DNA replication initiation timing is important for maintaining genome integrity.

DNA replication is regulated by factors that promote or inhibit initiation. In Bacillus subtilis, YabA is a negative regulator of DNA replication initiation while the newly identified kinase CcrZ is a positive regulator. The consequences of under-initiation or over-initiation of DNA replication to genome stability remain unclear. In this work, we measure origin to terminus ratios as a proxy for replication initiation activity. We show that ΔccrZ and several ccrZ alleles under-initiate DNA replication while ablation of yabA or overproduction of CcrZ leads to over-initiation. We find that cells under-initiating DNA replication have few incidents of replication fork stress as determined by low formation of RecA-GFP foci compared with wild type. In contrast, cells over-initiating DNA replication show levels of RecA-GFP foci formation analogous to cells directly challenged with DNA damaging agents. We show that cells under-initiating and over-initiating DNA replication were both sensitive to mitomycin C and that changes in replication initiation frequency cause increased sensitivity to genotoxic stress. With these results, we propose that cells under-initiating DNA replication are sensitive to DNA damage due to a shortage of DNA for repair through homologous recombination. For cells over-initiating DNA replication, we propose that an increase in the number of replication forks leads to replication fork stress which is further exacerbated by chromosomal DNA damage. Together, our study shows that DNA replication initiation frequency must be tightly controlled as changes in initiation influence replication fork fate and the capacity of cells to efficiently repair damage to their genetic material.

Two distinct regulatory systems control pulcherrimin biosynthesis in Bacillus subtilis.

Regulation of transcription is a fundamental process that allows bacteria to respond to external stimuli with appropriate timing and magnitude of response. In the soil bacterium Bacillus subtilis, transcriptional regulation is at the core of developmental processes needed for cell survival. Gene expression in cells transitioning from exponential phase to stationary phase is under the control of a group of transcription factors called transition state regulators (TSRs). TSRs influence numerous developmental processes including the decision between biofilm formation and motility, genetic competence, and sporulation, but the extent to which TSRs influence bacterial physiology remains to be fully elucidated. Here, we demonstrate two TSRs, ScoC and AbrB, along with the MarR-family transcription factor PchR negatively regulate production of the iron chelator pulcherrimin in B. subtilis. Genetic analysis of the relationship between the three transcription factors indicate that all are necessary to limit pulcherrimin production during exponential phase and influence the rate and total amount of pulcherrimin produced. Similarly, expression of the pulcherrimin biosynthesis gene yvmC was found to be under control of ScoC, AbrB, and PchR and correlated with the amount of pulcherrimin produced by each background. Lastly, our in vitro data indicate a weak direct role for ScoC in controlling pulcherrimin production along with AbrB and PchR. The layered regulation by two distinct regulatory systems underscores the important role for pulcherrimin in B. subtilis physiology.

Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB.

Mitomycin C (MMC) repair factor A (mrfA) and factor B (mrfB), encode a conserved helicase and exonuclease that repair DNA damage in the soil-dwelling bacterium Bacillus subtilis. Here we have focused on the characterization of MrfB, a DEDDh exonuclease in the DnaQ superfamily. We solved the structure of the exonuclease core of MrfB to a resolution of 2.1 Å, in what appears to be an inactive state. In this conformation, a predicted α-helix containing the catalytic DEDDh residue Asp172 adopts a random coil, which moves Asp172 away from the active site and results in the occupancy of only one of the two catalytic Mg2+ ions. We propose that MrfB resides in this inactive state until it interacts with DNA to become activated. By comparing our structure to an AlphaFold prediction as well as other DnaQ-family structures, we located residues hypothesized to be important for exonuclease function. Using exonuclease assays we show that MrfB is a Mg2+-dependent 3'-5' DNA exonuclease. We show that Leu113 aids in coordinating the 3' end of the DNA substrate, and that a basic loop is important for substrate binding. This work provides insight into the function of a recently discovered bacterial exonuclease important for the repair of MMC-induced DNA adducts.

