Dihydrocaffeic acid improves IL-1ß-induced inflammation and cartilage degradation via inhibiting NF-κB and MAPK signalling pathways.

Aims: Osteoarthritis (OA) is a prevalent joint disorder with inflammatory response and cartilage deterioration as its main features. Dihydrocaffeic acid (DHCA), a bioactive component extracted from natural plant (gynura bicolor), has demonstrated anti-inflammatory properties in various diseases. We aimed to explore the chondroprotective effect of DHCA on OA and its potential mechanism. Methods: In vitro, interleukin-1 beta (IL-1ß) was used to establish the mice OA chondrocytes. Cell counting kit-8 evaluated chondrocyte viability. Western blotting analyzed the expression levels of collagen II, aggrecan, SOX9, inducible nitric oxide synthase (iNOS), IL-6, matrix metalloproteinases (MMPs: MMP1, MMP3, and MMP13), and signalling molecules associated with nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Immunofluorescence analysis assessed the expression of aggrecan, collagen II, MMP13, and p-P65. In vivo, a destabilized medial meniscus (DMM) surgery was used to induce mice OA knee joints. After injection of DHCA or a vehicle into the injured joints, histological staining gauged the severity of cartilage damage. Results: DHCA prevented iNOS and IL-6 from being upregulated by IL-1ß. Moreover, the IL-1ß-induced upregulation of MMPs could be inhibited by DHCA. Additionally, the administration of DHCA counteracted IL-1ß-induced downregulation of aggrecan, collagen II, and SOX9. DHCA protected articular cartilage by blocking the NF-κB and MAPK pathways. Furthermore, DHCA mitigated the destruction of articular cartilage in vivo. Conclusion: We present evidence that DHCA alleviates inflammation and cartilage degradation in OA chondrocytes via suppressing the NF-κB and MAPK pathways, indicating that DHCA may be a potential agent for OA treatment.

BMP7 ameliorates intervertebral disc degeneration in type 1 diabetic rats by inhibiting pyroptosis of nucleus pulposus cells and NLRP3 inflammasome activity.

BACKGROUND: Accumulating evidence indicates that intervertebral disc degeneration (IDD) is associated with diabetes mellitus (DM), while the underlying mechanisms still remain elusive. Herein, the current study sought to explore the potential molecular mechanism of IDD in diabetic rats based on transcriptome sequencing data. METHODS: Streptozotocin (STZ)-induced diabetes mellitus type 1 (T1DM) rats were used to obtain the nucleus pulposus tissues for transcriptome sequencing. Next, differentially expressed genes (DEGs) in transcriptome sequencing data and GSE34000 microarray dataset were obtained and intersected to acquire the candidate genes. Moreover, GO and KEGG enrichment analyses were performed to analyze the cellular functions and molecular signaling pathways primarily regulated by candidate DEGs. RESULTS: A total of 35 key genes involved in IDD of T1DM rats were mainly enriched in the extracellular matrix (ECM) and cytokine adhesion binding-related pathways. NLRP3 inflammasome activation promoted the pyroptosis of nucleus pulposus cells (NPCs). Besides, BMP7 could affect the IDD of T1DM rats by regulating the inflammatory responses. Additionally, NPCs were isolated from STZ-induced T1DM rats to illustrate the effects of BMP7 on IDD of T1DM rats using the ectopic expression method. Both in vitro and in vivo experiments validated that BMP7 alleviated IDD of T1DM rats by inhibiting NLRP3 inflammasome activation and pyroptosis of NPCs. CONCLUSION: Collectively, our findings provided novel mechanistic insights for understanding of the role of BMP7 in IDD of T1DM, and further highlighted BMP7 as a potential therapeutic target for preventing IDD in T1DM.

Ferrostatin-1 Inhibits Toll-Like Receptor 4/NF-κB Signaling to Alleviate Intervertebral Disc Degeneration in Rats.

