Antigen-specific B cells direct T follicular-like helper cells into lymphoid follicles to mediate Mycobacterium tuberculosis control.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a global cause of death. Granuloma-associated lymphoid tissue (GrALT) correlates with protection during TB, but the mechanisms of protection are not understood. During TB, the transcription factor IRF4 in T cells but not B cells is required for the generation of the TH1 and TH17 subsets of helper T cells and follicular helper T (TFH)-like cellular responses. A population of IRF4+ T cells coexpress the transcription factor BCL6 during Mtb infection, and deletion of Bcl6 (Bcl6fl/fl) in CD4+ T cells (CD4cre) resulted in reduction of TFH-like cells, impaired localization within GrALT and increased Mtb burden. In contrast, the absence of germinal center B cells, MHC class II expression on B cells, antibody-producing plasma cells or interleukin-10-expressing B cells, did not increase Mtb susceptibility. Indeed, antigen-specific B cells enhance cytokine production and strategically localize TFH-like cells within GrALT via interactions between programmed cell death 1 (PD-1) and its ligand PD-L1 and mediate Mtb control in both mice and macaques.

Molecular insights into the activation of Mre11-Rad50 endonuclease activity by Sae2/CtIP.

In Saccharomyces cerevisiae (S. cerevisiae), Mre11-Rad50-Xrs2 (MRX)-Sae2 nuclease activity is required for the resection of DNA breaks with secondary structures or protein blocks, while in humans, the MRE11-RAD50-NBS1 (MRN) homolog with CtIP is needed to initiate DNA end resection of all breaks. Phosphorylated Sae2/CtIP stimulates the endonuclease activity of MRX/N. Structural insights into the activation of the Mre11 nuclease are available only for organisms lacking Sae2/CtIP, so little is known about how Sae2/CtIP activates the nuclease ensemble. Here, we uncover the mechanism of Mre11 activation by Sae2 using a combination of AlphaFold2 structural modeling of biochemical and genetic assays. We show that Sae2 stabilizes the Mre11 nuclease in a conformation poised to cleave substrate DNA. Several designs of compensatory mutations establish how Sae2 activates MRX in vitro and in vivo, supporting the structural model. Finally, our study uncovers how human CtIP, despite considerable sequence divergence, employs a similar mechanism to activate MRN.

Mechanisms of Bacterial Transcription Termination: All Good Things Must End.

Transcript termination is essential for accurate gene expression and the removal of RNA polymerase (RNAP) at the ends of transcription units. In bacteria, two mechanisms are responsible for proper transcript termination: intrinsic termination and Rho-dependent termination. Intrinsic termination is mediated by signals directly encoded within the DNA template and nascent RNA, whereas Rho-dependent termination relies upon the adenosine triphosphate-dependent RNA translocase Rho, which binds nascent RNA and dissociates the elongation complex. Although significant progress has been made in understanding these pathways, fundamental details remain undetermined. Among those that remain unresolved are the existence of an inactivated intermediate in the intrinsic termination pathway, the role of Rho-RNAP interactions in Rho-dependent termination, and the mechanisms by which accessory factors and nucleoid-associated proteins affect termination. We describe current knowledge, discuss key outstanding questions, and highlight the importance of defining the structural rearrangements of RNAP that are involved in the two mechanisms of transcript termination.

CDK4/6 inhibitor-mediated cell overgrowth triggers osmotic and replication stress to promote senescence.

Abnormal increases in cell size are associated with senescence and cell cycle exit. The mechanisms by which overgrowth primes cells to withdraw from the cell cycle remain unknown. We address this question using CDK4/6 inhibitors, which arrest cells in G0/G1 and are licensed to treat advanced HR+/HER2- breast cancer. We demonstrate that CDK4/6-inhibited cells overgrow during G0/G1, causing p38/p53/p21-dependent cell cycle withdrawal. Cell cycle withdrawal is triggered by biphasic p21 induction. The first p21 wave is caused by osmotic stress, leading to p38- and size-dependent accumulation of p21. CDK4/6 inhibitor washout results in some cells entering S-phase. Overgrown cells experience replication stress, resulting in a second p21 wave that promotes cell cycle withdrawal from G2 or the subsequent G1. We propose that the levels of p21 integrate signals from overgrowth-triggered stresses to determine cell fate. This model explains how hypertrophy can drive senescence and why CDK4/6 inhibitors have long-lasting effects in patients.