Neural control of fluid homeostasis is engaged below 10°C in hibernation.

Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) hibernate for several months each winter without access to water,1 but the mechanisms that maintain fluid homeostasis during hibernation are poorly understood. In torpor, when body temperature (TB) reaches 4°C, squirrels decrease metabolism, slow heart rate, and reduce plasma levels of the antidiuretic hormones arginine vasopressin (AVP) and oxytocin (OXT).1 Squirrels spontaneously undergo interbout arousal (IBA) every 2 weeks, temporarily recovering an active-like metabolism and a TB of 37°C for up to 48 h.1,2 Despite the low levels of AVP and OXT during torpor, profound increases in blood pressure and heart rate during the torpor-IBA transition are not associated with massive fluid loss, suggesting the existence of a mechanism that protects against diuresis at a low TB. Here, we demonstrate that the antidiuretic hormone release pathway is activated by hypothalamic supraoptic nucleus (SON) neurons early in the torpor-arousal transition. SON neuron activity, dense-core vesicle release from the posterior pituitary, and plasma hormone levels all begin to increase before TB reaches 10°C. In vivo fiber photometry of SON neurons from hibernating squirrels, together with RNA sequencing and c-FOS immunohistochemistry, confirms that SON is electrically, transcriptionally, and translationally active to monitor blood osmolality throughout the dynamic torpor-arousal transition. Our work emphasizes the importance of the antidiuretic pathway during the torpor-arousal transition and reveals that the neurophysiological mechanism that coordinates the hormonal response to retain fluid is active at an extremely low TB, which is prohibitive for these processes in non-hibernators.

Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB.

Mitomycin C (MMC) repair factor A (mrfA) and factor B (mrfB), encode a conserved helicase and exonuclease that repair DNA damage in the soil-dwelling bacterium Bacillus subtilis. Here we have focused on the characterization of MrfB, a DEDDh exonuclease in the DnaQ superfamily. We solved the structure of the exonuclease core of MrfB to a resolution of 2.1 Å, in what appears to be an inactive state. In this conformation, a predicted α-helix containing the catalytic DEDDh residue Asp172 adopts a random coil, which moves Asp172 away from the active site and results in the occupancy of only one of the two catalytic Mg2+ ions. We propose that MrfB resides in this inactive state until it interacts with DNA to become activated. By comparing our structure to an AlphaFold prediction as well as other DnaQ-family structures, we located residues hypothesized to be important for exonuclease function. Using exonuclease assays we show that MrfB is a Mg2+-dependent 3'-5' DNA exonuclease. We show that Leu113 aids in coordinating the 3' end of the DNA substrate, and that a basic loop is important for substrate binding. This work provides insight into the function of a recently discovered bacterial exonuclease important for the repair of MMC-induced DNA adducts.

Two Distinct Regulatory Systems Control Pulcherrimin Biosynthesis in Bacillus subtilis.

Regulation of transcription is a fundamental process that allows bacteria to respond to external stimuli with appropriate timing and magnitude of response. In the soil bacterium Bacillus subtilis, transcriptional regulation is at the core of developmental processes needed for cell survival. Gene expression in cells transitioning from exponential phase to stationary phase is under the control of a group of transcription factors called transition state regulators (TSRs). TSRs influence numerous developmental processes including the decision between biofilm formation and motility, genetic competence, and sporulation, but the extent to which TSRs influence bacterial physiology remains to be fully elucidated. Here, we demonstrate two TSRs, ScoC and AbrB, along with the MerR-family transcription factor PchR negatively regulate production of the iron chelator pulcherrimin in B. subtilis. Genetic analysis of the relationship between the three transcription factors indicate that all are necessary to limit pulcherrimin production during exponential phase and influence the rate and total amount of pulcherrimin produced. Similarly, expression of the pulcherrimin biosynthesis gene yvmC was found to be under control of ScoC, AbrB, and PchR and correlated with the amount of pulcherrimin produced by each background. Lastly, our in vitro data indicate a weak direct role for ScoC in controlling pulcherrimin production along with AbrB and PchR. The layered regulation by two distinct regulatory systems underscores the important, and somewhat enigmatic, role for pulcherrimin in B. subtilis physiology.

