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Predicting and understanding thorax injury is fundamental for the assessment and development of safety systems to mitigate injury risk to the increasing and vulnerable aged population. While computational human models have contributed to the understanding of injury biomechanics, contemporary human body models have struggled to predict rib fractures and explain the increased incidence of injury in the aged population. The present study enhanced young and aged human body models (HBMs) by integrating a biofidelic cortical bone constitutive model and population-based bone material properties. The HBMs were evaluated using side impact sled tests assessed using chest compression and number of rib fractures. The increase in thoracic kyphosis and the associated change in rib angle with increasing age, led to increased rib torsional moment increasing the rib shear stress. Coupled with and improved cortical bone constitutive model and aged material properties, the higher resulting shear stress led to an increased number of rib fractures in the aged model. The importance of shear stress resulting from torsional load was further investigated using an isolated rib model. In contrast, HBM chest compression, a common thorax injury-associated metric, was insensitive to the aging factors studied. This study proposes an explanation for the increased incidence of thorax injury with increasing age reported in epidemiological data, and provides an enhanced understanding of human rib mechanics that will benefit assessment and design of future safety systems.

Objective: Real-life car crashes are often preceded by an evasive maneuver, which can alter the occupant posture and muscle state. To simulate the occupant response in such maneuvers, human body models (HBMs) with active muscles have been developed. The aim of this study was to implement an omni-directional rotational head-neck muscle controller in the SAFER HBM and compare the bio-fidelity of the HBM with a rotational controller to the HBM with a translational controller, in simulations of evasive maneuvers.Methods: The rotational controller was developed using an axis-angle representation of head rotations, with x, y, and z components in the axis. Muscle load sharing was based on rotational direction in the simulation and muscle activity recorded in three volunteer experiments in these directions. The gains of the rotational and translational controller were tuned to minimize differences between translational and rotational head displacements of the HBM and volunteers in braking and lane change maneuvers using multi-objective optimizations. Bio-fidelity of the model with tuned controllers was evaluated objectively using CORrelation and Analysis (CORA).Results: The results indicated comparable performance for both controllers after tuning, with somewhat higher bio-fidelity for rotational kinematics with the translational controller. After tuning, good or excellent bio-fidelity was indicated for both controllers in the loading direction (forward in braking, and lateral in lane change), with CORA scores of 0.86−0.99 and 0.93−0.98 for the rotational and translational controllers, respectively. For rotational displacements, and translational displacements in the other directions, bio-fidelity ranged from poor to excellent, with slightly higher average CORA scores for the HBM with the translational controller in both braking and lane changing. Time-averaged muscle activity was within one standard deviation of time-averaged muscle activity from volunteers.Conclusion: Overall, the results show that when tuned, both the translational and rotational controllers can be used to predict the occupant response to an evasive maneuver, allowing for the inclusion of evasive maneuvers prior to a crash in evaluation of vehicle safety. The rotational controller shows potential in controlling omni-directional head displacements, but the translational controller outperformed the rotational controller. Thus, for now, the recommendation is to use the translational controller with tuned gains.

Peptide-loaded MHC class I (pMHC-I) multimers have revolutionized our capabilities to monitor disease-associated T cell responses with high sensitivity and specificity. To improve the discovery of T cell receptors (TCR) targeting neoantigens of individual tumor patients with recombinant MHC molecules, we developed a peptide-loadable MHC class I platform termed MediMer. MediMers are based on soluble disulfide-stabilized β2-microglobulin/heavy chain ectodomain single-chain dimers (dsSCD) that can be easily produced in large quantities in eukaryotic cells and tailored to individual patients’ HLA allotypes with only little hands-on time. Upon transient expression in CHO-S cells together with ER-targeted BirA biotin ligase, biotinylated dsSCD are purified from the cell supernatant and are ready to use. We show that CHO-produced dsSCD are free of endogenous peptide ligands. Empty dsSCD from more than 30 different HLA-A,B,C allotypes, that were produced and validated so far, can be loaded with synthetic peptides matching the known binding criteria of the respective allotypes, and stored at low temperature without loss of binding activity. We demonstrate the usability of peptide-loaded dsSCD multimers for the detection of human antigen-specific T cells with comparable sensitivities as multimers generated with peptide-tethered β2m-HLA heavy chain single-chain trimers (SCT) and wild-type peptide-MHC-I complexes prior formed in small-scale refolding reactions. Using allotype-specific, fluorophore-labeled competitor peptides, we present a novel dsSCD-based peptide binding assay capable of interrogating large libraries of in silico predicted neoepitope peptides by flow cytometry in a high-throughput and rapid format. We discovered rare T cell populations with specificity for tumor neoepitopes and epitopes from shared tumor-associated antigens in peripheral blood of a melanoma patient including a so far unreported HLA-C*08:02-restricted NY-ESO-1-specific CD8+ T cell population. Two representative TCR of this T cell population, which could be of potential value for a broader spectrum of patients, were identified by dsSCD-guided single-cell sequencing and were validated by cognate pMHC-I multimer staining and functional responses to autologous peptide-pulsed antigen presenting cells. By deploying the technically accessible dsSCD MHC-I MediMer platform, we hope to significantly improve success rates for the discovery of personalized neoepitope-specific TCR in the future by being able to also cover rare HLA allotypes.