Companies inadvertently fund online misinformation despite consumer backlash.

The financial motivation to earn advertising revenue has been widely conjectured to be pivotal for the production of online misinformation1-4. Research aimed at mitigating misinformation has so far focused on interventions at the user level5-8, with little emphasis on how the supply of misinformation can itself be countered. Here we show how online misinformation is largely financed by advertising, examine how financing misinformation affects the companies involved, and outline interventions for reducing the financing of misinformation. First, we find that advertising on websites that publish misinformation is pervasive for companies across several industries and is amplified by digital advertising platforms that algorithmically distribute advertising across the web. Using an information-provision experiment9, we find that companies that advertise on websites that publish misinformation can face substantial backlash from their consumers. To examine why misinformation continues to be monetized despite the potential backlash for the advertisers involved, we survey decision-makers at companies. We find that most decision-makers are unaware that their companies' advertising appears on misinformation websites but have a strong preference to avoid doing so. Moreover, those who are unaware and uncertain about their company's role in financing misinformation increase their demand for a platform-based solution to reduce monetizing misinformation when informed about how platforms amplify advertising placement on misinformation websites. We identify low-cost, scalable information-based interventions to reduce the financial incentive to misinform and counter the supply of misinformation online.

Mechanism of BRCA1-BARD1 function in DNA end resection and DNA protection.

DNA double-strand break (DSB) repair by homologous recombination is initiated by DNA end resection, a process involving the controlled degradation of the 5'-terminated strands at DSB sites1,2. The breast cancer suppressor BRCA1-BARD1 not only promotes resection and homologous recombination, but it also protects DNA upon replication stress1,3-9. BRCA1-BARD1 counteracts the anti-resection and pro-non-homologous end-joining factor 53BP1, but whether it functions in resection directly has been unclear10-16. Using purified recombinant proteins, we show here that BRCA1-BARD1 directly promotes long-range DNA end resection pathways catalysed by the EXO1 or DNA2 nucleases. In the DNA2-dependent pathway, BRCA1-BARD1 stimulates DNA unwinding by the Werner or Bloom helicase. Together with MRE11-RAD50-NBS1 and phosphorylated CtIP, BRCA1-BARD1 forms the BRCA1-C complex17,18, which stimulates resection synergistically to an even greater extent. A mutation in phosphorylated CtIP (S327A), which disrupts its binding to the BRCT repeats of BRCA1 and hence the integrity of the BRCA1-C complex19-21, inhibits resection, showing that BRCA1-C is a functionally integrated ensemble. Whereas BRCA1-BARD1 stimulates resection in DSB repair, it paradoxically also protects replication forks from unscheduled degradation upon stress, which involves a homologous recombination-independent function of the recombinase RAD51 (refs. 4-6,8). We show that in the presence of RAD51, BRCA1-BARD1 instead inhibits DNA degradation. On the basis of our data, the presence and local concentration of RAD51 might determine the balance between the pronuclease and the DNA protection functions of BRCA1-BARD1 in various physiological contexts.

Prevention of respiratory virus transmission by resident memory CD8+ T cells.

An ideal vaccine both attenuates virus growth and disease in infected individuals and reduces the spread of infections in the population, thereby generating herd immunity. Although this strategy has proved successful by generating humoral immunity to measles, yellow fever and polio, many respiratory viruses evolve to evade pre-existing antibodies1. One approach for improving the breadth of antiviral immunity against escape variants is through the generation of memory T cells in the respiratory tract, which are positioned to respond rapidly to respiratory virus infections2-6. However, it is unknown whether memory T cells alone can effectively surveil the respiratory tract to the extent that they eliminate or greatly reduce viral transmission following exposure of an individual to infection. Here we use a mouse model of natural parainfluenza virus transmission to quantify the extent to which memory CD8+ T cells resident in the respiratory tract can provide herd immunity by reducing both the susceptibility of acquiring infection and the extent of transmission, even in the absence of virus-specific antibodies. We demonstrate that protection by resident memory CD8+ T cells requires the antiviral cytokine interferon-γ (IFNγ) and leads to altered transcriptional programming of epithelial cells within the respiratory tract. These results suggest that tissue-resident CD8+ T cells in the respiratory tract can have important roles in protecting the host against viral disease and limiting viral spread throughout the population.

