Age dictates brain functional connectivity and axonal integrity following repetitive mild traumatic brain injuries in mice.

Traumatic brain injuries (TBIs) present a major public health challenge, demanding an in-depth understanding of age-specific symptoms and risk factors. Aging not only significantly influences brain function and plasticity but also elevates the risk of hospitalizations and death following TBIs. Repetitive mild TBIs (rmTBIs) compound these issues, resulting in cumulative and long-term brain damage in the brain. In this study, we investigate the impact of age on brain network changes and white matter properties following rmTBI employing a multi-modal approach that integrates resting-state functional magnetic resonance imaging (rsfMRI), graph theory analysis, diffusion tensor imaging (DTI), and neurite orientation dispersion and density imaging (NODDI). Our hypothesis is that the effects of rmTBI are worsened in aged animals, with this group showing more pronounced alterations in brain connectivity and white matter structure. Utilizing the CHIMERA model, we conducted rmTBIs or sham (control) procedures on young (2.5-3 months old) and aged (22-month-old) male and female mice to model high risk groups. Functional and structural imaging unveiled age-related reductions in communication efficiency between brain regions, while injuries induced opposing effects on the small-world index across age groups, influencing network segregation. Functional connectivity analysis also identified alterations in 79 out of 148 brain regions by age, treatment (sham vs. rmTBI), or their interaction. Injuries exerted pronounced effects on sensory integration areas, including insular and motor cortices. Age-related disruptions in white matter integrity were observed, indicating alterations in various diffusion directions (mean, radial, axial diffusivity, fractional anisotropy) and density neurite properties (dispersion index, intracellular and isotropic volume fraction). Neuroinflammation, assessed through Iba-1 and GFAP markers, correlated with higher dispersion in the optic tract, suggesting a neuroinflammatory response in injured aged animals compared to sham aged. These findings offer insight into the interplay between age, injuries, and brain connectivity, shedding light on the long-term consequences of rmTBIs.

Anti-IL23/12 agents and JAK inhibitors for inflammatory bowel disease.

IBD (inflammatory bowel disease) is a chronic inflammatory disease of the gastrointestinal tract with increasing incidence worldwide. Multiple factors, such as genetic background, environmental and luminal factors, and mucosal immune dysregulation, have been implicated in the cause of IBD, although the cause of the disease remains unknown. IL-12 and IL-23 and their downstream signaling pathways participate in the pathogenesis of inflammatory bowel disease. Early and aggressive treatment with biologic therapies or novel small molecules is needed to decrease complications and the need for hospitalization and surgery. The landscape of inflammatory bowel disease (IBD) treatment has tremendously improved with the development of biologics and small molecule drugs. Several novel biologics and small molecule drugs targeting IL-12 and IL-23 and their downstream targets have shown positive efficacy and safety data in clinical trials, and several drugs have been approved for the treatment of IBD. In the future, numerous potential emerging therapeutic options for IBD treatment are believed to come to the fore, achieving disease cure.

Targeting the mSWI/SNF complex in POU2F-POU2AF transcription factor-driven malignancies.

The POU2F3-POU2AF2/3 transcription factor complex is the master regulator of the tuft cell lineage and tuft cell-like small cell lung cancer (SCLC). Here, we identify a specific dependence of the POU2F3 molecular subtype of SCLC (SCLC-P) on the activity of the mammalian switch/sucrose non-fermentable (mSWI/SNF) chromatin remodeling complex. Treatment of SCLC-P cells with a proteolysis targeting chimera (PROTAC) degrader of mSWI/SNF ATPases evicts POU2F3 and its coactivators from chromatin and attenuates downstream signaling. B cell malignancies which are dependent on the POU2F1/2 cofactor, POU2AF1, are also sensitive to mSWI/SNF ATPase degraders, with treatment leading to chromatin eviction of POU2AF1 and IRF4 and decreased IRF4 signaling in multiple myeloma cells. An orally bioavailable mSWI/SNF ATPase degrader significantly inhibits tumor growth in preclinical models of SCLC-P and multiple myeloma without signs of toxicity. This study suggests that POU2F-POU2AF-driven malignancies have an intrinsic dependence on the mSWI/SNF complex, representing a therapeutic vulnerability.

