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Immunoglobulins are important building blocks in biology and biotechnology. With the emergence of comprehensive deep‐learning approaches, there are enormous opportunities for fast and accurate methods of classification of protein–protein interactions to arise. Herein, widely accessible image classification algorithms for species‐specific typification of a range of different immunoglobulin G (IgG) complexes are repurposed. Droplets of various immunoglobulins mixed with a B‐cell superantigen (SAg) (recombinant staphylococcal Protein A) are deposited onto hydrophobic polymer substrates and the resulting protein stains are imaged using polarized light microscopy. A comprehensive study based on 23 745 images finds that the pretrained convolutional neural network (CNN) InceptionV3 not only successfully categorizes IgGs from four different species but also predicts their binding affinity to Protein A: averaged over 36 binding pairs, the following are observed: 1) an overall accuracy of 81.4%, 2) the highest prediction accuracy for human IgG, the antibody with the highest binding affinity for Protein A, and 3) that the classification accuracy regarding the various IgG/Protein A ratios generally correlates with the binding strength of the protein–protein–complex as determined via circular dichroism spectroscopy. In addition, the CNN pretrained with IgG/Protein A stain images has been challenged with a new set of images using a different superantigen (SAg, Protein G). Despite the use of the unknown SAg, the CNN correctly classifies the images of human IgG and Protein G as indicated by a 94% accuracy over the various molar binding ratios. These findings are noteworthy because they demonstrate that appropriately pretrained CNNs can be used for the prediction of protein–protein interactions beyond the scope of the original training set. Aided by deep‐learning methods, simple stains of mixed protein solutions may serve as accurate predictors of the strength of protein–protein interactions with relevance to protein engineering, self‐aggregation, or protein stability in complex media.

Abstract Previously, we reported successful cellular expansion of a murine colorectal carcinoma cell line (CT-26) using a three-dimensional (3D) engineered extracellular matrix (EECM) fibrillar scaffold structure. CCL-247 were grown over a limited time period of 8 days on 3D EECM or tissue culture polystyrene (TCPS). Cells were then assayed for growth, electroporation efficiency and Vigil manufacturing release criteria. Using EECM scaffolds, we report an expansion of CCL-247 (HCT116), a colorectal carcinoma cell line, from a starting concentration of 2.45 × 105 cells to 1.9 × 106 cells per scaffold. Following expansion, 3D EECM-derived cells were assessed based on clinical release criteria of the Vigil manufacturing process utilized for Phase IIb trial operation with the FDA. 3D EECM-derived cells passed all Vigil manufacturing release criteria including cytokine expression. Here, we demonstrate successful Vigil product manufacture achieving the specifications necessary for the clinical trial product release of Vigil treatment. Our results confirm that 3D EECM can be utilized for the expansion of human cancer cell CCL-247, justifying further clinical development involving human tissue sample manufacturing including core needle biopsy and minimal ascites samples.

Developing scalable and accurate predictive analytical methods for the classification of protein‐DNA binding is critical for advancing our understanding of molecular biology, disease mechanisms, and a wide spectrum of biotechnological and medical applications. It is discovered that histone–DNA interactions can be stratified based on stain patterns created by the deposition of various nucleoprotein solutions onto a substrate. In this study, a deep‐learning neural network is applied to categorize polarized light microscopy images of drying droplet deposits originating from different histone–DNA mixtures. These DNA stain patterns featured high reproducibility across different species and thus enabled comprehensive DNA categorization (100% accuracy) and accurate prediction of their respective binding affinities to histones. Eukaryotic DNA, which has a higher binding affinity to mammalian histones than prokaryotic DNA, is associated with a higher overall prediction accuracy. For a given species, the average prediction accuracy increased with DNA size. To demonstrate generalizability, a pre‐trained CNN is challenged with unknown images that originated from DNA samples of species not included in the training set. The CNN classified these unknown histone‐DNA samples as either strong or medium binders with 84.4% and 96.25% accuracy, respectively.