Abstract Background HPV‐related oropharyngeal squamous cell carcinoma (OPSCC) is associated with a favorable prognosis, yet patients of color and low socioeconomic status (SES) continue to experience inferior outcomes. We aim to understand how the emergence of HPV has impacted race and SES survival disparities in OPSCC. Methods A retrospective cohort of 18,362 OPSCC cases from 2010 to 2017 was assembled using the SEER (Surveillance, Epidemiology, and End Results) database. Cox proportional regression and Fine and Gray regression models were used to calculate hazard ratios (HRs) adjusting for race, SES, age, subsite, stage, and treatment. Results Black patients had lower overall survival than patients of other races in HPV‐positive and HPV‐negative OPSCC (HR 1.31, 95% CI 1.13–1.53 and HR 1.23, 95% CI 1.09–1.39, respectively). Higher SES was associated with improved survival in all patients. Race had a diminished association with survival among high SES patients. Low SES Black patients had considerably worse survival than low SES patients of other races. Conclusion Race and SES interact variably across cohorts. High SES was protective of the negative effects of race, although there remains a disparity in outcomes among Black and non‐Black patients, even in high SES populations. The persistence of survival disparities suggests that the HPV epidemic has not improved outcomes equally across all demographic groups.

Finite element human body models (HBMs) are becoming increasingly important numerical tools for traffic safety. Developing a validated and reliable HBM from the start requires integrated efforts and continues to be a challenging task. Mesh morphing is an efficient technique to generate personalized HBMs accounting for individual anatomy once a baseline model has been developed. This study presents a new image registration–based mesh morphing method to generate personalized HBMs. The method is demonstrated by morphing four baseline HBMs (SAFER, THUMS, and VIVA+ in both seated and standing postures) into ten subjects with varying heights, body mass indices (BMIs), and sex. The resulting personalized HBMs show comparable element quality to the baseline models. This method enables the comparison of HBMs by morphing them into the same subject, eliminating geometric differences. The method also shows superior geometry correction capabilities, which facilitates converting a seated HBM to a standing one, combined with additional positioning tools. Furthermore, this method can be extended to personalize other models, and the feasibility of morphing vehicle models has been illustrated. In conclusion, this new image registration–based mesh morphing method allows rapid and robust personalization of HBMs, facilitating personalized simulations.

Pituitary tumor apoplexy (PTA) classically comprises sudden-onset headache, loss of vision, ophthalmoparesis, and decreased consciousness. It typically results from hemorrhage and/or infarction within a pituitary adenoma. Presentation is heterologous, and optimal management is debated. The time course of recovery of cranial nerve deficits (CNDs) and headaches is not well established. In this study, a retrospective series of consecutive patients with PTA managed at a single academic institution over a 22-year period is presented. Headaches at the time of surgery were more severe in the early and subacute surgical cohort and improved significantly within 72 h postoperatively (p < 0.01). At one year, 90% of CNDs affecting cranial nerves (CNs) 3, 4, and 6 had recovered, with no differences between early (14 d) time-to-surgery cohorts. Remarkably, half recovered within three days. In total, 56% of CN2 deficits recovered, with the early surgery cohort including more severe deficits and recovering at a lower rate (p = 0.01). No correlation of time-to-surgery and rapidity of recovery of CNDs was observed (p = 0.65, 0.72). Surgery for PTA is associated with rapid recovery of CNDs in the early, subacute, and delayed time frames, and with rapid headache improvement in the early and subacute time frames in 50% or more of patients.

Rib fractures remain a common injury for vehicle occupants in crashes. The risk of a human sustaining rib fractures from thorax loading is highly variable, potentially due to a variability in individual factors such as material properties and geometry of the ribs and ribcage. Human body models (HBMs) with a detailed ribcage can be used as occupant substitutes to aid in the prediction of rib injury risk at the tissue level in crash analysis. To improve this capability, model parametrization can be used to represent human variability in simulation studies. The aim of this study was to identify the variations in the physical properties of the human thorax that have the most influence on rib fracture risk for the population of vehicle occupants. A total of 15 different geometrical and material factors, sourced from published literature, were varied in a parametrized SAFER HBM. Parametric sensitivity analyses were conducted for two crash configurations, frontal and near-side impacts. The results show that variability in rib cortical bone thickness, rib cortical bone material properties, and rib cross-sectional width had the greatest influence on the risk for an occupant to sustain two or more fractured ribs in both impacts. Therefore, it is recommended that these three parameters be included in rib fracture risk analysis with HBMs for the population of vehicle occupants.

Introduction: Chest deformation has been proposed as the best predictor of thoracic injury risk in frontal impacts. Finite Element Human Body Models (FE-HBM) can enhance the results obtained in physical crash tests with Anthropometric Test Devices (ATD) since they can be exposed to omnidirectional impacts and their geometry can be modified to reflect specific population groups. This study aims to assess the sensitivity of two thoracic injury risk criteria (PC Score and Cmax) to several personalization techniques of FE-HBMs.Methods: Three 30° nearside oblique sled tests were reproduced using the SAFER HBM v8 and three personalization techniques were applied to this model to evaluate the influence on the risk of thoracic injuries. First, the overall mass of the model was adjusted to represent the weight of the subjects. Second, the model anthropometry and mass were modified to represent the characteristics of the post-mortem human subjects (PMHS). Finally, the spine alignment of the model was adapted to the PMHS posture at t = 0 ms, to conform to the angles between spinal landmarks measured in the PMHS. The following two metrics were used to predict three or more fractured ribs (AIS3+) of the SAFER HBM v8 and the effect of personalization techniques: the maximum posterior displacement of any studied chest point (Cmax), and the sum of the upper and lower deformation of selected rib points (PC score).Results: Despite having led to statistically significant differences in the probability of AIS3+ calculations, the mass-scaled and morphed version provided, in general, lower values for injury risk than the baseline model and the postured version being the latter, which exhibited the better approximation to the PMHS tests in terms of probability of injury. Additionally, this study found that the prediction of AIS3+ chest injuries based on PC Score resulted in higher probability values than the prediction based on Cmax for the loading conditions and personalization techniques analyzed within this study.Discussion: This study could demonstrate that the personalization techniques do not lead to linear trends when they are used in combination. Furthermore, the results included here suggest that these two criteria will result in significantly different predictions if the chest is loaded more asymmetrically.

