Automated Mass Photometry of Adeno-Associated Virus Vectors from Crude Cell Extracts.

Mass photometry (MP) is a fast and simple analysis method for the determination of the proportions of subpopulations in an AAV sample. It is label-free and requires minimal sample volumes between 5-10 µL, which makes it a promising candidate over orthogonal techniques such as analytical ultracentrifugation (AUC), cryo-transmission electron microscopy (Cryo-TEM) or charge-detection mass spectrometry (CDMS). However, these methods are limited in their application to purified samples only. Here we developed a purification step based on single-domain monospecific antibody fragments immobilised on either a poly(styrene-divinylbenzene) resin or on magnetic beads prior to MP analysis that allows the quantification of empty, partially filled, full and overfull AAV vectors in crude cell extracts. This is aimed at identifying potentially promising harvest conditions that yield large numbers of filled AAV vectors during the early stages of the viral vector development platform, e.g., the type of transfection reagent used. Furthermore, we provide a direct comparison of the automated and manual handling of the mass photometer with respect to the quantities of AAV subspecies, molar mass of the capsid and payload, and highlight the differences between the "buffer-free" sample measurement and the "buffer-dilution" mode. In addition, we provide information on which candidates to use for calibration and demonstrate the limitations of the mass photometer with respect to the estimation of the capsid titer.

Quantification of Empty, Partially Filled and Full Adeno-Associated Virus Vectors Using Mass Photometry.

Adeno-associated viruses (AAV) are one of the most commonly used vehicles in gene therapies for the treatment of rare diseases. During the AAV manufacturing process, particles with little or no genetic material are co-produced alongside the desired AAV capsid containing the transgene of interest. Because of the potential adverse health effects of these byproducts, they are considered impurities and need to be monitored carefully. To date, analytical ultracentrifugation (AUC), transmission electron microscopy (TEM) and charge-detection mass spectrometry (CDMS) are used to quantify these subspecies. However, they are associated with long turnaround times, low sample throughput and complex data analysis. Mass photometry (MP) is a fast and label-free orthogonal technique which is applicable to multiple serotypes without the adaption of method parameters. Furthermore, it can be operated with capsid titers as low as 8 × 1010 cp mL-1 with a CV < 5% using just 10 µL total sample volume. Here we demonstrate that mass photometry can be used as an orthogonal method to AUC to accurately quantify the proportions of empty, partially filled, full and overfull particles in AAV samples, especially in cases where ion-exchange chromatography yields no separation of the populations. In addition, it can be used to confirm the molar mass of the packaged genomic material in filled AAV particles.

Associations between occupational balance, subjective health, and well-being of informal caregivers of older persons based on a cross-sectional study.

OBJECTIVES: Population ageing leads to a noticeable increase in demand for informal care. Informal caregivers experience high caregiver burden, such as restricted subjective health and well-being. Occupational balance is associated with subjective health and well-being. However, associations between occupational balance and subjective health and well-being of informal caregivers of older persons have not been investigated yet. Thus, the objective of this study was to explore associations between occupational balance and subjective health and well-being of informal caregivers of older persons. METHODS: From September 2016 to July 2020, a cross-sectional multicenter study design was employed in Austria. Informal caregivers' occupational balance, subjective health, and well-being as well as comorbidity of persons to be cared for were assessed with seven self-reported questionnaires. Spearman's rank correlation coefficients rs were calculated to determine associations between occupational balance and subjective health and well-being of informal caregivers of older persons. RESULTS: In total 118 informal caregivers, 102 (86%) female, and their persons to be cared for, 70 (59%) female, were considered for analyses. Median age was 58 years for informal caregivers and 81 years for persons to be cared for. Informal caregivers reported restrictions in occupational balance, subjective health, and well-being. Persons to be cared for showed comorbid health conditions. Significant associations between occupational balance and determinants of subjective health and well-being were identified (rs - 0.30 - 0.69; p ≤ 0.01). CONCLUSIONS: As population ageing and the demand for informal care progress, efforts to support informal caregivers and to strengthen their occupational balance, subjective health and well-being are vital.

