Moreover, in vitro TRPV2 over-expression in Ishikawa cell line increased migratory ability and response to cisplatin. CBD reduced cell viability, activating predominantly apoptosis in type I cells and autophagy in mixed type EC cells. The CBD improved chemotherapeutic drugs cytotoxic effects, enhanced by TRPV2 over-expression. Hence, TRPV2 could be considered as a marker for optimizing the therapy and CBD might be a useful therapeutic option as adjuvant therapy.Several studies support, both in vitro and in vivo, the anti-cancer effects of cannabidiol (CBD), a transient receptor potential vanilloid 2 (TRPV2) ligand. TRPV2, often dysregulated in tumors, is associated with altered cell proliferation and aggressiveness. Endometrial cancer (EC) is historically divided in type I endometrioid EC and type II non-endometrioid EC, associated with poor prognosis. Treatment options with chemotherapy and combinations with radiation showed only limited efficacy. Since no data are reported concerning TRPV2 expression as well as CBD potential effects in EC, the aim of this study was to evaluate the expression of TRPV2 in biopsies and cell lines as well as the effects of CBD in in vitro models. Overall survival (OS), progression-free survival (PFS), cell viability, migration, and chemo-resistance have been evaluated. Results show that TRPV2 expression increased with the malignancy of the cancer tissue and correlated with shorter PFS ( Medical Cannabis (MC) and pure phyto-cannabinoids  have been largely studied for their ability to ameliorate some of the most common and debilitating symptoms in cancer patients, like nausea, vomiting and pain ,2.In addition to these effects, MC and THC/CBD have attracted attention as potential anti-tumoral drugs, mainly as adjuvant for current therapies in different type of cancers . The maiIt can exert its biological activities via dependent (D)- and independent (ID)-receptors mechanisms (RMs). D-RMs mainly include weak antagonistic effects on cannabinoid receptor type-1 and -2  , agonistRegarding TRPV2, its expression and activation by CBD have been associated with deregulation of proliferation, cell differentiation and invasiveness in different cancer cell lines and animal models . BrieflyEndometrial cancer (EC) is the most commonly diagnosed gynecological malignancy in developed countries. ECs are divided into two subtypes, type I endometrioid EC and type II non-endometrioid EC. Type I is well differentiated and is frequently associated with a hyper estrogenic environment. Type II carcinomas develop from atrophic endometrium and are classified in different histological subgroups . These subtypes are poorly differentiated and associated with dismal prognosis ,37. TreaSince no data have been reported about TRPV2 expression in EC and there is a relative lack of robust diagnostic/prognostic biomarkers especially for EC type II, we firstly analyzed TRPV2 in human type II EC biopsies, and then, by in vitro investigations, we analyzed the TRPV2-dependent effects on cell migration and chemo-sensitivity. In addition, we evaluated the anticancer effects of CBD on EC cell lines.CB receptors and TRPV1/2 gene expression in 506 EC data samples from TCGA, queried with cBioportal . Samples were divided in type I endometrioid (397 samples) and type II serous type (109 samples).We assessed CB1 receptor was highly expressed (p < 0.001), CB2 was not expressed in both types. TRPV1 and TRPV2 were expressed in EC samples of both types. TRPV1 was more expressed in serous subtype (p < 0.05) while TRPV2 was more expressed in endometrioid subtype (p < 0.05)  .According to evidences in patients and since no data were available about TRPV2 and EC, we focused the attention on this channel.TRPV2 mRNA, although PCEM004a and b display a higher amount compared to the others  while the lowest staining was found in clear cell type (71.43%). Regarding peritumoral and normal tissues, TRPV2 was predominantly moderate or low. Samples were classified according to FIGO staging system and we found that TRPV2 expression increases with increasing of tissue malignancy. No significant difference in TRPV2 expression was detected in patients ≤68 or >68 years, according to the median age. Furthermore, Kaplan–Meier analysis was performed, calculating overall survival (OS) and progression-free survival (PFS).high 37 months vs. TRPV2moderate 53 months, p = 0.9346, HR = 1.039, 95% CI = 0.4131 to 2.615, TRPV2high 37 months vs. TRPV2low 43 months, p = 1.326, HR = 1.039, 95% CI = 0.5579 to 3.149, TRPV2moderate 53 months vs. TRPV2low 43 months, p = 1.326, HR = 1.199, 95% CI = 0.5665 to 2.537). High TRPV2 expression correlated with a shorter PFS   .p > 0.05, HR = 0.6764, 95% CI = 0.3391 to 1.349) or PFS  . Stroma subgroup was excluded because there were too few patients for statistical analysis. Intra-tumoral TRPV2 distribution did not influence OS outcome (o 2.051) .Taken together, our data showed that TRPV2 expression correlates with malignancy in non-endometrioid EC and supported the investigation of CBD biological effects in EC.+ cells) and TRPV2 expression was subsequently confirmed by Western blot analysis   B.Additionally, since Protein kinase B (PKB), also known as Akt, pathway is involved in supporting several pro-tumoral processes, to assess the potential role of TRPV2 in regulating cancer cell migration, modulation of AKT/PKB pathway was evaluated through Western blot analysis.