The impact of AI on research.

Large and complex datasets have made artificial intelligence (AI) an invaluable tool for discovery across biological research. We asked experts how AI has impacted their work. Their experiences and perspectives offer thoughtful insights into potential offered by AI for their fields.

Self-Reporting Transposons Enable Simultaneous Readout of Gene Expression and Transcription Factor Binding in Single Cells.

Cellular heterogeneity confounds in situ assays of transcription factor (TF) binding. Single-cell RNA sequencing (scRNA-seq) deconvolves cell types from gene expression, but no technology links cell identity to TF binding sites (TFBS) in those cell types. We present self-reporting transposons (SRTs) and use them in single-cell calling cards (scCC), a novel assay for simultaneously measuring gene expression and mapping TFBS in single cells. The genomic locations of SRTs are recovered from mRNA, and SRTs deposited by exogenous, TF-transposase fusions can be used to map TFBS. We then present scCC, which map SRTs from scRNA-seq libraries, simultaneously identifying cell types and TFBS in those same cells. We benchmark multiple TFs with this technique. Next, we use scCC to discover BRD4-mediated cell-state transitions in K562 cells. Finally, we map BRD4 binding sites in the mouse cortex at single-cell resolution, establishing a new method for studying TF biology in situ.

CellNet: network biology applied to stem cell engineering.

Somatic cell reprogramming, directed differentiation of pluripotent stem cells, and direct conversions between differentiated cell lineages represent powerful approaches to engineer cells for research and regenerative medicine. We have developed CellNet, a network biology platform that more accurately assesses the fidelity of cellular engineering than existing methodologies and generates hypotheses for improving cell derivations. Analyzing expression data from 56 published reports, we found that cells derived via directed differentiation more closely resemble their in vivo counterparts than products of direct conversion, as reflected by the establishment of target cell-type gene regulatory networks (GRNs). Furthermore, we discovered that directly converted cells fail to adequately silence expression programs of the starting population and that the establishment of unintended GRNs is common to virtually every cellular engineering paradigm. CellNet provides a platform for quantifying how closely engineered cell populations resemble their target cell type and a rational strategy to guide enhanced cellular engineering.

Dissecting engineered cell types and enhancing cell fate conversion via CellNet.

Engineering clinically relevant cells in vitro holds promise for regenerative medicine, but most protocols fail to faithfully recapitulate target cell properties. To address this, we developed CellNet, a network biology platform that determines whether engineered cells are equivalent to their target tissues, diagnoses aberrant gene regulatory networks, and prioritizes candidate transcriptional regulators to enhance engineered conversions. Using CellNet, we improved B cell to macrophage conversion, transcriptionally and functionally, by knocking down predicted B cell regulators. Analyzing conversion of fibroblasts to induced hepatocytes (iHeps), CellNet revealed an unexpected intestinal program regulated by the master regulator Cdx2. We observed long-term functional engraftment of mouse colon by iHeps, thereby establishing their broader potential as endoderm progenitors and demonstrating direct conversion of fibroblasts into intestinal epithelium. Our studies illustrate how CellNet can be employed to improve direct conversion and to uncover unappreciated properties of engineered cells.

Dissecting cell identity via network inference and in silico gene perturbation.

Cell identity is governed by the complex regulation of gene expression, represented as gene-regulatory networks1. Here we use gene-regulatory networks inferred from single-cell multi-omics data to perform in silico transcription factor perturbations, simulating the consequent changes in cell identity using only unperturbed wild-type data. We apply this machine-learning-based approach, CellOracle, to well-established paradigms-mouse and human haematopoiesis, and zebrafish embryogenesis-and we correctly model reported changes in phenotype that occur as a result of transcription factor perturbation. Through systematic in silico transcription factor perturbation in the developing zebrafish, we simulate and experimentally validate a previously unreported phenotype that results from the loss of noto, an established notochord regulator. Furthermore, we identify an axial mesoderm regulator, lhx1a. Together, these results show that CellOracle can be used to analyse the regulation of cell identity by transcription factors, and can provide mechanistic insights into development and differentiation.

Stem cell dynamics and cellular heterogeneity across lineage subtypes of castrate-resistant prostate cancer.

