A Hyper-IgM Syndrome Mutation in Activation-Induced Cytidine Deaminase Disrupts G-Quadruplex Binding and Genome-wide Chromatin Localization.

Precise targeting of activation-induced cytidine deaminase (AID) to immunoglobulin (Ig) loci promotes antibody class switch recombination (CSR) and somatic hypermutation (SHM), whereas AID targeting of non-Ig loci can generate oncogenic DNA lesions. Here, we examined the contribution of G-quadruplex (G4) nucleic acid structures to AID targeting in vivo. Mice bearing a mutation in Aicda (AIDG133V) that disrupts AID-G4 binding modeled the pathology of hyper-IgM syndrome patients with an orthologous mutation, lacked CSR and SHM, and had broad defects in genome-wide AIDG133V chromatin localization. Genome-wide analyses also revealed that wild-type AID localized to MHCII genes, and AID expression correlated with decreased MHCII expression in germinal center B cells and diffuse large B cell lymphoma. Our findings indicate a crucial role for G4 binding in AID targeting and suggest that AID activity may extend beyond Ig loci to regulate the expression of genes relevant to the physiology and pathology of activated B cells.

A synergy between mechanosensitive calcium- and membrane-binding mediates tension-sensing by C2-like domains.

When nuclear membranes are stretched, the peripheral membrane enzyme cytosolic phospholipase A2 (cPLA2) binds via its calcium-dependent C2 domain (cPLA2-C2) and initiates bioactive lipid signaling and tissue inflammation. More than 150 C2-like domains are encoded in vertebrate genomes. How many of them are mechanosensors and quantitative relationships between tension and membrane recruitment remain unexplored, leaving a knowledge gap in the mechanotransduction field. In this study, we imaged the mechanosensitive adsorption of cPLA2 and its C2 domain to nuclear membranes and artificial lipid bilayers, comparing it to related C2-like motifs. Stretch increased the Ca2+ sensitivity of all tested domains, promoting half-maximal binding of cPLA2 at cytoplasmic resting-Ca2+ concentrations. cPLA2-C2 bound up to 50 times tighter to stretched than to unstretched membranes. Our data suggest that a synergy of mechanosensitive Ca2+ interactions and deep, hydrophobic membrane insertion enables cPLA2-C2 to detect stretched membranes with antibody-like affinity, providing a quantitative basis for understanding mechanotransduction by C2-like domains.

Maintenance of Lineage Identity: Lessons from a B Cell.

The maintenance of B cell identity requires active transcriptional control that enforces a B cell-specific program and suppresses alternative lineage genes. Accordingly, disrupting the B cell identity regulatory network compromises B cell function and induces cell fate plasticity by allowing derepression of alternative lineage-specific transcriptional programs. Although the B lineage is incredibly resistant to most differentiating factors, loss of just a single B lineage-specific transcription factor or the forced expression of individual non-B cell lineage transcription factors can radically disrupt B cell maintenance and allow dedifferentiation or transdifferentiation into entirely distinct lineages. B lymphocytes thereby offer an insightful and useful case study of how a specific cell lineage can maintain a stable identity throughout life and how perturbations of a single master regulator can induce cellular plasticity. In this article, we review the regulatory mechanisms that safeguard B cell identity, and we discuss how dysregulation of the B cell maintenance program can drive malignant transformation and enable therapeutic resistance.

Disentangling Mechanical and Sensory Modules in the Radiation of Noctilionoid Bats.

AbstractWith diverse mechanical and sensory functions, the vertebrate cranium is a complex anatomical structure whose shifts between modularity and integration, especially in mechanical function, have been implicated in adaptive diversification. Yet how mechanical and sensory systems and their functions coevolve, as well as how their interrelationship contributes to phenotypic disparity, remain largely unexplored. To examine the modularity, integration, and evolutionary rates of sensory and mechanical structures within the head, we analyzed hard and soft tissue scans from ecologically diverse bats in the superfamily Noctilionoidea, a clade that ranges from insectivores and carnivores to frugivores and nectarivores. We identified eight regions that evolved in a coordinated fashion, thus recognizable as evolutionary modules: five associated with bite force and three linked to olfactory, visual, and auditory systems. Interrelationships among these modules differ between Neotropical leaf-nosed bats (family Phyllostomidae) and other noctilionoids. Consistent with the hypothesis that dietary transitions begin with changes in the capacity to detect novel food items followed by adaptations to process them, peak rates of sensory module evolution predate those of some mechanical modules. We propose that the coevolution of structures influencing bite force, olfaction, vision, and hearing constituted a structural opportunity that allowed the phyllostomid ancestor to take advantage of existing ecological opportunities and contributed to the clade's remarkable radiation.

