By generating high-resolution electron density maps from atomic structures, this research presents an approach for predicting solution X-ray scattering profiles accurately at wide angles. Our approach incorporates the excluded volume of the bulk solvent by computing unique adjusted atomic volumes derived directly from atomic coordinate data. By employing this method, the necessity of a freely adjustable parameter, frequently incorporated in existing algorithms, is removed, leading to a more precise determination of the SWAXS profile. Water's form factor is utilized to construct an implicit model of the hydration shell. The two adjustable parameters, bulk solvent density and mean hydration shell contrast, are manipulated to generate the best possible fit to the experimental data. Eight publicly-available SWAXS profiles facilitated the generation of results showing high-quality fits to the data. Optimized parameter values, in each case, display minor variations, showcasing that default values are close to the optimal solution. Removing parameter optimization demonstrates a substantial improvement in the calculated scattering profiles, compared to the benchmark software. Demonstrating substantial computational efficiency, the algorithm executes in a time that is over ten times faster than the leading software. The algorithm is implemented in a command-line script, specifically denss.pdb2mrc.py. Open-source access to the DENSS v17.0 software package, encompassing this feature, is provided through the GitHub repository at https://github.com/tdgrant1/denss. These advancements in the field of comparing atomic models with experimental SWAXS data will also lead to more precise modeling algorithms that utilize SWAXS data, thus reducing the chance of overfitting.
Studying the solution state and conformational dynamics of biological macromolecules in solution hinges on the accurate calculation of small and wide-angle scattering (SWAXS) profiles from their atomic models. Utilizing high-resolution real-space density maps, we detail a new approach for calculating SWAXS profiles based on atomic models. By including novel calculations of solvent contributions, this approach eliminates a substantial fitting parameter. Multiple high-quality experimental SWAXS datasets were used to evaluate the algorithm, revealing enhanced precision in comparison with the most advanced software. Leveraging experimental SWAXS data, the algorithm, computationally efficient and resistant to overfitting, boosts the accuracy and resolution of modeling algorithms.
To gain insight into the solution state and conformational dynamics of biological macromolecules, accurate small- and wide-angle scattering (SWAXS) profile calculations from atomic models are essential. Using high-resolution real-space density maps, we present a fresh perspective on calculating SWAXS profiles, informed by atomic models. Solvent contribution calculations, a novel element of this approach, remove a substantial fitting parameter. The algorithm was tested on multiple high-quality SWAXS experimental datasets, revealing a marked improvement in accuracy over leading software. Because the algorithm is both computationally efficient and resistant to overfitting, it enhances the accuracy and resolution possible in modeling algorithms using experimental SWAXS data.
Researchers have undertaken large-scale sequencing of thousands of tumor specimens to characterize the mutational profile of the coding genome. While a minority of germline and somatic variants occur within coding regions, the vast majority are found in the non-coding regions of the genome. Cyclosporine These genomic domains, not directly tied to the creation of proteins, can nevertheless have critical roles in the development of cancer, as evidenced by their capacity to disrupt the precise regulation of gene expression. We have constructed a comprehensive computational and experimental platform to discover recurrently mutated non-coding regulatory regions that propel tumor development. This approach, applied to whole-genome sequencing (WGS) data from a diverse group of metastatic castration-resistant prostate cancer (mCRPC) patients, highlighted a substantial collection of recurrently mutated areas. By employing in silico prioritization of functional non-coding mutations, massively parallel reporter assays, and in vivo CRISPR-interference (CRISPRi) screens in xenografted mice, we successfully identified and validated driver regulatory regions as key factors in mCRPC development. We observed that the enhancer region GH22I030351 is instrumental in regulating a bidirectional promoter, impacting the simultaneous expression of U2-associated splicing factor SF3A1 and chromosomal protein CCDC157. Both SF3A1 and CCDC157 were found to promote tumor growth in xenograft models of prostate cancer. SOX6, along with a number of other transcription factors, was implicated in the upregulation of SF3A1 and CCDC157 expression. bacterial symbionts Our integrated computational and experimental approach has successfully mapped and confirmed the non-coding regulatory regions responsible for the development and progression of human cancers.
