Genomic Level Research Articles

Genomic and Transcriptomic Comparison Between Invasive Non-Typhoidal Salmonella and Non-Invasive Isolates

Invasive non-typhoidal Salmonella (iNTS) poses a significant threat to global public health. Salmonella enterica Enteritidis and Typhimurium are the primary serovars responsible for both invasive diseases and gastroenteritis. This study aims to investigate the genomic and transcriptomic differences between isolates associated with these contrasting clinical presentations. We retrieved genomes of Salmonella Enteritidis and Salmonella Typhimurium from Enterobase, utilizing blood and stool isolates as representatives for iNTS and non-iNTS, respectively. An indistinguishable phylogenetic relationship was revealed between the blood and stool isolates for both serovars. Few genes were specifically identified in iNTS. Random forest and principal coordinates analysis permitted moderate discrimination between the two sources of isolates based on overall genome content. Notably, the blood isolates of Salmonella Typhimurium displayed an elevated level of antimicrobial resistance and genome degradation compared to stool isolates. Meanwhile, transcriptome sequencing identified few genes that were differentially expressed between blood and stool isolates. Hierarchical clustering and principal component analysis did not effectively differentiate the expression profile of iNTS from non-iNTS. In summary, few genes could serve as reliable biomarkers to distinguish iNTS and non-iNTS at either the genomic or transcriptomic level. Nevertheless, iNTS has indeed accumulated subtle genomic differences from non-iNTS, which might contribute to invasiveness.

LINE-1 cfDNA Methylation as an Emerging Biomarker in Solid Cancers

Epigenetic dysregulation is a hallmark of many human malignancies, with DNA methylation being a primary mechanism influencing gene expression and maintaining genomic stability. Genome-wide hypomethylation, characteristic of many cancers, is partly attributed to the demethylation of repetitive elements, including LINE-1, a prevalent non-LTR retrotransposon. The methylation status of LINE-1 is closely associated with overall genomic methylation levels in tumors. cfDNA comprises extracellular DNA fragments found in bodily fluids such as plasma, serum, and urine, offering a dynamic snapshot of the genetic and epigenetic landscape of tumors. This real-time sampling provides a minimally invasive avenue for cancer diagnostics, prognostics, and monitoring. The methylation status of LINE-1 in cfDNA has emerged as a promising biomarker, with several studies highlighting its potential in diagnosing and predicting outcomes in cancer patients. Recent research also suggests that cfDNA-based LINE-1 methylation analysis could serve as a valuable tool in evaluating the efficacy of cancer therapies, including immunotherapy. The growing clinical significance of cfDNA calls for a closer examination of its components, particularly repetitive elements like LINE-1. Despite their importance, the role of LINE-1 elements in cfDNA has not been thoroughly gauged. We aim to address this gap by reviewing the current literature on LINE-1 cfDNA assays, focusing on their potential applications in diagnostics and disease monitoring.

Distinct molecular responses of mangrove plants to hypoxia and reoxygenation stresses contribute to their resilience in coastal wetland environment

Mangroves adapt to periodical submergence and constitute resilient ecosystems in coastal environments. The question is whether they can sustain long submergence stress when sea level rises as a consequence of climate change. To address this, seedlings of two representative mangrove species that acclimate to low to mid tide (Avicennia marina) and mid to high tide (Kandelia obovata) conditions were treated with continual submergence for 7 days as extended hypoxia, or semi-diurnal cyclic submergence and reoxygenation for 7 days. At specific time points, leaves were collected to construct RNA-Sequencing libraries for gene expression analysis. Through the lens of transcriptome, the initial response of A. marina to submergence was mild but more dramatic after prolong immersion. However, the initial response of K. obovata was drastic and reduced in latitude later, suggesting distinct species-specific responses. After adapting to diurnal cycles, both species minimized transcriptome fluctuations similarly. Metabolically, during initial response, sucrose and starch were converted into glucose for fermentation to increase glycolytic flux, coupled with regeneration of NAD+. The energy amelioration was accompanied by longer term phytohormone regulations where ethylene signal transduction pathway was enhanced, but abscisic acid biosynthesis was reduced. Notably, gibberellic acids biosynthesis increased in A. marina but decreased in K. obovata as a unique feature. Genomic level analysis indicated that only about 30 % of the conserved plant submergence responsive genes were expressed during submergence in these mangroves. The function of an ethylene responsive gene was validated in transgenic Arabidopsis. This research elucidates distinct molecular mechanisms and metabolic pathways that empower A. marina and K. obovata to endure prolonged submergence and hypoxia. By highlighting their unique adaptive strategies in response to rising sea levels, these findings enhance our understanding of mangrove resilience and provide insights for the conservation and management of these essential coastal ecosystems in the face of climate change.