Dietary Diversification and Specialization in Neotropical Bats Facilitated by Early Molecular Evolution.

Dietary adaptation is a major feature of phenotypic and ecological diversification, yet the genetic basis of dietary shifts is poorly understood. Among mammals, Neotropical leaf-nosed bats (family Phyllostomidae) show unmatched diversity in diet; from a putative insectivorous ancestor, phyllostomids have radiated to specialize on diverse food sources including blood, nectar, and fruit. To assess whether dietary diversification in this group was accompanied by molecular adaptations for changing metabolic demands, we sequenced 89 transcriptomes across 58 species and combined these with published data to compare ∼13,000 protein coding genes across 66 species. We tested for positive selection on focal lineages, including those inferred to have undergone dietary shifts. Unexpectedly, we found a broad signature of positive selection in the ancestral phyllostomid branch, spanning genes implicated in the metabolism of all major macronutrients, yet few positively selected genes at the inferred switch to plantivory. Branches corresponding to blood- and nectar-based diets showed selection in loci underpinning nitrogenous waste excretion and glycolysis, respectively. Intriguingly, patterns of selection in metabolism genes were mirrored by those in loci implicated in craniofacial remodeling, a trait previously linked to phyllostomid dietary specialization. Finally, we show that the null model of the widely-used branch-site test is likely to be misspecified, with the implication that the test is too conservative and probably under-reports true cases of positive selection. Our findings point to a complex picture of adaptive radiation, in which the evolution of new dietary specializations has been facilitated by early adaptations combined with the generation of new genetic variation.

Large-scale genome sampling reveals unique immunity and metabolic adaptations in bats.

Comprising more than 1,400 species, bats possess adaptations unique among mammals including powered flight, unexpected longevity, and extraordinary immunity. Some of the molecular mechanisms underlying these unique adaptations includes DNA repair, metabolism and immunity. However, analyses have been limited to a few divergent lineages, reducing the scope of inferences on gene family evolution across the Order Chiroptera. We conducted an exhaustive comparative genomic study of 37 bat species, one generated in this study, encompassing a large number of lineages, with a particular emphasis on multi-gene family evolution across immune and metabolic genes. In agreement with previous analyses, we found lineage-specific expansions of the APOBEC3 and MHC-I gene families, and loss of the proinflammatory PYHIN gene family. We inferred more than 1,000 gene losses unique to bats, including genes involved in the regulation of inflammasome pathways such as epithelial defence receptors, the natural killer gene complex and the interferon-gamma induced pathway. Gene set enrichment analyses revealed genes lost in bats are involved in defence response against pathogen-associated molecular patterns and damage-associated molecular patterns. Gene family evolution and selection analyses indicate bats have evolved fundamental functional differences compared to other mammals in both innate and adaptive immune system, with the potential to enhance antiviral immune response while dampening inflammatory signalling. In addition, metabolic genes have experienced repeated expansions related to convergent shifts to plant-based diets. Our analyses support the hypothesis that, in tandem with flight, ancestral bats had evolved a unique set of immune adaptations whose functional implications remain to be explored.

Molecular epidemiology and clinical impact of rhinovirus infections in adults during three epidemic seasons in 11 European countries (2007-2010).

BACKGROUND: Differences in clinical impact between rhinovirus (RVs) species and types in adults are not well established. The objective of this study was to determine the epidemiology and clinical impact of the different RV species. METHODS: We conducted a prospective study of RVs infections in adults with acute cough/lower respiratory tract infection (LRTI) and asymptomatic controls. Subjects were recruited from 16 primary care networks located in 11 European countries between 2007 and 2010. RV detection and genotyping was performed by means of real time and conventional reverse-transcriptase polymerase chain reaction assays, followed by sequence analysis. Clinical data were obtained from medical records and patient symptom diaries. RESULTS: RVs were detected in 566 (19%) of 3016 symptomatic adults, 102 (4%) of their 2539 follow-up samples and 67 (4%) of 1677 asymptomatic controls. Genotyping was successful for 538 (95%) symptomatic subjects, 86 (84%) follow-up infections and 62 (93%) controls. RV-A was the prevailing species, associated with an increased risk of LRTI as compared with RV-B (relative risk (RR), 4.5; 95% CI 2.5 to 7.9; p<0.001) and RV-C (RR 2.2; 95% CI 1.2 to 3.9; p=0.010). In symptomatic subjects, RV-A loads were higher than those of RV-B (p=0.015). Symptom scores and duration were similar across species. More RV-A infected patients felt generally unwell in comparison to RV-C (p=0·023). Of the 140 RV types identified, five were new types; asymptomatic infections were associated with multiple types. INTERPRETATION: In adults, RV-A is significantly more often detected in cases with acute cough/LRTI than RV-C, while RV-B infection is often found in asymptomatic patients.

