Generation and molecular characterization of human pluripotent stem cell-derived pharyngeal foregut endoderm.

Approaches to study human pharyngeal foregut endoderm-a developmental intermediate that is linked to various human syndromes involving pharynx development and organogenesis of tissues such as thymus, parathyroid, and thyroid-have been hampered by scarcity of tissue access and cellular models. We present an efficient stepwise differentiation method to generate human pharyngeal foregut endoderm from pluripotent stem cells. We determine dose and temporal requirements of signaling pathway engagement for optimized differentiation and characterize the differentiation products on cellular and integrated molecular level. We present a computational classification tool, "CellMatch," and transcriptomic classification of differentiation products on an integrated mouse scRNA-seq developmental roadmap confirms cellular maturation. Integrated transcriptomic and chromatin analyses infer differentiation stage-specific gene regulatory networks. Our work provides the method and integrated multiomic resource for the investigation of disease-relevant loci and gene regulatory networks and their role in developmental defects affecting the pharyngeal endoderm and its derivatives.

Perioperative Breast Analgesia: a Systematic Review of the Evidence for Perioperative Analgesic Medications.

PURPOSE OF REVIEW: Breast surgery is common and may result in significant acute as well as chronic pain. A wide range of pharmacologic interventions is available including opioids, non-steroidal anti-inflammatory drugs (NSAIDs), N-methyl-D-aspartate (NMDA) receptor antagonists, anticonvulsants, and other non-opioids with analgesic properties. We present a review of the evidence for these pharmacologic interventions. A literature search of the MEDLINE database was performed via PubMed with combined terms related to breast surgery, anesthesia, and analgesia. Articles were limited to randomized controlled trial (RCT) design, adult patients undergoing elective surgery on the breast (not including biopsy), and pharmacologic interventions only. Article titles and abstracts were screened, and risk of bias assessments were performed. RECENT FINDINGS: The search strategy initially captured 7254 articles of which 60 articles met the full inclusion criteria. Articles were organized according to intervention: 6 opioid agonists, 14 NSAIDs and acetaminophen, 4 alpha-2 agonists, 7 NMDA receptor antagonists, 6 local anesthetics, 7 steroids, 15 anticonvulsants (one of which also discussed an NMDA antagonist), 1 antiarrhythmic, and 2 serotonin reuptake inhibitors (one of which also studied an anticonvulsant). A wide variety of medications is effective for perioperative breast analgesia, but results vary by agent and dose. The most efficacious are likely NSAIDs and anticonvulsants. Some agents may also decrease the incidence of chronic postoperative pain, including flurbiprofen, gabapentin, venlafaxine, and memantine. While many individual agents are well studied, optimal combinations of analgesic medications remain unclear.

Systematic evaluation of chromosome conformation capture assays.

Chromosome conformation capture (3C) assays are used to map chromatin interactions genome-wide. Chromatin interaction maps provide insights into the spatial organization of chromosomes and the mechanisms by which they fold. Hi-C and Micro-C are widely used 3C protocols that differ in key experimental parameters including cross-linking chemistry and chromatin fragmentation strategy. To understand how the choice of experimental protocol determines the ability to detect and quantify aspects of chromosome folding we have performed a systematic evaluation of 3C experimental parameters. We identified optimal protocol variants for either loop or compartment detection, optimizing fragment size and cross-linking chemistry. We used this knowledge to develop a greatly improved Hi-C protocol (Hi-C 3.0) that can detect both loops and compartments relatively effectively. In addition to providing benchmarked protocols, this work produced ultra-deep chromatin interaction maps using Micro-C, conventional Hi-C and Hi-C 3.0 for key cell lines used by the 4D Nucleome project.

Development of Microbiome Biobanks - Challenges and Opportunities.

The microbiome research field is rapidly evolving, but the required biobanking infrastructure is currently fragmented and not prepared for the biobanking of microbiomes. The rapid advancement of technologies requires an urgent assessment of how biobanks can underpin research by preserving microbiome samples and their functional potential.