Ferrostatin-1 (Fer-1), an inhibitor of ferroptosis, is implicated in intervertebral disc degeneration (IDD). The current study explored the role of Fer-1 in IDD via the toll-like receptor 4 (TLR4)/NF-κB signaling pathway. IDD-related gene expression microarray GSE124272 and high-throughput sequencing data set GSE175710 were obtained through the Gene Expression Omnibus database. Differentially expressed genes in IDD were identified, followed by implementation of protein-protein interaction network analysis and receiver operating characteristic curve analysis. The main pathways in IDD were obtained through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional analyses, and target genes of Fer-1 were obtained through PubChem and PharmMapper websites. Finally, GPX4, FTH, and TLR4 expression was determined in a IDD rat model. Three key co-expression modules involved in IDD were obtained through Weighted Gene Co-Expression Network Analysis. Thirteen differentially expressed genes were found to be associated with IDD, and eight key genes (TLR4, BCL2A1, CXCL1, IL1R1, NAMPT, SOCS3, XCL1, and IRAK3) were found to affect IDD. These eight key genes had the diagnostic potential for IDD. The NF-κB signaling pathway was shown to play a predominant role in IDD development. Network pharmacologic analysis indicated a role of Fer-1 in suppressing ferroptosis and ameliorating IDD via the TLR4/NF-κB signaling pathway, which was verified by an in vivo animal experiment. The study showed that Fer-1 down-regulates TLR4 to inactivate NF-κB signaling pathway, suppressing ferroptosis and ultimately alleviating IDD in rats.

Mesenchymal stem cells shuttling miR-503 via extracellular vesicles enhance glioma immune escape.

Glioma is emerging as an aggressive type of glioma characterized by invasive growth pattern and dismal oncologic outcomes. microRNAs (miRNAs) have been attracting research attention in tumorigenesis. Herein, the aim of the current investigation was to explore the functional role of mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) containing miR-503 in glioma. The glioma tissues and corresponding normal brain tissues were collected from patients with glioma, followed by quantification of miR-503, kinesin family member 5A (KIF5A) and interleukin-7 (IL-7). EVs were isolated from bone marrow MSCs and identified by transmission electron microscope and nanoparticle tracking analysis. EVs from miR-503 mimic-transfected MSCs, miR-503 agomir,, oe-KIF5A, or sh-IL-7 was delivered into glioma cells to determine their effects on biological behaviors of glioma and T cells as well as the release of immunosuppressive factors. Lastly, a mouse model of glioma was developed to validate the function in vivo. miR-503 was expressed at a high level in glioma tissues while KIF5A was poorly expressed and targeted by miR-503. Furthermore, miR-503 loaded in MSC-EVs or upregulated miR-503 was demonstrated to facilitate glioma cell proliferation, migration and invasion accompanied by promoted release of immunosuppressive factors. Effects of overexpressed KIF5A on T cell behavior modulation were dependent on the IL-7 signaling pathway. Such results were reproduced in mice with glioma. Collectively, the discovery of miR-503 incorporated in MSC-EVs being a regulator that controls immune escape in glioma provides a novel molecular insight that holds promises to develop therapeutic strategies against glioma.

Long-term results of trans-scaphoid perilunate fracture dislocations treated by open reduction and internal fixation.

PURPOSE: The paper holds the research purpose of confirming the long-term results of trans-scaphoid perilunate fracture dislocations (TSPFD) under the treatment of open reduction and internal fixation. METHODS: Anteroposterial-lateral radiographs of the patient's wrist were taken before and after surgery. We use a dorsal approach for all cases. Postoperative clinical and radiographic assessments were performed routinely. The scapholunate angle (SLA), estradiol angle (RLA), as well as lunotriquetral distance (LTD) assisted in the radiographic assessment. Clinical assessment was performed using the Krimmer score, modified Mayo wrist score (MWS), active flexion extension arc (FEA), radial deviation and ulnar deviation arc (RUDA) and grip strength. A visual analog scale (VAS) assisted in the pain evaluation, the VAS score ranges from 0 to 10. RESULTS: Twenty-two TSPFD patients due to the wrist trauma received operative treatment and we retrospectively analyzed the surgical results, together with evaluating their clinical and radiological follow-up. These patients held a mean age of 30 years old. Herzberg's perilunate fracture-dislocation classification was taken into account to find that 19 males and 3 females suffered dorsal dislocation. The fellow-up time lasted 98.3 months on average. All cases obtained sufficient union after open reduction and internal fixation. The last follow-up found the median of grip strength was 20.00 (interquartile range, 20.00-21.25), which was 84.5% of the normal side. The modified Mayo wrist score evaluation scale considered 12 cases as excellent, and 10 good. The median of VAS and Krimmer scores at the final follow-up were 1.50 (interquartile range, 0.75-2.00) and 100.00 (interquartile range, 100.00-100.00), respectively, higher relative to the pre-operation (P < 0.001). No patients showed nerve damage preoperatively or postoperatively, or pin tract infection in any of the patient. CONCLUSIONS: It is necessary to diagnose such complicated biomechanical damage in early stage and adopt the open reduction and stable fixation for treatment; appropriate treatment can contribute to a functionally adequate and anatomically integrated wrist.

