Author Response: Linking genotypic and phenotypic changes in the E. coli Long-Term Evolution Experiment using metabolomics

Changes in an organism’s environment, genome, or gene expression patterns can lead to changes in its metabolism. The metabolic phenotype can be under selection and contributes to adaptation. However, the networked and convoluted nature of an organism’s metabolism makes relating mutations, metabolic changes, and effects on fitness challenging. To overcome this challenge, we use the Long-Term Evolution Experiment (LTEE) with E. coli as a model to understand how mutations can eventually affect metabolism and perhaps fitness. We used mass-spectrometry to broadly survey the metabolomes of the ancestral strains and all 12 evolved lines. We combined this metabolic data with mutation and expression data to suggest how mutations that alter specific reaction pathways, such as the biosynthesis of nicotinamide adenine dinucleotide, might increase fitness in the system. Our work provides a better understanding of how mutations might affect fitness through the metabolic changes in the LTEE and thus provides a major step in developing a complete genotype-phenotype map for this experimental system.

Background: Multiple myeloma (MM) is characterized by the neoplastic proliferation of a single clone of plasma cells that produce monoclonal immunoglobulins. Among its subtypes, immunoglobulin D multiple myeloma (IgD MM) is rare, accounting for less than 2% of all MM cases. IgD MM is often associated with rapid disease progression at diagnosis, an aggressive clinical course, and a poor prognosis compared with other MM subtypes. Methods: Samples were collected from patients diagnosed with MM through bone marrow examination, and myeloma cells were isolated using the CD138-positive marker. Paired datasets (n=37), including whole genome sequencing (WGS) and bulk RNA sequencing (RNA-seq), were generated and analyzed. We utilized whole exome sequencing (WES) and RNA-seq data from the CD138-positive fraction of bone marrow mononuclear cell (BMMC) samples provided by the Multiple Myeloma Research Foundation (MMRF) CoMMpass study. Results: The WGS data revealed an average of 8, 872 single nucleotide variants (SNVs) and 934 insertions and deletions (Indels) of somatic mutations in 37 MM patients, respectively. Using these somatic mutations, we performed a mutational signature analysis to infer the cause of the malignancy and were able to identify different proportions of mutational signatures in each MM patient. Interestingly, among the single base substitution (SBS) 9 signature varied widely between patients, four patients with non-IgG and non-IgA heavy chain and lambda light chain myeloma had significantly high SBS9 signature. Notably, three IgD MM patients were included exclusively in the high SBS9 signature group and not in the low SBS9 signature group (p = 0.001). Analyzing RNA-seq data, we found that the high and low SBS9 signature groups had different gene expression patterns. In particular, differentially expressed genes (DEGs) analysis suggested features of IgD myeloma, including high expression of IGHD, in high SBS9 signature group. Pathway gene expression analysis showed higher expression of genes associated with the DNA repair system and somatic hypermutation in IgD MM patients. We performed the identical mutation signature analysis on WES data of 784 MM patients from the MMRF database. Of the 30 patients for whom sufficient somatic mutations could be obtained, there were three IgD MM candidates with a high SBS9 signature, whereas there were no IgD MM candidates in the low SBS9 signature group. Similarly, three high SBS9 patients with suspected IgD MM showed characteristic gene expression patterns and upregulation of the DNA repair system. Conclusions: We found that IgD MM has distinctive features, including high SBS9 mutation signatures and upregulation of genes related to the DNA repair system and somatic hyper-mutation. Further research into IgD MM may reveal the detailed characteristics and underlying mechanisms of IgD MM and lead to tailored treatments. Citation Format: Sheehyun Kim, Hyundong Yoon, Youngil Koh, Sung-Soo Yoon. The characteristics of IgD subtype myeloma with distinctive genomic mutational signature and gene expression patterns [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6640.

