The effects of primary and secondary bacterial exposure on the seahorse (Hippocampus erectus) immune response.

Evolutionary adaptations in the Syngnathidae teleost family (seahorses, pipefish and seadragons) culminated in an array of spectacular morphologies, key immune gene losses, and the enigmatic male pregnancy. In seahorses, genome modifications associated with immunoglobulins, complement, and major histocompatibility complex (MHC II) pathway components raise questions concerning their immunological efficiency and the evolution of compensatory measures that may act in their place. In this investigation heat-killed bacteria (Vibrio aestuarianus and Tenacibaculum maritimum) were used in a two-phased experiment to assess the immune response dynamics of Hippocampus erectus. Gill transcriptomes from double and single-exposed individuals were analysed in order to determine the differentially expressed genes contributing to immune system responses towards immune priming. Double-exposed individuals exhibited a greater adaptive immune response when compared with single-exposed individuals, while single-exposed individuals, particularly with V. aestuarianus replicates, associated more with the innate branch of the immune system. T. maritimum double-exposed replicates exhibited the strongest immune reaction, likely due to their immunological naivety towards the bacterium, while there are also potential signs of innate trained immunity. MHC II upregulated expression was identified in selected V. aestuarianus-exposed seahorses, in the absence of other pathway constituents suggesting a possible alternative or non-classical MHC II immune function in seahorses. Gene Ontology (GO) enrichment analysis highlighted prominent angiogenesis activity following secondary exposure, which could be linked to an adaptive immune process in seahorses. This investigation highlights the prominent role of T-cell mediated adaptive immune responses in seahorses when exposed to sequential foreign bacteria exposures. If classical MHC II pathway function has been lost, innate trained immunity in syngnathids could be a potential compensatory mechanism.

Immunological tolerance in the evolution of male pregnancy.

The unique male pregnancy in pipefishes and seahorses ranges from basic attachment (pouch-less species: Nerophinae) of maternal eggs to specialized internal gestation in pouched species (e.g. Syngnathus and Hippocampus) with many transitions in between. Due to this diversity, male pregnancy offers a unique platform for assessing physiological and molecular adaptations in pregnancy evolution. These insights will contribute to answering long-standing questions of why and how pregnancy evolved convergently in so many vertebrate systems. To understand the molecular congruencies and disparities in male pregnancy evolution, we compared transcriptome-wide differentially expressed genes in four syngnathid species, at four pregnancy stages (nonpregnant, early, late and parturition). Across all species and pregnancy forms, metabolic processes and immune dynamics defined pregnancy stages, especially pouched species shared expression features akin to female pregnancy. The observed downregulation of adaptive immune genes in early-stage pregnancy and its reversed upregulation during late/parturition in pouched species, most notably in Hippocampus, combined with directionless expression in the pouch-less species, suggests immune modulation to be restricted to pouched species that evolved placenta-like systems. We propose that increased foeto-paternal intimacy in pouched syngnathids commands immune suppression processes in early gestation, and that the elevated immune response during parturition coincides with pouch opening and reduced progeny reliance. Immune response regulation in pouched species supports the recently described functional MHC II pathway loss as critical in male pregnancy evolution. The independent co-option of similar genes and pathways both in male and female pregnancy highlights immune modulation as crucial for the evolutionary establishment of pregnancy.

Comparative assessment of immunological tolerance in fish with natural immunodeficiency.

Natural occurrences of immunodeficiency by definition should lead to compromised immune function. The major histocompatibility complexes (MHC) are key components of the vertebrate adaptive immune system, charged with mediating allorecognition and antigen presentation functions. To this end, the genomic loss of the MHC II pathway in Syngnathus pipefishes raises questions regarding their immunological vigilance and allorecognition capabilities. Utilising allograft and autograft fin-transplants, we compared the allorecognition immune responses of two pipefish species, with (Nerophis ophidion) and without (Syngnathus typhle) a functional MHC II. Transcriptome-wide assessments explored the immunological tolerance and potential compensatory measures occupying the role of the absent MHC II. Visual observations suggested a more acute rejection response in N. ophidion allografts compared with S. typhle allografts. Differentially expressed genes involved in innate immunity, angiogenesis and tissue recovery were identified among transplantees. The intriguing upregulation of the cytotoxic T-cell implicated gzma in S. typhle allografts, suggests a prominent MHC I related response, which may compensate for the MHC II and CD4 loss. MHC I related downregulation in N. ophidion autografts hints at an immunological tolerance related reaction. These findings may indicate alternative measures evolved to cope with the MHC II genomic loss enabling the maintenance of appropriate tolerance levels. This study provides intriguing insights into the immune and tissue recovery mechanisms associated with syngnathid transplantation, and can be a useful reference for future studies focusing on transplantation transcriptomics in non-model systems.