(1) Background: The role of rare immune cell subtypes in many solid tumors, chief among them head and neck squamous cell carcinoma (HNSCC), has not been well defined. The objective of this study was to assess the association between proportions of common and rare immune cell subtypes and survival outcomes in HNSCC. (2) Methods: In this cohort study, we utilized a deconvolution approach based on the CIBERSORT algorithm and the LM22 signature matrix to infer proportions of immune cell subtypes from 517 patients with untreated HPV-negative HNSCC from The Cancer Genome Atlas. We performed univariate and multivariable survival analysis, integrating immune cell proportions with clinical, pathologic, and genomic data. (3) Results: We reliably deconvolved 22 immune cell subtypes in most patients and found that the most common immune cell types were M0 macrophages, M2 macrophages, and memory resting CD4 T cells. In the multivariable analysis, we identified advanced N stage and the presence of γδ T cells as independently predictive of poorer survival. (4) Conclusions: We uncovered that γδ T cells in the tumor microenvironment were a negative predictor of survival among patients with untreated HNSCC. Our findings underscore the need to better understand the role of γδ T cells in HNSCC, including potential pro-tumorigenic mechanisms, and whether their presence may predict the need for alternative therapy approaches.

In future fully automated vehicles, sleeping or resting will be desirable during a drive. While a horizontal position currently appears infeasible, a relaxed seating position with a reclined seatback and an inclined seat pan which enables a safe, comfortable position for sleeping or resting is possible. However, the inclined seat pan increases the forces and moments acting on the lumbar spine of the occupant and thereby the risk of lumbar vertebra fractures in a frontal crash. An energy management system integrated into the longitudinal seat adjustment (a seat track load limiter: STLL) that can reduce this risk should be investigated. When evaluating the injury reduction potential of a new restraint system such as a STLL it is important to include variations in both occupant size and crash severity. Otherwise, there is a risk of sub-optimizing, that is, the restraint system is only working for a limited number of situations. The restraint systems addressing these variations are normally referred to as adaptive restraint systems. The first objective of the study is to develop an activation strategy (adaptive release time of the STLL) for different crash severities and occupant sizes, making full use of the available stroke distance without bottoming out the STLL. The second objective is to evaluate the potential of the adaptive STLL to reduce the risk of lumbar vertebra fractures by comparing it to 1) a fixed seat and 2) a passive version of the STLL. Simulated frontal impacts were performed with two male SAFER human body models (HBMs) as occupant surrogates: mid-sized (80 kg and 1.8 m) and large (130 kg and 1.9 m). Three crash pulse severity levels were evaluated: low (40 km/h), medium (50 km/h), and high (56 km/h) impact speeds. The fracture risk was evaluated for the five lumbar vertebrae (L1–L5) in three different seat conditions: 1) a seat fixed to the sled, 2) a passive STLL that moves when a given force is exceeded, and 3) an adaptive STLL which moves at a time that depends on the occupant mass and crash pulse severity. The risk for lumbar vertebra fracture increased with crash pulse severity, while HBM size had no effect on risk. For all conditions, the passive STLL reduced injury risks compared to the fixed seat, and the adaptive STLL reduced risk even further.

In high-severity crashes, occupant protection is challenging. Automated Emergency Braking (AEB) and seatbelt pre-pretensioning (PPT) are means to improve occupant protection; the purpose of this study was to quantify their effects on occupant injury risks in high-severity full-frontal crashes by Finite Element (FE) simulations. The SAFER Active average male Human Body Model was used as an occupant substitute. The crash pulses used were from separate full-frontal crash simulations using a Honda Accord FE model. The vehicle interior model comprised a seat, an instrument panel, a three-point pretensioned seatbelt system with a load-limiter of 3.1 kN force level, and a frontal passenger airbag. The effects of AEB and PPT were evaluated by simulating a 1 g pre-crash braking scenario for 0.5 s, with and without AEB, for three different PPT force levels: 0, 300, and 600 N. The impact speed of 80 km/h was reduced to 69 km/h by AEB. When neither system was activated, the predicted risk for an occupant to sustain two or more fractured ribs (NFR2+) was 100% for both 45- and 65-year-old male occupants. The risks were reduced when the AEB was activated, particularly for the 45-year-old occupant. When the AEB was activated, the risks of concussion and rib fractures were reduced; upper neck tension forces, pelvis Anterior Superior Iliac Spine (ASIS) forces, and lower extremity forces were also reduced. Increasing the PPT forces reduced the rib fracture risk further (to about 48% for a 45-year-old occupant with 600 N PPT force). The reduced speed due to AEB resulted in a lower concussion risk (from 71.3% to 31%). However, the concussion risk increased slightly with increased PPT forces.

