Suspected clinical toxoplasmosis in a 12-week-old puppy in Singapore.

BACKGROUND: Toxoplasma gondii is traditionally known as a parasite of felids, with possible infection in intermediate hosts such as dogs and humans, and thus a disease of public health significance. Published data on the prevalence of toxoplasmosis in dogs and cats in Singapore is scanty, and this paper documents a suspect clinical case of toxoplasmosis in a free-roaming puppy trapped from an offshore island of Singapore. CASE PRESENTATION: A 12-week-old puppy presented with hindlimb weakness and sarcopenia, with rapidly progressing ascending paralysis and respiratory distress, one week after trapping. Toxoplasmosis was suspected after indirect fluorescence antibody testing (IFAT) revealed anti-T. gondii antibodies. The puppy responded quickly to clindamycin treatment and was discharged from hospital after 10 days. CONCLUSION: While rare and undocumented, veterinary clinicians in Singapore are advised to also include toxoplasmosis infection as a differential diagnosis in dogs presenting with similar clinical signs. This is especially so for dogs which have access to the outdoors.

Community-Derived Core Concepts for Neuroscience Higher Education.

Core concepts provide a framework for organizing facts and understanding in neuroscience higher education curricula. Core concepts are overarching principles that identify patterns in neuroscience processes and phenomena and can be used as a foundational scaffold for neuroscience knowledge. The need for community-derived core concepts is pressing, because both the pace of research and number of neuroscience programs are rapidly expanding. While general biology and many subdisciplines within biology have identified core concepts, neuroscience has yet to establish a community-derived set of core concepts for neuroscience higher education. We used an empirical approach involving more than 100 neuroscience educators to identify a list of core concepts. The process of identifying neuroscience core concepts was modeled after the process used to develop physiology core concepts and involved a nationwide survey and a working session of 103 neuroscience educators. The iterative process identified eight core concepts and accompanying explanatory paragraphs. The eight core concepts are abbreviated as communication modalities, emergence, evolution, gene-environment interactions, information processing, nervous system functions, plasticity, and structure-function. Here, we describe the pedagogical research process used to establish core concepts for the neuroscience field and provide examples on how the core concepts can be embedded in neuroscience education.

Introducing immunohistochemistry to the molecular biology laboratory.

Widely used in research laboratories, immunohistochemistry (IHC) is a transferable skill that prepares undergraduate students for a variety of careers in the biomedical field. We have developed an inquiry-based learning IHC laboratory exercise, which introduces students to the theory, procedure, and data interpretation of antibody staining. Students are tasked with performing IHC using an "unknown" antibody and then asked to identify the cells or molecular structures within the nervous systems specific for that unknown antibody. In two lab sessions, students are exposed to handling of delicate brain slices, fluorescent microscopy, and data analysis using the Allen Brain Atlas (ABA), an online freely accessible database of mRNA transcript expression patterns in the brain. Here, we present guidelines for easy implementation in the classroom and assess learning gains achieved by the students upon completion of the IHC laboratory module. Students clearly displayed an increase in knowledge in data interpretation, procedural knowledge, and theory surrounding IHC. Thus, this module works as an inquiry-based learning based method to introduce IHC principles to undergraduate students.

The Development of Core Concepts for Neuroscience Higher Education: From Beginning to Summer Virtual Meeting Satellite Session.

Neuroscience curricula vary widely across higher education institutions due to the lack of an accrediting body or a set of unified educational concepts or outcomes. Each institution has developed a unique set of fundamental knowledge, topical subdisciplines, and core competencies to be delivered in a neuroscience program. Core concepts would provide neuroscience departments and programs with a generally agreed upon set of overarching principles that organize knowledge and can be applied to all sub-disciplines of the field, providing a useful framework from which to approach neuroscience education. We set out to develop a consensus set of neuroscience core concepts to aid in higher education curricular development and assessment. Suggestions for neuroscience core concepts were solicited from neuroscience faculty in a nationwide survey and analyzed using an inductive, independent coding model to identify eight core concepts based upon survey responses. Accompanying explanatory paragraphs for each core concept were developed through an iterative process. We presented the resulting core concepts to 134 neuroscience educators at a satellite session of the Faculty for Undergraduate Neuroscience 2020 Summer Virtual Meeting (SVM). Individuals and groups of faculty provided feedback regarding the accuracy, comprehensiveness, and clarity of each concept and explanatory paragraph, as well as the structure of the document as a whole. We continue to refine the core concepts based upon this feedback and will distribute the final document in a subsequent publication. Following publication of the finalized list of core concepts, we will develop tools to help educators incorporate the core concepts into their curricula.