Foraging shifts and visual preadaptation in ecologically diverse bats.

Changes in behaviour may initiate shifts to new adaptive zones, with physical adaptations for novel environments evolving later. While new mutations are commonly considered engines of adaptive change, sensory evolution enabling access to new resources might also arise from standing genetic diversity, and even gene loss. We examine the relative contribution of molecular adaptations, measured by positive and relaxed selection, acting on eye-expressed genes associated with shifts to new adaptive zones in ecologically diverse bats from the superfamily Noctilionoidea. Collectively, noctilionoids display remarkable ecological breadth, from highly divergent echolocation to flight strategies linked to specialized insectivory, the parallel evolution of diverse plant-based diets (e.g., nectar, pollen and fruit) from ancestral insectivory, and-unusually for echolocating bats-often have large, well-developed eyes. We report contrasting levels of positive selection in genes associated with the development, maintenance and scope of visual function, tracing back to the origins of noctilionoids and Phyllostomidae (the bat family with most dietary diversity), instead of during shifts to novel diets. Generalized plant visiting was not associated with exceptional molecular adaptation, and exploration of these novel niches took place in an ancestral phyllostomid genetic background. In contrast, evidence for positive selection in vision genes was found at subsequent shifts to either nectarivory or frugivory. Thus, neotropical noctilionoids that use visual cues for identifying food and roosts, as well as for orientation, were effectively preadapted, with subsequent molecular adaptations in nectar-feeding lineages and the subfamily Stenodermatinae of fig-eating bats fine-tuning pre-existing visual adaptations for specialized purposes.

Single-molecule localization microscopy reveals molecular transactions during RAD51 filament assembly at cellular DNA damage sites.

RAD51 recombinase assembles on single-stranded (ss)DNA substrates exposed by DNA end-resection to initiate homologous recombination (HR), a process fundamental to genome integrity. RAD51 assembly has been characterized using purified proteins, but its ultrastructural topography in the cell nucleus is unexplored. Here, we combine cell genetics with single-molecule localization microscopy and a palette of bespoke analytical tools, to visualize molecular transactions during RAD51 assembly in the cellular milieu at resolutions approaching 30-40 nm. In several human cell types, RAD51 focalizes in clusters that progressively extend into long filaments, which abut-but do not overlap-with globular bundles of replication protein A (RPA). Extended filaments alter topographically over time, suggestive of succeeding steps in HR. In cells depleted of the tumor suppressor protein BRCA2, or overexpressing its RAD51-binding BRC repeats, RAD51 fails to assemble at damage sites, although RPA accumulates unhindered. By contrast, in cells lacking a BRCA2 carboxyl (C)-terminal region targeted by cancer-causing mutations, damage-induced RAD51 assemblies initiate but do not extend into filaments. We suggest a model wherein RAD51 assembly proceeds concurrently with end-resection at adjacent sites, via an initiation step dependent on the BRC repeats, followed by filament extension through the C-terminal region of BRCA2.

Limited Evidence for Parallel Molecular Adaptations Associated with the Subterranean Niche in Mammals: A Comparative Study of Three Superorders.

