Trial protocol for the Building Resilience through Socio-Emotional Training (ReSET) programme: a cluster randomised controlled trial of a new transdiagnostic preventative intervention for adolescents.

BACKGROUND: Adolescence is a period of heightened vulnerability to developing mental health problems, and rates of mental health disorder in this age group have increased in the last decade. Preventing mental health problems developing before they become entrenched, particularly in adolescents who are at high risk, is an important research and clinical target. Here, we report the protocol for the trial of the 'Building Resilience through Socioemotional Training' (ReSET) intervention. ReSET is a new, preventative intervention that incorporates individual-based emotional training techniques and group-based social and communication skills training. We take a transdiagnostic approach, focusing on emotion processing and social mechanisms implicated in the onset and maintenance of various forms of psychopathology. METHODS: A cluster randomised allocation design is adopted with randomisation at the school year level. Five-hundred and forty adolescents (aged 12-14) will be randomised to either receive the intervention or not (passive control). The intervention is comprised of weekly sessions over an 8-week period, supplemented by two individual sessions. The primary outcomes, psychopathology symptoms and mental wellbeing, will be assessed pre- and post-intervention, and at a 1-year follow-up. Secondary outcomes are task-based assessments of emotion processing, social network data based on peer nominations, and subjective ratings of social relationships. These measures will be taken at baseline, post-intervention and 1-year follow-up. A subgroup of participants and stakeholders will be invited to take part in focus groups to assess the acceptability of the intervention. DISCUSSION: This project adopts a theory-based approach to the development of a new intervention designed to target the close connections between young people's emotions and their interpersonal relationships. By embedding the intervention within a school setting and using a cluster-randomised design, we aim to develop and test a feasible, scalable intervention to prevent the onset of psychopathology in adolescence. TRIAL REGISTRATION: ISRCTN88585916. Trial registration date: 20/04/2023.

Racial, ethnic, and age disparities in the association of mental health symptoms and polysubstance use among persons in HIV care.

We characterized polysubstance use burden and associations with mental health problems across demographic subgroups of PWH. In 2018-2020, as part of a primary care-based intervention study, PWH in care at three medical centers in Kaiser Permanente Northern California were screened for depression (PHQ-9≥10), anxiety (GAD-2≥3), and substance use (Tobacco, Alcohol, Prescription medication, and other Substance use [TAPS]≥1 per substance). We used Poisson regression to estimate prevalence ratios (PRs) comparing polysubstance use prevalence (TAPS≥1 for ≥2 substances) between PWH with positive screens for depression or anxiety vs. neither, among all PWH, and stratified by race/ethnicity and age (restricted to men), adjusting for sociodemographics, CD4, and HIV load. Screened PWH (N = 2865) included 92% men, 56% White, 19% Black, and 15% Hispanic PWH, with a median age of 55 years. Overall, polysubstance use prevalence was 26.4% (95% CI 24.9%-28.1%). PWH with depression or anxiety (n = 515) had an adjusted polysubstance use PR of 1.26 (1.09-1.46) vs. PWH with neither (n = 2350). Adjusted PRs were 1.47 (1.11-1.96), 1.07 (0.74-1.54), and 1.10 (0.85-1.41) among Black, Hispanic, and White men, respectively. Adjusted PRs did not differ by age group. Interventions should consider jointly addressing mental health and substance use problems and potential drivers, e.g. stigma or socioeconomic factors.

Ethical Dilemmas Facing Substance Use Counselors During the COVID-19 Pandemic.

