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Abstract Radiotherapy of head and neck squamous cell carcinoma can lead to long-term complications like osteoradionecrosis, resulting in severe impairment of the jawbone. Current standard procedures require a 6-month wait after irradiation before dental reconstruction can begin. A comprehensive characterization of the irradiation-induced molecular and functional changes in bone cells could allow the development of novel strategies for an earlier successful dental reconstruction in patients treated by radiotherapy. The impact of ionizing radiation on the bone-forming alveolar osteoblasts remains however elusive, as previous studies have relied on animal-based models and fetal or animal-derived cell lines. This study presents the first in vitro data obtained from primary human alveolar osteoblasts. Primary human alveolar osteoblasts were isolated from healthy donors and expanded. After X-ray irradiation with 2, 6 and 10 Gy, cells were cultivated under osteogenic conditions and analyzed regarding their proliferation, mineralization, and expression of marker genes and proteins. Proliferation of osteoblasts decreased in a dose-dependent manner. While cells recovered from irradiation with 2 Gy, application of 6 and 10 Gy doses not only led to a permanent impairment of proliferation, but also resulted in altered cell morphology and a disturbed structure of the extracellular matrix as demonstrated by immunostaining of collagen I and fibronectin. Following irradiation with any of the examined doses, a decrease of marker gene expression levels was observed for most of the investigated genes, revealing interindividual differences. Primary human alveolar osteoblasts presented a considerably changed phenotype after irradiation, depending on the dose administered. Mechanisms for these findings need to be further investigated. This could facilitate improved patient care by re-evaluating current standard procedures and investigating faster and safer reconstruction concepts, thus improving quality of life and social integrity.

Objectives This study aimed to identify main sex-specific correlates of cardiorespiratory fitness (CRF) in a population-based, urban sample of Swedish adults.Design Cross-sectional.Setting Multi-site study at university hospitals, data from the Gothenburg site.Participants A total of 5308 participants (51% women, aged 50–64 years) with a valid estimated VO2max, from submaximal cycle test, in the Swedish CArdioPulmonary bioImage Study (SCAPIS), were included.Primary and secondary outcomes A wide range of correlates were examined including (a) sociodemographic and lifestyle behaviours, (b) perceived health, anthropometrics and chronic conditions and (c) self-reported as well as accelerometer-derived physical activity and sedentary behaviours. Both continuous levels of estimated VO2max as well as odds ratios (OR) and confidence intervals (CI)s of low VO2max (lowest sex-specific tertile) were reported.Results In multivariable regression analyses, higher age, being born abroad, short education, high waist circumference, poor perceived health, high accelerometer-derived time in sedentary and low in vigorous physical activity, as well as being passive commuter, correlated independently and significantly with low VO2max in both men and women (OR range 1.31–9.58). Additionally in men, financial strain and being an ex-smoker are associated with higher odds for low VO2max (OR 2.15; 95% CI 1.33 to 3.48 and OR 1.40; 95% CI 1.09 to 1.80), while constant stress with lower odds (OR 0.61; 95% CI 0.43 to 0.85). Additionally in women, being a regular smoker is associated with lower odds for low VO2max (OR 0.64; 95% CI 0.45 to 0.92).Conclusions The present study provides important reference material on CRF and correlates of CRF in a general middle-aged population, which can be valuable for future research, clinical practice and public health work. If relations are causal, increased knowledge about specific subgroups will aid in the development of appropriate, targeted interventions.

Abstract Lactate is a central metabolite in brain physiology but also contributes to tumor development. Glioblastoma (GB) is the most common and malignant primary brain tumor in adults, recognized by angiogenic and invasive growth, in addition to its altered metabolism. We show herein that lactate fuels GB anaplerosis by replenishing the tricarboxylic acid (TCA) cycle in absence of glucose. Lactate dehydrogenases (LDHA and LDHB), which we found spatially expressed in GB tissues, catalyze the interconversion of pyruvate and lactate. However, ablation of both LDH isoforms, but not only one, led to a reduction in tumor growth and an increase in mouse survival. Comparative transcriptomics and metabolomics revealed metabolic rewiring involving high oxidative phosphorylation (OXPHOS) in the LDHA/B KO group which sensitized tumors to cranial irradiation, thus improving mouse survival. When mice were treated with the antiepileptic drug stiripentol, which targets LDH activity, tumor growth decreased. Our findings unveil the complex metabolic network in which both LDHA and LDHB are integrated and show that the combined inhibition of LDHA and LDHB strongly sensitizes GB to therapy.

Due to their advantages in applicability, patient-specific (CAD/CAM) reconstruction plates are increasingly used in fibula free flap mandible reconstruction. In addition, recently, CAD/CAM miniplates, with further advantages in postoperative management, have been introduced. However, biomechanical conditions induced by CAD/CAM systems remain partially unknown. This study aimed to evaluate the primary fixation stability of CAD/CAM fixators. For a patient-specific scenario, the biomechanical conditions induced in a one segmental fibula free flap stabilized using either a CAD/CAM reconstruction plate or CAD/CAM miniplates were determined using finite element analysis. The main output parameters were the strains between intersegmental bone surfaces and stresses in the fixation systems due to different biting scenarios. CAD/CAM miniplates resulted in higher mechanical strains in the mesial interosseous gap, whereas CAD/CAM reconstruction plate fixation resulted in higher strains in the distal interosseous gap. For all investigated fixation systems, stresses in the fixation systems were below the material yield stress and thus material failure would not be expected. While the use of CAD/CAM miniplates resulted in strain values considered adequate to promote bone healing in the mesial interosseous gap, in the distal interosseous gap CAD/CAM reconstruction plate fixation might result in more beneficial tissue straining. A mechanical failure of the fixation systems would not be expected.

Animal behaviour is governed by planning and executing movements. Here, using non-stereotypical movements to separate planning and executional processes, the authors find evidence for slow processes for planning and fast processes for movement execution.