Human Impacts on Great Lakes Walleye Sander vitreus Structure, Diversity and Local Adaptation.

Artificial propagation and wild release may influence the genetic integrity of wild populations. This practice has been prevalent in fisheries for centuries and is often termed 'stocking'. In the Laurentian Great Lakes (Great Lakes here-on), walleye populations faced declines from the 1950s to the 1970s, prompting extensive stocking efforts for restoration. By the mid-2010s, walleye populations showed signs of recovery, but the genetic legacy of stocking on population structure at the genomic level remains unclear. Using a dataset of 45,600 genome-aligned SNP loci genotyped in 1075 walleye individuals, we investigated the genetic impacts of over 50 years of stocking across the Great Lakes. Population structure was associated with both natural geographic barriers and stocking from non-native sources. Admixture between Lake Erie walleye and walleye from the re-populated Tittabawassee River indicate that stocking may have re-distributed putatively adaptive alleles around the Great Lakes. Genome scans identified FST outliers and evidence of selective sweeps, indicating local adaptation of spawning populations is likely. Notably, one genomic region showed strong differentiation between Muskegon River and walleye from the Tittabawassee River, which was re-populated by Muskegon strain walleye, suggesting admixture and selection both impact the observed genetic diversity. Overall, our study underscores how artificial propagation and translocations can significantly alter the evolutionary trajectory of populations. The findings highlight the complex interplay between stocking practices and population genetic diversity, emphasising the need for careful management strategies to preserve the genetic integrity of wild populations amidst conservation efforts.

Transcription factor Pofst3 regulates Pleurotus ostreatus development by targeting multiple biological pathways

Pleurotus ostreatus is a popular edible mushroom cultivated worldwide. However, the mechanism of P. ostreatus primordia formation is unclear. Pofst3 is a MHR superfamily transcription factor, which has the function of regulating primordia formation. In this study, the target genes of Pofst3 in P. ostreatus were identified by DAP-Seq approach at the genome level, 1481 peaks were obtained and the Pofst3 binding motif sequence was GARGRVGARGAR. The interaction between transcription factor Pofst3 and this motif was confirmed in vitro and in vivo through electrophoretic mobility shift (EMSA) and yeast one-hybrid screening (Y1H) assays. Among the top 20 GO enrichment results, most were related to transcriptional regulation, and some transcription factor encoding genes, such as HMG-box (gene_5346), MADS-box (gene_86), FOG (gene_6211) and RFX (gene_3496) were obtained. Besides basic metabolism, MAPK signaling pathway, Inositol phosphate metabolism, Glycosylphosphatidylinositol (GPI)-anchor biosynthesis and Pentose phosphate pathway were significantly enriched in the KEGG pathway analysis. The expression levels of randomly selected 11 genes, some transcription factor genes, and genes involved in metabolic pathways in wild and Pofst3 transgenic P. ostreatus strains indicated that target genes likely involved in the development of the P. ostreatus primordia. These results indicated that transcription factor Pofst3 ultimately negatively regulated the development of P. ostreatus primordia very likely through regulating a series of biological pathways.