Characterization of Pipefish Immune Cell Populations Through Single-Cell Transcriptomics.

Teleost adaptive immune systems have evolved with more flexibility than previously assumed. A particularly enigmatic system to address immune system modifications in the evolutionary past is represented by the Syngnathids, the family of pipefishes, seahorses and seadragons. These small fishes with their unique male pregnancy have lost the spleen as an important immune organ as well as a functional major histocompatibility class II (MHC II) pathway. How these evolutionary changes have impacted immune cell population dynamics have up to this point remained unexplored. Here, we present the first immune cell repertoire characterization of a syngnathid fish (Syngnathus typhle) using single-cell transcriptomics. Gene expression profiles of individual cells extracted from blood and head-kidney clustered in twelve putative cell populations with eight belonging to those with immune function. Upregulated cell marker genes identified in humans and teleosts were used to define cell clusters. While the suggested loss of CD4+ T-cells accompanied the loss of the MHC II pathway was supported, the upregulation of specific subtype markers within the T-cell cluster indicates subpopulations of regulatory T-cells (il2rb) and cytotoxic T-cells (gzma). Utilizing single-cell RNA sequencing this report is the first to characterize immune cell populations in syngnathids and provides a valuable foundation for future cellular classification and experimental work within the lineage.

Physical separation from the mate diminishes male's attentiveness towards other females: a study in monogamous prairie voles Microtus ochrogaster.

We tested whether continuous cohabitation in monogamous voles affects the mated male's attentiveness to his breeding partner versus another female. Each male was housed in a 3-chamber apparatus with a Focal female (FF) and a Control female (CF) for 13 days then placed in a T-maze to assess his attentiveness to and memory of those females. The Distal male remained physically separated from both females, but received their distal cues. The Separate male cohabited with the FF for 3 days then remained physically separated from both females. The Disrupt male's continuous cohabitation with the FF was disrupted by having him physically separated from her after 10 days and placed with the CF for the last 3 days. The Continuous male cohabited continuously with the FF for 13 days. With females in the T-maze, the Separate and Disrupt males spent more time near the FF's box and the Disrupt males spent more time manipulating the FF's box than the CF's box. The Separate males groomed themselves more when near the FF's box than the CF's box. The Distal and Continuous males' attentiveness to the two females did not differ. Results suggest that physical distance from the partner may reduce male's attentiveness toward other potential mates. Prairie voles might be similar to socially monogamous primates in using tactile cues as a signal for maintaining their social bonds.

Puzzle-based versus traditional lecture: comparing the effects of pedagogy on academic performance in an undergraduate human anatomy and physiology II lab.

BACKGROUND: A traditional lecture-based pedagogy conveys information and content while lacking sufficient development of critical thinking skills and problem solving. A puzzle-based pedagogy creates a broader contextual framework, and fosters critical thinking as well as logical reasoning skills that can then be used to improve a student's performance on content specific assessments. This paper describes a pedagogical comparison of traditional lecture-based teaching and puzzle-based teaching in a Human Anatomy and Physiology II Lab. METHODS: Using a single subject/cross-over design half of the students from seven sections of the course were taught using one type of pedagogy for the first half of the semester, and then taught with a different pedagogy for the second half of the semester. The other half of the students were taught the same material but with the order of the pedagogies reversed. Students' performance on quizzes and exams specific to the course, and in-class assignments specific to this study were assessed for: learning outcomes (the ability to form the correct conclusion or recall specific information), and authentic academic performance as described by (Am J Educ 104:280-312, 1996). RESULTS: Our findings suggest a significant improvement in students' performance on standard course specific assessments using a puzzle-based pedagogy versus a traditional lecture-based teaching style. Quiz and test scores for students improved by 2.1 and 0.4% respectively in the puzzle-based pedagogy, versus the traditional lecture-based teaching. Additionally, the assessments of authentic academic performance may only effectively measure a broader conceptual understanding in a limited set of contexts, and not in the context of a Human Anatomy and Physiology II Lab. CONCLUSION: In conclusion, a puzzle-based pedagogy, when compared to traditional lecture-based teaching, can effectively enhance the performance of students on standard course specific assessments, even when the assessments only test a limited conceptual understanding of the material.