Abstract Glioblastoma (GBM), the most malignant tumor of the central nervous system, is marked by its dynamic response to microenvironmental niches. In particular, this cellular plasticity contributes to the development of an immediate resistance during tumor treatment. Novel insights into the developmental trajectory exhibited by GBM show a strong capability to respond to its microenvironment by clonal selection of specific phenotypes. Using the same mechanisms, malignant GBM do develop intrinsic mechanisms to resist chemotherapeutic treatments. This resistance was reported to be sustained by the paracrine and autocrine glutamate signaling via ionotropic and metabotropic receptors. However, the extent to which glutamatergic signaling modulates the chemoresistance and transcriptional profile of the GBM remains unexplored. In this study we aimed to map the manifold effects of glutamate signaling in GBM as the basis to further discover the regulatory role and interactions of specific receptors, within the GBM microenvironment. Our work provides insights into glutamate release dynamics, representing its importance for GBM growth, viability, and migration. Based on newly published multi-omic datasets, we explored the and characterized the functions of different ionotropic and metabotropic glutamate receptors, of which the metabotropic receptor 3 (GRM3) is highlighted through its modulatory role in maintaining the ability of GBM cells to evade standard alkylating chemotherapeutics. We addressed the clinical relevance of GRM3 receptor expression in GBM and provide a proof of concept where we manipulate intrinsic mechanisms of chemoresistance, driving GBM towards chemo-sensitization through GRM3 receptor inhibition. Finally, we validated our findings in our novel human organotypic section-based tumor model, where GBM growth and proliferation was significantly reduced when GRM3 inhibition was combined with temozolomide application. Our findings present a new picture of how glutamate signaling via mGluR3 interacts with the phenotypical GBM transcriptional programs in light of recently published GBM cell-state discoveries.

The role of human papillomavirus (HPV) status for the prognosis of oropharyngeal cancers (OPCs) is discussed controversially. Here, we present an analysis of 144,969 head and neck cancer cases (ICD-10 codes: C00–C13) with a sub-cohort of 62,775 tumor cases of the oropharynx (C01, C09, and C10). To this end, de-identified data from electronic health records of about 60 healthcare organizations from 30 different countries were used. Odds ratios, hazard ratios (HRs), and Kaplan–Meier analyses were used to compare outcomes between different cancer entities of neoplasms of the base of the tongue (C01), of tonsils (C09), and of the oropharynx (C10) of women and men with and without HPV infection. To avoid the bias from different age distributions, the cohorts were balanced using propensity score matching. The 5-year survival rate for HPV-positive patients is somewhat better than that for HPV-negative patients, but for age- and sex-balanced cohorts, there remains no significant advantage for HPV-positive patients [HR, 1.126 (0.897–1.413)]. Looking at the different entities and HPV status for age-matched male and female patients separately, HPV is a significantly positive prognostic factor for female patients in some entities, whereas for male patients, it is only a positive prognostic factor for malignant neoplasms of oropharynx (C10) [HR, 1.077 (0.602–1.926)].

Summary: Proteolytic cleavage is an important post-translational mechanism to increase protein variability and functionality. In cancer, this process can be deregulated to shut off tumor-suppressive functions. Here, we report that in glioblastoma (GBM), the tumor suppressor ZBTB18 is targeted for protein cleavage by the intracellular protease calpain. The N-terminal (Nte) ZBTB18 cleaved fragment localizes to the cytoplasm and thus, is unable to exert the gene expression repressive function of the uncleaved protein. Mass spectrometry (MS) analysis indicates that the Nte ZBTB18 short form (SF) interacts with C-terminal (Cte) binding proteins 1 and 2 (CTBP1/2), which appear to be involved in HIF1A signaling activation. In fact, we show that the new ZBTB18 product activates HIF1A-regulated genes, which in turn lead to increased lipid uptake, lipid droplets (LD) accumulation, and enhanced metabolic activity. We propose that calpain-mediated ZBTB18 cleavage represents a new mechanism to counteract ZBTB18 tumor suppression and increase tumor-promoting functions in GBM cells.

Introduction: The overall survival among head and neck cancer patients is still low, even in a time of new therapy regimes. Regarding cancer patients’ survival, statin use has already proven to be associated with favorable survival outcomes. Our objective was to investigate the influence of statin medication on the overall survival of head and neck cancer patients. Methods: Retrospective clinical data of patients diagnosed with head and neck cancer (International Classification of Diseases codes: C00–C14) were retrieved from a real-world evidence database. The initial cohort was divided into patients with statin medication, who were assigned to building cohort I, and subjects without statin medication, who were assigned to cohort II, both matched by age, gender, and risk factors (nicotine and alcohol abuse/dependence). Subsequently, Kaplan–Meier and risk analyses were performed, and odds and hazard ratios were calculated. Results: After matching, each cohort contained 48,626 patients (cohort I = females: 15,409; (31.7%), males 33,212 (68.3%); mean age ± standard deviation (SD) at diagnosis 66.3 ± 11.4 years; cohort II = females: 15,432; (31.7%), males 33,187 (68.2%); mean age ± standard deviation (SD) at diagnosis 66.4 ± 11.5 years). Five-year survival was found to be significantly higher for cohort I, with 75.19%, respectively 70.48% for cohort II. These findings were correlated significantly with a risk of death of 15.9% (cohort I) and 17.2% (cohort II); the odds ratio was 0.91 (95% CI: 0.881–0.942) and the hazard ratio 0.80 (0.777–0.827). Conclusions: The results indicate that the five-year survival of head and neck cancer patients is significantly improved by statin medication. As this study was conducted retrospectively, our data must be interpreted with caution, especially since other potential influencing factors and the initial tumor stage were not available.

Objective: The human gut microbiome is strongly influenced by the administration of drugs, namely antibiotics. We hypothesized that the effectiveness of immunotherapy with pembrolizumab in oral squamous cell carcinoma patients is decreased by the administration of antibiotics three months before and after immunotherapy. Methods: We retrieved data from patients diagnosed with head and neck squamous cell carcinoma (HNSCC) (International Classification of Diseases [ICD]-10 codes C00-C14) and receiving immunotherapy with pembrolizumab from the TriNetX network. Two cohorts were built: patients in cohort I did not receive any antibiotics within three months before or up to three months after immunotherapy, while patients in cohort II were administered antibiotics at least once within three months before or after immunotherapy. To exclude confounders, we matched cohorts 1:1 for age, sex, secondary lymph node metastases, nicotine dependence, the insertion of feeding devices, body mass index (BMI) and severe sepsis. After defining the primary outcome as “death”, a Kaplan–Meier analysis was performed, and the risk ratio (RR), odds ratio (OR) and hazard ratio (HR) were calculated. Results: A total of 3651 patients were enrolled, and after matching, each cohort consisted of 1362 patients. Among cohorts I and II, 346 and 511 patients were deceased within one year (risk of death = 25.5 and 38.3%, respectively), whereby the risk difference was significant (p = 0.000; log-rank test). The RR was 0.68 (95% confidence interval: 0.60–0.76), OR was 0.57 (0.48–0.67) and HR was 0.58 (0.51–0.67). Conclusions: Our hypothesis was confirmed: administering antibiotics significantly decreases the drug effectiveness of immunotherapy. We hypothesize that this finding is associated with antibiotic-related changes in the gut microbiome. Prospective clinical studies on the gut microbiome in cancer patients are necessary to understand the complex ecosystem of microbiota during immunotherapy. Trial Registration: Due to the retrospective nature of the study, no registration was necessary.