+ cells (p < 0.001) supporting the hypothesis that TRPV2 expression increases cancer aggressiveness , and PAC (from 0.00025 µg/mL to 4 µg/mL) for 72 h. Results showed that TRPV2 expression increased chemo-sensitivity in Ishikawa cells for CIS (p < 0.05), while it did not particularly modulate the effect of DOX and PAC  and percentage of cell viability was evaluated by a cell viability MTT assay . The res50 in daily administration, up to 72 h. Then, fluorescence was analyzed by flow cytometry. The results evidenced that CBD treatment increased the percentage of Annexin V+ or Annexin V+/PI+ cells in MFE-280, HEC-1a and in primary PCEM002 cell lines, indicating apoptotic cell death, but only PI+ cells were detected in Ishikawa cell line, indicating a necrotic cell death  and Propidium Iodide (PI) staining. Each cell line was treated with an appropriate dose of CBD according to the ICll death . In PCEMIn order to investigate the reduction in cell viability in mixed type I/II PCEM004a and PCEM004b, the role of CBD treatment in regulating cell cycle was evaluated. The results showed that CBD was able to induce cell accumulation in the G1 phase . These dSince it has been demonstrated that CBD is able to induce autophagy in cancer , we inveTo confirm autophagy activation, we used acridine orange dye, which accumulates in acidic vesicular organelles, considered as an indicative marker of autophagy. These organelles emit bright red fluorescence, the intensity of which is proportional to the degree of the acidity and the volume of these structures, thus we assessed this fluorescence at the microscope. As depicted in p < 0.05)  .p < 0.001)  .The effect of CBD combined with chemotherapeutic drugs was evaluated in TRPV2-transfected Ishikawa cells, compared with untransfected cells. Both models were exposed to CBD at 3.92 µg/mL, in combination with two non-cytotoxic doses of each chemotherapeutic drug: CIS 0.25 and 0.5 µg/mL, DOX 0.015 and 0.03 µg/mL, PAC 0.0015 and 0.003 µg/mL for 72 h. Chemotherapeutic drugs were administered once, while CBD was administered daily. The combined treatment induced an increased level of cytotoxicity, as compared with either CBD or all chemotherapeutic drugs alone, in both models . AdditioThe non-endometrioid type II EC is responsible for most EC-related deaths because it is characterized by an aggressive behavior and high resistance to the common therapies. Furthermore, there are no targeted therapies approved so far for this subtype, and it is still treated in the same way as endometrioid type I EC, which is generally characterized by good prognosis and good response to therapy ,43. The + EC cells have a high migratory ability, and its expression induces an increase of pAKT/AKT ratio, supporting the activation of PI3K pathway in TRPV2+ cells that leads to high migratory ability and survival. These data could support the evidence that high expression of TRPV2 in patients correlates with shorter PFS.Herein we show that TRPV2 expression augmented with the increasing of a non-endometrioid component and its expression in human EC samples of type II and mixed type was associated with a reduced PFS. Additionally, its expression increases with malignancy, according to FIGO stage. Previously, it has been demonstrated that TRPV2 expression correlates with worse OS and PFS for triple negative breast cancer , esophag+ EC cells with CIS, DOX or PAC, the presence of TRPV2 expression increased CIS cytotoxic effect, suggesting that TRPV2 can influence chemo-sensitivity to this drug. Compared with GBM cells, Ishikawa cells are more sensitive to DOX (~50-fold), and its increase in uptake and cytotoxicity TRPV2-dependent, as previously described in GBM -2,5 diphenyl tetrazolium bromide (MTT)  to the media. The absorbance of samples, solubilized in dimethyl sulfoxide (DMSO), against a background control  was measured at 570 nm using a reader microliter plate .Cell lines .Cell death was evaluated using Annexin V-FITC Apoptosis detection Kit  followed by biparametric FACS analysis. Cells, at a density of 3 × 104 cells/mL, were treated with CBD for up to 48 h, in daily administration. Cells were fixed by adding ice-cold 70% ethanol for 1 h and then washed with staining buffer . Next, 100 µg/mL ribonuclease A solution  was added for 30 min at 37 °C, stained with 20 µg/mL propidium iodide (PI)  for 30 min at room temperature and finally analyzed by flow cytometry using linear amplification.Cells, at a density of 3 × 104 cells/mL, were seeded in 12-well plates and were treated with CBD at 3.92 and 7.85 µg/mL for 48 h. Then, cells were stained with medium containing 1 μg/mL AO for 15 min at 37 °C, washed twice in PBS and immediately examined with Nikon Eclipse E800 fluorescence microscope and NIS-Elements 4.0 software . Cytoplasm and nuclei of AO-stained cells fluoresced bright green, whereas the acidic autophagic vacuoles fluoresced bright red.To detect the development of acidic vesicular organelles, which are the hallmarks of autophagy, the vital staining of PCEM004 cells with acridine orange  was performed. The cells, at density 3 × 10p < 0.05. Patients were divided in three groups according to high, moderate or low expression of the target protein. The Kaplan–Meier (KM) method was used for Overall Survival and Progression-Free Survival analysis. For Univariate analysis of significance , long-rank test was used. * p < 0.05 was considered as statistically significant. Statistical analysis of IC50 levels was performed using Prism 5.0a .The data presented represent the mean with standard deviation (SD) of at least 3 independent experiments. The statistical significance was determined by Student’s t-test and by One Way-Anova and Two Way-Anova with Bonferroni’s post-test; * In conclusion, TRPV2 could be considered as a new potential marker for type II EC, especially serous subtype and high-grade tumors. In addition, its expression increased EC aggressiveness, enhancing migratory capacity through AKT/mTOR activation. CBD, a TRPV2 ligand, might be a potentially useful therapeutic option as adjuvant therapy to increase the efficacy of chemotherapeutic drugs to reduce cancer cell spreading.


