Long-molecule scars of backup DNA repair in BRCA1- and BRCA2-deficient cancers.

Homologous recombination (HR) deficiency is associated with DNA rearrangements and cytogenetic aberrations1. Paradoxically, the types of DNA rearrangements that are specifically associated with HR-deficient cancers only minimally affect chromosomal structure2. Here, to address this apparent contradiction, we combined genome-graph analysis of short-read whole-genome sequencing (WGS) profiles across thousands of tumours with deep linked-read WGS of 46 BRCA1- or BRCA2-mutant breast cancers. These data revealed a distinct class of HR-deficiency-enriched rearrangements called reciprocal pairs. Linked-read WGS showed that reciprocal pairs with identical rearrangement orientations gave rise to one of two distinct chromosomal outcomes, distinguishable only with long-molecule data. Whereas one (cis) outcome corresponded to the copying and pasting of a small segment to a distant site, a second (trans) outcome was a quasi-balanced translocation or multi-megabase inversion with substantial (10 kb) duplications at each junction. We propose an HR-independent replication-restart repair mechanism to explain the full spectrum of reciprocal pair outcomes. Linked-read WGS also identified single-strand annealing as a repair pathway that is specific to BRCA2 deficiency in human cancers. Integrating these features in a classifier improved discrimination between BRCA1- and BRCA2-deficient genomes. In conclusion, our data reveal classes of rearrangements that are specific to BRCA1 or BRCA2 deficiency as a source of cytogenetic aberrations in HR-deficient cells.

Xenoprotein engineering via synthetic libraries.

Chemical methods have enabled the total synthesis of protein molecules of ever-increasing size and complexity. However, methods to engineer synthetic proteins comprising noncanonical amino acids have not kept pace, even though this capability would be a distinct advantage of the total synthesis approach to protein science. In this work, we report a platform for protein engineering based on the screening of synthetic one-bead one-compound protein libraries. Screening throughput approaching that of cell surface display was achieved by a combination of magnetic bead enrichment, flow cytometry analysis of on-bead screens, and high-throughput MS/MS-based sequencing of identified active compounds. Direct screening of a synthetic protein library by these methods resulted in the de novo discovery of mirror-image miniprotein-based binders to a ∼150-kDa protein target, a task that would be difficult or impossible by other means.

Chimeras of Cell-Penetrating Peptides Demonstrate Synergistic Improvement in Antisense Efficacy.

Phosphorodiamidate morpholino oligonucleotides (PMOs) make up a promising class of therapeutics for genetic disease. PMOs designed for "exon skipping" must be internalized into cells, reach the nucleus, and act on pre-mRNA to mediate their effects. One tactic for improving PMO delivery and exon skipping is to covalently conjugate PMOs to cell-penetrating peptides (CPPs). Here, we report the synthesis of PMOs conjugated to CPP chimeras, constructed by combining multiple CPPs into one sequence. The chimeric CPPs synergistically improve PMO activity up to 70-fold compared to that of the PMO alone and beyond the expected effects of each component peptide. By investigating the design space of CPP chimeras, we demonstrate that all components must be covalently attached, that the order of the two sequences matters, and that peptide identity can tune activity. We identified one chimera (pVEC-Bpep) to investigate in more detail and found that it engages mechanisms of endocytosis different from those of its parent peptides. We also examined the extent to which the beneficial effect comes from improved cellular uptake as opposed to the downstream steps required for exon skipping. Given the complexity of intracellular delivery, we anticipate this work will lead researchers to consider combining molecules with different physicochemical properties to aid in the delivery of biologic cargoes.

Mendelian Randomization Analysis Supports Causal Effect of Type II Diabetes Mellitus on Onychomycosis.

Introduction: Onychomycosis is common among adults with diabetes mellitus (DM). We used two-sample Mendelian randomization to estimate the causal effect of genetic risk for DM on onychomycosis and tinea skin infections in the All of Us Research Program. Methods: Onychomycosis and tinea corporis, pedis, manus, and cruris cases were identified using electronic health record data, and genetic instrument variables and summary statistics were collected from a type II DM (T2DM) genome-wide association study (GWAS) meta-analysis. Results: Inverse variance weighted regression showed positive effect of T2DM genetic risk on onychomycosis (beta = 0.135, p = 1.86E-2), and weighted median regression produced a comparable estimate of effect size (beta = 0.148). There was no significant effect of T2DM on skin dermatophytosis. Conclusions: Our results suggest that T2DM has a positive causal effect on onychomycosis but not tinea skin infection risk. As onychomycosis may impair occupational function and increase risk for secondary soft tissue infections, patients with diabetes should be screened for onychomycosis and counseled on mitigating infection risk.