RNase H genes cause distinct impacts on RNA:DNA hybrid formation and mutagenesis genome wide.

RNA:DNA hybrids compromise replication fork progression and genome integrity in all cells. The overall impacts of naturally occurring RNA:DNA hybrids on genome integrity, and the relative contributions of ribonucleases H to mitigating the negative effects of hybrids, remain unknown. Here, we investigate the contributions of RNases HII (RnhB) and HIII (RnhC) to hybrid removal, DNA replication, and mutagenesis genome wide. Deletion of either rnhB or rnhC triggers RNA:DNA hybrid accumulation but with distinct patterns of mutagenesis and hybrid accumulation. Across all cells, hybrids accumulate strongly in noncoding RNAs and 5'-UTRs of coding sequences. For ΔrnhB, hybrids accumulate preferentially in untranslated regions and early in coding sequences. We show that hybrid accumulation is particularly sensitive to gene expression in ΔrnhC cells. DNA replication in ΔrnhC cells is disrupted, leading to transversions and structural variation. Our results resolve the outstanding question of how hybrids in native genomic contexts cause mutagenesis and shape genome organization.

Bacillus subtilis encodes a discrete flap endonuclease that cleaves RNA-DNA hybrids.

The current model for Okazaki fragment maturation in bacteria invokes RNA cleavage by RNase H, followed by strand displacement synthesis and 5' RNA flap removal by DNA polymerase I (Pol I). RNA removal by Pol I is thought to occur through the 5'-3' flap endo/exonuclease (FEN) domain, located in the N-terminus of the protein. In addition to Pol I, many bacteria encode a second, Pol I-independent FEN. The contribution of Pol I and Pol I-independent FENs to DNA replication and genome stability remains unclear. In this work we purified Bacillus subtilis Pol I and FEN, then assayed these proteins on a variety of RNA-DNA hybrid and DNA-only substrates. We found that FEN is far more active than Pol I on nicked double-flap, 5' single flap, and nicked RNA-DNA hybrid substrates. We show that the 5' nuclease activity of B. subtilis Pol I is feeble, even during DNA synthesis when a 5' flapped substrate is formed modeling an Okazaki fragment intermediate. Examination of Pol I and FEN on DNA-only substrates shows that FEN is more active than Pol I on most substrates tested. Further experiments show that ΔpolA phenotypes are completely rescued by expressing the C-terminal polymerase domain while expression of the N-terminal 5' nuclease domain fails to complement ΔpolA. Cells lacking FEN (ΔfenA) show a phenotype in conjunction with an RNase HIII defect, providing genetic evidence for the involvement of FEN in Okazaki fragment processing. With these results, we propose a model where cells remove RNA primers using FEN while upstream Okazaki fragments are extended through synthesis by Pol I. Our model resembles Okazaki fragment processing in eukaryotes, where Pol δ catalyzes strand displacement synthesis followed by 5' flap cleavage using FEN-1. Together our work highlights the conservation of ordered steps for Okazaki fragment processing in cells ranging from bacteria to human.

RNase H genes cause distinct impacts on RNA:DNA hybrid formation and mutagenesis genome-wide.

