes  in the 315 accessions . The average PL of the three genotypes, Hap1/Hap1, Hap1/Hap2 and Hap2/Hap2, were 54.02 ± 12.03, 50.65 and 21.35 ± 10.98 cm . These data suggested that genomic variants in the PIN1 gene showed a significant association with PL variations although the corresponding genomic region did not show a significant association in further GWAS. These data further suggested that genomic differentiation between subsp. sesquipedalis and unguiculata might be attributed to a number of trait variations between these two subspecies.Subsp. t Figure . Interest Figure . A comprns Table . There wns Table , respectcm Table , respectFst genomic regions were identified and considered highly differentiated regions. A total of 1347 genes were located in both the top 5% XP‐CLR and Fst genomic regions  model implemented in GEMMA software  and 4.63 × 10−8  after Bonferroni correction. The Manhattan plot revealed that two QTLs showed an association with PL  and 22.00 ± 5.90 (n = 30) cm . To determine the genetic variation between subsp. sesquipedalis and unguiculata in the two PL QTLs, haplotype analysis of the ten significant SNPs in Chr03:61899120‐62027427 was performed, and a total of fourteen haplotypes that were assigned as Hap1 to Hap14 were calculated  and Fst (Pop1/Pop9) were also calculated. The data revealed that the QTL at Chr03:61899120‐62027427 was located in the genomic regions that had >top 5% Fst (Pop1/Pop8) and Fst (Pop1/Pop9)  , suggesting that PW was not a challenge in selection. The GWAS showed that there were two major QTLs for PW located on Chr01 and Chr11   and evm.TU.Chr11.1814 (EXLA2) regulate the variation in PW in V. unguiculata.An additional important agricultural trait pod width (PW) was also recorded in the five successive years 2017–2021 for the 278 germplasm accessions. A comparison of the average PW between the subsp. 1 Figure . The Q–Q1 Figure . For the2 Figure . A homol2 Figure . In this) Figure . In Arabsesquipedalis A147. Although there was a chromosome genome sequence for subsp. unguiculata in a previous report  showed that V. unguiculata subsp. unguiculata is closer to the outgroup V. angularis. This result suggested that this subspecies was originally domesticated from its progenitor and that V. unguiculata subsp. sesquipedalis evolved from V. unguiculata subsp. unguiculata. Thus, our study is highly consistent with previous results . Also, we found a greater LD distance and lower genetic diversity in s Figure . Thus, tV. unguiculata subsp. sesquipedalis, while IT97K is regarded as a representative plant of V. unguiculata subsp. unguiculata. The most prominent difference was the large variation in R gene numbers between the two genomes according to our comparisons. There were 180 more R genes in the genome of IT97K, and a total of 138 R genes were lost in A147 via SV deletions , which is widely grown in China, was used as plant material for genome assembly. It was grown in the field in Hannan district, Wuhan, Hubei Province of China  with normal irrigation, and young seedlings were collected as samples for DNA extraction. Different tissues, including seedlings, young flowers, roots, young pods and young leaves, were collected for RNA preparation. A total of 278 germplasm accessions were collected and grown in the same field where A147 was grown for five successive years (2017–2021), including autumn 2017 (August 1st to November 15th), spring 2018 (April 18th to July 30th), autumn 2019 (August 1st to November 15th), spring 2021 (April 18th to July 30th) and autumn 2021 (August 1st to November 15th). The locations where these germplasm accessions were collected are listed in Table et al., et al., et al., et al., et al., et al., et al., et al., et al., For genome assembly, two types of genomic data were generated. High‐quality genomic DNA for A147 was isolated using the modified cetyltrimethylammonium bromide (CTAB) method (Porebski de novo approaches was applied for transposable element (TE) identification. Briefly, the TE database was first built with the software program RepeatModeler‐2.0.2a; then, the TEs were searched with the software program RepeatMasker v.4.0.5 . Functional enrichment was performed by using TBtools software  pipelines to assign the GO terms to each gene . Single‐copy genes were identified among eight plants, including V. vinifera, P. trichocarpa, G. max, V. angularis, V. radiata, P. vulgaris, A. thaliana, IT97K and A147, by using the OrthoFinder program , while genomes of G. max, V. angularis, V. radiata, P. vulgaris and IT97K were downloaded from the V. unguiculata database on 2022‐05‐01 (https://phytozome‐next.jgi.doe.gov/). The phylogenetic trees were constructed with the software RAxML (the model parameter was set as “PROTGAMMAAUTO”)   . The PE reads for another 37 accessions were downloaded from the SRA database in GenBank (v) Table . The rawK from 1 to 20 , setting V. unguiculata accessions were used