A new tau dephosphorylation-targeting chimera for the treatment of tauopathies.

Abnormal accumulation of hyperphosphorylated tau protein plays a pivotal role in a collection of neurodegenerative diseases named tauopathies, including Alzheimer's disease (AD). We have recently conceptualized the design of hetero-bifunctional chimeras for selectively promoting the proximity between tau and phosphatase, thus specifically facilitating tau dephosphorylation and removal. Here, we sought to optimize the construction of tau dephosphorylating-targeting chimera (DEPTAC) and obtained a new chimera D14, which had high efficiency in reducing tau phosphorylation both in cell and tauopathy mouse models, while showing limited cytotoxicity. Moreover, D14 ameliorated neurodegeneration in primary cultured hippocampal neurons treated with toxic tau-K18 fragments, and improved cognitive functions of tauopathy mice. These results suggested D14 as a cost-effective drug candidate for the treatment of tauopathies.

O-GlcNAcylated RALY Contributes to Hepatocellular Carcinoma Cells Proliferation by Regulating USP22 mRNA Nuclear Export.

Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly tumors; however, its pathogenic mechanism remains largely elusive. In-depth researches are needed to reveal the expression regulatory mechanisms and functions of the RNA-binding protein RALY in HCC. Here, we identify RALY as a highly expressed oncogenic factor that affects HCC cells proliferation both in vitro and in vivo. O-GlcNAcylation of RALY at Ser176 enhances its stability by protecting RALY from TRIM27-mediated ubiquitination, thus maintaining hyper-expression of the RALY protein. Mechanistically, RALY interacts with USP22 messenger RNA, as revealed by RNA immunoprecipitation, to increase their cytoplasmic localization and protein expression, thereby promoting the proliferation of HCC cells. Furthermore, we develop a novel RALY protein degrader based on peptide proteolysis-targeting chimeras, named RALY-PROTAC, which we chemically synthesize by linking a RALY-targeting peptide with the E3 ubiquitin ligase recruitment ligand pomalidomide. In conclusion, our findings demonstrate a novel mechanism by which O-GlcNAcylation/RALY/USP22 mRNA axis aggravates HCC cells proliferation. RALY-PROTACs as degraders of the RALY protein exhibit potential as therapeutic drugs for RALY-overexpressing HCC.

A modular inquiry-based semester theme that integrates data science education and bioinformatics in protein structure function courses.

BACKGROUND: With an exponential growth in biological data and computing power, familiarity with bioinformatics has become a demanding and popular skill set both in academia and industry. There is a need to increase students' competencies to be able to take on bioinformatic careers, to get them familiarized with scientific professions in data science and the academic training required to pursue them, in a field where demand outweighs the supply. METHODS: Here we implemented a set of bioinformatic activities into a protein structure and function course of a graduate program. Concisely, students were given hands-on opportunities to explore the bioinformatics-based analyses of biomolecular data and structural biology via a semester-long case study structured as inquiry-based bioinformatics exercises. Towards the end of the term, the students also designed and presented an assignment project that allowed them to document the unknown protein that they identified using bioinformatic knowledge during the term. RESULTS: The post-module survey responses and students' performances in the lab module imply that it furthered an in-depth knowledge of bioinformatics. Despite having not much prior knowledge of bioinformatics prior to taking this module students indicated positive feedback. CONCLUSION: The students got familiar with cross-indexed databases that interlink important data about proteins, enzymes as well as genes. The essential skillsets honed by this research-based bioinformatic pedagogical approach will empower students to be able to leverage this knowledge for their future endeavours in the bioinformatics field.

