pyDarwin machine learning algorithms application and comparison in nonlinear mixed-effect model selection and optimization.

Forward addition/backward elimination (FABE) has been the standard for population pharmacokinetic model selection (PPK) since NONMEM® was introduced. We investigated five machine learning (ML) algorithms (Genetic algorithm [GA], Gaussian process [GP], random forest [RF], gradient boosted random tree [GBRT], and particle swarm optimization [PSO]) as alternatives to FABE. These algorithms were applied to PPK model selection with a focus on comparing the efficiency and robustness of each of them. All machine learning algorithms included the combination of ML algorithms with a local downhill search. The local downhill search consisted of systematically changing one or two "features" at a time (a one-bit or a two-bit local search), alternating with the ML methods. An exhaustive search (all possible combinations of model features, N = 1,572,864 models) was the gold standard for robustness, and the number of models examined leading prior to identification of the final model was the metric for efficiency.All algorithms identified the optimal model when combined with the two-bit local downhill search. GA, RF, GBRT, and GP identified the optimal model with only a one-bit local search. PSO required the two-bit local downhill search. In our analysis, GP was the most efficient algorithm as measured by the number of models examined prior to finding the optimal (495 models), and PSO exhibited the least efficiency, requiring 1710 unique models before finding the best solution. Additionally, GP was also the algorithm that needed the longest elapsed time of 2975.6 min, in comparison with GA, which only required 321.8 min.

Workplace Violence in the Pediatric Emergency Department: A National Survey of Physicians in the United States.

OBJECTIVES: Previous work shows that health care workers, and particularly emergency department (ED) staff, are at an above average risk of workplace violence (WPV), defined as verbal threats or physical violence in the workplace. Previous data suggest that staff of a single pediatric ED frequently feel unsafe. The objective of this study was to conduct the first national survey study to assess the prevalence and incidence of WPV in the pediatric ED. METHODS: In this cross-sectional survey study, a representative sample of US pediatric emergency medicine physicians were invited to participate in a national survey adapted from the previously validated Workplace Violence in the Health Sector questionnaire from the World Health Organization. The primary outcome measure was the proportion of physicians who have been exposed to WPV. Rates of exposure to violence, reporting habits, and physician perception of various violence prevention strategies were studied secondarily. RESULTS: Surveys were completed by 207 (45%) invited participants, representing 31 US children's hospitals. The prevalence of WPV exposure was 96%. Physical violence with a weapon was witnessed by 20% of participants. Injuries requiring medical attention or time off work were experienced by 10% of participants. Half of participants never reported violent events. Security guards were welcome by 99%, armed law enforcement officers by 70%, and metal detectors by 81% of physicians. Self-arming was opposed by 85% of respondents. CONCLUSIONS: Exposure to WPV is frequent among pediatric emergency medicine physicians with a prevalence similar to that of general emergency departments. Workplace violence remains underreported. This national survey contributes to the objective evaluation of individual- and systems-level violence prevention interventions.

Advancing in vivo reprogramming with synthetic biology.

Reprogramming cells will play a fundamental role in shaping the future of cell therapies by developing new strategies to engineer cells for improved performance and higher-order physiological functions. Approaches in synthetic biology harness cells' natural ability to sense diverse signals, integrate environmental inputs to make decisions, and execute complex behaviors based on the health of the organism or tissue. In this review, we highlight strategies in synthetic biology to reprogram cells, and discuss how recent approaches in the delivery of modified mRNA have created new opportunities to alter cell function in vivo. Finally, we discuss how combining concepts from synthetic biology and the delivery of mRNA in vivo could provide a platform for innovation to advance in vivo cellular reprogramming.

Hepatocyte-specific mitogen-activated protein kinase phosphatase 1 in sexual dimorphism and susceptibility to alcohol induced liver injury.