The anterolateral ligament (ALL) is subject of the current debate concerning rotational stability in case of anterior cruciate ligament (ACL) injuries. Today, reliable anatomical and biomechanical evidence for its existence and course is available. Some radiologic studies claim to be able to identify the ALL on standard coronal plane MRI sections. In the experience of the authors, however, ALL identification on standard MRI sequences frequently fails and is prone to errors. The reason for this mainly lies in the fact, that the entire ALL often cannot be identified on a single MRI image. This study aimed to establish an MRI evaluation protocol improving the visualization of the ALL, using multiplanar reformation (MPR) with the goal to be able to evaluate the ALL on one MRI image. A total of 47 knee MRIs performed due to atraumatic knee pain between 2018 and 2019 without any pathology were analyzed. Identification of the ALL was performed twice by an orthopedic surgeon and a radiologist on standard coronal plane and after MPR. For the latter axial and coronal alignment was obtained with the femoral condyles as a reference. Then the coronal plane was adjusted to the course of the ALL with the lateral epicondyle as proximal reference. Visualization of the ALL was rated as “complete” , “partial”  and “not visible”. The distances of its tibial insertion to the bony joint line, Gerdy’s tubercle and the tip of the fibular head were measured. On standard coronal images the ALL was fully visible in 17/47, partially visible in 27/47, and not visible in 3/47 cases. With MPR the ALL was fully visible in 44/47 and not visible in 3/47 cases. The median distance of its tibial insertion to the bony joint line, Gerdy’s tubercle and the tip of the fibular head were 9, 21 and 25 mm, respectively. The inter-  and intraobserver  reliability was good to excellent. Complete visualization of the ALL on a single MRI image is critical for its identification and evaluation. Applying multiplanar reformation achieved reliable full-length visualization of the ALL in 94% of cases. The described MPR technique can be applied easily and fast in clinical routine. It is a reliable tool to improve the assessment of the ALL. Although reliable anatomical and biomechanical evidence for its existence is available today2, the impact of the ALL on rotational stability has not been fully understood. Furthermore, the indication to reconstruct the ALL has been discussed controversially3.The anterolateral ligament (ALL) is subject of the current debate on rotational stability in association with anterior cruciate ligament (ACL) injuries4. Another study that supports the important role regarding rotatory stability found a significant increase of rotatory instability when the ALL was sectioned additional to an ACL deficiency and therefore, claimed the ALL to be an important secondary stabilizer of the knee5. A recent study with a minimum 2 years follow-up was able to link this biomechanical findings to clinical results. They reported a lower reconstruction failure rate and better clinical outcomes in patients without ALL injury compared to patients with ALL injury on the initial MRI. Anterior stability and pivot-shift interestingly did not differ between those two groups6. The surgical decision to additionally perform an anterolateral stabilizing procedure is primarily made based on the clinical finding of rotational instability, i.e. a high-grade pivot shift and the type and level of the sporting activity of the patients7. An international consensus paper summarized the possible indications for additional anterolateral procedures. They stated that revision ACL reconstruction, high grade pivot-shift, generalized ligamentous laxity, genu recurvatum and young patients returning to pivoting activities may be good candidates for such additional procedures8.A biomechanical cadaver study reported abnormal knee kinematics in ALL deficient knees when only the ACL was reconstructed and found normalization of the kinematics if an additional modified deep Lemaire or MacIntosh tenodesis was added10. This should ultimately guide the surgical and non-surgical treatment of these patients. For radiologic assessment, an MRI is usually obtained in addition to standard radiographs. Previous studies report ALL identification rates on standard coronal plane MRI images ranging between 11 and 100%13. A recent review on the identification of the ALL on MRI highlights this heterogeneity and attributes this to different protocols used for the assessment. Moreover, knee sizes plays an important role regarding length, width and thickness of the ALL14. This is further supported by a study of patients younger than 18 years, were the authors stated that identification of the ALL was not possible on MRI in females younger than 7 years and males younger than 6 year. In this study a visualization of the ALL in 70% was reached after the age of 13 years in both sexes15. Most of the studies reporting on the ALL used standard coronal images for the assessment, while some tried to enhance ALL visualization by slightly flexing and externally rotating the knee during image acquisition14.The radiological assessment is crucial to distinguish whether rotational instability is due to an ALL injury or another reason such as a meniscal root tear16. To assess the ALL on MRI, a very good anatomical understanding of the anterolateral complex of the knee is required because the capsule, the meniscotibial and meniscofemoral