Diffusive lensing as a mechanism of intracellular transport and compartmentalization.

While inhomogeneous diffusivity has been identified as a ubiquitous feature of the cellular interior, its implications for particle mobility and concentration at different length scales remain largely unexplored. In this work, we use agent-based simulations of diffusion to investigate how heterogeneous diffusivity affects the movement and concentration of diffusing particles. We propose that a nonequilibrium mode of membrane-less compartmentalization arising from the convergence of diffusive trajectories into low-diffusive sinks, which we call 'diffusive lensing,' is relevant for living systems. Our work highlights the phenomenon of diffusive lensing as a potentially key driver of mesoscale dynamics in the cytoplasm, with possible far-reaching implications for biochemical processes.

Sex-Based Differences in the Epidemiology, Clinical Characteristics, and Outcomes Associated with Worsening Heart Failure Events in a Learning Health System.

BACKGROUND: Differences in demographics, risk factors, and clinical characteristics may contribute to variations in men and women in terms of the prevalence, clinical setting, and outcomes associated with worsening heart failure (WHF) events. We sought to describe sex-based differences in the epidemiology, clinical characteristics, and outcomes associated with WHF events across clinical settings. METHODS AND RESULTS: We examined adults diagnosed with HF from 2010 to 2019 within a large, integrated health care delivery system. Electronic health record data were accessed for hospitalizations, emergency department (ED) visits and observation stays, and outpatient encounters. WHF was identified using validated natural language processing algorithms and defined as ≥1 symptom, ≥2 objective findings (including ≥1 sign), and ≥1 change in HF-related therapy. Incidence rates and associated outcomes for WHF were compared across care setting by sex. We identified 1,122,368 unique clinical encounters with a diagnosis code for HF, with 124,479 meeting WHF criteria. These WHF encounters existed among 102,116 patients, of whom 48,543 (47.5%) were women and 53,573 (52.5%) were men. Women experiencing WHF were older and more likely to have HF with preserved ejection fraction compared with men. The clinical settings of WHF were similar among women and men: hospitalizations (36.8% vs 37.7%), ED visits or observation stays (11.8% vs 13.4%), and outpatient encounters (4.4% vs 4.9%). Women had lower odds of 30-day mortality after an index hospitalization (adjusted odds ratio 0.88, 95% confidence interval 0.83-0.93) or ED visit or observation stay (adjusted odds ratio 0.86, 95% confidence interval 0.75-0.98) for WHF. CONCLUSIONS: Women and men contribute similarly to WHF events across diverse clinical settings despite marked differences in age and left ventricular ejection fraction.

Collective good and individual choice: Perceptions on COVID-19 vaccine mandate among COVID-19 vaccinated individuals.

INTRODUCTION: Vaccine mandates are controversial, and people vary widely in their preferences to support or reject vaccine mandates. For some, vaccine mandates represent a commitment to reduce harm and support public health. For others, vaccine mandates are viewed as a threat to individual freedom and a violation of personal choice. This manuscript investigated support for a COVID-19 vaccine mandate among COVID-19-vaccinated individuals and identified differences by demographic characteristics and COVID-19 experience. METHODS: Cross-sectional surveys were given to COVID-19-vaccinated individuals at a vaccination clinic in South Texas in the U.S. with the goal of identifying attitudes, beliefs, and perceptions about COVID-19 vaccination and willingness to support a COVID-19 vaccination mandate. Associations of interest were analyzed using descriptive statistics. KEY RESULTS: Approximately half of the sample was of Hispanic or Latino origin (48 %); most respondents identified as White (59 %), followed by 12 % who identified as Asian. Overall, 59 % of participants supported the possibility for a COVID-19 vaccine mandate. Preliminary data showed significant racial differences in willingness to support a possible COVID-19 vaccine mandate (χ2 (1, n = 893) = 26.7, p < .001, phi = .17); 80 % of Asian people reported support for COVID-19 vaccination mandate compared to 50 % to 57 % for other racial groups. Significant differences also emerged by ethnicity (χ2 (4, n = 1033) = 7.12, p = .008, phi = .08) whereby a higher percentage of Latino participants (66 %) reported willingness to support a COVID-19 vaccine mandate. Similarly, significant differences were found by age (χ2 (4, n = 1045) = 20.92, p < .001, phi = .21), yet no significant differences were found by sex or previous COVID-19 diagnosis. CONCLUSION: Support for a COVID-19 vaccination mandate is controversial even among vaccinated people. Identifying and understanding cultural and contextual factors that underlie differences in attitudes and beliefs about COVID-19 vaccination mandates is essential to advance dialogue and inform educational health campaigns to increase COVID-19 vaccination rates.