Oncologic and functional outcomes of different reconstruction modalities after resection of chondrosarcoma of the scapula: a medium- to long-term follow-up study.

OBJECTIVES: To evaluate the oncologic and functional results of scapular reconstruction after partial or total scapulectomy for chondrosarcoma. MATERIALS AND METHODS: Twenty-one patients with chondrosarcoma who underwent partial or total scapulectomy between January 2005 and July 2019 were reviewed retrospectively. RESULTS: At a mean follow-up of 62.6 months (range, 13-123 months), four patients developed local recurrence, and three developed distant metastases, one of which developed both recurrence and metastasis. The overall survival rate of patients at 5 years was 84.6%, the disease-free survival rate was 69.3%, and the complication rate was 19% (4/21). The 1993 American Musculoskeletal Tumor Society (MSTS93) scores of patients in the partial scapulectomy group, total scapulectomy + humeral suspension group and prosthetic reconstruction group were 26.50 ± 1.38, 19.00 ± 2.58, and 21.38 ± 2.62, respectively. There was a statistically significant difference between the partial scapulectomy group and the total scapulectomy + humeral suspension or prosthetic reconstruction group ( P = 0.006 and 0.0336, respectively). The range of motion of the shoulder joint for forward flexion was 80.83° ± 11.14°, 51.25° ± 21.36°, and 52.50° ± 11.02°, respectively. The p-values for the comparison between the partial scapulectomy group and the total scapulectomy + humeral suspension or prosthetic reconstruction group were 0.0493 and 0.0174, respectively. And the range of motion of abduction was 75.00° ± 10.49°, 32.50° ± 11.90°, 41.88° ± 11.63°, respectively. Patients in the partial scapulectomy group had significantly better postoperative shoulder abduction function than the total scapulectomy + humeral suspension or prosthetic reconstruction group (P = 0.0035 and 0.0304, respectively). There was no significant difference in MSTS93 scores and flexion and abduction function of the shoulder joint in the upper extremity after total scapulectomy with humeral suspension or prosthetic reconstruction (P > 0.05). CONCLUSIONS: Surgical treatment of chondrosarcoma of the scapula can achieve a satisfactory prognosis and shoulder function. Total scapulectomy followed by prosthetic reconstruction or humeral suspension are both feasible treatments.

Bone Marrow Mesenchymal Stem Cell-Derived Extracellular Vesicles Carrying circ_0050205 Attenuate Intervertebral Disc Degeneration.

Objective: It has been reported that bone marrow mesenchymal stem cells (BMSCs) are a potential source of autologous stem cells to support the nucleus pulposus (NP) regeneration in intervertebral disc degeneration (IDD). Herein, we aim to study the mechanism underlying the effects of BMSC-derived extracellular vesicles (BMSC-EVs) on nucleus pulposus cells (NPCs) in IDD. Methods: EVs were isolated from BMSCs. An IDD model was surgically established in C57BL/6J mice. NPCs were exposed to tBHP to establish an IDD cell model. RNA sequencing was performed to identify differentially expressed circRNAs in NP tissues harvested from mice with IDD. Interactions among circ_0050205, miR-665, and GPX4 were validated, and different interventions were used to study the roles of these molecules in NPC biological functions. Results: BMSC-EVs promoted NPC survival and inhibited NPC apoptosis and extracellular matrix (ECM) degradation. circ_0050205 expression was downregulated in the NP tissues of IDD mice, and BMSC-EVs facilitated NPC survival and suppressed ECM degradation in NPCs by transferring circ_0050205. circ_0050205 sponged miR-665 and upregulated GPX4 expression. BMSC-EVs expressing circ_0050205 promoted NPC survival-inhibited ECM degradation in NPCs and alleviated IDD in mice via the miR-665/GPX4 axis. Conclusion: In conclusion, BMSC-EVs promoted NPC survival-inhibited ECM degradation in NPCs and attenuated IDD progression via the circ_0050205/miR-665/GPX4 axis.