Birdlike growth and mixed-age flocks in avimimids (Theropoda, Oviraptorosauria).

Avimimids were unusual, birdlike oviraptorosaurs from the Late Cretaceous of Asia. Initially enigmatic, new information has ameliorated the understanding of their anatomy, phylogenetic position, and behaviour. A monodominant bonebed from the Nemegt Formation of Mongolia showed that some avimimids were gregarious, but the site is unusual in the apparent absence of juveniles. Here, a second monodominant avimimid bonebed is described from the Iren Dabasu Formation of northern China. Elements recovered include numerous vertebrae and portions of the forelimbs and hindlimbs, representing a minimum of six individuals. Histological sampling of two tibiotarsi from the bonebed reveals rapid growth early in ontogeny followed by unexpectedly early onset of fusion and limited subsequent growth. This indicates that avimimids grew rapidly to adult size, like most extant birds but contrasting other small theropod dinosaurs. The combination of adults and juveniles in the Iren Dabasu bonebed assemblage provides evidence of mixed-age flocking in avimimids and the onset of fusion in young individuals suggests that some of the individuals in the Nemegt Formation bonebed may have been juveniles. Regardless, these individuals were likely functionally analogous to adults, and this probably facilitated mixed-age flocking by reducing ontogenetic niche shift in avimimids.

Diverse repertoire of human adipocyte subtypes develops from transcriptionally distinct mesenchymal progenitor cells.

Single-cell sequencing technologies have revealed an unexpectedly broad repertoire of cells required to mediate complex functions in multicellular organisms. Despite the multiple roles of adipose tissue in maintaining systemic metabolic homeostasis, adipocytes are thought to be largely homogenous with only 2 major subtypes recognized in humans so far. Here we report the existence and characteristics of 4 distinct human adipocyte subtypes, and of their respective mesenchymal progenitors. The phenotypes of these distinct adipocyte subtypes are differentially associated with key adipose tissue functions, including thermogenesis, lipid storage, and adipokine secretion. The transcriptomic signature of "brite/beige" thermogenic adipocytes reveals mechanisms for iron accumulation and protection from oxidative stress, necessary for mitochondrial biogenesis and respiration upon activation. Importantly, this signature is enriched in human supraclavicular adipose tissue, confirming that these cells comprise thermogenic depots in vivo, and explain previous findings of a rate-limiting role of iron in adipose tissue browning. The mesenchymal progenitors that give rise to beige/brite adipocytes express a unique set of cytokines and transcriptional regulators involved in immune cell modulation of adipose tissue browning. Unexpectedly, we also find adipocyte subtypes specialized for high-level expression of the adipokines adiponectin or leptin, associated with distinct transcription factors previously implicated in adipocyte differentiation. The finding of a broad adipocyte repertoire derived from a distinct set of mesenchymal progenitors, and of the transcriptional regulators that can control their development, provides a framework for understanding human adipose tissue function and role in metabolic disease.

Fungal biological resources to support international development: challenges and opportunities.

Exploitation of microbes, especially fungi, has the potential to help humankind meet the UN's sustainable development goals, help feed the worlds growing population and improve bioeconomies of poorer nations. The majority of the world's fungal genetic resources are held in collections in developed countries, primarily within the USA, Europe and Japan. Very little capacity exists in low to middle income countries, which are often rich in biodiversity but lack resources to be able to conserve and exploit their own microbial resources. In this paper we review the current challenges facing culture collections and the challenges of integrating new approaches, the worth of collaborative networks, and the importance of technology, taxonomy and data handling. We address the need to underpin research and development in developing countries through the need to build 'in country' infrastructure to address these challenges, whilst tackling the global challenges to meet the requirements of the research community through the impacts of legislation and the Nagoya protocol on access to biological resources.

MALDI-TOF MS Spectral Variation Is Observed between Fungal Samples Grown under Identical Conditions after Long-term Storage by Cryopreservation, Freeze-drying, and under Oil.