Abstract Background The anatomical features of the gingiva and the clinical crowns and their interrelation, especially in aesthetically and functionally demanding areas, are important in complex dental or implant-retained prosthetic rehabilitations. This observational cross-sectional study was designed to evaluate gender- and age-related differences in the width of attached gingiva (WAG), the clinical crown length (CCL), and their interrelation in the anterior teeth to determine the relationship between the pink and white aesthetics. Methods Eighty (54 females, 26 males) fully dentate Caucasian participants between the ages of 20 and 25 years and 36 probands (23 females, 13 males) between the ages of 45 and 55 years were included in the present study. The CCL of the maxillary and mandibular canines, as well as the central incisors of the maxilla and mandible, were determined with a dental sliding caliper measuring from the middle margin of the gingiva at its deepest point to the incisal edge. The clinical investigation of the WAG was performed by inserting a periodontal probe into the gingival sulcus in the middle of the buccal surface to firstly measure the probing pocket depth. The distance between the gingival margin and mucogingival junction (MGJ) was then measured with a Beerendonk sliding caliper in the middle of the labial curvature, and the clinical WAG was determined by subtraction of the measured probing depth. For the statistical analysis, the Mann–Whitney U test, the Wilcoxon-Test, the Spearman’s rank correlation, and a two-factorial non-parametric analysis were used. Results There was no correlation between the CCL and the WAG in a healthy periodontium. Gender influenced the CCL, with men having significantly longer teeth than women in both maxilla (P ≤ 0.01) and mandible (P ≤ 0.05). Age did not influence the CCL significantly neither in the upper (P = 0.06) nor in the lower jaw (P = 0.33). Gender did not show to have a significant influence on the WAG of maxilla (P = 0.69) and mandible (P = 0.26). But differences in the WAG between young and old participants were observed in both upper (P ≤ 0.01) and lower jaw (P ≤ 0.05). Conclusion The present observational study demonstrated that the mean values of cohorts with mixed age groups and genders should not be considered when attempting to determine the ideal relationships between the pink and white aesthetics since the statistical analyses showed significant differences between different age groups and genders.

In order to investigate the effect of grain boundaries on the mechanical response in the micrometer and submicrometer levels, complementary experiments and molecular dynamics simulations were conducted on a model bcc metal, tantalum. Microscale pillar experiments (diameters of 1 and 2 μm) with a grain size of ~100–200 nm revealed a mechanical response characterized by a yield stress of ~1500 MPa. The hardening of the structure is reflected in the increase in the flow stress to 1700 MPa at a strain of ~0.35. Molecular dynamics simulations were conducted for nanocrystalline tantalum with grain sizes in the range of 20–50 nm and pillar diameters in the same range. The yield stress was approximately 6000 MPa for all specimens and the maximum of the stress–strain curves occurred at a strain of 0.07. Beyond that strain, the material softened because of its inability to store dislocations. The experimental results did not show a significant size dependence of yield stress on pillar diameter (equal to 1 and 2 um), which is attributed to the high ratio between pillar diameter and grain size (~10–20). This behavior is quite different from that in monocrystalline specimens where dislocation ‘starvation’ leads to a significant size dependence of strength. The ultrafine grains exhibit clear ‘pancaking’ upon being plastically deformed, with an increase in dislocation density. The plastic deformation is much more localized for the single crystals than for the nanocrystalline specimens, an observation made in both modeling and experiments. In the molecular dynamics simulations, the ratio of pillar diameter (20–50 nm) to grain size was in the range 0.2–2, and a much greater dependence of yield stress to pillar diameter was observed. A critical result from this work is the demonstration that the important parameter in establishing the overall deformation is the ratio between the grain size and pillar diameter; it governs the deformation mode, as well as surface sources and sinks, which are only important when the grain size is of the same order as the pillar diameter.

ObjectiveThe pathogenesis of diabetic polyneuropathy (DN) is poorly understood and given the increasing prevalence of DN, there is a need for clinical or imaging biomarkers that quantify structural and functional nerve damage. While clinical studies have found evidence of an association between elevated levels of troponin T (hsTNT) and N-terminal pro brain natriuretic peptide (proBNP) with microvascular compromise in type 2 diabetes (T2D), their implication in mirroring DN nerve perfusion changes remains unclear. The objective of this study was, therefore, to investigate whether hsTNT and proBNP assays are associated with MRI nerve perfusion in T2D.MethodsIn this prospective cross-sectional single-center case-control study, 56 participants (44 with T2D, 12 healthy control subjects) consented to undergo magnetic resonance neurography (MRN) including dynamic contrast-enhanced (DCE) perfusion imaging of the right leg. Using the extended Tofts model, primary outcome parameters that were quantified are the sciatic nerve’s microvascular permeability (Ktrans), the extravascular extracellular volume fraction (ve), and the plasma volume fraction (vp), as well as hsTNT and proBNP values from serological workup. Further secondary outcomes were clinical, serological, and electrophysiological findings.ResultsIn T2D patients, hsTNT was negatively correlated with Ktrans (r=-0.38; p=0.012) and ve (r=-0.30; p=0.048) but not with vp (r=-0.16; p=0.294). HsTNT, Ktrans, and ve were correlated with peroneal nerve conduction velocities (NCVs; r=-0.44; p=0.006, r=0.42; p=0.008, r=0.39; p=0.014), and tibial NCVs (r=-0.38;p=0.022, r=0.33; p=0.048, r=0.37; p=0.025). No such correlations were found for proBNP.ConclusionsThis study is the first to find that hsTNT is correlated with a decrease of microvascular permeability and a reduced extravascular extracellular volume fraction of nerves in patients with T2D. The results indicate that hsTNT may serve as a potential marker for the assessment of nerve perfusion in future studies on DN.

Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with a median survival of 15 months that has remained unchanged despite advances in the standard of care. GBM cells express human cytomegalovirus (HCMV) proteins, providing a unique opportunity for targeted therapy. We utilized our UNITE (UNiversal Intracellular Targeted Expression) platform to develop a multi-antigen DNA vaccine (ITI-1001) that codes for the HCMV proteins pp65, gB, and IE-1. The UNITE platform involves lysosomal targeting technology, fusing lysosome-associated membrane protein 1 (LAMP1) with target ntigens. We demonstrate evidence of increased antigen presentation by both MHC-I and -II, delivering a robust antigen-specific CD4 and CD8 T-cell response in addition to a strong humoral response. Using a syngeneic orthotopic GBM mouse model, therapeutic treatment with the ITI-1001 vaccine resulted in ~56% survival of tumor-bearing mice. Investigation of the tumor microenvironment showed significant CD4 infiltration as well as enhanced Th1 and cytotoxic CD8 T activation. Regulatory T cells were also upregulated after ITI-1001 vaccination. In addition, tumor burden negatively correlated with activated interferon (IFN)γ+ CD4 T cells, reiterating the importance of CD4 activation in ITI-1001 efficacy and in identifying treatment responders and non-responders. Further characterization of these two groups showed high infiltration of CD3+, CD4+, and CD8+ T cells in responders compared to non-responders. Thus, we show that vaccination with HCMV antigens using the ITI-1001-UNITE platform generates strong cellular and humoral immune responses, triggering significant antitumor activity, leading to enhanced survival in a mouse model of GBM.

Background and PurposeOutcome prediction after mechanical thrombectomy (MT) in patients with acute ischemic stroke (AIS) and large vessel occlusion (LVO) is commonly performed by focusing on favorable outcome (modified Rankin Scale, mRS 0–2) after 3 months but poor outcome representing severe disability and mortality (mRS 5 and 6) might be of equal importance for clinical decision-making.MethodsWe retrospectively analyzed patients with AIS and LVO undergoing MT from 2009 to 2018. Prognostic variables were grouped in baseline clinical (A), MRI-derived variables including mismatch [apparent diffusion coefficient (ADC) and time-to-maximum (Tmax) lesion volume] (B), and variables reflecting speed and extent of reperfusion (C) [modified treatment in cerebral ischemia (mTICI) score and time from onset to mTICI]. Three different scenarios were analyzed: (1) baseline clinical parameters only, (2) baseline clinical and MRI-derived parameters, and (3) all baseline clinical, imaging-derived, and reperfusion-associated parameters. For each scenario, we assessed prediction for favorable and poor outcome with seven different machine learning algorithms.ResultsIn 210 patients, prediction of favorable outcome was improved after including speed and extent of recanalization [highest area under the curve (AUC) 0.73] compared to using baseline clinical variables only (highest AUC 0.67). Prediction of poor outcome remained stable by using baseline clinical variables only (highest AUC 0.71) and did not improve further by additional variables. Prediction of favorable and poor outcomes was not improved by adding MR-mismatch variables. Most important baseline clinical variables for both outcomes were age, National Institutes of Health Stroke Scale, and premorbid mRS.ConclusionsOur results suggest that a prediction of poor outcome after AIS and MT could be made based on clinical baseline variables only. Speed and extent of MT did improve prediction for a favorable outcome but is not relevant for poor outcome. An MR mismatch with small ischemic core and larger penumbral tissue showed no predictive importance.

The control of general nonlinear systems is a challenging task in particular for large-scale models as they occur in the semi-discretization of partial differential equations (PDEs) of, say, fluid flow. In order to employ powerful methods from linear numerical algebra and linear control theory, one may embed the nonlinear system in the class of linear parameter varying (LPV) systems. In this work, we show how convolutional neural networks can be used to design LPV approximations of incompressible Navier-Stokes equations. In view of a possibly low-dimensional approximation of the parametrization, we discuss the use of deep neural networks (DNNs) in a semi-discrete PDE context and compare their performance to an approach based on proper orthogonal decomposition (POD). For a streamlined training of DNNs directed to the PDEs in a Finite Element (FEM) framework, we also discuss algorithmical details of implementing the proper norms in general loss functions.

IntroductionPetroclival meningioma (PCM) remains a major neurosurgical challenge. There are still controversial strategic treatment concepts about surgical approach, the extent of resection, and postoperative radiotherapy. We aimed to evaluate prognostic factors influencing the progression-free survival (PFS) rates of PCM, with a particular focus on the retrosigmoidal approach, the role of the extent of resection, and postoperative radiotherapy.MethodsEighty-nine patients with complete follow-up data were included. All patients were operated on via a retrosigmoidal approach, of whom 19 underwent gross total resection (GTR) and 70 underwent subtotal resection (STR). In the subgroups of tumors with infiltration of the cavernous sinus, 41 patients received near total resection (NTR) and 24 STR. Thirty-one patients received postoperative radiotherapy of the residual tumor and 58 were treated with surgery alone. Kaplan–Meier analyses and Cox regression were used to identify significant factors associated with treatment.ResultsGTR (p=0.0107) and postoperative radiotherapy (p=0.014) were associated with significantly improved PFS. Even the subgroup analysis of extended PCM with infiltration of the cavernous sinus (CS) showed an advantage for PFS after near total resection (NTR) (p=0.0017). The additional radiotherapy of the residual tumor in the CS in this subgroup also showed a beneficial effect on PFS (p=0.012).ConclusionThe extension of surgical resection remains the most important prognostic factor in relation to oncological outcomes. However, the GTR of extended PCM with infiltration of the CS is associated with significant neurological morbidity and requires additional adjuvant therapy concepts. Postoperative radiotherapy is an important element in the treatment of the residual tumor after surgery.

Abstract Background Meningiomas are common brain tumours that are usually defined by benign clinical course. However, some meningiomas undergo a malignant transformation and recur within a short time period regardless of their World Health Organization (WHO) grade. The current study aimed to identify potential markers that can discriminate between benign and malignant meningioma courses. Methods We profiled the metabolites from 43 patients with low- and high-grade meningiomas. Tumour specimens were analyzed by nuclear magnetic resonance analysis; 270 metabolites were identified and clustered with the AutoPipe algorithm. Results We observed two distinct clusters marked by alterations in glycine/serine and choline/tryptophan metabolism. Glycine/serine cluster showed significantly lower WHO grades and proliferation rates. Also progression-free survival was significantly longer in the glycine/serine cluster. Conclusion Our findings suggest that alterations in glycine/serine metabolism are associated with lower proliferation and more recurrent tumours. Altered choline/tryptophan metabolism was associated with increases proliferation, and recurrence. Our results suggest that tumour malignancy can be reflected by metabolic alterations, which may support histological classifications to predict the clinical outcome of patients with meningiomas.

