Memory Sequencing Reveals Heritable Single-Cell Gene Expression Programs Associated with Distinct Cellular Behaviors.

Non-genetic factors can cause individual cells to fluctuate substantially in gene expression levels over time. It remains unclear whether these fluctuations can persist for much longer than the time of one cell division. Current methods for measuring gene expression in single cells mostly rely on single time point measurements, making the duration of gene expression fluctuations or cellular memory difficult to measure. Here, we combined Luria and Delbrück's fluctuation analysis with population-based RNA sequencing (MemorySeq) for identifying genes transcriptome-wide whose fluctuations persist for several divisions. MemorySeq revealed multiple gene modules that expressed together in rare cells within otherwise homogeneous clonal populations. These rare cell subpopulations were associated with biologically distinct behaviors like proliferation in the face of anti-cancer therapeutics. The identification of non-genetic, multigenerational fluctuations can reveal new forms of biological memory in single cells and suggests that non-genetic heritability of cellular state may be a quantitative property.

Corrigendum: Rare cell variability and drug-induced reprogramming as a mode of cancer drug resistance.

This corrects the article DOI: 10.1038/nature22794.

Rare cell variability and drug-induced reprogramming as a mode of cancer drug resistance.

Therapies that target signalling molecules that are mutated in cancers can often have substantial short-term effects, but the emergence of resistant cancer cells is a major barrier to full cures. Resistance can result from secondary mutations, but in other cases there is no clear genetic cause, raising the possibility of non-genetic rare cell variability. Here we show that human melanoma cells can display profound transcriptional variability at the single-cell level that predicts which cells will ultimately resist drug treatment. This variability involves infrequent, semi-coordinated transcription of a number of resistance markers at high levels in a very small percentage of cells. The addition of drug then induces epigenetic reprogramming in these cells, converting the transient transcriptional state to a stably resistant state. This reprogramming begins with a loss of SOX10-mediated differentiation followed by activation of new signalling pathways, partially mediated by the activity of the transcription factors JUN and/or AP-1 and TEAD. Our work reveals the multistage nature of the acquisition of drug resistance and provides a framework for understanding resistance dynamics in single cells. We find that other cell types also exhibit sporadic expression of many of these same marker genes, suggesting the existence of a general program in which expression is displayed in rare subpopulations of cells.

SAVER: gene expression recovery for single-cell RNA sequencing.

In single-cell RNA sequencing (scRNA-seq) studies, only a small fraction of the transcripts present in each cell are sequenced. This leads to unreliable quantification of genes with low or moderate expression, which hinders downstream analysis. To address this challenge, we developed SAVER (single-cell analysis via expression recovery), an expression recovery method for unique molecule index (UMI)-based scRNA-seq data that borrows information across genes and cells to provide accurate expression estimates for all genes.

Structural imprints in vivo decode RNA regulatory mechanisms.

Visualizing the physical basis for molecular behaviour inside living cells is a great challenge for biology. RNAs are central to biological regulation, and the ability of RNA to adopt specific structures intimately controls every step of the gene expression program. However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles include only two of the four nucleotides that make up RNA. Here we present a novel biochemical approach, in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE), which enables the first global view, to our knowledge, of RNA secondary structures in living cells for all four bases. icSHAPE of the mouse embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguish different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved from in vitro conditions, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA-protein interactions and N(6)-methyladenosine (m(6)A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.

Gene expression distribution deconvolution in single-cell RNA sequencing.

Single-cell RNA sequencing (scRNA-seq) enables the quantification of each gene's expression distribution across cells, thus allowing the assessment of the dispersion, nonzero fraction, and other aspects of its distribution beyond the mean. These statistical characterizations of the gene expression distribution are critical for understanding expression variation and for selecting marker genes for population heterogeneity. However, scRNA-seq data are noisy, with each cell typically sequenced at low coverage, thus making it difficult to infer properties of the gene expression distribution from raw counts. Based on a reexamination of nine public datasets, we propose a simple technical noise model for scRNA-seq data with unique molecular identifiers (UMI). We develop deconvolution of single-cell expression distribution (DESCEND), a method that deconvolves the true cross-cell gene expression distribution from observed scRNA-seq counts, leading to improved estimates of properties of the distribution such as dispersion and nonzero fraction. DESCEND can adjust for cell-level covariates such as cell size, cell cycle, and batch effects. DESCEND's noise model and estimation accuracy are further evaluated through comparisons to RNA FISH data, through data splitting and simulations and through its effectiveness in removing known batch effects. We demonstrate how DESCEND can clarify and improve downstream analyses such as finding differentially expressed genes, identifying cell types, and selecting differentiation markers.

Angiokeratoma-like purpuric palmar nodules following chemotherapy.

We describe a patient with leukemia undergoing chemotherapy who developed painful purpuric nodules of the digits. These findings were concerning for endocarditis (clinically) and angiokeratomas on gross histology. After extensive evaluation, we report the development of painful purpuric nodules as a likely side effect of the patient's therapeutic regimen (hydroxyurea, danorubicin, cytarabine, and methotrexate).

