Retinoid orphan receptor gamma t (rorγt) promotes inflammatory eosinophilia but is dispensable for innate immune-mediated colitis.

Inflammatory bowel diseases (IBD) result from uncontrolled inflammation in the intestinal mucosa leading to damage and loss of function. Both innate and adaptive immunity contribute to the inflammation of IBD and innate and adaptive immune cells reciprocally activate each other in a forward feedback loop. In order to better understand innate immune contributions to IBD, we developed a model of spontaneous 100% penetrant, early onset colitis that occurs in the absence of adaptive immunity by crossing villin-TNFAIP3 mice to RAG1-/- mice (TRAG mice). This model is driven by microbes and features increased levels of innate lymphoid cells in the intestinal mucosa. To investigate the role of type 3 innate lymphoid cells (ILC3) in the innate colitis of TRAG mice, we crossed them to retinoid orphan receptor gamma t deficient (Rorγt-/-) mice. Rorγt-/- x TRAG mice exhibited markedly reduced eosinophilia in the colonic mucosa, but colitis persisted in these mice. Colitis in Rorγt-/- x TRAG mice was characterized by increased infiltration of the intestinal mucosa by neutrophils, inflammatory monocytes, macrophages and other innate cells. RNA and cellular profiles of Rorγt-/- x TRAG mice were consistent with a lack of ILC3 and ILC3 derived cytokines, reduced antimicrobial factors, increased activation oof epithelial repair processes and reduced activation of epithelial cell STAT3. The colitis in Rorγt-/- x TRAG mice was ameliorated by antibiotic treatment indicating that microbes contribute to the ILC3-independent colitis of these mice. Together, these gene expression and cell signaling signatures reflect the double-edged sword of ILC3 in the intestine, inducing both proinflammatory and antimicrobial protective responses. Thus, Rorγt promotes eosinophilia but Rorγt and Rorγt-dependent ILC3 are dispensable for the innate colitis in TRAG mice.

Handling Difficult Cryo-ET Samples: A Study with Primary Neurons from Drosophila melanogaster.

Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons having been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems.

Handling difficult cryo-ET samples: A study with primary neurons from Drosophila melanogaster.

Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems.

Altered microbial biogeography in an innate model of colitis.

Changes in the spatial organization, or biogeography, of colonic microbes have been observed in human inflammatory bowel disease (IBD) and mouse models of IBD. We have developed a mouse model of IBD that occurs spontaneously and consistently in the absence of adaptive immunity. Mice expressing tumor necrosis factor-induced protein 3 (TNFAIP3) in intestinal epithelial cells (villin-TNFAIP3) develop colitis when interbred with Recombination Activating 1-deficient mice (RAG1^-/-). The colitis in villin-TNFAIP3 × RAG1^-/- (TRAG) mice is prevented by antibiotics, indicating a role for microbes in this innate colitis. We therefore explored the biogeography of microbes and responses to antibiotics in TRAG colitis. Laser capture microdissection and 16S rRNA sequencing revealed altered microbial populations across the transverse axis of the colon as the inner mucus layer of TRAG, but not RAG1^-/-, mice was infiltrated by microbes, which included increased abundance of the classes Gammaproteobacteria and Actinobacteria. Along the longitudinal axis differences in the efficacy of antibiotics to prevent colitis were evident. Neomycin was most effective for prevention of inflammation in the cecum, while ampicillin was most effective in the proximal and distal colon. RAG1^-/-, but not TRAG, mice exhibited a structured pattern of bacterial abundance with decreased Firmicutes and Proteobacteria but increased Bacteroidetes along the proximal to distal axis of the gut. TRAG mice exhibited increased relative abundance of potential pathobionts including <i>Bifidobacterium animalis</i> along the longitudinal axis of the gut whereas others, like <i>Helicobacter hepaticus</i> were increased only in the cecum. Potential beneficial organisms including <i>Roseburia</i> were decreased in the proximal regions of the TRAG colon, while <i>Bifidobacterium pseudolongulum</i> was decreased in the TRAG distal colon. Thus, the innate immune system maintains a structured, spatially organized, gut microbiome along the transverse and longitudinal axis of the gut, and disruption of this biogeography is a feature of innate immune colitis.

Transcriptional regulation of MGE progenitor proliferation by PRDM16 controls cortical GABAergic interneuron production.