Objective: The Inventory to Measure and Assess imaGe disturbance – Head and Neck (IMAGE-HN) is a validated patient-reported outcome measure of head and neck cancer-related body image-related distress (BID). However, the IMAGE-HN score corresponding to clinically relevant BID is unknown. The study objective is to determine the IMAGE-HN cutoff score that identifies head and neck cancer patients with clinically relevant BID.Methods: We conducted a cross-sectional study at six academic medical centers. Individuals ≥18 years old with a history of head and neck cancer treated with definitive intent were included. The primary outcome measure was the IMAGE-HN. A Receiver Operating Characteristic curve analysis was performed to identify the IMAGE-HN score that maximized sensitivity and specificity relative to a Body Image Scale score of ≥10 (which indicates clinically relevant BID in a general oncology population). To confirm the validity of the IMAGE-HN cutoff score, we compared the severity of depressive [Patient Health Questionnaire-9 (PHQ-9)] and anxiety symptoms [Generalized Anxiety Disorder-7 (GAD-7)], and quality of life [University of Washington-QOL (UW-QOL)] in patients with IMAGE-HN scores above and below the cutoff.Results: Of the 250 patients, 70.4% were male and the mean age was 62.3 years. An IMAGE-HN score of ≥22 was the optimal cutoff score relative to a Body Image Scale score of ≥10 and represents a clinically relevant level of head and neck cancer-related BID. Relative to those with an IMAGE-HN score of

To evaluate vehicle occupant injury risk, finite element human body models (HBMs) can be used in vehicle crash simulations. HBMs can predict tissue loading levels, and the risk for fracture can be estimated based on a tissue-based risk curve. A probabilistic framework utilizing an age-adjusted rib strain-based risk function was proposed in 2012. However, the risk function was based on tests from only twelve human subjects. Further, the age adjustment was based on previous literature postulating a 5.1% decrease in failure strain for femur bone material per decade of aging. The primary aim of this study was to develop a new strain-based rib fracture risk function using material test data spanning a wide range of ages. A second aim was to update the probabilistic framework with the new risk function and compare the probabilistic risk predictions from HBM simulations to both previous HBM probabilistic risk predictions and to approximate real-world rib fracture outcomes. Tensile test data of human rib cortical bone from 58 individuals spanning 17–99 years of ages was used. Survival analysis with accelerated failure time was used to model the failure strain and age-dependent decrease for the tissue-based risk function. Stochastic HBM simulations with varied impact conditions and restraint system settings were performed and probabilistic rib fracture risks were calculated. In the resulting fracture risk function, sex was not a significant covariate—but a stronger age-dependent decrease than previously assumed for human rib cortical bone was evident, corresponding to a 12% decrease in failure strain per decade of aging. The main effect of this difference is a lowered risk prediction for younger individuals than that predicted in previous risk functions. For the stochastic analysis, the previous risk curve overestimated the approximate real-world rib fracture risk for 30-year-old occupants; the new risk function reduces the overestimation. Moreover, the new function can be used as a direct replacement of the previous one within the 2012 probabilistic framework.

In patients with cervical necrotizing fasciitis undergoing extensive surgical debridement, vascularized reconstruction may be required. Little is known regarding the outcomes for free tissue transfer in the setting of a prior necrotizing infection in the head and neck. A retrospective chart review identified patients treated for cervicofacial necrotizing fasciitis at a single academic tertiary care center between 2008 and 2017. Disease and functional data were collected on patients requiring free tissue transfer. Among 23 patients treated for necrotizing fasciitis of the head and neck, two patients required free tissue transfer (each with an anterolateral thigh free flap) after appropriate control of their infectious process. There were no postoperative complications and both patients recovered well with good functional outcomes. These cases demonstrate that in select patients with extensive cervicofacial soft tissue defects after resolution of necrotizing fasciitis, free tissue transfer may be a safe and viable option.

Multidisciplinary involvement in radiation therapy (RT) treatment planning is currently underused. A radiation oncologist sought input for generating target contours from a neuro-radiologist (NR) and otolaryngologist (OL) for 3 patients requiring skull-base RT during the coronavirus disease 2019 pandemic. A Health Insurance Portability and Accountability Act compliant virtual meeting between the radiation oncologist, NR, and OL was arranged. Involvement of the OL and NR led to significant changes in the clinical target volume for all patients. Our experience highlights the feasibility of using commercially available video-conference platforms for multidisciplinary target volume delineation for complex RT cases. Further applications include interdisciplinary contour review for RT cases requiring special expertise and joint attending/resident physician contour review for resident education. The video-conference platform technology has demonstrated benefit during the coronavirus disease 2019 pandemic, and we believe it will remain an integral component of our field moving forward.

Introduction: Lymphatic malformations (LM) are non-malignant, congenital masses that contain fluid-filled lymph channels. These lesions typically present in childhood, but they can rarely present as a new head and neck mass in adults, which may not be on the differential diagnoses of clinicians. We present a case of an intraparotid mass that expanded rapidly secondary to hematoma after a diagnostic fine needle aspiration was performed. Case details: A 24 year-old male presented with a right parotid mass. CT imaging revealed a 3.2 × 1.8cm cystic lesion in the parotid gland. Ultrasound-guided fine needle aspiration was performed, resulting in a parotid hematoma and compression of the buccal branch of the right facial nerve. The patient was taken to the operating room urgently for evacuation of hematoma, and a superficial parotidectomy was performed. Pathology revealed an intraparotid lymphatic malformation with organized hematoma. Conclusions: We present a case of hematoma following fine needle aspiration of a lymphatic malformation. While these lesions typically present in the pediatric population, they may be first diagnosed in adulthood. Hematoma following FNA is exceedingly rare and may increase the clinical suspicion for LM.

