Cutaneous TRPV1+ Neurons Trigger Protective Innate Type 17 Anticipatory Immunity.

Cutaneous TRPV1+ neurons directly sense noxious stimuli, inflammatory cytokines, and pathogen-associated molecules and are required for innate immunity against some skin pathogens. Important unanswered questions are whether TRPV1+ neuron activation in isolation is sufficient to initiate innate immune responses and what is the biological function for TRPV1+ neuron-initiated immune responses. We used TRPV1-Ai32 optogenetic mice and cutaneous light stimulation to activate cutaneous neurons in the absence of tissue damage or pathogen-associated products. We found that TRPV1+ neuron activation was sufficient to elicit a local type 17 immune response that augmented host defense to C. albicans and S. aureus. Moreover, local neuron activation elicited type 17 responses and augmented host defense at adjacent, unstimulated skin through a nerve reflex arc. These data show the sufficiency of TRPV1+ neuron activation for host defense and demonstrate the existence of functional anticipatory innate immunity at sites adjacent to infection that depends on antidromic neuron activation.

Extreme genome scrambling in marine planktonic Oikopleura dioica cryptic species.

Genome structural variations within species are rare. How selective constraints preserve gene order and chromosome structure is a central question in evolutionary biology that remains unsolved. Our sequencing of several genomes of the appendicularian tunicate Oikopleura dioica around the globe reveals extreme genome scrambling caused by thousands of chromosomal rearrangements, although showing no obvious morphological differences between these animals. The breakpoint accumulation rate is an order of magnitude higher than in ascidian tunicates, nematodes, Drosophila, or mammals. Chromosome arms and sex-specific regions appear to be the primary unit of macrosynteny conservation. At the microsyntenic level, scrambling did not preserve operon structures, suggesting an absence of selective pressure to maintain them. The uncoupling of the genome scrambling with morphological conservation in O. dioica suggests the presence of previously unnoticed cryptic species and provides a new biological system that challenges our previous vision of speciation in which similar animals always share similar genome structures.

An Electronic Health Record-Based Automated Self-Rescheduling Tool to Improve Patient Access: Retrospective Cohort Study.

BACKGROUND: In many large health centers, patients face long appointment wait times and difficulties accessing care. Last-minute cancellations and patient no-shows leave unfilled slots in a clinician's schedule, exacerbating delays in care from poor access. The mismatch between the supply of outpatient appointments and patient demand has led health systems to adopt many tools and strategies to minimize appointment no-show rates and fill open slots left by patient cancellations. OBJECTIVE: We evaluated an electronic health record (EHR)-based self-scheduling tool, Fast Pass, at a large academic medical center to understand the impacts of the tool on the ability to fill cancelled appointment slots, patient access to earlier appointments, and clinical revenue from visits that may otherwise have gone unscheduled. METHODS: In this retrospective cohort study, we extracted Fast Pass appointment offers and scheduling data, including patient demographics, from the EHR between June 18, 2022, and March 9, 2023. We analyzed the outcomes of Fast Pass offers (accepted, declined, expired, and unavailable) and the outcomes of scheduled appointments resulting from accepted Fast Pass offers (completed, canceled, and no-show). We stratified outcomes based on appointment specialty. For each specialty, the patient service revenue from appointments filled by Fast Pass was calculated using the visit slots filled, the payer mix of the appointments, and the contribution margin by payer. RESULTS: From June 18 to March 9, 2023, there were a total of 60,660 Fast Pass offers sent to patients for 21,978 available appointments. Of these offers, 6603 (11%) were accepted across all departments, and 5399 (8.9%) visits were completed. Patients were seen a median (IQR) of 14 (4-33) days sooner for their appointments. In a multivariate logistic regression model with primary outcome Fast Pass offer acceptance, patients who were aged 65 years or older (vs 20-40 years; P=.005 odds ratio [OR] 0.86, 95% CI 0.78-0.96), other ethnicity (vs White; P<.001, OR 0.84, 95% CI 0.77-0.91), primarily Chinese speakers (P<.001; OR 0.62, 95% CI 0.49-0.79), and other language speakers (vs English speakers; P=.001; OR 0.71, 95% CI 0.57-0.87) were less likely to accept an offer. Fast Pass added 2576 patient service hours to the clinical schedule, with a median (IQR) of 251 (216-322) hours per month. The estimated value of physician fees from these visits scheduled through 9 months of Fast Pass scheduling in professional fees at our institution was US $3 million. CONCLUSIONS: Self-scheduling tools that provide patients with an opportunity to schedule into cancelled or unfilled appointment slots have the potential to improve patient access and efficiently capture additional revenue from filling unfilled slots. The demographics of the patients accepting these offers suggest that such digital tools may exacerbate inequities in access.