Onychogryphosis Is Associated with Dermatologic and Vascular Disease: A Case-Control Study of the All of Us Research Program.

Introduction: Onychogryphosis is a nail condition characterized clinically by a thickened, curved, yellow-brown, and opaque nail plate and may result in pain, paronychia, and onychogryphosis. Methods: We performed a nested case-control study of 1,114 onychogryphosis patients and 3,423 matched controls to quantify the association between onychogryphosis and self-care limitations, chronic foot injury, dermatologic conditions, and vascular disease. Results and Conclusion: Onychogryphosis was positively associated with increased age, activity limitations (difficulty running errands alone, bathing, and concentrating), psoriasis, onychomycosis, hallux malleus, hallux valgus, peripheral vascular disease, lower extremity ulcers, venous varices, and type II diabetes mellitus. Therefore, physicians should screen patients presenting with onychogryphosis for these conditions.

Risk Factors and Treatment Trends for Onychomycosis: A Case-Control Study of Onychomycosis Patients in the All of Us Research Program.

Introda significant: Onychomycosis is the most common nail disorder seen in clinical practice, and it may have significant impact on patient quality of life. Understanding risk factors for onychomycosis may help to devise screening and treatment guidelines for populations that are more susceptible to this infection. Using a national database, we aimed to explore associations between onychomycosis and age, sex, and underlying medical conditions, as well as to examine current onychomycosis treatment trends. Materials and Methods: We performed a nested, matched, case-control study of patients in the All of Us database aged ≥ 18 years (6 May 2018-1 January 2022). Onychomycosis cases were identified using International Classification of Diseases (ICD) and Systematized Nomenclature of Medicine (SNOMED) diagnostic codes (ICD-9 110.1, ICD-10 B35.1, SNOMED 414941008). Demographic information (i.e., age, sex, and race), treatments, and co-diagnoses for onychomycosis patients and case-controls were recorded. Wald's test applied to multivariate logistic regression was used to calculate odds ratios and p-values between onychomycosis and co-diagnoses. Additionally, 95% confidence intervals were calculated with a proportion test. Results: We included 15,760 onychomycosis patients and 47,280 matched controls. The mean age of onychomycosis patients was 64.9 years, with 54.2% female, 52.8% Non-Hispanic White, 23.0% Black, 17.8% Hispanic, and 6.3% other, which was similar to controls. Patients with onychomycosis vs. controls were more likely to have a co-diagnosis of obesity (46.4%, OR 2.59 [2.49-2.69]), tinea pedis (21.5%, OR 10.9 [10.1-11.6]), peripheral vascular disease (PVD) (14.4%, OR 3.04 [2.86-3.24]), venous insufficiency (13.4%, OR 3.38 [3.15-3.59]), venous varices (5.6%, OR 2.71 [2.47-2.97]), diabetes mellitus (5.6%, OR 3.28 [2.98-3.61]), and human immunodeficiency virus (HIV) (3.5%, OR 1.8 [1.61-2.00]) (p < 0.05, all). The most frequently prescribed oral and topical medications were terbinafine (20.9%) and ciclopirox (12.4%), respectively. The most common therapeutic procedure performed was debridement (19.3%). Over the study period, ciclopirox prescriptions (Spearman correlation 0.182, p = 0.0361) and fluconazole prescriptions increased (Spearman correlation 0.665, p = 2.44 × 10-4), and griseofulvin (Spearman correlation -0.557, p = 0.0131) and itraconazole prescriptions decreased (Spearman correlation -0.681, p = 3.32 × 10-6). Conclusions: Our study demonstrated that age, obesity, tinea pedis, PVD, venous insufficiency, diabetes mellitus, and HIV were significant risk factors for onychomycosis. In addition, the most frequent oral and topical onychomycosis medications prescribed were terbinafine and ciclopirox, likely reflective of efficacy and cost considerations. Identifying and managing these risk factors is essential to preventing onychomycosis' primary infections and recurrences and improving treatment efficacy.