The aim of this work was to evaluate results of a remote electronic portal imaging based dosimetric auditing method using Task-Group 218 clinical gamma evaluation criteria (3%,2 mm, 10% dose threshold). For intensity modulated radiation therapy the results were (mean ± 1 SD) 97.9 ± 4.5% with 31/34 audits passing (optimal level, ≥ 95%) and 3/34 audits failing (action level, < 90%). For volumetric modulated arc therapy the results were 98.5 ± 2.3% with 32/36 audits passing (optimal level) and 4/36 passing (tolerance level, ≥ 90% and < 95%). The audit has been successfully applied globally for clinical trial quality assurance.

Abstract Objective Brain organoids are miniaturized in vitro brain models generated from pluripotent stem cells, which resemble full‐sized brain more closely than conventional two‐dimensional cell cultures. Although brain organoids mimic the human brain's cell‐to‐cell network interactions, they generally fail to faithfully recapitulate cell‐to‐matrix interactions. Here, an engineered framework, called an engineered extracellular matrix (EECM), was developed to provide support and cell‐to‐matrix interactions to developing brain organoids. Methods We generated brain organoids using EECMs comprised of human fibrillar fibronectin supported by a highly porous polymer scaffold. The resultant brain organoids were characterized by immunofluorescence microscopy, transcriptomics, and proteomics of the cerebrospinal fluid (CSF) compartment. Results The interstitial matrix‐mimicking EECM enhanced neurogenesis, glial maturation, and neuronal diversity from human embryonic stem cells versus conventional protein matrix (Matrigel). Additionally, EECMs supported long‐term culture, which promoted large‐volume organoids containing over 250 μL of CSF. Proteomics analysis of the CSF found it superseded previous brain organoids in protein diversity, as indicated by 280 proteins spanning 500 gene ontology pathways shared with adult CSF. Interpretation Engineered EECM matrices represent a major advancement in neural engineering as they have the potential to significantly enhance the structural, cellular, and functional diversity that can be achieved in advanced brain models.

Abstract Chemical vapor deposition (CVD) polymerization is a prevalent technique for fabricating conformal, defect-free, and systematically adjustable organic thin films. CVD is particularly beneficial for barrier coatings due to its ability to eliminate solvent-related environmental, health, and safety risk factors and provide a wide spectrum of post-polymerization modification strategies. This review discusses poly-p-xylylene and its functional derivatives. CVD polymerization of [2.2]paracyclophane precursors has undergone a recent renaissance due to advancements in chemical and morphological surface manipulation. This review summarizes emerging trends based on the following outline: Table of content: 1 Introduction 2 CVD Polymerization as a Sustainable Coating Technology 3 CVD Instrumentation 4 Poly-p-xylylene Coatings: Background of Polymerization Process and Functionalized Films 5 Main Applications of Poly-p-xylylenes 6 Area-Selective CVD Polymerization 7 Fabrication and Applications of Topological Structures 8 Conclusions and Outlook

The potential of therapeutically loaded nanoparticles (NPs) has been successfully demonstrated during the last decade, with NP-mediated nonviral gene delivery gathering significant attention as highlighted by the broad clinical acceptance of mRNA-based COVID-19 vaccines. A significant barrier to progress in this emerging area is the wild variability of approaches reported in published literature regarding nanoparticle characterizations. Here, we provide a brief overview of the current status and outline important concerns regarding the need for standardized protocols to evaluate NP uptake, NP transfection efficacy, drug dose determination, and variability of nonviral gene delivery systems. Based on these concerns, we propose wide adherence to multimodal, multiparameter, and multistudy analysis of NP systems. Adoption of these proposed approaches will ensure improved transparency, provide a better basis for interlaboratory comparisons, and will simplify judging the significance of new findings in a broader context, all critical requirements for advancing the field of nonviral gene delivery.