Multi-omics evaluation of clinical-grade human umbilical cord-derived mesenchymal stem cells in synergistic improvement of aging related disorders in a senescence-accelerated mouse model

BackgroundThe prevalence of age-related disorders, particularly in neurological and cardiovascular systems, is an increasing global health concern. Mesenchymal stem cell (MSC) therapy, particularly using human umbilical cord-derived MSCs (HUCMSCs), has shown promise in mitigating these disorders. This study investigates the effects of HUCMSCs on aging-related conditions in a senescence-accelerated mouse model (SAMP8), with a focus on DNA damage, gut microbiota alterations, and metabolic changes.MethodsSAMP8 mice were treated with clinical-grade HUCMSCs via intraperitoneal injections. Behavioral and physical assessments were conducted to evaluate cognitive and motor functions. The Single-Strand Break Mapping at Nucleotide Genome Level (SSiNGLe) method was employed to assess DNA single-strand breaks (SSBs) across the genome, with particular attention to exonic regions and transcription start sites. Gut microbiota composition was analyzed using 16S rRNA sequencing, and carboxyl metabolomic profiling was performed to identify changes in circulating metabolites.ResultsHUCMSC treatment significantly improved motor coordination and reduced anxiety in SAMP8 mice. SSiNGLe analysis revealed a notable reduction in DNA SSBs in MSC-treated mice, especially in critical genomic regions, suggesting that HUCMSCs may mitigate age-related DNA damage. The functional annotation of the DNA breaktome indicated a potential link between reduced DNA damage and altered metabolic pathways. Additionally, beneficial alterations in gut microbiota were observed, including an increase in short-chain fatty acid (SCFA)-producing bacteria, which correlated with improved metabolic profiles.ConclusionThe administration of HUCMSCs in SAMP8 mice not only reduces DNA damage but also induces favorable changes in gut microbiota and metabolism. The observed alterations in DNA break patterns, along with specific changes in microbiota and metabolic profiles, suggest that these could serve as potential biomarkers for evaluating the efficacy of HUCMSCs in treating age-related disorders. This highlights a promising avenue for the development of new therapeutic strategies that leverage these biomarkers, to enhance the effectiveness of HUCMSC-based treatments for aging-associated diseases.

Zinc oxide nanoparticles at low dose mitigate lead toxicity in pea (Pisum sativum L.) seeds during germination by modulating metabolic and cellular defense systems

Addressing lead (Pb) pollution and contamination in soil and agriculture is urgent due to its widespread and long-lasting impacts on human health, food safety, and the environment. In the current study, we assessed the potential use of Zinc oxide nanoparticles (ZnONPs) in alleviating Pb toxicity in germinating seedlings. Pea (Pisum sativum L.) seedlings were exposed to Pb (83 mg/L) over a 7-day period, without and with ZnONPs amendment. Our results showed that the application of ZnONPs at 10 mg/L restored the morphological parameters affected by Pb, 34 %, 26 % and 23 %, for the length, fresh, and dry biomass of embryonic axis, respectively. ZnONPs decreased the activities of antioxidative enzymes of catalase (87.5 %), guaiacol peroxidase (60 %), and glutathione peroxidase (25 %), but increased the activities of ascorbate peroxidase (60 %) and glutathione reductase (25 %). These changes were associated with the increase (7 %) in the thiol groups, and reductions in the malondialdehyde (23 %), hydrogen peroxide (33 %), and carbonyl group levels (78 %), in addition to the stimulation of the activities of ROS-associated enzymes, including glycolate oxidase (40 %) and NADPH oxidase (by 89 %). Consequently, cell viability was increased by 66 %, while cellular death was reduced by 10 %, with ZnONPs, relative to the Pb-stressed seedlings. These findings highlight the mechanisms surrounding the ability of nanosized ZnO to mitigate the harmful effects of Pb on the growth and physiology of plants. Further investigation is needed to understand the mitigating effects of ZnONPs on Pb on the molecular and genomic levels in seedlings and plants, and ensure the sustainability of agricultural practices and food safety. Besides, site and source-specific integrated approaches must be practiced to formulate suitable remediation strategies.