Among mammals, several lineages have independently adapted to a subterranean niche and possess similar phenotypic traits for burrowing (e.g., cylindrical bodies, short limbs, and absent pinnae). Previous research on mole-rats has revealed molecular adaptations for coping with reduced oxygen, elevated carbon dioxide, and the absence of light. In contrast, almost nothing is known regarding molecular adaptations in other subterranean lineages (e.g., true moles and golden moles). Therefore, the extent to which the recurrent phenotypic adaptations of divergent subterranean taxa have arisen via parallel routes of molecular evolution remains untested. To address these issues, we analyzed ∼8,000 loci in 15 representative subterranean taxa of four independent transitions to an underground niche for signatures of positive selection and convergent amino acid substitutions. Complementary analyses were performed in nonsubterranean "control" taxa to assess the biological significance of results. We found comparable numbers of positively selected genes in each of the four subterranean groups; however, correspondence in terms of gene identity between gene sets was low. Furthermore, we did not detect evidence of more convergent amino acids among subterranean species pairs compared with levels found between nonsubterranean controls. Comparisons with nonsubterranean taxa also revealed loci either under positive selection or with convergent substitutions, with similar functional enrichment (e.g., cell adhesion, immune response, and coagulation). Given the limited indication that positive selection and convergence occurred in the same loci, we conclude that selection may have acted on different loci across subterranean mammal lineages to produce similar phenotypes.

Assessing Soft-Tissue Shrinkage Estimates in Museum Specimens Imaged With Diffusible Iodine-Based Contrast-Enhanced Computed Tomography (diceCT).

The increased accessibility of soft-tissue data through diffusible iodine-based contrast-enhanced computed tomography (diceCT) enables comparative biologists to increase the taxonomic breadth of their studies with museum specimens. However, it is still unclear how soft-tissue measurements from preserved specimens reflect values from freshly collected specimens and whether diceCT preparation may affect these measurements. Here, we document and evaluate the accuracy of diceCT in museum specimens based on the soft-tissue reconstructions of brains and eyes of five bats. Based on proxies, both brains and eyes were roughly 60% of the estimated original sizes when first imaged. However, these structures did not further shrink significantly over a 4-week staining interval, and 1 week in 2.5% iodine-based solution yielded sufficient contrast for differentiating among soft-tissues. Compared to six "fresh" bat specimens imaged shortly after field collection (not fixed in ethanol), the museum specimens had significantly lower relative volumes of the eyes and brains. Variation in field preparation techniques and conditions, and long-term storage in ethanol may be the primary causes of shrinkage in museum specimens rather than diceCT staining methodology. Identifying reliable tissue-specific correction factors to adjust for the shrinkage now documented in museum specimens requires future work with larger samples.

Assessment of executive functioning in binge-eating disorder independent of weight status.

OBJECTIVE: Executive functioning (EF) problems may serve as vulnerability or maintenance factors for Binge-Eating Disorder (BED). However, it is unclear if EF problems observed in BED are related to overweight status or BED status. The current study extends this literature by examining EF in overweight and normal-weight BED compared to weight-matched controls. METHOD: Participants were normal-weight women with BED (n = 23), overweight BED (n = 32), overweight healthy controls (n = 48), and normal-weight healthy controls (n = 29). The EF battery utilized tests from the National Institutes of Health (NIH) Toolbox and Delis-Kaplan Executive Function System (D-KEFS). RESULTS: After controlling for years of education and minority status, overweight individuals performed more poorly than normal-weight individuals on a task of cognitive flexibility requiring generativity (p < .01), and speed on psychomotor performance tasks (p = .01). Normal-weight and overweight BED performed worse on working memory tasks compared to controls (p = .04). Unexpectedly, normal-weight BED individuals out-performed all other groups on an inhibitory control task (p < .01). No significant differences were found between the four groups on tasks of planning. DISCUSSION: Regardless of weight status, BED is associated with working memory problems. Replication of the finding that normal-weight BED is associated with enhanced inhibitory control is needed.

Quantitative expression of regulatory and differentiation-related genes in the key steps of human hematopoiesis: The LeukoStage Database.

Differentiation during hematopoiesis leads to the generation of many cell types with specific functions. At various stages of maturation, the cells may change pathologically, leading to diseases including acute leukemias (ALs). Expression levels of regulatory molecules (such as the IKZF, GATA, HOX, FOX, NOTCH and CEBP families, as well as SPI-1/PU1 and PAX5) and lineage-specific molecules (including CD2, CD14, CD79A, and BLNK) may be compared between pathological and physiological cells. Although the key steps of differentiation are known, the available databases focus mainly on fully differentiated cells as a reference. Precursor cells may be a more appropriate reference point for diseases that evolve at immature stages. Therefore, we developed a quantitative real-time polymerase chain reaction (qPCR) array to investigate 90 genes that are characteristic of the lymphoid or myeloid lineages and/or are thought to be involved in their regulation. Using this array, sorted cells of granulocytic, monocytic, T and B lineages were analyzed. For each of these lineages, 3-5 differentiation stages were selected (17 stages total), and cells were sorted from 3 different donors per stage. The qPCR results were compared to similarly processed AL cells of lymphoblastic (n=18) or myeloid (n=6) origins and biphenotypic AL cells of B cell origin with myeloid involvement (n=5). Molecules characteristic of each lineage were found. In addition, cells of a newly discovered switching lymphoblastic AL (swALL) were sorted at various phases during the supposed transdifferentiation from an immature B cell to a monocytic phenotype. As demonstrated previously, gene expression changed along with the immunophenotype. The qPCR data are publicly available in the LeukoStage Database in which gene expression in malignant and non-malignant cells of different lineages can be explored graphically and differentially expressed genes can be identified. In addition, the LeukoStage Database can aid the functional analyses of next-generation sequencing data.