RNA:DNA hybrids such as R-loops affect genome integrity and DNA replication fork progression. The overall impacts of naturally occurring RNA:DNA hybrids on genome integrity, and the relative contributions of ribonucleases H to mitigating the negative effects of hybrids, remain unknown. Here, we investigate the contributions of RNases HII (RnhB) and HIII (RnhC) to hybrid removal, DNA replication, and mutagenesis genome-wide. Deletion of either rnhB or rnhC triggers RNA:DNA hybrid accumulation, but with distinct patterns of mutagenesis and hybrid accumulation. Across all cells, hybrids accumulate most strongly in non-coding RNAs and 5'-UTRs of coding sequences. For Δ rnhB , hybrids accumulate preferentially in untranslated regions and early in coding sequences. Hybrid accumulation is particularly sensitive to gene expression in Δ rnhC ; in cells lacking RnhC, DNA replication is disrupted leading to transversions and structural variation. Our results resolve the outstanding question of how hybrids in native genomic contexts interact with replication to cause mutagenesis and shape genome organization.

Diverse Mechanisms of Helicase Loading during DNA Replication Initiation in Bacteria.

Initiation of DNA replication is required for cell viability and passage of genetic information to the next generation. Studies in Escherichia coli and Bacillus subtilis have established ATPases associated with diverse cellular activities (AAA+) as essential proteins required for loading of the replicative helicase at replication origins. AAA+ ATPases DnaC in E. coli and DnaI in B. subtilis have long been considered the paradigm for helicase loading during replication in bacteria. Recently, it has become increasingly clear that most bacteria lack DnaC/DnaI homologs. Instead, most bacteria express a protein homologous to the newly described DciA (dnaC/dnaI antecedent) protein. DciA is not an ATPase, and yet it serves as a helicase operator, providing a function analogous to that of DnaC and DnaI across diverse bacterial species. The recent discovery of DciA and of other alternative mechanisms of helicase loading in bacteria has changed our understanding of DNA replication initiation. In this review, we highlight recent discoveries, detailing what is currently known about the replicative helicase loading process across bacterial species, and we discuss the critical questions that remain to be investigated.

Green synthesis of silica and silicon from agricultural residue sugarcane bagasse ash - a mini review.

Silicon dioxide (SiO2), also known as silica, has received attention in recent years due to wide range of capable applications including biomedical/pharmaceutical, energy, food, and personal care products. This has accelerated research in the extraction of materials from various agricultural wastes; this review investigates the extraction of silica and silicon nanoparticles from sugarcane bagasse ash with potential applications in electronic devices. Specific properties of silica have attracted the interest of researchers, which include surface area, size, biocompatibility, and high functionality. The production of silica from industrial agricultural waste exhibits sustainability and potential reduction in waste production. Bagasse is sustainable and environmentally friendly; though considered waste, it could be a helpful component for sustainable progress and further technological advancement. The chemical, biogenic and green synthesis are discussed in detail for the production of silica. In green synthesis, notable attempts have been made to replace toxic counterparts and decrease energy usage with the same quantity and quality of silica obtained. Methods of reducing silica to silicon are also discussed with the potential application-specific properties in electronic devices, and modern technological applications, such as batteries, supercapacitors, and solar cells.

DNA Methylation and RNA-DNA Hybrids Regulate the Single-Molecule Localization of a DNA Methyltransferase on the Bacterial Nucleoid.