Background: It is well established that females are more susceptible to the toxic effects of alcohol, although the exact mechanisms are still poorly understood. Previous studies noted that alcohol reduces the expression of mitogen-activated protein kinase phosphatase 1 (MKP1), a negative regulator of mitogen-activated protein kinases (MAPK) in the liver. However, the role of hepatocyte- specific MKP1 in the pathogenesis of alcohol-associated liver disease (ALD) remains uncharacterized. This study aimed to evaluate the role of hepatocyte-specific MKP1 in the susceptibility and sexual dimorphism in alcohol-induced liver injury. Methods: C57Bl/6 mice were used in an intragastric ethanol feeding model of alcohol-associated steatohepatitis (ASH). Hepatocyte-specific Mkp1-/- knockout and (Mkp1+/+ "f/f" male and female mice were subjected to the NIAAA chronic plus binge model. Primary mouse hepatocytes were used for in vitro studies. Liver RNA sequencing was performed on an Illumina NextSeq 500. Liver injury was evaluated by plasma alanine transaminase (ALT), hepatic ER stress and inflammation markers. Statistical analysis was carried out using ANOVA and the unpaired Student's t-test. Results: ASH was associated with the severe injury accompanied by increased endoplasmic reticulum (ER) stress and significant downregulation of Dusp1 mRNA expression. In vitro, ethanol treatment resulted in a time-dependent decrease in Dusp1 mRNA and protein expression in primary hepatocytes in both males and females; however, this effect was significantly more pronounced in hepatocytes from females. In vivo, female mice developed more liver injury in a chronic plus binge model which was accompanied by a significant decrease in liver Dusp1 mRNA expression. In comparison, liver Dusp1 was not changed in male mice, while they developed milder injury to alcohol. Mkp1 deletion in hepatocytes led to increased alcohol induced liver injury, ER stress and inflammation in both sexes. Conclusion: Hepatocyte Mkp1 plays a significant role in alcohol induced liver injury. Alcohol downregulates Mkp1 expression in hepatocytes in a sex dependent manner and could play a role in sexual dimorphism in increased female susceptibility to alcohol.

pyDarwin: A Machine Learning Enhanced Automated Nonlinear Mixed-Effect Model Selection Toolbox.

pyDarwin is an open-source Python package for nonlinear mixed-effect model selection. pyDarwin combines machine-learning algorithms and NONMEM to perform a global search for the optimal model in a user-defined model search space. Compared with traditional stepwise search, pyDarwin provides an efficient platform for conducting an objective, robust, less labor-intensive model selection process without compromising model interpretability. In this tutorial, we will begin by introducing the essential components and concepts within the package. Subsequently, we will provide an overview of the pyDarwin modeling workflow and the necessary files needed for model selection. To illustrate the entire process, we will conclude with an example utilizing quetiapine clinical data.

Organometallic Pillarplexes That Bind DNA 4-Way Holliday Junctions and Forks.

Holliday 4-way junctions are key to important biological DNA processes (insertion, recombination, and repair) and are dynamic structures that adopt either open or closed conformations, the open conformation being the biologically active form. Tetracationic metallo-supramolecular pillarplexes display aryl faces about a cylindrical core, an ideal structure to interact with open DNA junction cavities. Combining experimental studies and MD simulations, we show that an Au pillarplex can bind DNA 4-way (Holliday) junctions in their open form, a binding mode not accessed by synthetic agents before. Pillarplexes can bind 3-way junctions too, but their large size leads them to open up and expand that junction, disrupting the base pairing, which manifests in an increased hydrodynamic size and lower junction thermal stability. At high loading, they rearrange both 4-way and 3-way junctions into Y-shaped forks to increase the available junction-like binding sites. Isostructural Ag pillarplexes show similar DNA junction binding behavior but lower solution stability. This pillarplex binding contrasts with (but complements) that of metallo-supramolecular cylinders, which prefer 3-way junctions and can rearrange 4-way junctions into 3-way junction structures. The pillarplexes' ability to bind open 4-way junctions creates exciting possibilities to modulate and switch such structures in biology, as well as in synthetic nucleic acid nanostructures. In human cells, the pillarplexes do reach the nucleus, with antiproliferative activity at levels similar to those of cisplatin. The findings provide a new roadmap for targeting higher-order junction structures using a metallo-supramolecular approach, as well as expanding the toolbox available to design bioactive junction binders into organometallic chemistry.

I-CARE: Feasibility, Acceptability, and Appropriateness of a Digital Health Intervention for Youth Experiencing Mental Health Boarding.

