MCMICRO: a scalable, modular image-processing pipeline for multiplexed tissue imaging.

Highly multiplexed tissue imaging makes detailed molecular analysis of single cells possible in a preserved spatial context. However, reproducible analysis of large multichannel images poses a substantial computational challenge. Here, we describe a modular and open-source computational pipeline, MCMICRO, for performing the sequential steps needed to transform whole-slide images into single-cell data. We demonstrate the use of MCMICRO on tissue and tumor images acquired using multiple imaging platforms, thereby providing a solid foundation for the continued development of tissue imaging software.

Genome Sequencing and Transposon Mutagenesis of Burkholderia seminalis TC3.4.2R3 Identify Genes Contributing to Suppression of Orchid Necrosis Caused by B. gladioli.

From a screen of 36 plant-associated strains of Burkholderia spp., we identified 24 strains that suppressed leaf and pseudobulb necrosis of orchid caused by B. gladioli. To gain insights into the mechanisms of disease suppression, we generated a draft genome sequence from one suppressive strain, TC3.4.2R3. The genome is an estimated 7.67 megabases in size, with three replicons, two chromosomes, and the plasmid pC3. Using a combination of multilocus sequence analysis and phylogenomics, we identified TC3.4.2R3 as B. seminalis, a species within the Burkholderia cepacia complex that includes opportunistic human pathogens and environmental strains. We generated and screened a library of 3,840 transposon mutants of strain TC3.4.2R3 on orchid leaves to identify genes contributing to plant disease suppression. Twelve mutants deficient in suppression of leaf necrosis were selected and the transposon insertions were mapped to eight loci. One gene is in a wcb cluster that is related to synthesis of extracellular polysaccharide, a key determinant in bacterial-host interactions in other systems, and the other seven are highly conserved among Burkholderia spp. The fundamental information developed in this study will serve as a resource for future research aiming to identify mechanisms contributing to biological control.

Mutualistic co-evolution of type III effector genes in Sinorhizobium fredii and Bradyrhizobium japonicum.

Two diametric paradigms have been proposed to model the molecular co-evolution of microbial mutualists and their eukaryotic hosts. In one, mutualist and host exhibit an antagonistic arms race and each partner evolves rapidly to maximize their own fitness from the interaction at potential expense of the other. In the opposing model, conflicts between mutualist and host are largely resolved and the interaction is characterized by evolutionary stasis. We tested these opposing frameworks in two lineages of mutualistic rhizobia, Sinorhizobium fredii and Bradyrhizobium japonicum. To examine genes demonstrably important for host-interactions we coupled the mining of genome sequences to a comprehensive functional screen for type III effector genes, which are necessary for many Gram-negative pathogens to infect their hosts. We demonstrate that the rhizobial type III effector genes exhibit a surprisingly high degree of conservation in content and sequence that is in contrast to those of a well characterized plant pathogenic species. This type III effector gene conservation is particularly striking in the context of the relatively high genome-wide diversity of rhizobia. The evolution of rhizobial type III effectors is inconsistent with the molecular arms race paradigm. Instead, our results reveal that these loci are relatively static in rhizobial lineages and suggest that fitness conflicts between rhizobia mutualists and their host plants have been largely resolved.

Exceptional Response to Trastuzumab Deruxtecan in a Patient With Recurrent Ovarian Clear Cell Carcinoma With Human Epidermal Growth Factor Receptor 2 Expression.

Case report of a HER2-expressed ovarian clear cell carcinoma with exceptional response to trastuzumab deruxtecan.

Addressing persistent challenges in digital image analysis of cancerous tissues.

The National Cancer Institute (NCI) supports many research programs and consortia, many of which use imaging as a major modality for characterizing cancerous tissue. A trans-consortia Image Analysis Working Group (IAWG) was established in 2019 with a mission to disseminate imaging-related work and foster collaborations. In 2022, the IAWG held a virtual hackathon focused on addressing challenges of analyzing high dimensional datasets from fixed cancerous tissues. Standard image processing techniques have automated feature extraction, but the next generation of imaging data requires more advanced methods to fully utilize the available information. In this perspective, we discuss current limitations of the automated analysis of multiplexed tissue images, the first steps toward deeper understanding of these limitations, what possible solutions have been developed, any new or refined approaches that were developed during the Image Analysis Hackathon 2022, and where further effort is required. The outstanding problems addressed in the hackathon fell into three main themes: 1) challenges to cell type classification and assessment, 2) translation and visual representation of spatial aspects of high dimensional data, and 3) scaling digital image analyses to large (multi-TB) datasets. We describe the rationale for each specific challenge and the progress made toward addressing it during the hackathon. We also suggest areas that would benefit from more focus and offer insight into broader challenges that the community will need to address as new technologies are developed and integrated into the broad range of image-based modalities and analytical resources already in use within the cancer research community.

An omic and multidimensional spatial atlas from serial biopsies of an evolving metastatic breast cancer.