Bevacizumab attenuates osteosarcoma angiogenesis by suppressing MIAT encapsulated by serum-derived extracellular vesicles and facilitating miR-613-mediated GPR158 inhibition.

Targeting angiogenesis has been considered a promising treatment for a large number of malignancies, including osteosarcoma. Bevacizumab (Bev) is an anti-vascular endothelial growth factor being used for this purpose. We herein investigate the therapeutic potential of Bev in angiogenesis during osteosarcoma and the related mechanisms. Bioinformatics were performed for identification of osteosarcoma-related microarray dataset to collect related lncRNA and miRNA, with MIAT and miR-613 obtained. The predicted binding site between miR-613 and GPR158 3'UTR region was further confirmed by luciferase assay. Then, their effects combined with treatment with Bev on osteosarcoma cells were explored by the gain- and loss-of-function. After extraction from osteosarcoma patients' serum (serum-EVs) and identification, EVs were co-cultured with osteosarcoma cells, the biological behaviors of which were detected by CCK-8 assay and microtubule formation in vitro. A mouse tumor xenograft model was used to determine the effect of Bev on tumor angiogenesis in vivo. Bev inhibited osteosarcoma cell proliferation and angiogenesis in vivo and in vitro. Besides, serum-EVs could transfer MIAT (EV-MIAT) into osteosarcoma cells, where it is competitively bound to miR-613 to elevate GPR158, thus promoting osteosarcoma cell proliferation and angiogenesis. Furthermore, Bev arrested osteosarcoma cell proliferation and angiogenesis by inhibiting EV-MIAT and inducing miR-613-mediated GPR158 inhibition. In conclusion, the Bev-mediated MIAT/miR-613/GPR158 regulatory feedback revealed a new molecular mechanism in the pathogenesis of osteosarcoma angiogenesis.

Risk factors for lymph node metastasis of soft tissue sarcomas of the head, neck, and extremities, and the clinical significance of negative lymph node dissection.

BACKGROUND: This study sought to define the risk factors for lymph node metastasis (LNM) of soft tissue sarcomas (STS) of the head, neck, and extremities, and the clinical significance of negative lymph node dissection (NLND). METHODS: STS patient data in the Surveillance, Epidemiology, and End Results (SEER) database from 1988 to 2015 were extracted and pooled. Logistics regression analysis was used to identify risk factors for LNM, Cox proportional hazards and Fine-Grey's models were used for survival analysis, and Propensity score matching analysis (PSM) was used to assess the impact of NLND on patient prognosis. RESULTS: A total of 3276 patients were enrolled in the study, of whom 283 (8.6%) developed LNM. Rhabdomyosarcoma had the highest rate of LNM (25.3%), followed by clear cell sarcoma (16.8%) and epithelioid sarcoma (12.4%), while leiomyosarcoma had the lowest rate of LNM (1.3%). Sex, tumor size, grade, histology, and site were significantly associated with LNM. For specific histologic subtypes of STS, NLND significantly improves overall survival (HR: 0.718, 95%CI 0.535-0.962; P = 0.026) and cancer-specific survival (HR: 0.699, 95%CI 0.506-0.967; P = 0.031) and reduces cancer-specific mortality (Gray's test, P = 0.017). However, NLND did not improve overall survival (P = 0.46) or reduce cancer-specific mortality (Gray's test, P = 0.772) of patients with leiomyosarcoma. CONCLUSIONS: Histology is an independent risk factor for LNM in STS of the head, neck, and extremities. Prophylactic NLND treatment was necessary and had a clinical benefit for patients with STS who were at high risk for LNM but had no significant impact on the prognosis of patients with leiomyosarcoma.

[Effects of ERα gene overexpression on bone mineral density and calcium and phosphorus metabolism inovariectomized osteoporosis mice].