Complexities in cell-type composition have rightfully led to skepticism and caution in the interpretation of bulk transcriptomic analyses. Recent studies have shown that deconvolution algorithms can be utilized to computationally estimate cell-type proportions from the gene expression data of bulk blood samples, but their performance when applied to tumor tissues, including those from head and neck, remains poorly characterized. Here, we use single-cell data (~6000 single cells) collected from 21 head and neck squamous cell carcinoma (HNSCC) samples to generate cell-type-specific gene expression signatures. We leverage bulk RNA-seq data from >500 HNSCC samples profiled by The Cancer Genome Atlas (TCGA), and using single-cell data as a reference, apply two newly developed deconvolution algorithms (CIBERSORTx and MuSiC) to the bulk transcriptome data to quantitatively estimate cell-type proportions for each tumor in TCGA. We show that these two algorithms produce similar estimates of constituent/major cell-type proportions and that a high T-cell fraction correlates with improved survival. By further characterizing T-cell subpopulations, we identify that regulatory T-cells (Tregs) were the major contributor to this improved survival. Lastly, we assessed gene expression, specifically in the Treg population, and found that TNFRSF4 (Tumor Necrosis Factor Receptor Superfamily Member 4) was differentially expressed in the core Treg subpopulation. Moreover, higher TNFRSF4 expression was associated with greater survival, suggesting that TNFRSF4 could play a key role in mechanisms underlying the contribution of Treg in HNSCC outcomes.

Covariance mapping [L. J. Frasinski, K. Codling, and P. A. Hatherly, Science 246, 1029 (1989)SCIEAS0036-807510.1126/science.246.4933.1029] is a well-established technique used for the study of mechanisms of laser-induced molecular ionization and decomposition. It measures statistical correlations between fluctuating signals of pairs of detected species (ions, fragments, electrons). A positive correlation identifies pairs of products originating from the same dissociation or ionization event. A major challenge for covariance-mapping spectroscopy is accessing decompositions of large polyatomic molecules, where true physical correlations are overwhelmed by spurious signals of no physical significance induced by fluctuations in experimental parameters. As a result, successful applications of covariance mapping have so far been restricted to low-mass systems, e.g., organic molecules of around 50 daltons (Da). Partial-covariance mapping was suggested to tackle the problem of spurious correlations by taking into account the independently measured fluctuations in the experimental conditions. However, its potential has never been realized for the decomposition of large molecules, because in these complex situations, determining and continuously monitoring multiple experimental parameters affecting all the measured signals simultaneously becomes unfeasible. We introduce, through deriving theoretically and confirming experimentally, a conceptually new type of partial-covariance mapping—self-correcting partial-covariance spectroscopy—based on a parameter extracted from the measured spectrum itself. We use the readily available total ion count as the self-correcting partial-covariance parameter, thus eliminating the challenge of determining experimental parameter fluctuations in covariance measurements of large complex systems. The introduced self-correcting partial covariance enables us to successfully resolve correlations of molecules as large as 10^{3}–10^{4} Da, 2 orders of magnitude above the state of the art. This opens new opportunities for mechanistic studies of large molecule decompositions through revealing their fragment-fragment correlations. Moreover, we demonstrate that self-correcting partial covariance is applicable to solving the inverse problem: reconstruction of a molecular structure from its fragment spectrum, within two-dimensional partial-covariance mass spectrometry.

Sarcomas are highly aggressive cancers that have a high propensity for metastasis, fail to respond to conventional therapies, and carry a poor 5-year survival rate. This is particularly true for patients with neurofibromatosis type 1 (NF1), in which 8%–13% of affected individuals will develop a malignant peripheral nerve sheath tumor (MPNST). Despite continued research, no effective therapies have emerged from recent clinical trials based on preclinical work. One explanation for these failures could be the lack of attention to intra-tumoral heterogeneity. Prior studies have relied on a single sample from these tumors, which may not be representative of all subclones present within the tumor. In the current study, samples were taken from three distinct areas within a single tumor from a patient with an NF1-MPNST. Whole exome sequencing, RNA sequencing, and copy number analysis were performed on each sample. A blood sample was obtained as a germline DNA control. Distinct mutational signatures were identified in different areas of the tumor as well as significant differences in gene expression among the spatially distinct areas, leading to an understanding of the clonal evolution within this patient. These data suggest that multi-regional sampling may be important for driver gene identification and biomarker development in the future.

With the increase in molecular diagnostics and patient-specific therapeutic approaches, the delivery and targeting of imaging molecules and pharmacologically active agents gain increasing importance. The ideal delivery system does not exist yet. The realization of two features is indispensable: first, a locally high concentration of target-specific diagnostic and therapeutic molecules; second, the broad development of effective and safe carrier systems. Here we characterize the transport properties of the peptide-based BioShuttle transporter using FFM and CLSM methods. The modular design of BioShuttle-based formulations results in a multi-faceted field of applications, also as a theranostic tool.

Progress in genomics and proteomics attended to the door for better understanding the recent rapid expanding complex research field of metabolomics. This trend in biomedical research increasingly focuses to the development of patient-specific therapeutic approaches with higher efficiency and sustainability. Simultaneously undesired adverse reactions are avoided. In parallel, the development of molecules for molecular imaging is required not only for the imaging of morphological structures but also for the imaging of metabolic processes like the aberrant expression of the cysteine protease cathepsin B (CtsB) gene and the activity of the resulting product associated with metastasis and invasiveness of malign tumors. Finally the objective is to merge imaging and therapy at the same level. The design of molecules which fulfil these responsibilities is pivotal and requires proper chemical methodologies. In this context our modified solid phase peptide chemistry using temperature shifts during synthesis is considered as an appropriate technology. We generated highly variable conjugates which consist of molecules useful as diagnostically and therapeutically active molecules. As an example the modular PNA products with the complementary sequence to the CtsB mRNA and additionally with a cathepsin B cleavage site had been prepared as functional modules for distinction of cell lines with different CtsB gene expression. After ligation to the modular peptide-based BioShuttle carrier, which was utilized to facilitate the delivery of the functional modules into the cells' cytoplasm, the modules were scrutinized.