Neuroimmune interactions in atopic and allergic contact dermatitis.

The skin is a barrier organ populated by many types of skin-resident immune cells and sensory neurons. It has become increasingly appreciated that neuroimmune interactions are an important component of inflammatory diseases such as atopic dermatitis and allergic contact dermatitis. Neuropeptides secreted from nerve terminals play an important role in mediating cutaneous immune cell function, and soluble mediators derived from immune cells interact with neurons to induce itch. In this review article, we will explore emerging research describing neuronal effector functions on skin immune cells in mouse models of atopic and contact dermatitis. We will also discuss the contributions of both specific neuronal subsets and secreted immune factors to itch induction and the associated inflammatory processes. Finally, we will explore how treatment strategies have emerged around these findings and discuss the relationship between scratching and dermatitis.

Telomere-to-telomere assembly of the genome of an individual Oikopleura dioica from Okinawa using Nanopore-based sequencing.

BACKGROUND: The larvacean Oikopleura dioica is an abundant tunicate plankton with the smallest (65-70 Mbp) non-parasitic, non-extremophile animal genome identified to date. Currently, there are two genomes available for the Bergen (OdB3) and Osaka (OSKA2016) O. dioica laboratory strains. Both assemblies have full genome coverage and high sequence accuracy. However, a chromosome-scale assembly has not yet been achieved. RESULTS: Here, we present a chromosome-scale genome assembly (OKI2018_I69) of the Okinawan O. dioica produced using long-read Nanopore and short-read Illumina sequencing data from a single male, combined with Hi-C chromosomal conformation capture data for scaffolding. The OKI2018_I69 assembly has a total length of 64.3 Mbp distributed among 19 scaffolds. 99% of the assembly is contained within five megabase-scale scaffolds. We found telomeres on both ends of the two largest scaffolds, which represent assemblies of two fully contiguous autosomal chromosomes. Each of the other three large scaffolds have telomeres at one end only and we propose that they correspond to sex chromosomes split into a pseudo-autosomal region and X-specific or Y-specific regions. Indeed, these five scaffolds mostly correspond to equivalent linkage groups in OdB3, suggesting overall agreement in chromosomal organization between the two populations. At a more detailed level, the OKI2018_I69 assembly possesses similar genomic features in gene content and repetitive elements reported for OdB3. The Hi-C map suggests few reciprocal interactions between chromosome arms. At the sequence level, multiple genomic features such as GC content and repetitive elements are distributed differently along the short and long arms of the same chromosome. CONCLUSIONS: We show that a hybrid approach of integrating multiple sequencing technologies with chromosome conformation information results in an accurate de novo chromosome-scale assembly of O. dioica's highly polymorphic genome. This genome assembly opens up the possibility of cross-genome comparison between O. dioica populations, as well as of studies of chromosomal evolution in this lineage.

The nucleus accumbens and inhibition in the ventral tegmental area play a causal role in the Kamin blocking effect.