Hydroxyacid Oxidase 1, a Glutamine Metabolism-Associated Protein, Predicts Poor Patient Outcome in Luminal Breast Cancer

Breast cancer (BC), which remains the most prevalent malignancy among women, is characterised by significant heterogeneity across its molecular subtypes. Oestrogen receptor-positive (ER+) (luminal) BC represents approximately 75% of cases, and despite advancements in treatment there remains around a 40% recurrence rate. Cellular uptake of glutamine is conducted by solute carriers (SLCs), which are significantly associated with outcome in luminal BC. In this study, differential gene expression analysis was carried out using The Cancer Genome Atlas BC dataset. This identified hydroxyacid oxidase 1 (HAO1) as significantly overexpressed in luminal BC with a high expression of SLCs. Extended analysis in the METABRIC (n = 1980) and Breast Cancer Gene-Expression Miner (n = 4421) transcriptomic databases and the Nottingham (n = 952) BC tissue cohort showed a varied survival outcome for HAO1 expression at the genomic, transcriptomic, and proteomic levels. HAO1 copy number (CN) gain (p = 0.002) and high HAO1 protein expression (p = 0.019) were associated with poor prognosis in luminal BC, whereas high HAO1 mRNA expression correlated with better survival outcomes (p = 0.023) suggesting a complex regulatory mechanism affecting HAO1 at different biological levels. Importantly, in luminal BC patients treated with endocrine therapy, high protein expression of HAO1 predicted shorter distant-metastasis free survival (p = 0.042). The knockdown of SLC1A5 and SLC7A5 significantly reduced HAO1 expression in MCF-7 and ZR-751 BC cell lines. Protein analysis confirmed significant associations between HAO1 and SLC7A5 and SLC1A5, emphasising a potential role for the enzyme in glutamine metabolism and its potential as a therapeutic target. This study underscores the prognostic significance of HAO1 in luminal BC and its relationship with patient outcomes.

Evolution of the pheV-tRNA integrated genomic island in Escherichia coli.

Escherichia coli exhibit extensive genetic diversity at the genome level, particularly within their accessory genome. The tRNA integrated genomic islands (GIs), a part of the E. coli accessory genome, play an important role in pathogenicity. However, studies examining the evolution of GIs have been challenging due to their large size, considerable gene content variation and fragmented assembly in draft genomes. Here we examined the evolution of the GI integrated at pheV-tRNA (GI-pheV), with a primary focus on uropathogenic E. coli (UPEC) and the globally disseminated multidrug resistant ST131 clone. We show the gene content of GI-pheV is highly diverse and arranged in a modular configuration, with the P4 integrase encoding gene intP4 the only conserved gene. Despite this diversity, the GI-pheV gene content displayed conserved features among strains from the same pathotype. In ST131, GI-pheV corresponding to the reference strain EC958 (EC958_GI-pheV) was found in ~90% of strains. Phylogenetic analyses suggested that GI-pheV in ST131 has evolved together with the core genome, with the loss/gain of specific modules (or the entire GI) linked to strain specific events. Overall, we show GI-pheV exhibits a dynamic evolutionary pathway, in which modules and genes have evolved through multiple events including insertions, deletions and recombination.

Transcription-dependent mobility of single genes and genome-wide motions in live human cells

The human genome is highly dynamic across all scales. At the gene level, chromatin is persistently remodeled and rearranged during active processes such as transcription, replication and DNA repair. At the genome level, chromatin moves in micron-scale domains that break up and re-form over seconds, but the origin of these coherent motions is unknown. Here, we investigate the connection between genomic motions and gene-level activity. Simultaneous mapping of single-gene and genome-wide motions shows that the coupling of gene transcriptional activity to flows of the nearby genome is modulated by chromatin compaction. A motion correlation analysis suggests that a single active gene drives larger-scale motions in low-compaction regions, but high-compaction chromatin drives gene motion regardless of its activity state. By revealing unexpected connections among gene activity, spatial heterogeneities of chromatin and its emergent genome-wide motions, these findings uncover aspects of the genome’s spatiotemporal organization that directly impact gene regulation and expression.

