Higher Accuracy of Arthroscopy Compared to MRI in the Diagnosis of Chondral Lesions in Acute Ankle Fractures: A Prospective Study.

Even after successful surgery for acute ankle fractures, many patients continue having complaints. A possible explanation is the presence of concomitant chondral lesions. The aim of this study is to investigate the accuracy of MRI compared to that of arthroscopy in the assessment of chondral lesions in acute ankle fractures. In this prospective single-center study, patients presenting with acute ankle fractures over a period of three years were identified. A preoperative MRI was performed within a maximum of 10 days after trauma. During surgery, ankle arthroscopy was also performed. The International Cartilage Repair Society (ICRS) cartilage lesion classification was used to grade the detected chondral lesions. To localize the chondral lesions, the talar dome was divided into eight zones and the tibial/fibular articular surfaces into three zones. In total, 65 patients (28 females) with a mean age of 41.1 ± 15 years were included. In the MRI scans, 70 chondral lesions were detected (69.2% of patients) affecting mostly the tibial plafond (30%) and mostly graded as ICRS 3. The mean lesion area measured was 20.8 mm2. In the arthroscopy, 85 chondral lesions were detected (70.8% of patients) affecting mostly the medial surface of the talar dome (25.9%) and mostly graded ICRS 3. The mean lesion area measured was 43.4 mm2. The highest agreement between the two methods was observed in the size estimation of the chondral lesions. The present study shows the reduced accuracy of MRI when compared to arthroscopy in the assessment of traumatic chondral lesions in the setting of acute ankle fractures especially regarding lesion size. MRI remains an essential instrument in the evaluation of such lesions; however, surgeons should take this discrepancy into consideration, particularly the underestimation of chondral lesions' size in the preoperative planning of surgical treatment and operative technique.

Titanium induces pro-inflammatory and tissue-destructive responses in primary human macrophages.

Implants and medical devices are efficient and practical therapeutic solutions for a multitude of pathologies. Titanium and titanium alloys are used in orthopedics, dentistry, and cardiology. Despite very good mechanical properties, and corrosion resistance titanium implants can fail due to inflammatory or tissue-degradation related complications. Macrophages are major immune cells that control acceptance of failure of the implant. In this study, for the first time, we have performed a systematic analysis of the response of differentially activated human macrophages (M(Control), M(IFNγ) and M(IL-4)) to the polished and porous titanium surfaces in order to identify the detrimental effect of titanium leading to the tissue destruction and chronic inflammation. Transcriptome analysis revealed that the highest number of differences between titanium and control settings are found in M(IL-4) that model healing type of macrophages. RT-qPCR analysis confirmed that both polished and porous titanium affected expression of cytokines, chitinases/chitinase-like proteins and matrix metalloproteinases. Titanium-induced release and activation of MMP7 by macrophages was enhanced by fibroblasts in both juxtacrine and paracrine cell interaction models. Production of titanium-induced MMPs and cytokines associated with chronic inflammation were independent of the presence of Staphylococcus aureus. MMP7, one of the most pronounced tissue-destroying factor and chitinase-like protein YKL-40 were expressed in CD68+ macrophages in peri-implant tissues of patients with orthopedic implants. In summary, we demonstrated that titanium induces pro-inflammatory and tissue-destructing responses mainly in healing macrophages, and the detrimental effects of titanium surfaces on implant-adjacent macrophages are independent on the bacterial contamination.

ASXL1 mutations are associated with a response to alvocidib and 5-azacytidine combination in myelodysplastic neoplasms.

Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMAs) are promising therapeutic approaches in acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS). Alvocidib, a cyclin-dependent kinase 9 (CDK9) inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has previously shown clinical activity in AML. Availability of biomarkers for response to the alvocidib + 5- AZA could also extend the rationale of this treatment concept to high-risk MDS. In this study, we performed a comprehensive in vitro assessment of alvocidib and 5-AZA effects in n=45 high-risk MDS patients. Our data revealed additive cytotoxic effects of the combination treatment. Mutational profiling of MDS samples identified ASXL1 mutations as predictors of response. Further, increased response rates were associated with higher gene-expression of the pro-apoptotic factor NOXA in ASXL1 mutated samples. The higher sensitivity of ASXL1 mutant cells to the combination treatment was confirmed in vivo in ASXL1Y588X transgenic mice. Overall, our study demonstrated augmented activity for the alvocidib + 5-AZA combination in higher-risk MDS and identified ASXL1 mutations as a biomarker of response for potential stratification studies.