Introduction: During the COVID-19 pandemic, substance use disorder (SUD) treatment settings experienced several abrupt changes, including decreased admissions, reduction in services, and modified requirements for medication for substance use disorder. While these changes were implemented to facilitate the maintenance of important treatment options, the ethical consequences of such changes remained unknown. The current study aimed to explore ethical issues related to COVID-19-related changes reported by counselors in SUD treatment facilities. Method: From May to August 2020, we conducted 60 to 90 minutes in-depth interviews with 18 front-line staff in 1 residential and 1 outpatient treatment program, exploring issues drawn from the ethical principles of the national organization representing SUD counselors. Counselors volunteered to participate via phone or email, and participation was confidential. Interviews were conducted via videoconferencing. Topics included day-to-day experiences of ethical dilemmas in the workplace, particularly during the COVID-19 era. Interviews were recorded, transcribed, and checked for accuracy and a trained team of analysts then coded transcripts using thematic analysis. Results: As a result of the COVID-19 pandemic, SUD treatment programs quickly modified procedures to adhere to public health mandates while also continuing to offer care to clients. SUD counselors reported several ways their programs adapted new and creative procedures to reduce the risk of COVID-19 transmission. SUD counselors also identified several novel ethical dilemmas that occurred during the COVID-19 pandemic, often resulting from the counselor balancing the needs for responding to public health mandates with providing services to clients. There were several ways that COVID-19 related changes resulted in therapeutic challenges for some clients, and the SUD counselors highlighted ways that changes resulted in more flexible services for other clients. Conclusions: This study highlights the quick response to COVID-19 that occurred within SUD treatment. While these changes resulted in novel ethical dilemmas, they also offered more flexible and client-centered approaches to treatment.

Evaluation of the Fast Prior Authorization Technology Highway.

BACKGROUND: Prior authorization (PA) is a utilization management tool used by health plans and pharmacy benefit managers where the payer requires additional documentation from health care providers before authorization of payment for a medication or procedure. PA processes are hypothesized to be more efficient if electronic transmission is utilized instead of manual submission. OBJECTIVE: To evaluate the impact of electronic PA (ePA) on approval rate and time to decision and to assess health care provider perception of using ePA. METHODS: America's Health Insurance Plans selected 2 technology companies, Availity and Surescripts, and used an independent research organization (Research Triangle Institute [RTI]) to conduct a provider survey and analyze over 40,000 PA transactions from participating health plans. RTI examined processing time, provider experience, and other measures for PAs both before and after provider implementation of ePA. RESULTS: Providers used these tools for roughly 62% of PAs in the 6 months after implementation. The median time from PA request to decision fell from 18.7 hours to 5.7 hours. Providers using ePA reported observing some benefits relative to the number of phone calls and faxes required after ePA implementation. CONCLUSION: The primary benefit of ePA implementation was reduced time to decision. Additional benefits may occur with greater adoption since 38% of PAs were still manual after implementation of ePA.

Association of pregnancy attitudes and intentions with sexual activity and psychiatric symptoms in justice-involved youth.

Context: With over one-third of detained girls experiencing teenage pregnancy, it is critical that the juvenile justice system better addresses the sexual and reproductive health (SRH) needs of youth. Although pregnancy attitudes and intentions (PAI) are associated with pregnancy outcomes among the general adolescent population, this relationship has not been examined among justice-involved youth. Methods: Participants were drawn from a longitudinal study characterizing trajectories of behavioral and reproductive health and recidivism among newly justice-involved youth in a Northeast family court. Baseline and four-month follow-up data from 288 justice-involved youth (JIY) were analyzed to characterize PAI; examine associations between pregnancy intentions and unprotected sexual activity (i.e., no hormonal, intrauterine, or barrier protection against pregnancy); and explore the relationship between pregnancy intentions and psychiatric symptoms. Results: At baseline, 39% of JIY youth were sexually active, 44% of these youth reported inconsistent condom use and 14% had not used birth control at last sexual intercourse. Nearly half of sexually active youth reported some intent around pregnancy and those with any pregnancy intentions were more likely to report depression, low self-esteem, substance use, and trauma history. Pregnancy intentions at baseline predicted higher rates of unprotected sexual activity at four months (OR: 16.9, CI = 2.48-115.7). Conclusions: This study highlights the importance of developing and implementing more comprehensive SRH assessments and brief interventions for youth entering the justice system.

Embedding interagency working between schools and mental health specialists: A service evaluation of the Mental Health Services and Schools and Colleges Link Programme workshops.

There is increasing focus on the need for schools to work more effectively with specialist mental health providers, but there have been historic challenges in embedding closer interagency working. This article reports the results of a service evaluation of a 2-day workshop designed to facilitate improved working between schools and children and young people's mental health services (CYPMHS). Mental health leads from 255 schools, mental health professionals and other key stakeholders all took part in one of 26 two-day workshops across the United Kingdom. The impact on interagency working was examined using changes in pre- and post-survey results, changes in self-reported aspects of interagency working and 10 local reviews of practice. The pre-post questionnaires showed improvements in interagency working (e.g. 55% of school leads reported being in 'monthly' or 'continuous' contact with the National Health Service (NHS) CYPMHS1 at follow-up, compared with 24% at baseline). The group-completed CASCADE framework showed an overall increase in collaborative working, although some areas continued to report significant challenges such as in relation to common outcome measures. The local reviews found positive changes in interagency working, in terms of building relationships, improved communication and sharing good practice. This service evaluation of the workshops found some evidence of improved interagency working between schools and CYPMHS, but more controlled research is needed to consider generalisability and scalability.