as plant materials for the GWAS. The PL and PW were recorded in the field for five successive years. The best linear unbiased prediction (BLUP) values of PL and PW were used as trait data for the GWAS. The BLUP was conducted by using the “lme4” package implemented in R version 4.0.5 (https://www.r‐project.org/). Genomic variants, including SNPs and InDels, were used as genotypic data. Genomic variants showing a minor allele frequency (MAF) were excluded from further analysis. GEMMA  pipelines were used to perform the GWAS, setting the parameter LMM as 1 .The 278 Lei Pan, Minghui Liu, Yan Kang, Xiang Mei, Gege Hu, Chun Bao, Yu Zheng, Huixia Zhao and Chanyou Chen performed the field experiments. Nian Wang and Lei Pan conducted data analyses, manuscript writing, and editing. Lei Pan and Nian Wang supervised this project.The authors declare no conflict of interest.Figure S1 Plot of the high‐throughput chromosome conformation capture (Hi‐C) matrix for the chromosome‐scale genome of Vigna unguiculata subsp. sesquipedalis line A147.Figure S2 Synteny comparisons of the chromosome‐scale genomes for two pairs of IT97K/A147 and IT97K/V. angularis.Figure S3 Synteny comparisons of the chromosome‐scale genomes for two pairs of A147/IT97K and A147/V. radiata.Figure S4 Determination of K values for population structure analysis.Figure S5 Genetic differentiation analysis between Pop1 and Pop8.Figure S6 Genetic differentiation analysis between Pop1 and Pop9.Click here for additional data file.Table S1 Summary of sequence data used for genome assembly.Table S2 Summary of contigs assembled with different programs.Table S3 Summary of A147 pseudochromosomes.Table S4 Summary of the mapping rate for RNA‐Seq data.Table S5 Functional annotation of the A147 genome.Table S6 Genome comparisons of two subspecies in Vigna.Table S7 Structural variations (SVs) between the A147 and IT97K genomes.Table S8 Gene loss resulting from insertion and deletion of SVs in the IT97K genome.Table S9 Gene loss resulting from insertion and deletion of SVs in the IT97K genome.Table S10 The V. unguiculata germplasm and the classification of subspecies and subpopulations.Table S11 Summary of genomic variant effects by type and region.Table S12 Functions of genomic variants.Table S13 Analysis of genomic variants on PIN1 (evm.TU.Chr04.258).Table S14 Genes located in highly differentiated regions.Table S15 Gene information in the pod length QTL region Chr03:61899120‐62027427.Click here for additional data file.


Pagiophloeus tsushimanus (Coleoptera: Curculionidae) feeds exclusively on camphor trees  in China, causing substantial economic and ecological losses. It is unclear how the larvae of P. tsushimanus outcome the main secondary metabolites of C. camphora such as D-camphor, eucalyptol, and linalool. In this study, we isolated terpenoid-degrading bacteria from the gut of P. tsushimanus larvae by using selective culture medium. Maximum likelihood phylogenetic analyses were performed with 16S rDNA sequences to identify the bacteria, and results showed ten strains belonged to four genera, including Pseudomonas, Enterobacter, Serratia, and Corynebacterium. Then, gas chromatography was employed to determine the degradability of D-camphor, eucalyptol, and linalool by the isolated strains, results showed that Z5 strain , F1 strain , and A3 strain  had the highest degradation rates of D-camphor, linalool, and eucalyptol, respectively. The intestinal bacteria were capable of terpenoid degradation in vitro, which suggested that these gut bacteria associated with P. tsushimanus play an important role in overcoming host plant secondary metabolite defense, thereby facilitating the host specialization of this pest.Insect intestinal bacteria play an important role in resisting defensive substances of host plants.  The diversity and evolutionary success of insects depends partly on their inextricable relationships with beneficial microorganisms . InsectsSolanum nigrum, such as 7-epizingiberene , have been shown to be toxic to the silver leaf whitefly Bemisia tabaci, thereby reducing insect feeding on plants  were proven to be able to reduce concentrations of terpenes, which directly or indirectly contribute to the capacity of the mountain pine beetle D. ponderosae to overcome host tree defenses  is a phloem-feeding weevil that attacks camphor trees, Cinnamomum camphora   feed on the phloem, while 3rd–5th instar can bore into the phloem and the cambium (P. tsushimanus larvae by gas chromatography–mass spectrometry (GC–MS) and found 19 main secondary metabolites. Among them, the relative content of the top three substances were eucalyptol (31.89%), D-camphor (19.22%), and linalool (10.16%) (C. camphora are considered to have a high degree of resistance to a variety of herbivorous insects (P. tsushimanus (P. tsushimanus larvae play an important role in detoxifying and metabolizing these hosts’ toxins.uraceae) . This weuraceae) . Over