PURPOSE: Youth with suicidality requiring psychiatric hospitalization may first experience boarding at acute care hospitals. Given infrequent provision of therapy during this period, we developed a modular digital intervention (I-CARE; Improving Care, Accelerating Recovery and Education) to facilitate delivery of evidence-based psychosocial skills by non-mental health clinicians. This pilot study describes changes in emotional distress, severity of illness, and readiness for engagement following I-CARE participation, and evaluates the feasibility, acceptability, and appropriateness of I-CARE. METHODS: A mixed-methods approach was used to evaluate I-CARE, offered to youth 12-17 years from 11/21 to 06/22. Changes in emotional distress, severity of illness, and engagement readiness were evaluated using paired t-tests. Semistructured interviews with youth, caregivers, and clinicians were conducted concurrently with collection of validated implementation outcome measures. Quantitative measure results were linked to interview transcripts, which were analyzed thematically. RESULTS: Twenty-four adolescents participated in I-CARE; median length of stay was 8 days (IQR:5-12 days). Emotional distress decreased significantly by 6.3 points (63-point scale) following participation (p = .02). The increase in engagement readiness and decrease in youth-reported illness severity were not statistically significant. Among 40 youth, caregivers, and clinicians who participated in the mixed-methods evaluation, 39 (97.5%) rated I-CARE as feasible, 36 (90.0%) as acceptable, and 31 (77.5%) as appropriate. Adolescents' prior knowledge of psychosocial skills and clinicians' competing demands were reported barriers. DISCUSSION: I-CARE was feasible to implement and youth reported reduced levels of distress following participation. I-CARE has the potential to teach evidence-based psychosocial skills during boarding, which may provide a head-start on recovery before psychiatric hospitalization.

Lunchroom-Specific Peer Acceptance and Children's Internalizing Symptoms.

Prior research suggests the elementary school lunchroom is an important context for children's social development. Using a sample of 659 fourth-grade students in 10 public schools (50.7% female; 42.7% Hispanic/Latinx, 30.3% White, 10% Pacific Islander, 7.8% bi/multiracial, 2.2% American Indian, 2.2% Black, 1.9% Asian, and 2.9% other), we examined the association between lunchroom-specific peer acceptance and internalizing symptoms (i.e., depression and social anxiety symptoms). We hypothesized that lunchroom peer acceptance would predict self-rated depression and social anxiety symptoms when controlling for social preference scores. Using hierarchical linear modeling, results indicated self-rated lunchtime peer acceptance scores in December significantly predicted depression symptoms in May when controlling social preference scores and accounted for changes in depression scores across a school year. However, some significant gender differences emerged. Results suggest that elementary school lunchroom interventions should attend to children's perceptions of lunchroom likability and their experiences of depression symptoms.

Multistakeholder Perspectives on Interventions to Support Youth During Mental Health Boarding.

OBJECTIVE: To identify and prioritize opportunities to improve the psychiatric boarding experience for youth awaiting admission or transfer to inpatient psychiatric care. STUDY DESIGN: This study utilized an exploratory mixed methods design. The study team convened multidisciplinary stakeholder focus groups to discuss proposed hospital-based solutions to mental health boarding, potential psychosocial interventions deliverable during boarding, and outcomes measurement. Focus group responses were transcribed and analyzed to extract themes pertaining to these improvement opportunities. These results informed a follow-up survey which was then sent to the stakeholders to rate the feasibility and importance of modifications using a modified RAND-UCLA Appropriateness Method. RESULTS: Qualitative analyses revealed 9 themes across 2 domains related to psychiatric boarding care: in-hospital improvements and transitions of care. The follow-up survey identified 6 improvement opportunities rated as both feasible and important. Additionally, 6 psychosocial interventions, 2 delivery modalities, and 5 outcomes were rated as both feasible and important. CONCLUSIONS: Stakeholders concerned with the psychiatric boarding of youth identified numerous opportunities for improving the boarding process within 2 domains of in-hospital improvements and transitions of care. Most of the improvements were considered feasible and important with several serving as particularly viable strategies. These have the potential for implementation to improve the care of this vulnerable population and inform local and national quality improvement efforts.

Oral Hydration Before and After Hip Replacement: The Notion Behind Every Action.