Bacterial DNA methyltransferases (MTases) function in restriction modification systems, cell cycle control, and the regulation of gene expression. DnmA is a recently described DNA MTase that forms N6-methyladenosine at nonpalindromic 5'-GACGAG-3' sites in Bacillus subtilis, yet how DnmA activity is regulated is unknown. To address DnmA regulation, we tested substrate binding in vitro and found that DnmA binds poorly to methylated DNA and to an RNA-DNA hybrid with the DNA recognition sequence. Further, DnmA variants with amino acid substitutions that disrupt cognate sequence recognition or catalysis also bind poorly to DNA. Using superresolution fluorescence microscopy and single-molecule tracking of DnmA-PAmCherry, we characterized the subcellular DnmA diffusion and detected its preferential localization to the replisome region and the nucleoid. Under conditions where the chromosome is highly methylated, upon RNA-DNA hybrid accumulation, or with a DnmA variant with severely limited DNA binding activity, DnmA is excluded from the nucleoid, demonstrating that prior methylation or accumulation of RNA-DNA hybrids regulates the association of DnmA with the chromosome in vivo. Furthermore, despite the high percentage of methylated recognition sites and the proximity to putative endonuclease genes conserved across bacterial species, we find that DnmA fails to protect B. subtilis against phage predation, suggesting that DnmA is functionally an orphan MTase involved in regulating gene expression. Our work explores the regulation of a bacterial DNA MTase and identifies prior methylation and RNA-DNA hybrids as regulators of MTase localization. These MTase regulatory features could be common across biology. IMPORTANCE DNA methyltransferases (MTases) influence gene expression, cell cycle control, and host defense through DNA modification. Predicted MTases are pervasive across bacterial genomes, but the vast majority remain uncharacterized. Here, we show that in the soil microorganism Bacillus subtilis, the DNA MTase dnmA and neighboring genes are remnants of a phage defense system that no longer protects against phage predation. This result suggests that portions of the bacterial methylome may originate from inactive restriction modification systems that have maintained methylation activity. Analysis of DnmA movement in vivo shows that active DnmA localizes in the nucleoid, suggesting that DnmA can search for recognition sequences throughout the nucleoid region with some preference for the replisome. Our results further show that prior DNA methylation and RNA-DNA hybrids regulate DnmA dynamics and nucleoid localization, providing new insight into how DNA methylation is coordinated within the cellular environment.

Machine learning multi-omics analysis reveals cancer driver dysregulation in pan-cancer cell lines compared to primary tumors.

Cancer cell lines have been widely used for decades to study biological processes driving cancer development, and to identify biomarkers of response to therapeutic agents. Advances in genomic sequencing have made possible large-scale genomic characterizations of collections of cancer cell lines and primary tumors, such as the Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA). These studies allow for the first time a comprehensive evaluation of the comparability of cancer cell lines and primary tumors on the genomic and proteomic level. Here we employ bulk mRNA and micro-RNA sequencing data from thousands of samples in CCLE and TCGA, and proteomic data from partner studies in the MD Anderson Cell Line Project (MCLP) and The Cancer Proteome Atlas (TCPA), to characterize the extent to which cancer cell lines recapitulate tumors. We identify dysregulation of a long non-coding RNA and microRNA regulatory network in cancer cell lines, associated with differential expression between cell lines and primary tumors in four key cancer driver pathways: KRAS signaling, NFKB signaling, IL2/STAT5 signaling and TP53 signaling. Our results emphasize the necessity for careful interpretation of cancer cell line experiments, particularly with respect to therapeutic treatments targeting these important cancer pathways.

The Impact of RNA-DNA Hybrids on Genome Integrity in Bacteria.

During the essential processes of DNA replication and transcription, RNA-DNA hybrid intermediates are formed that pose significant risks to genome integrity when left unresolved. To manage RNA-DNA hybrids, all cells rely on RNase H family enzymes that specifically cleave the RNA portion of the many different types of hybrids that form in vivo. Recent experimental advances have provided new insight into how RNA-DNA hybrids form and the consequences to genome integrity that ensue when persistent hybrids remain unresolved. Here we review the types of RNA-DNA hybrids, including R-loops, RNA primers, and ribonucleotide misincorporations, that form during DNA replication and transcription and discuss how each type of hybrid can contribute to genome instability in bacteria. Further, we discuss how bacterial RNase HI, HII, and HIII and bacterial FEN enzymes contribute to genome maintenance through the resolution of hybrids.

Structure and kinase activity of bacterial cell cycle regulator CcrZ.