Access To E-Prescriptions And Related Technologies Before And After Hurricanes Harvey, Irma, And Maria.

Hurricanes Harvey, Irma, and Maria devastated parts of the US mainland and Puerto Rico during the 2017 hurricane season, causing loss of life and substantial damage and interrupting access to health care for many people in the hurricane impact zones. Using data on e-prescriptions and medication history transactions from a large health information network for the period August 2017-May 2018, we examined providers' access to these technologies across affected areas. Although e-prescribing and medication history transactions decreased considerably during each hurricane, transaction volumes returned to normal levels in the days immediately following Hurricanes Harvey and Irma in Texas and Florida. E-prescribing activity in Puerto Rico did not return to baseline levels at all during the study period, and medication history transactions returned to normal only after an extended period following Hurricane Maria.

TMEM266 is a functional voltage sensor regulated by extracellular Zn2.

Voltage-activated ion channels contain S1-S4 domains that sense membrane voltage and control opening of ion-selective pores, a mechanism that is crucial for electrical signaling. Related S1-S4 domains have been identified in voltage-sensitive phosphatases and voltage-activated proton channels, both of which lack associated pore domains. hTMEM266 is a protein of unknown function that is predicted to contain an S1-S4 domain, along with partially structured cytoplasmic termini. Here we show that hTMEM266 forms oligomers, undergoes both rapid (µs) and slow (ms) structural rearrangements in response to changes in voltage, and contains a Zn2+ binding site that can regulate the slow conformational transition. Our results demonstrate that the S1-S4 domain in hTMEM266 is a functional voltage sensor, motivating future studies to identify cellular processes that may be regulated by the protein. The ability of hTMEM266 to respond to voltage on the µs timescale may be advantageous for designing new genetically encoded voltage indicators.

A pedometer-based walking intervention in 45- to 75-year-olds, with and without practice nurse support: the PACE-UP three-arm cluster RCT.