ligaments as well as fibers of the LCL or the iliotibial band may be mistaken as the ALL due to their immediate proximity. Visualization of the whole ALL including its femoral and tibial attachment on one MRI layer seems to be critical for correct identification of the ligament, because if only parts of the ALL are visible, a confusion with other anterolateral structures like the posterior fibers of the iliotibial band, the joint capsule or the meniscotibial and meniscofemoral ligaments is possible. Considering this, a standardized imaging approach using well-defined anatomical landmarks is key but lacking to date. The mentioned anatomic structures are displayed in Fig. However, identification of the ALL on standard coronal MRI images is challenging and associated with a high level of uncertainty in the discrimination of the ALL and its surrounding structures. This is because the ALL is a fine structure which is usually not aligned precisely to the coronal plane. Furthermore, it merges with the lateral collateral ligament (LCL) near its femoral insertion complicating the discrimination of the two structures proximally. Muramatsu et al. reported 3D-MRI in contrast to the above mentioned 2D analysis allows reliable full length identification of the ALLThis study aimed to establish an MRI evaluation protocol improving the visualization of the ALL using a standardized multiplanar reformation (MPR) protocol. It was hypothesized that visualization of the ALL can be improved significantly by applying this protocol when compared to the evaluation on standard coronal plane MRI.All patients undergoing an MRI performed due to atraumatic knee pain between 2018 and 2019 were retrospectively identified within the picture archiving and communication system (PACS). Only patients analyzed in a 3 T MRI with a 3-dimensional intermediate weighted (proton weighted) fat-suppressed sequence  and with a standard coronal reformation (slice thickness 1 mm) of this acquisition directly performed at the MRI console were included. MRIs revealing any injury or pathology as well as previous surgery around the knee were subsequently excluded. Due to technical reasons not anonymized MRI data was analyzed first, the anonymization was done when documenting the measurement results. The study was approved by the local ethics committee and handling of the data was performed in accordance with the guidelines of the Declaration of Helsinki and the Swiss human research act. The ethics committee  waived the need to obtain informed consent in this study, according to Article 34 of the Swiss human research act.19. Second, the 3D acquisition was analyzed using MPR allowing for free orientation of the axial, coronal, and sagittal image planes.First, the ALL was identified on standard coronal reformations and classified as completely visible, partially visible and not visible Fig. . Complet20. Then the z-axis was tilted in the sagittal image . Data was not normally distributed and are given as median and range. Intraclass correlation (ICC) was calculated to assess inter- and intra-observer reliability. Two-way mixed measures checked for consistency and ICC is presented with 95% confidence interval. ICC values of < 0.40 were rated as poor, between 0.40 and 0.59 fair, between 0.60 and 0.74 good and between 0.75 and 1.00 excellentThe study was approved by the local ethical review board  Application BASEC-Nr. 2020-01559.Informed consent was not necessary for this retrospective study according to the swiss human research act.47 patients could be included for detailed analysis. 28 patients were women and 19 men, the mean age was 29 (range 20–39) and 24/23 were left/right knees, respectively. On standard coronal images Reader 1 rated the ALL as fully visible in 26% (12/47), partially visible in 66% (31/47), and not visible in 9% (4/47) cases in the first read and as fully visible in 28% (13/47), partially visible in 64% (30/47), and not visible in 9% (4/47) cases in the second read  and fair to excellent for reader 2 . The interobserver reliability regarding the same distances was good to excellent .This study aimed to establish an MRI evaluation protocol improving the visualization of the ALL using a standardized multiplanar reformation (MPR) protocol. It was hypothesized that visualization of the ALL can be improved significantly by applying this protocol when compared to the evaluation on standard coronal plane MRI. This analysis of 47 knee MRIs shows a significantly better visualization of the ALL after applying the MPR protocol described above, which confirms our hypothesis.22. More recently, the ALL related debate has shifted towards the clinical relevance of this structure2 including controversial discussions on the need for ALL reconstruction when anterior cruciate ligament reconstruction is performed24. Other possibilities to address the anterolateral rotatory instability are lateral tenodesis of parts of the Iliotibial band with modified Lemaire’s or modified MacIntosh’s techniques being the most referred ones26. However, there is no clear data available to guide surgical planning yet.For many years the existence of a distinct ligament in the anterolateral knee capsule called the ALL has been questioned. Today, reliable anatomic, histologic and biomechanical data exist proofing the existence of the ALL28. After acute ACL injuries Ferretti et al. showed a high prevalence (88%) of ALL abnormalities on MRI, which were significantly associated with lateral joint capsule tears. There were no problems reported in identifying the ALL independent of whether the ALL was injured or not28. This is in concordance with many other studies reporting that it is possible to