Novel imported clades accelerated the RSV surge in Beijing, China, 2023-2024

ObjectivesDespite the optimisation of the zero-COVID policy in late 2022, there was a subsequent increase noted in the number of respiratory syncytial virus (RSV) cases in Northern China. In this study, we investigated and characterised the dynamics of this surge at the genomic level in Beijing, China. MethodsPatients with acute respiratory tract infections (ARTIs) were enrolled from 35 sentinel hospitals in Beijing, China. Epidemiological investigations, G gene amplification, and whole-genome sequencing were performed, followed by epidemiological analysis, imported clade detection, and mutation identification. We also combined global data to illustrate the biological and epidemiological characteristics of the emerging clades. ResultsA total of 60,423 patients with ARTIs were recruited between January 2015 and January 2024. The RSV peak observed in the winter of 2023 was the highest in the past 9 years. Two novel imported clades, A.D.5.2 and B.D.E.1, were detected for the first time in China. This surge was mainly driven by B.D.E.1, which exhibited a significantly higher proportion of older individuals both in Beijing and globally. Seven non-synonymous mutations in B.D.E.1 were found in Beijing. B.D.E.1 had more sites suffering from positive selection than its parent. ConclusionsThe novel imported clade B.D.E.1 accelerated an unprecedented RSV surge in Beijing, presenting noteworthy epidemiological and biological characteristics. Continuous RSV genome monitoring has important implications for RSV outbreak identification, genetic diversity tracking, vaccine development, and strategy implementation.

DeOri 10.0: An Updated Database of Experimentally Identified Eukaryotic Replication Origins.

DNA replication is a complex and crucial biological process in eukaryotes. To facilitate the study of eukaryotic replication events, we present a database of eukaryotic DNA replication origins (DeOri), which collects scattered data and integrates extensive sequencing data on eukaryotic DNA replication origins. With continuous updates of DeOri, the number of datasets in the new release increased from 10 to 151 and the number of sequences increased from 16,145 to 9,742,396. Besides nucleotide sequences and bed files, corresponding annotation files, such as coding sequences (CDS), mRNA, and other biological elements within replication origins, are also provided. The experimental techniques used for each dataset, as well as other statistical data, are also presented on web page. Differences in experimental methods, cell lines, and sequencing technologies have resulted in distinct replication origins, making it challenging to differentiate between cell-specific and non-specific replication. We combined multiple replication origins at the species level, scored them, and screened them. The screened regions were considered as species-conservative origins. They are integrated and presented as reference replication origins (rORIs), including Homo sapiens, Gallus gallus, Mus musculus, Drosophila melanogaster, and Caenorhabditis elegans. Additionally, we analyzed the distribution of relevant genomic elements associated with replication origins at the genome level, such as CpG island (CGI), transcription start site (TSS), and G-quadruplex (G4). These analysis results allow users to select the desired data based on it. DeOri is available at http://tubic.tju.edu.cn/deori/.

PARP1 acetylation at K119 is essential in regulating the progression and proliferation of cervical cancer cells.

Cervical cancer, CC, is one of the malignant cancers in women worldwide. Many studies about the genesis and progression of CC have been done at genomic, transcriptional, translational, and epigenetic levels. However, much less is done at post-translational modification (PTM) level. We first used pan-PTM antibodies to compare the pan PTM levels between clinical normal cervical tissues and CC tissues; we then sent the selected samples for label-free identification of acetylation sites. Next, we employed WT or K119A mutant PARP1-EGFP-STREPII plasmid transfection in Hela cells and examined various indexes including colony formation, wound healing, ROS generation, early apoptosis, and immunofluorescence and quantification of proliferation markers (Ki67, PCNA, and p-P53). Last, we examined the levels of multiple important kinases regulating cervical cancer progression. We found that pan-acetylation was the most downregulated in clinical CC samples, whereas the acetylation of PARP1, Poly(ADP-ribose) polymerase-1, was upregulated at K119. Next, we showed that PARP1-WT overexpression significantly suppressed the proliferation and progression in CC cell line Hela, while K119A overexpression didn't show any impact. Finally, PARP1-WT overexpression significantly decreased p-ERK1/2 while didn't affect the phosphorylation levels of other important kinases such as AKT, MTOR, and RPS6. This study discovered a new type of PTM of PARP1 in CC, and showed that PARP1 acetylation at K119 is essential in regulating the proliferation and progression of CC through ERK1/2. Further studies are required to investigate how PARP1 acetylation impact its function.