BACKGROUND: Guidelines recommend walking to increase moderate to vigorous physical activity (MVPA) for health benefits. OBJECTIVES: To assess the effectiveness, cost-effectiveness and acceptability of a pedometer-based walking intervention in inactive adults, delivered postally or through dedicated practice nurse physical activity (PA) consultations. DESIGN: Parallel three-arm trial, cluster randomised by household. SETTING: Seven London-based general practices. PARTICIPANTS: A total of 11,015 people without PA contraindications, aged 45-75 years, randomly selected from practices, were invited. A total of 6399 people were non-responders, and 548 people self-reporting achieving PA guidelines were excluded. A total of 1023 people from 922 households were randomised to usual care (n = 338), postal intervention (n = 339) or nurse support (n = 346). The recruitment rate was 10% (1023/10,467). A total of 956 participants (93%) provided outcome data. INTERVENTIONS: Intervention groups received pedometers, 12-week walking programmes advising participants to gradually add '3000 steps in 30 minutes' most days weekly and PA diaries. The nurse group was offered three dedicated PA consultations. MAIN OUTCOME MEASURES: The primary and main secondary outcomes were changes from baseline to 12 months in average daily step counts and time in MVPA (in ≥ 10-minute bouts), respectively, from 7-day accelerometry. Individual resource-use data informed the within-trial economic evaluation and the Markov model for simulating long-term cost-effectiveness. Qualitative evaluations assessed nurse and participant views. A 3-year follow-up was conducted. RESULTS: Baseline average daily step count was 7479 [standard deviation (SD) 2671], average minutes per week in MVPA bouts was 94 minutes (SD 102 minutes) for those randomised. PA increased significantly at 12 months in both intervention groups compared with the control group, with no difference between interventions; additional steps per day were 642 steps [95% confidence interval (CI) 329 to 955 steps] for the postal group and 677 steps (95% CI 365 to 989 steps) for nurse support, and additional MVPA in bouts (minutes per week) was 33 minutes per week (95% CI 17 to 49 minutes per week) for the postal group and 35 minutes per week (95% CI 19 to 51 minutes per week) for nurse support. Intervention groups showed no increase in adverse events. Incremental cost per step was 19p and £3.61 per minute in a ≥ 10-minute MVPA bout for nurse support, whereas the postal group took more steps and cost less than the control group. The postal group had a 50% chance of being cost-effective at a £20,000 per quality-adjusted life-year (QALY) threshold within 1 year and had both lower costs [-£11M (95% CI -£12M to -£10M) per 100,000 population] and more QALYs [759 QALYs gained (95% CI 400 to 1247 QALYs)] than the nurse support and control groups in the long term. Participants and nurses found the interventions acceptable and enjoyable. Three-year follow-up data showed persistent intervention effects (nurse support plus postal vs. control) on steps per day [648 steps (95% CI 272 to 1024 steps)] and MVPA bouts [26 minutes per week (95% CI 8 to 44 minutes per week)]. LIMITATIONS: The 10% recruitment level, with lower levels in Asian and socioeconomically deprived participants, limits the generalisability of the findings. Assessors were unmasked to the group. CONCLUSIONS: A primary care pedometer-based walking intervention in 45- to 75-year-olds increased 12-month step counts by around one-tenth, and time in MVPA bouts by around one-third, with similar effects for the nurse support and postal groups, and persistent 3-year effects. The postal intervention provides cost-effective, long-term quality-of-life benefits. A primary care pedometer intervention delivered by post could help address the public health physical inactivity challenge. FUTURE WORK: Exploring different recruitment strategies to increase uptake. Integrating the Pedometer And Consultation Evaluation-UP (PACE-UP) trial with evolving PA monitoring technologies. TRIAL REGISTRATION: Current Controlled Trials ISRCTN98538934. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 22, No. 37. See the NIHR Journals Library website for further project information.

Correction: An App to Help Young People Self-Manage When Feeling Overwhelmed (ReZone): Protocol of a Cluster Randomized Controlled Trial.

[This corrects the article DOI: 10.2196/resprot.7019.].

An App to Help Young People Self-Manage When Feeling Overwhelmed (ReZone): Protocol of a Cluster Randomized Controlled Trial.

BACKGROUND: The association between behavioral difficulties and academic attainment is well established. Recent policy advising schools on managing behavior has promoted the early identification of behavioral difficulties. There is also increasing research into mHealth interventions to provide support for emotional and behavioral difficulties for young people. OBJECTIVE: The primary aim of the proposed research is to examine the effectiveness of an mHealth intervention, ReZone, in reducing emotional and behavioral difficulties in young people. METHODS: The protocol is a cluster trial of 12 classes with N=120 students with classes randomized to ReZone or management as usual. Multilevel modeling will be used to compare ReZone versus management as usual accounting for classroom-level variation. RESULTS: Baseline data collection started in February 2017 and ended in April 2017. Follow-up data collection started in April 2017 and ended in June 2017. CONCLUSIONS: The proposed research will provide evidence as to whether ReZone is effective at helping young people to self-manage when feeling overwhelmed. TRIAL REGISTRATION: ISRCTN 13425994; http://www.isrctn.com/ISRCTN13425994 (Archived by WebCite at http://www.webcitation.org/6tePwwiHk).

Effect of a Primary Care Walking Intervention with and without Nurse Support on Physical Activity Levels in 45- to 75-Year-Olds: The Pedometer And Consultation Evaluation (PACE-UP) Cluster Randomised Clinical Trial.