Genomic subtypes of cutaneous melanoma have distinct metabolic profiles: A single-cell transcriptomic analysis.

OBJECTIVE: Genomic profiling previously classified melanoma into distinct subtypes based on the presence or absence of mutations in driver genes, but metabolic differences between and within these groups have yet to be thoroughly analyzed. Thus, the objective of the present study is to provide the first effort to holistically characterize the metabolic landscape of qualified melanoma genomic subtypes at single-cell resolution. METHODS: Expression data for a total of 1145 malignant cells sourced from NRAS(Q61L), BRAF(V600E), and NRAS/BRAF WT melanomas were retrieved from the Broad Single Cell Portal. Metabolic activity was interrogated by pathway scoring and gene set enrichment analysis. RESULTS: A total of 53 metabolic pathways were differentially regulated in at least one melanoma genomic subtype. Some notable findings include: BRAF/NRAS WT cells were enriched for fatty acid biosynthesis and depleted for metabolism of alanine, aspartate, and glutamate; BRAF(V600E) melanoma cells were enriched for beta-alanine metabolism and depleted for phenylalanine metabolism; NRAS(Q61L) melanoma cells were enriched for steroid biosynthesis and depleted for linoleic acid metabolism. CONCLUSION: Primary limitations include the total quantity of single cells and breadth of available genomic subtypes plus inherent noisiness of the applied methodologies. Nonetheless, these findings nominate novel, testable therapeutic targets.

SEACells infers transcriptional and epigenomic cellular states from single-cell genomics data.

Metacells are cell groupings derived from single-cell sequencing data that represent highly granular, distinct cell states. Here we present single-cell aggregation of cell states (SEACells), an algorithm for identifying metacells that overcome the sparsity of single-cell data while retaining heterogeneity obscured by traditional cell clustering. SEACells outperforms existing algorithms in identifying comprehensive, compact and well-separated metacells in both RNA and assay for transposase-accessible chromatin (ATAC) modalities across datasets with discrete cell types and continuous trajectories. We demonstrate the use of SEACells to improve gene-peak associations, compute ATAC gene scores and infer the activities of critical regulators during differentiation. Metacell-level analysis scales to large datasets and is particularly well suited for patient cohorts, where per-patient aggregation provides more robust units for data integration. We use our metacells to reveal expression dynamics and gradual reconfiguration of the chromatin landscape during hematopoietic differentiation and to uniquely identify CD4 T cell differentiation and activation states associated with disease onset and severity in a Coronavirus Disease 2019 (COVID-19) patient cohort.

Most large structural variants in cancer genomes can be detected without long reads.

Short-read sequencing is the workhorse of cancer genomics yet is thought to miss many structural variants (SVs), particularly large chromosomal alterations. To characterize missing SVs in short-read whole genomes, we analyzed 'loose ends'-local violations of mass balance between adjacent DNA segments. In the landscape of loose ends across 1,330 high-purity cancer whole genomes, most large (>10-kb) clonal SVs were fully resolved by short reads in the 87% of the human genome where copy number could be reliably measured. Some loose ends represent neotelomeres, which we propose as a hallmark of the alternative lengthening of telomeres phenotype. These pan-cancer findings were confirmed by long-molecule profiles of 38 breast cancer and melanoma cases. Our results indicate that aberrant homologous recombination is unlikely to drive the majority of large cancer SVs. Furthermore, analysis of mass balance in short-read whole genome data provides a surprisingly complete picture of cancer chromosomal structure.

Epigenetic plasticity cooperates with cell-cell interactions to direct pancreatic tumorigenesis.

The response to tumor-initiating inflammatory and genetic insults can vary among morphologically indistinguishable cells, suggesting as yet uncharacterized roles for epigenetic plasticity during early neoplasia. To investigate the origins and impact of such plasticity, we performed single-cell analyses on normal, inflamed, premalignant, and malignant tissues in autochthonous models of pancreatic cancer. We reproducibly identified heterogeneous cell states that are primed for diverse, late-emerging neoplastic fates and linked these to chromatin remodeling at cell-cell communication loci. Using an inference approach, we revealed signaling gene modules and tissue-level cross-talk, including a neoplasia-driving feedback loop between discrete epithelial and immune cell populations that was functionally validated in mice. Our results uncover a neoplasia-specific tissue-remodeling program that may be exploited for pancreatic cancer interception.