BACKGROUND: Matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) offers a powerful, versatile, and relatively-inexpensive tool for the characterization and/or identification of protein-containing samples. OBJECTIVE: In the case of filamentous fungi, significant variation in MALDI-TOF MS spectra can be observed for growth under different conditions on agar plates, as well as over time for growth on the same medium. We therefore sought to investigate the possibility of additional variance as a result of the preservation conditions prior to culturing. MATERIALS AND METHODS: We investigated Fusarium nygamai IMI 383003, previously preserved by liquid nitrogen cryopreservation, freeze-drying, and storage under oil. RESULTS: Significant spectral differences are observed as a function of preservation conditions prior to culturing on agar plates, under experimental conditions that show high reproducibility between replicate sample preparations and between replicate agar plates used for culturing. CONCLUSION: Storage and cryopreservation conditions can affect MALDI-TOF MS spectra.

Single-Cell RNA-Sequencing-Based CRISPRi Screening Resolves Molecular Drivers of Early Human Endoderm Development.

Studies in vertebrates have outlined conserved molecular control of definitive endoderm (END) development. However, recent work also shows that key molecular aspects of human END regulation differ even from rodents. Differentiation of human embryonic stem cells (ESCs) to END offers a tractable system to study the molecular basis of normal and defective human-specific END development. Here, we interrogated dynamics in chromatin accessibility during differentiation of ESCs to END, predicting DNA-binding proteins that may drive this cell fate transition. We then combined single-cell RNA-seq with parallel CRISPR perturbations to comprehensively define the loss-of-function phenotype of those factors in END development. Following a few candidates, we revealed distinct impairments in the differentiation trajectories for mediators of TGFß signaling and expose a role for the FOXA2 transcription factor in priming human END competence for human foregut and hepatic END specification. Together, this single-cell functional genomics study provides high-resolution insight on human END development.

Inferring population dynamics from single-cell RNA-sequencing time series data.

Recent single-cell RNA-sequencing studies have suggested that cells follow continuous transcriptomic trajectories in an asynchronous fashion during development. However, observations of cell flux along trajectories are confounded with population size effects in snapshot experiments and are therefore hard to interpret. In particular, changes in proliferation and death rates can be mistaken for cell flux. Here we present pseudodynamics, a mathematical framework that reconciles population dynamics with the concepts underlying developmental trajectories inferred from time-series single-cell data. Pseudodynamics models population distribution shifts across trajectories to quantify selection pressure, population expansion, and developmental potentials. Applying this model to time-resolved single-cell RNA-sequencing of T-cell and pancreatic beta cell maturation, we characterize proliferation and apoptosis rates and identify key developmental checkpoints, data inaccessible to existing approaches.

A Single-Cell Transcriptomic Atlas of Thymus Organogenesis Resolves Cell Types and Developmental Maturation.

Thymus development is critical to the adaptive immune system, yet a comprehensive transcriptional framework capturing thymus organogenesis at single-cell resolution is still needed. We applied single-cell RNA sequencing (RNA-seq) to capture 8 days of thymus development, perturbations of T cell receptor rearrangement, and in vitro organ cultures, producing profiles of 24,279 cells. We resolved transcriptional heterogeneity of developing lymphocytes, and genetic perturbation confirmed T cell identity of conventional and non-conventional lymphocytes. We characterized maturation dynamics of thymic epithelial cells in vivo, classified cell maturation state in a thymic organ culture, and revealed the intrinsic capacity of thymic epithelium to preserve transcriptional regularity despite exposure to exogenous retinoic acid. Finally, by integrating the cell atlas with human genome-wide association study (GWAS) data and autoimmune-disease-related genes, we implicated embryonic thymus-resident cells as possible participants in autoimmune disease etiologies. This resource provides a single-cell transcriptional framework for biological discovery and molecular analysis of thymus organogenesis.

Perioperative Breast Analgesia: A Qualitative Review of Anatomy and Regional Techniques.