The mammalian cortex is populated by neurons derived from neural progenitors located throughout the embryonic telencephalon. Excitatory neurons are derived from the dorsal telencephalon, whereas inhibitory interneurons are generated in its ventral portion. The transcriptional regulator PRDM16 is expressed by radial glia, neural progenitors present in both regions; however, its mechanisms of action are still not fully understood. It is unclear whether PRDM16 plays a similar role in neurogenesis in both dorsal and ventral progenitor lineages and, if so, whether it regulates common or unique networks of genes. Here, we show that Prdm16 expression in mouse medial ganglionic eminence (MGE) progenitors is required for maintaining their proliferative capacity and for the production of proper numbers of forebrain GABAergic interneurons. PRDM16 binds to cis-regulatory elements and represses the expression of region-specific neuronal differentiation genes, thereby controlling the timing of neuronal maturation. PRDM16 regulates convergent developmental gene expression programs in the cortex and MGE, which utilize both common and region-specific sets of genes to control the proliferative capacity of neural progenitors, ensuring the generation of correct numbers of cortical neurons.

Age predicts the absence of caloric-induced vertigo.

INTRODUCTION: The absence of vertigo during the caloric test, despite a robust response, has been suggested to represent a central vestibular system phenomenon. The purpose of this investigation was to determine the prevalence of absent caloric-induced vertigo perception in an unselected group of patients and to assess possible predicting variables. METHODS: Prospective investigation of 92 unselected patients who underwent caloric testing. Inclusion criteria were that each patient generate a maximum slow phase velocity (maxSPV) ≥ 15 deg/sec and a caloric asymmetry of ≤10%. Following the caloric, patients were asked, "Did you have any sensation of motion?" RESULTS: Results showed 75% of patients reported motion with a mean age of 56.51 years compared to a mean age of 66.55 in the 25% of patients reporting an absence of motion. A logistic regression was performed and the overall model was statistically significant accounting for 29% of the variance in caloric perception. The significant predictor variables were patient age and maxSPV of the caloric response. The effect size for both variables was small with an odds ratio of .9 for maxSPV and 1.06 for age. CONCLUSIONS: The current investigation showed that both age and maxSPV of the caloric response were significant predictors of vertigo perception during the caloric exam. However, the association between age and caloric perception is not conclusive. Although there is evidence to suggest that these findings represent age-related changes in the central processing of vestibular system stimulation, there are additional unmeasured factors that influence the perception of caloric-induced vertigo.

Assessment of Subjective Visual Vertical (SVV) Using the "Bucket Test" and the Virtual SVV System.

OBJECTIVE: The purpose of this study was to describe the variability and test-retest reliability of a commercially available subjective visual vertical (SVV) system known as Virtual SVV (Interacoustics). In addition, the study aimed to compare the reliability of the Virtual system with a previously established bucket test of SVV. STUDY DESIGN: Fifteen participants with normal hearing, normal middle ear function, and normal utricular function were included in the study. Each participant underwent static SVV testing using both the Virtual system and the bucket test. Subjects completed 2 testing sessions to determine test-retest reliability. For each test, data were collected with the head at 0°, tilted 45° to the right, and tilted 45° to the left. SETTING: This study was conducted in a balance function laboratory embedded in a large, tertiary care otology clinic. RESULTS: The mean SVV values obtained with the Virtual system were within 1°-2° from 0 with the head positioned at 0°, which is in agreement with many other studies of SVV with the head at 0° (Akin & Murnane, 2009; Halmagyi & Curthoys, 1999; Zwergal, Rettinger, Frenzel, Dieterich, & Strupp, 2009). Using the intraclass correlation coefficient, test-retest reliability of the Virtual system was excellent in the 45° left position and fair to good in the 45° right and 0° position. Test-retest reliability of the bucket test was poor in all head positions. CONCLUSIONS: The Virtual system is a more reliable measure of static SVV than the bucket test. Therefore, the Virtual system could be utilized as a screening device for utricular dysfunction in busy clinical settings.

Variation in crocodilian dorsal scute organization and geometry with a discussion of possible functional implications.

Dermal ossifications, including osteoderms, are present in many vertebrates and are frequently interpreted as a defense against predators. Nevertheless, osteoderms remain ubiquitous in adult crocodilians while being absent in hatchlings, even though adults rarely experience predation. In other biological systems, increased variation, particularly fluctuating asymmetry, have proven useful for identifying biological structures likely to have evolved under relaxed selection, which in turn may inform their function. Therefore, using the keratinous scutes as proxies for the underlying osteoderm morphology, I investigated the average intraspecific variability of geometry and fluctuating asymmetry in dorsal scutes in five species of crocodilians. I first tested for differences in variability of scute length and width, then for differences in bilateral fluctuating asymmetry of scute number, before finally investigating scute distribution patterns for each species compared to hypothetical rectangular and hexagonal scute arrangements. The American crocodile, Crocodylus acutus, shows significantly more asymmetry than other species, which is consistent with relaxed selection on osteoderms in this species. A suspected decrease in intraspecific aggression within Crocodylus acutus, in conjunction with the inferred relaxed selection, suggests that, in general, crocodilian osteoderms function primarily as defensive armor in aggressive encounters with conspecifics. The smooth-fronted caiman, Paleosuchus trigonatus, exhibits increased variation in scute dimensions linked to the mediolateral offset of osteoderms in adjacent rows, possibly resulting in a more rigid carapace. Unfortunately, comparative data on crocodilian behavior, physiology, and development is extremely limited and restricts the ability to explore other potential explanations for the patterns observed, highlighting the need for more research on rare and cryptic crocodylians.