Lineage plasticity in prostate cancer depends on JAK/STAT inflammatory signaling.

Drug resistance in cancer is often linked to changes in tumor cell state or lineage, but the molecular mechanisms driving this plasticity remain unclear. Using murine organoid and genetically engineered mouse models, we investigated the causes of lineage plasticity in prostate cancer and its relationship to antiandrogen resistance. We found that plasticity initiates in an epithelial population defined by mixed luminal-basal phenotype and that it depends on increased Janus kinase (JAK) and fibroblast growth factor receptor (FGFR) activity. Organoid cultures from patients with castration-resistant disease harboring mixed-lineage cells reproduce the dependency observed in mice by up-regulating luminal gene expression upon JAK and FGFR inhibitor treatment. Single-cell analysis confirms the presence of mixed-lineage cells with increased JAK/STAT (signal transducer and activator of transcription) and FGFR signaling in a subset of patients with metastatic disease, with implications for stratifying patients for clinical trials.

Mapping the evolution of T cell states during response and resistance to adoptive cellular therapy.

To elucidate mechanisms by which T cells eliminate leukemia, we study donor lymphocyte infusion (DLI), an established immunotherapy for relapsed leukemia. We model T cell dynamics by integrating longitudinal, multimodal data from 94,517 bone marrow-derived single T cell transcriptomes in addition to chromatin accessibility and single T cell receptor sequencing from patients undergoing DLI. We find that responsive tumors are defined by enrichment of late-differentiated T cells before DLI and rapid, durable expansion of early differentiated T cells after treatment, highly similar to "terminal" and "precursor" exhausted subsets, respectively. Resistance, in contrast, is defined by heterogeneous T cell dysfunction. Surprisingly, early differentiated T cells in responders mainly originate from pre-existing and novel clonotypes recruited to the leukemic microenvironment, rather than the infusion. Our work provides a paradigm for analyzing longitudinal single-cell profiling of scenarios beyond adoptive cell therapy and introduces Symphony, a Bayesian approach to infer regulatory circuitry underlying T cell subsets, with broad relevance to exhaustion antagonists across cancers.

HyperTRIBE uncovers increased MUSASHI-2 RNA binding activity and differential regulation in leukemic stem cells.

The cell-context dependency for RNA binding proteins (RBPs) mediated control of stem cell fate remains to be defined. Here we adapt the HyperTRIBE method using an RBP fused to a Drosophila RNA editing enzyme (ADAR) to globally map the mRNA targets of the RBP MSI2 in mammalian adult normal and malignant stem cells. We reveal a unique MUSASHI-2 (MSI2) mRNA binding network in hematopoietic stem cells that changes during transition to multipotent progenitors. Additionally, we discover a significant increase in RNA binding activity of MSI2 in leukemic stem cells compared with normal hematopoietic stem and progenitor cells, resulting in selective regulation of MSI2's oncogenic targets. This provides a basis for MSI2 increased dependency in leukemia cells compared to normal cells. Moreover, our study provides a way to measure RBP function in rare cells and suggests that RBPs can achieve differential binding activity during cell state transition independent of gene expression.

Machine Learning To Predict Cell-Penetrating Peptides for Antisense Delivery.

Cell-penetrating peptides (CPPs) can facilitate the intracellular delivery of large therapeutically relevant molecules, including proteins and oligonucleotides. Although hundreds of CPP sequences are described in the literature, predicting efficacious sequences remains difficult. Here, we focus specifically on predicting CPPs for the delivery of phosphorodiamidate morpholino oligonucleotides (PMOs), a compelling type of antisense therapeutic that has recently been FDA approved for the treatment of Duchenne muscular dystrophy. Using literature CPP sequences, 64 covalent PMO-CPP conjugates were synthesized and evaluated in a fluorescence-based reporter assay for PMO activity. Significant discrepancies were observed between the sequences that performed well in this assay and the sequences that performed well when conjugated to only a small-molecule fluorophore. As a result, we envisioned that our PMO-CPP library would be a useful training set for a computational model to predict CPPs for PMO delivery. We used the PMO activity data to fit a random decision forest classifier to predict whether or not covalent attachment of a given peptide would enhance PMO activity at least 3-fold. To validate the model experimentally, seven novel sequences were generated, synthesized, and tested in the fluorescence reporter assay. All computationally predicted positive sequences were positive in the assay, and one sequence performed better than 80% of the tested literature CPPs. These results demonstrate the power of machine learning algorithms to identify peptide sequences with particular functions and illustrate the importance of tailoring a CPP sequence to the cargo of interest.