Widespread Distribution of Xylazine Detected Throughout the United States in Healthcare Patient Samples.

OBJECTIVES: Xylazine is a tranquilizer commonly added into the illicit drug supply and a likely contributor to overdoses because it does not respond to naloxone reversal. The objective of this study was to perform a retrospective data analysis on xylazine-positive samples collected from patients in various outpatient healthcare settings to illustrate geographic distribution and common copositive substances, which may also contribute to risk of adverse events. METHODS: Samples for which providers ordered testing for xylazine were subjected to enzymatic hydrolysis, extracted, and analyzed using liquid chromatography-tandem mass spectrometry. Retrospective analysis was performed on xylazine-positive samples collected from April 2021 to March 2022, to include geographic location and copositive substances. RESULTS: Xylazine was identified in 413 of 59,498 samples from adults aged 20-73 years and originated from 25 of the 39 states where xylazine testing was ordered. The most common routine substances detected with xylazine were fentanyl, buprenorphine, naloxone, cocaine, d -methamphetamine, and delta-9-tetrahydrocannabinol. The most common designer drugs detected included fentanyl analogs, isotonitazene, and designer benzodiazepines. CONCLUSIONS: Xylazine is geographically spread throughout the United States, indicative of a wide incorporation into the illicit drug supply. These findings differ from previous studies in that these samples originated from healthcare providers in routine care settings, where other reports typically involve overdose deaths. This analysis illustrates that routine testing for xylazine in outpatient settings can afford providers the opportunity to educate individuals and adjust harm reduction measures to potentially mitigate overdose risk.

Learning Through Doing: Comprehensive Programming for a Training Program in Cancer Disparities.

In the United States, preparing researchers and practitioners for careers in cancer requires multiple components for success. In this reflection article, we discuss our approach to designing a comprehensive research training program in cancer disparities. We focused on elements that provide students and early career scientists a deep understanding of disparities through first-hand experiences and skills training necessary to build a research career in the area. Our Educational Program sits within the framework of an NCI P20 program, "UHAND (University of Houston/MD Anderson Cancer Center)", jointly established by an NCI-designated comprehensive cancer center and a minority-serving university as a collaborative partnership devoted to the elimination of cancer inequities among disproportionately affected racial and ethnic groups (UHAND Program to Reduce Cancer Disparities; NCI P20CA221696/ P20CA221697). The Education Program was designed to build on and enhance skills that are critical to pursuing a career in cancer disparities research at the undergraduate, doctoral, and post-doctoral levels-such as scientific communication, career planning and development, professional and community-based collaboration, and resilience in addition to solid scientific training. As such, our program integrates (1) opportunities for learning through service to community organizations providing resources to populations with documented cancer disparities, (2) a tailored curriculum of learning activities with program leadership and mentored research with scientists focused on cancer disparities and cancer prevention, (3) professional development training critical to career success in disparities research, and (4) support to address unique challenges faced by trainees from backgrounds that are historically underrepresented in research.

Activation of the EGFR/MAPK pathway drives transdifferentiation of quiescent niche cells to stem cells in the Drosophila testis niche.