The discovery of Kamin blocking led to the idea that associative learning occurs only when there is a mismatch between actual and predicted outcomes, or prediction error. The neural substrates involved in regulating this prediction error during behavioral learning are still not fully elucidated. We investigated in rats the role of the ventral tegmental area and the nucleus accumbens in Kamin blocking. Our blocking paradigm involved three phases: appetitive classical conditioning of a lever cue, conditioning of a compound of the lever cue plus an auditory cue, and testing response to the auditory cue in extinction. We found that disruption of inhibition in the ventral tegmental area by bicuculline, or designer receptor mediated inactivation of the nucleus accumbens, during compound cue conditioning, attenuated Kamin blocking. These results suggest that inhibition in the ventral tegmental area and inhibitory output from the nucleus accumbens are necessary for blocking and make behaviorally significant contributions to the computation of reward prediction error. In addition, we found that inactivating the neurons in the nucleus accumbens during classical conditioning of the lever cue also attenuated blocking, without affecting classical conditioning of the lever. This indicates that learning in the nucleus accumbens is necessary for blocking and reward estimation. Our results reveal a causal role for nucleus accumbens modulated inhibitory inputs to the ventral tegmental area in the blocking effect and suggest that they contribute to computation of reward prediction error during associative learning.

Cholinergic interneurons in the rat striatum modulate substitution of habits.

Behavioural flexibility is crucial for adaptive behaviour, and recent evidence suggests that cholinergic interneurons of the striatum play a distinct role. Previous studies of cholinergic function have focused on strategy switching by the dorsomedial or ventral striatum. We here investigated whether cholinergic interneurons in the dorsolateral striatum play a similar role at the level of switching of habitual responses. Because the dorsolateral striatum is particularly involved in habitual responding, we developed a habit substitution task that involved switching habitual lever-press responses to one side to another. We first measured the effect of cholinergic activation in the dorsolateral striatum on this task. Chemogenetic activation of cholinergic interneurons caused an increase in the response rate for the substituted response that was significantly greater than the increase normally seen in control animals. The increase was due to burst-like responses with shorter inter-press intervals. However, there was no effect on inhibiting the old habit, or on habitual responding that did not require a switch. There was also no effect on lever-press performance and its reversal before lever-press responses became habitual. Conversely, neurochemically specific ablation of cholinergic interneurons did not significantly change habitual responding or response substitution. Thus, activation -but not ablation -of cholinergic interneurons in the dorsolateral striatum modulates expression of a new habit when an old habit is replaced by a new one. Together with previous work, this suggests that striatal cholinergic interneurons facilitate behavioural flexibility in both dorsolateral striatum in addition to dorsomedial and ventral striatum.

Role of Striatal Cholinergic Interneurons in Set-Shifting in the Rat.

The ability to change strategies in different contexts is a form of behavioral flexibility that is crucial for adaptive behavior. The striatum has been shown to contribute to certain forms of behavioral flexibility such as reversal learning. Here we report on the contribution of striatal cholinergic interneurons-a key element in the striatal neuronal circuit-to strategy set-shifting in which an attentional shift from one stimulus dimension to another is required. We made lesions of rat cholinergic interneurons in dorsomedial or ventral striatum using a specific immunotoxin and investigated the effects on set-shifting paradigms and on reversal learning. In shifting to a set that required attention to a previously irrelevant cue, lesions of dorsomedial striatum significantly increased the number of perseverative errors. In this condition, the number of never-reinforced errors was significantly decreased in both types of lesions. When shifting to a set that required attention to a novel cue, rats with ventral striatum lesions made more perseverative errors. Neither lesion impaired learning of the initial response strategy nor a subsequent switch to a new strategy when response choice was indicated by a previously relevant cue. Reversal learning was not affected. These results suggest that in set-shifting the striatal cholinergic interneurons play a fundamental role, which is dissociable between dorsomedial and ventral striatum depending on behavioral context. We propose a common mechanism in which cholinergic interneurons inhibit neurons representing the old strategy and enhance plasticity underlying exploration of a new rule.

Behavioral flexibility is increased by optogenetic inhibition of neurons in the nucleus accumbens shell during specific time segments.