th cambium . Therefo(10.16%) . Studies insects . In our shimanus , suggestP. tsushimanus larvae and identified the strains by combining morphological characteristics as well as phylogenetic analysis for the first time. We further tested the terpenoids degradability of isolated strains by GC measurements. Our results may further elucidate interactions between P. tsushimanus and its gut bacteria in terms of host plant adaptations.In this study, we isolated the gut bacteria of Pagiophloeus tsushimanus larvae were collected from a camphor plantation in Songjiang District, Shanghai, China. The larvae were subjected to starvation in an incubator at a temperature of 28 ± 0.5 °C and a relative humidity of 70 ± 5% in the dark for 24 h. Thirty 4th-instar larvae were randomly selected, and the surface of the larvae was disinfected with 70% alcohol and soaked in phosphate buffer solution (PBS), which was repeated three times to avoid external contamination  , and linalool   were added to 50 ml of Bushnell Haas broth liquid medium at a ratio of 1% (w/v), and the liquid BHB medium consisted of 0.20 g/l MgSO4, 0.02 g/l CaCl2, 1.0 g/l KH2PO4, 1.0 g/l K2HPO4, 1.0 g/l (NH4)2SO4, 0.05 g/l FeCl3 (pH 7.0–7.2). Then, the larval intestinal abrasive was inoculated into the medium at a ratio of 3% (v/v), without intestinal abrasive medium as a control. After inoculation, the medium was incubated at 28 °C and 150 rpm. When the bacterial liquid was cloudy, the culture was serially diluted from 10−1 to 10−6, and 100 μl of dilution was coated on LB, NA, and TYA solid media, with three replicates per medium. The LB, NA, and TYA solid media consisted of 10 g/l peptone, 5 g/l yeast extract, 10 g/l NaCl, and 18 g/l agar (pH 7.2); 10 g/l peptone, 3 g/l beef extract, 5 g/l NaCl, and 18 g/l agar (pH 7.2); and 2.0 g/l yeast extract, 2.0 g/l beef paste, 6.0 g/l peptone, 40 g/l glucose, 3.0 g/l ammonium acetate, 0.5 g/l KH2PO4, 0.2 g/l neutral red, 0.2 g/l MgSO4·7H2O, 0.02 g/l FeSO4·7H2O, and 15 g/l agar (pH 6.5), respectively. After coating, the Petri dishes were incubated at 28 °C for 48 h in a bacteriological incubator. After selecting single colonies, the isolated and purified strains were preserved in 25% glycerol at −80 °C.Eucalyptol  , Referring to The purified bacterial strains were inoculated in LB liquid medium. After inoculation, these media were incubated at 28 °C, with shaking at 220 rpm for 12 h, and 1 ml of the cloudy bacterial solution was taken. Then, the bacterial genomic DNA was extracted by the E.Z.N.A.Soil DNA Kit  following the manufacturer’s instructions.2O, 12.5 μl 2 × Accurate Taq Master Mix, 1.0 μl gDNA (50 ng/μl), 1 μl primer 27F (10 μmol/l), and 1 μl primer 1492R (10 μmol/l). The PCR program included 30 s at 94 °C; 30 cycles of 98 °C for 10 s, 55 °C for 30 s, 72 °C for 2 min; and 72 °C for 2 min. Then, the PCR products were separated on a 1% agarose gel and sequenced by Jie Li Biology Co. Ltd .PCR amplification was performed using the extracted total bacterial DNA as a template with Taq DNA polymerase  and 16S rDNA universal primers 27F (5ʹ-AGAGTTTGATCCTGG CTCAG-3ʹ) and 1492R (5ʹ-GGTTACCTTGTTACGACTT-3ʹ). The PCR reaction volume was 25 μl: 9.5 μl ddHhttp://www.ncbi.nih.gov/blast). Multiple sequence alignments of 16S rDNA gene sequences of terpenoids-degrading bacteria and their closest relatives or members were performed by MAFFT version 7 software (https://www.ebi.ac.uk/Tools/msa/mafft/). Phylogenetic analyses were conducted using Bayesian inference (BI) and Maximum likelihood (ML). ModelFinder  eucalyptol, and the BHB medium without bacterial liquid was used as the blank control group. After shaking culture at 28 °C and 150 r/min for 10 days, 2 ml of chromatographically pure n-hexane   was added, treated in a sealed shaker for 3 h, placed upright, and treated overnight at −20 °C. After thawing at room temperature, 1 ml of hexane extract was transferred into a 2 ml autosampler vial . There were five replicates in the experimental group and the control group.Ten microlitres of n-hexane solution with 50% isobutylbenzene   was added as the internal standard (IS) for each sample, GC was used for quantitative analysis, and the sample was stored in a −20 °C refrigerator before analysis. Standard treatment: eucalyptol, D-camphor, and linalool standards with different masses were added to the n-hexane solution. Then, 10 μl n-hexane solution with 50% isobutylbenzene was added as the IS to prepare the standard with mass ratios of 1:1, 2:1, 3:1, 4:1, and 5:1. After mixing evenly, the standard was placed in a refrigerator at −20 °C to be tested.The degradation capacity of each strain for D-camphor, eucalyptol, and linalool was determined by a GC system  equipped with a flame ionization detector with an ionization energy of 70 eV and a fused silica capillary column . The carrier gas was nitrogen, with a flow rate of 1 ml/min. The injector and mass transfer line temperatures were