Biophysical Characterization of Adeno-Associated Virus Vectors Using Ion-Exchange Chromatography Coupled to Light Scattering Detectors.

Ion-exchange chromatography coupled to light scattering detectors represents a fast and simple analytical method for the assessment of multiple critical quality attributes (CQA) in one single measurement. The determination of CQAs play a crucial role in Adeno-Associated Virus (AAV)-based gene therapies and their applications in humans. Today, several different analytical techniques, including size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), qPCR or ELISA, are commonly used to characterize the gene therapy product regarding capsid titer, packaging efficiency, vector genome integrity, aggregation content and other process-related impurities. However, no universal method for the simultaneous determination of multiple CQAs is currently available. Here, we present a novel robust ion-exchange chromatography method coupled to multi-angle light scattering detectors (IEC-MALS) for the comprehensive characterization of empty and filled AAVs concerning capsid titer, full-to-total ratio, absolute molar mass of the protein and nucleic acid, and the size and polydispersity without baseline-separation of both species prior to data analysis. We demonstrate that the developed IEC-MALS assay is applicable to different serotypes and can be used as an orthogonal method to other established analytical techniques.

Impact of COVID-19 lockdown on routine oncology versus emergency care at a high volume cancer centre.

BACKGROUND: We investigated the influence of population-wide COVID-19 lockdown measures implemented on 16, March 2020 on routine and emergency care of cancer outpatients at a tertiary care cancer centre in Vienna, Austria. METHODS: We compared the number/visits of cancer outpatients receiving oncological therapies at the oncologic day clinic (DC) and admissions at the emergency department (ED) of our institution in time periods before (pre-lockdown period: 1 January - 15 March 2020) and after (post-lockdown period: 16 March- 31 May 2020) lockdown implementation with the respective reference periods of 2018 and 2019. Additionally, we analysed Emergency Severity Index (ESI) score of unplanned cancer patient presentations to the ED in the same post-lockdown time periods. Patient outcome was described as 3-month mortality rate (3-MM). RESULTS: In total, 16 703 visits at the DC and 2664 patient visits for the respective time periods were recorded at the ED. No decrease in patient visits was observed at the DC after lockdown implementation (P = .351), whereas a substantial decrease in patient visits at the ED was seen (P < .001). This translates into a 26%-31% reduction of cancer-related patient visits per half month after the lockdown at the ED (P < .001 vs. 2018 + 2019). There was no difference in the distribution of ESI scores at ED presentation (P = .805), admission rates or 3-MM in association with lockdown implementation (P = .086). CONCLUSION: We demonstrate the feasibility of maintaining antineoplastic therapy administration during the COVID-19 pandemic. However, our data underline the need for adapted management strategies for emergency presentations of cancer patients.

FGF2 and EGF induce epithelial-mesenchymal transition in malignant pleural mesothelioma cells via a MAPKinase/MMP1 signal.

Malignant pleural mesothelioma (MPM), an aggressive malignancy affecting pleural surfaces, occurs in three main histological subtypes. The epithelioid and sarcomatoid subtypes are characterized by cuboid and fibroblastoid cells, respectively. The biphasic subtype contains a mixture of both. The sarcomatoid subtype expresses markers of epithelial-mesenchymal transition (EMT) and confers the worst prognosis, but the signals and pathways controlling EMT in MPM are not well understood. We demonstrate that treatment with FGF2 or EGF induced a fibroblastoid morphology in several cell lines from biphasic MPM, accompanied by scattering, decreased cell adhesion and increased invasiveness. This depended on the MAP-kinase pathway but was independent of TGFß or PI3-kinase signaling. In addition to changes in known EMT markers, microarray analysis demonstrated differential expression of MMP1, ESM1, ETV4, PDL1 and BDKR2B in response to both growth factors and in epithelioid versus sarcomatoid MPM. Inhibition of MMP1 prevented FGF2-induced scattering and invasiveness. Moreover, in MPM cells with sarcomatoid morphology, inhibition of FGF/MAP-kinase signaling induced a more epithelioid morphology and gene expression pattern. Our findings suggest a critical role of the MAP-kinase axis in the morphological and behavioral plasticity of mesothelioma.