Adult stem cells are maintained in niches, specialized microenvironments that regulate their self-renewal and differentiation. In the adult Drosophila testis stem cell niche, somatic hub cells produce signals that regulate adjacent germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Hub cells are normally quiescent, but after complete genetic ablation of CySCs, they can proliferate and transdifferentiate into new CySCs. Here we find that Epidermal growth factor receptor (EGFR) signaling is upregulated in hub cells after CySC ablation and that the ability of testes to recover from ablation is inhibited by reduced EGFR signaling. In addition, activation of the EGFR pathway in hub cells is sufficient to induce their proliferation and transdifferentiation into CySCs. We propose that EGFR signaling, which is normally required in adult cyst cells, is actively inhibited in adult hub cells to maintain their fate but is repurposed to drive stem cell regeneration after CySC ablation.

Cleanability of Metal Surface Finishes Found in Medical Devices and the Environment of Care.

The surface finish of reusable medical devices often is a consideration during design, with attention paid to utility, aesthetics, and cost. To study the cleanability of metal surfaces of varying roughness and finish, soil made of bovine blood and egg yolk was placed on nickel alloy surface of varying average roughness (Ra) values (nominal values of 2-500 µin) and finishes (lapped, ground, milled, profiled, and shape turned). A commercially available wipe consisting of quaternary ammonium compound and less than 20% alcohol was applied for a total of eight wipe cycles to remove the soil. The wipe was applied in both horizontal and vertical directions. Evaluation methods for cleanliness include visual inspection and adenosine triphosphate (ATP) measurement. Rougher surfaces above nominal Ra of 250 were found to have higher ATP readings when wiped in both horizontal and vertical directions. In addition, different surface finishes have different cleanabilities despite similar nominal Ra. To ensure optimal cleaning, surfaces should be cleaned in multiple directions. In the future, similar studies will be coupled with efficacy studies and surfaces made with other materials will be investigated.

Smoking Intervention Practices in Texas Healthcare Centers with Sexual and Gender Minority Patients.

Objectives: This study evaluated the use of evidence-based practices (EBPs) for smoking cessation in centers providing behavioral healthcare for patient populations that included some proportion of sexual and gender minorities (SGMs). Methods: Healthcare providers from 75 healthcare centers across Texas serving SGMs with behavioral health needs participated in a survey assessing their center's tobacco control policies and practices. Results: Nearly half (N = 36) of participating centers had a comprehensive tobacco-free workplace policy, 30.67% employed ≥1 tobacco treatment specialist, 73.91% employed ≥1 prescriber, 80.82% mandated screening for patient tobacco use at intake, and 57.53% provided a template for tobacco use assessments. Overall, 70.67% of providers asked patients about smoking status, 69.33% advised patients to quit, 64.00% assessed patients' interest in quitting, 58.67% assisted patients with quit attempts, and 36.00% arranged follow-up. Providers' ability to tailor interventions for special populations like SGMs ranged from very low/0 to very high/10 (M = 4.63 ± 2.59). Conclusions: There are opportunities to improve policy implementation, standardization and usage of evidence-based interventions, and intervention tailoring within settings providing care to SGM patients in Texas to better address their tobacco use inequities.

Organizational Factors Moderating Changes in Tobacco Use Dependence Care Delivery Following a Comprehensive Tobacco-Free Workplace Intervention in Non-Profit Substance Use Treatment Centers.

Although tobacco use is the leading preventable cause of death and is elevated among patients with substance use disorders, many substance use treatment centers (SUTCs) do not offer tobacco use interventions (i.e., screening and treatment). This study examined a key outcome of the implementation of a tobacco-free workplace program that provided education and specialized training to employees; namely, changes in clinician provision of the five As (Asking about tobacco use; Advising to quit; Assessing willingness to quit; Assisting with quitting; Arranging follow-up) from before to after the larger program implementation. The five As are a brief tobacco screening and treatment protocol that was taught as part of the program and that formed the basis for further intervention (e.g., provision of nicotine replacement therapies, Motivational Interviewing to enhance desire and willingness to make a quit attempt). Moreover, we also examined organizational moderators that may have impacted changes in the delivery of the five As over time among clinicians from 15 participating SUTCs. The number of the centers' total and unique annual patient visits; full-time employees; and organizational readiness for implementing change were assessed as potential moderators of change in clinicians' behaviors over time. Clinicians completed pre- and post-program implementation surveys assessing their provision of the five As. Results demonstrated significant increases in Asking (p = 0.0036), Advising (p = 0.0176), Assisting (p < 0.0001), and Arranging (p < 0.0001). SUTCs with higher Change Efficacy (p = 0.025) and lower Resource Availability (p = 0.019) had greater increases in Asking. SUTCs with lower Resource Availability had greater increases in Assessing (p = 0.010). These results help guide tobacco control program implementation to increase the provision of tobacco use interventions (i.e., the five As) to SUTC patients and elucidate Change Efficacy and Resource Availability as organizational factors promoting this clinician behavior change.