The PAediatric Risk Assessment (PARA) Mobile App to Reduce Postdischarge Child Mortality: Design, Usability, and Feasibility for Health Care Workers in Uganda.

BACKGROUND: Postdischarge death in children is increasingly being recognized as a major contributor to overall child mortality. The PAediatric Risk Assessment (PARA) app is an mHealth tool developed to aid health care workers in resource-limited settings such as Sub-Saharan Africa to identify pediatric patients at high risk of both in-hospital and postdischarge mortality. The intended users of the PARA app are health care workers (ie, nurses, doctors, and clinical officers) with varying levels of education and technological exposure, making testing of this clinical tool critical to successful implementation. OBJECTIVE: Our aim was to summarize the usability evaluation of the PARA app among target users, which consists of assessing the ease of use, functionality, and navigation of the interfaces and then iteratively improving the design of this clinical tool. METHODS: Health care workers (N=30) were recruited to participate at Mbarara Regional Referral Hospital and Holy Innocents Children's Hospital in Mbarara, Southwestern Uganda. This usability study was conducted in two phases to allow for iterative improvement and testing of the interfaces. The PARA app was evaluated using quantitative and qualitative measures, which were compared between Phases 1 and 2 of the study. Participants were given two patient scenarios that listed hypothetical information (ie, demographic, social, and clinical data) to be entered into the app and to determine the patient's risk of in-hospital and postdischarge mortality. Time-to-completion and user errors were recorded for each participant while using the app. A modified computer system usability questionnaire was utilized at the end of each session to elicit user satisfaction with the PARA app and obtain suggestions for future improvements. RESULTS: The average time to complete the PARA app decreased by 30% from Phase 1 to Phase 2, following user feedback and modifications. Participants spent the longest amount of time on the oxygen saturation interface, but modifications following Phase 1 cut this time by half. The average time-to-completion (during Phase 2) for doctors/medical students was 3 minutes 56 seconds. All participants agreed they would use the PARA app if available at their health facility. Given a high PARA risk score, participants suggested several interventions that would be appropriate for the sociocultural context in southwestern Uganda, which involved strengthening discharge and referral procedures within the current health care system. CONCLUSIONS: Through feedback and modifications made during this usability study, the PARA app was developed into a user-friendly app, encompassing user expectations and culturally intuitive interfaces for users with a range of technological exposure. Doctors and medical students had shorter task completion times, though all participants reported the usefulness of this tool to improve postdischarge outcomes.

The development of the vanderbilt pediatric dizziness handicap inventory for patient caregivers (DHI-PC).

PURPOSE: The purpose of the present investigation was to develop a psychometrically sound dizziness disability/handicap outcome measure for use with a pediatric population between 5 and 12 years of age. METHODS: Items comprising Phase 1 of the DHI-PC were created based on reports from parents, providers and patients. This version was administered to the caregivers (mean age 31.6 years, sd 5 years, 74 female) of 86 pediatric patients (mean age 9 years, sd=2.83 years, 45 female). The caregiver's responses to each item were limited to "yes" (scored as 4 points), "sometimes" (scored as 2 points) or "no" (scored as zero points). RESULTS: A factor analysis for Phase 1 of the scale showed there to be a single factor (eigenvalue of 11.51) that explained 29% of the total variance. The results of Cronbach's alpha analysis enabled us to eliminate 15 items reducing the scale to 25 items (i.e. Phase 2 of the DHI-PC). Following elimination of the items with low item-total coefficients, the second phase of the DHI-PC was administered to 56 legal guardians (mean patient age 8 years, sd 4.65 years, 37 female). The analysis of this data again showed there to be a single factor (eigenvalue of 8.30) that explained 33% of the variance. Four items demonstrated item-total correlations less than 0.40. The final version of the DHI-PC has 21 items and a maximum score of 84%. Short-term test-retest reliability (i.e. three week interval between test and retest) of this DHI-PC was assessed for a subset of 10 patients (caregivers, mean age 38 years, sd=7 years, 10 female). The results indicated the short-term, test-retest reliability to be strong (r=0.98, p≤0.001). CONCLUSION: The DHI-PC represents a new tool for assessing the impact of pediatric dizziness on the patient (as viewed through the perspective of the caregiver). This tool may be incorporated into the comprehensive evaluation of children suffering from dizziness.