In utero exposure to cigarette smoke and effects across generations: A conference of animals on asthma.

BACKGROUND: The prevalence of asthma and chronic obstructive pulmonary disease (COPD) has risen markedly over the last decades and is reaching epidemic proportions. However, underlying molecular mechanisms are not fully understood, hampering the urgently needed development of approaches to prevent these diseases. It is well established from epidemiological studies that prenatal exposure to cigarette smoke is one of the main risk factors for aberrant lung function development or reduced fetal growth, but also for the development of asthma and possibly COPD later in life. Of note, recent evidence suggests that the disease risk can be transferred across generations, that is, from grandparents to their grandchildren. While initial studies in mouse models on in utero smoke exposure have provided important mechanistic insights, there are still knowledge gaps that need to be filled. OBJECTIVE: Thus, in this review, we summarize current knowledge on this topic derived from mouse models, while also introducing two other relevant animal models: the fruit fly Drosophila melanogaster and the zebrafish Danio rerio. METHODS: This review is based on an intensive review of PubMed-listed transgenerational animal studies from 1902 to 2018 and focuses in detail on selected literature due to space limitations. RESULTS: This review gives a comprehensive overview of mechanistic insights obtained in studies with the three species, while highlighting the remaining knowledge gaps. We will further discuss potential (dis)advantages of all three animal models. CONCLUSION/CLINICAL RELEVANCE: Many studies have already addressed transgenerational inheritance of disease risk in mouse, zebrafish or fly models. We here propose a novel strategy for how these three model organisms can be synergistically combined to achieve a more detailed understanding of in utero cigarette smoke-induced transgenerational inheritance of disease risk.

Retention Load of Telescopic Crowns with Different Taper Angles between Cobalt-Chromium and Polyetheretherketone Made with Three Different Manufacturing Processes Examined by Pull-Off Test.

PURPOSE: To investigate the retention loads of differently fabricated secondary telescopic polyetheretherketone (PEEK) crowns on cobalt-chromium primary crowns with different tapers. MATERIALS AND METHODS: Cobalt-chromium primary crowns with 0°, 1°, and 2° tapers were constructed, milled, and sintered. Corresponding secondary crowns were fabricated by milling, pressing from pellets, and pressing from granules. For these nine test groups, the pull-off tests of each crown combination were performed 20 times, and the retention loads were measured (Zwick 1445, 50 mm/min). Data were analyzed using linear regression, covariance analysis, mixed models, Kruskal-Wallis, and Mann-Whitney U-test, together with the Benferroni-Holm correction. RESULTS: The mixed models covariance analysis reinforced stable retention load values (p = 0.162) for each single test sequence. There was no interaction between the groups and the separation cycles (p = 0.179). Milled secondary crowns with 0° showed the lowest mean retention load values compared to all tested groups (p = 0.003) followed by those pressed form pellets with 1°. Regarding the different tapers, no effect of manufacturing method on the results was observed within 1° and 2° groups (p = 0.540; p = 0.052); however, among the 0° groups, the milled ones showed significantly the lowest retention load values (p = 0.002). Among the manufacturing methods, both pressed groups showed no impact of taper on the retention load values (p > 0.324 and p > 0.123, respectively), whereas among the milled secondary crowns, the 0° taper showed significantly lower retention load values than the 1° and 2° taper (p < 0.002). CONCLUSION: Based on these results, telescopic crowns made of PEEK seem to show stable retention load values for each test sequence; however, data with thermo-mechanical aging are still required. In addition, further developments in CAD/CAM manufacturing of PEEK materials for telescopic crowns are warranted, especially for 0°.

Airway remodeling in asthma: what really matters.