characterize the ALL on MRI in acute case in a high percentage and could also link the MRI findings to the results of surgical exploration during ACL reconstruction30. Another study investigating the same topic stated that the ALL was not visible in 24% of cases27. Helito et al. examined MRI scans of uninjured knees and reported a visibility of the whole ALL in 72% on standard MRI images12. A comparative table showing the visualization rates of recent MRI studies about the ALL can be found in the review of Andrade et al.14. Porrino et al. stated that in many MRI evaluations different anatomical structures are named as ALL and that a reliable discrimination of the anterolateral structures is very difficult if not impossible on routine MRI31. Another drawback of the studies mentioned above is their lack to specify the MRI layer thickness. MRI layer thickness may be a critical factor in identifying the ALL. Large slice thickness can obscure the delicate ALL due to partial volume artifacts/effects by adjacent ligamentous and capsule structures falling within the same voxels during MRI acquisition32. In the authors’ opinion it should not be more than 1 mm. This is in agreement with a study by Taneja et al. which reported a visibility rate of the ALL of no more than 11% when MRI with a layer thickness of 3 mm were performed13.Several authors have claimed to be able to visualize the ALL on standard MRI sequences34. Therefore, on MRI this may give the impression of a continuous structure inserting at the tibia and femur. However, the femoral insertion of these fibers is slightly proximal and posterior to the origin of the ALL, but confusion of these structures on MRI is possible. Therefore, in our opinion, a reliable identification and evaluation can only be ensured, if the whole ALL is visible as a distinct ligamentous structure with clear tibial and femoral attachment on a single MRI image and with a femoral attachment right next to the lateral epicondyle. To reach this goal we applied multiplanar reformation and a novel MRI evaluation protocol based on anatomic landmarks of the ALL. With this technique, the full visibility of the ALL could be improved from 36% on standard coronal MRI images to 94%. However, it must be noted, that even using this technique the visualization of the ALL remains challenging and can only be accomplished by applying the knowledge of anatomical studies.This overview of MRI studies on the ALL points out the problems associated with its visualization and explains the varying results. The ALL is a very thin structure that may be mistaken easily for the capsule or the meniscotibial and meniscofemoral attachments. Moreover, the ALL lies in very close proximity to the capsulo-osseus layer of the ITB, which is connected to the femur at the intermuscular septum by Kaplan fibers19. A similar study using a 3D protocol was introduced by Muramatsu et al. in 2018, in this study a reference plane through the lateral epicondyle and a point midway between the posterior edge of Gerdy’s tubercle and the anterior margin of the fibula was chosen. The authors claimed to be able to visualize the ALL in 100% of the uninjured cases using this technique16. Our study confirms this findings that the ALL can be visualized in a high percentage using a 3D technique. An advantage of the technique described in our work is, that it primarily uses the femoral condyles to align the MRI in a neutral rotation and neutral varus/valgus and therefore creates a reproducible starting point. Moreover, our reference plane did not go exactly through defined anatomical landmarks, but was oriented exactly to the course of the ALL using those landmarks only as a starting point. With this flexible reference plane the authors tried to address the variations of the femoral and tibial insertions of the ALL.To be sure that the ALL was identified correctly, we rechecked our results by measuring the distances of its tibial insertion to the bony joint line, the middle of the Gerdy’s tubercle and to the tip of the fibular head. These were found to be 9 mm, 21 mm and 25 mm, respectively, which is within the range of the reported distances in anatomic studiesA major limitation of this study is, that the authors only had access to the MRI data and not to any clinical evaluation or additional demographic data like weight and height. Therefore absolute measurements of the dimensions of the ALL were not reasonable because they are directly linked for example to patient’s height and need to be interpreted in that context. Another limitation is, that the described MPR technique cannot be utilized in standard 3 mm 2D MRI sequences, but needs 3D sequences with maximum 1 mm slice thickness. Moreover, we did not dived the visualization of the ALL in tibial, menical and femoral part as some authors proposed. The reason for this is that many authors claimed that the meniscal and femoral part of the ALL cannot be reliably identified on MRI. Therefor we decided to stay with “partially visible” which is less precise but can be stated more reliable. The goal of this study was to visualize the entire ALL on one MRI image, which could be achieved in 94% of the case using our MPR technique. The question about the parts of the ALL will become more important when the technique described here is used for the assessment of ALL injuries. We will address this in further studies.Complete visualization of the ALL is critical for its identification and evaluation. Applying multiplanar reformation achieved reliable full-length visualization of the ALL in 94% of cases. The described MPR technique can be applied easily and fast in clinical routine. It is a reliable tool to improve the assessment of the ALL.