Objective: To investigate the effects of estrogen receptor α (ERα) gene overexpression on bone metabolism and calcium and phosphorus metabolism in ovariectomized osteoporosis mice, and to provide experimental basis for targeted gene therapy of osteoporosis. Methods: Thirty SPF female mice were randomly divided into sham operation group, model group and ERα overexpression group with 10 mice in each group. After the model was established, the ERα overexpression group was transfected with recombinant adenovirus vector carrying mouse ERα gene by intraspinal injection. The model group was transfected with empty virus, and the sham operation group was not treated. The expression of ERα gene in bone tissue of mice was detected by quantitative Real-time PCR (qRT-PCR). Bone mineral density (BMD) of mouse femur was measured after modeling. Trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular segregation (Tb.Sp), bone volume fraction (BV/TV) and biomechanical strength of femur were measured by micro-CT scanning. Serum levels of calcium (Ca), phosphorus (P), osteocalcin (BGP) and alkaline phosphatase (ALP) were measured by automatic biochemical analyzer. The expressions of tissue inhibitor of metalloproteinases 1 (TIMP-1) and monocyte chemotactic protein 1 (MCP-1) in bone homogenate were detected by Immunohistochemistry. Results: Compared with sham operation group, the expression level of ERα gene in bone tissue of model group was decreased significantly, the levels of BMD, BV/TV, Tb. Th, maximum load, rigidity coefficient, Ca and P were decreased, while the levels of Tb. Sp, BGP and ALP were increased significantly (P＜0.05). Compared with the sham operation group, the expression level of TIMP-1 protein in the bone tissue of the model group was significantly decreased, while that of MCP-1 protein was increased, while that of the ERα overexpression group was increased while that of MCP-1 was decreased (P＜0.05).The levels of ERα gene expression, BMD, BV/TV, TB. Th, maximum load, rigidity coefficient, Ca and P in the ERα overexpression group were significantly higher than those in the model group, while Tb. Sp, BGP and ALP were significantly lower (P＜0.05). Compared with the sham operation group, mean optical density of TIMP-1 in the bone tissue of the model group was significantly decreased, while that of MCP-1 was significantly increased, and that of the ERα overexpression group was significantly increased while that of MCP-1 was significantly decreased (P＜0.05). Conclusion: ERα gene overexpression can improve osteoporosis by regulating bone mineral density, bone parameters, bone metabolism, calcium and phosphorus metabolic indicators and the expression levels of TIMP-1 and MCP-1 in tissues.

Early and mid-term results of transarticular external fixation in the treatment of supination-external rotation type IV equivalent ankle fractures.

PURPOSE: To investigate the early and mid-term results of open reduction and internal fixation (ORIF) with transarticular external fixation (TEF) but no deltoid ligament repair (DLR) in the treatment of supination-external rotation type IV equivalent (SER IV E) ankle fractures (AO/OTA classification 44-B 3.1) and provide evidence for clinical practice. METHODS: This study cohort consisted of 22 patients with SER IV E ankle fractures that underwent ORIF with TEF but no DLR between December 2011 and December 2014. There were 13 males and 9 females, mean age 38.9 years (range, 17-73 years). Eight cases involved the left side and 14 the right side. The causes of fractures included road traffic accidents (11 cases), falling from height (6 cases) and sports injuries (5 cases). The mean period of hospitalization was 9.8 days (range, 6-14 days). For all the patients, MRI and three-dimensional CT were done before surgery and X-rays done preoperatively and during follow-ups. The external frame was kept for 8-10 weeks. The preoperative American Orthopedic Foot and Ankle Society (AOFAS) ankle-hindfoot score was 56.86 ± 4.400, the Medical Outcomes Short Form 36-item (SF-36) questionnaire score was 57.41 ± 4.102 and the visual analog score (VAS) was 5.50 ± 1.058. Patients' main complaints about inconvenience of daily life were also recorded. RESULTS: All the 22 patients were followed up for 24-63 months (mean, 33.6 months). None of them developed nonunion during the follow-up; pin site infection was observed in one patient and posttraumatic osteoarthritis in another. At the final follow-up, the average AOFAS score, SF-36 score and VAS score were respectively 90.59 ± 5.096, 79.59 ± 5.394 and 1.82 ± 1.181, which were significantly improved compared with the preoperative data (t = 26.221, p < 0.001; t = 11.910, p < 0.001; t = 11.571, p < 0.001). The therapeutic effect was excellent in 13 cases, good in 7 cases and fair in 2 cases, with a good-excellent rate of 90.9%. Patients' main complaints were inconvenience of clothing (17 cases) and extremity cleaning (5 cases). CONCLUSION: In the treatment of SER IV E ankle fractures, ORIF with TEF but no DLR can achieve satisfactory outcome, but long-term effect should be confirmed by large sample randomized controlled trials.