To resist lineage-dependent therapies such as androgen receptor inhibition, prostate luminal epithelial adenocarcinoma cells often adopt a stem-like state resulting in lineage plasticity and phenotypic heterogeneity. Castrate-resistant prostate adenocarcinoma can transition to neuroendocrine (NE) and occasionally to amphicrine, co-expressed luminal and NE, phenotypes. We developed castrate-resistant prostate cancer (CRPC) patient-derived organoid models that preserve heterogeneity of the originating tumor, including an amphicrine model displaying a range of luminal and NE phenotypes. To gain biological insight and to identify potential treatment targets within heterogeneous tumor cell populations, we assessed the lineage hierarchy and molecular characteristics of various CRPC tumor subpopulations. Transcriptionally similar stem/progenitor (St/Pr) cells were identified for all lineage populations. Lineage tracing in amphicrine CRPC showed that heterogeneity originated from distinct subclones of infrequent St/Pr cells that produced mainly quiescent differentiated amphicrine progeny. By contrast, adenocarcinoma CRPC progeny originated from St/Pr cells and self-renewing differentiated luminal cells. Neuroendocrine prostate cancer (NEPC) was composed almost exclusively of self-renewing St/Pr cells. Amphicrine subpopulations were enriched for secretory luminal, mesenchymal, and enzalutamide treatment persistent signatures that characterize clinical progression. Finally, the amphicrine St/Pr subpopulation was specifically depleted with an AURKA inhibitor, which blocked tumor growth. These data illuminate distinct stem cell (SC) characteristics for subtype-specific CRPC in addition to demonstrating a context for targeting differentiation-competent prostate SCs.

Single-cell mapping of lineage and identity in direct reprogramming.

Direct lineage reprogramming involves the conversion of cellular identity. Single-cell technologies are useful for deconstructing the considerable heterogeneity that emerges during lineage conversion. However, lineage relationships are typically lost during cell processing, complicating trajectory reconstruction. Here we present 'CellTagging', a combinatorial cell-indexing methodology that enables parallel capture of clonal history and cell identity, in which sequential rounds of cell labelling enable the construction of multi-level lineage trees. CellTagging and longitudinal tracking of fibroblast to induced endoderm progenitor reprogramming reveals two distinct trajectories: one leading to successfully reprogrammed cells, and one leading to a 'dead-end' state, paths determined in the earliest stages of lineage conversion. We find that expression of a putative methyltransferase, Mettl7a1, is associated with the successful reprogramming trajectory; adding Mettl7a1 to the reprogramming cocktail increases the yield of induced endoderm progenitors. Together, these results demonstrate the utility of our lineage-tracing method for revealing the dynamics of direct reprogramming.

Chemical Stability of Lorazepam Oral Solution Repackaged in Plastic Oral Syringes at Room and Refrigerated Temperature.

Purpose: Generic lorazepam oral solution is supplied in a 30 mL multi-dose bottle requiring protection from light and refrigeration, with a beyond use date of 90 days once the bottle is opened. The repackaging of 1 mL doses of lorazepam oral solution into oral syringes allows for facilitated dispensing, yet no available data supports repackaging and storing lorazepam oral solution in syringes. The validation and application of a stability-indicating high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method for the quantification of lorazepam allowed for the determination of the stability of lorazepam oral solution when stored in oral syringes. Methods: A stability-indicating HPLC-UV method was developed for the quantification of lorazepam in oral solution. The method was validated using guidance from USP < 1225 >. For the stability investigation, 2 mg/mL lorazepam oral solution was aliquoted into clear plastic oral syringes in 1 mLmilliliter doses from 2 multi-dose stock bottles and randomly allocated for storage in room temperature or refrigerated environment. Baseline lorazepam concentrations were measured on the day the study was initiated and designated as 100% initial concentration samples. Subsequent samples were analyzed in triplicate at time points of 24, 48, and 96 hours and 7, 10, 14, 21, 30, and 60 days. Results: The calibration curves on three non-consecutive days met the linearity criteria of R 2 > 0.99. Inter- day and intra-day precision and accuracy (percent relative standard deviation and percent error) were ≤2% over three days. During the stability investigation, percent initial concentration of lorazepam from room and refrigerated syringes remained above 90% for the duration of the study. Conclusion: The stability-indicating HPLC-UV method was successfully applied to the investigation of lorazepam oral solution stability when stored in syringes at room and refrigerated temperatures. The emergent need for use of lorazepam concentrate for inpatients and the restrictions of how the medication is supplied necessitated a need for the evaluation of repackaging into unit dose syringes for immediate availability from automated dispensing cabinets. Lorazepam oral solution stored in clear plastic syringes maintained greater than 90% initial concentration at both room and refrigerated temperatures for 60 days.

