High-resolution electron density maps generated from atomic models are employed in this study to formulate an approach enabling accurate prediction of solution X-ray scattering profiles at wide angles. Unique adjusted atomic volumes, directly calculated from atomic coordinates, are used in our method to account for the excluded volume of bulk solvent. The proposed method eliminates the need for a free fitting parameter, typically included in existing algorithms, resulting in improved precision of the small-angle X-ray scattering (SWAXS) analysis. A hydration shell's implicit model, whose design draws upon the form factor of water, is produced. The experimental data is best matched by suitably altering the bulk solvent density and the mean hydration shell contrast. Analysis of eight publicly available SWAXS profiles produced results with excellent agreement to the data. The optimized parameter values demonstrate minimal adjustments, thereby highlighting the proximity of default values to the true solution. The act of disabling parameter optimization produces a substantial advancement in the calculated scattering profiles, resulting in superior output over prevailing software. The algorithm's computational efficiency results in a more than tenfold decrease in execution time when contrasted with the leading software. Encoded within the command-line script denss.pdb2mrc.py is the algorithm. This feature, part of the open-source DENSS v17.0 software package, is obtainable via the GitHub repository at https://github.com/tdgrant1/denss. Besides bolstering the capability of aligning atomic models with experimental SWAXS data, these innovations pave the way for the development of more accurate modeling algorithms that use SWAXS data, reducing the likelihood of overfitting.
The utility of accurate small and wide-angle scattering (SWAXS) profile calculations from atomic models for understanding solution state and conformational dynamics of biological macromolecules is undeniable. Utilizing high-resolution real-space density maps, we detail a new approach for calculating SWAXS profiles based on atomic models. In this approach, novel calculations regarding solvent contributions eliminate a substantial fitting parameter. High-quality experimental SWAXS datasets were used to evaluate the algorithm, showcasing improved accuracy relative to leading software programs. Experimental SWAXS data allows for modeling algorithms with increased accuracy and resolution, facilitated by the computationally efficient and overfitting-resistant algorithm.
The examination of biological macromolecules in solution, specifically concerning their solution state and conformational dynamics, benefits from the accurate calculation of small and wide-angle scattering (SWAXS) profiles using atomic models. From atomic models, and utilizing high-resolution real-space density maps, we introduce a new approach to calculating SWAXS profiles. This approach employs novel solvent contribution calculations, thereby eliminating a considerable fitting parameter. The algorithm's accuracy surpasses that of leading software, as evidenced by its testing on numerous high-quality SWAXS experimental datasets. By being computationally efficient and robust to overfitting, the algorithm empowers modeling algorithms using experimental SWAXS data to achieve increased accuracy and resolution.
Researchers have undertaken large-scale sequencing of thousands of tumor specimens to characterize the mutational profile of the coding genome. Yet, the majority of genetic alterations in germline and somatic cells lie outside the coding regions of the genome. selleck inhibitor These genomic locales, lacking the direct function of protein encoding, can nevertheless profoundly affect cancer progression, particularly by causing abnormal control of gene expression. We established a computational and experimental framework that systematically identifies recurrently mutated non-coding regulatory regions driving tumor development. This approach, when utilized on whole-genome sequencing (WGS) data from a sizable cohort of metastatic castration-resistant prostate cancer (mCRPC) cases, led to the identification of a sizable quantity of recurrently mutated segments. Through in silico prioritization of functional non-coding mutations, coupled with massively parallel reporter assays and in vivo CRISPR-interference (CRISPRi) screens in xenografted mice, we methodically recognized and authenticated driver regulatory regions that cause mCRPC. Our investigation revealed that the enhancer region GH22I030351 impacts a bidirectional promoter, leading to the coordinated regulation of U2-associated splicing factor SF3A1 and the chromosomal protein CCDC157 expression. Studies of xenograft models of prostate cancer identified SF3A1 and CCDC157 as promoters of tumor growth. SOX6, along with a number of other transcription factors, was implicated in the upregulation of SF3A1 and CCDC157 expression. MRI-directed biopsy Our computational and experimental methodology, when integrated, has led to the identification and validation of the non-coding regulatory regions driving the course of human cancer development.