A Multi-Disciplinary MRI Assessment May Optimize the Evaluation of Chondral Lesions in Acute Ankle Fractures: A Prospective Study.

Chondral lesions (CL) in the ankle following acute fractures are frequently overlooked immediately after the injury or diagnosed at a later stage, leading to persistent symptoms despite successful surgery. The literature presents a wide range of discrepancies in the reported incidence of CLs in acute ankle fractures. The objective of this prospective study is to provide a precise assessment of the occurrence of chondral lesions (CLs) in acute ankle fractures through MRI scans conducted immediately after the trauma and prior to scheduled surgery. Furthermore, the study aims to highlight the disparities in the interpretation of these MRI scans, particularly concerning the size and extent of chondral damage, between radiologists and orthopedic surgeons. Over the period of three years, all patients presenting with an unstable ankle fracture that underwent operative treatment were consecutively included in this single-center prospective study. Preoperative MRIs were obtained for all included patients within 10 days of the trauma and were evaluated by a trauma surgeon and a radiologist specialized in musculoskeletal MRI blinded to each other's results. The location of the lesions was documented, as well as their size and ICRS classification. Correlations and kappa coefficients as well as the p-values were calculated. A total of 65 patients were included, with a mean age of 41 years. The evaluation of the orthopedic surgeon showed CLs in 52.3% of patients. CLs occurred mainly on the tibial articular surface (70.6%). Most talar lesions were located laterally (11.2%). The observed CLs were mainly ICRS grade 4. According to the radiologist, 69.2% of the patients presented with CLs. The most common location was the talar dome (48.9%), especially laterally. Most detected CLs were graded ICRS 3a. The correlation between the two observers was weak/fair regarding the detection and classification of CLs and moderate regarding the size of the detected CLs. To enhance the planning of surgical treatment for ankle chondral lesions (CLs), it may be beneficial to conduct an interdisciplinary preoperative assessment of the performed scans. This collaborative approach can optimize the evaluation of ankle CLs and improve overall treatment strategies.

Inhibition of lysyl oxidases synergizes with 5-azacytidine to restore erythropoiesis in myelodysplastic and myeloid malignancies.

Limited response rates and frequent relapses during standard of care with hypomethylating agents in myelodysplastic neoplasms (MN) require urgent improvement of this treatment indication. Here, by combining 5-azacytidine (5-AZA) with the pan-lysyl oxidase inhibitor PXS-5505, we demonstrate superior restoration of erythroid differentiation in hematopoietic stem and progenitor cells (HSPCs) of MN patients in 20/31 cases (65%) versus 9/31 cases (29%) treated with 5-AZA alone. This effect requires direct contact of HSPCs with bone marrow stroma components and is dependent on integrin signaling. We further confirm these results in vivo using a bone marrow niche-dependent MN xenograft model in female NSG mice, in which we additionally demonstrate an enforced reduction of dominant clones as well as significant attenuation of disease expansion and normalization of spleen sizes. Overall, these results lay out a strong pre-clinical rationale for efficacy of combination treatment of 5-AZA with PXS-5505 especially for anemic MN.

Anthropometric measurements of the pediatric hip using CT-based simulated anteroposterior radiographs of the pelvis.

Normal anatomical variants and pathological deformities of the pediatric hip can only be differentiated after a prior definition of normal ranges for anthropometric parameters with increasing age. Aim of the present study was to provide reliable reference values of the pediatric hip morphometry, using computed tomography (CT)-based rotation-corrected summation images of the pelvis that simulate the widely available plain radiograph-based measurements, but offer the higher precision of the CT technique. This retrospective study included 85 patients (170 hips) under 15 years of age (0-15). The measured anthropometric parameters included femur head extrusion index, lateral center-edge angle, acetabular inclination, Tönnis angle, and femoral neck-shaft angle. Mean values, range, SD, P values, intra-rater, and inter-rater reliability were calculated. All measurements correlated with age. None of the measurements correlated with gender or side. Rapid growth phases were noted in all measurements at the age of 12 (14 in males and 11 in females). The inter-rater and intra-rater reliability was high (range inter/intraclass correlation coefficient 0.926-0.998 Cronbach's alpha 0.986-0.998). The present work provides age- and gender-related normative values of the classically used hip measurements as well as growth phases describing pediatric hip morphology in a broad age range. A discrepancy was noted between the values measured in the current study and the classical X-ray-based reference values in the literature especially for the Tönnis angle and LCEA values. This suggests that the rotation and inclination correction in the CT-based techniques might have the advantage of compensating for a possible overestimation in the conventional X-ray-based methods.