FPGA-Based High-Performance Embedded Systems for Adaptive Edge Computing in Cyber-Physical Systems: The ARTICo³ Framework.

Cyber-Physical Systems are experiencing a paradigm shift in which processing has been relocated to the distributed sensing layer and is no longer performed in a centralized manner. This approach, usually referred to as Edge Computing, demands the use of hardware platforms that are able to manage the steadily increasing requirements in computing performance, while keeping energy efficiency and the adaptability imposed by the interaction with the physical world. In this context, SRAM-based FPGAs and their inherent run-time reconfigurability, when coupled with smart power management strategies, are a suitable solution. However, they usually fail in user accessibility and ease of development. In this paper, an integrated framework to develop FPGA-based high-performance embedded systems for Edge Computing in Cyber-Physical Systems is presented. This framework provides a hardware-based processing architecture, an automated toolchain, and a runtime to transparently generate and manage reconfigurable systems from high-level system descriptions without additional user intervention. Moreover, it provides users with support for dynamically adapting the available computing resources to switch the working point of the architecture in a solution space defined by computing performance, energy consumption and fault tolerance. Results show that it is indeed possible to explore this solution space at run time and prove that the proposed framework is a competitive alternative to software-based edge computing platforms, being able to provide not only faster solutions, but also higher energy efficiency for computing-intensive algorithms with significant levels of data-level parallelism.

Defining the identity of mouse embryonic dermal fibroblasts.

Embryonic dermal fibroblasts in the skin have the exceptional ability to initiate hair follicle morphogenesis and contribute to scarless wound healing. Activation of the Wnt signaling pathway is critical for dermal fibroblast fate selection and hair follicle induction. In humans, mutations in Wnt pathway components and target genes lead to congenital focal dermal hypoplasias with diminished hair. The gene expression signature of embryonic dermal fibroblasts during differentiation and its dependence on Wnt signaling is unknown. Here we applied Shannon entropy analysis to identify the gene expression signature of mouse embryonic dermal fibroblasts. We used available human DNase-seq and histone modification ChiP-seq data on various cell-types to demonstrate that genes in the fibroblast cell identity signature can be epigenetically repressed in other cell-types. We found a subset of the signature genes whose expression is dependent on Wnt/ß-catenin activity in vivo. With our approach, we have defined and validated a statistically derived gene expression signature that may mediate dermal fibroblast identity and function in development and disease. genesis 54:415-430, 2016. © 2016 Wiley Periodicals, Inc.

RNA SHAPE analysis in living cells.

RNA structure has important roles in practically every facet of gene regulation, but the paucity of in vivo structural probes limits current understanding. Here we design, synthesize and demonstrate two new chemical probes that enable selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) in living cells. RNA structures in human, mouse, fly, yeast and bacterial cells are read out at single-nucleotide resolution, revealing tertiary contacts and RNA-protein interactions.

Self-attempted labioplasty with elastic bands resulting in severe necrosis.

BACKGROUND: Labial hypertrophy is protuberant labial tissue extending beyond the labia majora. Self-perception of poor cosmetic appearance is common in young patients and not necessarily pathologic. Labioplasty is indicated for patients with persistent symptoms including entrapment and painful intercourse. CASE: A 26-year-old woman presented with genital pain and foul odor after self-applying elastic bands to her labia minora. The bands were applied for a self-perceived abnormal appearance and lack of insurance for medical consultation. Surgical debridement and revision of the labia were performed using a straight vertical approach. CONCLUSIONS: Self-attempted labioplasty can result in necrosis and infection. Education and counseling of patients on the normal variants of labial anatomy and the recommended therapeutic methods will lead to better cosmetic results and prevent self-mutilation.

Incidence of Abnormal Findings During Pelvic Examinations in Women Aged 21-35 Years.

To describe the incidence of abnormal gynecologic examination findings in asymptomatic compared with symptomatic patients during preventive visits, we conducted a retrospective study of 1,121 visits for patients between the ages of 21 and 35 years from January 2017 to March 2017. Only 1.2% (95% CI, 0.5%,1.9%) of asymptomatic patients had abnormal findings on pelvic examination, compared with 32.4% (95% CI, 27.0%, 37.8%) of those with symptoms ( P ≤.001). In symptomatic patients, the most common symptoms were vaginal discharge (25.1%), pelvic pain (16.4%), and vaginal bleeding (15.7%). In asymptomatic patients, the most common findings were bacterial vaginosis and Candida infection. Asymptomatic patients presenting for a routine preventive visit have low rates of abnormalities detected on examination, and routine pelvic examinations should be re-considered.

Single-site laparoscopic management of a large adnexal mass.