Optic Nerve Head Characteristics in Chronic Angle Closure Glaucoma Detected by Swept-Source OCT.

PURPOSE: To compare structural features in prelaminar and laminar tissues of the optic nerve head (ONH) in chronic angle closure glaucoma (CACG), primary open angle glaucoma (POAG), and control subjects. MATERIALS AND METHODS: ONH imaging was performed using swept-source optical coherence tomography (SS-OCT) for measurements of minimum rim width at Bruch's membrane opening (BMO-MRW), horizontal, and vertical lamina cribrosa depth (LCD). Prelaminar defects, categorized as hole and wedge, and lamina cribrosa (LC) defects were identified. Enhanced depth imaging spectral domain OCT (EDI-OCT) customized to perform high-resolution volume scans was used in conjunction to further characterize prelaminar holes. One eye per subject was analyzed. RESULTS: Eighty subjects (20 CACG, 40 POAG, and 20 controls) were included in the study. CACG and POAG groups had similar mean deviation on Humphrey visual field testing (-6.9 ± 5.1 vs. -6.3 ± 6.0 dB, p > 0.05) and IOP on the day of imaging (14.0 ± 3.1 vs. 13.8 ± 2.7 mmHg, p > 0.05). Thinnest and global BMO-MRW in CACG (120.3 ± 44.8, 225.5 ± 53.9 µm) and POAG (109.7 ± 56.3, 213.8 ± 59.7 µm) groups were lower than controls (200.1 ± 40.8, 308.3 ± 70.8 µm; p < 0.001 for both). Prelaminar holes were most frequent in CACG (65.0%) than POAG (25.0%, p=0.008) or control groups (20.0%, p=0.01). After adjusting for demographic and ophthalmic covariates, CACG was associated with increased odds of having prelaminar holes compared to POAG (odds ratio, 9.79; 95% CI, 2.12-45.19; p=0.003). Hole volume was similar between CACG and POAG (p > 0.05), but the CACG group had more holes per scan than POAG (maximum 2.5 ± 1.9 vs. 1.2 ± 0.4, p=0.02). Prelaminar wedge defects were less common in the CACG than the POAG group (5.0% vs. 37.5%, p=0.02). The CACG group did not differ from controls in laminar characteristics, such as LCD and LC defects. CONCLUSIONS: SS-OCT evaluation of the ONH revealed more frequent prelaminar holes in CACG compared to POAG and control patients.

Macrocyclization of Unprotected Peptide Isocyanates.

A chemistry for the facile two-component macrocyclization of unprotected peptide isocyanates is described. Starting from peptides containing two glutamic acid γ-hydrazide residues, isocyanates can be readily accessed and cyclized with hydrazides of dicarboxylic acids. The choice of a nucleophilic linker allows for the facile modulation of biochemical properties of a macrocyclic peptide. Four cyclic NYAD-1 analogues were synthesized using the described method and displayed a range of biological activities.

Divergent responses of viral and bacterial communities in the gut microbiome to dietary disturbances in mice.

To improve our understanding of the stability of mammalian intestinal communities, we characterized the responses of both bacterial and viral communities in murine fecal samples to dietary changes between high- and low-fat (LF) diets. Targeted DNA extraction methods for bacteria, virus-like particles and induced prophages were used to generate bacterial and viral metagenomes as well as 16S ribosomal RNA amplicons. Gut microbiome communities from two cohorts of C57BL/6 mice were characterized in a 6-week diet perturbation study in response to high fiber, LF and high-refined sugar, milkfat (MF) diets. The resulting metagenomes from induced bacterial prophages and extracellular viruses showed significant overlap, supporting a largely temperate viral lifestyle within these gut microbiomes. The resistance of baseline communities to dietary disturbances was evaluated, and we observed contrasting responses of baseline LF and MF bacterial and viral communities. In contrast to baseline LF viral communities and bacterial communities in both diet treatments, baseline MF viral communities were sensitive to dietary disturbances as reflected in their non-recovery during the washout period. The contrasting responses of bacterial and viral communities suggest that these communities can respond to perturbations independently of each other and highlight the potentially unique role of viruses in gut health.