FGF23 is elevated in multiple myeloma and increases heparanase expression by tumor cells.

Multiply myeloma (MM) grows in and destroys bone, where osteocytes secrete FGF23, a hormone which affects phosphate homeostasis and aging. We report that multiple myeloma (MM) cells express receptors for and respond to FGF23. FGF23 increased mRNA for EGR1 and its target heparanase, a pro-osteolytic factor in MM. FGF23 signals through a complex of klotho and a classical FGF receptor (FGFR); both were expressed by MM cell lines and patient samples. Bone marrow plasma cells from 42 MM patients stained positively for klotho, while plasma cells from 8 patients with monoclonal gammopathy of undetermined significance (MGUS) and 6 controls were negative. Intact, active FGF23 was increased 2.9X in sera of MM patients compared to controls. FGF23 was not expressed by human MM cells, but co-culture with mouse bone increased its mRNA. The FGFR inhibitor NVP-BGJ398 blocked the heparanase response to FGF23. NVP-BGJ398 did not inhibit 8226 growth in vitro but significantly suppressed growth in bone and induction of the osteoclast regulator RANK ligand, while decreasing heparanase mRNA. The bone microenvironment provides resistance to some anti-tumor drugs but increased the activity of NVP-BGJ398 against 8226 cells. The FGF23/klotho/heparanase signaling axis may offer targets for treatment of MM in bone.

Branching and intercellular communication in the Section V cyanobacterium Mastigocladus laminosus, a complex multicellular prokaryote.

The filamentous Section V cyanobacterium Mastigocladus laminosus is one of the most morphologically complex prokaryotes. It exhibits cellular division in multiple planes, resulting in the formation of true branches, and cell differentiation into heterocysts, hormogonia and necridia. Here, we investigate branch formation and intercellular communication in M. laminosus. Monitoring of membrane rearrangement suggests that branch formation results from a randomized direction of cell growth. Transmission electron microscopy reveals cell junction structures likely to be involved in intercellular communication. We identify a sepJ gene, coding for a potential key protein in intercellular communication, and show that SepJ is localized at the septa. To directly investigate intercellular communication, we loaded the fluorescent tracer 5-carboxyfluorescein diacetate into the cytoplasm, and quantified its intercellular exchange by fluorescence recovery after photobleaching. Results demonstrate connectivity of the main trichome and branches, enabling molecular exchange throughout the filament network. Necridia formation inhibits further molecular exchange, determining the fate of a branch likely to become a hormogonium. Cells in young, narrow trichomes and hormogonia exhibited faster exchange rates than cells in older, wider trichomes. Signal transduction to co-ordinate movement of hormogonia might be accelerated by reducing cell volume.

Mating increases male's interest in other females: a cognitive study in socially monogamous prairie voles (Microtus ochrogaster).

To determine whether socio-sexual interactions with females influence the male prairie vole's cognitive processing, three groups of males were simultaneously exposed to sensory stimuli of a control and a focal female then tested for their behavioral and neuronal responsiveness to the female cues. From the control female, all males received distal cues. From the focal female, the Unmated males received distal cues, the Unmated-Contact males received all cues but did not mate with her, and the Mated-Contact males received all cues and mated with her. Males were tested for their attentiveness to enclosures holding each female and for their memory of the females' previous location. Males' brains were then examined to localize activated regions following exposure to the odor of familiar versus unfamiliar focal females. The Mated-Contact males spent more time in the cage of the control female attending to her enclosure than in the cage of the focal female. Exposure to odors of unfamiliar focal females activated the cingulate cortex of Unmated-Contact males. There was a negative correlation between the level of neuronal activation in the retrosplenial cortex of males that were exposed to the odors of a familiar focal female and their attentiveness to the enclosure of the control female. The data suggest that the effect of socio-sexual interactions with a female on males' cognition depends on the type of sensory signals males receive from females and how individual males perceive those signals.