Abstract Electrospinning has received a lot of attention in recent years because it can create nonwoven nanofiber webs with high surface area and porosity. However, the typical needle and syringe‐based electrospinning systems feature poor productivity that has limited their usefulness in the industrial field. Here, current developments in the creation of nanofibers employing nonconventional electrospinning methods, such as needleless electrospinning and syringeless electrospinning, are examined. These alternate electrospinning techniques, which are dependent on numerous polymer droplets of varied shapes, have the potential to match the productivity required for industry‐scale manufacturing of nanofibers. Additionally, they make it possible to produce nanofibers that are difficult to spin using traditional techniques, like electrospinning of colloidal suspensions.

Background and purpose: Real-time treatment monitoring with the electronic portal imaging device (EPID) can conceptually provide a more accurate assessment of the quality of deep inspiration breath-hold (DIBH) and patient movement during tangential breast radiotherapy (RT). A system was developed to measure two geometrical parameters, the lung depth (LD) and the irradiated width (named here skin distance, SD), along three user-selected lines in MV EPID images of breast tangents. The purpose of this study was to test the system during tangential breast RT with DIBH. Materials and methods: Measurements of LDs and SDs were carried out in real time. DIBH was guided with a commercial system using a marker block. Results from 17 patients were assessed. Mean midline LDs, , per tangent were compared to the planned mLDs; differences between the largest and smallest observed () per tangent were calculated. Results: For 56% (162/288) of the tangents tested, were outside the tolerance window. All but one patient had at least one fraction showing this behaviour. The largest difference found between an and its planned mLD was −16.9 mm. The accuracy of patient positioning and the quality of marker-block-based DIBH guidance contributed to the differences. Fractions with patient position verification using a single EPID image taken before treatment showed a lower rate (34%), suggesting reassessment of setup procedures. Conclusions: Real-time treatment monitoring of the internal anatomy during DIBH delivery of tangential breast RT is feasible and useful. The new system requires no additional radiation for the patient.

The demand for high‐throughput and scalable cell expansion platforms that can accommodate diverse cell types remains a critical requirement across various biomedical fields. Fibronectin (Fn), an essential component of the extracellular matrix (ECM), has been used as a conformal surface coating for two‐dimensional (2D) cell culture systems. However, the soluble, globular Fn used for 2D coatings differs structurally from the native Fn, which possesses a three‐dimensional (3D) fibrillar structure. Herein, a large‐scale engineered ECM (EECM) cell expansion platform based on a 3D fibrillar Fn network spanning over centimeters is presented. Extended fibrillar networks are formed by shearing dilute Fn solutions over tessellated polymeric scaffolds, which are conveniently prepared by 3D printing. The structure and size of the Fn‐based 3D EECM scaffold are optimized by evaluating the proliferation of a colorectal tumor cell line, CT26, commonly used in the in vivo tumor immunotherapy models. The 3D EECM scaffolds support a fourfold more efficient tumor cell expansion than a conventional 2D culture system, demonstrating the potential efficacy in supporting the robust expansion of cancer cells ex vivo with an eye on cancer immunotherapy.

Although river ecosystems constitute a small fraction of Earth’s total area, they are critical modulators of microbially and virally orchestrated global biogeochemical cycles. However, most studies either use data that is not spatially resolved or is collected at timepoints that do not reflect the short life cycles of microorganisms. To address this gap, we assessed how viral and microbial communities change over a 48-hour period by sampling surface water and pore water compartments of the wastewater-impacted River Erpe in Germany. We sampled every 3 hours resulting in 32 samples for which we obtained metagenomes along with geochemical and metabolite measurements. From our metagenomes, we identified 6,500 viral and 1,033 microbial metagenome assembled genomes (MAGs) and found distinct community membership and abundance associated with each river compartment (e.g., Competibacteraceae in surfacewater and Sulfurimonadaceae in pore water). We show that 17% of our viral MAGs clustered to viruses from other ecosystems like wastewater treatment plants and rivers. Our results also indicated that 70% of the viral community was persistent in surface waters, whereas only 13% were persistent in the pore waters taken from the hyporheic zone. Finally, we predicted linkages between 73 viral genomes and 38 microbial genomes. These putatively linked hosts included members of the Competibacteraceae, which we suggest are potential contributors to river carbon and nitrogen cycling via denitrification and nitrogen fixation. Together, these findings demonstrate that members of the surface water microbiome from this urban river are stable over multiple diurnal cycles. These temporal insights raise important considerations for ecosystem models attempting to constrain dynamics of river biogeochemical cycles.