Double-stranded DNA binding function of RAD51 in DNA protection and its regulation by BRCA2.

RAD51 and the breast cancer suppressor BRCA2 have critical functions in DNA double-strand (dsDNA) break repair by homologous recombination and the protection of newly replicated DNA from nucleolytic degradation. The recombination function of RAD51 requires its binding to single-stranded DNA (ssDNA), whereas binding to dsDNA is inhibitory. Using reconstituted MRE11-, EXO1-, and DNA2-dependent nuclease reactions, we show that the protective function of RAD51 unexpectedly depends on its binding to dsDNA. The BRC4 repeat of BRCA2 abrogates RAD51 binding to dsDNA and accordingly impairs the function of RAD51 in protection. The BRCA2 C-terminal RAD51-binding segment (TR2) acts in a dominant manner to overcome the effect of BRC4. Mechanistically, TR2 stabilizes RAD51 binding to dsDNA, even in the presence of BRC4, promoting DNA protection. Our data suggest that RAD51's dsDNA-binding capacity may have evolved together with its function in replication fork protection and provide a mechanistic basis for the DNA-protection function of BRCA2.

Recurrent repeat expansions in human cancer genomes.

Expansion of a single repetitive DNA sequence, termed a tandem repeat (TR), is known to cause more than 50 diseases1,2. However, repeat expansions are often not explored beyond neurological and neurodegenerative disorders. In some cancers, mutations accumulate in short tracts of TRs, a phenomenon termed microsatellite instability; however, larger repeat expansions have not been systematically analysed in cancer3-8. Here we identified TR expansions in 2,622 cancer genomes spanning 29 cancer types. In seven cancer types, we found 160 recurrent repeat expansions (rREs), most of which (155/160) were subtype specific. We found that rREs were non-uniformly distributed in the genome with enrichment near candidate cis-regulatory elements, suggesting a potential role in gene regulation. One rRE, a GAAA-repeat expansion, located near a regulatory element in the first intron of UGT2B7 was detected in 34% of renal cell carcinoma samples and was validated by long-read DNA sequencing. Moreover, in preliminary experiments, treating cells that harbour this rRE with a GAAA-targeting molecule led to a dose-dependent decrease in cell proliferation. Overall, our results suggest that rREs may be an important but unexplored source of genetic variation in human cancer, and we provide a comprehensive catalogue for further study.

Dopamine facilitates associative memory encoding in the entorhinal cortex.

Mounting evidence shows that dopamine in the striatum is critically involved in reward-based reinforcement learning1,2. However, it remains unclear how dopamine reward signals influence the entorhinal-hippocampal circuit, another brain network that is crucial for learning and memory3-5. Here, using cell-type-specific electrophysiological recording6, we show that dopamine signals from the ventral tegmental area and substantia nigra control the encoding of cue-reward association rules in layer 2a fan cells of the lateral entorhinal cortex (LEC). When mice learned novel olfactory cue-reward associations using a pre-learned association rule, spike representations of LEC fan cells grouped newly learned rewarded cues with a pre-learned rewarded cue, but separated them from a pre-learned unrewarded cue. Optogenetic inhibition of fan cells impaired the learning of new associations while sparing the retrieval of pre-learned memory. Using fibre photometry, we found that dopamine sends novelty-induced reward expectation signals to the LEC. Inhibition of LEC dopamine signals disrupted the associative encoding of fan cells and impaired learning performance. These results suggest that LEC fan cells represent a cognitive map of abstract task rules, and that LEC dopamine facilitates the incorporation of new memories into this map.

Disruption of Telomerase RNA Maturation Kinetics Precipitates Disease.