The use of CRISPR-Cas9 technology for targeted gene therapy in Parkinson's disease focusing on LRRK2 and SNCA genes

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta, leading to the start of motor and non-motor symptoms. These include bradykinesia, resting tremor, rigidity, and postural instability, alongside cognitive impairments, mood disorders, and autonomic dysfunctions. The hallmark of PD is the accumulation of α-synuclein protein aggregates, forming Lewy bodies within the neurons. Given the numerous causes of Parkinson’s, involving genetic, epigenetic, and environmental factors, therapeutic treatments are hard to come by. Traditional pharmacological treatments, such as levodopa and dopamine agonists, primarily offer symptomatic relief and are associated with diminishing efficacy over time. Therefore, there is a need for new therapeutic approaches that target the disease at its roots. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, with the RNA-guided Cas9 endonuclease enables precise, targeted modifications at the genomic level. This technology offers potential for terminating the genetic factors causing parkinsons and aid in developing gene-based therapies. By attempting to regulate gene expression, CRISPR holds promise for addressing the fundamental causes of neurodegeneration in Parkinsons. One of the key genetic targets in PD is the leucine-rich repeat kinase 2 (LRRK2) gene, where specific gain-of- function mutations, such as G2019S, lead to hyperactivation of its kinase activity, contributing to neurotoxicity. CRISPR-Cas9 can be employed to introduce precise double-strand breaks at the mutation site, followed by homology-directed repair (HDR) to restore the wild-type sequence, thereby normalizing LRRK2 function and preventing neuronal death. Similarly, CRISPR technology can target the SNCA gene, which encodes α-synuclein. Pathogenic duplicates or triplicates of SNCA result in overproduction of α-synuclein and subsequent aggregation. Utilizing CRISPR to disrupt these extra copies can reduce α-synuclein levels, alleviating its toxic effects.CRISPR activation (CRISPRa) and interference (CRISPRi) systems enable fine-tuned upregulation or downregulation of gene expression, offering strategies to increase the expression of neuroprotective factors or suppress the expression of deleterious genes.

Understanding the role of ten-eleven translocation family proteins in kidney diseases.

Epigenetic mechanisms play a critical role in the pathogenesis of human diseases including kidney disorders. As the erasers of DNA methylation, Ten-eleven translocation (TET) family proteins can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), thus leading to passive or active DNA demethylation. Similarly, TET family proteins can also catalyze the same reaction on RNA. In addition, TET family proteins can also regulate chromatin structure and gene expression in a catalytic activity-independent manner through recruiting the SIN3A/HDAC co-repressor complex. In 2012, we reported for the first time that the genomic 5-hydroxymethylcytosine level and the mRNA levels of Tet1 and Tet2 were significantly downregulated in murine kidneys upon ischemia and reperfusion injury. Since then, accumulating evidences have eventually established an indispensable role of TET family proteins in not only acute kidney injury but also chronic kidney disease. In this review, we summarize the upstream regulatory mechanisms and the pathophysiological role of TET family proteins in major types of kidney diseases and discuss their potential values in clinical diagnosis and treatment.