Whether the perivascular niche (PVN) and the integration into multicellular networks by tumor microtubes (TMs) have a different role in glioblastoma progression and resistance to therapies is currently unclear. Here, the authors, by long-term tracking of individual glioma, demonstrate that both niches can partially compensate for each other and that glioma cells localized in both niches are resistant to radio- and chemotherapy.

This article focuses on the development of a theoretical framework for jazz dance in which researchers hypothesized how motif notation concepts from Laban Movement Analysis (LMA) and Language of Dance® (LOD) could be used to help learners understand how the roots and branches of jazz dance styles are related. We began by investigating the Africanist aesthetics of jazz dance to better grasp how each element influences the feel of jazz as it exists within various jazz dance styles. To differentiate between the styles, we deciphered how the Africanist aesthetics blend with Europeanist aesthetics and movement vocabulary to merge into a variety of distinctly American dance forms. Notation-based dance theory offers a unique lens for analytical observation that helps to support theory discussion surrounding the essence of jazz as it is felt in the body. The Africanist aesthetic framework we created provides conceptual tools for comprehending the scope of jazz dance and a movement analysis perspective that might contribute to social, concert, and commercial or entertainment research. We hope this framework proves useful to others in their jazz inquiries by offering fertile conceptual ground for jazz dance research and pedagogical development within academic and artistic practices.

This statistical study of 272 dancers, ages 18-96, using Kolb Learning Styles Inventory 4.0, identifies dominant learning cycles, learning styles, and learning style flexibility of the dance participants, and subgroups of notation users, and then compares these results to existing results from other disciplines. The research reveals the depth and breadth of learning cycles and styles among the dancers (notation users and non-notation users) and provides implications suggested by comparing to other disciplines. The results provide insight into the value of shifting approaches to teaching by using motif notation or structured dance notation, some of which closely match disciplines that are seemingly quite different from dance. Motif notation results hint that motif notation pushes students to be much more flexible in their learning styles. This distinction represents motif notation users' abilities to switch to a 'back up' learning style, when their dominant learning style seems not to fulfill every learning task. Structured notation users results point to ability to focus deeply into analysis. The results of this study point to two considerations for the dance classroom. Engaging in motif notation may be an ideal tool to stimulate flexibility for lifelong learning and structured notation encourages analytical skill building.

Background: The aim of this study was to assess the phenotype of multifocal motor neuropathy (MMN) and amyotrophic lateral sclerosis (ALS) in quantitative MR neurography. Methods: In this prospective study, 22 patients with ALS, 8 patients with MMN, and 10 healthy volunteers were examined with 3T MR neurography, using a high-resolution fat-saturated T2-weighted sequence, diffusion-tensor imaging (DTI), and a multi-echo T2-relaxometry sequence. The quantitative biomarkers fractional anisotropy (FA), radial and axial diffusivity (RD, AD), mean diffusivity (MD), cross-sectional area (CSA), T2-relaxation time, and proton spin density (PSD) were measured in the tibial nerve at the thigh and calf, and in the median, radial, and ulnar nerves at the mid-upper arm. Results: MMN showed a characteristic imaging pattern of decreased FA (p = 0.018), increased RD (p = 0.014), increased CSA (p < 0.001), increased T2-relaxation time (p < 0.001), and increased PSD (p = 0.025) in the upper arm nerves compared to ALS and controls. ALS patients did not differ from controls in any imaging marker, nor were there any group differences in the tibial nerve (p > 0.05). Conclusions: MMN shows a characteristic pattern of quantitative DTI and T2-relaxometry parameters in the upper-arm nerves, primarily indicating demyelination. Peripheral nerve changes in ALS seem to be below the detection level of current state-of-the-art quantitative MR neurography.

Background: MR image classification in datasets collected from multiple sources is complicated by inconsistent and missing DICOM metadata. Therefore, we aimed to establish a method for the efficient automatic classification of MR brain sequences. Methods: Deep convolutional neural networks (DCNN) were trained as one-vs-all classifiers to differentiate between six classes: T1 weighted (w), contrast-enhanced T1w, T2w, T2w-FLAIR, ADC, and SWI. Each classifier yields a probability, allowing threshold-based and relative probability assignment while excluding images with low probability (label: unknown, open-set recognition problem). Data from three high-grade glioma (HGG) cohorts was assessed; C1 (320 patients, 20,101 MRI images) was used for training, while C2 (197, 11,333) and C3 (256, 3522) were for testing. Two raters manually checked images through an interactive labeling tool. Finally, MR-Class’ added value was evaluated via radiomics model performance for progression-free survival (PFS) prediction in C2, utilizing the concordance index (C-I). Results: Approximately 10% of annotation errors were observed in each cohort between the DICOM series descriptions and the derived labels. MR-Class accuracy was 96.7% [95% Cl: 95.8, 97.3] for C2 and 94.4% [93.6, 96.1] for C3. A total of 620 images were misclassified; manual assessment of those frequently showed motion artifacts or alterations of anatomy by large tumors. Implementation of MR-Class increased the PFS model C-I by 14.6% on average, compared to a model trained without MR-Class. Conclusions: We provide a DCNN-based method for the sequence classification of brain MR images and demonstrate its usability in two independent HGG datasets.