Breast surgery is exceedingly common and may result in significant acute as well as chronic pain. Numerous options exist for the control of perioperative breast pain, including several newly described regional anesthesia techniques, but anesthesiologists have an insufficient understanding of the anatomy of the breast, the anatomic structures disrupted by the various breast surgeries, and the theoretical and experimental evidence supporting the use of the various analgesic options. In this article, we review the anatomy of the breast, common breast surgeries and their potential anatomic sources of pain, and analgesic techniques for managing perioperative pain. We performed a systematic review of the evidence for these analgesic techniques, including intercostal block, epidural administration, paravertebral block, brachial plexus block, and novel peripheral nerve blocks.

Patient Eligibility for Randomized Controlled Trials in Critical Care Medicine: An International Two-Center Observational Study.

OBJECTIVE: We conducted this study to determine the generalizability of information gained from randomized controlled trials in critically ill patients by assessing the incidence of eligibility for each trial. DESIGN: Prospective, observational cohort study. We identified the 15 most highly cited randomized controlled trials in critical care medicine published between 1998 and 2008. We examined the inclusion and exclusion criteria for each randomized controlled trial and then assessed the eligibility of each patient admitted to a study ICU for each randomized controlled trial and calculated rates of potential trial eligibility in the cohort. SETTING: Three ICUs in two academic medical centers in Canada and the United States. PATIENTS: Adults admitted to participating medical or surgical ICU in November 2010 or July 2011. MEASUREMENTS AND MAIN RESULTS: Among the 15 trials, the most common trial inclusion criteria were clinical criteria for sepsis (six trials) or acute respiratory distress syndrome (four trials), use of invasive mechanical ventilation (five trials) or related to ICU type or duration of ICU stay (five trials). Of the 93 patients admitted to a study ICU, 52% of patients (n = 48) did not meet enrollment criteria for any studied randomized controlled trial and 30% (n = 28) were eligible for only one of the 15. Trial ineligibility was mostly due to failure to meet inclusion criteria (87% of screening assessments) rather than meeting specific exclusion criteria (52% of screening assessments). Of the positive screening assessments, 85% occurred on the first day of ICU admission. CONCLUSIONS: Slightly more than half of the patients assessed were not eligible for enrollment in any of 15 major randomized controlled trials in critical care, most often due to the absence of the specific clinical condition of study. The majority of patients who met criteria for a randomized controlled trial did so on the first day of ICU admission.

Bats perceptually weight prey cues across sensory systems when hunting in noise.

Anthropogenic noise can interfere with environmental information processing and thereby reduce survival and reproduction. Receivers of signals and cues in particular depend on perceptual strategies to adjust to noisy conditions. We found that predators that hunt using prey sounds can reduce the negative impact of noise by making use of prey cues conveyed through additional sensory systems. In the presence of masking noise, but not in its absence, frog-eating bats preferred and were faster in attacking a robotic frog emitting multiple sensory cues. The behavioral changes induced by masking noise were accompanied by an increase in active localization through echolocation. Our findings help to reveal how animals can adapt to anthropogenic noise and have implications for the role of sensory ecology in driving species interactions.

Selective arterial embolisation for intractable vaginal haemorrhage in genital tract malignancies.

PURPOSE OF INVESTIGATION: Embolisation of the internal iliac artery has been described as an effective and safe method of treating massive vaginal haemorrhage in small series of advanced uterine cancer and case reports of cervical cancer. Selective embolization of the bleeding vessel is potentially less morbid. The aim of this study was to assess the efficacy of selective arterial embolisation (SAE) in controlling intractable haemorrhage due to gynaecological malignancy. MATERIALS AND METHODS: This retrospective observational study comes from in a tertiary cancer center with 300 new gynecologic cancers per annum. The authors reviewed all gynecology cancer patients who had intractable major vaginal haemorrhage in the first five years following the introduction of selective arterial embolisation at their unit. The outcomes measured were the control of acute haemorrhage and discharge to planned pathway of treatment. RESULTS: SAE was successful in all cases. Identification of the bleeding point facilitated highly selective embolisation in more than half of the patients. The uterine arteries were embolised in the remaining cases. Bleeding stopped immediately. The expedient control of haemorrhage facilitated early discharge to commencement/continuation of radiation treatment or palliative care as appropriate. CONCLUSIONS: Since the introduction of SAE the authors have avoided emergency radiotherapy, surgery, and repeat vaginal packing in patients with intractable vaginal bleeding due to gynaecological cancer. Patients were discharged to their appropriate treatment pathways in a timely manner. The authors recommend the application of SAE.