The association between levator plate integrity and pelvic floor defaecatory dysfunction.

AIMS: Levator ani deficiency has been implicated in anterior pelvic floor pathology but its association with pelvic floor defaecatory dysfunction is less clear. The aim was to examine the relationship of levator ani deficiency with anatomical abnormalities (rectocoele, intussusception, enterocoele, perineal descent) and patient symptoms (bowel, vagina) in patients with pelvic floor defaecatory dysfunction. METHODS: The prospective observational case series of 223 women presenting to a tertiary colorectal pelvic floor unit with defaecatory dysfunction. Each underwent assessment with symptom severity and quality of life (QoL) scores, integrated total pelvic floor ultrasound (PFUS) (transvaginal, transperineal) and defaecation proctography (DP). Rectocoele, intussusception, enterocoele and perineal descent were assessed on both. Levator ani deficiency was scored using endovaginal ultrasound (score 0-18; mild [0-6], moderate [>6-12], severe [>12-18]). RESULTS: The proportion of patients with rectocoele, enterocoele, and intussusception increased with increasing levator ani damage (mild, moderate, severe). There was a weakly positive correlation between size of rectocoele and levator ani deficiency. On PFUS, there was a weakly positive correlation between severity of intussusception and enterocoele with levator ani deficiency. On DP, there was a weakly positive correlation between perineal descent and levator ani deficiency. There was no association between bowel symptom and QoL scores and levator ani deficiency. Vaginal symptoms were associated with levator ani deficiency. CONCLUSIONS: Anatomical abnormalities which are implicated in pelvic floor defaecatory dysfunction (rectocoele, intussusception, enterocoele, perineal descent) were associated with worsening levator ani deficiency. There was no association between bowel symptoms and levator ani deficiency. Vaginal symptoms were associated with levator ani deficiency.

Pelvic floor imaging in asymptomatic subjects.

AIM: The aim of this work was to determine the range of normal imaging features during total pelvic floor ultrasound (TPFUS) (transperineal, transvaginal, endovaginal and endoanal) and defaecation MRI (dMRI). METHOD: Twenty asymptomatic female volunteers (mean age 36.5 years) were prospectively investigated with dMRI and TPFUS. Subjects were screened with symptom questionnaires (ICIQ-B, St Mark's faecal incontinence score, obstructed defaecation syndrome score, ICIQ-V, BSAQ). dMRI and TPFUS were performed and interpreted by blinded clinicians according to previously published methods. RESULTS: The subjects comprised six parous and 14 nulliparous women, of whom three were postmenopausal. There were three with a rectocoele on both modalities and one with a rectocoele on dMRI only. There was one with intussusception on TPFUS. Two had an enterocoele on both modalities and one on TPFUS only. There were six with a cystocoele on both modalities, one on dMRI only and one on TPFUS only. On dMRI, there were 12 with functional features. Four also displayed functional features on TPFUS. Two displayed functional features on TPFUS only. CONCLUSION: This study demonstrates the presence of abnormal findings on dMRI and TPFUS without symptoms. There was a high rate of functional features on dMRI. This series is not large enough to redefine normal parameters but is helpful for appreciating the wide range of findings seen in health.

The evolving concept of cell identity in the single cell era.

Fueled by recent advances in single cell biology, we are moving away from qualitative and undersampled assessments of cell identity, toward building quantitative, high-resolution cell atlases. However, it remains challenging to precisely define cell identity, leading to renewed debate surrounding this concept. Here, I present three pillars that I propose are central to the notion of cell identity: phenotype, lineage and state. I explore emerging technologies that are enabling the systematic and unbiased quantification of these properties, and outline how these efforts will enable the construction of a high-resolution, dynamic landscape of cell identity, potentially revealing its underlying molecular regulation to provide new opportunities for understanding and manipulating cell fate.

Is there any association between symptoms and findings on imaging in pelvic floor defaecatory dysfunction? A prospective study.