Mechanisms of therapeutic resistance and vulnerability evolve in metastatic cancers as tumor cells and extrinsic microenvironmental influences change during treatment. To support the development of methods for identifying these mechanisms in individual people, here we present an omic and multidimensional spatial (OMS) atlas generated from four serial biopsies of an individual with metastatic breast cancer during 3.5 years of therapy. This resource links detailed, longitudinal clinical metadata that includes treatment times and doses, anatomic imaging, and blood-based response measurements to clinical and exploratory analyses, which includes comprehensive DNA, RNA, and protein profiles; images of multiplexed immunostaining; and 2- and 3-dimensional scanning electron micrographs. These data report aspects of heterogeneity and evolution of the cancer genome, signaling pathways, immune microenvironment, cellular composition and organization, and ultrastructure. We present illustrative examples of how integrative analyses of these data reveal potential mechanisms of response and resistance and suggest novel therapeutic vulnerabilities.

Characterizing advanced breast cancer heterogeneity and treatment resistance through serial biopsies and comprehensive analytics.

Molecular heterogeneity in metastatic breast cancer presents multiple clinical challenges in accurately characterizing and treating the disease. Current diagnostic approaches offer limited ability to assess heterogeneity that exists among multiple metastatic lesions throughout the treatment course. We developed a precision oncology platform that combines serial biopsies, multi-omic analysis, longitudinal patient monitoring, and molecular tumor boards, with the goal of improving cancer management through enhanced understanding of the entire cancer ecosystem within each patient. We describe this integrative approach using comprehensive analytics generated from serial-biopsied lesions in a metastatic breast cancer patient. The serial biopsies identified remarkable heterogeneity among metastatic lesions that presented clinically as discordance in receptor status and genomic alterations with mixed treatment response. Based on our study, we highlight clinical scenarios, such as rapid progression or mixed response, that indicate consideration for repeat biopsies to evaluate intermetastatic heterogeneity (IMH), with the objective of refining targeted therapy. We present a framework for understanding the clinical significance of heterogeneity in breast cancer between metastatic lesions utilizing multi-omic analyses of serial biopsies and its implication for effective personalized treatment.

An improved, high-quality draft genome sequence of the Germination-Arrest Factor-producing Pseudomonas fluorescens WH6.

BACKGROUND: Pseudomonas fluorescens is a genetically and physiologically diverse species of bacteria present in many habitats and in association with plants. This species of bacteria produces a large array of secondary metabolites with potential as natural products. P. fluorescens isolate WH6 produces Germination-Arrest Factor (GAF), a predicted small peptide or amino acid analog with herbicidal activity that specifically inhibits germination of seeds of graminaceous species. RESULTS: We used a hybrid next-generation sequencing approach to develop a high-quality draft genome sequence for P. fluorescens WH6. We employed automated, manual, and experimental methods to further improve the draft genome sequence. From this assembly of 6.27 megabases, we predicted 5876 genes, of which 3115 were core to P. fluorescens and 1567 were unique to WH6. Comparative genomic studies of WH6 revealed high similarity in synteny and orthology of genes with P. fluorescens SBW25. A phylogenomic study also placed WH6 in the same lineage as SBW25. In a previous non-saturating mutagenesis screen we identified two genes necessary for GAF activity in WH6. Mapping of their flanking sequences revealed genes that encode a candidate anti-sigma factor and an aminotransferase. Finally, we discovered several candidate virulence and host-association mechanisms, one of which appears to be a complete type III secretion system. CONCLUSIONS: The improved high-quality draft genome sequence of WH6 contributes towards resolving the P. fluorescens species, providing additional impetus for establishing two separate lineages in P. fluorescens. Despite the high levels of orthology and synteny to SBW25, WH6 still had a substantial number of unique genes and represents another source for the discovery of genes with implications in affecting plant growth and health. Two genes are demonstrably necessary for GAF and further characterization of their proteins is important for developing natural products as control measure against grassy weeds. Finally, WH6 is the first isolate of P. fluorescens reported to encode a complete T3SS. This gives us the opportunity to explore the role of what has traditionally been thought of as a virulence mechanism for non-pathogenic interactions with plants.

Phytopathogenic Rhodococcus Have Diverse Plasmids With Few Conserved Virulence Functions.

Rhodococcus is a genus of Gram-positive bacteria with species that can cause growth deformations to a large number of plant species. This ability to cause disease is hypothesized to be dependent on a cluster of three gene loci on an almost 200 kb-sized linear plasmid. To reevaluate the roles of some of the genes in pathogenicity, we constructed and characterized deletion mutants of fasR and four fas genes. Findings confirmed that fasR, which encodes a putative transcriptional regulator, is necessary for pathogenesis. However, three of the fas genes, implicated in the metabolism of plant growth promoting cytokinins, are dispensable for the ability of the pathogen to cause disease. We also used long-read sequencing technology to generate high quality genome sequences for two phytopathogenic strains in which virulence genes are diverged in sequence and/or hypothesized to have recombined into the chromosome. Surprisingly, findings showed that the two strains carry extremely diverse virulence plasmids. Ortholog clustering identified only 12 genes present on all three virulence plasmids. Rhodococcus requires a small number of horizontally acquired traits to be pathogenic and the transmission of the corresponding genes, via recombination and conjugation, has the potential to rapidly diversify plasmids and bacterial populations.