Amynthas corticis) were buried in two soils where the earthworms inhabited, or not, until more than 50% of the earthworm mass was lost. For both soils, earthworms decomposed faster during the early stage (between 0 and 3 days), as reflected by the higher rate of decomposition and increased accumulation of dissolved organic matter (DOM). This decomposition pattern was paralleled by bacterial community dynamics, where bacterial richness and diversity were significantly higher during early decomposition (p < 0.05) with the relative abundances of many genera decreasing as decomposition progressed. The succession of the bacterial community composition was significantly correlated with time-course changes in DOM composition (p < 0.05). Particularly, more functional groups  were identified to be linked with the change of a specific DOM type during the early decomposition phase. By exploring the ecologically important process of soil invertebrate decomposition and its associated bacterial communities, this study provides evidence, e.g., a statistically significant positive correlation between bacterial community and DOM compositions, which supports the widely recognized yet less-tested microbial community structure–function relationship hypothesis in invertebrate decomposition.Soil invertebrate corpse decomposition is an ecologically significant, yet poorly understood, process affecting nutrient biogeochemical cycling in terrestrial ecosystems. Here, we attempted to answer how the substrate chemistry and microbial community change during soil invertebrate (earthworm) decomposition and what roles microbes play in this process. Specifically, the dead earthworms ( Decomposition is a fundamental process to energy flow and nutrient cycling within ecosystems, with the balance between decomposition and net primary production responsible for ecosystem carbon content . Compare1, account for up to 90% of invertebrate biomass present in soil  how the substrate chemistry and bacterial communities change during earthworm decomposition and (2) what roles bacteria play in the decomposition process. We hypothesized that the total concentration of DOM would first increase and then decrease, but with different magnitudes of change for various components, thus causing the changes in DOM composition during earthworm decomposition. Prior to these shifts in DOM, the bacterial community composition may also change. For example, nitrogen-cycling-related functional groups, stimulated by the protein-rich decomposing earthworm, would dominate in the initial stage of decomposition and then would be replaced by generalists  as decomposition proceeds. Thus, the microbial community structure–function relationship would be observed during the decomposition of earthworm tissues.Amynthas corticis is an epi-endogeic earthworm species with global distribution  were retained for use in the experimental setup in order to circumvent the effects of minor factors  on results. Target individuals were brought to the laboratory, and non-targets were returned to the field. Then the target earthworms were surface-washed with sterile water and euthanatized by freezing at −40°C for 10 min , where the earthworms were originally collected from, and Xuchang , which is a non-native environment for the collected earthworms. The soils collected from Beijing and Xuchang were denoted as native soil and non-native soil, respectively. Both soils were classified as fluvo-aquic  but withFor each microcosm, sieved (mesh size 2 mm) and homogenized soil  was placed in polypropylene bottles . Three euthanatized earthworms were put in a nylon mesh bag (mesh size < 1 mm), and their weights were recorded. The mesh bags allowed for efficient sampling and nutrient exchange. Then the decomposition bag containing dead earthworms was buried in the middle layer of soil (4 cm from the bottom), mimicking a realistic situation. Soil moisture was adjusted to 40% of water-holding capacity by adding sterile deionized water. All microcosms were incubated at 20°C in the dark in a climate chamber. Based on a preliminary experiment, approximately 80% of earthworm mass is lost in native soil due to decomposition within 10 days. Therefore, to ensure sufficient samples for subsequent analysis, the incubation period was set as 8 days. To explore earthworm decomposition dynamics and bacterial community shifts, five replicates were destructively sampled on days 0, 1, 3, 5, and 8 for the following DNA extraction and DOM analyses. In total, 50 decomposition bags (2 soil types × 5 time points × 5 replicates = 50) and 50 soil samples (collected underneath the decomposing earthworm) were collected.The mass loss was determined from the difference of dry weight between the initial and final mass of each sampling time. At each sampling time, decomposition bags were removed from the microcosms and were freeze-dried at −40°C for 24 h to a constant weight using a vacuum freeze dryer . After drying, the undigested soil in earthworm gut was carefully removed from earthworm tissues using tweezers. Then the tissue samples were weighed and manually ground to fine powder with a mortar. The freeze-dried samples were also used for subsequent DOM analysis and DNA extraction. Because fresh earthworms were used for the decomposition experiment, the initial tissue dry mass (three euthanatized earthworms) in each microcosm was estimated based on the ratio of tissue dry mass to total wet mass . To determine the ratio, three replicates of fresh earthworms collected from the same batch were freeze-dried, and the tissue dry mass of these three earthworms was determined following the same procedure described above to