AIM: To compare features on imaging (integrated total pelvic floor ultrasound (transperineal, transvaginal) and defaecation proctography) with bowel, bladder and vaginal symptoms in pelvic floor defaecatory dysfunction. METHOD: A prospective observational case series of 216 symptomatic women who underwent symptom severity scoring (bowel, bladder and vaginal), integrated total pelvic floor ultrasound and defaecation proctography. Anatomical (rectocele, intussusception, enterocele, cystocele) and functional (co-ordination, evacuation) features were examined. RESULTS: Irrespective of imaging modality, patients with a rectocele had higher International Consultation on Incontinence Modular Questionnaire - Vaginal Symptoms (ICIQ-VS) scores than patients without. On integrated total pelvic floor ultrasound, ICIQ-VS quality of life scores were higher in those with a rectocele. There was a higher International Consultation on Incontinence Modular Questionnaire - Bowel Symptoms (ICIQ-BS) bowel pattern score in those with a rectocele, and a lower ICIQ-BS bowel pattern and sexual impact score in those with intussusception. Poor co-ordination was associated with increased ICIQ-BS bowel control scores and obstructed defaecation symptom scores. On defaecation proctography, ICIQ-VS symptom scores were lower in patients with poor co-ordination. CONCLUSION: Patients with a rectocele on either imaging modality may have qualitative vaginal symptoms on assessment. In patients with bowel symptoms but no vaginal symptoms, it is not possible to predict which anatomical abnormalities will be present on imaging.

Direct lineage reprogramming via pioneer factors; a detour through developmental gene regulatory networks.

Although many approaches have been employed to generate defined fate in vitro, the resultant cells often appear developmentally immature or incompletely specified, limiting their utility. Growing evidence suggests that current methods of direct lineage conversion may rely on the transition through a developmental intermediate. Here, I hypothesize that complete conversion between cell fates is more probable and feasible via reversion to a developmentally immature state. I posit that this is due to the role of pioneer transcription factors in engaging silent, unmarked chromatin and activating hierarchical gene regulatory networks responsible for embryonic patterning. Understanding these developmental contexts will be essential for the precise engineering of cell identity.

Sediment exchange to mitigate pollutant exposure in urban soil.

Urban soil is an ongoing source for lead (Pb) and other pollutant exposure. Sources of clean soil that are locally-available, abundant and inexpensive are needed to place a protective cover layer over degraded urban soil to eliminate direct and indirect pollutant exposures. This study evaluates a novel sediment exchange program recently established in New York City (NYC Clean Soil Bank, CSB) and found that direct exchange of surplus sediment extracted from urban construction projects satisfies these criteria. The CSB has high total yield with 4.2 × 105 t of sediment exchanged in five years. Average annual yield (8.5 × 104 t yr-1) would be sufficient to place a 15-cm (6-in.) sediment cover layer over 3.2 × 105 m2 (80 acres) of impacted urban soil or 1380 community gardens. In a case study of sediment exchange to mitigate community garden soil contamination, Pb content in sediment ranged from 2 to 5 mg kg-1. This sediment would reduce surface Pb concentrations more than 98% if it was used to encapsulate soil with Pb content exceeding USEPA residential soil standards (400 mg kg-1). The maximum observed sediment Pb content is a factor of 42 and 71 lower than median surface soil and garden soil in NYC, respectively. All costs (transportation, chemical testing, etc.) in the CSB are paid by the donor indicating that urban sediment exchange could be an ultra-low-cost source for urban soil mitigation. Urban-scale sediment exchange has advantages over existing national- or provincial-scale sediment exchanges because it can retain and upcycle local sediment resources to attain their highest and best use (e.g. lowering pollutant exposure), achieve circular urban materials metabolism, improve livability and maximize urban sustainability.

What was hot at ICS 2016?

The 46th annual conference of the International Continence Society was held in Tokyo, Japan, between September 13th and 16th, 2016. In this article, we present selected highlights of the broad range of excellent research presented by colleagues from around the world from a variety of areas of continence research, from cellular models to population-based epidemiological studies.

Making a firm decision: multifaceted regulation of cell fate in the early mouse embryo.

The preimplantation mammalian embryo offers a striking opportunity to address the question of how and why apparently identical cells take on separate fates. Two cell fate decisions are taken before the embryo implants; these decisions set apart a group of pluripotent cells, progenitors for the future body, from the distinct extraembryonic lineages of trophectoderm and primitive endoderm. New molecular, cellular and developmental insights reveal the interplay of transcriptional regulation, epigenetic modifications, cell position and cell polarity in these two fate decisions in the mouse. We discuss how mechanisms proposed in previously distinct models might work in concert to progressively reinforce cell fate decisions through feedback loops.