Anthropometric three-dimensional computed tomography reconstruction measurements of the acetabulum in children/adolescents.

The key element for differentiation between normal anatomical variants and pathological deformities is the prior definition of normal ranges for anthropometric parameters of acetabulum according to each age group. Aim of the present study is to analyze the development of the acetabulum in children/adolescents by accurate anthropometric measurements using 3D-CT scans and determine the variations occurring depending on age, gender and/or side. This retrospective observational study included 85 patients (170 hips) under 15 years of age (0-15) undergoing 1.5mm CT scanning for non-hip related reasons. The measurements were performed by 2 board-certified orthopaedic surgeons. Each year of life represented an age group forming a total of 16 groups. Median number of patients per age group was 12 (range 4-16). The anthropometric parameters included acetabular volume, inclination, version, depth (coronal and axial), width (coronal and axial), Tönnis angle as well as anterior and posterior acetabular sector angles. Mean values, range, standard deviation, p-values, intra- and interrater reliability were calculated. All measurement values correlated significantly with age. Statistically, there was no side or gender related difference. Rapid growth phases were observed at the age of 11-12. The inter- and intrarater reliability was high (range ICC 0.8-0.99, Cronbach alpha 0.86-0.99, Bland-Altman good agreement). The present data provides age- and gender-related normative values as well as growth phases describing acetabular morphology. It should help paediatricians as well as paediatric and orthopaedic surgeons as a tool for early diagnosis of deformity and guidance for possible procedures.

Preclinical evaluation of eltrombopag in a PDX model of myelodysplastic syndromes.

Preclinical research of myelodysplastic syndromes (MDSs) is hampered by a lack of feasible disease models. Previously, we have established a robust patient-derived xenograft (PDX) model for MDS. Here we demonstrate for the first time that this model is applicable as a preclinical platform to address pending clinical questions by interrogating the efficacy and safety of the thrombopoietin receptor agonist eltrombopag. Our preclinical study included n = 49 xenografts generated from n = 9 MDS patient samples. Substance efficacy was evidenced by FACS-based human platelet quantification and clonal bone marrow evolution was reconstructed by serial whole-exome sequencing of the PDX samples. In contrast to clinical trials in humans, this experimental setup allowed vehicle- and replicate-controlled analyses on a patient-individual level deciphering substance-specific effects from natural disease progression. We found that eltrombopag effectively stimulated thrombopoiesis in MDS PDX without adversely affecting the patients' clonal composition. In conclusion, our MDS PDX model is a useful tool for testing new therapeutic concepts in MDS preceding clinical trials.

Humanized three-dimensional scaffold xenotransplantation models for myelodysplastic syndromes.

Patient-derived xenograft (PDX) models have emerged as versatile preclinical platforms for investigation of functional pathomechanisms in myelodysplastic syndromes (MDS) and other myeloid neoplasms. However, despite increasingly improved methodology, engraftment efficiencies frequently remain low. Humanized three-dimensional scaffold models (ossicle xenotransplantation models) in immunocompromised mice have recently been found to enable improved engraftment rates of healthy and malignant human hematopoiesis. We therefore interrogated the feasibility of using four different three-dimensional ossicle-based PDX models for application with primary MDS samples. In a fully standardized comparison, we evaluated scaffold materials such as Gelfoam, extracellular matrix (ECM), and human or xenogenous bone substance in comparison to intrafemoral (IF) co-injection of bone marrow (BM)-derived mesenchymal stromal cells (MSCs) and CD34+ hematopoietic stem and progenitor cells (HSPCs). Our study included13 primary MDS patient samples transplanted in parallel according to these five different conditions. Engraftment of MDS samples was assessed by flow cytometry, immunohistological staining, and molecular validation. We determined that three-dimensional ossicle-based methods achieved higher relative rates of engraftment and enabled long-term retrievability of patient-derived MSCs from implanted ossicles. In summary, HSPCs and MSCs derived from MDS BM, which did not significantly engraft in NSG mice after intrafemoral injection, were able to colonize humanized scaffold models. Therefore, these models are promising new xenotransplantation techniques for addressing preclinical and functional questions of the interaction between hematopoiesis and the BM niche in MDS.