BACKGROUND: Pedometers can increase walking and moderate-to-vigorous physical activity (MVPA) levels, but their effectiveness with or without support has not been rigorously evaluated. We assessed the effectiveness of a pedometer-based walking intervention in predominantly inactive adults, delivered by post or through primary care nurse-supported physical activity (PA) consultations. METHODS AND FINDINGS: A parallel three-arm cluster randomised trial was randomised by household, with 12-mo follow-up, in seven London, United Kingdom, primary care practices. Eleven thousand fifteen randomly selected patients aged 45-75 y without PA contraindications were invited. Five hundred forty-eight self-reporting achieving PA guidelines were excluded. One thousand twenty-three people from 922 households were randomised between 2012-2013 to one of the following groups: usual care (n = 338); postal pedometer intervention (n = 339); and nurse-supported pedometer intervention (n = 346). Of these, 956 participants (93%) provided outcome data (usual care n = 323, postal n = 312, nurse-supported n = 321). Both intervention groups received pedometers, 12-wk walking programmes, and PA diaries. The nurse group was offered three PA consultations. Primary and main secondary outcomes were changes from baseline to 12 mo in average daily step-counts and time in MVPA (in ≥10-min bouts), respectively, measured objectively by accelerometry. Only statisticians were masked to group. Analysis was by intention-to-treat. Average baseline daily step-count was 7,479 (standard deviation [s.d.] 2,671), and average time in MVPA bouts was 94 (s.d. 102) min/wk. At 12 mo, mean steps/d, with s.d. in parentheses, were as follows: control 7,246 (2,671); postal 8,010 (2,922); and nurse support 8,131 (3,228). PA increased in both intervention groups compared with the control group; additional steps/d were 642 for postal (95% CI 329-955) and 677 for nurse support (95% CI 365-989); additional MVPA in bouts (min/wk) were 33 for postal (95% CI 17-49) and 35 for nurse support (95% CI 19-51). There were no significant differences between the two interventions at 12 mo. The 10% (1,023/10,467) recruitment rate was a study limitation. CONCLUSIONS: A primary care pedometer-based walking intervention in predominantly inactive 45- to 75-y-olds increased step-counts by about one-tenth and time in MVPA in bouts by about one-third. Nurse and postal delivery achieved similar 12-mo PA outcomes. A primary care pedometer intervention delivered by post or with minimal support could help address the public health physical inactivity challenge. CLINICAL TRIAL REGISTRATION: isrctn.com ISRCTN98538934.

Dispatch from the non-HITECH-incented Health IT world: electronic medication history adoption and utilization.

OBJECTIVE: To document national trends of electronic medication history use in the ambulatory setting and describe the characteristics and predicting factors of providers who regularly use medication history transaction capabilities through their e-prescribing systems. MATERIALS AND METHODS: The study used provider-initiated medication history data requests, electronically sent over an e-prescribing network from all 50 states and the District of Columbia. Data from 138,000 prescribers were evaluated using multivariate analyses from 2007 to 2013. RESULTS: Medication history use showed significant growth, increasing from 8 to 850 million history requests during the study period. Prescribers on the network for <5 years had a lower likelihood of requests than those on the network for 5 or more years. Although descriptive analyses showed that prescribers in rural areas were alongside e-prescribing, and requesting medication histories more often than those in large and small cities, these findings were not significant in multivariate analyses. Providers in orthopedic surgery and internal medicine had a higher likelihood of more requests than family practice prescribers, with 12% and 7% higher likelihood, respectively. DISCUSSION: Early adopters of e-prescribing have remained medication history users and have continually increased their volume of requests for medication histories. CONCLUSION: Despite the fact that the use of medication histories through e-prescribing networks in the ambulatory care setting has not been encouraged through federal incentive programs, there has been substantial growth in the use of medication histories offered through e-prescribing networks.

Family and professionals underestimate quality of life across diverse cultures and health conditions: systematic review.

OBJECTIVES: To examine how accurately proxies evaluate quality of life (QoL) in people they know, using cross-cultural data from the multidimensional, multilingual World Health Organization Quality of Life assessment short-form (the WHOQOL-BREF) and whether accuracy varies by health condition or proxy type (eg, family/professional). STUDY DESIGN AND SETTING: Systematic review with meta-analysis: We searched five databases for reports of proxy-completed WHOQOL-BREF scores and aggregated results using a random-effects model. Minimal clinically important difference values were calculated. RESULTS: Analyses included nine studies (1980 dyads) of physical (n = 762) or mental (n = 604) health conditions, or intellectual disability (n = 614), in 10 countries. Mean person-proxy correlations ranged from 0.28 (social QoL) to 0.44 (physical QoL). Proxy measures were underestimates (ie, significantly lower than persons reported for themselves) for social [mean difference (MD) = 4.7, 95% confidence interval (CI): 1.8, 7.6], psychological (MD = 3.7, 95% CI: 0.6, 6.8), and physical (MD = 3.1, 95% CI: 0.6, 5.6) QoL. Underestimates varied significantly between health conditions for social (P < 0.001), psychological (P = 0.009), and physical (P = 0.02) QoL. Family members assessed psychological and environmental QoL better than professionals. CONCLUSION: Proxies tend to be imprecise, underestimating QoL, and should be aware of this tendency. Where health care is decided for others, family members' views about QoL should be prioritized.