Persistent propagative plant viruses are usually transmitted between a vector insect and a host plant. To adapt to the two different organisms, viruses may show distinct genomic replication or gene expression patterns. To verify this hypothesis, we applied an aboslute real-time quantitative PCR method to measure and compare the replication levels of four genomic RNA segments and the expression levels of seven genes of rice stripe virus (RSV) according to the infection time in the small brown planthopper and rice plant, respectively. In the vector insect, RNA3 began replicating later than the other segments, and RNA2 remained nearly constant during the infection process. RNA1 was the dominant segment, and a difference of over 300-fold appeared among the four segments. In rice plants, the size of the four segments increased with infection time, but decreased to a low level in the late infection period. The ratios of the four segments varied by no more than 15-fold. In planthoppers, three expression patterns were observed for the seven viral genes during viral infection, while in rice plants, the expression patterns of the seven viral genes were similar. These results reflect distinct genomic replication and gene expression patterns in a persistent propagative plant virus in adapting to vector insects and host plants.

Synonymous genetic differences vary by more than 20-fold among genes in natural isolates of Escherichia coli. One hypothesis to explain this heterogeneity is that genes with high levels of synonymous variation mutate at higher rates than genes with low synonymous variation. If so, then one would expect to observe similar mutational patterns in evolution experiments. In fact, however, the pattern of synonymous substitutions in a long-term evolution experiment with E. coli does not support this hypothesis. In particular, the extent of synonymous variation across genes in that experiment does not reflect the variation observed in natural isolates of E. coli. Instead, gene length alone predicts with high accuracy the prevalence of synonymous changes in the experimental populations. We hypothesize that patterns of synonymous variation in natural E. coli populations are instead caused by differences across genomic regions in their effective population size that, in turn, reflect different histories of recombination, horizontal gene transfer, selection, and population structure.

Macrophage is a critical microenvironmental component which can control tumor progression, and dynamic changes in cellular metabolism accompany the polarization. As well as other immune cells, the activation of macrophages can be modulated by cancer cells. Cancer-derived exosome is a newly emerging microenvironmental factor enables cancer cells to communicate with macrophages. In this study, examined were the activation-associated bioenergetic changes and the effect of cancer cell-derived exosome on the changes by a) monitoring the early metabolic kinetics and b) profiling bioenergetic phenotype resulted. In vitro activation of RAW264.7 macrophage cell line by injecting lipopolysaccharide (LPS) and interferon γ (IFNγ) in a Seahorse XF analyzer induced an immediate early increase in glycolytic rate. It also showed a delayed down-regulation of mitochondrial respiration, which resulted in a secondary elevation of glycolytic rate. This secondary glycolytic response was only obtained by LPS-IFNγ co-stimulation and mediated by induced nitric oxide synthase (iNOS) signaling. In contrast to the dynamic changes in metabolic phenotype, the total ATP production rate appeared to be stably maintained according to the XF data normalized by cell number. However, macrophage activation significantly increased glycolysis-dependent portion of ATP production. ATPs were generated almost solely by glycolysis after the delayed mitochondrial down-regulation by iNOS. Co-injection of cancer cell-derived exosomes with LPS slightly but significantly accelerated the immediate early glycolytic response as well as the delayed mitochondrial down-regulation. Furthermore, exosomes induced the immediate early glycolytic response even in the absence of LPS. These results imply that presence of cancer cell-derived exosomes may promote macrophage activation and accelerate pro-inflammatory immune response in a tumor microenvironment. Citation Format: Yoonseok Kam, Brian P. Dranka. Effect of cancer cell-derived exosome on energy metabolism associated with macrophage activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 62.

New mutations leading to structural variation (SV) in genomes—in the form of mobile element insertions, large deletions, gene duplications, and other chromosomal rearrangements—can play a key role in microbial evolution. Yet, SV is considerably more difficult to predict from short-read genome resequencing data than single-nucleotide substitutions and indels (SN), so it is not yet routinely identified in studies that profile population-level genetic diversity over time in evolution experiments. We implemented an algorithm for detecting polymorphic SV as part of the breseq computational pipeline. This procedure examines split-read alignments, in which the two ends of a single sequencing read match disjoint locations in the reference genome, in order to detect structural variants and estimate their frequencies within a sample. We tested our algorithm using simulated Escherichia coli data and then applied it to 500- and 1000-generation population samples from the Lenski E. coli long-term evolution experiment (LTEE). Knowledge of genes that are targets of selection in the LTEE and mutations present in previously analyzed clonal isolates allowed us to evaluate the accuracy of our procedure. Overall, SV accounted for ~25% of the genetic diversity found in these samples. By profiling rare SV, we were able to identify many cases where alternative mutations in key genes transiently competed within a single population. We also found, unexpectedly, that mutations in two genes that rose to prominence at these early time points always went extinct in the long term. Because it is not limited by the base-calling error rate of the sequencing technology, our approach for identifying rare SV in whole-population samples may have a lower detection limit than similar predictions of SNs in these data sets. We anticipate that this functionality of breseq will be useful for providing a more complete picture of genome dynamics during evolution experiments with haploid microorganisms.