Evolutionary transitions between beneficial and phytopathogenic Rhodococcus challenge disease management.

Understanding how bacteria affect plant health is crucial for developing sustainable crop production systems. We coupled ecological sampling and genome sequencing to characterize the population genetic history of Rhodococcus and the distribution patterns of virulence plasmids in isolates from nurseries. Analysis of chromosome sequences shows that plants host multiple lineages of Rhodococcus, and suggested that these bacteria are transmitted due to independent introductions, reservoir populations, and point source outbreaks. We demonstrate that isolates lacking virulence genes promote beneficial plant growth, and that the acquisition of a virulence plasmid is sufficient to transition beneficial symbionts to phytopathogens. This evolutionary transition, along with the distribution patterns of plasmids, reveals the impact of horizontal gene transfer in rapidly generating new pathogenic lineages and provides an alternative explanation for pathogen transmission patterns. Results also uncovered a misdiagnosed epidemic that implicated beneficial Rhodococcus bacteria as pathogens of pistachio. The misdiagnosis perpetuated the unnecessary removal of trees and exacerbated economic losses.

The ABCs and 123s of bacterial secretion systems in plant pathogenesis.

Bacteria have many export and secretion systems that translocate cargo into and across biological membranes. Seven secretion systems contribute to pathogenicity by translocating proteinaceous cargos that can be released into the extracellular milieu or directly into recipient cells. In this review, we describe these secretion systems and how their complexities and functions reflect differences in the destinations, states, functions, and sizes of the translocated cargos as well as the architecture of the bacterial cell envelope. We examine the secretion systems from the perspective of pathogenic bacteria that proliferate within plant tissues and highlight examples of translocated proteins that contribute to the infection and disease of plant hosts.

Use of whole genome sequences to develop a molecular phylogenetic framework for Rhodococcus fascians and the Rhodococcus genus.

The accurate diagnosis of diseases caused by pathogenic bacteria requires a stable species classification. Rhodococcus fascians is the only documented member of its ill-defined genus that is capable of causing disease on a wide range of agriculturally important plants. Comparisons of genome sequences generated from isolates of Rhodococcus associated with diseased plants revealed a level of genetic diversity consistent with them representing multiple species. To test this, we generated a tree based on more than 1700 homologous sequences from plant-associated isolates of Rhodococcus, and obtained support from additional approaches that measure and cluster based on genome similarities. Results were consistent in supporting the definition of new Rhodococcus species within clades containing phytopathogenic members. We also used the genome sequences, along with other rhodococcal genome sequences to construct a molecular phylogenetic tree as a framework for resolving the Rhodococcus genus. Results indicated that Rhodococcus has the potential for having 20 species and also confirmed a need to revisit the taxonomic groupings within Rhodococcus.

Analysis of genome sequences from plant pathogenic Rhodococcus reveals genetic novelties in virulence loci.

Members of Gram-positive Actinobacteria cause economically important diseases to plants. Within the Rhodococcus genus, some members can cause growth deformities and persist as pathogens on a wide range of host plants. The current model predicts that phytopathogenic isolates require a cluster of three loci present on a linear plasmid, with the fas operon central to virulence. The Fas proteins synthesize, modify, and activate a mixture of growth regulating cytokinins, which cause a hormonal imbalance in plants, resulting in abnormal growth. We sequenced and compared the genomes of 20 isolates of Rhodococcus to gain insights into the mechanisms and evolution of virulence in these bacteria. Horizontal gene transfer was identified as critical but limited in the scale of virulence evolution, as few loci are conserved and exclusive to phytopathogenic isolates. Although the fas operon is present in most phytopathogenic isolates, it is absent from phytopathogenic isolate A21d2. Instead, this isolate has a horizontally acquired gene chimera that encodes a novel fusion protein with isopentyltransferase and phosphoribohydrolase domains, predicted to be capable of catalyzing and activating cytokinins, respectively. Cytokinin profiling of the archetypal D188 isolate revealed only one activate cytokinin type that was specifically synthesized in a fas-dependent manner. These results suggest that only the isopentenyladenine cytokinin type is synthesized and necessary for Rhodococcus phytopathogenicity, which is not consistent with the extant model stating that a mixture of cytokinins is necessary for Rhodococcus to cause leafy gall symptoms. In all, data indicate that only four horizontally acquired functions are sufficient to confer the trait of phytopathogenicity to members of the genetically diverse clade of Rhodococcus.