Welding PROxAb Shuttles: A Modular Approach for Generating Bispecific Antibodies via Site-Specific Protein-Protein Conjugation.

Targeted protein degradation is an innovative therapeutic strategy to selectively eliminate disease-causing proteins. Exemplified by proteolysis-targeting chimeras (PROTACs), they have shown promise in overcoming drug resistance and targeting previously undruggable proteins. However, PROTACs face challenges, such as low oral bioavailability and limited selectivity. The recently published PROxAb Shuttle technology offers a solution enabling the targeted delivery of PROTACs using antibodies fused with PROTAC-binding domains derived from camelid single-domain antibodies (VHHs). Here, a modular approach to quickly generate PROxAb Shuttles by enzymatically coupling PROTAC-binding VHHs to off-the-shelf antibodies was developed. The resulting conjugates retained their target binding and internalization properties, and incubation with BRD4-targeting PROTACs resulted in formation of defined PROxAb-PROTAC complexes. These complexes selectively induced degradation of the BRD4 protein, resulting in cytotoxicity specifically to cells expressing the antibody's target. The chemoenzymatic approach described herein provides a versatile and efficient solution for generating antibody-VHH conjugates for targeted protein degradation applications, but it could also be used to combine antibodies and VHH binders to generate bispecific antibodies for further applications.

Artificial intelligence's contribution to early pulmonary lesion detection in chest X-rays: insights from two retrospective studies on a Czech population.

In recent years healthcare is undergoing significant changes due to technological innovations, with Artificial Intelligence (AI) being a key trend. Particularly in radiodiagnostics, according to studies, AI has the potential to enhance accuracy and efficiency. We focus on AI's role in diagnosing pulmonary lesions, which could indicate lung cancer, based on chest X-rays. Despite lower sensitivity in comparison to other methods like chest CT, due to its routine use, X-rays often provide the first detection of lung lesions. We present our deep learning-based solution aimed at improving lung lesion detection, especially during early-stage of illness. We then share results from our previous studies validating this model in two different clinical settings: a general hospital with low prevalence findings and a specialized oncology center. Based on a quantitative comparison with the conclusions of radiologists of different levels of experience, our model achieves high sensitivity, but lower specificity than comparing radiologists. In the context of clinical requirements and AI-assisted diagnostics, the experience and clinical reasoning of the doctor play a crucial role, therefore we currently lean more towards models with higher sensitivity over specificity. Even unlikely suspicions are presented to the doctor. Based on these results, it can be expected that in the future artificial intelligence will play a key role in the field of radiology as a supporting tool for evaluating specialists. To achieve this, it is necessary to solve not only technical but also medical and regulatory aspects. It is crucial to have access to quality and reliable information not only about the benefits but also about the limitations of machine learning and AI in medicine.

3D printing traceability in healthcare using 3Diamond software.

Background: 3D printing is one of the fastest-growing technologies in medicine, but it is essential to have a system for 3D printing documentation that is accessible for not only clinical engineers and surgeons, but also quality managers and data-privacy officers in hospitals. Dedicated software such as product lifecycle management (PLM) software could enable comprehensive management and traceability of all data relevant to 3D printing tasks in a hospital and would highly beneficial. Therefore, customizable software called 3Diamond was developed for 3D printing in medicine. Methods: The software development process involved several stages, including setting specifications based on end-user requirements, design, implementation, and testing. In order to ensure the software's long-term success and smooth operation, critical phases were also considered, such as deployment and maintenance. Results: The developed software provides immediate and complete traceability of all preparations and controls, as well as management of reports, orders, stock, and post-operative follow-up of tasks related to 3D printing in a hospital. Based on user requirements, software testing is provided automatically with each release. The software was implemented in a natural clinical environment with a developed 3D printing center. Conclusion: Although 3D printing has potential for innovation in the medical profession, it is nevertheless subject to regulations. Even though there are exemptions for patient-specific products, the effects of their local legal implementations related to 3D printing cannot be fully overseen. To this end, 3Diamond provides a robust system for 3D printing documentation that is accessible to different personnel in hospitals.

Cryo-EM Structures of CRAF2/14-3-32 and CRAF2/14-3-32/MEK12 Complexes.

RAF protein kinases are essential effectors in the MAPK pathway and are important cancer drug targets. Structural understanding of RAF activation is so far based on cryo-electron microscopy (cryo-EM) and X-ray structures of BRAF in different conformational states as inactive or active complexes with KRAS, 14-3-3 and MEK1. In this study, we have solved the first cryo-EM structures of CRAF2/14-3-32 at 3.4 Å resolution and CRAF2/14-3-32/MEK12 at 4.2 Å resolution using CRAF kinase domain expressed as constitutively active Y340D/Y341D mutant in insect cells. The overall architecture of our CRAF2/14-3-32 and CRAF2/14-3-32/MEK12 cryo-EM structures is highly similar to corresponding BRAF structures in complex with 14-3-3 or 14-3-3/MEK1 and represent the activated dimeric RAF conformation. Our CRAF cryo-EM structures provide additional insights into structural understanding of the activated CRAF2/14-3-32/MEK12 complex.

Diffusion tensor imaging in anisotropic tissues: application of reduced gradient vector schemes in peripheral nerves.