both 250 °C. The injection volume was 1 μl, with a hydrogen flow of 40 ml/min, air flow of 450 ml/min, and nitrogen flow of 15 ml/min. The starting temperature of the heating program was kept at 40 °C for 1 min, 20 °C/min to 110 °C for 1 min, 4 °C/min to 120 °C for 1 min, and 20 °C/min to 200 °C. The total running time was 13 min.Calculation of the Degradation Rate. A 1 μl standard sample was taken for GC analysis. The peak retention time of each component in the sample to be measured is compared with that in the standard sample to determine the component to be tested. The standard samples with different mass ratios were determined by GC. According to the peak area ratio of standard samples to IS, the standard curve fitting was plotted by Origin 2021  to obtain the linear regression equation. The quantifications were carried out using the internal standard method  of the tested component, the peak area ratio of the internal standard, and the relative correction factor, the mass (mi) of the tested component was calculated as follows:According to the peak area (DE) was calculated according to the following formula:Finally, the degradation rate of the strain , followed by Tukey’s test (P < 0.05). Difference in the linalool degradation rate between F1 strain and F2 strain was determined by independent sample t-test (P < 0.05). All statistical analyses were performed using SPSS 22.0  and plotted with Origin 2021 .Differences in the degradation rates of D-camphor-degrading bacteria were coded as Z1, Z2, Z3, and Z4 , the degradation rate of Z5 was the highest (11.44 ± 2.88%), which was significantly different from that of the other four bacteria ; the degradation rates of Z4, Z1, and Z2 were 6.07 ± 3.27%, 5.59 ± 1.89%, and 3.61 ± 2.19%, respectively. The degradation rate of Z3 was the lowest, which was 2.00 ± 1.59% . Namely, the degradation rates of A1 and A2 were 9.49 ± 1.94% and 11.80 ± 1.45%, respectively   ʹ.Pagiophloeus tsushimanus is an emerging insect pest exclusively infesting camphor trees. Its outbreak is largely due to its evolution of a high degree of adaptability to the chemical defense of specific host plants . In this context, clarifying the biological metabolic function of microbial symbioses in P. tsushimanus is not only helpful to understand the multiple interactions of insect–plant–microbes, but also provides a new insight for the comprehensive control of this pest. So, it’s of great interest to explore the microbial symbionts of P. tsushimanus that contribute to the detoxification of plant secondary metabolites. In this study, we first isolated and identified terpenoid-degrading bacteria in the gut of P. tsushimanus larvae. These bacteria were proven to degrade terpenoids when cultured separately on medium containing one terpenoid . Although A1, F1, and Z1 strain have almost no differences in morphology  and molecular phylogenetic traits, but since there is no in-depth analysis, we initially treated them as different strains for the GC experiments, which does not rule out the possibility that they are consistent. Similarly, A2, F2, and Z3 are also that. After determining the terpenoid degradation rate of each bacterial strain by GC, we found that each strain could efficiently degrade on terpenoids , and that the degradation efficiencies of Pseudomonas and Serratia were relatively significant. In summary, these results revealed that the intestinal bacteria of P. tsushimanus have the capacity in detoxifying the host’s defensive substances and sequentially help P. tsushimanus adapt to host plants.Herbivorous insects mitigate the effects of toxic plant secondary metabolites by seeking help from symbiotic gut microbes to adapt to plant secondary metabolites . The facPseudomonas and Enterobacter species, for example P. aeruginosa  capacity of P. tsushimanus gut bacteria and have not yet been tested with other terpenoids, such as sesquiterpenes, and diterpenes.Some intestinal bacteria can help host insects defend against toxic secondary metabolites of plants and play an important role in the co-evolution of phytophagous insects and host plants . In partradation . The genlinalool . Pseudomterpenes . Pseudomn source . The intSpodoptera exigua larvae can produce N-acyl amino acid hydrolases, which helps to decompose these defense-induced substances, thereby reducing the toxic effects of secondary metabolites on larvae  and D. rhyzophagus  can fix nitrogen and provide nutrition , which highlighted the important role of intestinal bacteria in degrading secondary metabolites of C. camphora. It must be noted that our current work is not sufficient, and in-depth studies should focus on testing the physiological role in vivo and clarifying the metabolic pathways of these bacteria in degrading secondary metabolites. In conclusion, similar to other herbivorous insects, the intestinal microbiota of iead019_suppl_Supplementary_MaterialClick here for additional data file.