Behavioral flexibility is vital for survival in an environment of changing contingencies. The nucleus accumbens may play an important role in behavioral flexibility, representing learned stimulus-reward associations in neural activity during response selection and learning from results. To investigate the role of nucleus accumbens neural activity in behavioral flexibility, we used light-activated halorhodopsin to inhibit nucleus accumbens shell neurons during specific time segments of a bar-pressing task requiring a win-stay/lose-shift strategy. We found that optogenetic inhibition during action selection in the time segment preceding a lever press had no effect on performance. However, inhibition occurring in the time segment during feedback of results--whether rewards or nonrewards--reduced the errors that occurred after a change in contingency. Our results demonstrate critical time segments during which nucleus accumbens shell neurons integrate feedback into subsequent responses. Inhibiting nucleus accumbens shell neurons in these time segments, during reinforced performance or after a change in contingencies, increases lose-shift behavior. We propose that the activity of nucleus shell accumbens shell neurons in these time segments plays a key role in integrating knowledge of results into subsequent behavior, as well as in modulating lose-shift behavior when contingencies change.

Patient Engagement With and Perspectives on a Mobile Health Home Spirometry Intervention: Mixed Methods Study.

BACKGROUND: Patient engagement attrition in mobile health (mHealth) remote patient monitoring (RPM) programs decreases program benefits. Systemic disparities lead to inequities in RPM adoption and use. There is an urgent need to understand patients' experiences with RPM in the real world, especially for patients who have stopped using the programs, as addressing issues faced by patients can increase the value of mHealth for patients and subsequently decrease attrition. OBJECTIVE: This study sought to understand patient engagement and experiences in an RPM mHealth intervention in lung transplant recipients. METHODS: Between May 4, 2020, and November 1, 2022, a total of 601 lung transplant recipients were enrolled in an mHealth RPM intervention to monitor lung function. The predictors of patient engagement were evaluated using multivariable logistic and linear regression. Semistructured interviews were conducted with 6 of 39 patients who had engaged in the first month but stopped using the program, and common themes were identified. RESULTS: Patients who underwent transplant more than 1 year before enrollment in the program had 84% lower odds of engaging (odds ratio [OR] 0.16, 95% CI 0.07-0.35), 82% lower odds of submitting pulmonary function measurements (OR 0.18, 95% CI 0.09-0.33), and 78% lower odds of completing symptom checklists (OR 0.22, 95% CI 0.10-0.43). Patients whose primary language was not English had 78% lower odds of engaging compared to English speakers (OR 0.22, 95% CI 0.07-0.67). Interviews revealed 4 prominent themes: challenges with devices, communication breakdowns, a desire for more personal interactions and specific feedback with the care team about their results, understanding the purpose of the chat, and understanding how their data are used. CONCLUSIONS: Care delivery and patient experiences with RPM in lung transplant mHealth can be improved and made more equitable by tailoring outreach and enhancements toward non-English speakers and patients with a longer time between transplant and enrollment. Attention to designing programs to provide personalization through supplementary provider contact, education, and information transparency may decrease attrition rates.

Impact of an automated peri-procedural digital health intervention on rates of emergency department visits and readmissions.

BACKGROUND: Providing timely peri-procedural education, reminders, and check-ins can improve patient adherence and clinical outcomes. We sought to retrospectively evaluate the impact of a peri-procedural digital health tool on emergency department (ED) visits and readmissions. METHODS: A digital health tool for peri-procedural care engaged patients at scheduled intervals, resulting in an overall engagement score. Multivariate models determined predictors of tool engagement and post-procedural 30- and 90-day rehospitalizations and ED visits. RESULTS: 11,737 unique completed procedures were analyzed from 10,438 patients. Patients of Black and Latinx race/ethnicity (vs White), those with Medicare and Medicaid insurance (vs commercial), and those with non-activated patient portals (vs activated) were less likely to engage. After adjustment for confounders, higher engagement with the tool was associated with lower rates of 30-day hospitalizations (OR 0.64), 90-day hospitalizations (OR 0.65), and 90-day ED visits (OR 0.77). CONCLUSIONS: Highly engaged patients had fewer 30-day and 90-day ED visit and readmissions, even after adjustment for key confounders. Engagement, and thus the resulting benefits, were not equitably distributed.