Instructor facilitation mediates students' negative perceptions of active learning instruction.

Studies have demonstrated students' resistance to active learning, despite evidence illustrating that their learning is improved relative to students in lectures. Specifically, while active learning and group work are effective at engaging students in their learning process, studies report that students' perceptions of active learning approaches are not always positive. What remains underexplored is whether students' perceptions of active learning improve with effective instructor facilitation and whether there exists differential perceptions between racially minoritized students and represented students. Here, we estimate students' perceptions of effective instructor facilitation as the mediator in the relationship between active learning and perceptions of learning and perceived utility for class activities (task value). Then, we examine differences by racial identification. We collected classroom observation data to empirically categorize courses as active learning or lecture-based and surveyed 4,257 college students across 25 STEM classrooms at a research-intensive university. We first examined the relationship between active learning on student perceptions and found a negative relationship between active learning and perceptions of learning and task value for both racially minoritized students and represented students. Next, we assessed whether students' perceptions of instructor effectiveness in facilitating group activities mediate these negative relationships. We found that, on average, students of all races were more likely to positively perceive instructor facilitation in active learning classes relative to lectures. In turn, the positive perceptions of instructor facilitation partially suppressed the negative relationship between active learning and perceptions of learning and task value. These results demonstrate that effective instructor facilitation can influence both students' self-assessment of learning and perceived utility of the learning activities, and underscores the importance of developing pedagogical competence among college instructors.

Characterizing Biology Education Research: Perspectives from Practitioners and Scholars in the Field.

Biology education research (BER) is a recently emerging field mainly focused on the learning and teaching of biology in postsecondary education. As BER continues to grow, exploring what goals, questions, and scholarship the field encompasses will provide an opportunity for the community to reflect on what new lines of inquiry could be pursued in the future. There have been top-down approaches at characterizing BER, such as aims and scope provided by professional societies or peer-reviewed journals, and literature analyses with evidence for current and historical research trends. However, there have not been previous attempts with a bottom-up approach at characterizing BER by directly surveying practitioners and scholars in the field. Here, we share survey results that asked participants at the Society for the Advancement of Biology Education Research (SABER) annual meeting what they perceive as current scholarship in BER as well as what areas of inquiry in the field that they would like to see pursued in the future. These survey responses provide us with information directly from BER practitioners and scholars, and we invite colleagues to reflect on how we can collectively and collaboratively continue to promote BER as a field.

Manual wheelchair tilt-rest skill: a cross-sectional survey of awareness and capacity among wheelchair users.

Purpose: The tilt-rest skill consists of tipping the wheelchair back and allowing it to rest against a solid object with the wheel locks applied (e.g., for pressure redistribution, neck comfort or hands-free activities). The objective of this study was to determine the proportion of experienced manual wheelchair users who are aware of this skill and who can perform it. Materials and methods: We conducted a cross-sectional survey of 49 manual wheelchair users using a questionnaire developed for the purpose. The tilt-rest skill was attempted by those who reported that they were capable of performing it. Results: Participants' mean (SD) age was 55.1 (18.2) years, 38 (77.6%) were male, their median (IQR) duration of wheelchair use was 2 (7.2) years and their mean (SD) daily time spent in the wheelchair was 9.5 (4.6) hours. Twenty-seven (55.1%) participants were aware of the skill, 19 (38.8%) reported being able to perform the skill and 16 of 47 (34.0%) were able to demonstrate the skill. Multivariate modelling with the question "Can you complete the tilt-rest skill?" as the dependent measure revealed an inverse relationship with age - Odds Ratio (95% Confidence Interval) of 0.476 (0.293, 0.774) (p = .0028) for each 10 year increase in age. Conclusions: Only just over half of manual wheelchair users are aware of the tilt-rest skill and one-third of users can perform it. Older people are less likely to report being able to complete the skill. These findings have implications for wheelchair skills training during the wheelchair-provision process. Implications for Rehabilitation Only just over half of manual wheelchair users are aware of the tilt-rest skill and only about one-third of users can perform it. Older people are less likely to report being able to complete the skill. These findings have clinical implications for wheelchair skills training during the, specifically that clinicians responsible for manual wheelchair-provision process should ensure that appropriate wheelchair users have the opportunity to learn this skill.