Innovative and personalized therapeutic approaches result from the identification and control of individual aberrantly expressed genes at the transcriptional and post-transcriptional level. Therefore, it is of high interest to establish diagnostic, therapeutic and theranostic strategies at these levels. In the present study, we used the Diels-Alder Reaction with inverse electron demand (DARinv) click chemistry to prepare a series of cyclic RGD-BioShuttle constructs. These constructs carry the near-infrared (NIR) imaging agent Cy7 and the chemotherapeutic agent temozolomide (TMZ). We evaluated their uptake by and their efficacy against integrin αvβ3-expressing MCF7 human breast carcinoma cells. In addition, using a mouse phantom, we analyzed the suitability of this targeted theranostic agent for NIR optical imaging. We observed that the cyclic RGD-based carriers containing TMZ and/or Cy7 were effectively taken up by αvβ3-expressing cells, that they were more effective than free TMZ in inducing cell death, and that they could be quantitatively visualized using NIR fluorescence imaging. Therefore, these targeted theranostic agents are considered to be highly suitable systems for improving disease diagnosis and therapy.

In the near future personalized medicine with nucleic acids will play a key role in molecular diagnostics and therapy, which require new properties of the nucleic acids, like stability against enzymatic degradation. Here we demonstrate that the replacement of nucleobases with PNA by functional molecules harbouring either a dienophile or a diene reactivity is feasible and confers all new options for functionalization. These newly developed derivatives allow independent multi-ligations of multi-faceted components by use of the inverse Diels Alder technology. The high chemical stability and the ease of synthesis qualify these polyamide building blocks as favourites for intracellular delivery and targeting applications. This allows local drug concentrations sufficient for imaging and therapy and simultaneously a reduction of the application doses. It is important to point out that this technology is not restricted to ligation of medicament material; it is also a candidate to develop new and highly efficient active compounds for a “sustainable pharmacy”.

Progress in genome research led to new perspectives in diagnostic applications and to new promising therapies. On account of their specificity and sensitivity, nucleic acids (DNA/RNA) increasingly are in the focus of the scientific interest. While nucleic acids were a target of therapeutic interventions up to now, they could serve as excellent tools in the future, being highly sequence-specific in molecular diagnostics. Examples for imaging modalities are the representation of metabolic processes (Molecular Imaging) and customized therapeutic approaches (“Targeted Therapy”). In the individualized medicine nucleic acids could play a key role; this requires new properties of the nucleic acids, such as stability. Due to evolutionary reasons natural nucleic acids are substrates for nucleases and therefore suitable only to a limited extent as a drug. To use DNA as an excellent drug, modifications are required leading e.g. to a peptide nucleic acid (PNA). Here we show that an easy substitution of nucleobases by functional molecules with different reactivity like the Reppe anhydride and pentenoic acid derivatives is feasible. These derivatives allow an independent multi-ligation of functionalized compounds, e.g. pharmacologically active ones together with imaging components, leading to local concentrations sufficient for therapy and diagnostics at the same time. The high chemical stability and ease of synthesis could enhance nucleic chemistry applications and qualify PNA as a favourite for delivery. This system is not restricted to medicament material, but appropriate for the development of new and highly efficient drugs for a sustainable pharmacy.

Among the applications of fullerene technology in health sciences the expanding field of magnetic resonance imaging (MRI) of molecular processes is most challenging. Here we present the synthesis and application of a GdxSc3-xN@C80-BioShuttle-conjugate referred to as Gd-cluster@-BioShuttle, which features high proton relaxation and, in comparison to the commonly used contrast agents, high signal enhancement at very low Gd concentrations. This modularly designed contrast agent represents a new tool for improved monitoring and evaluation of interventions at the gene transcription level. Also, a widespread monitoring to track individual cells is possible, as well as sensing of microenvironments. Furthermore, BioShuttle can also deliver constructs for transfection or active pharmaceutical ingredients, and scaffolding for incorporation with the host's body. Using the Gd-cluster@-BioShuttle as MRI contrast agent allows an improved evaluation of radio- or chemotherapy treated tissues.

Clinical experiences often document, that a successful tumor control requires high doses of drug applications. It is widely believed that unavoidable adverse reactions could be minimized by using gene-therapeutic strategies protecting the tumor-surrounding healthy tissue as well as the bone-marrow. One new approach in this direction is the use of “Targeted Therapies” realizing a selective drug targeting to gain effectual amounts at the target site, even with drastically reduced application doses. MCF-7 breast cancer cells expressing the αvβ3 [alpha(v)beta(3)] integrin receptor are considered as appropriate candidates for such a targeted therapy. The modularly composed BioShuttle carrier consisting of different units designed to facilitate the passage across the cell membranes and for subcellular addressing of diagnostic and/or therapeutic molecules could be considered as an eligible delivery platform. Here we used the cyclic RGD-BioShuttle as a carrier for temozolomide (TMZ) at the αvβ3 integrin receptor realizing local TMZ concentrations sufficient for cell killing. The IC50 values are 12 µMol/L in the case of cRGD-BioShuttle-TMZ and 100 µMol/L for underivatized TMZ, which confirms the advantage of TMZ reformulation to realize local concentrations sufficient for cell killing.Our paper focuses on the design, synthesis and application of the cRGD-BioShuttle conjugate composed of the cyclic RGD, a αvβ3 integrin-ligand, ligated to the cytotoxic drug TMZ. The ligation was carried out by the Diels Alder Reaction with inverse electron demand (DARinv).