Open Access Series of Imaging Studies (OASIS): cross-sectional MRI data in young, middle aged, nondemented, and demented older adults.

The Open Access Series of Imaging Studies is a series of magnetic resonance imaging data sets that is publicly available for study and analysis. The initial data set consists of a cross-sectional collection of 416 subjects aged 18 to 96 years. One hundred of the included subjects older than 60 years have been clinically diagnosed with very mild to moderate Alzheimer's disease. The subjects are all right-handed and include both men and women. For each subject, three or four individual T1-weighted magnetic resonance imaging scans obtained in single imaging sessions are included. Multiple within-session acquisitions provide extremely high contrast-to-noise ratio, making the data amenable to a wide range of analytic approaches including automated computational analysis. Additionally, a reliability data set is included containing 20 subjects without dementia imaged on a subsequent visit within 90 days of their initial session. Automated calculation of whole-brain volume and estimated total intracranial volume are presented to demonstrate use of the data for measuring differences associated with normal aging and Alzheimer's disease.

Dissociable but inter-related systems of cognitive control and reward during decision making: evidence from pupillometry and event-related fMRI.

Decision making involves the allocation of cognitive resources in response to expectations and feedback. Here we explored how frontal networks respond in a gambling paradigm in which uncertainty was manipulated to increase demands for cognitive control. In one experiment, pupil diameter covaried with uncertainty during decision making and with the degree to which subsequent outcomes violated reward expectations. In a second experiment, fMRI showed that both uncertainty and unexpected outcomes modulated activation in a network of frontal regions. Thus, the frontal network supports multiple phases of the decision-making process including information regarding reward uncertainty and reward outcome. In contrast, striatal activation only tracked reward delivery, suggesting a distinct reward pathway that might, under certain circumstances, oppose the frontal network. These results are consistent with the interpretation that reward signals may bias recruitment of frontal networks that are linked to allocation of cognitive resources.

A unified approach for morphometric and functional data analysis in young, old, and demented adults using automated atlas-based head size normalization: reliability and validation against manual measurement of total intracranial volume.

Atlas normalization, as commonly used by functional data analysis, provides an automated solution to the widely encountered problem of correcting for head size variation in regional and whole-brain morphometric analyses, so long as an age- and population-appropriate target atlas is used. In the present article, we develop and validate an atlas normalization procedure for head size correction using manual total intracranial volume (TIV) measurement as a reference. The target image used for atlas transformation consisted of a merged young and old-adult template specifically created for cross age-span normalization. Automated atlas transformation generated the Atlas Scaling Factor (ASF) defined as the volume-scaling factor required to match each individual to the atlas target. Because atlas normalization equates head size, the ASF should be proportional to TIV. A validation analysis was performed on 147 subjects to evaluate ASF as a proxy for manual TIV measurement. In addition, 19 subjects were imaged on multiple days to assess test-retest reliability. Results indicated that the ASF was (1) equivalent to manual TIV normalization (r = 0.93), (2) reliable across multiple imaging sessions (r = 1.00; mean absolute percentage of difference = 0.51%), (3) able to connect between-gender head size differences, and (4) minimally biased in demented older adults with marked atrophy. Hippocampal volume differences between nondemented (n = 49) and demented (n = 50) older adults (measured manually) were equivalent whether corrected using manual TIV or automated ASF (effect sizes of 1.29 and 1.46, respectively). To provide normative values, ASF was used to automatically derive estimated TIV (eTIV) in 335 subjects aged 15-96 including both clinically characterized nondemented (n = 77) and demented (n = 90) older adults. Differences in eTIV between nondemented and demented groups were negligible, thus failing to support the hypothesis that large premorbid brain size moderates Alzheimer's disease. Gender was the only robust factor that influenced eTIV. Men showed an approximately approximately 12% larger eTIV than women. These results demonstrate that atlas normalization using appropriate template images provides a robust, automated method for head size correction that is equivalent to manual TIV correction in studies of aging and dementia. Thus, atlas normalization provides a common framework for both morphometric and functional data analysis.