A Digital Health Intervention to Improve the Clinical Care of Inflammatory Bowel Disease Patients.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic condition that requires close monitoring. Digital health virtual care platforms can enable self-monitoring and allow providers to remotely surveil patients and efficiently identify those with active disease. OBJECTIVES: The primary aim was to design and implement an IBD remote monitoring program, identify predictors of patient engagement, and determine who found the chat to be a valuable tool. METHODS: We developed the IBD Virtual Care Chat, an electronic health record (EHR)-integrated chat to monitor electronic patient reported outcomes (ePROs), medication changes, and disease activity, and subsequently report concerning findings to providers via the EHR. All patients in the IBD practice over age 18 with a clinical encounter in the preceding 12 months were eligible to be enrolled. The primary aim was to identify predictors of patient engagement and determine who found the chat to be a valuable tool. RESULTS: Between May 2021 and March 2022, 2,934 patients were enrolled. A total of 1,160 engaged at least once and 687 (23.4%) continually engaged, submitting at least three ePROs. Disease severity (based on Harvey-Bradshaw Index or Simple Clinical Colitis Activity Index) did not impact ePRO submissions. Patients were significantly more likely to be continually engaged if they self-reported the presence of extraintestinal manifestations (7%, 95% confidence interval: 0.01-0.14; p = 0.04). Patient satisfaction remained moderately high with a median score of 8 (interquartile range: 5-10) on a scale of 1 (poor) to 10 (good). CONCLUSION: Our program demonstrates the potential for EHR-integrated digital health as part of routine IBD care to achieve sustained engagement with high patient satisfaction.

Design and implementation of a digital health home spirometry intervention for remote monitoring of lung transplant function.

BACKGROUND: We developed an automated, chat-based, digital health intervention using Bluetooth-enabled home spirometers to monitor for complications of lung transplantation in a real-world application. METHODS: A chat-based application prompted patients to perform home spirometry, enter their forced expiratory volume in 1 second (FEV1), answer symptom queries, and provided patient education. The program alerted patients and providers to substantial FEV1 decreases and concerning symptoms. Data was integrated into the electronic health record (EHR) system and dashboards were developed for program monitoring. RESULT: Between May 2020 and December 2021, 544 patients were invited to enroll, of whom 427 were invited remotely and 117 were enrolled in-person. 371 (68%) participated by submitting ≥1 FEV1 values. Overall engagement was high, with an average of 197 unique patients submitting FEV1 data per month. In-person enrollees submitted an average of 4.6 FEV1 values per month and responded to 55% of scheduled chats. Home and laboratory FEV1 values correlated closely (rho = 0.93). There was an average of 133 ± 59 FEV1 decline alerts and 59 ± 23 symptom alerts per month. 72% of patients accessed education modules, and the program had a high net promoter score (53) amongst users. CONCLUSIONS: We demonstrate that a novel, automated, chat-based, and EHR-integrated home spirometry intervention is well accepted, generates reliable assessments of graft function, and can deliver automated feedback and education resulting in moderately-high adherence rates. We found that in-person onboarding yields better engagement and adherence. Future work will aim to demonstrate the impact of remote care monitoring on early detection of lung transplant complications.

Automated phenol-chloroform extraction of high molecular weight genomic DNA for use in long-read single-molecule sequencing.

Background: Automation has increasingly become more commonplace in the research laboratory workspace. The introduction of articulated robotic arms allows the researcher more flexibility in the tasks a single piece of automated machinery can perform. We set out to incorporate automation in processing of genomic DNA organic extractions to increase throughput and limit researchers to the exposure of organic solvents. Methods: In order to automate the genome sequencing pipeline in our laboratory, we programmed a dual-arm anthropomorphic robot, the Robotic Biology Institute's Maholo LabDroid, to perform organic solvent-based genomic DNA extraction from cell lysates. To the best of our knowledge, this is the first time that automation of phenol-chloroform extraction has been reported. Results: We achieved routine extraction of high molecular weight genomic DNA (>100 kb) from diverse biological samples including algae cultured in sea water, bacteria, whole insects, and human cell lines. The results of pulse-field electrophoresis size analysis and the N50 sequencing metrics of reads obtained from Nanopore MinION runs verified the presence of intact DNA suitable for direct sequencing. Conclusions: We present the workflow that can be used to program similar robots and discuss the problems and solutions we encountered in developing the workflow. The protocol can be adapted to analogous methods such as RNA extraction, and there is ongoing work to incorporate further post-extraction steps such as library construction. This work shows the potential for automated robotic workflows to free molecular biological researchers from manual interventions in routine experimental work. A time-lapse movie of the entire automated run is included in this report.