Organizational-Level Moderators Impacting Tobacco-Related Knowledge Change after Tobacco Education Training in Substance Use Treatment Centers.

Tobacco use is disproportionately elevated among patients with substance use disorders relative to the general U.S. population. Tobacco interventions are lacking within substance use treatment centers (SUTCs) due to lack of knowledge and training. This study examined knowledge gain and the organizational factors that might moderate knowledge gains following tobacco education training provided to employees (N = 580) within 15 SUTCs that were participating in a tobacco-free workplace program. The number of total annual patient visits, unique annual patient visits, number of full-time employees, and organizational readiness for implementing change (ORIC) as assessed prior to implementation were examined as potential moderators. Results demonstrated significant knowledge gain (p < 0.001) after training overall; individually, 13 SUTCs had significant knowledge gain (p's < 0.014). SUTCs with fewer total annual patient visits and fewer full-time employees showed greater knowledge gains. The ORIC total score and all but one of its subscales (Resource Availability) moderated knowledge gain. SUTCs with greater initial Change Efficacy (p = 0.029), Valence (p = 0.027), and Commitment (p < 0.001) had greater knowledge gain than SUTCs with lower scores on these constructs; SUTCs with greater Task Knowledge (p < 0.001) regarding requirements for change exhibited less knowledge gain. Understanding the organizational-level factors impacting training effectiveness can inform efforts in organizational change and tobacco control program implementation.

Addressing Smoking Cessation among Women in Substance Use Treatment: A Qualitative Approach to Guiding Tailored Interventions.

Intersecting socially marginalized identities and unique biopsychosocial factors place women with substance use disorders (SUDs) experiencing myriad disadvantages at higher risk for smoking and stigmatization. Here, based on our work with women receiving care for SUDs in four participating treatment/women-serving centers (N = 6 individual clinics), we: (1) describe the functions of smoking for women with SUDs; and (2) explore participants' experiences of a comprehensive tobacco-free workplace (TFW) program, Taking Texas Tobacco-Free (TTTF), that was implemented during their SUD treatment. Ultimately, information gleaned was intended to inform the development of women-tailored tobacco interventions. Data collection occurred pre- and post-TTTF implementation and entailed conducting client (7) and clinician (5) focus groups. Using thematic analysis, we identified four main themes: "the social context of smoking," "challenges to finding support and better coping methods," "addressing underlying conditions: building inner and outer supportive environments," and "sustaining support: TFW program experiences." Women reported that: smoking served as a "coping mechanism" for stress and facilitated socialization; stigmatization hindered quitting; non-stigmatizing counseling cessation support provided alternative coping strategies; and, with clinicians, the cessation opportunities TTTF presented are valuable. Clinicians reported organizational support, or lack thereof, and tobacco-related misconceptions as the main facilitator/barriers to treating tobacco addiction. Effective tobacco cessation interventions for women with SUDs should be informed by, and tailored to, their gendered experiences, needs, and recommendations. Participants recommended replacing smoking with healthy stress alleviating strategies; the importance of adopting non-judgmental, supportive, cessation interventions; and the support of TFW programs and nicotine replacement therapy to aid in quitting.

The Surprise Question and Serious Illness Conversations: A pilot study.