Double-stranded DNA break polarity skews repair pathway choice during intrachromosomal and interchromosomal recombination.

Activation-induced cytidine deaminase (AID) inflicts DNA damage at Ig genes to initiate class switch recombination (CSR) and chromosomal translocations. However, the DNA lesions formed during these processes retain an element of randomness, and thus knowledge of the relationship between specific DNA lesions and AID-mediated processes remains incomplete. To identify necessary and sufficient DNA lesions in CSR, the Cas9 endonuclease and nickase variants were used to program DNA lesions at a greater degree of predictability than is achievable with conventional induction of CSR. Here we show that Cas9-mediated nicks separated by up to 250 nucleotides on opposite strands can mediate CSR. Staggered double-stranded breaks (DSBs) result in more end resection and junctional microhomology than blunt DSBs. Moreover, Myc-Igh chromosomal translocations, which are carried out primarily by alternative end joining (A-EJ), were preferentially induced by 5' DSBs. These data indicate that DSBs with 5' overhangs skew intrachromosomal and interchromosomal end-joining toward A-EJ. In addition to lending potential insight to AID-mediated phenomena, this work has broader carryover implications in DNA repair and lymphomagenesis.

Genetic Pathways of Neuroregeneration in a Novel Mild Traumatic Brain Injury Model in Adult Zebrafish.

Mild traumatic brain injuries (mTBIs) are one of the most prevalent neurological disorders, and humans are severely limited in their ability to repair and regenerate central nervous system (CNS) tissue postinjury. However, zebrafish (Danio rerio) maintain the remarkable ability to undergo complete and functional neuroregeneration as an adult. We wish to extend knowledge of the known mechanisms of neuroregeneration by analyzing the differentially expressed genes (DEGs) in a novel adult zebrafish model of mTBI. In this study, a rodent weight drop model of mTBI was adapted to the adult zebrafish. A memory test showed significant deficits in spatial memory in the mTBI group. We identified DEGs at 3 and 21 days postinjury (dpi) through RNA-sequencing analysis. The resulting DEGs were categorized according to gene ontology (GO) categories. At 3 dpi, GO categories consisted of peak injury response pathways. Significantly, at 21 dpi, GO categories consisted of neuroregeneration pathways. Ultimately, these results validate a novel zebrafish model of mTBI and elucidate significant DEGs of interest in CNS injury and neuroregeneration.

Genetic and neuronal regulation of sleep by neuropeptide VF.

Sleep is an essential and phylogenetically conserved behavioral state, but it remains unclear to what extent genes identified in invertebrates also regulate vertebrate sleep. RFamide-related neuropeptides have been shown to promote invertebrate sleep, and here we report that the vertebrate hypothalamic RFamide neuropeptide VF (NPVF) regulates sleep in the zebrafish, a diurnal vertebrate. We found that NPVF signaling and npvf-expressing neurons are both necessary and sufficient to promote sleep, that mature peptides derived from the NPVF preproprotein promote sleep in a synergistic manner, and that stimulation of npvf-expressing neurons induces neuronal activity levels consistent with normal sleep. These results identify NPVF signaling and npvf-expressing neurons as a novel vertebrate sleep-promoting system and suggest that RFamide neuropeptides participate in an ancient and central aspect of sleep control.

Genetic Analysis of Histamine Signaling in Larval Zebrafish Sleep.