Middle-term follow-up results of Pipkin type IV femoral head fracture patients treated by reconstruction plate and bioabsorbable screws.

PURPOSE: To investigate the mid-term curative effects of the treatment of Pipkin type IV femoral head fractures using a reconstruction plate and bioabsorbable screws and provide the evidence for clinical practice. METHODS: From February 2010 to September 2014, 21 patients with Pipkin type IV femoral head fractures were treated surgically. There were 13 males and 8 females with an average age of 41.1 years (range, 20-65 years). The causes of the fractures included traffic accidents (13 cases), falls from a height (four cases), heavy lifting injuries (three cases), and sport injury (one case). All patients were followed up with radiography and three-dimensional reconstruction computed tomography and other checks and any complications were actively managed. Closed reduction of fracture-dislocation of the hip was attempted under general anesthesia using the Kocher-Langenbeck approach. Femoral head fractures were treated with internal fixation or excision based on the size of the fracture fragments, whereas acetabular fractures were fixed with a reconstruction plate and screws following anatomic reduction. RESULTS: The incisions healed by primary intention in all patients after surgery, without any infection, deep venous thrombosis, or other complications. All 21 patients were followed up for 36-76 months, with an average follow-up duration of 49 months. Postoperative imaging data showed that all dislocations and fractures were anatomically reduced, and bony union of the fractures was achieved. Heterotopic ossification was found in four patients, post-traumatic osteoarthritis in three, and avascular necrosis of the femoral head in two. At the final follow-up, the assessment of hip joint function according to the Thompson-Epstein scoring scale was excellent in 10 cases, good in six cases, fair in three cases, and poor in two cases. The rate of excellent and good functional outcomes was 76.1%. CONCLUSION: The mid-term curative effects of a reconstruction plate and bioabsorbable screws in the treatment of Pipkin type IV femoral head fractures is significant, and such the treatment can significantly improve the patient's joint function and quality of life.

[Study on the association between total plasma homocysteine levels, dietary habits and the risk of gastric cancer].

OBJECTIVE: To explore the relationship between total plasma homocysteine (tHcy) levels, dietary habits and susceptibility of gastric cancer (CGC) in Yangzhong and Yixing cities, the two high GC risk areas in Jiangsu province. METHODS: A population-based case-control study was conducted including 391 histologically-confirmed adenocarcinoma GC cases and 608 age and sex frequency-matched cancer-free controls. The plasma tHcy concentration was measured by enzymatic biochemical assay of homocysteine on microtiter plates, using crude lysate containing recombinant methionine 7-lyase. The relationship between different tHcy levels and risk of GC was analyzed and factors as vegetables and fruits intake, smoking and drinking status were also evaluated together with tHey levels on the risk of GC. RESULTS: The average tHcy levels in GC cases were significantly higher than that in controls (P = 0.002). In addition, according to the quartile levels (7.9, 10.1, 13.7 micromol/L) in the controls, the risks of GC had an increase of 67% (adjusted OR = 1.67, 95% CI: 1.12-2.48), 98% (adjusted OR = 1.98, 95% CI: 1.33-2.94) and 112% (adjusted OR = 2.12, 95% CI: 1.44-3.15) compared to the lowest quartile of tHcy (< or = 7.9 micromol/L), respectively while the increasing trend was significantly noticed (chi2 = 15.78, P < 0.001). The increase of vegetables and fruits intake could decrease the risk of GC. Results from crossover analyses indicated that subjects with less vegetables and fruits intake or both smoking drinking together with plasma tHcy >15.0 micromol/L could increase the GC risk, when compared to the effect on GC risk of each factor. CONCLUSION: These findings supported the hypothesis that the high level of plasma tHcy and the badness dietary habits were associated to the increased risk of GC. Further larger scale and genetics involved studies on the environment and genetic factors were needed to confirm our findings.