Airway remodeling is generally quite broadly defined as any change in composition, distribution, thickness, mass or volume and/or number of structural components observed in the airway wall of patients relative to healthy individuals. However, two types of airway remodeling should be distinguished more clearly: (1) physiological airway remodeling, which encompasses structural changes that occur regularly during normal lung development and growth leading to a normal mature airway wall or as an acute and transient response to injury and/or inflammation, which ultimately results in restoration of a normal airway structures; and (2) pathological airway remodeling, which comprises those structural alterations that occur as a result of either disturbed lung development or as a response to chronic injury and/or inflammation leading to persistently altered airway wall structures and function. This review will address a few major aspects: (1) what are reliable quantitative approaches to assess airway remodeling? (2) Are there any indications supporting the notion that airway remodeling can occur as a primary event, i.e., before any inflammatory process was initiated? (3) What is known about airway remodeling being a secondary event to inflammation? And (4), what can we learn from the different animal models ranging from invertebrate to primate models in the study of airway remodeling? Future studies are required addressing particularly pheno-/endotype-specific aspects of airway remodeling using both endotype-specific animal models and "endotyped" human asthmatics. Hopefully, novel in vivo imaging techniques will be further advanced to allow monitoring development, growth and inflammation of the airways already at a very early stage in life.

Poly(inosinic-cytidylic) acid-triggered exacerbation of experimental asthma depends on IL-17A produced by NK cells.

Viral infection of the respiratory tract represents the major cause of acute asthma exacerbations. dsRNA is produced as an intermediate during replication of respiratory viruses and triggers immune responses via TLR3. This study aimed at clarifying the mechanisms underlying TLR3 triggered exacerbation of experimental allergic asthma. The TLR3 ligand poly(inosinic-cytidylic) acid was applied intranasally to mice with already established experimental allergic asthma. Airway inflammation, cytokine expression, mucus production, and airway reactivity was assessed in wild-type, IL-17A, or IL-23p19-deficient, and in NK cell-depleted mice. Local application of poly(inosinic-cytidylic) acid exacerbated experimental allergic asthma in mice as characterized by enhanced release of proinflammatory cytokines, aggravated airway inflammation, and increased mucus production together with pronounced airway hyperresponsiveness. This was further associated with augmented production of IL-17 by Th17 cells and NK cells. Whereas experimental exacerbation could be induced in IL-23p19-deficient mice lacking mature, proinflammatory Th17 cells, this was not possible in mice lacking IL-17A or in NK cell-depleted animals. These experiments indicate a central role for IL-17 derived from NK cells but not from Th17 cells in the pathogenesis of virus-triggered exacerbation of experimental asthma.

Maternal depression research: socioeconomic analysis and density-equalizing mapping of the global research architecture.

Maternal depression can be accounted for one of the most common complications during pregnancy and the postpartum period affecting women all over the world. So far, no detailed map of the worldwide maternal depression research architecture has been constructed, which encompasses aspects of research activity, quality, and also socioeconomic features. Using the NewQIS platform, density-equalizing mapping projections, scientometric techniques, and economic benchmarking procedures were applied to evaluate global maternal depression research for the period between 1900 and 2012. In total, 7330 related publications and 3335 international collaborations were identified. The USA was the most active country concerning collaborations and total research activity. In the socioeconomic analysis of research activity in high-income countries, Australia was ranked first with an average of 412.05 maternal depression-related publications per 1000 billion US$ GDP (Q1), followed by the UK (Q1 = 373.51) and Canada (Q1 = 306.32). The group of upper-middle-income countries was led by South Africa (Q1 = 145.67), followed by Turkey (Q1 = 91.8). China authored 11.95 maternal depression-related publications per 1000 billion US$ GDP. The USA had the highest activity of maternal depression research per GDP in billion US$ per capita (Q2 = 60.86). When research activity was related to population size (Q3 = publications per Mio. inhabitants), Australia (Q3 = 26.44) was leading the field, followed by Norway (Q3 = 18.48). Gender analysis revealed a relatively high degree of female scientists involved in this field of research with pronounced differences between single subject areas. In summary, we here present the first picture of the global scientific development in maternal depression research over a period of more than 100 years. The research landscape is clearly dominated by North American and Western European countries, with only minor contribution of Asian or South American countries.