Numbers are not the whole story: a qualitative exploration of barriers and facilitators to increased physical activity in a primary care based walking intervention.

BACKGROUND: The majority of mid-life and older adults in the UK are not achieving recommended physical activity levels and inactivity is associated with many health problems. Walking is a safe, appropriate exercise. The PACE-UP trial sought to increase walking through the structured use of a pedometer and handbook, with and without support from a practice nurse trained in behaviour change techniques (BCTs). Understanding barriers and facilitators to engagement with a primary care based physical activity intervention is essential for future trials and programmes. METHODS: We conducted semi-structured telephone interviews using a topic guide with purposive samples of participants who did and did not increase their walking from both intervention groups. Interviews were audio-recorded, transcribed and coded independently by researchers prior to performing a thematic analysis. Responsiveness to the specific BCTs used was also analysed. RESULTS: Forty-three trial participants were interviewed in early 2014. Almost all felt they had benefitted, irrespective of their change in step-count, and that primary care was an appropriate setting.Important facilitators included a desire for a healthy lifestyle, improved physical health, enjoyment of walking in the local environment, having a flexible routine allowing for an increase in walking, appropriate self and external monitoring and support from others.Important barriers included physical health problems, an inflexible routine, work and other commitments, the weather and a mistrust of the monitoring equipment.BCTs that were reported to have the most impact included: providing information about behaviour-health link; prompting self-monitoring and review of goals and outcomes; providing feedback; providing specific information about how to increase walking; planning social support/change; and relapse prevention. Rewards were unhelpful. CONCLUSIONS: Despite our expectation that there would be a difference between the experiences of those who did and did not objectively increase their walking, we found that most participants considered themselves to have succeeded in the trial and benefitted from taking part. Barriers and facilitators were similar across demographic groups and trial outcomes. Findings indicated several BCTs on which PA trial and programme planners could focus efforts with the expectation of greatest impact as well as strong support for primary care as an appropriate venue. TRIAL REGISTRATION: ISRCTN98538934.

Molecular interactions of the gating modifier toxin ProTx-II with NaV 1.5: implied existence of a novel toxin binding site coupled to activation.

Voltage-gated Na(+) channels are critical components in the generation of action potentials in excitable cells, but despite numerous structure-function studies on these proteins, their gating mechanism remains unclear. Peptide toxins often modify channel gating, thereby providing a great deal of information about these channels. ProTx-II is a 30-amino acid peptide toxin from the venom of the tarantula, Thrixopelma pruriens, that conforms to the inhibitory cystine knot motif and which modifies activation kinetics of Na(v) and Ca(v), but not K(v), channels. ProTx-II inhibits current by shifting the voltage dependence of activation to more depolarized potentials and, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. Despite this difference in functional effects, ProTx-II has been proposed to bind to neurotoxin site 4 because it modifies activation. Here, we investigate the bioactive surface of ProTx-II by alanine-scanning the toxin and analyzing the interactions of each mutant with the cardiac isoform, Na(v)1.5. The active face of the toxin is largely composed of hydrophobic and cationic residues, joining a growing group of predominantly K(v) channel gating modifier toxins that are thought to interact with the lipid environment. In addition, we performed extensive mutagenesis of Na(v)1.5 to locate the receptor site with which ProTx-II interacts. Our data establish that, contrary to prior assumptions, ProTx-II does not bind to the previously characterized neurotoxin site 4, thus making it a novel probe of activation gating in Na(v) channels with potential to shed new light on this process.

ProTx-I and ProTx-II: gating modifiers of voltage-gated sodium channels.