Pharmacological studies in mammals and zebrafish suggest that histamine plays an important role in promoting arousal. However, genetic studies using rodents with disrupted histamine synthesis or signaling have revealed only subtle or no sleep/wake phenotypes. Studies of histamine function in mammalian arousal are complicated by its production in cells of the immune system and its roles in humoral and cellular immunity, which can have profound effects on sleep/wake states. To avoid this potential confound, we used genetics to explore the role of histamine in regulating sleep in zebrafish, a diurnal vertebrate in which histamine production is restricted to neurons in the brain. Similar to rodent genetic studies, we found that zebrafish that lack histamine due to mutation of histidine decarboxylase (hdc) exhibit largely normal sleep/wake behaviors. Zebrafish containing predicted null mutations in several histamine receptors also lack robust sleep/wake phenotypes, although we are unable to verify that these mutants are completely nonfunctional. Consistent with some rodent studies, we found that arousal induced by overexpression of the neuropeptide hypocretin (Hcrt) or by stimulation of hcrt-expressing neurons is not blocked in hdc or hrh1 mutants. We also found that the number of hcrt-expressing or histaminergic neurons is unaffected in animals that lack histamine or Hcrt signaling, respectively. Thus, while acute pharmacological manipulation of histamine signaling has been shown to have profound effects on zebrafish and mammalian sleep, our results suggest that chronic loss of histamine signaling due to genetic mutations has only subtle effects on sleep in zebrafish, similar to rodents.

Pediatric Sepsis.

Pediatric sepsis is distinct from adult sepsis in its definitions, clinical presentations, and management. Recognition of pediatric sepsis is complicated by the various pediatric-specific comorbidities that contribute to its mortality and the age- and development-specific vital sign and clinical parameters that obscure its recognition. This article outlines the clinical presentation and management of sepsis in neonates, infants, and children, and highlights some key populations who require specialized care.

QRFP and Its Receptors Regulate Locomotor Activity and Sleep in Zebrafish.

The hypothalamus plays an important role in regulating sleep, but few hypothalamic sleep-promoting signaling pathways have been identified. Here we demonstrate a role for the neuropeptide QRFP (also known as P518 and 26RFa) and its receptors in regulating sleep in zebrafish, a diurnal vertebrate. We show that QRFP is expressed in ∼10 hypothalamic neurons in zebrafish larvae, which project to the hypothalamus, hindbrain, and spinal cord, including regions that express the two zebrafish QRFP receptor paralogs. We find that the overexpression of QRFP inhibits locomotor activity during the day, whereas mutation of qrfp or its receptors results in increased locomotor activity and decreased sleep during the day. Despite the restriction of these phenotypes to the day, the circadian clock does not regulate qrfp expression, and entrained circadian rhythms are not required for QRFP-induced rest. Instead, we find that QRFP overexpression decreases locomotor activity largely in a light-specific manner. Our results suggest that QRFP signaling plays an important role in promoting sleep and may underlie some aspects of hypothalamic sleep control. SIGNIFICANCE STATEMENT: The hypothalamus is thought to play a key role in regulating sleep in vertebrate animals, but few sleep-promoting signaling pathways that function in the hypothalamus have been identified. Here we use the zebrafish, a diurnal vertebrate, to functionally and anatomically characterize the neuropeptide QRFP. We show that QRFP is exclusively expressed in a small number of neurons in the larval zebrafish hypothalamus that project widely in the brain. We also show that QRFP overexpression reduces locomotor activity, whereas animals that lack QRFP signaling are more active and sleep less. These results suggest that QRFP signaling participates in the hypothalamic regulation of sleep.

Mechanisms of amphetamine action illuminated through optical monitoring of dopamine synaptic vesicles in Drosophila brain.

Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. Here we show in rodents that acute pharmacological inhibition of the vesicular monoamine transporter (VMAT) blocks amphetamine-induced locomotion and self-administration without impacting cocaine-induced behaviours. To study VMAT's role in mediating amphetamine action in dopamine neurons, we have used novel genetic, pharmacological and optical approaches in Drosophila melanogaster. In an ex vivo whole-brain preparation, fluorescent reporters of vesicular cargo and of vesicular pH reveal that amphetamine redistributes vesicle contents and diminishes the vesicle pH-gradient responsible for dopamine uptake and retention. This amphetamine-induced deacidification requires VMAT function and results from net H(+) antiport by VMAT out of the vesicle lumen coupled to inward amphetamine transport. Amphetamine-induced vesicle deacidification also requires functional dopamine transporter (DAT) at the plasma membrane. Thus, we find that at pharmacologically relevant concentrations, amphetamines must be actively transported by DAT and VMAT in tandem to produce psychostimulant effects.