Randomized controlled pilot trial of mindfulness-based stress reduction compared to psychoeducational support for persistently fatigued breast and colorectal cancer survivors.

PURPOSE: Cancer-related fatigue (CRF) is a disruptive symptom for many survivors. Despite promising evidence for efficacy of mindfulness-based stress reduction (MBSR) in reducing CRF, no trials comparing it to an active comparator for fatigued survivors have been published. The purpose of this trial was to compare MBSR to psychoeducation for CRF and associated symptoms. METHODS: Breast (n = 60) and colorectal (n = 11) cancer survivors (stage 0-III) with clinically significant CRF after completing chemotherapy and/or radiation therapy an average of 28 months prior to enrollment were randomized to MBSR or psychoeducation/support groups (PES). MBSR focused on mindfulness training; PES focused on CRF self-management. Outcomes included CRF interference (primary), CRF severity and global improvement, vitality, depression, anxiety, sleep disturbance, and pain. Outcomes were assessed at baseline (T1), post-intervention (T2), and 6-month follow-up (T3) using intent-to-treat analysis. RESULTS: Between-group differences in CRF interference were not significant at any time point; however, there was a trend favoring MBSR (d = -0.46, p = 0.073) at T2. MBSR participants reported significantly greater improvement in vitality (d = 0.53, p = 0.003) and were more likely to report CRF as moderately to completely improved compared to the PES group (χ2 (1) = 4.1765, p = 0.041) at T2. MBSR participants also reported significantly greater reductions in pain at T2 (d = 0.53, p = 0.014). In addition, both MBSR and PES produced moderate-to-large and significant within-group improvements in all fatigue outcomes, depression, anxiety, and sleep at T2 and T3 compared to T1. CONCLUSION: MBSR and PES appear efficacious for CRF and related symptoms. Larger trials including a usual care arm are warranted. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01724333.

Fibroblast growth factor receptor inhibition is active against mesothelioma and synergizes with radio- and chemotherapy.

RATIONALE: Malignant pleural mesothelioma is an aggressive malignancy characterized by frequent resistance to chemo- and radiotherapy, poor outcome, and limited therapeutic options. Fibroblast growth factors (FGFs) and their receptors are potential targets for cancer therapy, but their significance in mesothelioma has remained largely undefined. OBJECTIVES: To investigate the antimesothelioma potential of FGF receptor 1 (FGFR1) inhibition. METHODS: Expression of FGFs and their receptors was analyzed in mesothelioma cell lines and tissue specimens. Several cell models were used to investigate FGFR1 inhibition in vitro and in combination with cisplatin and irradiation. Mouse intraperitoneal xenotransplant models were used for in vivo validation. MEASUREMENTS AND MAIN RESULTS: FGFR1, FGF2, and FGF18 were overexpressed in mesothelioma. Stimulation with FGF2 led to increased cell proliferation, migration, and transition to a more sarcomatoid phenotype in subsets of mesothelioma cell lines. In contrast, inhibition of FGFR1 by a specific kinase inhibitor or a dominant-negative FGFR1 construct led to significantly decreased proliferation, clonogenicity, migration, spheroid formation, and G1 cell cycle arrest in several mesothelioma cell lines, accompanied by apoptosis induction and decreased mitogen-activated protein kinase pathway activity. Reduced tumor growth, proliferation, mitogenic signaling, and apoptosis induction were observed in vivo. Inhibition of FGFR1 synergistically enhanced the cytotoxic effects of ionizing radiation and cisplatin. CONCLUSIONS: Our data suggest that the malignant phenotype of mesothelioma cells depends on intact FGF signals, which should be considered as therapeutic targets with a promising chemo- and radiosensitizing potential.