CcrZ is a recently discovered cell cycle regulator that connects DNA replication initiation with cell division in pneumococci and may have a similar function in related bacteria. CcrZ is also annotated as a putative kinase, suggesting that CcrZ homologs could represent a novel family of bacterial kinase-dependent cell cycle regulators. Here, we investigate the CcrZ homolog in Bacillus subtilis and show that cells lacking ccrZ are sensitive to a broad range of DNA damage. We demonstrate that increased expression of ccrZ results in over-initiation of DNA replication. In addition, increased expression of CcrZ activates the DNA damage response. Using sensitivity to DNA damage as a proxy, we show that the negative regulator for replication initiation (yabA) and ccrZ function in the same pathway. We show that CcrZ interacts with replication initiation proteins DnaA and DnaB, further suggesting that CcrZ is important for replication timing. To understand how CcrZ functions, we solved the crystal structure bound to AMP-PNP to 2.6 Å resolution. The CcrZ structure most closely resembles choline kinases, consisting of a bilobal structure with a cleft between the two lobes for binding ATP and substrate. Inspection of the structure reveals a major restructuring of the substrate-binding site of CcrZ relative to the choline-binding pocket of choline kinases, consistent with our inability to detect activity with choline for this protein. Instead, CcrZ shows activity on D-ribose and 2-deoxy-D-ribose, indicating adaptation of the choline kinase fold in CcrZ to phosphorylate a novel substrate. We show that integrity of the kinase active site is required for ATPase activity in vitro and for function in vivo. This work provides structural, biochemical, and functional insight into a newly identified, and conserved group of bacterial kinases that regulate DNA replication initiation.

Genome Editing Methods for Bacillus subtilis.

Bacillus subtilis is a widely studied Gram-positive bacterium that serves as an important model for understanding processes critical for several areas of biology including biotechnology and human health. B. subtilis has several advantages as a model organism: it is easily grown under laboratory conditions, it has a rapid doubling time, it is relatively inexpensive to maintain, and it is nonpathogenic. Over the last 50 years, advancements in genetic engineering have continued to make B. subtilis a genetic workhorse in scientific discovery. In this chapter, we describe methods for traditional gene disruptions, use of gene deletion libraries from the Bacillus Genetic Stock Center, allelic exchange, CRISPRi, and CRISPR/Cas9. Additionally, we provide general materials and equipment needed, strengths and limitations, time considerations, and troubleshooting notes to perform each method. Use of the methods outlined in this chapter will allow researchers to create gene insertions, deletions, substitutions, and RNA interference strains through a variety of methods custom to each application.

Bacterial DNA excision repair pathways.

Bacteria are continuously exposed to numerous endogenous and exogenous DNA-damaging agents. To maintain genome integrity and ensure cell survival, bacteria have evolved several DNA repair pathways to correct different types of DNA damage and non-canonical bases, including strand breaks, nucleotide modifications, cross-links, mismatches and ribonucleotide incorporations. Recent advances in genome-wide screens, the availability of thousands of whole-genome sequences and advances in structural biology have enabled the rapid discovery and characterization of novel bacterial DNA repair pathways and new enzymatic activities. In this Review, we discuss recent advances in our understanding of base excision repair and nucleotide excision repair, and we discuss several new repair processes including the EndoMS mismatch correction pathway and the MrfAB excision repair system.

The Bacillus subtilis PriA Winged Helix Domain Is Critical for Surviving DNA Damage.