calculate the proportion of tissue dry mass to total wet mass.g and 4°C for 10 min and then filtered through a 0.45-μm cellulose acetate membrane. The collected filtrate was diluted 20-fold with ultrapure water to an appropriate concentration  before excitation-emission matrix (EEM) spectroscopy analysis. The EEM analysis is a widely used tool to characterize the composition of complex DOM mixture (–1. The spectrum of ultrapure water was recorded as the blank (Freeze-dried earthworm powder (0.02 g) was dissolved in ultrapure water with a ratio of 1:10. The suspensions were centrifuged at 8,000  mixture , since dhe blank . Raman she blank : tyrosin2+; Takara, Dalian, China), 1 μl of template DNA (10 ng), and 8 μl of nuclease-free PCR-grade water. The PCR was performed on a T100 Thermal Cycler . The thermal cycling conditions were as follows: 94°C for 5 min, followed by 28 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 45 s, and a final extension at 72°C for 10 min. Negative controls, other ingredients not changed except the DNA templates replaced with sterilized water, were included for detecting potential contamination . The quality of DNA was assessed using the ND-1000 spectrophotometer . Primer pairs 338F (5′-ACT CCT ACG GGA GGC AGC AG-3′) and 806R (5′-GGA CTA CHV GGG TWT CTA AT-3′), targeting the V3–V4 region of 16S RNA genes, were used for amplification . An addimination . PCR proThe resulting sequence reads were first sorted based on orientation, and the barcodes were extracted by using extract.barcodes.py in QIIME . Then tht-test  was performed based on the Bray–Curtis distance of the DOM fluorescence spectra. Also, permutational multivariate analysis of variance (PERMANOVA) was conducted to test the significant distinctions in the DOM constituent across different sampling times.We estimated the temporal dynamics of mass loss and the bacterial alpha diversity of decomposing earthworms using linear regression analysis. The slopes of linear fits were compared by bootstrapping followed by t-test . The difPrincipal coordinate analysis based on the Bray–Curtis matrix was performed to explore changes in the bacterial community composition, and the shift patterns were confirmed by PERMANOVA. To examine how the identity of bacterial taxa changed during earthworm decomposition, the shared genera between different sampling times and the unique genera at a specific sampling time were visualized via a network graph using Gephi v0.9.2 . To furtp-values of correlation analysis to control the false discovery rate caused by multiple testing. PCoA, PERMANOVA, and Mantel test were conducted using the “vegan” package in R platform2.Potential functions for bacterial taxa were evaluated with the program “functional annotation of prokaryotic taxa” (FAPROTAX). The FAPROTAX defines the metabolic functions in terms of prokaryotic taxa  . Currentp = 0.33). At each sampling time, the mass loss was also unaffected by soil origin (p < 0.01 for both soils); then the decomposition rate significantly decreased after day 3 . However, there was no significant difference in decomposition rate for soil either before day 3 (p = 0.19) or after day 3 (p = 0.40).After 8 days of incubation, a decrease in the total mass of approximately 60% was detected (l origin . Decompol origin  and non-l origin . In natil origin . In non-l origin . A signip < 0.01) and a subsequent decrease after day 3 (p < 0.01). More specifically, in native soil, the fluorescent intensities of all five DOM types increased during the first 3 days (p < 0.01), with a peak of ∼2.2 × 105 arbitrary units on day 3 and then a decrease to ∼1.3 × 105 arbitrary units by day 8 (p < 0.01). In non-native soil, the fluorescent intensities increased to a peak of ∼2.0 × 105 arbitrary units during the first 3 days (p < 0.01) and then decreased to ∼1.1 × 105 arbitrary units over time (p < 0.01). Both in native soil and non-native soil, the extracted DOM was dominated by tyrosine-like proteins , tryptophan-like proteins , and microbial byproduct-like materials .Throughout the study period, the total amount of earthworm DOM showed a clear temporal pattern in both native and non-native soils, with a distinct increase before day 3 (p < 0.01), as shown by the progressive changes of DOM composition over time in native soil (p > 0.05).The shifts in DOM composition over time were also visualized in the PCoA based on the Bray–Curtis distance, and the significance of differences was confirmed by PERMANOVA. The decomposition time had a significant effect on DOM composition in both soils (ive soil  and non-ive soil . Similarp = 0.02) and then decreased  and then decreased during later stages of decomposition . The temporal changes of bacterial richness were marginally different in both soils either before day 3 (p = 0.06) or after day 3 . The Simpson index was significantly affected by soil origin only on days 5 and 8  as well as in the non-native soil treatment (p < 0.01). The bacterial communities displayed significant differences between native soil and non-native soil on most sampling days .The succession of the bacterial community on the decaying earthworms was also clearly detected, as indicated by PCoA performed on the entire dataset . The comProteobacterium) and two (Oscillochloris and Terrisporobacter), were present on days 5 and 8, respectively. The unique genera in the non-native soil (Brevibacillus and Romboutsia) and seven  on days 5 and 8, respectively. As decomposition proceeded, the number of genera shared by the two adjacent days also decreased. The number dropped to four and two, from 29 and 22 genera in native soil . Among the different compartments analyzed in this study, the contribution