BACKGROUND: Serious Illness Conversations aim to discuss patient goals. However, on acute medicine units, seriously ill patients may undergo distressing interventions until death. OBJECTIVES: To investigate the feasibility of using the Surprise Question, "Would you be surprised if this patient died within the next year?" to identify patients who would benefit from early Serious Illness Conversations and study any changes in the interdisciplinary team's beliefs, confidence, and engagement as a result of asking the Surprise Question. DESIGN: A prospective cohort pilot study with two Plan-Do-Study-Act cycles. PARTICIPANTS/CONTEXT: Fifty-eight healthcare professionals working on Acute Medicine Units participated in pre- and post-intervention questionnaires. The intervention involved asking participants the Surprise Question for each patient. Patient charts were reviewed for Serious Illness Conversation documentation. ETHICAL CONSIDERATIONS: Ethical approval was granted by the institutions involved. FINDINGS: Equivocal overall changes in the beliefs, confidence, and engagement of healthcare professionals were observed. Six out of 23 patients were indicated as needing a Serious Illness Conversation; chart review provided some evidence that these patients had more Serious Illness Conversation documentation compared with the 17 patients not flagged for a Serious Illness Conversation. Issues were identified in equating the Surprise Question to a Serious Illness Conversation. DISCUSSION: Appropriate support for seriously ill patients is both a nursing professional and ethical duty. Flagging patients for conversations may act as a filtering process, allowing healthcare professionals to focus on conversations with patients who need them most. There are ethical and practical issues as to what constitutes a "serious illness" and if answering "no" to the Surprise Question always equates to a conversation. CONCLUSION: The barriers of time constraints and lack of training call for institutional change in order to prioritise the moral obligation of Serious Illness Conversations.

Increased intrinsic excitability and decreased synaptic inhibition in aged somatosensory cortex pyramidal neurons.

Sensorimotor performance declines during advanced age, partially due to deficits in somatosensory acuity. Cortical receptive field expansion contributes to somatosensory deficits, suggesting increased excitability or decreased inhibition in primary somatosensory cortex (S1) pyramidal neurons. To ascertain changes in excitability and inhibition, we measured both properties in neurons from vibrissal S1 in brain slices from young and aged mice. Because adapting and non-adapting neurons-the principal pyramidal types in layer 5 (L5)-differ in intrinsic properties and inhibitory inputs, we determined age-dependent changes according to neuron type. We found an age-dependent increase in intrinsic excitability in adapting neurons, caused by a decrease in action potential threshold. Surprisingly, in non-adapting neurons we found both an increase in excitability caused by increased input resistance, and a decrease in synaptic inhibition. Spike frequency adaptation, already small in non-adapting neurons, was further reduced by aging, whereas sag, a manifestation of Ih, was increased. Therefore, aging caused both decreased inhibition and increased intrinsic excitability, but these effects were specific to pyramidal neuron type.

PyRosetta Jupyter Notebooks Teach Biomolecular Structure Prediction and Design.

Biomolecular structure drives function, and computational capabilities have progressed such that the prediction and computational design of biomolecular structures is increasingly feasible. Because computational biophysics attracts students from many different backgrounds and with different levels of resources, teaching the subject can be challenging. One strategy to teach diverse learners is with interactive multimedia material that promotes self-paced, active learning. We have created a hands-on education strategy with a set of sixteen modules that teach topics in biomolecular structure and design, from fundamentals of conformational sampling and energy evaluation to applications like protein docking, antibody design, and RNA structure prediction. Our modules are based on PyRosetta, a Python library that encapsulates all computational modules and methods in the Rosetta software package. The workshop-style modules are implemented as Jupyter Notebooks that can be executed in the Google Colaboratory, allowing learners access with just a web browser. The digital format of Jupyter Notebooks allows us to embed images, molecular visualization movies, and interactive coding exercises. This multimodal approach may better reach students from different disciplines and experience levels as well as attract more researchers from smaller labs and cognate backgrounds to leverage PyRosetta in their science and engineering research. All materials are freely available at https://github.com/RosettaCommons/PyRosetta.notebooks.

Adapting and Evaluating Implementation of a Tobacco-Free Workplace Program in Behavioral Health Centers.