Introduction: Even though nearly 20 patients undergo hip replacement every hour just in Italy and the United Kingdom, it is unclear what are the most appropriate oral hydration practices that patients should follow before and after surgery. Improper administration can cause postoperative fluid disturbances or exacerbate pre-existing conditions, which are not an uncommon find in older subjects. Significance: Considering that the number of hip operations is expected to increase in the next years as well as the age of patients, it is important to recall the notions behind water balance, especially in light of modern surgical and anesthetic practices. This technical perspective discusses the perioperative changes in the hydration status that occur during hip replacement and provides the concepts that help clinicians to better manage how much water the patient can drink. Results: The points of view of the surgeon, the anesthetist, and the nurse are offered together with the description of mineral waters intended for human consumption. Before surgery, water should be always preferred over caffeinated, sugar-sweetened, and alcoholic beverages. The drinking requirements on the day of surgery should consider the water output from urine, feces, respiration, exudation, and bleeding along with the water input from metabolic production and intravenous administration of fluids and medications. Healthy eating habits provide water and should be promoted before and after surgery. Conclusions: The judgment on which is the most appropriate approach to oral hydration practices must be the responsibility of the multidisciplinary perioperative team. Nevertheless, it is reasonable to argue that, in the presence of a patient with no relevant illness and who follows a healthy diet, it is more appropriate to stay closer to dehydration than liberalizing water intake both prior to surgery and in the early postoperative hours until the resumption of normal physiological functions.

Liver-specific drug delivery platforms: Applications for the treatment of alcohol-associated liver disease.

Alcohol-associated liver disease (ALD) is a common chronic liver disease and major contributor to liver disease-related deaths worldwide. Despite its pre-valence, there are few effective pharmacological options for the severe stages of this disease. While much pre-clinical research attention is paid to drug development in ALD, many of these experimental therapeutics have limitations such as poor pharmacokinetics, poor efficacy, or off-target side effects due to systemic administration. One means of addressing these limitations is through liver-targeted drug delivery, which can be accomplished with different platforms including liposomes, polymeric nanoparticles, exosomes, bacteria, and adeno-associated viruses, among others. These platforms allow drugs to target the liver passively or actively, thereby reducing systemic circulation and increasing the 'effective dose' in the liver. While many studies, some clinical, have applied targeted delivery systems to other liver diseases such as viral hepatitis or hepatocellular carcinoma, only few have investigated their efficacy in ALD. This review provides basic information on these liver-targeting drug delivery platforms, including their benefits and limitations, and summarizes the current research efforts to apply them to the treatment of ALD in rodent models. We also discuss gaps in knowledge in the field, which when addressed, may help to increase the efficacy of novel therapies and better translate them to humans.

Using their heads - A novel, collaborative approach between industry and scientists to monitor a commercial mullet fishery as a result of COVID-19 restrictions.

The COVID-19 global pandemic-related restrictions during 2020 severely impacted the Australian seafood industry, including essential scientific monitoring to support stock assessment and to demonstrate sustainability. Here we detail a novel, collaborative monitoring program between scientists and the seafood industry to generate length and age compositions that were representative of one of the largest, most valuable, and controversial fisheries along eastern Australia, the pre-spawning ocean run fishery for Sea Mullet Mugil cephalus that is predominantly a roe fishery. The standard approach to monitoring this fishery has been to base trained scientific staff at the major processing facility for M. cephalus, where they access whole fish from entire catches to generate representative length and age compositions during the peak season, April to May. Covid-19 restrictions prevented this approach for 2020 in eastern Australia. In recognition that in addition to the high-value roe, all components of the female fish are utilized (heads and guts for bait, bodies for human consumption), a multi-stage, spatially stratified sampling design was investigated. Female heads were retained from randomly selected catches from each of the three major fishing zones and transported to the New South Wales Department of Primary Industries fish laboratory based in Sydney. Head lengths (HLs) were measured and converted to Fork Lengths (FLs) using a HL to FL relationship. The resulting fish length compositions from each catch were subsequently combined based on: (i) relative catch size of females within an ocean zone, and; (ii) the relative reported landings of females in each ocean fishing zone. Otoliths were randomly collected from heads sampled from each ocean zone and used to estimate age. The resulting ocean zone to age matrix was combined with the relative reported landings of female fish in each ocean fishing zone to generate a total female age composition for the fishery. The estimated age composition of females were typical in being mainly between ages 3 and 6, with a strong presence of 4-year olds. This stronger cohort was present as 3-year olds in 2018/19 and 5-year olds in 2020/21, thus providing confidence that our sampling was representative of the fishery. The study reinforces the positive outcomes that can be generated through co-management between scientists and the seafood industry.