(1) Background: Evaluation of impact of adjuvant radiation therapy (RT) in patients with oral squamous cell carcinoma of the oral cavity/oropharynx (OSCC) of up to 4 cm (pT1/pT2) and solitary ipsilateral lymph node metastasis (pN1). A non-irradiated group with clinical follow-up was chosen for control, and survival and quality of life (QL) were compared; (2) Methods: This prospective multicentric comprehensive cohort study included patients with resected OSCC (pT1/pT2, pN1, and cM0) who were allocated into adjuvant radiation therapy (RT) or observation. The primary endpoint was overall survival. Secondary endpoints were progression-free survival and QL after surgery; (3) Results: Out of 27 centers, 209 patients were enrolled with a median follow-up of 3.4 years. An amount of 137 patients were in the observation arm, and 72 received adjuvant irradiation. Overall survival did not differ between groups (hazard ratio (HR) 0.98 [0.55–1.73], p = 0.94). There were fewer neck metastases (HR 0.34 [0.15–0.77]; p = 0.01), as well as fewer local recurrences (HR 0.41 [0.19–0.89]; p = 0.02) under adjuvant RT. For QL, irradiated patients showed higher values for the symptom scale pain after 0.5, two, and three years (all p < 0.05). After six months and three years, irradiated patients reported higher symptom burdens (impaired swallowing, speech, as well as teeth-related problems (all p < 0.05)). Patients in the RT group had significantly more problems with mouth opening after six months, one, and two years (p < 0.05); (4) Conclusions: Adjuvant RT in patients with early SCC of the oral cavity and oropharynx does not seem to influence overall survival, but it positively affects progression-free survival. However, irradiated patients report a significantly decreased QL up to three years after therapy compared to the observation group.

Smoking is a leading cause of head and neck squamous cell carcinoma (HNSCC). However, non-smokers are also affected by HNSCC, and the prognostic factors applicable to older non-smokers with HNSCC are largely unknown. The aim of this study was to determine predictors of overall survival (OS) in patients both with and without a smoking history aged 70 and over at initial diagnosis. Retrospective data of patients aged ≥70 (initial diagnoses 2004–2018) were examined. Evaluated predictors included tumour stage, biological age, health and therapy. A total of 688 patients (520 smokers, 168 non-smokers) were included with a median age of 74. The 5-year OS was 39.6%. Non-smokers had significantly improved OS compared to smokers (52.0% versus 36.0%, p < 0.001). Disease-free survival (DFS) differed significantly between both groups (hazard ratio = 1.3; 95%CI 1.04–1.626). TNM stage and the recommended therapies (curative versus palliative) were comparable. The proportion of p16-positive oropharyngeal carcinomas was significantly higher in non-smokers (76.7% versus 43.8%, p < 0.001). Smokers were significantly more likely to be men (p < 0.001), drinkers (p < 0.001), and have poorer health status (Karnofsky performance status, KPS, p = 0.023). They were also more likely to have additional tumours (p = 0.012) and lower treatment adherence (p = 0.038). Important predictors of OS identified in both groups, were, among others, alcohol abuse, KPS, Charlson comorbidity index, site of primary tumour, UICC stage and treatment received. Elderly non-smokers are also affected by HNSCC, however, both OS and DFS are increased compared to smokers.

Bite force measuring devices that are generally suitable for edentulous patients or patients undergoing mandibular reconstruction are missing. This study assesses the validity of a new bite force measuring device (prototype of loadpad®, novel GmbH) and evaluates its feasibility in patients after segmental mandibular resection. Accuracy and reproducibility were analyzed with two different protocols using a universal testing machine (Z010 AllroundLine, Zwick/Roell, Ulm, Germany). Four groups were tested to evaluate the impact of silicone layers around the sensor: no silicone (“pure”), 2.0 mm soft silicone (“2-soft”), 7.0 mm soft silicone (“7-soft”) and 2.0 mm hard silicone (“2-hard”). Thereafter, the device was tested in 10 patients prospectively who underwent mandibular reconstruction using a fibula free flap. Average relative deviations of the measured force in relation to the applied load reached 0.77% (“7-soft”) to 5.28% (“2-hard”). Repeated measurements in “2-soft” revealed a mean relative deviation of 2.5% until an applied load of 600 N. Maximum bite force decreased postoperatively by 51.8% to a maximum mean bite force of 131.5 N. The novel device guarantees a high accuracy and degree of reproducibility. Furthermore, it offers new opportunities to quantify perioperative oral function after reconstructive surgery of the mandible also in edentulous patients.

Introduction: In lateralized oral squamous cell carcinoma (OSCC) with ipsilateral cervical lymph node metastasis (CLNM), the surgical management of the unsuspicious contralateral neck remains a matter of debate. The aim of this study was to analyze this cohort and to compare the outcomes of patients with and without contralateral elective neck dissection (END). Material and Methods: A retrospective analysis of patients with lateralized OSCC, ipsilateral CLNM (pN+) and contralateral cN0-stage was performed. Patients were divided into two groups according to the surgical management of the contralateral neck: I: END; and II: no END performed. Adjuvant radiotherapy was applied bilaterally in both groups according to individual risk. Results: A total of 65 patients (group I: 16 (24.6%); group II: 49 (75.4%)) with a median follow-up of 28 months were included. Initially, there was no case of contralateral CLNM after surgery. During follow-up, 6 (9.2%) patients presented with recurrent CLNM. In 5 of these cases (7.7%), the contralateral neck (group I: 3/16 (18.8%); group II: 2/49 (4.1%)) was affected. Increased ipsilateral lymph node ratio was associated with contralateral CLNM (p = 0.07). END of the contralateral side showed no significant benefit regarding OS (p = 0.59) and RFS (p = 0.19). Conclusions: Overall, the risk for occult contralateral CLNM in patients with lateralized OSCC ipsilateral CLNM is low. Our data suggest that END should not be performed routinely in this cohort. Risk-adapted radiotherapy of the contralateral neck alone seems to be sufficient from the oncological point of view.

Purpose: This study investigates the impact of different intensity normalization (IN) methods on the overall survival (OS) radiomics models’ performance of MR sequences in primary (pHGG) and recurrent high-grade glioma (rHGG). Methods: MR scans acquired before radiotherapy were retrieved from two independent cohorts (rHGG C1: 197, pHGG C2: 141) from multiple scanners (15, 14). The sequences are T1 weighted (w), contrast-enhanced T1w (T1wce), T2w, and T2w-FLAIR. Sequence-specific significant features (SF) associated with OS, extracted from the tumour volume, were derived after applying 15 different IN methods. Survival analyses were conducted using Cox proportional hazard (CPH) and Poisson regression (POI) models. A ranking score was assigned based on the 10-fold cross-validated (CV) concordance index (C-I), mean square error (MSE), and the Akaike information criterion (AICs), to evaluate the methods’ performance. Results: Scatter plots of the 10-CV C-I and MSE against the AIC showed an impact on the survival predictions between the IN methods and MR sequences (C1/C2 C-I range: 0.62–0.71/0.61–0.72, MSE range: 0.20–0.42/0.13–0.22). White stripe showed stable results for T1wce (C1/C2 C-I: 0.71/0.65, MSE: 0.21/0.14). Combat (0.68/0.62, 0.22/0.15) and histogram matching (HM, 0.67/0.64, 0.22/0.15) showed consistent prediction results for T2w models. They were also the top-performing methods for T1w in C2 (Combat: 0.67, 0.13; HM: 0.67, 0.13); however, only HM achieved high predictions in C1 (0.66, 0.22). After eliminating IN impacted SF using Spearman’s rank-order correlation coefficient, a mean decrease in the C-I and MSE of 0.05 and 0.03 was observed in all four sequences. Conclusion: The IN method impacted the predictive power of survival models; thus, performance is sequence-dependent.