Family Wide Molecular Adaptations to Underground Life in African Mole-Rats Revealed by Phylogenomic Analysis.

During their evolutionary radiation, mammals have colonized diverse habitats. Arguably the subterranean niche is the most inhospitable of these, characterized by reduced oxygen, elevated carbon dioxide, absence of light, scarcity of food, and a substrate that is energetically costly to burrow through. Of all lineages to have transitioned to a subterranean niche, African mole-rats are one of the most successful. Much of their ecological success can be attributed to a diet of plant storage organs, which has allowed them to colonize climatically varied habitats across sub-Saharan Africa, and has probably contributed to the evolution of their diverse social systems. Yet despite their many remarkable phenotypic specializations, little is known about molecular adaptations underlying these traits. To address this, we sequenced the transcriptomes of seven mole-rat taxa, including three solitary species, and combined new sequences with existing genomic data sets. Alignments of more than 13,000 protein-coding genes encompassed, for the first time, all six genera and the full spectrum of ecological and social variation in the clade. We detected positive selection within the mole-rat clade and along ancestral branches in approximately 700 genes including loci associated with tumorigenesis, aging, morphological development, and sociality. By combining these results with gene ontology annotation and protein-protein networks, we identified several clusters of functionally related genes. This family wide analysis of molecular evolution in mole-rats has identified a suite of positively selected genes, deepening our understanding of the extreme phenotypic traits exhibited by this group.

Family-Based Therapy for Young Adults with Anorexia Nervosa Restores Weight.

OBJECTIVE: We examined the preliminary acceptability and efficacy of family-based therapy (FBT) for weight restoration in young adults (FBTY) with Anorexia Nervosa (AN). METHOD: Twenty-two primarily female participants ranging from age 18 to 26, with AN or atypical AN (ICD-10) and their support adults were enrolled in a 6-month open trial of FBTY. Participants were assessed at baseline, after treatment, and at six and 12 month follow-up visits. The primary outcome was BMI and secondary outcomes included eating disorder psychopathology, current eating disorder obsessions, and compulsions, number of other Axis I disorders and global assessment of functioning. RESULTS: Although FBTY was rated as suitable by participants and their support adults, during FBTY, 9/22 participants dropped out and 3/22 dropped out at follow-up assessments. Despite being offered 18-20 sessions over six months, a mean of 12 FBTY sessions (SD = 6) were attended. After FBTY, 15 of the intent-to-treat sample of 22 were no longer underweight (BMIs ≥ 19 kg/m(2) ) and 12 months after treatment, 13/22 were no longer underweight. The magnitude of the BMI increase during FBTY (Hedges g = 1.20, 95th percentile CI = 0.55-1.85) was comparable to findings for adolescent FBT for AN. Secondary outcomes also improved. DISCUSSION: FBTY for young adults with AN and atypical AN, which involves support adults participants have chosen, results in weight restoration that is sustained up to a year after treatment. © 2016 Wiley Periodicals, Inc. (Int J Eat Disord 2016; 49:701-707).

Fast and simple spectral FLIM for biochemical and medical imaging.

Spectrally resolved fluorescence lifetime imaging microscopy (λFLIM) has powerful potential for biochemical and medical imaging applications. However, long acquisition times, low spectral resolution and complexity of λFLIM often narrow its use to specialized laboratories. Therefore, we demonstrate here a simple spectral FLIM based on a solid-state detector array providing in-pixel histrogramming and delivering faster acquisition, larger dynamic range, and higher spectral elements than state-of-the-art λFLIM. We successfully apply this novel microscopy system to biochemical and medical imaging demonstrating that solid-state detectors are a key strategic technology to enable complex assays in biomedical laboratories and the clinic.