The sensitivity of simulated streamflow to individual hydrologic processes across North America.

Streamflow sensitivity to different hydrologic processes varies in both space and time. This sensitivity is traditionally evaluated for the parameters specific to a given hydrologic model simulating streamflow. In this study, we apply a novel analysis over more than 3000 basins across North America considering a blended hydrologic model structure, which includes not only parametric, but also structural uncertainties. This enables seamless quantification of model process sensitivities and parameter sensitivities across a continuous set of models. It also leads to high-level conclusions about the importance of water cycle components on streamflow predictions, such as quickflow being the most sensitive process for streamflow simulations across the North American continent. The results of the 3000 basins are used to derive an approximation of sensitivities based on physiographic and climatologic data without the need to perform expensive sensitivity analyses. Detailed spatio-temporal inputs and results are shared through an interactive website.

The association between work hours, shift work, and job latitude with fecundability: A preconception cohort study.

The role of occupational stress on male fertility is understudied. We examined associations between male occupational stress and fecundability. We used data from Pregnancy Study Online (PRESTO), a North American preconception cohort study. At baseline (2013-2019), male participants aged ≥ 21 years completed a baseline questionnaire on employment status, hours worked per week, time of day worked (daytime, evening, nights, and changing or rotating shifts), and job title. We used the O*NET occupational database to rate independence by job title. Female partners were followed via bimonthly follow-up questionnaires for 12 months or until pregnancy. We restricted analyses to 1,818 couples attempting conception for ≤ 6 cycles at enrollment. We used proportional probabilities regression to estimate fecundability ratios (FRs) and 95% confidence intervals (CIs). The FR comparing unemployed with employed men was0.84 (95% CI: 0.62-1.14). Among employed men, FRs and 95% CIs for evening shift work, night shift work, and rotating shift work were 0.89 (95% CI: 0.68-1.17), 0.94 (95% CI: 0.66-1.33), and 0.91 (95% CI: 0.75-1.11) relative to daytime shift work. The FR for any nondaytime shift work was 0.91 (95% CI: 0.78-1.07). Total work hours (long or short) and job independence scores were not appreciably associated with fecundability. In conclusion, working nondaytime shifts and being unemployed were associated with slightly decreased fecundability. However, the variability in these estimates was substantial and the results were reasonably consistent with chance. Little association was observed for other occupation measures. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Great Lakes Runoff Intercomparison Project Phase 3: Lake Erie (GRIP-E).

Hydrologic model intercomparison studies help to evaluate the agility of models to simulate variables such as streamflow, evaporation, and soil moisture. This study is the third in a sequence of the Great Lakes Runoff Intercomparison Projects. The densely populated Lake Erie watershed studied here is an important international lake that has experienced recent flooding and shoreline erosion alongside excessive nutrient loads that have contributed to lake eutrophication. Understanding the sources and pathways of flows is critical to solve the complex issues facing this watershed. Seventeen hydrologic and land-surface models of different complexity are set up over this domain using the same meteorological forcings, and their simulated streamflows at 46 calibration and seven independent validation stations are compared. Results show that: (1) the good performance of Machine Learning models during calibration decreases significantly in validation due to the limited amount of training data; (2) models calibrated at individual stations perform equally well in validation; and (3) most distributed models calibrated over the entire domain have problems in simulating urban areas but outperform the other models in validation.

So your patient is a gamer: an exploration of benefits and harms.

PURPOSE OF REVIEW: The appropriate amount and type of screen time for children remains a persistent topic of discussion between parents and pediatricians. During the COVID-19 pandemic, screen time has considerably increased. The need to quarantine and utilize remote learning prompted our review of the basis for current screen time recommendations and more recent research. RECENT FINDINGS: The broad stroke of recent research points to an association of increased video game screen time with sedentary lifestyle and its related harms. However, there also are specific instances where video games have been found to be therapeutically useful for certain populations. SUMMARY: In light of these findings, it is prudent for the pediatrician to explore more of the 'why' of video game use as opposed to purely the 'how much'. Given the future beneficial therapeutic uses of some video games, clinicians should keep their eyes on this space for its continued development.

Identification of a lichen depside polyketide synthase gene by heterologous expression in Saccharomyces cerevisiae.