The aim of this bibliometric analysis was to benchmark the publication activities of German university departments of oral and maxillofacial surgery. The publication performance of staff surgeons (chief and consultants), documented by first or last authorship, from 37 German university departments was captured over a 10-year period (January 1, 2010, to December 31, 2019). All publications listed in PubMed were included. Additionally, the Impact Factor (IF) was determined. A total of 213 surgeons were identified, of whom 158 (74.2%) were publishing. The number of publications was 1,777, published in 311 journals. Publication activity ranged from an average of 23.3 publications per staff surgeon in the top-ranked department to 0 publications in the last-ranked. The same trend was observed for the total cumulative IFs (CIFs) per member (range from 56.2 to 0). The most common used journal was the Journal of Cranio-Maxillofacial Surgery (19.7%), with focus on "dentoalveolar surgery" (24%) and "operative techniques and procedures" (28.3%). Women constituted 19.2% of the staff, contributing to 8.5% of the publications. The publication performance of German university departments of oral and maxillofacial surgery exhibits a high variance, which did not correlate with the number of personnel and could only be explained by different research motivations. [ABSTRACT FROM AUTHOR]

This study describes the morphologic and phenotypic spatial heterogeneity of tumor cells and the tissue microenvironment (TME), focusing on immune infiltration in OSCCs. Patients with OSCCs and planned surgical tumor resection were eligible for the study. Two biopsies each from the tumor center and the tumor rim were obtained. Immunohistochemical characterization of tumor and immune cells was performed using a panel of immunohistochemical markers. Thirty-six biopsies were obtained from the 9 patients. All patients showed an individual marker expression profile with ITH. Within the same biopsy, the CPS and TPS scores showed relevant variations in PD-L1 expression. Comparisons between the tumor center and rim revealed significant differences in the up/downregulation of p53. Marker expression of patients with recurrences clustered similarly, with the higher expression of FoxP3, IDO, CD4, CD68, and CD163 at the tumor rim. OSCCs were found to exhibit relevant ITH involving both tumor cells and TME, suggesting that biomarker analysis of multiple tumor regions may be helpful for clinical decision making and tumor characterization. The analysis of multiple spots within a biopsy is recommended for a reliable determination of PD-L1 expression and other biomarkers, impacting current clinical assessments. [ABSTRACT FROM AUTHOR]

BackgroundThis study was conducted to evaluate the feasibility, clinical outcomes, and accuracy of patient-specific 3D-printed miniplates for mandible reconstruction with fibula free flaps.MethodsA feasibility study was conducted with 8 patients. Following virtual planning, patient-specific 1.0 mm titanium non-locking miniplates were produced via laser selective melting. 3D-printed cutting and drilling guides were used for segmental mandible resection and flap harvesting. Flap fixation was performed with two 4-hole miniplates and 2.0 mm non-locking screws (screw length 7 mm) for each intersegmental gap. Clinical follow-up was at least 6 months. Preoperative and postoperative CT/cone beam CT data were used for 3D accuracy analysis and evaluation of bone healing. Plate-related complications were monitored clinically.ResultsPatient-specific miniplate fixation of all flaps was successfully conducted (4 mono-segmental, 4 dual-segmental) with high accuracy (3.64 ± 1.18 mm) between the virtual plan and postoperative result. No technical complications were encountered intraoperatively. Osseous union occurred in all intersegmental gaps (1 partial, 18 complete) after 10 ± 2 months. No material fracture, dislocation, or plate exposure was observed.ConclusionsBased on this pilot observational study including a limited number of patients, free flap fixation for mandibular reconstruction with patient-specific 3D-printed miniplates is feasible and associated with high accuracy, bone healing, and remote soft tissue complications.

BackgroundGlioblastoma is the most common and the most challenging to treat adult primary central nervous system tumor. Although modern management strategies modestly improved the overall survival, the prognosis remains dismal associated with poor life quality and the clinical course often dotted by treatment side effects and cognitive decline. Functional deterioration might be caused by obstructive or communicating hydrocephalus but due to poor overall prognosis surgical treatment options are often limited and its optimal management strategies remain elusive. We aimed to investigate risk factors, treatment options and outcomes for tumor-associated hydrocephalus in a contemporary 10 years cohort of glioblastoma patients.MethodsWe reviewed electronic health records of 1800 glioblastoma patients operated at the Department of Neurosurgery, Medical Center – University of Freiburg from 2009 to 2019. Demographics, clinical characteristics and radiological features were analyzed. Univariate analysis for nominal variables was performed either by Fisher’s exact test or Chi-square test, as appropriate.ResultsWe identified 39 glioblastoma patients with symptomatic communicating hydrocephalus treated by ventricular shunting (incidence 2.1%). Opening of the ventricular system during a previous tumor resection was associated with symptomatic hydrocephalus (p

Titanium fixation devices are the gold standard for the treatment of mandibular fractures; however, they present serious limitations, such as non-degradability and generation of imaging artifacts. As an alternative, biodegradable magnesium alloys have lately drawn attention due to their biodegradability and biocompatibility. In addition, magnesium alloys offer a relatively high modulus of elasticity in comparison to biodegradable polymers, being a potential option to substitute titanium in highly loaded anatomical areas, such as the mandible. This study aimed to evaluate the biomechanical competence of magnesium alloy WE43 plates for mandibular fracture fixation in comparison to the clinical standard or even softer polymer solutions. A 3D finite element model of the human mandible was developed, and four different fracture scenarios were simulated, together with physiological post-operative loading and boundary conditions. In a systematic comparison, the material properties of titanium alloy Ti-6Al-4V, magnesium alloy WE43, and polylactic acid (PLA) were assigned to the fixation devices, and two different plate thicknesses were tested. No failure was predicted in the fixation devices for any of the tested materials. Moreover, the magnesium and titanium fixation devices induced a similar amount of strain within the healing regions. On the other hand, the PLA devices led to higher mechanical strains within the healing region. Plate thickness only slightly influenced the primary fixation stability. Therefore, magnesium alloy WE43 fixation devices seem to provide a suitable biomechanical environment to support mandibular fracture healing in the early stages of bone healing. Magnesium WE43 showed a biomechanical performance similar to clinically used titanium devices with the added advantages of biodegradability and radiopacity, and at the same time it showed a remarkably higher primary stability compared to PLA fixation devices, which appear to be too unstable, especially in the posterior and more loaded mandibular fracture cases.