Multimodal cues improve prey localization under complex environmental conditions.

Predators often eavesdrop on sexual displays of their prey. These displays can provide multimodal cues that aid predators, but the benefits in attending to them should depend on the environmental sensory conditions under which they forage. We assessed whether bats hunting for frogs use multimodal cues to locate their prey and whether their use varies with ambient conditions. We used a robotic set-up mimicking the sexual display of a male túngara frog (Physalaemus pustulosus) to test prey assessment by fringe-lipped bats (Trachops cirrhosus). These predatory bats primarily use sound of the frog's call to find their prey, but the bats also use echolocation cues returning from the frog's dynamically moving vocal sac. In the first experiment, we show that multimodal cues affect attack behaviour: bats made narrower flank attack angles on multimodal trials compared with unimodal trials during which they could only rely on the sound of the frog. In the second experiment, we explored the bat's use of prey cues in an acoustically more complex environment. Túngara frogs often form mixed-species choruses with other frogs, including the hourglass frog (Dendropsophus ebraccatus). Using a multi-speaker set-up, we tested bat approaches and attacks on the robofrog under three different levels of acoustic complexity: no calling D. ebraccatus males, two calling D. ebraccatus males and five D. ebraccatus males. We found that bats are more directional in their approach to the robofrog when more D. ebraccatus males were calling. Thus, bats seemed to benefit more from multimodal cues when confronted with increased levels of acoustic complexity in their foraging environments. Our data have important consequences for our understanding of the evolution of multimodal sexual displays as they reveal how environmental conditions can alter the natural selection pressures acting on them.

The effect of compressive loading magnitude on in situ chondrocyte calcium signaling.

Chondrocyte metabolism is stimulated by deformation and is associated with structural changes in the cartilage extracellular matrix (ECM), suggesting that these cells are involved in maintaining tissue health and integrity. Calcium signaling is an initial step in chondrocyte mechanotransduction that has been linked to many cellular processes. Previous studies using isolated chondrocytes proposed loading magnitude as an important factor regulating this response. However, calcium signaling in the intact cartilage differs compared to isolated cells. The purpose of this study was to investigate the effect of loading magnitude on chondrocyte calcium signaling in intact cartilage. We hypothesized that the percentage of cells exhibiting at least one calcium signal increases with increasing load. Fully intact rabbit femoral condyle and patellar bone/cartilage samples were incubated in calcium-sensitive dyes and imaged continuously under compressive loads of 10-40 % strain. Calcium signaling was primarily associated with the dynamic loading phase and greatly increased beyond a threshold deformation of about 10 % nominal tissue strain. There was a trend toward more cells exhibiting calcium signaling as loading magnitude increased (p = 0.133). These results provide novel information toward identifying mechanisms underlying calcium-dependent signaling pathways related to cartilage homeostasis and possibly the onset and progression of osteoarthritis.

Risky ripples allow bats and frogs to eavesdrop on a multisensory sexual display.

Animal displays are often perceived by intended and unintended receivers in more than one sensory system. In addition, cues that are an incidental consequence of signal production can also be perceived by different receivers, even when the receivers use different sensory systems to perceive them. Here we show that the vocal responses of male túngara frogs (Physalaemus pustulosus) increase twofold when call-induced water ripples are added to the acoustic component of a rival's call. Hunting bats (Trachops cirrhosus) can echolocate this signal by-product and prefer to attack model frogs when ripples are added to the acoustic component of the call. This study illustrates how the perception of a signal by-product by intended and unintended receivers through different sensory systems generates both costs and benefits for the signaler.