Identifying Barriers to Successful Completion of Video Telemedicine Visits in Urology.

OBJECTIVE: The utilization of video telemedicine has dramatically increased due to the COVID-19 pandemic. However, significant social and technological barriers have led to disparities in access. We aimed to identify factors associated with patient inability to successfully initiate a video visit across a high-volume urologic practice. MATERIALS AND METHODS: Video visit completion rates and patient characteristics were extracted from the electronic medical record and linked with census-level socioeconomic data. Associations between video visit failure were identified using multivariate regression modeling and random forest ensemble classification modeling. RESULTS: Six thousand eighty six patients and their first video visits were analyzed. On multivariate logistic regression analysis, Hispanic or Latino patients (OR 0.52, 95%CI 0.31-0.89), patients insured by Medicare (OR 0.46, 95%CI 0.26-0.79) or Medicaid (OR 0.50, 95%CI 0.29-0.87), patients of low socioeconomic status (OR 0.98, 95%CI 0.98-0.99), patients with an un-activated MyChart patient portal (OR 0.43, 95%CI 0.29-0.62), and patients unconfirmed at appointment reminder (OR 0.68, 95%CI 0.48-0.96) were significantly associated with video visit failure. Patients with primary diagnosis category of men's health (OR 47.96, 95%CI 10.24-856.35), and lower urinary tract syndromes (OR 2.69, 95%CI 1.66-4.51) were significantly associated with video visit success. Random forest analyses identified insurance status and socioeconomic status as the top predictors of video visit failure. CONCLUSION: An analysis of a urology video telemedicine cohort reveals clinical and demographic disparities in video visit completion and priorities for future interventions to ensure equity of access. Our study further suggests that specific urologic indications may play a role in success or failure of video visits.

Patient Experience and Feedback After Using an Electronic Health Record-Integrated COVID-19 Symptom Checker: Survey Study.

BACKGROUND: Symptom checkers have been widely used during the COVID-19 pandemic to alleviate strain on health systems and offer patients a 24-7 self-service triage option. Although studies suggest that users may positively perceive web-based symptom checkers, no studies have quantified user feedback after use of an electronic health record-integrated COVID-19 symptom checker with self-scheduling functionality. OBJECTIVE: In this paper, we aimed to understand user experience, user satisfaction, and user-reported alternatives to the use of a COVID-19 symptom checker with self-triage and self-scheduling functionality. METHODS: We launched a patient-portal-based self-triage and self-scheduling tool in March 2020 for patients with COVID-19 symptoms, exposures, or questions. We made an optional, anonymous Qualtrics survey available to patients immediately after they completed the symptom checker. RESULTS: Between December 16, 2021, and March 28, 2022, there were 395 unique responses to the survey. Overall, the respondents reported high satisfaction across all demographics, with a median rating of 8 out of 10 and 288/395 (47.6%) of the respondents giving a rating of 9 or 10 out of 10. User satisfaction scores were not associated with any demographic factors. The most common user-reported alternatives had the web-based tool not been available were calling the COVID-19 telephone hotline and sending a patient-portal message to their physician for advice. The ability to schedule a test online was the most important symptom checker feature for the respondents. The most common categories of user feedback were regarding other COVID-19 services (eg, telephone hotline), policies, or procedures, and requesting additional features or functionality. CONCLUSIONS: This analysis suggests that COVID-19 symptom checkers with self-triage and self-scheduling functionality may have high overall user satisfaction, regardless of user demographics. By allowing users to self-triage and self-schedule tests and visits, tools such as this may prevent unnecessary calls and messages to clinicians. Individual feedback suggested that the user experience for this type of tool is highly dependent on the organization's operational workflows for COVID-19 testing and care. This study provides insight for the implementation and improvement of COVID-19 symptom checkers to ensure high user satisfaction.

Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica.

Oikopleura dioica is a planktonic chordate with exceptional filter-feeding ability, rapid generation time, conserved early development, and a compact genome. For these reasons, it is considered a useful model organism for marine ecological studies, evolutionary developmental biology, and genomics. As research often requires a steady supply of animal resources, it is useful to establish a reliable, low-maintenance culture system. Here we describe a step-by-step method for establishing an O. dioica culture. We describe how to select potential sampling sites, collection methods, target animal identification, and the set-up of the culturing system. We provide troubleshooting advice based on our own experiences. We also highlight critical factors that help sustain a robust culture system. Although the culture protocol provided here is optimized for O. dioica, we hope our sampling technique and culture setup will inspire new ideas for maintaining other fragile pelagic invertebrates.

H3S28P Antibody Staining of Okinawan Oikopleura dioica Suggests the Presence of Three Chromosomes.

Oikopleura dioica is a ubiquitous marine tunicate of biological interest due to features that include dioecious reproduction, short life cycle, and vertebrate-like dorsal notochord while possessing a relatively compact genome. The use of tunicates as model organisms, particularly with these characteristics, offers the advantage of facilitating studies in evolutionary development and furthering understanding of enduring attributes found in the more complex vertebrates. At present, we are undertaking an initiative to sequence the genomes of Oikopleura individuals in populations found among the seas surrounding the Ryukyu Islands in southern Japan. To facilitate and validate genome assemblies, karyotyping was employed to count individual animals' chromosomes in situ using centromere-specific antibodies directed against H3S28P, a prophase-metaphase cell cycle-specific marker of histone H3. New imaging data of embryos and oocytes stained with two different antibodies were obtained; interpretation of these data lead us to conclude that the Okinawan Oikopleura dioica has three pairs of chromosomes, akin to previous results from genomic assemblies in Atlantic populations. The imaging data have been deposited to the open-access EBI BioImage Archive for reuse while additionally providing representative images of two commercially available anti-H3S28P antibodies' staining properties for use in epifluorescent and confocal based fluorescent microscopy.

A Streamlined Method for the Preparation of Gelatin Embedded Brains and Simplified Organization of Sections for Serial Reconstructions.

Gelatin embedding of whole brains for sectioning is a critical procedure used in neuroscience to ensure all morphological and spatial details are preserved intact. Here, we describe an inexpensive, reproducible and efficient means to embed post-fixed brains ready for sectioning in gelatin within a week's time. The sections obtained are distortion-free and their fragile internal structures preserved which can be used for serial reconstructions for lesion studies and mapping of viral expression after stereotaxic injections. In addition, the separation of adjacent slices into a series of 3-4 vials facilitates subsequent organization and assembly of serial sections at the mounting step.

New Variations for Strategy Set-shifting in the Rat.

Behavioral flexibility is crucial for survival in changing environments. Broadly defined, behavioral flexibility requires a shift of behavioral strategy based on a change in governing rules. We describe a strategy set-shifting task that requires an attentional shift from one stimulus dimension to another. The paradigm is often used for testing cognitive flexibility in primates. However, the rodent version has not been as extensively developed. We have recently extended an established set-shifting task in the rat1 by requiring attention to different stimuli according to context. All the experimental conditions required animals to choose either a left or right lever. Initially, all animals had to choose on the basis of the location of the lever. Subsequently, a change in the rule occurred, which required a shift in set from location-based rule to a rule in which the correct lever was indicated by a light cue. We compared performance on three different versions of the task, in which the light stimulus was either novel, previously relevant, or previously irrelevant. We found that specific neurochemical lesions selectively impaired the ability to make particular types of set shift as measured by the performance on the different versions of the task.