DNA replication forks regularly encounter lesions or other impediments that result in a blockage to fork progression. PriA is one of the key proteins used by virtually all eubacteria to survive conditions that result in a blockage to replication fork movement. PriA directly binds stalled replication forks and initiates fork restart allowing for chromosomes to be fully duplicated under stressful conditions. We used a CRISPR-Cas gene editing approach to map PriA residues critical for surviving DNA damage induced by several antibiotics in B. subtilis. We find that the winged helix (WH) domain in B. subtilis PriA is critical for surviving DNA damage and participates in DNA binding. The important in vivo function of the WH domain mapped to distinct surfaces that were also conserved among several Gram-positive human pathogens. In addition, we identified an amino acid linker neighboring the WH domain that is greatly extended in B. subtilis due to an insertion. Shortening this linker induced a hypersensitive phenotype to DNA damage, suggesting that its extended length is critical for efficient replication fork restart in vivo. Because the WH domain is dispensable in E. coli PriA, our findings demonstrate an important difference in the contribution of the WH domain during fork restart in B. subtilis. Furthermore, with our results we suggest that this highly variable region in PriA could provide different functions across diverse bacterial organisms. IMPORTANCE PriA is an important protein found in virtually all bacteria that recognizes stalled replication forks orchestrating fork restart. PriA homologs contain a winged helix (WH) domain. The E. coli PriA WH domain is dispensable and functions in a fork restart pathway that is not conserved outside of E. coli and closely related proteobacteria. We analyzed the importance of the WH domain and an associated linker in B. subtilis and found that both are critical for surviving DNA damage. This function mapped to a small motif at the C-terminal end of the WH domain, which is also conserved in pathogenic bacteria. The motif was not required for DNA binding and therefore may perform a novel function in the replication fork restart pathway.

Distinct heterochromatin-like domains promote transcriptional memory and silence parasitic genetic elements in bacteria.

There is increasing evidence that prokaryotes maintain chromosome structure, which in turn impacts gene expression. We recently characterized densely occupied, multi-kilobase regions in the E. coli genome that are transcriptionally silent, similar to eukaryotic heterochromatin. These extended protein occupancy domains (EPODs) span genomic regions containing genes encoding metabolic pathways as well as parasitic elements such as prophages. Here, we investigate the contributions of nucleoid-associated proteins (NAPs) to the structuring of these domains, by examining the impacts of deleting NAPs on EPODs genome-wide in E. coli and B. subtilis. We identify key NAPs contributing to the silencing of specific EPODs, whose deletion opens a chromosomal region for RNA polymerase binding at genes contained within that region. We show that changes in E. coli EPODs facilitate an extra layer of transcriptional regulation, which prepares cells for exposure to exotic carbon sources. Furthermore, we distinguish novel xenogeneic silencing roles for the NAPs Fis and Hfq, with the presence of at least one being essential for cell viability in the presence of domesticated prophages. Our findings reveal previously unrecognized mechanisms through which genomic architecture primes bacteria for changing metabolic environments and silences harmful genomic elements.

A Functional Precision Medicine Pipeline Combines Comparative Transcriptomics and Tumor Organoid Modeling to Identify Bespoke Treatment Strategies for Glioblastoma.

Li Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in TP53. TP53 is the most common mutated gene in human cancer, occurring in 30-50% of glioblastomas (GBM). Here, we highlight a precision medicine platform to identify potential targets for a GBM patient with LFS. We used a comparative transcriptomics approach to identify genes that are uniquely overexpressed in the LFS GBM patient relative to a cancer compendium of 12,747 tumor RNA sequencing data sets, including 200 GBMs. STAT1 and STAT2 were identified as being significantly overexpressed in the LFS patient, indicating ruxolitinib, a Janus kinase 1 and 2 inhibitors, as a potential therapy. The LFS patient had the highest level of STAT1 and STAT2 expression in an institutional high-grade glioma cohort of 45 patients, further supporting the cancer compendium results. To empirically validate the comparative transcriptomics pipeline, we used a combination of adherent and organoid cell culture techniques, including ex vivo patient-derived organoids (PDOs) from four patient-derived cell lines, including the LFS patient. STAT1 and STAT2 expression levels in the four patient-derived cells correlated with levels identified in the respective parent tumors. In both adherent and organoid cultures, cells from the LFS patient were among the most sensitive to ruxolitinib compared to patient-derived cells with lower STAT1 and STAT2 expression levels. A spheroid-based drug screening assay (3D-PREDICT) was performed and used to identify further therapeutic targets. Two targeted therapies were selected for the patient of interest and resulted in radiographic disease stability. This manuscript supports the use of comparative transcriptomics to identify personalized therapeutic targets in a functional precision medicine platform for malignant brain tumors.