INTRODUCTION: Single-site laparoscopy is gaining acceptance in many surgical fields including gynecology. The purpose of this report is to demonstrate the technique and outcome for removing a large adnexal mass through a single site. CASE DESCRIPTION: A 41-y-old female was referred to gynecology oncology for increased abdominal girth for 3 mo. An ultrasound confirmed a benign-appearing, 37-cm left adnexal mass. The mass was removed through a single-site laparoscopic incision with the aid of drainage and a morcellator. The operating time was 84 min. The patient was discharged 2 h and 35 min later with full return to normal activity in 5 d. CONCLUSION: Large, benign-appearing adnexal masses can be managed safely with superior cosmetic results using single-site laparoscopy.

A Retrospective Study of Implant Placement Lateral to the Inferior Alveolar Nerve (ILIAN) in Severely Atrophic Posterior Mandibular Ridges

Currently, there are several techniques being used in the posterior mandible to increase alveolar bone height and width. However, each of these has potential complications and limitations. The purpose of the current study was to present the surgical technique and restorative considerations for implant placement lateral to the inferior alveolar nerve (IAN) in cases of severely atrophic edentulous posterior mandibles. In the current study, 26 implants in 16 patients were successfully placed lateral to IAN and restored with splinted screw-retained prostheses with a follow-up time after loading ranging from 3 months to 6 years. Two patients reported complications. One patient had a temporary paresthesia that resolved 3 months after implant placement and the second patient had minor paresthesia which was reduced after implant removal but remained in a small area on the left corner of her lip.

Levels of Gynecologic Care: A Task Force Consensus Statement.

Systems of care have been established for obstetrics, trauma, and neonatology. An American College of Obstetricians and Gynecologists Presidential Task Force was established to develop a care system for gynecologic surgery. A group of experts who represent diverse perspectives in gynecologic practice proposed definitions of levels of gynecologic care using the Delphi method. The goal is to improve the quality of gynecologic surgical care performed in the United States by providing a framework of minimal institutional requirements for each level. Subgroups developed draft criteria for each level of care. The entire Task Force then met to reach consensus regarding the levels of care final definitions and parameters. The levels of gynecologic care framework focuses on systems of care by considering institutional resources and expertise, providing guidance on the provision of care in appropriate level facilities. These levels were defined by the ability to care for patients of increasing risk, complexity, and comorbidities, organizing gynecologic care around hospital capability. This framework can also be used to inform the escalation of care to appropriate facilities by identifying patients at risk and guiding them to facilities with the skills, expertise, and capabilities to safely and effectively meet their needs. The levels of gynecologic care framework is intended for use by patients, hospitals, and clinicians in the United States to guide where elective surgery can be done most safely and effectively by specialists and subspecialists in obstetrics and gynecology. The key features of the levels of gynecologic care include ensuring provision of risk-appropriate care and regionalization of care by facility capabilities.

Adolescent gynecology: special considerations for special patients.

Developments in the field of adolescent gynecology highlight the specific expertise and care required by this population. Given the ability to shape their future health choices, adolescents are a critical target for preventative health care. The approach to the evaluation and management of this unique population rests not only on the practitioner's adept ability to recognize the unique clinical challenges that may occur, but also rests on his/her understanding of these problems. Here, we review recent guidelines and practice patterns in the evaluation and management of issues in adolescent gynecology.

Using SRAM based FPGAs for power-aware high performance wireless sensor networks.

While for years traditional wireless sensor nodes have been based on ultra-low power microcontrollers with sufficient but limited computing power, the complexity and number of tasks of today's applications are constantly increasing. Increasing the node duty cycle is not feasible in all cases, so in many cases more computing power is required. This extra computing power may be achieved by either more powerful microcontrollers, though more power consumption or, in general, any solution capable of accelerating task execution. At this point, the use of hardware based, and in particular FPGA solutions, might appear as a candidate technology, since though power use is higher compared with lower power devices, execution time is reduced, so energy could be reduced overall. In order to demonstrate this, an innovative WSN node architecture is proposed. This architecture is based on a high performance high capacity state-of-the-art FPGA, which combines the advantages of the intrinsic acceleration provided by the parallelism of hardware devices, the use of partial reconfiguration capabilities, as well as a careful power-aware management system, to show that energy savings for certain higher-end applications can be achieved. Finally, comprehensive tests have been done to validate the platform in terms of performance and power consumption, to proof that better energy efficiency compared to processor based solutions can be achieved, for instance, when encryption is imposed by the application requirements.

Improving Free Gingival Graft Success Around Implants Using a Completed Implant Restoration.

Keratinized mucosa around implants is considered essential for maintaining peri-implant health. Clinicians may find it necessary to augment keratinized tissue after implant loading when complications arise. Immobilizing the graft can be challenging when there is a complete absence of attached gingiva or when the vestibule is shallow creating an opportunity for muscle forces to move the graft. To overcome these limitations, various stents have been created aimed at improving the stability of soft-tissue grafts around implants; however, many of these stents have drawbacks. This case report presents a novel approach for improving free gingival graft immobility and success around implants that utilizes a completed implant restoration.