of soil-derived microbes was lower than that of earthworm-derived microbes . In the native soil treatment, the soil-derived microbes accounted for 4.8% on day 1 and then decreased to 0.8% (p < 0.05 on days 1 and 3). This result suggests that earthworm-derived microbes contributed primarily to the decomposition of earthworm tissues.Source tracking was used to quantify the contribution of the likely source environments (source) to the microbial community on decaying earthworms . The “so to 0.8% . In the  to 0.8% . In contR = 0.54, p = 0.001) and non-native  soils. The effects of the bacterial community were still maintained when we merged the two datasets from different soils . In addition, the bacterial community compositions were significantly correlated to mass loss in native  and non-native  soils. The effects of bacterial community on mass loss were still maintained when we merged the two datasets from the different soils .Shifts in the DOM composition were significantly correlated to the changes in bacterial community composition for earthworms in native , were defined as those intimately associated with temporal DOM shift during earthworm decomposition. Among these functional groups, 29 were shared in both native soil and non-native soils. The direction of correlation between the abundance change of specific functional group and the amount change of each DOM type was consistent, within a specific sampling time in each soil treatment. Moreover, the direction of correlations between changes in specific functional group abundance and DOM amount was consistent at different sampling times except for the fermentation group.Among the 90 functional groups in the FAPROTAX database, 42 and 40 groups were present in at least one of the samples in the native soil and non-native soil treatments, respectively. After false discovery rate correction, 35 functional groups in native soil and 34 functional groups in non-native soil, whose relative abundance changes were significantly correlated to the amount change of a specific DOM type at a specific sampling time , while 13 functional groups showed the opposite directions between the two soils . The posAnimal decomposition, creating resource islands and nutrient pulses, is one of the main drivers of the biogeochemical cycle and consequently has bottom-up importance for ecosystem functions. Studies addressing the role of bacteria during animal decomposition are still rare . As far We conducted a controlled laboratory study to decipher the microbial-mediated process of earthworm decomposition. After 8 days of incubation in native soil and non-native soil, a decrease in earthworm mass of nearly 60% was detected . The decp < 0.05). In theory, the bacterial community composition in soil would be different between soils with earthworm additions and those without, because of the nutrients from decomposing earthworm tissues. In this regard, when the study focuses on how soil bacterial communities change during earthworm decomposition, it is best to set controls without earthworm addition.Multiple lines of evidence showed that the bacterial community on decaying earthworm changed as decomposition proceeded. For example, the bacterial richness and Simpson index showed higher values at the early decomposition phase and then decreased . Also, aR = 0.54, p = 0.001; R = 0.66, p = 0.001) correlated to the chemical composition of decaying earthworm. Bacterial community composition correlated weakly but significantly with mass loss , while it strongly correlated with DOM composition. These results indicated that microbial communities play an irreplaceable role in the progression of decomposition. These results also provided evidence regarding the microbial structure–function relationship in invertebrate decomposition: the difference in DOM composition increased with the increasing dissimilarity in bacterial community composition.Although community succession occurred continuously, we identified two discrete phases of decomposition based upon changes in DOM composition and bacterial community diversity over time , 3. The Microbial taxa vary in their substrate preferences and strategies of nutrient acquisition, whereby some microbes may have higher acquisition ability and thus an advantage in the community . Based oThe main limitation of applying FAPROTAX to our data is the implicit assumption in FAPROTAX that if the cultured members of a taxon can perform a particular function, then all members of the taxon (both the cultured and non-cultured) can perform that function . HoweverAs far as we know, this is a novel study regarding the microbial-mediated process of earthworm decomposition by applying high-throughput sequencing. In this controlled laboratory study, we found that earthworms decomposed faster during the early stage (between 0 and 3 days), as reflected by the higher rate of decomposition and increased accumulation of DOM. We also found that the succession of the bacterial community composition was significantly correlated with time-course changes in DOM composition. In fact, the decomposition of soil animal tissue is a complex process because of many interacting factors related to the chemical, physical, and biological properties of the earthworm, as well as to soil environment and climate. This study provides evidence supporting the microbial community structure–function relationship hypothesis in invertebrate decomposition.https://www.ncbi.nlm.nih.gov/, PRJNA637816.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: YG designed research. Y-QS performed the research. Y-QS and YG analyzed the data and wrote the manuscript. Both authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.