Ziram, a pesticide associated with increased risk for Parkinson's disease, differentially affects the presynaptic function of aminergic and glutamatergic nerve terminals at the Drosophila neuromuscular junction.

Multiple populations of aminergic neurons are affected in Parkinson's disease (PD), with serotonergic and noradrenergic loci responsible for some non-motor symptoms. Environmental toxins, such as the dithiocarbamate fungicide ziram, significantly increase the risk of developing PD and the attendant spectrum of both motor and non-motor symptoms. The mechanisms by which ziram and other environmental toxins increase the risk of PD, and the potential effects of these toxins on aminergic neurons, remain unclear. To determine the relative effects of ziram on the synaptic function of aminergic versus non-aminergic neurons, we used live-imaging at the Drosophila melanogaster larval neuromuscular junction (NMJ). In contrast to nearly all other studies of this model synapse, we imaged presynaptic function at both glutamatergic Type Ib and aminergic Type II boutons, the latter responsible for storage and release of octopamine, the invertebrate equivalent of noradrenalin. To quantify the kinetics of exo- and endo-cytosis, we employed an acid-sensitive form of GFP fused to the Drosophila vesicular monoamine transporter (DVMAT-pHluorin). Additional genetic probes were used to visualize intracellular calcium flux (GCaMP) and voltage changes (ArcLight). We find that at glutamatergic Type Ib terminals, exposure to ziram increases exocytosis and inhibits endocytosis. By contrast, at octopaminergic Type II terminals, ziram has no detectable effect on exocytosis and dramatically inhibits endocytosis. In contrast to other reports on the neuronal effects of ziram, these effects do not appear to result from perturbation of the Ubiquitin Proteasome System (UPS) or calcium homeostasis. Unexpectedly, ziram also caused spontaneous and synchronized bursts of calcium influx (measured by GCaMP) and electrical activity (measured by ArcLight) at aminergic Type II, but not glutamatergic Type Ib, nerve terminals. These events are sensitive to both tetrodotoxin and cadmium chloride, and thus appear to represent spontaneous depolarizations followed by calcium influx into Type II terminals. We speculate that the differential effects of ziram on Type II versus Type Ib terminals may be relevant to the specific sensitivity of aminergic neurons in PD, and suggest that changes in neuronal excitability could contribute to the increased risk for PD caused by exposure to ziram. We also suggest that the fly NMJ will be useful to explore the synaptic effects of other pesticides associated with an increased risk of PD.

The redistribution of Drosophila vesicular monoamine transporter mutants from synaptic vesicles to large dense-core vesicles impairs amine-dependent behaviors.

Monoamine neurotransmitters are stored in both synaptic vesicles (SVs), which are required for release at the synapse, and large dense-core vesicles (LDCVs), which mediate extrasynaptic release. The contributions of each type of vesicular release to specific behaviors are not known. To address this issue, we generated mutations in the C-terminal trafficking domain of the Drosophila vesicular monoamine transporter (DVMAT), which is required for the vesicular storage of monoamines in both SVs and LDCVs. Deletion of the terminal 23 aa (DVMAT-Δ3) reduced the rate of endocytosis and localization of DVMAT to SVs, but supported localization to LDCVs. An alanine substitution mutation in a tyrosine-based motif (DVMAT-Y600A) also reduced sorting to SVs and showed an endocytic deficit specific to aminergic nerve terminals. Redistribution of DVMAT-Y600A from SV to LDCV fractions was also enhanced in aminergic neurons. To determine how these changes might affect behavior, we expressed DVMAT-Δ3 and DVMAT-Y600A in a dVMAT null genetic background that lacks endogenous dVMAT activity. When expressed ubiquitously, DVMAT-Δ3 showed a specific deficit in female fertility, whereas DVMAT-Y600A rescued behavior similarly to DVMAT-wt. In contrast, when expressed more specifically in octopaminergic neurons, both DVMAT-Δ3 and DVMAT-Y600A failed to rescue female fertility, and DVMAT-Y600A showed deficits in larval locomotion. DVMAT-Y600A also showed more severe dominant effects than either DVMAT-wt or DVMAT-Δ3. We propose that these behavioral deficits result from the redistribution of DVMAT from SVs to LDCVs. By extension, our data suggest that the balance of amine release from SVs versus that from LDCVs is critical for the function of some aminergic circuits.