The tarantula venom peptides ProTx-I and ProTx-II inhibit voltage-gated sodium channels by shifting their voltage dependence of activation to a more positive potential, thus acting by a mechanism similar to that of potassium channel gating modifiers such as hanatoxin and VSTX1. ProTx-I and ProTx-II inhibit all sodium channel (Nav1) subtypes tested with similar potency and represent the first potent peptidyl inhibitors of TTX-resistant sodium channels. Like gating modifiers of potassium channels, ProTx-I and ProTx-II conform to the inhibitory cystine knot motif, and ProTx-II was demonstrated to bind to sodium channels in the closed state. Both toxins have been synthesized chemically, and ProTx-II, produced by recombinant means, has been used to map the interaction surface of the peptide with the Nav1.5 channel. In comparison, beta-scorpion toxins activate sodium channels by shifting the voltage dependence of activation to more negative potentials, and together these peptides represent valuable tools for exploring the gating mechanism of sodium channels.

Site-3 sea anemone toxins: molecular probes of gating mechanisms in voltage-dependent sodium channels.

Sea anemone toxins, whose biological function is the capture of marine prey, are invaluable tools for studying the structure and function of mammalian voltage-gated sodium channels. Their high degree of specificity and selectivity have allowed for detailed analysis of inactivation gating and assignment of molecular entities responsible for this process. Because of their ability to discriminate among channel isoforms, and their high degree of structural conservation, these toxins could serve as important lead compounds for future pharmaceutical design.

Comparison of mammalian cell lines expressing distinct isoforms of divalent metal transporter 1 in a tetracycline-regulated fashion.

DMT1 (divalent metal transporter; also known as SLC11A2, DCT1 or Nramp2) is responsible for ferrous iron uptake in the duodenum, iron exit from endosomes during the transferrin cycle and some transferrin-independent iron uptake in many cells. Four protein isoforms differ by starting in exon 1A or 2 and ending with alternative peptides encoded by mRNA that contains or lacks an IRE (iron responsive element; +/-IRE). We have compared 1A/+IRE and 2/-IRE DMT1 during regulated ectopic expression. HEK-293-F (human embryonic kidney-293-fast growing variant) cells were stably transfected with each construct expressed from a tetracycline-regulated CMV promoter. Reverse transcriptase-PCR analysis showed that construct expression responded to doxycycline. Immunofluorescence staining of cells, using antibodies specific for DMT1 isoforms, confirmed an increase in expression in the plasma membrane and cytosolic vesicles after doxycycline treatment, but with isoform specific distributions. Immunoblotting also revealed stimulation of expression. Nevertheless, both DMT1 isoforms performed similarly in assays for functional properties based on 54Mn2+ and 59Fe2+ uptake. Mn incorporation after doxycycline treatment was approximately 10-fold greater than that of untreated cells, while expression in the untreated cells was approximately 5-fold greater than in the untransfected cells. Uptake of Mn depended on addition of doxycycline, with half maximal response at approximately 1 nM doxycycline. Doxycycline-stimulated Mn and Fe uptake was linear with time for 10 min but not over longer periods. Transport exhibited a pH optimum at approximately 5.5 and dependence on incubation temperature and Mn or Fe concentration. The new cell lines should prove useful for research on metal homoeostasis, toxicological studies and efforts to identify distinctive properties of the isoforms.

Characterization of a novel modification to monoclonal antibodies: thioether cross-link of heavy and light chains.

A novel, nonreducible thioether bridge between the light and heavy chains of different IgG1 monoclonal antibodies has been characterized. An additional band with an apparent molecular weight of 92 kDa was detected when monoclonal antibodies were analyzed by reducing capillary gel electrophoresis (rCGE) and reducing SDS-PAGE. To further investigate this observation, an early-eluting peak in the size exclusion chromatogram of a reduced and alkylated monoclonal antibody was collected and characterized by liquid chromatography, mass spectrometry, and gel electrophoresis. The reduced and alkylated Mab was shown to be a cross-linked adduct with a molecular weight of 75 kDa. In the adduct, the heavy and light chains of the antibody were cross-linked by a nonreducible thioether bond between Cys-223 of the heavy chain and the C-terminal Cys residue of the light chain. The thioether bond modification was confirmed in the Fab fragment of a monoclonal antibody by LC-MS and nonreduced Lys-C peptide mapping with tandem mass spectrometry. The data show that the disulfide bond modification occurred under nonreducing conditions and was not an artifact of sample preparation for the rCGE analysis. The thioether bond modification was observed in several IgG1 monoclonal antibody products. Structural characterization of this novel modification is important in understanding the mechanism of thioether bond formation.