Mutations in RNA-processing enzymes are increasingly linked to human disease. Telomerase RNA and related noncoding RNAs require 3' end-processing steps, including oligoadenylation. Germline mutations in poly(A)ribonuclease (PARN) cause accumulation of extended human telomerase RNA (hTR) species and precipitate dyskeratosis congenita and pulmonary fibrosis. Here, we develop nascent RNAend-seq to measure processing rates of RNA precursors. We find that mature hTR derives from extended precursors but that in PARN-mutant cells hTR maturation kinetically stalls and unprocessed precursors are degraded. Loss of poly(A)polymerase PAPD5 in PARN-mutant cells accelerates hTR maturation and restores hTR processing, indicating that oligoadenylation and deadenylation set rates of hTR maturation. The H/ACA domain mediates hTR maturation by precisely defining the 3' end, recruiting poly(A)polymerase activity, and conferring sensitivity to PARN regulation. These data reveal a feedforward circuit in which post-transcriptional oligoadenylation controls RNA maturation kinetics. Similar alterations in RNA processing rates may contribute to mechanisms of RNA-based human disease.

The GATA transcriptional program dictates cell fate equilibrium to establish the maternal-fetal exchange interface and fetal development.

The placenta establishes a maternal-fetal exchange interface to transport nutrients and gases between the mother and the fetus. Establishment of this exchange interface relies on the development of multinucleated syncytiotrophoblasts (SynT) from trophoblast progenitors, and defect in SynT development often leads to pregnancy failure and impaired embryonic development. Here, we show that mouse embryos with conditional deletion of transcription factors GATA2 and GATA3 in labyrinth trophoblast progenitors (LaTPs) have underdeveloped placenta and die by ~embryonic day 9.5. Single-cell RNA sequencing analysis revealed excessive accumulation of multipotent LaTPs upon conditional deletion of GATA factors. The GATA factor-deleted multipotent progenitors were unable to differentiate into matured SynTs. We also show that the GATA factor-mediated priming of trophoblast progenitors for SynT differentiation is a conserved event during human placentation. Loss of either GATA2 or GATA3 in cytotrophoblast-derived human trophoblast stem cells (human TSCs) drastically inhibits SynT differentiation potential. Identification of GATA2 and GATA3 target genes along with comparative bioinformatics analyses revealed that GATA factors directly regulate hundreds of common genes in human TSCs, including genes that are essential for SynT development and implicated in preeclampsia and fetal growth retardation. Thus, our study uncovers a conserved molecular mechanism, in which coordinated function of GATA2 and GATA3 promotes trophoblast progenitor-to-SynT commitment, ensuring establishment of the maternal-fetal exchange interface.

Schizophrenia risk-associated SNPs affect expression of microRNA 137 host gene: a postmortem study.

Common variants in the MicroRNA 137 host gene MIR137HG and its adjacent gene DPYD have been associated with schizophrenia risk and the latest Psychiatric Genomics Consortium (PGC). Genome-Wide Association Study on schizophrenia has confirmed and extended these findings. To elucidate the association of schizophrenia risk-associated SNPs in this genomic region, we examined the expression of both mature and immature transcripts of the miR-137 host gene (MIR137HG) in the dorsolateral prefrontal cortex (DLPFC) and subgenual anterior cingulate cortex (sgACC) of postmortem brain samples of donors with schizophrenia and psychiatrically-unaffected controls using qPCR and RNA-Seq approaches. No differential expression of miR-137, MIR137HG, or its transcripts was observed. Two schizophrenia risk-associated SNPs identified in the PGC study, rs11165917 (DLPFC: P = 2.0e-16; sgACC: P = 6.4e-10) and rs4274102 (DLPFC: P = 0.036; sgACC: P = 0.002), were associated with expression of the MIR137HG long non-coding RNA transcript MIR137HG-203 (ENST00000602672.2) in individuals of European ancestry. Carriers of the minor (risk) allele of rs11165917 had significantly lower expression of MIR137HG-203 compared with those carrying the major allele. However, we were unable to validate this result by short-read sequencing of RNA extracted from DLPFC or sgACC tissue. This finding suggests that immature transcripts of MIR137HG may contribute to genetic risk for schizophrenia.

Mice expressing nonpolymerizable fibrinogen have reduced arterial and venous thrombosis with preserved hemostasis.