Objectives: State-administered community behavioral health centers (CBHCs) rarely treat tobacco dependence, despite high client tobacco use. Using a mixed-methods approach we examine the adaptation and implementation of an evidence-based tobacco-free workplace (TFW) program in 2 CBHCs (17 individual clinics). Methods: Varied data collection included pre- and post-implementation leader, clinician, and staff surveys; pre-, mid-, and post-implementation staff and client focus groups; and monthly implementation logs. The RE-AIM framework guided translation of behavioral interventions into sustainable practice. Results: Pre- to post-implementation increases were seen in training receipt among clinicians and employees. Both CBHCs adopted a 100% TFW policy, integrated tobacco screenings into routine practice, and delivered evidence-based practices (EBPs). Qualitative methods enlisted key stakeholders contributing towards adapting program strategies to local contexts, addressing barriers, adjusting tobacco screening administration, and understanding reasons for success or failure to implement specific components. Conclusions: Program implementation at both CBHCs increased organizational capacity in the provision of EBPs to treat tobacco dependence through successfully meeting the majority of our RE-AIM targets. Findings contribute to the development of flexible strategies and interventions responsive to variable implementation contexts and barriers; enhancing the effectiveness and sustainability of a TFW program.

Tobacco-free Workplace Program at a Substance Use Treatment Center.

Objectives: About 65%-87% of substance use disorder patients smoke cigarettes, compared to 14% of the general adult population. Few substance use treatment centers (SUTCs) have comprehensive tobacco-free workplace (TFW) policies or offer tobacco interventions. Taking Texas Tobacco Free (TTTF) implements an evidence-based TFW program in SUTCs, including at the Billy T. Cattan Recovery Outreach Center (BTC). We present a mixed methods case study of BTC's TTTF implementation, success factors, and challenges. Methods: TTTF provided policy development assistance, training, treatment resources, and technical assistance over ∼9 months. Implementation was tailored using mixed methods. Quantitative data included surveys to stakeholders (Nmax = 7), a pre- and post-training questionnaire assessing knowledge gain, and reported quantities of tobacco use assessments (TUAs) administered and nicotine replacement therapy (NRT) provided. Qualitative data included stakeholder focus groups and interviews (18 participants). Results: All employees reported TFW policy compliance. Employees exhibited a 20% knowledge gain. Clinicians increased self-report of NRT provision and tobacco cessation counseling. During implementation, BTC administered TUAs to 171 patients and dispensed NRT to 70 of 110 tobacco-using patients. Conclusion: Qualitative findings contextualized quantitative outcomes. TTTF implementation changed clinician attitudes, knowledge, and practices regarding tobacco treatment, facilitating patient quit attempts.

Clinician Training in the Adaptation of a Comprehensive Tobacco-Free Workplace Program in Agencies Serving the Homeless and Vulnerably Housed.

Tobacco use is exceedingly high among those who are homeless or at risk of homelessness but not commonly addressed by clinicians. Taking Texas Tobacco Free (TTTF) is a tobacco control program that addresses known clinician barriers to intervention (e.g., low training receipt, limited resources). Here, we examine the process and outcomes of TTTF's adaptation within four agencies that provide housing or other services to individuals who are homeless or vulnerably housed. Pre- and post-implementation data were collected from clinicians (N = 68) to assess changes in training receipt, knowledge, and intervention behaviors, relative to program goals. Results indicated significant gains in clinicians' receipt of training in 9 (of 9) target areas (p's ≤ 0.0042) and a 53% knowledge gain (p < 0.0001). From pre- to post-implementation, there were mean increases in the use of the 5As (ask, advise, assess, assist, and arrange) and other evidence-based interventions for tobacco cessation, with significant gains seen in assisting residents/clients to quit, arranging follow-ups, and providing or referring for non-nicotine medications (p's ≤ 0.0491). All program goals, except gains related to advising smokers to quit and the use of specific interventions (behavioral counseling), were met. Overall, TTTF improved clinicians' capacity to address tobacco use among homeless and vulnerably housed individuals and can serve as a model for tobacco control efforts in similar agencies.