Recent advances in the molecular design and applications of viral RNA-targeting antiviral modalities.

Pathogenic viruses are a profound threat to global public health, underscoring the urgent need for the development of efficacious antiviral therapeutics. The advent of RNA-targeting antiviral strategies has marked a significant paradigm shift in the management of viral infections, offering a potent means of control and potential cure. In this review, we delve into the cutting-edge progress in RNA-targeting antiviral agents, encompassing antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), small and bifunctional molecules. We provide an in-depth examination of their strategic molecular design and elucidate the underlying mechanisms of action that confer their antiviral efficacy. By synthesizing recent findings, we shed light on the innovative potential of RNA-targeting approaches and their pivotal role in advancing the frontiers of antiviral drug discovery.

Conquering the beyond Rule of Five Space with an Optimized High-Throughput Caco-2 Assay to Close Gaps in Absorption Prediction.

Current drug development tends towards complex chemical molecules, referred to as "beyond rule of five" (bRo5) compounds, which often exhibit challenging physicochemical properties. Measuring Caco-2 permeability of those compounds is difficult due to technical limitations, including poor recovery and detection sensitivity. We implemented a novel assay, with optimized incubation and analytics, to measure permeability close to equilibrium. In this setup an appropriate characterization of permeability for bRo5 compounds is achievable. This equilibrated Caco-2 assay was verified with respect to data validity, compound recovery, and in vitro to in vivo correlation for human absorption. Compared to a standard assay, it demonstrated comparable performance in predicting the human fraction absorbed (fa) for reference compounds. The equilibrated assay also successfully characterized the permeability of more than 90% of the compounds analyzed, the majority of which were bRo5 (68%). These compounds could not be measured using the standard assay. Permeability and efflux ratio (ER) were highly predictive for in vivo absorption for a large set of internal bRo5 compounds. Reference cut-offs enabled the correct classification of high, moderate, and low absorption. This optimized equilibrated Caco-2 assay closes the gap for a high-throughput cellular permeability method in the bRo5 chemical space.

Effects of heterologous kinase domains on growth factor receptor specificity.

The kinase domains of receptor tyrosine kinases (RTKs) are highly conserved, yet they are able to discriminate among potential substrates to selectively activate downstream signaling pathways. In this study, we tested the importance of catalytic domain specificity by creating two series of chimeric RTKs. In one set, the kinase domain of insulin-like growth factor I receptor (IGF1R) was replaced by the kinase domains from insulin receptor (IR), macrophage stimulating protein 1 receptor/Ron (Ron) or Src. In the other set of chimeras, the kinase domain of epidermal growth factor receptor (EGFR) was similarly replaced by the kinase domains of IR, Ron, or Src. We expressed the wild-type and chimeric forms of the receptors in mammalian cells. For some signaling events, such as recognition of IRS1, the identity of the tyrosine kinase catalytic domain did not appear to be crucial. In contrast, recognition of some sites, such as the C-terminal autophosphorylation sites on EGFR, did depend on the identity of the kinase domain. Our data also showed that ligand dependence was lost when the native kinase domains were replaced by Src, suggesting that the identity of the kinase domains could be important for proper receptor regulation. Overall, the results are consistent with the idea that the fidelity of RTK signaling depends on co-localization and targeting with substrates, as well as on the intrinsic specificity of the kinase domain.

Approach for Phased Sequence-Based Genotyping of the Critical Pharmacogene Dihydropyrimidine Dehydrogenase (DPYD).