Anthropometry of the proximal femur and femoral head in children/adolescents using three-dimensional computed tomography-based measurements.

PURPOSE: Defining normal anthropometric ranges of proximal femur and femoral head for each age group in children/adolescents is a necessity when differentiating normal anatomical variants from pathological deformities. Aim of this study is to define a set of normal anthropometric parameters based on 3D-CT measurements in normal asymptomatic children/adolescents and analyse the variations arising depending on age, side, and/or gender. METHODS: Morphology of the proximal femur was retrospectively assessed in 170 hips (85 children, < 15 years). Measurements included covered femoral head volume (CFHV), femoral head diameter (FHD), femoral head extrusion index (FHEI), coronal alpha angle (CAA), lateral centre-edge angle (LCEA), anterior (AOS) and posterior head-neck offset (POS) and femoral neck-shaft angle (FNSA). Correlation analyses as well as inter- and intra-rater reliability were performed. RESULTS: CFHV, LCEA, FHD and AOS/POS increased with age and FHEI, CAA, and FNSA decreased with age. None of the measurements correlated with the side. AOS showed a poor correlation with gender. Rapid growth phases were observed at the age of 1, 7 and 11. The inter- and intra-rater reliability was high (range ICC 0.8-0.99 Cronbach alpha 0.86-0.99). CONCLUSION: This data delivers a description of growth phases as well as gender and age-correlated reference values of the proximal femoral morphology that could be used by paediatricians and orthopaedic/paediatric surgeons to early diagnose proximal femur deformities and provide guidance in the planning of possible operations.

Bone marrow derived stromal cells from myelodysplastic syndromes are altered but not clonally mutated in vivo.

The bone marrow (BM) stroma in myeloid neoplasms is altered and it is hypothesized that this cell compartment may also harbor clonal somatically acquired mutations. By exome sequencing of in vitro expanded mesenchymal stromal cells (MSCs) from n = 98 patients with myelodysplastic syndrome (MDS) and n = 28 healthy controls we show that these cells accumulate recurrent mutations in genes such as ZFX (n = 8/98), RANK (n = 5/98), and others. MDS derived MSCs display higher mutational burdens, increased replicative stress, senescence, inflammatory gene expression, and distinct mutational signatures as compared to healthy MSCs. However, validation experiments in serial culture passages, chronological BM aspirations and backtracking of high confidence mutations by re-sequencing primary sorted MDS MSCs indicate that the discovered mutations are secondary to in vitro expansion but not present in primary BM. Thus, we here report that there is no evidence for clonal mutations in the BM stroma of MDS patients.

Proteins Marking the Sequence of Genotoxic Signaling from Irradiated Mesenchymal Stromal Cells to CD34+ Cells.

Non-targeted effects (NTE) of ionizing radiation may initiate myeloid neoplasms (MN). Here, protein mediators (I) in irradiated human mesenchymal stromal cells (MSC) as the NTE source, (II) in MSC conditioned supernatant and (III) in human bone marrow CD34+ cells undergoing genotoxic NTE were investigated. Healthy sublethal irradiated MSC showed significantly increased levels of reactive oxygen species. These cells responded by increasing intracellular abundance of proteins involved in proteasomal degradation, protein translation, cytoskeleton dynamics, nucleocytoplasmic shuttling, and those with antioxidant activity. Among the increased proteins were THY1 and GNA11/14, which are signaling proteins with hitherto unknown functions in the radiation response and NTE. In the corresponding MSC conditioned medium, the three chaperones GRP78, CALR, and PDIA3 were increased. Together with GPI, these were the only four altered proteins, which were associated with the observed genotoxic NTE. Healthy CD34+ cells cultured in MSC conditioned medium suffered from more than a six-fold increase in γH2AX focal staining, indicative for DNA double-strand breaks, as well as numerical and structural chromosomal aberrations within three days. At this stage, five proteins were altered, among them IQGAP1, HMGB1, and PA2G4, which are involved in malign development. In summary, our data provide novel insights into three sequential steps of genotoxic signaling from irradiated MSC to CD34+ cells, implicating that induced NTE might initiate the development of MN.