Publisher Correction: Marked bias towards spontaneous synaptic inhibition distinguishes non-adapting from adapting layer 5 pyramidal neurons in the barrel cortex.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Marked bias towards spontaneous synaptic inhibition distinguishes non-adapting from adapting layer 5 pyramidal neurons in the barrel cortex.

Pyramidal neuron subtypes differ in intrinsic electrophysiology properties and dendritic morphology. However, do different pyramidal neuron subtypes also receive synaptic inputs that are dissimilar in frequency and in excitation/inhibition balance? Unsupervised clustering of three intrinsic parameters that vary by cell subtype - the slow afterhyperpolarization, the sag, and the spike frequency adaptation - split layer 5 barrel cortex pyramidal neurons into two clusters: one of adapting cells and one of non-adapting cells, corresponding to previously described thin- and thick-tufted pyramidal neurons, respectively. Non-adapting neurons presented frequencies of spontaneous inhibitory postsynaptic currents (sIPSCs) and spontaneous excitatory postsynaptic currents (sEPSCs) three- and two-fold higher, respectively, than those of adapting neurons. The IPSC difference between pyramidal subtypes was activity independent. A subset of neurons were thy1-GFP positive, presented characteristics of non-adapting pyramidal neurons, and also had higher IPSC and EPSC frequencies than adapting neurons. The sEPSC/sIPSC frequency ratio was higher in adapting than in non-adapting cells, suggesting a higher excitatory drive in adapting neurons. Therefore, our study on spontaneous synaptic inputs suggests a different extent of synaptic information processing in adapting and non-adapting barrel cortex neurons, and that eventual deficits in inhibition may have differential effects on the excitation/inhibition balance in adapting and non-adapting neurons.

Agonist-dependent internalization of the human melanocortin-4 receptors in human embryonic kidney 293 cells.

A chimeric protein comprised of melanocortin-4 receptor (MC4R) and the green fluorescent protein (GFP) was created for studying receptor/ligand localization and trafficking. The ligand binding affinities and second messenger stimulation induced by MC4R-GFP closely resembled those of the wild-type receptor, suggesting functional integrity of the chimeric protein. As observed with a confocal microscope, in human embryonic kidney (HEK)-293 cells MC4R/GFP was distributed evenly along the cell membrane. Addition of [Nle4-d-Phe7]-alpha-melanocyte-stimulating hormone (NDP-MSH), a peptide MC4R agonist, induced receptor translocation into intracellular compartments in a time- and concentration-dependent manner. [Ac-Nle-c[Asp-His-d-Nal(2')-Arg-Trp-Lys]-NH2] (SHU9119), a potent MC4R antagonist, completely inhibited NDP-MSH-mediated internalization. MC4R-GFP internalization was unaffected by a protein kinase A inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), but was impaired by pretreatment with inhibitors of endocytosis through clathrin-coated pits, hypertonic sucrose, or concanavalin A. Time-dependent colocalization of MC4R-GFP with rhodamine-transferrin, an early endosome marker, and with LysoTraker, a lysosome marker, was observed after short-term (45 min) and prolonged (20 h) agonist exposure, respectively. Rhodamine-[AcNle-c[Asp-His-d-Phe-Arg-Trp-Lys]-NH2] (MTII), a fluorescent derivative of an MC4R agonist, was found to cointernalize with MC4R-GFP into intracellular vesicles. No significant receptor recycling or segregation from the ligand was observed 60 min after removal of the agonist. In contrast, an antagonist rhodamine-Ac-Cys-Glu-His-(d-Nal)-Arg-Trp-Gly-Cys-Pro-Pro-Lys-Asp-NH2 (HS014) bound to and colocalized with MC4R-GFP on the cell surface and did not stimulate receptor internalization. In sum, these results suggest that MC4R is subject to agonist-dependent endocytosis via clathrin-coated pits. Prolonged agonist exposure directs MC4R into lysosomes, possibly for degradation. Receptor and ligand recycling is not efficient for MC4R in HEK-293 cells.