Congenomics (United States)

Abstract Multiplexed protein analysis in native cellular contexts holds great promise for uncovering the composition, interactions, and functions of distinct cell types in complex biological systems. However, current multiplexed protein imaging technologies often face limitations in detection sensitivity or involve technically demanding protocols. To address these issues, we have developed an ultra-sensitive, multiplexed in situ protein profiling approach that combines reiterative staining with off-the-shelf antibodies and the newly developed Cleavable Fluorescent Probe (CFP™) Fluor cleavable TSA fluorophores. Target proteins are recognized by primary antibodies followed by secondary antibodies conjugated with horseradish peroxidase (HRP), which catalyzes the covalent deposition of CFP fluorophores on or close to the target proteins. After imaging, the fluorophores are chemically cleaved, and antibodies stripped, enabling subsequent rounds of staining, imaging, cleavage, and stripping. This approach allows for the interrogation of many proteins in individual cells while maintaining spatial resolution. In addition to protein analysis, glycan modifications can also be profiled using this approach. Glycan modifications also play critical roles in cell function in both healthy and diseased states from controlling proliferation to modulating the immune response. Lectins are part of a broader group called glycan-binding proteins that recognize glycan chains and mediate their functionality. Their glycan-specific nature makes them important tools to profile, characterize, and capture complex glycans in biological systems. We demonstrated the compatibility of CFP Fluor cleavable TSA fluorophores with lectins developed by Vector Laboratories. This assay facilitates the exploration of glycan complexity, enabling the detection of major glycan motifs, including sialylation, fucosylation, and galactosylation, for characterizing glycan distributions in target specimens. These methods are highly effective for detecting multiplexed proteins and glycans in the same biological samples, including FFPE tissues. We developed a novel protocol which enabled us to stain more than 10 protein markers on the same tissue section of human tonsil FFPE samples using the cleavable TSA fluorophores. We have also applied this approach to stain 7 neural cell markers and 4 varied glycans together on 5 normal and 5 Alzheimer’s human hippocampus FFPE tissues. The obtained results enable us to explore the altered glycosylation in individual cells of Alzheimer’s brains and/or other tissues across various disease indications such as cancers. Citation Format: Nishinki Muthumuni, Jia Guo, Jing Zhou, Xiaoshan Wang, Shuhui Chen, Erika Leonard. Multiplexed in situ detection of protein and glycan expression using CFP Fluor cleavable TSA fluorophores [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB199.

JSBML is a community-driven project to create a free, open-source, pure Java library for reading, writing, and manipulating SBML files and data streams. It is an alternative to the mixed Java/native code-based interface provided in libSBML. For more details, please visit the JSBML project home page: http://sbml.org/Software/JSBML You can find a detailed list of the user-visible new features and bug fixes since JSBML version 1.4 on the NEWS file. Thank you for your interest and support of JSBML and SBML in general. The JSBML team.

List of software packages for single-cell data analysis, including RNA-seq, ATAC-seq, etc.

144 Background: Although cell free DNA (cfDNA) profiling by next generation sequencing (NGS) holds great promise for precision oncology, the inability to estimate tumor content a priori typically results in ultra-deep sequencing (e.g. 10,000x) based approaches—often at limited loci—to ensure accurate assessment of cfDNA samples where tumor content can be < 0.1%. However, a large subset of patients with advanced cancers, where most precision oncology is applied, have much greater cfDNA tumor content. Methods: Here we demonstrate the utility of a pan-cancer, rapid, inexpensive, whole genome NGS of cfDNA approach (PRINCe) on benchtop sequencers as a precision medicine screening strategy based on ultra-low coverage (~0.005x) tumor content determination through genome-wide copy number alteration (CNA) profiling using 48 plasma cfDNA samples from patients with advanced cancer. Results: Using this approach, we identified therapeutically relevant focal CNAs in 13 of 48 (27%) cfDNA samples from patients with metastatic cancer, including 11 of 36 (31%) from patients with mCRPC. We further show that PRINCe is effective at whole genome coverages as low as 0.005x. Combining PRINCe with targeted multiplexed-PCR NGS of the same cfDNA enables mutation and CNA assessment using effective coverage as low as 25x based on calibrating sequencing depth to cfDNA tumor content. Lastly, PRINCe identifies pre-treatment cfDNA copy number profiles that can be used for inexpensive disease monitoring. Conclusions: Taken together, our screening approach enables broadly applicable cfDNA based precision oncology for patients with advanced cancer through rapid, inexpensive identification of therapeutically relevant CNAs and pre-treatment genomic profiles for disease monitoring, while also guiding additional cfDNA profiling approaches to reserve costly ultra-deep approaches for patients with very low cfDNA tumor content.

Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and analysis of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products. At the enzyme level, active sites of key biosynthetic enzymes are now pinpointed through a curated pattern-matching procedure and Enzyme Commission numbers are assigned to functionally classify all enzyme-coding genes. Additionally, chemical structure prediction has been improved by incorporating polyketide reduction states. Finally, in order for users to be able to organize and analyze multiple antiSMASH outputs in a private setting, a new XML output module allows offline editing of antiSMASH annotations within the Geneious software.

A new cyclic decadepsipeptide was isolated from Chaetosphaeria tulasneorum with potent bioactivity on mammalian and yeast cells. Chemogenomic profiling in S. cerevisiae indicated that the Sec61 translocon complex, the machinery for protein translocation and membrane insertion at the endoplasmic reticulum, is the target. The profiles were similar to those of cyclic heptadepsipeptides of a distinct chemotype (including HUN-7293 and cotransin) that had previously been shown to inhibit cotranslational translocation at the mammalian Sec61 translocon. Unbiased, genome-wide mutagenesis followed by full-genome sequencing in both fungal and mammalian cells identified dominant mutations in Sec61p (yeast) or Sec61α1 (mammals) that conferred resistance. Most, but not all, of these mutations affected inhibition by both chemotypes, despite an absence of structural similarity. Biochemical analysis confirmed inhibition of protein translocation into the endoplasmic reticulum of both co- and post-translationally translocated substrates by both chemotypes, demonstrating a mechanism independent of a translating ribosome. Most interestingly, both chemotypes were found to also inhibit SecYEG, the bacterial Sec61 translocon homolog. We suggest 'decatransin' as the name for this new decadepsipeptide translocation inhibitor.