7615 Genomic Consequences of GRHL2 Overexpression in ER+ Breast Cancer Cells

Abstract Disclosure: K.O. Allen: None. C. Zheng: None. N. Solodin: None. M.S. Ozers: Owner/Co-Owner; Self; Proteovista, LLC. C.L. Warren: None. P.E. Messa: None. D.T. Vogt: None. E.T. Alarid: None. Breast cancer progression is characterized by the presence and prevalence of metastatic disease. At a genomic level, metastasis can be facilitated via rearrangement of the chromatin landscape to expose oncogenic genes for transcription. Grainyhead like protein 2 (GRHL2) is a nuclear transcription factor implicated in epithelial cell differentiation. The GRHL2 motif has been found near activated estrogen receptor (ER) binding sites, and elevated levels of GRHL2 have been linked to worse prognosis in hormone receptor positive breast cancer patients. In its capacity to contribute to a more severe disease state when overexpressed, GRHL2 may function as either a transcription or pioneer factor to aid in the expression of metastasis-associated genes. Our lab previously showed that GRHL2 genomic binding precedes ER binding at co-regulated binding sites. Also, overexpression of GRHL2 in MCF7 cells for 24 hours did not appreciably alter gene expression by RNA-Seq. These data taken together indicate that GRHL2 may be exhibiting pioneer factor activity. However, the specific role of GRHL2 overexpression in hormone positive breast cancer progression remains unknown. In order to examine the mechanism of GRHL2 action, GRHL2 genomic binding was investigated using a tetracycline-inducible MCF7 model that allows controlled overexpression of GRHL2. ChIP studies were performed to determine GRHL2 binding intensity at specific and genome wide GRHL2 binding sites. Specific focus was given to established GRHL2 binding sites near oncogenic genes responsible for epithelial to mesenchymal transition (EMT), dormancy, and proto-oncogenic behavior. A similar analysis examined H3K27 acetylation to validate active gene transcription. An orthogonal technology, Specificity and Affinity for Protein (SNAP) microarrays, which characterizes direct GRHL2 binding to tiled genomic regions identified in ChIP-Seq analyses from multiple labs, was utilized to discriminate direct and indirect GRHL2 binding sites. The requirement for GRHL2 transactivation function in regulation of EMT related genes was further interrogated using transactivation GRHL2 dominant negative mutants. This work aims to distinguish the transcriptional and non-transcriptional activities of GRHL2 in breast cancer cells on a genome wide level and thereby, provide a deeper understanding of how overexpressed GRHL2 contributes to hormone positive breast cancer. Presentation: 6/3/2024

7324 Regulation of Super Enhancers by Phosphorylated Progesterone Receptors Drives Breast Cancer Stem Cell Expansion

Abstract Disclosure: N. Gillis: None. C.A. Lange: None. Current treatments for estrogen receptor (ER) positive breast cancer largely target rapidly dividing tumor cells. However, aggressive breast cancers often contain long-lived and weakly proliferative cancer stem cells (CSCs) that readily escape treatments aimed at cycling cells. We previously reported that progesterone receptors (PR) and their target genes aid the growth of these CSCs and help them evade anti-estrogen therapy. As proliferation and stemness are directed by separate signaling pathways that lead to the expression of distinct gene programs, it's crucial to understand how PR facilitates this cellular transformation at the genomic level. Establishing a gene expression program that may limit proliferation but encourages stemness requires coordination between transcription factors and their coregulators to alter the nuclear microenvironment and chromatin landscape.To reveal these mechanisms, we profiled PR and ER genomic binding using CUT&RUN in the T47D luminal breast cancer cell line grown in ultra-low attachment (3D) mammosphere conditions. Our analysis of genomic binding profiles displayed a pattern of PR/ER co-occupancy at distant sites more than 10 kilobases away from the nearest transcriptional start site. We used Rank Ordering of Super Enhancer (ROSE) analysis on these PR/ER binding sites to predict which are likely super enhancer elements capable of coordinating the regulation of multi-gene subsets. We employed a genetic approach to directly test the function of novel enhancer sites linked to PR-target genes by disrupting the PR/ER binding with a dCas9-KRAB construct directed to the predicted enhancer elements. We propose that abnormal growth factor signaling in breast cancer cells permits PR/ER complex formation which promotes expression of genes that induce endocrine resistance and CSC expansion through regulation of super enhancer elements. Further studies to elucidate these mechanisms are ongoing. Understanding the intricacies of PR/ER crosstalk is the crucial next step in developing new therapies that target PR-driven processes in ER+ breast cancers. Presentation: 6/3/2024

Inhibition of phototrophic iron oxidation by nitric oxide in ferruginous environments

Anoxygenic phototrophic Fe(II) oxidizers (photoferrotrophs) are thought to have thrived in Earth’s ancient ferruginous oceans and played a primary role in the precipitation of Archaean and Palaeoproterozoic (3.8–1.85-billion-year-old) banded iron formations (BIFs). The end of BIF deposition by photoferrotrophs has been interpreted as the result of a deepening of water-column oxygenation below the photic zone, concomitant with the proliferation of cyanobacteria. However, photoferrotrophs may have experienced competition from other anaerobic Fe(II)-oxidizing microorganisms, altering the formation mechanism of BIFs. Here we utilize microbial incubations to show that nitrate-reducing Fe(II) oxidizers metabolically outcompete photoferrotrophs for dissolved Fe(II). Moreover, both experiments and numerical modelling show that the nitrate-reducing Fe(II) oxidizers inhibit photoferrotrophy via the production of toxic intermediates. Four different photoferrotrophs, representing both green sulfur and purple non-sulfur bacteria, are susceptible to this toxic effect despite having genomic capabilities for nitric oxide detoxification. Indeed, despite nitric oxide detoxification mechanisms being ubiquitous in some groups of phototrophs at the genomic level (for example, Chlorobi and Cyanobacteria) it is likely that they would still be affected. We suggest that the production of reactive nitrogen species during nitrate-reducing Fe(II) oxidation in ferruginous environments may have inhibited the activity of photoferrotrophs in the ancient oceans and thus impeded their role in the precipitation of BIFs.