Pre-treatment genotyping of four well-characterized toxicity risk-variants in the dihydropyrimidine dehydrogenase gene (DPYD) has been widely implemented in Europe to prevent serious adverse effects in cancer patients treated with fluoropyrimidines. Current genotyping practices are largely limited to selected commonly studied variants and are unable to determine phasing when more than one variant allele is detected. Recent evidence indicates that common DPYD variants modulate the functional impact of deleterious variants in a phase-dependent manner, where a cis- or a trans-configuration translates into different toxicity risks and dosing recommendations. DPYD is a large gene with 23 exons spanning nearly a mega-base of DNA, making it a challenging candidate for full-gene sequencing in the diagnostic setting. Herein, we present a time- and cost-efficient long-read sequencing approach for capturing the complete coding region of DPYD. We demonstrate that this method can reliably produce phased genotypes, overcoming a major limitation with current methods. This method was validated using 21 subjects, including two cancer patients, each of whom carried multiple DPYD variants. Genotype assignments showed complete concordance with conventional approaches. Furthermore, we demonstrate that the method is robust to technical challenges inherent in long-range sequencing of PCR products, including reference alignment bias and PCR chimerism.

Profiling Cellular Morphological Changes Induced by Dual-Targeting PROTACs of Aurora Kinase and RNA-Binding Protein YTHDF2.

Proteolysis targeting chimeras (PROTACs) are new chemical modalities that degrade proteins of interest, including established kinase targets and emerging RNA-binding proteins (RBPs). Whereas diverse sets of biochemical, biophysical and cellular assays are available for the evaluation and optimizations of PROTACs in understanding the involved ubiquitin-proteasome-mediated degradation mechanism and the structure-degradation relationship, a phenotypic method profiling the cellular morphological changes is rarely used. In this study, first, we reported the only examples of PROTACs degrading the mRNA-binding protein YTHDF2 via screening of multikinase PROTACs. Second, we reported the profiling of cellular morphological changes of the dual kinase- and RBP-targeting PROTACs using the unbiased cell painting assay (CPA). The CPA analysis revealed the high biosimilarity with the established aurora kinase cluster and annotated aurora kinase inhibitors, which reflected the association between YTHDF2 and the aurora kinase signaling network. Broadly, the results demonstrated that the cell painting assay can be a straightforward and powerful approach to evaluate PROTACs. Complementary to the existing biochemical, biophysical and cellular assays, CPA provided a new perspective in characterizing PROTACs at the cellular morphology.

Expanding the horizons of targeted protein degradation: A non-small molecule perspective.

Targeted protein degradation (TPD) represented by proteolysis targeting chimeras (PROTACs) marks a significant stride in drug discovery. A plethora of innovative technologies inspired by PROTAC have not only revolutionized the landscape of TPD but have the potential to unlock functionalities beyond degradation. Non-small-molecule-based approaches play an irreplaceable role in this field. A wide variety of agents spanning a broad chemical spectrum, including peptides, nucleic acids, antibodies, and even vaccines, which not only prove instrumental in overcoming the constraints of conventional small molecule entities but also provided rapidly renewing paradigms. Herein we summarize the burgeoning non-small molecule technological platforms inspired by PROTACs, including three major trajectories, to provide insights for the design strategies based on novel paradigms.

Ancestral ribonucleases back in motion for evolutionary-dynamics guided protein design.

The dynamics behavior of a protein is essential for its functionality. Here, Doucet et al. demonstrate how the evolutionary analysis of conformational pathways within a protein family serves to identify common core scaffolds that accommodate branch-specific functional regions controlled by flexibility switches, offering a model for evolutionary-dynamics based protein design.

Therapeutic potential of human microglia transplantation in a chimeric model of CSF1R-related leukoencephalopathy.

Microglia replacement strategies are increasingly being considered for the treatment of primary microgliopathies like adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). However, available mouse models fail to recapitulate the diverse neuropathologies and reduced microglia numbers observed in patients. In this study, we generated a xenotolerant mouse model lacking the fms-intronic regulatory element (FIRE) enhancer within Csf1r, which develops nearly all the hallmark pathologies associated with ALSP. Remarkably, transplantation of human induced pluripotent stem cell (iPSC)-derived microglial (iMG) progenitors restores a homeostatic microglial signature and prevents the development of axonal spheroids, white matter abnormalities, reactive astrocytosis, and brain calcifications. Furthermore, transplantation of CRISPR-corrected ALSP-patient-derived iMG reverses pre-existing spheroids, astrogliosis, and calcification pathologies. Together with the accompanying study by Munro and colleagues, our results demonstrate the utility of FIRE mice to model ALSP and provide compelling evidence that iMG transplantation could offer a promising new therapeutic strategy for ALSP and perhaps other microglia-associated neurological disorders.