Genetics has long been regarded as the cornerstone of biological understanding—explaining how life reproduces, evolves, and sustains human health. Mendelism, including Johannsen’s 1911 genotype conception, which posits that ‘genes are the units of heredity’, supplanted Galton’s earlier phenotype-based statistical law of ancestral heredity. Yet, this shift was not the result of decisive empirical validation. Evidence accumulated over the past three decades challenges the genotype conception. In 1992, the discovery of radiation-induced genomic instability and the bystander effect undermined the assumption that heritable change is strictly gene-dependent. In 2009, genetic variance failed to account for the fitness trajectory observed in the Long-Term Evolution Experiment (LTEE) with _E. coli_. By 2013, it became evident that evolution in the LTEE was thermodynamically—not genetically—driven. This paper traces the historiography of heredity from 1880 onwards, critically re-evaluates Mendelism, and reviews recent empirical findings. It concludes that Mendelism fails as a scientific framework. Consequently, there is no valid consensus underpinning the life sciences, human health, or evolutionary theory. This represents a profound failure in twentieth-century biology.

Introduction. Colorectal cancer (CRC) is the second leading cause of cancer related deaths, due to the development of distant metastases in many CRC patients. To detect metastatic disease in early stage, the identification of tumor cells circulating in the peripheral blood of patients (CTCs) is of outmost importance. Several techniques have recently been developed which can detect CTCs, two of them are the CellSearch® system and the EpiSpot assay. We have investigated genomic aberrations and gene expression patterns in primary colorectal tumors in correlation with CTC status of patients. This may lead to the identification of the underlying mechanisms of metastatic spread and potential new markers for detecting the tumor's capability to disseminate. Methods. Peripheral blood of 48 colorectal patients was investigated by CellSearch® and EpiSpot for CTCs. Samples with at least one CTC, detected with either the CellSearch® system or the EpiSpot assay were considered as positive. Genomic DNA and mRNA extracted from the primary tumors of the same patients were investigated with Illumina based Comparative Genomic Hybridization (CGH) and Gene Expression profiling (GEP) microarrays. CGH arrays covered the whole genome with an average resolution of 5kb and GEP arrays covered more than 25,000 annotated genes. Results. Twenty-nine patients were diagnosed to be positive for CTCs, either with the EpiSpot assay or the CellSearch® system; negative for CTCs in both assays were 17 blood samples. Array-CGH showed that primary tumors from CTC positive patients harbored significantly more often loss of chromosome 15q11 (Fisher's exact test, p<0.01). The Significance Analysis of Microarrays (SAM) revealed 13 genes to be differentially expressed between both groups. These genes are mainly involved in kinase activity and ATP-binding, and are located in the plasma membrane. Furthermore, CYFIP1 was downregulated in colorectal tumors from CTC positive patients compared with CTC negative patients which correlated with its loss detected with array-CGH. CYFIP1 is a known putative invasion suppressor gene in epithelial cancers. Conclusion. Our results suggest that several molecular and genomic profiles can be identified as marker for tumor cell dissemination in CRC. More over, significant correlation between CTC status and the primary tumor's genomic profile indicates a biological rationale for tumor cell dissemination rather than a random effect or shedding of cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2165.