Protein O-GlcNAcylation, a post-translational modification (PTM) of proteins by O-linked – N -acetyl-D-glucosamine, is present across the entire proteome of all multicellular organisms across their entire lifespan. Nevertheless, practically all functional studies have concentrated on single protein modifications, ignoring the abundance of concurrent O-GlcNAcylation events that collaborate to control cellular functions. We present NISE, a novel systems-level approach to rapidly and comprehensively monitor O-GlcNAcylation across the entire proteome, focusing on the networking of interactors and substrates. Utilizing a combined approach of affinity purification-mass spectrometry (AP-MS), site-specific chemoproteomic techniques, network construction, and unsupervised clustering, our method identifies connections between potential upstream regulators and downstream targets of O-GlcNAcylation. A data-rich network structure unveils both conserved O-GlcNAcylation functions, such as epigenetic regulation, and tissue-specific roles, including the characteristics of synaptic morphology. This systems-level, unbiased, and comprehensive approach, going beyond O-GlcNAc, provides a widely applicable framework for exploring post-translational modifications (PTMs) and uncovering their diverse functions in particular cell types and biological scenarios.
Understanding the mechanisms of injury and repair in pulmonary fibrosis demands a focus on the varying spatial distribution of the disease's effects. In preclinical animal model studies, the modified Ashcroft score, a semi-quantitative rubric evaluating macroscopic resolution, is employed to assess fibrotic remodeling. Manual pathohistological grading is inherently limited, necessitating a standardized, unbiased approach to consistently evaluate the extent of fibroproliferative tissue. Utilizing computer vision on immunofluorescent laminin images of the extracellular matrix, we created a robust and repeatable quantitative remodeling score (QRS). In the bleomycin-induced lung injury model, QRS analysis demonstrated a strong correlation with the modified Ashcroft scoring, as evidenced by a significant Spearman rank correlation coefficient of 0.768. Multiplex immunofluorescent experiments easily accommodate this antibody-based approach, enabling us to investigate the spatial arrangement of tertiary lymphoid structures (TLS) adjacent to fibroproliferative tissue. The application in this manuscript is autonomous and operates independently, requiring no coding.
A persistent presence of the COVID-19 virus within the human population is indicated by the continued emergence of new variants, which, coupled with millions of deaths, is a lasting impact of the pandemic. The current era of readily available vaccines and the emergence of antibody-based therapies present a wealth of questions regarding the long-term establishment and strength of immunity and protective measures. Highly specialized assays, such as functional neutralizing assays, are often used to identify protective antibodies in individuals; however, such assays are typically unavailable in typical clinical settings. Thus, a pressing need exists for the development of fast, clinically practical assays that correlate with neutralizing antibody tests, identifying individuals who could benefit from additional immunization or specific COVID-19 therapies. Using a newly developed semi-quantitative lateral flow assay (sqLFA), we investigated in this report the functionality and detection of neutralizing antibodies present in the serum of individuals recovered from COVID-19. biosafety analysis Our findings revealed a strong positive correlation linking sqLFA to neutralizing antibody levels. At lower assay thresholds, the sqLFA assay exhibits high sensitivity in detecting various levels of neutralizing antibodies. Increased cutoff values lead to the detection of elevated levels of neutralizing antibodies with a high degree of specificity. A screening tool for neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this sqLFA can also pinpoint individuals with high levels of these antibodies, potentially not requiring further antibody therapies or vaccinations.
In mice, we previously reported a process, transmitophagy, where mitochondria detached from retinal ganglion cell (RGC) axons are transported to and broken down by surrounding astrocytes within the optic nerve head. Because Optineurin (OPTN), a crucial mitophagy receptor, is frequently identified as a significant genetic contributor to glaucoma, and the optic nerve head experiences axonal damage in glaucoma, this study investigated whether OPTN mutations could affect transmitophagy. Live-imaging of Xenopus laevis optic nerves revealed an increase in stationary mitochondria and mitophagy machinery colocalization within RGC axons, driven by diverse human mutant OPTN, but absent in wild-type OPTN; glaucoma-associated OPTN mutations further expanded this colocalization to outside of the axons. The degradation of extra-axonal mitochondria is carried out by astrocytes. Investigations into RGC axons under standard conditions indicate a low level of mitophagy, yet glaucoma-related modifications in OPTN increase axonal mitophagy, including the release and subsequent astrocytic breakdown of mitochondria.