The Chimeric LFC and DCIA Flap in Combined Mandibular and Condylar Head and Neck Reconstruction-A Case Series.

Background: Defects of the ascending ramus of the mandible, including the condylar head and neck or the whole temporomandibular joint (TMJ), are difficult to reconstruct. Reconstruction is mainly based on the use of alloplastic joint prosthesis, costochondral grafting, distraction osteogenesis of the dorsal part of the mandibular ramus, or osseous microvascular flaps of various origin. With the objective of developing a method that overcomes the restrictions of these methods, we recently introduced a sequential chimeric flap consisting of a lateral femoral condyle flap (LFC) and deep circumflex iliac artery flap (DCIA) for reconstruction of up to half of the mandible and the condylar head and neck. Methods: The chimeric flap was used in four patients with the following diagnoses: therapy-refractory osteomyelitis, extended recurrent odontogenic keratozyst, Goldenhar syndrome, and adenocarcinoma of the parotid gland. After a diagnostic workup, LFC and DCIA flaps were harvested in all patients and used in a sequential chimeric design for the reconstruction of the mandibular body and condylar head and neck. Results: Follow-up from at least 24 months up to 70 month after surgery showed a successful reconstruction in all four patients. The LFC provided a cartilaginous joint surface, allowing for a satisfactory masticatory function with a stable occlusion and unrestricted mouth opening and preserved or regained lateral and medial excursions in all patients. The DCIA allowed for a bony reconstruction anatomically resembling a non-atrophied mandibular body. No flap-related complications were observed. Conclusions: The sequential chimeric LFC and DCIA flap is an appropriate method for reconstructing up to half of the mandible and the condylar head and neck. It is suitable in cases where alloplastic joint replacement cannot be used or where other methods have failed. Due to the necessity of harvesting two flaps, the burden of care is increased, and a careful indication is required. The technique is reserved for maxillofacial surgeons who have already gained significant experience in the field of microsurgery.

Cyclosporin A-Based PROTACs Can Deplete Abundant Cellular Cyclophilin A without Suppressing T Cell Activation.

Cyclophilin A (CypA), the cellular receptor of the immunosuppressant cyclosporin A (CsA), is an abundant cytosolic protein and is involved in a variety of diseases. For example, CypA supports cancer proliferation and mediates viral infections, such as the human immunodeficiency virus 1 (HIV-1). Here, we present the design of PROTAC (proteolysis targeting chimera) compounds against CypA to induce its intracellular proteolysis and to investigate their effect on immune cells. Interestingly, upon connecting to E3 ligase ligands, both peptide-based low-affinity binders and CsA-based high-affinity binders can degrade CypA at nM concentration in HeLa cells and fibroblast cells. As the immunosuppressive effect of CsA is not directly associated with the binding of CsA to CypA but the inhibition of phosphatase calcineurin by the CypA:CsA complex, we investigated whether a CsA-based PROTAC compound could induce CypA degradation without affecting the activation of immune cells. P3, the most efficient PROTAC compound discovered from this study, could deplete CypA in lymphocytes without affecting cell proliferation and cytokine production. This work demonstrates the feasibility of the PROTAC approach in depleting the abundant cellular protein CypA at low drug dosage without affecting immune cells, allowing us to investigate the potential therapeutic effects associated with the endogenous protein in the future.

Effect of Domain Manipulation in the Staphylococcal Phage Endolysin, Endo88, on Lytic Efficiency and Host Range.