Dispensable, redundant, complementary, and cooperative roles of dopamine, octopamine, and serotonin in Drosophila melanogaster.

To investigate the regulation of Drosophila melanogaster behavior by biogenic amines, we have exploited the broad requirement of the vesicular monoamine transporter (VMAT) for the vesicular storage and exocytotic release of all monoamine neurotransmitters. We used the Drosophila VMAT (dVMAT) null mutant to globally ablate exocytotic amine release and then restored DVMAT activity in either individual or multiple aminergic systems, using transgenic rescue techniques. We find that larval survival, larval locomotion, and female fertility rely predominantly on octopaminergic circuits with little apparent input from the vesicular release of serotonin or dopamine. In contrast, male courtship and fertility can be rescued by expressing DVMAT in octopaminergic or dopaminergic neurons, suggesting potentially redundant circuits. Rescue of major aspects of adult locomotion and startle behavior required octopamine, but a complementary role was observed for serotonin. Interestingly, adult circadian behavior could not be rescued by expression of DVMAT in a single subtype of aminergic neurons, but required at least two systems, suggesting the possibility of unexpected cooperative interactions. Further experiments using this model will help determine how multiple aminergic systems may contribute to the regulation of other behaviors. Our data also highlight potential differences between behaviors regulated by standard exocytotic release and those regulated by other mechanisms.

Induction of immunotolerance via mPEG grafting to allogeneic leukocytes.

The induction of anergy or tolerance to prevent allorecognition is of clinical interest. To this end, the effects of methoxypoly(ethylene glycol) [mPEG] grafting to allogeneic lymphocytes on proliferation and phenotype (Th17 and Treg) was examined in vitro and in vivo. Control studies demonstrated that PEGylation did not affect cells viability or proliferation (mitogen) potential. Conditioned media (1° MLR) collected at 72 h from resting PBMC demonstrated no immunomodulatory effects whereas the control MLR demonstrated significant (p < 0.001) pro-proliferative potential and significantly increased in IL-2, TNF-α, and INF-γ. However, 1° media from either resting mPEG-PBMC or the PEGylated MLR resulted in a significant inhibitory effect (p < 0.001) in the 2° MLR and no increase in cytokines. PEGylation of donor murine splenocytes resulted in significant in vivo immunosuppressive effects in H2-disparate mice. While unmodified allogeneic splenocytes resulted in a significant in vivo decrease in Treg and increased Th17 lymphocytes, PEGylated allogeneic splenocytes yielded significantly increased Tregs and baseline levels of Th17 lymphocytes. This effect was persistent to at least 30 days post challenge and was not reversed by unmodified allogeneic cells. These studies demonstrate that PEGylation of allogeneic lymphocytes induced an immunoquiescent state both in vitro and in vivo.

Vesicular neurotransmitter transporter trafficking in vivo: moving from cells to flies.

During exocytosis, classical and amino acid neurotransmitters are released from the lumen of synaptic vesicles to allow signaling at the synapse. The storage of neurotransmitters in synaptic vesicles and other types of secretory vesicles requires the activity of specific vesicular transporters. Glutamate and monoamines such as dopamine are packaged by VGLUTs and VMATs respectively. Changes in the localization of either protein have the potential to up- or down regulate neurotransmitter release, and some of the mechanisms for sorting these proteins to secretory vesicles have been investigated in cultured cells in vitro. We have used Drosophila molecular genetic techniques to study vesicular transporter trafficking in an intact organism and have identified a motif required for localizing Drosophila VMAT (DVMAT) to synaptic vesicles in vivo. In contrast to DVMAT, large deletions of Drosophila VGLUT (DVGLUT) show relatively modest deficits in localizing to synaptic vesicles, suggesting that DVMAT and DVGLUT may undergo different modes of trafficking at the synapse. Further in vivo studies of DVMAT trafficking mutants will allow us to determine how changes in the localization of vesicular transporters affect the nervous system as a whole and complex behaviors mediated by aminergic circuits.