Thermal drift of optical systems employed for surface guided radiation therapy (SGRT) adds uncertainty to patient setup and monitoring. This work describes methods to measure the drift of individual camera pods as well as the drift of the combined clinical signal. It presents results for four clinical C-Rad Catalyst+ HD systems. Based on the measured clinical drift, recipes are provided on how to calculate relevant uncertainties in patient setup and patient position monitoring with SGRT. Strategies to reduce the impact of drift are explained. While the results are specific to the systems investigated, the methodology is transferable and the clinical recipes are universally applicable.

Nanoparticles are frequently pursued as drug delivery carriers due to their potential to alter the pharmacological profiles of drugs, but their broader utility in nanomedicine hinges upon exquisite control of critical nanoparticle properties, such as shape, size, or monodispersity. Electrohydrodynamic (EHD) jetting is a probate method to formulate synthetic protein nanoparticles (SPNPs), but a systematic understanding of the influence of crucial processing parameters, such as protein composition, on nanoparticle morphologies is still missing. Here, we address this knowledge gap by evaluating formulation trends in SPNPs prepared by EHD jetting based on a series of carrier proteins and protein blends (hemoglobin, transferrin, mucin, or insulin). In general, blended SPNPs presented uniform populations with minimum diameters between 43 and 65 nm. Size distributions of as-jetted SPNPs approached monodispersity as indicated by polydispersity indices (PDISEM) ranging from 0.11–0.19. Geometric factor analysis revealed high circularities (0.82–0.90), low anisotropy (

Background and purpose: Synthetic diamond detectors offer real time measurement of dose in radiotherapy applications which require high spatial resolution. Additional considerations and corrections are required for measurements where the diamond detector is orientated at various angles to the incident beam. This study investigated diamond detectors for end-to-end testing of Stereotactic Body Radiotherapy (SBRT) and Stereotactic Radiosurgery (SRS) in the context of dosimetry audits. Material and methods: Seven individual diamond detectors were investigated and compared with respect to warm up stability, dose-rate dependence, linearity, detector shadowing, energy response, cross-calibration, angular dependence and positional sensitivity in SBRT and SRS. Results: Large variation in the cross calibration factors was found between the seven individual detectors. For each detector, the energy dependence in the cross calibration factor was on average

Introduction Treatment with biologics for severe asthma is informed by international and national guidelines and defined by national regulating bodies, but how these drugs are used in real-life is unknown. Materials and methods The European Respiratory Society (ERS) SHARP Clinical Research Collaboration conducted a three-step survey collecting information on asthma biologics use in Europe. Five geographically distant countries defined the survey questions, focusing on seven end-points: biologics availability and financial issues, prescription and administration modalities, inclusion criteria, continuation criteria, switching biologics, combining biologics and evaluation of corticosteroid toxicity. The survey was then sent to SHARP National Leads of 28 European countries. Finally, selected questions were submitted to a broad group of 263 asthma experts identified by national societies. Results Availability of biologics varied between countries, with 17 out of 28 countries having all five existing biologics. Authorised prescribers (pulmonologists and other specialists) also differed. In-hospital administration was the preferred deliverance modality. While exacerbation rate was used as an inclusion criterion in all countries, forced expiratory volume in 1 s was used in 46%. Blood eosinophils were an inclusion criterion in all countries for interleukin-5 (IL-5)-targeted and IL-4/IL-13-targeted biologics, with varying thresholds. There were no formally established criteria for continuing biologics. Reduction in exacerbations represented the most important benchmark, followed by improvement in asthma control and quality of life. Only 73% (191 out of 263) of surveyed clinicians assessed their patients for corticosteroid-induced toxicity. Conclusion Our study reveals important heterogeneity in the use of asthma biologics across Europe. To what extent this impacts on clinical outcomes relevant to patients and healthcare services needs further investigation.