Hyperactive STAT5 hijacks T cell receptor signaling and drives immature T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive immature T cell cancer. Mutations in IL7R have been analyzed genetically, but downstream effector functions such as STAT5A and STAT5B hyperactivation are poorly understood. Here, we studied the most frequent and clinically challenging STAT5BN642H driver in T cell development and immature T cell cancer onset and compared it with STAT5A hyperactive variants in transgenic mice. Enhanced STAT5 activity caused disrupted T cell development and promoted an early T cell progenitor-ALL phenotype, with upregulation of genes involved in T cell receptor (TCR) signaling, even in absence of surface TCR. Importantly, TCR pathway genes were overexpressed in human T-ALL and mature T cell cancers and activation of TCR pathway kinases was STAT5 dependent. We confirmed STAT5 binding to these genes using ChIP-Seq analysis in human T-ALL cells, which were sensitive to pharmacologic inhibition by dual STAT3/5 degraders or ZAP70 tyrosine kinase blockers in vitro and in vivo. We provide genetic and biochemical proof that STAT5A and STAT5B hyperactivation can initiate T-ALL through TCR pathway hijacking and suggest similar mechanisms for other T cell cancers. Thus, STAT5 or TCR component blockade are targeted therapy options, particularly in patients with chemoresistant clones carrying STAT5BN642H.

Toso regulates the balance between apoptotic and nonapoptotic death receptor signaling by facilitating RIP1 ubiquitination.

The regulation of cellular survival and apoptosis is of critical importance for the immune system to maintain immune homeostasis and to establish tolerance. Here, we demonstrate that the immune specific cell surface molecule Toso exhibits antiapoptotic effects on death receptor signaling by a novel regulatory mechanism involving the adaptor kinase RIP1. The antiapoptotic function of Toso depends on RIP1 ubiquitination and involves the recruitment of the death adaptor FADD to a Toso/RIP1 protein complex. In response to CD95L and TNFα, Toso promotes the activation of MAPK and NF-κB signaling pathways. Because of this relative augmentation of survival versus apoptotic signals, Toso raises the threshold for death receptor-mediated apoptosis. Our analysis of Toso-deficient mice revealed that Toso is essential for TNFα-mediated liver damage. Furthermore, the antiapoptotic function of Toso could be blocked by a Toso-specific monoclonal antibody, opening up new therapeutic prospects for the treatment of immune disorders and hematologic malignancies.

A Cancer-Indicative microRNA Pattern in Normal Prostate Tissue.

We analyzed the levels of selected micro-RNAs in normal prostate tissue to assess their potential to indicate tumor foci elsewhere in the prostate. Histologically normal prostate tissue samples from 31 prostate cancer patients and two cancer negative control groups with either unsuspicious or elevated prostate specific antigen (PSA) levels (14 and 17 individuals, respectively) were analyzed. Based on the expression analysis of 157 microRNAs in a pool of prostate tissue samples and information from data bases/literature, we selected eight microRNAs for quantification by real-time polymerase chain reactions (RT-PCRs). Selected miRNAs were analyzed in histologically tumor-free biopsy samples from patients and healthy controls. We identified seven microRNAs (miR-124a, miR-146a & b, miR-185, miR-16 and let-7a & b), which displayed significant differential expression in normal prostate tissue from men with prostate cancer compared to both cancer negative control groups. Four microRNAs (miR-185, miR-16 and let-7a and let-7b) remained to significantly discriminate normal tissues from prostate cancer patients from those of the cancer negative control group with elevated PSA levels. The transcript levels of these microRNAs were highly indicative for the presence of cancer in the prostates, independently of the PSA level. Our results suggest a microRNA-pattern in histologically normal prostate tissue, indicating prostate cancer elsewhere in the organ.

Transfer Function for Relative Blast Overpressure Through Porcine and Human Skulls In Situ.