Genomic characteristics of antimicrobial resistance and virulence factors of carbapenem-resistant Stutzerimonas nitrititolerans isolated from the clinical specimen

BackgroundStutzerimonas nitrititolerans (S. nitrititolerans) is a rare human pathogenic bacterium and has been inadequately explored at the genomic level. Here, we report the first case of carbapenem-resistant S. nitrititolerans isolated from the peritoneal dialysis fluid of a patient with chronic renal failure. This study analyzed the genomic features, antimicrobial resistance, and virulence factors of the isolated strain through whole genome sequencing (WGS).MethodsThe bacterial isolate from the peritoneal dialysis fluid was named PDI170223, and preliminary identification was conducted through Matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS). WGS of the strain PDI170223 was performed using the Illumina platform, and a phylogenetic tree was constructed based on the 16S rRNA gene sequences. Antimicrobial susceptibility test (AST) was conducted using the TDR-200B2 automatic bacteria identification/drug sensitivity tester.ResultsS. nitrititolerans may emerge as a human pathogen due to its numerous virulence genes, including those encoding toxins, and those involved in flagellum and biofilm formation. The AST results revealed that the strain is multidrug- and carbapenem-resistant. The antimicrobial resistance genes of S. nitrititolerans are complex and diverse, including efflux pump genes and β⁃lactam resistance genes.ConclusionThe analysis of virulence factors and antimicrobial resistance of S. nitrititolerans provides clinical insight into the pathogenicity and potential risks of this bacterium. It is crucial to explore the mechanisms through which S. nitrititolerans causes diseases and maintains its antimicrobial resistance, thereby contributing to development of effective treatment and prevention strategies.

Genomic and phenotypic polymorphism of Clostridium botulinum Group II strain Beluga through laboratory domestication

Laboratory domestication is the result of genetic and physiological changes of organisms acquired during numerous passages in vitro. This phenomenon has been observed in bacteria as well as in higher organisms. In an effort to understand the impact of laboratory domestication on the foodborne pathogen Clostridium botulinum and related microbial food safety research, we investigated multiple spore stocks of C. botulinum Group II Beluga from our collection, as that is a widely applied model strain used in laboratories over decades. An acquired nutrient auxotrophy was confirmed as thymidine dependency using phenotypic microarrays. In parallel, whole-genome re-sequencing of all stocks revealed a mutation in thyA encoding thymidylate synthase essential for de-novo synthesis of dTMP from dUMP in the auxotrophic stocks. A thyA-deficient Beluga variant stock was successfully complemented by introducing an intact variant of thyA and thymidine prototrophy was restored, indicating that the thymidine auxotrophy was solely due to the presence of a SNP in thyA. Our data suggested that this mutation, deleterious under nutrient-poor growth conditions in a chemically defined medium, has been present and maintained in laboratory stocks for nearly 30 years. Yet, the mutation remained unidentified since receiving the strain, most likely due to routine use of culture conditions optimized for growth performance. This work pinpoints the need for careful monitoring of model strains extensively used in laboratory settings at both phenotypic and genomic level. In applications like food safety challenge tests, compromised strains could cause incorrect predictions and thereby have deleterious consequences. To mitigate the risk of acquiring mutations, we recommend keeping passage numbers of laboratory strains low and to avoid single-colony passaging. In addition, relevant strains should be subjected to regular WGS checks and physiological validation to exclude DNA mutations with potential negative impacts on research data integrity and reproducibility.