Introduction The European Sleep Apnea Database was used to identify distinguishable obstructive sleep apnoea (OSA) phenotypes and to investigate the clinical outcome during positive airway pressure (PAP) treatment. Method Prospective OSA patient data were recruited from 35 sleep clinics in 21 European countries. Unsupervised cluster analysis (anthropometrics, clinical variables) was performed in a random sample (n=5000). Subsequently, all patients were assigned to the clusters using a conditional inference tree classifier. Responses to PAP treatment change in apnoea severity and Epworth sleepiness scale (ESS) were assessed in relation to baseline patient clusters and at short- and long-term follow-up. Results At baseline, 20 164 patients were assigned (mean age 54.1±12.2 years, 73% male, median apnoea–hypopnoea index (AHI) 27.3 (interquartile range (IQR) 14.1–49.3) events·h−1, and ESS 9.8±5.3) to seven distinct clusters based on anthropometrics, comorbidities and symptoms. At PAP follow-up (median 210 [IQR 134–465] days), the observed AHI reduction (n=1075) was similar, whereas the ESS response (n=3938) varied: largest reduction in cluster 3 (young healthy symptomatic males) and 6 (symptomatic males with psychiatric disorders, −5.0 and −5.1 units, respectively (all p

Abstract Trace organic compounds (TrOCs) enter rivers with discharge of treated wastewater. These effluents can contain high loads of dissolved organic matter (DOM). In a 48 h field study, we investigated changes in molecular composition of seven DOM compound classes (FTICR-MS) and attenuation of 17 polar TrOCs in a small urban stream receiving treated wastewater. Correlations between TrOCs and DOM were used to identify simultaneous changes in surface water and the hyporheic zone. Changes in TrOC concentrations in surface water ranged between a decrease of 29.2% for methylbenzotriazole and an increase of 152.2% for the transformation product gabapentin-lactam. In the hyporheic zone, only decreasing TrOC concentrations were observed, ranging from 4.9% for primidone to 93.8% for venlafaxine . TrOC attenuation coincided with a decline of molecular diversity of easily biodegradable DOM compound classes while molecular diversity of poorly biodegradable DOM compound classes increased. This concurrence indicates similar or linked attenuation pathways for biodegradable DOM and TrOCs. Strong correlations between TrOCs and DOM compound classes as well as high attenuation of TrOCs primarily occurred in the hyporheic zone. This suggests high potential for DOM turnover and TrOC mitigation in rivers if hyporheic exchange is sufficient.

O ensaio inicia com uma revisão das incertezas que permeiam o conceito de “direitos culturais”, questão que é fortemente ligada à indeterminação do próprio conceito de “cultura”. Realiza-se um estudo comparativo filológico sobre o conceito de cultura no sentido do que é relevante quanto à universalidade dos direitos culturais. O artigo enfoca na análise do conceito de “cultura legal de culturas”, da obra de Peter Häberle, que é necessário para o estudo do papel dos direitos culturais no constitucionalismo contemporâneo. Analisa-se a origem dos direitos culturais, através de uma revisão das legislações, que frequentemente são fragmentadas e podem estar sujeitas a interpretações sistemáticas e evolutivas. Em seguida, prossegue-se com a análise das garantias legais de proteção aos direitos culturais no estado da arte. O ensaio termina com a exposição de alguns dos desafios futuros que definirão o debate sobre a lei e a cultura dos direitos fundamentais culturais.