The bacteriophage endolysin Endo88 targeting Staphylococcus aureus PS88 consists of the CHAP and Amidase-2 enzymatic domains and one SH3b targeting domain. In this study, the effects of domain manipulations on Endo88 functionality were determined. Three truncated mutants of Endo88 (CHAP, CHAPAmidase and CHAPSH3) and two chimeras (CHAPAmidase-Cpl7Cpl7 and Endo88-Cpl7Cpl7) containing the Cpl7Cpl7 targeting domains of the streptococcal LambdaSa2-ECC endolysin were cloned in E. coli (pET28a), expressed, and then purified. Lytic efficiency and host range were assessed through plate lysis assays and turbidity reduction assays. Endo88 required all domains for maximum functionality, with activity detected against Staphylococcus aureus PS88 (host strain), S. aureus Mu50 (VISA), CoNS (Staphylococcus epidermidis and Staphylococcus hominis), and Enterococcus faecalis. The truncated constructs maintained the original host range but with reduced lytic efficiency. The Amidase-2 and SH3b domains are interdependent in maximizing functionality. The chimera constructs demonstrated reduced functionality, without activity against Streptococcus agalactiae in both assays. This study provides insights into domain function in a staphylococcal endolysin, which could enable the development of prospective engineered antimicrobials against multidrug-resistant pathogens.

The emerging role of targeted protein degradation to treat and study cancer.

The evolution of cancer treatment has provided increasingly targeted strategies both in the upfront and relapsed disease settings. Small-molecule inhibitors and immunotherapy have risen to prominence with chimeric antigen receptor T-cells, checkpoint inhibitors, kinase inhibitors, and monoclonal antibody therapies being deployed across a range of solid organ and haematological malignancies. However, novel approaches are required to target transcription factors and oncogenic fusion proteins that are central to cancer biology and have generally eluded successful drug development. Thalidomide analogues causing protein degradation have been a cornerstone of treatment in multiple myeloma, but a lack of in-depth mechanistic understanding initially limited progress in the field. When the protein cereblon (CRBN) was found to mediate thalidomide analogues' action and CRBN's neo-targets were identified, existing and novel drug development accelerated, with applications outside multiple myeloma, including non-Hodgkin's lymphoma, myelodysplastic syndrome, and acute leukaemias. Critically, transcription factors were the first canonical targets described. In addition to broadening the application of protein-degrading drugs, resistance mechanisms are being overcome and targeted protein degradation is widening the scope of druggable proteins against which existing approaches have been ineffective. Examples of targeted protein degraders include molecular glues and proteolysis targeting chimeras (PROTACs): heterobifunctional molecules that bind to proteins of interest and cause proximity-induced ubiquitination and proteasomal degradation via a linked E3 ligase. Twenty years since their inception, PROTACs have begun progressing through clinical trials, with early success in targeting the oestrogen receptor and androgen receptor in breast and prostate cancer respectively. This review explores important developments in targeted protein degradation to both treat and study cancer. It also considers the potential advantages and challenges in the translational aspects of developing new treatments. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Generation of salivary glands derived from pluripotent stem cells via conditional blastocyst complementation.

Whole salivary gland generation and transplantation offer potential therapies for salivary gland dysfunction. However, the specific lineage required to engineer complete salivary glands has remained elusive. In this study, we identify the Foxa2 lineage as a critical lineage for salivary gland development through conditional blastocyst complementation (CBC). Foxa2 lineage marking begins at the boundary between the endodermal and ectodermal regions of the oral epithelium before the formation of the primordial salivary gland, thereby labeling the entire gland. Ablation of Fgfr2 within the Foxa2 lineage in mice leads to salivary gland agenesis. We reversed this phenotype by injecting donor pluripotent stem cells into the mouse blastocysts, resulting in mice that survived to adulthood with salivary glands of normal size, comparable to those of their littermate controls. These findings demonstrate that CBC-based salivary gland regeneration serves as a foundational experimental approach for future advanced cell-based therapies.