Genotoxic Bystander Signals from Irradiated Human Mesenchymal Stromal Cells Mainly Localize in the 10-100 kDa Fraction of Conditioned Medium.

Genotoxic bystander signals released from irradiated human mesenchymal stromal cells (MSC) may induce radiation-induced bystander effects (RIBEs) in human hematopoietic stem and progenitor cells (HSPC), potentially causing leukemic transformation. Although the source of bystander signals is evident, the identification and characterization of these signals is challenging. Here, RIBEs were analyzed in human CD34+ cells cultured in distinct molecular size fractions of medium, conditioned by 2 Gy irradiated human MSC. Specifically, γH2AX foci (as a marker of DNA double-strand breaks) and chromosomal instability were evaluated in CD34+ cells grown in approximate (I) < 10 kDa, (II) 10-100 kDa and (III) > 100 kDa fractions of MSC conditioned medium and un-/fractionated control medium, respectively. Hitherto, significantly increased numbers of γH2AX foci (p = 0.0286) and aberrant metaphases (p = 0.0022) were detected in CD34+ cells grown in the (II) 10-100 kDa fraction (0.67 ± 0.10 γH2AX foci per CD34+ cell ∨ 3.8 ± 0.3 aberrant metaphases per CD34+ cell sample; mean ± SEM) when compared to (I) < 10 kDa (0.19 ± 0.01 ∨ 0.3 ± 0.2) or (III) > 100 kDa fractions (0.23 ± 0.04 ∨ 0.4 ± 0.4) or un-/fractionated control medium (0.12 ± 0.01 ∨ 0.1 ± 0.1). Furthermore, RIBEs disappeared after heat inactivation of medium at 75 °C. Taken together, our data suggest that RIBEs are mainly mediated by the heat-sensitive (II) 10-100 kDa fraction of MSC conditioned medium. We postulate proteins as RIBE mediators and in-depth proteome analyses to identify key bystander signals, which define targets for the development of next-generation anti-leukemic drugs.

[Risk factors affecting mortality in patients with hip fractures at a regional trauma center]. / Einflussfaktoren auf die Mortalität bei Patienten mit hüftgelenknahen Frakturen an einem regionalen Traumazentrum.

BACKGROUND: Primary care hospitals and regional trauma centers play an essential role in the treatment of hip fractures. OBJECTIVE: This study investigated the relationship between patient-related parameters and in-hospital mortality as well as complications of hip fractures at a regional trauma center. METHODS: In a retrospective study, data were collected from all patients > 60 years admitted over 2 years to a regional trauma center with a hip fracture. Patient-related parameters included age, sex, fracture location, method of surgical treatment, time of surgery, duration of surgery, length of inpatient stay, blood transfusion, complications, comorbidities, use of anticoagulant medication and need for postoperative intensive care treatment. The relationship between these parameters and hospital mortality as well as complications was investigated. RESULTS: Data were collected from 360 patients undergoing 335 surgeries (f:m 225:110) with a mean age of 83 ± 8 years. The total in-hospital mortality rate was 7.76% (n = 26). Factors increasing in-hospital mortality included: age > 85 years (odds ratio [OR] 5.126; 95% confidence interval [CI] 0.665-39.498; p = 0.1167); male sex (OR 1.85 95%-CI [0.82-4.14]; p = 0.0555); time of surgery > 24 h (OR 1.896 95%-CI [0.661-5.441]; p = 0.2341); ≥ 3 comorbidities (OR 10.61 95%-CI [3.681-27.501]; p < 0.0001); intake of anticoagulants (OR 6.19 95%-CI [2.69-14.24]; p < 0.0001) and postoperative intensive care (OR 5.9 95%-CI [2.56-13.76]; p < 0.0001). CONCLUSION: In the present study a statistically significant influence of the number of comorbidities or Charlson comorbidity index, the intake of anticoagulant drugs and need for postoperative intensive care treatment on the in-hospital mortality of patients with proximal femoral fractures in a regional trauma center was found.