The ligation of active pharmaceutical ingredients (API) for working with image processing systems in diagnostics (MRT) attracts increasing notice and scientific interest. The Diels-Alder ligation Reaction with inverse electron demand (DARinv) turns out to be an appropriate candidate. The DARinv is characterized by a specific distribution of electrons of the diene and the corresponding dienophile counterpart. Whereas the reactants in the classical Diels-Alder Reaction feature electron-rich diene and electron-poor dienophile compounds, the DARinv exhibits exactly the opposite distribution of electrons. Substituents with pushing electrones increase and, with pulling electrons reduce the electron density of the dienes as used in the DARinv. We report here that the DARinv is an efficient route for coupling of multifunctional molecules like active peptides, re-formulated drugs or small molecules like the alkyalting agent temozolomide (TMZ). This is an example of our contribution to the "Click chemistry" technology. In this case TMZ is ligated by DARinv as a cargo to transporter molecules facilitating the passage across the cell membranes into cells and subsequently into subcellular components like the cell nucleus by using address molecules. With such constructs we achieved high local concentrations at the desired target site of pharmacological action. The DARinv ligation was carried out using the combination of several technologies, namely: the organic chemistry and the solid phase peptide synthesis which can produce 'tailored' solutions for questions not solely restricted to the medical diagnostics or therapy, but also result in functionalizations of various surfaces qualified amongst others also for array development. We like to acquaint you with the DARinv and we like to exemplify that all ligation products were generated after a rapid and complete reaction in organic solutions at room temperature, in high purity, but also, hurdles and difficulties on the way to the TMZ-BioShuttle conjugate should be mentioned. With this report we would like to stimulate scientists working with the focus on "Click chemistry" to intensify research with this expanding DARinv able to open the door for new solutions inconceivable so far.

If metastatic prostate cancer gets resistant to antiandrogen therapy, there are few treatment options, because prostate cancer is not very sensitive to cytostatic agents. Temozolomide (TMZ) as an orally applicable chemotherapeutic substance has been proven to be effective and well tolerated with occasional moderate toxicity especially for brain tumors and an application to prostate cancer cells seemed to be promising. Unfortunately, TMZ was inefficient in the treatment of symptomatic progressive hormone-refractory prostate cancer (HRPC). The reasons could be a low sensitivity against TMZ the short plasma half-life of TMZ, non-adapted application regimens and additionally, the aneuploid DNA content of prostate cancer cells suggesting different sensitivity against therapeutical interventions e.g. radiation therapy or chemotherapy. Considerations to improve this unsatisfying situation resulted in the realization of higher local TMZ concentrations, sufficient to kill cells regardless of intrinsic cellular sensitivity and cell DNA-index. Therefore, we reformulated the TMZ by ligation to a peptide-based carrier system called TMZ-BioShuttle for intervention. The modular-composed carrier consists of a transmembrane transporter (CPP), connected to a nuclear localization sequence (NLS) cleavably-bound, which in turn was coupled with TMZ. The NLS-sequence allows an active delivery of the TMZ into the cell nucleus after transmembrane passage of the TMZ-BioShuttle and intra-cytoplasm enzymatic cleavage and separation from the CPP. This TMZ-BioShuttle could contribute to improve therapeutic options exemplified by the hormone refractory prostate cancer. The next step was to syllogize a qualified method monitoring cell toxic effects in a high sensitivity under consideration of the ploidy status. The high-resolution flow cytometric analysis showed to be an appropriate system for a better detection and distinction of several cell populations dependent on their different DNA-indices as well as changes in proliferation of cell populations after chemotherapeutical treatment.

Klaus Braun1, Manfred Wiessler1, Volker Ehemann2, Ruediger Pipkorn3, Herbert Spring4, Juergen Debus5, Bernd Didinger5, Mario Koch3, Gabriele Muller6, Waldemar Waldeck61German Cancer Research Center, Dept of Imaging and Radiooncology, Heidelberg, Germany; 2University of Heidelberg, Institute of Pathology, Heidelberg, Germany; 3German Cancer Research Center, Central Peptide Synthesis Unit, Heidelberg, Germany; 4German Cancer Research Center, Dept of Structural Analysis of Gene Structure and Function, Heidelberg, Germany; 5University of Heidelberg, Dept of Radiation Oncology, Heidelberg, Germany; 6German Cancer Research Center,Division of Biophysics of Macromolecules, Heidelberg, GermanyAbstract: Recurrent glioblastoma multiforme (GBM), insensitive against most therapeutic interventions, has low response and survival rates. Temozolomide (TMZ) was approved for second-line therapy of recurrent anaplastic astrocytoma. However, TMZ therapy in GBM patients reveals properties such as reduced tolerability and inauspicious hemogram. The solution addressed here concerning GBM therapy consolidates and uses the potential of organic and peptide chemistry with molecular medicine. We enhanced the pharmacologic potency with simultaneous reduction of unwanted adverse reactions of the highly efficient chemotherapeutic TMZ. The TMZ connection to transporter molecules (TMZ-BioShuttle) was investigated, resulting in a much higher pharmacological effect in glioma cell lines and also with reduced dose rate. From this result we can conclude that a suitable chemistry could realize the ligation of pharmacologically active, but sensitive and highly unstable pharmaceutical ingredients without functional deprivation. The TMZ-BioShuttle dramatically enhanced the potential of TMZ for the treatment of brain tumors and is an attractive drug for combination chemotherapy.Keywords: drug delivery, carrier molecules, facilitated transport, glioblastoma multiforme, temozolomide