ABSTRACT: Elevated circulating fibrinogen levels correlate with increased risk for both cardiovascular and venous thromboembolic diseases. In vitro studies show that formation of a highly dense fibrin matrix is a major determinant of clot structure and stability. Here, we analyzed the impact of nonpolymerizable fibrinogen on arterial and venous thrombosis as well as hemostasis in vivo using FgaEK mice that express normal levels of a fibrinogen that cannot be cleaved by thrombin. In a model of carotid artery thrombosis, FgaWT/EK and FgaEK/EK mice were protected from occlusion with 4% ferric chloride (FeCl3) challenges compared with wild-type (FgaWT/WT) mice, but this protection was lost, with injuries driven by higher concentrations of FeCl3. In contrast, fibrinogen-deficient (Fga-/-) mice showed no evidence of occlusion, even with high-concentration FeCl3 challenge. Fibrinogen-dependent platelet aggregation and intraplatelet fibrinogen content were similar in FgaWT/WT, FgaWT/EK, and FgaEK/EK mice, consistent with preserved fibrinogen-platelet interactions that support arterial thrombosis with severe challenge. In an inferior vena cava stasis model of venous thrombosis, FgaEK/EK mice had near complete protection from thrombus formation. FgaWT/EK mice also displayed reduced thrombus incidence and a significant reduction in thrombus mass relative to FgaWT/WT mice after inferior vena cava stasis, suggesting that partial expression of nonpolymerizable fibrinogen was sufficient for conferring protection. Notably, FgaWT/EK and FgaEK/EK mice had preserved hemostasis in multiple models as well as normal wound healing times after skin incision, unlike Fga-/- mice that displayed significant bleeding and delayed healing. These findings indicate that a nonpolymerizable fibrinogen variant can significantly suppress occlusive thrombosis while preserving hemostatic potential in vivo.

A CRISPR activation screen identifies FBXO22 supporting targeted protein degradation.

Targeted protein degradation (TPD) represents a potent chemical biology paradigm that leverages the cellular degradation machinery to pharmacologically eliminate specific proteins of interest. Although multiple E3 ligases have been discovered to facilitate TPD, there exists a compelling requirement to diversify the pool of E3 ligases available for such applications. Here we describe a clustered regularly interspaced short palindromic repeats (CRISPR)-based transcriptional activation screen focused on human E3 ligases, with the goal of identifying E3 ligases that can facilitate heterobifunctional compound-mediated target degradation. Through this approach, we identified a candidate proteolysis-targeting chimera (PROTAC), 22-SLF, that induces the degradation of FK506-binding protein 12 when the transcription of FBXO22 gene is activated. Subsequent mechanistic investigations revealed that 22-SLF interacts with C227 and/or C228 in F-box protein 22 (FBXO22) to achieve target degradation. Lastly, we demonstrated the versatility of FBXO22-based PROTACs by effectively degrading additional endogenous proteins, including bromodomain-containing protein 4 and the echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion protein.

ADAR-mediated regulation of PQM-1 expression in neurons impacts gene expression throughout C. elegans and regulates survival from hypoxia.

The ability to alter gene expression programs in response to changes in environmental conditions is central to the ability of an organism to thrive. For most organisms, the nervous system serves as the master regulator in communicating information about the animal's surroundings to other tissues. The information relay centers on signaling pathways that cue transcription factors in a given cell type to execute a specific gene expression program, but also provide a means to signal between tissues. The transcription factor PQM-1 is an important mediator of the insulin signaling pathway contributing to longevity and the stress response as well as impacting survival from hypoxia. Herein, we reveal a novel mechanism for regulating PQM-1 expression specifically in neural cells of larval animals. Our studies reveal that the RNA-binding protein (RBP), ADR-1, binds to pqm-1 mRNA in neural cells. This binding is regulated by the presence of a second RBP, ADR-2, which when absent leads to reduced expression of both pqm-1 and downstream PQM-1 activated genes. Interestingly, we find that neural pqm-1 expression is sufficient to impact gene expression throughout the animal and affect survival from hypoxia, phenotypes that we also observe in adr mutant animals. Together, these studies reveal an important posttranscriptional gene regulatory mechanism in Caenorhabditis elegans that allows the nervous system to sense and respond to environmental conditions to promote organismal survival from hypoxia.

Regulation of the MLH1-MLH3 endonuclease in meiosis.