ObjectiveIt is controversially discussed in how far smoking contributes to diabetic polyneuropathy (DPN) in type 2 diabetes (T2D). Diffusion-weighted magnetic resonance neurography (MRN) at 3 Tesla has been shown to provide objective values for structural nerve integrity in patients with T2D. The aim of this study was to investigate the contribution of cigarette smoking on structural nerve integrity in T2D.MethodsThis cross-sectional prospective cohort study investigated the structural integrity of the sciatic nerve in 10 smokers, 40 never-smokers, and 20 ex-smokers with T2D and 10 healthy control subjects, using diffusion tensor imaging MRN at 3 Tesla and semi-automated nerve fiber tracking. Results were correlated with clinical, electrophysiological, and serological data.ResultsThe sciatic nerve’s fractional anisotropy (FA), a parameter for structural nerve integrity, was significantly lower in smokers with T2D when compared to controls (p = 0.002) and never-smokers (p = 0.015), and lower in ex-smokers when compared to controls (p = 0.015). In addition, sciatic nerve radial diffusivity, a marker of myelin damage, was increased in smokers versus controls and never-smokers (p = 0.048, p = 0.049, respectively). Furthermore, FA in T2D patients was negatively correlated with clinical and electrophysiological markers of DPN. FA also showed negative correlations with the pulse wave velocity, a marker of arterial stiffness and associated microangiopathy, in controls (r = −0.70; p = 0.037), never-smokers (r = −0.45; p = 0.004), ex-smokers (r = −0.55; p = 0.009), and a similar trend in smokers (r = −0.63; p = 0.076). Negative correlations were found between FA and skin auto-fluorescence, a marker of tissue advanced glycation end product accumulation and therefore long-term glycemic stress in T2D, in never-smokers (r = −0.39; p = 0.020) and smokers (r = −0.84; p = 0.004), but not in ex-smokers (r = −0.07; p = 0.765).ConclusionThe findings indicate that smoking contributes to sciatic nerve damage in T2D, potentially worsening DPN due to glycemic stress and less microangiopathy-associated myelin damage in active smokers, while angiopathic effects predominate in ex-smokers. To stop smoking may therefore pose a promising preventive measure to slow the progression of DPN in T2D.

BackgroundQuantitative MR-neurography (MRN) is increasingly applied, however, the impact of the MR-scanner on the derived parameters is unknown. Here, we used different 3.0T MR scanners and applied comparable MR-sequences in order to quantify the inter-scanner reproducibility of various MRN parameters of the sciatic nerve.MethodsTen healthy volunteers were prospectively examined at three different 3.0T MR scanners and underwent MRN of their sciatic nerve using comparable imaging protocols including diffusion tensor imaging (DTI) and T2 relaxometry. Subsequently, inter-scanner agreement was assessed for seven different parameters by calculating the intraclass correlation coefficients (ICCs) and the standard error of measurement (SEM).ResultsAssessment of inter-scanner reliability revealed good to excellent agreement for T2 (ICC: 0.846) and the quantitative DTI parameters, such as fractional anisotropy (FA) (ICC: 0.876), whereas moderate agreement was observed for proton spin density (PD) (ICC: 0.51). Analysis of variance identified significant inter-scanner differences for several parameters, such as FA (p < 0.001; p = 0.02), T2 (p < 0.01) and PD (p = 0.02; p < 0.01; p = 0.02). Calculated SEM values were mostly within the range of one standard deviation of the absolute mean values, for example 0.033 for FA, 4.12 ms for T2 and 27.8 for PD.ConclusionThis study quantifies the measurement imprecision for peripheral nerve DTI and T2 relaxometry, which is associated with the use of different MR scanners. The here presented values may serve as an orientation of the possible scanner-associated fluctuations of MRN biomarkers, which can occur under similar conditions.

Indonesia has nearly doubled its urban population in the past three decades. In this period, the prevalence of overweight and obesity in Indonesia has also nearly doubled. We examined 1993-2014 panel data from the Indonesian Family Life Survey (IFLS) to determine the extent to which the increase in one's built environment contributed to a corresponding increase in adult overweight and obesity during this period. We estimated longitudinal regression models for body mass index (BMI) and being overweight or obese using novel matched geospatial measures of built-up land area. Living in a more built-up area was associated with greater BMI and risk of being overweight or obese. The contribution of the built environment was estimated to be small but statistically significant even after accounting for individuals' initial BMI. We discuss the findings considering the evidence on nutritional and technological transitions affecting food consumption patterns and physical activity levels in urban and rural areas.

Drug-resistant epilepsy remains a significant clinical and societal burden, with one third of people with epilepsy continuing to experience seizures despite the availability of around 30 anti-seizure drugs (ASDs). Further, ASDs often have substantial adverse effects, including impacts on learning and memory. Therefore, it is important to develop new ASDs, which may be more potent or better tolerated. Here, we report the preliminary preclinical evaluation of BICS01, a synthetic product based on a natural compound, as a potential ASD. To model seizure-like activity in vitro, we prepared hippocampal slices from adult male Sprague Dawley rats, and elicited epileptiform bursting using high extracellular potassium. BICS01 (200 μM) rapidly and reversibly reduced the frequency of epileptiform bursting but did not change broad measures of network excitability or affect short-term synaptic facilitation. BICS01 was well tolerated following systemic injection at up to 1,000 mg/kg. However, we did not observe any protective effect of systemic BICS01 injection against acute seizures evoked by pentylenetetrazol. These results indicate that BICS01 is able to acutely reduce epileptiform activity in hippocampal networks. Further preclinical development studies to enhance pharmacokinetics and accumulation in the brain, as well as studies to understand the mechanism of action, are now required.