BACKGROUND: In contrast to the brain, fibers within peripheral nerves have distinct monodirectional structure questioning the necessity of complex multidirectional gradient vector schemes for DTI. This proof-of-concept study investigated the diagnostic utility of reduced gradient vector schemes in peripheral nerve DTI. METHODS: Three-Tesla magnetic resonance neurography of the tibial nerve using 20-vector DTI (DTI20) was performed in 10 healthy volunteers, 12 patients with type 2 diabetes, and 12 age-matched healthy controls. From the full DTI20 dataset, three reduced datasets including only two or three vectors along the x- and/or y- and z-axes were built to calculate major parameters. The influence of nerve angulation and intraneural connective tissue was assessed. The area under the receiver operating characteristics curve (ROC-AUC) was used for analysis. RESULTS: Simplified datasets achieved excellent diagnostic accuracy equal to DTI20 (ROC-AUC 0.847-0.868, p ≤ 0.005), but compared to DTI20, the reduced models yielded mostly lower absolute values of DTI scalars: median fractional anisotropy (FA) ≤ 0.12; apparent diffusion coefficient (ADC) ≤ 0.25; axial diffusivity ≤ 0.96, radial diffusivity ≤ 0.07). The precision of FA and ADC with the three-vector model was closest to DTI20. Intraneural connective tissue was negatively correlated with FA and ADC (r ≥ -0.49, p < 0.001). Small deviations of nerve angulation had little effect on FA accuracy. CONCLUSIONS: In peripheral nerves, bulk tissue DTI metrics can be approximated with only three predefined gradient vectors along the scanner's main axes, yielding similar diagnostic accuracy as a 20-vector DTI, resulting in substantial scan time reduction. RELEVANCE STATEMENT: DTI bulk tissue parameters of peripheral nerves can be calculated with only three predefined gradient vectors at similar diagnostic performance as a standard DTI but providing a substantial scan time reduction. KEY POINTS: • In peripheral nerves, DTI parameters can be approximated using only three gradient vectors. • The simplified model achieves a similar diagnostic performance as a standard DTI. • The simplified model allows for a significant acceleration of image acquisition. • This can help to introduce multi-b-value DTI techniques into clinical practice.

Quantitative magnetic resonance neurography in chronic inflammatory demyelinating polyradiculoneuropathy: A longitudinal study over 6 years.

OBJECTIVE: To evaluate magnetic resonance neurography (MRN) for the longitudinal assessment of patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). METHODS: Prospective examination of twelve CIDP patients by neurological assessment, MRN, and nerve conduction studies in 2016 and 6 years later in 2022. Imaging parameters were compared with matched healthy controls and correlated with clinical and electrophysiological markers. The MRN protocol included T2-weighted imaging, diffusion tensor imaging (DTI), T2 relaxometry, and magnetization transfer imaging (MTI). RESULTS: Nerve cross-sectional area (CSA) was increased in CIDP patients compared to controls (plexus: p = 0.003; sciatic nerve: p < 0.001). Over 6 years, nerve CSA decreased in CIDP patients, most pronounced at the lumbosacral plexus (p = 0.015). Longitudinally, changes in CSA correlated with changes in the inflammatory neuropathy cause and treatment validated overall disability sum score (INCAT/ODSS) (p = 0.006). High initial nerve CSA was inversely correlated with changes in the INCAT/ODSS over 6 years (p < 0.05). The DTI parameter fractional anisotropy (FA) showed robust correlations with electrodiagnostic testing both cross-sectionally and longitudinally (p < 0.05). MTI as a newly added imaging technique revealed a significantly reduced magnetization transfer ratio (MTR) in CIDP patients (p < 0.01), suggesting underlying changes in macromolecular tissue composition, and correlated significantly with electrophysiological parameters of demyelination (p < 0.05). INTERPRETATION: This study provides evidence that changes in nerve CSA and FA reflect the clinical and electrophysiological course of CIDP patients. Initial nerve hypertrophy might predict a rather benign course or better therapy response.

Inferior long-term Results of a Prospective Randomized Controlled Trial initially demonstrating enhanced Sensory Nerve Recovery using a Chitosan Nerve Tube.

INTRODUCTION: Traumatic peripheral nerve injuries can result in significant functional impairments and long-term sequelae. This study evaluated the long-term outcomes of a chitosan tube implantation protecting the epineural coaptation after peripheral nerve injuries using two different tube versions (V 1.0 and V 2.0 with different wall thickness and resorption characteristics) compared to a control group. The study focused on pain levels, sensory function, and overall functional outcomes. METHODS: Patients who received tube implantation around direct coaptation sites of digital nerves were prospectively randomized and compared to control patients without additional tube protection. Pain levels, sensory function, grip force, and functional scores were assessed at different time points, ranging from three months to five years after the procedure. Furthermore, biodegradation of the tubes was measured via high-resolution MR-neurography (MRN) and categorized. RESULTS: Long-term evaluation revealed that patients with V 1.0 had higher pain levels compared to the control group after five years. They also reported more symptoms of numbness and hypersensitivity. V 2.0 patients exhibited higher pain levels at three months, which did not persist at six months. However, they showed compromised sensory function, with higher values of two-point discrimination compared to V 1.0 and the control group. No differences were found in grip force or functional scores between the groups. MRI displayed remnants of implants even in long-term follow-up. DISCUSSION: The findings suggest potential limitations due to pain increase and impaired sensory function associated with tube implantation in the long term. However, in the short term, the material seemed to have a protective effect (as published previously). The resorption process was not completed at the end of the observation period of five years. This might explain the prolonged scarring and inferior long-term results. Future research should focus on improving tube materials and design to minimize adverse effects and enhance functional outcomes in patients with peripheral nerve injuries.