The Human immunodeficiency 1 derived capsid assembly inhibitor peptide (HIV-1 CAI-peptide) is a promising lead candidate for anti-HIV drug development. Its drawback, however, is that it cannot permeate cells directly. Here we report the transport of the pharmacologically active CAI-peptide into human lymphocytes and Human Embryonic Lung cells (HEL) using the BioShuttle platform. Generally, the transfer of pharmacologically active substances across membranes, demonstrated by confocal laser scanning microscopy (CLSM), could lead to a loss of function by changing the molecule's structure. Molecular dynamics (MD) simulations and circular dichroism (CD) studies suggest that the CAI-peptide has an intrinsic capacity to form a helical structure, which seems to be critical for the pharmacological effect as revealed by intensive docking calculations and comparison with control peptides. This coupling of the CAI-peptide to a BioShuttle-molecule additionally improved its solubility. Under the conditions described, the HIV-1 CAI peptide was transported into living cells and could be localized in the vicinity of the mitochondria.

There is a large number of effective cytotoxic drugs whose side effect profile, efficacy, and long-term use in man are well understood and documented over decades of use in clinical routine e.g. in the treatment of recurrent glioblastoma multiforme (GBM) and the hormone-refractory prostate cancer (HRPC). Both cancers are insensitive against most chemotherapeutic interventions; they have low response rates and poor prognoses. Some cytotoxic agents can be significantly improved by using modern technology of drug delivery or formulation. We succeeded to enhance the pharmacologic potency with simultaneous reduction of unwanted adverse reactions of the highly efficient chemotherapeutic temozolomide (TMZ) as an example. The TMZ connection to transporter molecules (TMZ-BioShuttle) resulted in a much higher pharmacological effect in glioma cell lines while using reduced doses. This permits the conclusion that a suitable chemistry could realize the ligation of pharmacologically active, but sensitive and highly unstable pharmaceutical ingredients without functional deprivation. The re-formulation of TMZ to TMZ-BioShuttle achieved a nearly 10-fold potential of the established pharmaceutic TMZ far beyond the treatment of brain tumors cells and results in an attractive reformulated drug with enhanced therapeutic index.

An efficient gene transfer into target tissues and cells is needed for safe and effective treatment of genetic diseases like cancer. In this paper, we describe the development of a transport system and show its ability for transporting plasmids. This non-viral peptide-based BioShuttle-mediated transfer system consists of a nuclear localization address sequence realizing the delivery of the plasmid phNIS-IRES-EGFP coding for two independent reporter genes into nuclei of HeLa cells. The quantification of the transfer efficiency was achieved by measurements of the sodium iodide symporter activity. EGFP gene expression was measured with Confocal Laser Scanning Microscopy and quantified with biostatistical methods by analysis of the frequency of the amplitude distribution in the CLSM images. The results demonstrate that the “BioShuttle”-Technology is an appropriate tool for an effective transfer of genetic material carried by a plasmid.

Myalgic encephalomyelitis (ME, also called Chronic Fatigue Syndrome), a common disease with chronic fatigability, cognitive dysfunction and myalgia of unknown etiology, often starts with an infection. The chaperonin human heat shock protein 60 (HSP60) occurs in mitochondria and in bacteria, is highly conserved, antigenic and a major autoantigen. The anti-HSP60 humoral (IgG and IgM) immune response was studied in 69 ME patients and 76 blood donors (BD) (the Training set) with recombinant human and E coli HSP60, and 136 30-mer overlapping and targeted peptides from HSP60 of humans, Chlamydia, Mycoplasma and 26 other species in a multiplex suspension array. Peptides from HSP60 helix I had a chaperonin-like activity, but these and other HSP60 peptides also bound IgG and IgM with an ME preference, theoretically indicating a competition between HSP60 function and antibody binding. A HSP60-based panel of 25 antigens was selected. When evaluated with 61 other ME and 399 non-ME samples (331 BD, 20 Multiple Sclerosis and 48 Systemic Lupus Erythematosus patients), a peptide from Chlamydia pneumoniae HSP60 detected IgM in 15 of 61 (24%) of ME, and in 1 of 399 non-ME at a high cutoff (p

Gammaretrovirus-like sequences occur in most vertebrate genomes. Murine Leukemia Virus (MLV) like retroviruses (MLLVs) are a subset, which may be pathogenic and spread cross-species. Retroviruses highly similar to MLLVs (xenotropic murine retrovirus related virus (XMRV) and Human Mouse retrovirus-like RetroViruses (HMRVs)) reported from patients suffering from prostate cancer (PC) and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) raise the possibility that also humans have been infected. Structurally intact, potentially infectious MLLVs occur in the genomes of some mammals, especially mouse. Mouse MLLVs contain three major groups. One, MERV G3, contained MLVs and XMRV/HMRV. Its presence in mouse DNA, and the abundance of xenotropic MLVs in biologicals, is a source of false positivity. Theoretically, XMRV/HMRV could be one of several MLLV transspecies infections. MLLV pathobiology and diversity indicate optimal strategies for investigating XMRV/HMRV in humans and raise ethical concerns. The alternatives that XMRV/HMRV may give a hard-to-detect “stealth” infection, or that XMRV/HMRV never reached humans, have to be considered.