Lichen-forming fungi produce a variety of secondary metabolites including bioactive polyketides. Advances in DNA and RNA sequencing have led to a growing database of new lichen gene clusters encoding polyketide synthases (PKS) and associated ancillary activities. Definitive assignment of a PKS gene to a metabolic product has been challenging in the lichen field due to a lack of established gene knockout or heterologous gene expression systems. Here, we report the reconstitution of a non-reducing PKS gene from the lichen Pseudevernia furfuracea and successful heterologous expression of the synthetic lichen PKS gene in engineered Saccharomyces cerevisiae. We show that P. furfuracea PFUR17_02294 produces lecanoric acid, the depside dimer of orsellinic acid, at 360 mg/L in small-scale yeast cultures. Our results unequivocally identify PFUR17_02294 as a lecanoric acid synthase and establish that a single lichen PKS synthesizes two phenolic rings and joins them by an ester linkage to form the depside product.

Supramolecular Cylinders Target Bulge Structures in the 5' UTR of the RNA Genome of SARS-CoV-2 and Inhibit Viral Replication*.

The untranslated regions (UTRs) of viral genomes contain a variety of conserved yet dynamic structures crucial for viral replication, providing drug targets for the development of broad spectrum anti-virals. We combine in vitro RNA analysis with molecular dynamics simulations to build the first 3D models of the structure and dynamics of key regions of the 5' UTR of the SARS-CoV-2 genome. Furthermore, we determine the binding of metallo-supramolecular helicates (cylinders) to this RNA structure. These nano-size agents are uniquely able to thread through RNA junctions and we identify their binding to a 3-base bulge and the central cross 4-way junction located in stem loop 5. Finally, we show these RNA-binding cylinders suppress SARS-CoV-2 replication, highlighting their potential as novel anti-viral agents.

"The Back-up Vasectomy Reversal." Simultaneous Sperm Retrieval and Vasectomy Reversal in the Couple With Advanced Maternal Age: A Cost-Effectiveness Analysis.

OBJECTIVE: To determine the cost-effectiveness of different fertility options in men who have undergone vasectomy in couples with a female of advanced maternal age (AMA). The options include vasectomy reversal (VR), sperm retrieval (SR) with in vitro fertilization (IVF), and the combination of VR and SR with IVF, which is a treatment pathway that has been understudied. MATERIALS AND METHODS: Using TreeAge software, a model-based cost-utility analysis was performed estimating the cost per quality-adjusted life years (QALY) in couples with infertility due to vasectomy and advanced female age over a period of one year. The model stratified for female age (35-37, 38-40, >40) and evaluated four strategies: VR followed by natural conception (NC), SR with IVF, VR and SR followed by failed NC and then IVF, and VR and SR followed by failed IVF and then NC. QALY estimates and outcome probabilities were obtained from the literature and average patient charges were calculated from high-volume centers. RESULTS: The most cost-effective fertility strategy was to undergo VR and try for NC (cost-per-QALY: $7,150 (35-37 y), $7,203 (38-40 y), and $7,367 (>40 y)). The second most cost-effective strategy was the "back-up vasectomy reversal": undergo VR and SR, attempt IVF and switch to NC if IVF fails. CONCLUSION: In couples with a history of vasectomy and female of AMA, it is most cost-effective to undergo a VR. If the couple opts for SR for IVF, it is more cost-effective to undergo a concomitant VR than SR alone.

Supramolecular Cylinders Target Bulge Structures in the 5' UTR of the RNA Genome of SARS-CoV-2 and Inhibit Viral Replication.

The untranslated regions (UTRs) of viral genomes contain a variety of conserved yet dynamic structures crucial for viral replication, providing drug targets for the development of broad spectrum anti-virals. We combine in vitro RNA analysis with molecular dynamics simulations to build the first 3D models of the structure and dynamics of key regions of the 5' UTR of the SARS-CoV-2 genome. Furthermore, we determine the binding of metallo-supramolecular helicates (cylinders) to this RNA structure. These nano-size agents are uniquely able to thread through RNA junctions and we identify their binding to a 3-base bulge and the central cross 4-way junction located in stem loop 5. Finally, we show these RNA-binding cylinders suppress SARS-CoV-2 replication, highlighting their potential as novel anti-viral agents.