High erythroferrone expression in CD71+ erythroid progenitors predicts superior survival in myelodysplastic syndromes.

Ineffective erythropoiesis and iron overload are common in myelodysplastic syndromes (MDS). Erythroferrone (ERFE) and growth/differentiation factor 15 (GDF15) are two regulators of iron homeostasis produced by erythroid progenitors. Elevated systemic levels of ERFE and GDF15 in MDS are associated with dysregulated iron metabolism and iron overload, which is especially pronounced in MDS with SF3B1 gene mutations. However, the role of ERFE and GDF15 in MDS pathogenesis and their influence on disease progression are largely unknown. Here, we analyzed the expression of ERFE and GDF15 in CD71+ erythroid progenitors of n = 111 MDS patients and assessed their effects on patient survival. The expression of ERFE and GDF15 in MDS was highly aberrant. Unexpectedly, ERFE expression in erythroprogenitors was highly relevant for MDS prognosis and independent of International Prognostic Scoring System (IPSS) stratification. Although ERFE expression was increased in patients with SF3B1 mutations, it predicted overall survival (OS) in both the SF3B1wt and SF3B1mut subgroups. Of note, ERFE overexpression predicted superior OS in the IPSS low/Int-1 subgroup and in patients with normal karyotype. Similar observations were made for GDF15, albeit not reaching statistical significance. In summary, our results revealed a strong association between ERFE expression and MDS outcome, suggesting a possible involvement of ERFE in molecular MDS pathogenesis.

Incidence of concomitant chondral/osteochondral lesions in acute ankle fractures and their effect on clinical outcome: a systematic review and meta-analysis.

INTRODUCTION: Despite successful osteosynthesis, some patients report residual symptoms after ankle fractures. One of the reasons behind the postoperative complaints might be traumatic concomitant chondral lesions (CL) and/or osteochondral lesions (OCL) within the ankle joint. The study aims to systematically review the incidence of CL and/or OCL in ankle fractures and to assess their effect on the clinical outcome. MATERIALS AND METHODS: This work was conducted according to PRISMA checklists. A systematic literature search was performed using following keywords: "Ankle Fractures" OR "Trimalleolar Fracture" OR "Bimalleolar Fracture" OR "Maisonneuve fracture" OR "Malleolus Fracture" AND "Cartilage" OR "Cartilage Diseases" OR "Cartilage, Articular" OR "chondral" up to March 2020. The identified articles were analysed to determine the incidence of CL and/or OCL. Included studies in the meta-analysis assessed possible cartilage damage through arthroscopy or MRI immediately after traumatic ankle fractures and described the postoperative clinical outcome. RESULTS: The search identified a total of 111 publications; 19 described the incidence of CL and/or OCL after ankle fractures; six met the criteria to be included in the meta-analysis: five (n = 293) diagnosed CL and/or OCL through arthroscopy during ORIF and one study (n = 153) used preoperative MRI. The clinical outcome was evaluated in four studies (n = 177) using AOFAS score and in two (n = 269) using FAOS score. The mean incidence of arthroscopically detected CL and/or OCL was 65 ± 21% [95% CI 53.9 to 76.72]. The cumulative meta-analysis sample size comprised a total of 400 Patients (170 with and 230 without CL and/or OCL) available for a mean follow-up of 23.9 ± 11.5 months [95% CI 11.79 to 36.07]. The average age was 44.3 ± 5.5 years [95% CI 38.57 to 50.13]. The meta-analysis revealed a mean AOFAS score of 91.2 ± 4.8 [95% CI 83.53 to 98.93] with versus 94.4 ± 4.7 [95% CI 86.81 to 102.07] without CL and/or OCL (p = 0.15) and a mean FAOS score of 73.2 ± 11.31 [95% CI - 28.44 to 174.85] with versus 79.0 ± 18.4 [95% CI - 86.77 to 244.87] without CL and/or OCL (p = 0.18). CONCLUSIONS: CL and/or OCL appear very frequently after ankle fractures. A tendency towards a favourable short- to mid-term clinical outcome was noticed in ankle fractures without CL and/or OCL, however without reaching statistical significance. LEVEL OF EVIDENCE: Level I.