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.

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.

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.

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.

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.

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.

Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells.

The identification of the trans-acting factors and cis-regulatory modules that are involved in human pluripotent stem cell (hPSC) maintenance and differentiation is necessary to dissect the operating regulatory networks in these processes and thereby identify nodes where signal input will direct desired cell fate decisions in vitro or in vivo. To deconvolute these networks, we established a method to influence the differentiation state of hPSCs with a CRISPR-associated catalytically inactive dCas9 fused to an effector domain. In human embryonic stem cells, we find that the dCas9 effectors can exert positive or negative regulation on the expression of developmentally relevant genes, which can influence cell differentiation status when impinging on a key node in the regulatory network that governs the cell state. This system provides a platform for the interrogation of the underlying regulators governing specific differentiation decisions, which can then be employed to direct cellular differentiation down desired pathways.

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.

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.

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.

Animal communication: complex call production in the túngara frog.

Animals' sound-producing organs often act as an integrated whole--particular vocal structure are not directly associated with the creation of discrete syllables. But here we show that the 'chuck' of the 'whine-chuck' mating call of the túngara frog, Physalaemus pustulosus, is caused by a fibrous mass attached to the vocal folds; the chuck is eliminated by removal of this structure, although the frog still tries to produce the sound. Sexual selection affects the acoustic complexity of the frog's call, so evolution may have shaped this unusual vocalization, which is akin to the two-voiced song of songbirds.

Candida keroseneae sp. nov., a novel contaminant of aviation kerosene.

AIMS: To characterize and identify a novel contaminant of aviation fuel. METHODS AND RESULTS: Micro-organisms (yeasts and bacteria) were isolated from samples of aviation fuel. A yeast that proved to have been unrecorded previously was isolated from more than one fuel sample. This novel yeast proved to be a new species of Candida and is described here. Ribosomal RNA gene sequence analyses of internal transcribed spacer (ITS) regions (including 5·8S subunit) plus the 26S D1/D2 domains showed the strains to cluster within the Candida membranifaciens clade nearest to, but distinct from, Candida tumulicola. Phenotypic tests were identical for both isolates. Physiological and biochemical tests supported their position as a separate taxon. The yeast was assessed for its effect on the main constituent hydrocarbons of aviation fuel. CONCLUSIONS: Two strains (IMI 395605(T) and IMI 395606) belonging to the novel yeast species, Candida keroseneae, were isolated from samples of aircraft fuel (kerosene), characterized and described herein with reference to their potential as contaminants of aviation fuel. SIGNIFICANCE AND IMPACT OF THE STUDY: As a result of isolating a novel yeast from aviation fuel, the implications for microbial contamination of such fuel should be considered more widely than previously thought.

Categorical perception of a natural, multivariate signal: mating call recognition in túngara frogs.

Categorical perception is common in humans, but it is not known whether animals perceive continuous variation in their own multidimensional social signals categorically. There are two components to categorical perception: labeling and discrimination. In the first, continuously variable stimuli on each side of a category boundary are labeled. In the second, there is strong discrimination between stimuli from opposite sides of the boundary, whereas stimuli on the same side of the boundary are not discriminated. Here, we show that female túngara frogs respond categorically to complex mating calls that vary simultaneously along multiple dimensions and are within the natural range of signal variation. In response to a transect of synthetic stimuli that varied continuously and systematically in seven dimensions, female túngara frogs label mating calls as either conspecific or not conspecific. For pairs of stimuli that differed by the same magnitude, females discriminate those in different categories but not those in the same category. In addition, latency to respond was significantly shorter when stimuli were in the same versus different categories. Because responses to mating calls are critical in generating species recognition and sexual selection, this finding has implications for both animal perception and the influences of mate choice on the tempo and mode of evolution.

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.

Evolution of sperm quality but not quantity in the internally fertilized fish Xiphophorus nigrensis.

Species with alternative reproductive strategies are characterized by discrete differences among males in suites of traits related to competition for fertilizations. Models predict sneaker males should allocate more resources to their ejaculates because they experience sperm competition more frequently and often occupy a disfavoured 'role' owing to subordinance in intramale competition and female preferences for larger males. We examined whether sperm number and quality differed between male strategies in the internally fertilized fish Xiphophorus nigrensis and explored the relationship between sperm morphology and performance. We found sneaker males had similar testes sizes compared to courting males but ejaculates with both more viable and longer lived sperm. Sneaker sperm also had longer midpieces, which was positively correlated with both velocity and longevity. Our study suggests that the evolution of sperm quantity and quality can be decoupled and that the sperm morphology is likely to play an important role in mediating sperm competition through its effects on sperm performance.

Genetic divergence is more tightly related to call variation than landscape features in the Amazonian frogs Physalaemus petersi and P. freibergi.

Behavioural isolation from divergence in male advertisement calls and female preferences is hypothesized to cause genetic divergence and speciation in the Amazonian frogs Physalaemus petersi and P. freibergi, yet the importance of call variation and landscape features in genetic divergence is unresolved. We tested for correlations between genetic divergence at microsatellite loci and (1) call variables; and (2) landscape variables among 10 populations of these frogs. Genetic divergence was not correlated with geographical distance, rivers or elevation. There was a strong positive relationship, however, between genetic divergence and inter-population differences in one call variable, whine dominant frequency. Effective population sizes varied among sites (range = 15-846) and were often small, suggesting that genetic drift could influence call evolution. Evidence for fine-scale genetic structure within sites was also found. Our results support the hypothesis that behavioural isolation from divergence in male calls and female preferences causes genetic divergence and speciation.

Coevolution of sender and receiver: effect on local mate preferecnce in cricket frogs.

Mate recognition in frogs requires congruence of call characters, such as dominant frequency, and properties ofthe auditory system, such as frequency sensitivity of inner ear organs. Two neighboring populations of cricket frogs (Acri crepitans) exhibit statistically significant differences in the dominant frequency of the advertisement call and the frequency to which the basilar papilla of the inner ear is most sensitive. Call frequency and frequency sensitivity are matched within but differ between populations. These characters usually are negatively correlated with body size, and thus their congruence and coevolution often is explained by pleiotropic effects of size. However, within this species call frequency and frequency sensitivity ofthe basilar papilla evolved independent of body size, yielding local mate preferences that could contribute to genetic differentiation among neighboring populations.

Female mate choice in a neotropical frog.

Female Physalaemus pustulosus choose their mates and are more likely to choose larger males. There is a significant negative correlation between the size of the male and the fundamental frequency of one of the components of its advertisement call. Playback experiments demonstrate that females are capable of choosing larger males by distinguishing among differences in spectral components of the advertisement call.

Sexual selection, receiver biases, and the evolution of sex differences.

REVIEW Recent approaches to analyzing the evolution of female mating preferences emphasize how historical influences on female receiver systems can bias the evolution of male traits that females find attractive. These studies combine animal behavior, sensory biology, phylogenetics, and artificial neural network models. They attempt to understand why specific phenotypes involved in sexual selection have evolved, rather than merely determining whether such traits and preferences are adaptive. It is now clear that traits and preferences often do not coevolve via genetic correlations, that female mating preferences for a given male trait are influenced by adaptations and constraints outside of the context of female responses to that particular trait, and that receiver biases can explain much of the diversity in male signaling phenotypes. It also appears that an understanding of historical effects will prove valuable in investigating why neural and cognitive systems respond to sensory stimuli as they do.