Introduction: Severe trauma(ST) is a cause of significant morbidity and mortality which is exacerbated in the elderly. Despite theories as to why this may be true, the exact cause has yet to be fully understood. We feel that this failure can be explained by the diversity of the host response to ST. The objective was to determine whether the elderly have unique genomic patterns in neutrophils in the acute/subacute periods after ST consistent with either ‘hyperinflammation’ or ‘immuno-senescence’ explaining the worse outcomes seen in the elderly. Methods: Microarray data collected in the Glue Grant from 34 severely traumatized adult patients were used to evaluate genome-wide expression from blood neutrophils on days 0.5, 1, 4, 7, 14, and 21 after ST. 17 elderly (≥55yo) and young (<55yo) patients with complicated ICU outcomes were matched based on gender and AIS score for comparison to non-injured controls (n=17). Subsequent analysis consisted of identifying differences in gene expression (p<0.001), individual fold gene changes (vs. control, p<0.05), gene ontologies, as well as functional pathways among elderly and young ST patients, and healthy subjects. Results: In the young, genome-wide expression patterns at 0.5 and 7 days were significantly more aberrant from control subjects than those in the elderly. Among 61 genes whose dysregulation is known to predict complicated outcomes after ST in young patients (CCM 2013 41(5):1175–85), we found that the aberrant genome expression was significantly less in the elderly populations at 0.5 and 1 days, and greater at 4 days. Fold changes in genes involved in inflammation, myeloid derived suppressor cells, and chemotaxis were also increased from control more in the young than the elderly at 0.5 days (Z>2.0). By day 4, young patients’ gene expression patterns appeared to return to baseline, while the dysregulated expression of the elderly patients persisted. The top canonical pathways involved in innate immunity in the young compared to elderly were significantly upregulated at 0.5 days and then downregulated by day 4. Conclusions: Our findings reveal a unique early genomic expression pattern of elderly patients after ST consistent with ‘immuno-senescence’. Initially, there is a diminished immune response compared to their younger counterparts, followed by prolonged up regulation of inflammatory genes associated with ‘hyperinflammation’. This research indicates that interventions to improve outcomes in the elderly will need to be distinct from younger cohorts, and address multiple components of protective immunity.

BackgroundIsobutanol is considered a potential biofuel, thanks to its high-energy content and octane value, limited water solubility, and compatibility with gasoline. As its biosynthesis pathway is known, a microorganism, such as Saccharomyces cerevisiae, that inherently produces isobutanol, can serve as a good engineering host. Isobutanol’s toxicity, however, is a major obstacle for bioproduction. This study is to understand how yeast tolerates isobutanol.ResultsA S. cerevisiae gene-deletion library with 5006 mutants was used to screen genes related to isobutanol tolerance. Image recognition was efficiently used for high-throughput screening via colony size on solid media. In enrichment analysis of the 161 isobutanol-sensitive clones identified, more genes than expected were mapped to tryptophan biosynthesis, ubiquitination, and the pentose phosphate pathway (PPP). Interestingly, adding exogenous tryptophan enabled both tryptophan biosynthesis and PPP mutant strains to overcome the stress. In transcriptomic analysis, cluster analysis of differentially expressed genes revealed the relationship between tryptophan and isobutanol stress through some specific cellular functions, such as biosynthesis and transportation of amino acids, PPP, tryptophan metabolism, nicotinate/nicotinamide metabolism (e.g., nicotinamide adenine dinucleotide biosynthesis), and fatty acid metabolism.ConclusionsThe importance of tryptophan in yeast’s tolerance to isobutanol was confirmed by the recovery of isobutanol tolerance in defective strains by adding exogenous tryptophan to the growth medium. Transcriptomic analysis showed that amino acid biosynthesis- and transportation-related genes in a tryptophan biosynthesis-defective host were up-regulated under conditions similar to nitrogen starvation. This may explain why ubiquitination was required for the protein turnover. PPP metabolites may serve as precursors and cofactors in tryptophan biosynthesis to enhance isobutanol tolerance. Furthermore, the tolerance mechanism may also be linked to tryptophan downstream metabolism, including the kynurenine pathway and nicotinamide adenine dinucleotide biosynthesis. Both pathways are responsible for cellular redox balance and anti-oxidative ability. Our study highlights the central role of tryptophan in yeast’s isobutanol tolerance and offers new clues for engineering a yeast host with strong isobutanol tolerance.