During prophase of the first meiotic division, cells deliberately break their DNA1. These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables the reciprocal exchange of DNA segments between homologous chromosomes2. A pathway that depends on the MLH1-MLH3 (MutLγ) nuclease has been implicated in the biased processing of meiotic recombination intermediates into crossovers by an unknown mechanism3-7. Here we have biochemically reconstituted key elements of this pro-crossover pathway. We show that human MSH4-MSH5 (MutSγ), which supports crossing over8, binds branched recombination intermediates and associates with MutLγ, stabilizing the ensemble at joint molecule structures and adjacent double-stranded DNA. MutSγ directly stimulates DNA cleavage by the MutLγ endonuclease. MutLγ activity is further stimulated by EXO1, but only when MutSγ is present. Replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) are additional components of the nuclease ensemble, thereby triggering crossing-over. Saccharomyces cerevisiae strains in which MutLγ cannot interact with PCNA present defects in forming crossovers. Finally, the MutLγ-MutSγ-EXO1-RFC-PCNA nuclease ensemble preferentially cleaves DNA with Holliday junctions, but shows no canonical resolvase activity. Instead, it probably processes meiotic recombination intermediates by nicking double-stranded DNA adjacent to the junction points9. As DNA nicking by MutLγ depends on its co-factors, the asymmetric distribution of MutSγ and RFC-PCNA on meiotic recombination intermediates may drive biased DNA cleavage. This mode of MutLγ nuclease activation might explain crossover-specific processing of Holliday junctions or their precursors in meiotic chromosomes4.

Climate change framing and innovator attention: Evidence from an email field experiment.

Drawing the attention of innovators to climate change is important for green innovation. We report an email field experiment with MIT using messages about the impact of climate change to invite innovators (SBIR grantees) to apply to a technology competition. We vary our messages on the time frame and scale of the human cost of climate change across scientifically valid scenarios. Innovator attention (clicks) is sensitive to climate change messaging. These changes in clicks also predict higher application rates. The response varies by individual characteristics such as location-based exposure to climate change risks and whether innovators have climate-related innovations. Finally, using a structural model of innovator attention, we provide estimates of the implied discount rate of time and the elasticity of attention to lives at stake.

Light-dependent signal transduction in the marine diatom Phaeodactylum tricornutum.

Unlike most higher plants, unicellular algae can acclimate to changes in irradiance on time scales of hours to a few days. The process involves an enigmatic signaling pathway originating in the plastid that leads to coordinated changes in plastid and nuclear gene expression. To deepen our understanding of this process, we conducted functional studies to examine how the model diatom, Phaeodactylum tricornutum, acclimates to low light and sought to identify the molecules responsible for the phenomenon. We show that two transformants with altered expression of two putative signal transduction molecules, a light-specific soluble kinase and a plastid transmembrane protein, that appears to be regulated by a long noncoding natural antisense transcript, arising from the opposite strand, are physiologically incapable of photoacclimation. Based on these results, we propose a working model of the retrograde feedback in the signaling and regulation of photoacclimation in a marine diatom.

Using evolutionary constraint to define novel candidate driver genes in medulloblastoma.

Current knowledge of cancer genomics remains biased against noncoding mutations. To systematically search for regulatory noncoding mutations, we assessed mutations in conserved positions in the genome under the assumption that these are more likely to be functional than mutations in positions with low conservation. To this end, we use whole-genome sequencing data from the International Cancer Genome Consortium and combined it with evolutionary constraint inferred from 240 mammals, to identify genes enriched in noncoding constraint mutations (NCCMs), mutations likely to be regulatory in nature. We compare medulloblastoma (MB), which is malignant, to pilocytic astrocytoma (PA), a primarily benign tumor, and find highly different NCCM frequencies between the two, in agreement with the fact that malignant cancers tend to have more mutations. In PA, a high NCCM frequency only affects the BRAF locus, which is the most commonly mutated gene in PA. In contrast, in MB, >500 genes have high levels of NCCMs. Intriguingly, several loci with NCCMs in MB are associated with different ages of onset, such as the HOXB cluster in young MB patients. In adult patients, NCCMs occurred in, e.g., the WASF-2/AHDC1/FGR locus. One of these NCCMs led to increased expression of the SRC kinase FGR and augmented responsiveness of MB cells to dasatinib, a SRC kinase inhibitor. Our analysis thus points to different molecular pathways in different patient groups. These newly identified putative candidate driver mutations may aid in patient stratification in MB and could be valuable for future selection of personalized treatment options.