INTRODUCTION: The overarching objective of the Office of Naval Research sponsored Blast Load Assessment Sense and Test (BLAST) program was to quantify neurofunctional risk from repeated blast exposure. However, human studies have limitations in data collection that can only be addressed by animal models. To utilize a large animal model in this work, researchers developed an approach for scaling blast exposure data from animal to human-equivalent loading. For this study, energy interacting with the brain tissue was selected as a translation metric because of the hypothesized association between observed neurological changes and energy transmitted through the skull. This article describes the methodology used to derive an energy-based transfer function capable of serving as a global correspondence rule for primary blast injury exposure, allowing researchers to derive human-appropriate thresholds from animal data. METHODS AND MATERIALS: To generate data for the development of the transfer functions, three disarticulated cadaveric Yucatan minipigs and three postmortem human surrogate heads were exposed to blast overpressure using a large bore, compressed-gas shock tube. Pressure gauges in the free field, on the skull surface, and pressure probes within the brain cavity filled with Sylgard silicone gel recorded the pressure propagation through the skull of each specimen. The frequency components of the freefield and brain cavity measurements from the pig and human surrogates were interrogated in the frequency domain. Doing so quantifies the differences in the amount of energy, in each frequency band, transmitted through both the porcine and the human skull, and the transfer function was calculated to quantify those differences. RESULTS: Nonlinear energy transmission was observed for both the porcine and human skulls, indicating that linear scaling would not be appropriate for developing porcine to human transfer functions. This study demonstrated similar responses between species with little to no attenuation at frequencies below 30 Hz. The phase of the pressure transmission to the brain is also similar for both species up to approximately 10 kHz. There were two notable differences between the porcine and human surrogates. First, in the 40-100 Hz range, human subjects have approximately 8 dB more pressure transmitted through the skull relative to porcine subjects. Second, in the 1-10 kHz range, human subjects have up to 10 dB more pressure transmitted into the brain (10 dB more attenuation) relative to the porcine subjects. CONCLUSIONS: The fundamental goal of this study was to develop pig-to-human transfer functions to allow researchers to interpret data collected from large animal studies and aid in deriving risk functions for repeated blast exposures. Similarities in porcine and human brain physiology make the minipig experimental model an excellent candidate for blast research. However, differences in the skull geometry have historically made the interpretation of animal data difficult for the purposes of characterizing potential neurological risk in humans. Human equivalent loading conditions are critical so that the thresholds are not over- or underpredicted due to differences in porcine skull geometry. This research provides a solution to this challenge, providing a robust methodology for interpreting animal data for blast research.

The Application of Machine Learning to Identify Large Animal Blast Exposure Thresholds.

INTRODUCTION: The Office of Naval Research sponsored the Blast Load Assessment Sense and Test program to develop a rapid, in-field solution that could be used by team leaders, commanders, and medical personnel to make science-based stand-down decisions for service members exposed to blast overpressure. Toward this goal, the authors propose an ensemble approach based on machine learning (ML) methods to derive a threshold surface for potential neurological deficits that encompasses the intensity of the blast events, the number of exposures, and the period over which the exposures occurred. Because of collection challenges presented by human subjects, the authors utilized data representing a comprehensive set of measures, including structural, behavioral, and cellular changes, from preclinical large animal studies on minipig models. This article describes the development process used to procure the resulting methodology from these studies. METHODS AND MATERIALS: Using an ensemble of ML methods applied to experimental data obtained from 71 Yucatan minipigs, the relationship between blast exposure and neurological deficits was delineated. Despite a relatively small sample size, ML methods with k-fold cross-validation (with k = 5) were justified because of the complexity of the dataset reflecting numerous nonlinear relationships between cellular, structural, and behavioral markers. Based on the physiological responses and environmental measures collected during the large animal study, two models were developed to investigate the relationship between multiple outcome measures and exposure to blast. The histological features model was trained on single-exposure animal data to predict a binary injury response (injured or not) using histological features. The environmental features model related the observed behavioral changes to the environmental parameters collected. RESULTS: The histological features model predicted a binary injury outcome from cellular and physiological measurements. Features identified in developing this classification model showed some level of correlation to observed behavioral changes, suggesting that glial activation inflammation and neurodegenerative responses occur even at the lowest levels of blast exposures tested. The results of the environmental features model, which estimated injury risk from environmental blast exposure characteristics, suggested that the observed changes are not just a function of impulse but an average dynamic impulse rate. Noticeable behavioral deficits were observed at loading rates of 100 kPa (impulse/positive duration) or peak pressures of 300-350 kPa, with an approximate positive phase duration of 3.4 ms for single exposure. Based on this analysis, a 3D threshold surface was developed to characterize the potential risk of neurological deficits. CONCLUSIONS: The ensemble approach facilitated the identification of a pattern of changes across multiple variables to predict the occurrence of changes in brain function. Many changes observed after blast exposure were subtle, making them difficult to measure in human subjects. ML methodologies applied to minipig data demonstrated the value of these techniques in analyzing complex datasets to complement human studies. Importantly, the threshold surface supports the development of science-based blast exposure guidelines.