Novel genotype-phenotype associations in human cancers enabled by advanced molecular platforms and computational analysis of whole slide images

Divergent selection of populations in contrasting environments leads to functional genomic divergence. However, the genomic architecture underlying heterogeneous genomic differentiation remains poorly understood. Here, we de novo assembled two high-quality wild barley (Hordeum spontaneum K. Koch) genomes and examined genomic differentiation and gene expression patterns under abiotic stress in two populations. These two populations had a shared ancestry and originated in close geographic proximity but experienced different selective pressures due to their contrasting micro-environments. We identified structural variants that may have played significant roles in affecting genes potentially associated with well-differentiated phenotypes such as flowering time and drought response between two wild barley genomes. Among them, a 29-bp insertion into the promoter region formed a cis-regulatory element in the HvWRKY45 gene, which may contribute to enhanced tolerance to drought. A single SNP mutation in the promoter region may influence HvCO5 expression and be putatively linked to local flowering time adaptation. We also revealed significant genomic differentiation between the two populations with ongoing gene flow. Our results indicate that SNPs and small SVs link to genetic differentiation at the gene level through local adaptation and are maintained through divergent selection. In contrast, large chromosome inversions may have shaped the heterogeneous pattern of genomic differentiation along the chromosomes by suppressing chromosome recombination and gene flow. Our research offers novel insights into the genomic basis underlying local adaptation and provides valuable resources for the genetic improvement of cultivated barley.

Hox genes specify the structures that form along the anteroposterior (AP) axis of bilateria. Within the genome, they often form clusters where, remarkably enough, their position within the clusters reflects the relative positions of the structures they specify along the AP axis. This correspondence between genomic organization and gene expression pattern has been conserved through evolution and provides a unique opportunity to study how chromosomal context affects gene regulation. In Drosophila, a general rule, often called “posterior dominance,” states that Hox genes specifying more posterior structures repress the expression of more anterior Hox genes. This rule explains the apparent spatial complementarity of Hox gene expression patterns in Drosophila. Here we review a noticeable exception to this rule where the more-posteriorly expressed Abd-B Hox gene fails to repress the more-anterior abd-A gene in cells of the central nervous system (CNS). While Abd-B is required to repress ectopic expression of abd-A in the posterior epidermis, abd-A repression in the posterior CNS is accomplished by a different mechanism that involves a large 92 kb long non-coding RNA (lncRNA) encoded by the intergenic region separating abd-A and Abd-B (the iab8ncRNA). Dissection of this lncRNA revealed that abd-A is repressed by the lncRNA using two redundant mechanisms. The first mechanism is mediated by a microRNA (mir-iab-8) encoded by intronic sequence within the large iab8-ncRNA. Meanwhile, the second mechanism seems to involve transcriptional interference by the long iab-8 ncRNA on the abd-A promoter. Recent work demonstrating CNS-specific regulation of genes by ncRNAs in Drosophila, seem to highlight a potential role for the iab-8-ncRNA in the evolution of the Drosophila Hox complexes.

Craniopharyngioma presents malignant clinical manifestations with severe symptoms and higher incidence of hypothalamic dysfunction. The pathogenesis of craniopharyngioma remains unclear, which impedes the development of effective treatments and preventive measures. Comprehensively understanding the neoplastic programming and tumoral evolution may contribute to the improvement of prognosis for craniopharyngioma. Using the single cell RNA-sequencing and high-resolution spatial transcriptomics analysis on adamantinomatous craniopharyngioma (ACP), papillary craniopharyngioma (PCP) and Rathke cleft cyst (RCC) to depict the cellular hierarchies and neoplastic evolution. Multiplex immunohistochemistry and ex vivo pituitary culture from animal model were used to identify the putative therapeutic targets. Cellular hierarchy analysis uncovered a more inflammatory tumor microenvironment in PCP compared to ACP. Parallel genomic variation and gene expression patterns were revealed between PCP and RCC, suggesting a continuum of the same disease spectrum that evolutes from RCC to PCP. Furthermore, we identified the disease-type-unique structures composed of various cellular states, including the tumoral progenitors surrounding the fibrovascular cores in PCP and senescence associated secretory phenotype (SASP)-related cellular hierarchy in ACP, respectively. In addition, SASP-related fibroblast growth factor (FGF) signaling was identified as a potential therapeutic target for ACP and inhibition of it led to decreased tumor cell proliferation in murine model. Our study reveals the neoplastic programming and evolution of craniopharyngioma at single cell and spatial levels. Notably, SASP-related FGF signaling is identified as a potential therapeutic target for ACP and inhibition of it reduces tumor cell proliferation in murine model.

High‐Throughput Lung Cancer Cell Line Screening for Genotype‐Correlated Sensitivity to an EGFR Kinase Inhibitor