O-linked – N -acetyl-D-glucosamine (O-GlcNAcylation), a post-translational protein modification (PTM), is ubiquitous across the proteome in all multicellular organisms throughout their lives. Still, almost all functional studies have been centered on single protein modifications, neglecting the considerable number of simultaneous O-GlcNAcylation events that interact to orchestrate cellular processes. NISE, a novel systems-level method, is presented to comprehensively and rapidly monitor O-GlcNAcylation throughout the proteome, concentrating on the interrelationships of interactors and substrates. By integrating affinity purification-mass spectrometry (AP-MS) with site-specific chemoproteomics, our method leverages network generation and unsupervised partitioning to associate potential upstream regulators with downstream targets of O-GlcNAcylation. From the data-rich network, both conserved O-GlcNAcylation activities, including epigenetic regulation, and tissue-specific functions, such as synaptic structure, are demonstrably exhibited. This systems-level approach, encompassing O-GlcNAc and beyond, provides a widely applicable framework for investigating post-translational modifications and unearthing their diverse functions in particular cell types and biological situations.
The study of injury and repair in pulmonary fibrosis requires an acknowledgement of the differing spatial patterns of the disease throughout the lung. To evaluate fibrotic remodeling in preclinical animal models, the modified Ashcroft score, a semi-quantitative macroscopic resolution scoring rubric, is routinely applied. Manually grading pathohistological samples suffers from inherent limitations, leading to a persistent need for an objective, reproducible system for quantifying fibroproliferative tissue. By employing computer vision methods on immunofluorescent images of the extracellular matrix protein laminin, we created a repeatable and robust quantitative remodeling scorer (QRS). The modified Ashcroft score and QRS readings showed a substantial agreement (Spearman correlation coefficient r = 0.768) in the bleomycin lung injury model. A straightforward integration of this antibody-based strategy is possible within large multiplex immunofluorescent studies, providing us with a study of the spatial adjacency of tertiary lymphoid structures (TLS) and fibroproliferative tissue. Without programming experience, the application outlined in this manuscript can be readily used.
The ongoing COVID-19 pandemic has taken the lives of millions, and the persistent appearance of novel variants underscores the virus's sustained presence in the human population. Despite the current accessibility of vaccines and the burgeoning field of antibody-based therapies, the long-term effects on immune response and protective capabilities remain uncertain. The identification of protective antibodies in individuals is frequently reliant on highly specialized, challenging assays, like functional neutralizing assays, which are generally not available in clinical laboratories. Practically speaking, there is an urgent demand for producing fast, clinically useful assays which align with neutralizing antibody tests, thereby identifying subjects who might profit from additional vaccination or bespoke COVID-19 therapies. A novel semi-quantitative lateral flow assay (sqLFA) is introduced in this report, assessing its performance in detecting functional neutralizing antibodies from the serum of COVID-19 convalescent individuals. hepato-pancreatic biliary surgery A substantial positive correlation was observed between sqLFA and neutralizing antibody levels. The sqLFA assay displays remarkable sensitivity at reduced assay cutoffs for identifying a spectrum of neutralizing antibody concentrations. Increased cutoff values lead to the detection of elevated levels of neutralizing antibodies with a high degree of specificity. The sqLFA, capable of identifying any level of neutralizing antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), serves as a versatile tool for identifying individuals with high levels of neutralizing antibodies who potentially do not need antibody-based therapies or additional vaccinations.
Our prior description of transmitophagy involved the shedding of mitochondria from retinal ganglion cell (RGC) axons, which are then subsequently transported to and degraded by neighboring astrocytes situated in the optic nerve head of mice. Considering the prominent role of Optineurin (OPTN), a mitophagy receptor and a significant glaucoma gene, and the axonal damage prevalent at the optic nerve head in glaucoma, this study explores the potential effect of OPTN mutations on transmitophagy. Live imaging of Xenopus laevis optic nerves highlighted a difference in the effect of human mutant OPTN versus wild-type OPTN. Mutant OPTN, but not wild-type OPTN, increased stationary mitochondria and mitophagy machinery, showing colocalization within and, in the context of glaucoma-associated OPTN mutations, beyond RGC axons. Extra-axonal mitochondria are targeted for degradation by astrocytes. Our research affirms the observation that, in RGC axons under normal circumstances, mitophagy is limited, but glaucoma-induced disruptions in OPTN result in enhanced axonal mitophagy, characterized by mitochondrial shedding and subsequent astrocytic degradation.