The microbial composition of larval airways from Drosophila melanogaster differ between specimens from laboratory and natural habitats.

BACKGROUND: The fruit fly Drosophila melanogaster lives in natural habitats and has also long been used as a model organism in biological research. In this study, we used a molecular barcoding approach to analyse the airways microbiome of larvae of D. melanogaster, which were obtained from eggs of flies of the laboratory strain w1118 and from immune deficient flies (NF-kB-K), and from wild-caught flies. To assess intergenerational transmission of microbes, all eggs were incubated under the same semi-sterile conditions. RESULTS: The airway microbiome of larvae from both lab-strains was dominated by the two families Acetobacteraceae and Lactobacillaceae, while larvae from wild-caught flies were dominated by Lactobacillaceae, Anaplasmataceae and Leuconostocaceae. Barcodes linked to Anaplasmataceae could be further assigned to Wolbachia sp., which is a widespread intracellular pathogen in arthropods. For Leuconostoceae, the most abundant reads were assigned to Weissella sp. Both Wolbachia and Weissella affect the development of the insects. Finally, a relative high abundance of Serratia sp. was found in larvae from immune deficient relish-/- compared to w1118 and wild-caught fly airways. CONCLUSIONS: Our results show for the first time that larvae from D. melanogaster harbor an airway microbiome, which is of low complexity and strongly influenced by the environmental conditions and to a lesser extent by the immune status. Furthermore, our data indicate an intergenerational transmission of the microbiome as shaped by the environment.

Real-world Data of Palliative First-line Checkpoint Inhibitor Therapy for Head and Neck Cancer.

BACKGROUND/AIM: Pembrolizumab alone or combined with chemotherapy is now approved in PD-L1-positive patients with recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN). Since real-world data are pending, our goal was to evaluate the efficacy and safety of immune checkpoint inhibitor (CPI) therapy in an unselected cohort of patients with SCCHN. PATIENTS AND METHODS: We analyzed 78 patients with recurrent or metastatic SCCHN from three Austrian cancer centers that received CPI therapy alone or with chemotherapy as palliative first-line systemic treatment for this retrospective study. Patient characteristics, details on treatment, and survival were analyzed by a chart-based review. RESULTS: Of the 78 patients analyzed, 55 patients were treated with CPI alone (45 with Pembrolizumab, 10 with Nivolumab) and 23 patients received chemotherapy with a platinum and 5-FU in addition to CPI. With a median follow-up of twelve months, the median PFS of all patients was 4 months [95% confidence interval (CI)=2.2-5.8] and the median OS was 11 months (95% CI=7.1-14.9). The overall response and disease control rates were 20.5% and 46.1%, respectively. There was no statistically significant difference in clinical outcome between patient groups with a different combined positive score (CPS). The rate of reported immune related adverse events was comparable to existing data. CONCLUSION: Our findings confirm the results of the KEYNOTE-048 trial that CPI therapy alone or together with chemotherapy is an effective treatment for patients with recurrent or metastatic CPS-positive SCCHN.