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Abstract While a low degree of energy compensation is typically reported over the 24 h following a session of exercise, the prolonged impact of a bout of exercise on energy intake remains unclear. To overcome the challenge associated with accurately measuring energy intake in a free‐living environment, this study employed the use of a meal replacement beverage to assess the 3 day impact of an exercise session on energy intake. In a randomized, crossover study, 14 participants (8 male, 6 female) completed two trials: (1) EX: 75 min exercise on a motorized treadmill (75% VO2peak); and (2) SED: 75 min sedentary control session. Each condition was followed by 3 days of exclusive ad libitum consumption of a meal replacement beverage. Appetite‐regulating hormones, subjective appetite, energy intake, and energy expenditure were assessed. Exercise transiently suppressed the orexigenic hormone acyl‐ghrelin (p

Summary: Olanzapine is a second-generation antipsychotic (AP) used in the management of schizophrenia. Although effective at reducing psychoses, APs cause rapid hyperglycemia, insulin resistance, and dyslipidemia, an effect mediated in part by glucagon. We tested if amylin, a hormone that reduces glucagon, or the amylin receptor agonist pramlintide would protect against acute olanzapine-induced impairments in glucose and lipid homeostasis alone or in combination with other glucose-lowering agents such as liraglutide. We demonstrated that pramlintide lowered olanzapine-induced increases in glucagon:insulin ratio with a trend to protect against excursions in blood glucose. There was an additive effect of pramlintide and liraglutide in protecting against olanzapine-induced hyperglycemia, which was mirrored by reductions in glucagon and attenuated markers of dyslipidemia. Our findings provide evidence that pramlintide, although moderately protective against some aspects of olanzapine-induced metabolic dysfunction, can be used to enhance the protective effects of other interventions against acute olanzapine-induced metabolic dysfunction.

Antipsychotic medications are used in the management of schizophrenia and a growing number of off-label conditions. While effective at reducing psychoses, these drugs possess noted metabolic side effects including weight gain, liver lipid accumulation and disturbances in glucose and lipid metabolism. To counter the side effects of antipsychotics standard of care has typically included metformin. Unfortunately, metformin does not protect against antipsychotic induced metabolic disturbances in all patients and thus additional treatment approaches are needed. One potential candidate could be salsalate, the prodrug of salicylate, which acts synergistically with metformin to improve indices of glucose and lipid metabolism in obese mice. The purpose of the current investigation was to compare the effects of salsalate, metformin and a combination of both drugs, on weight gain and indices of metabolic health in female mice treated with the antipsychotic, olanzapine. Herein we demonstrate that salsalate was equally as effective as metformin in protecting against olanzapine induced weight gain and liver lipid accumulation with no additional benefit of combining both drugs. Conversely, metformin treatment, either alone or in combination with salsalate, improved indices of glucose metabolism and increased energy expenditure in olanzapine treated mice. Collectively, our findings provide evidence that dual therapy with both metformin and salsalate could be an efficacious approach with which to dampen the metabolic consequences of antipsychotic medications.

Abstract Species distribution models (SDM) are widely applied to understand changing species geographical distribution and abundance patterns. However, existing SDM tools are inherently static and inadequate for modelling species distributions that are driven by dynamic environmental conditions. dynamicSDM provides novel tools that explicitly consider the temporal dimension at key SDM stages, including functions for: (a) Cleaning and filtering species occurrence records by spatial and temporal qualities; (b) Generating pseudo‐absence records through space and time; (c) Extracting spatiotemporally buffered explanatory variables; (d) Fitting SDMs whilst accounting for temporal biases and autocorrelation and (e) Projecting intra‐ and inter‐ annual geographical distributions and abundances at high spatiotemporal resolution. Package functions have been designed to be: flexible for targeting specific study species; compatible with other SDM tools; and, by utilising Google Earth Engine and Google Drive, to have low computing power and storage needs. We illustrate dynamicSDM functions with an example of a nomadic bird in southern Africa, the red‐billed quelea Quelea quelea. As dynamicSDM functions are flexible and easily applied, we suggest that these tools could be readily applied to other taxa and systems globally.

Agro-technologies such as irrigation and new crop varieties can reduce climate risk for agricultural production in sub-Sahara Africa (SSA). SSA has the highest maize yield gaps globally, despite its importance as a staple crop in the region. Reducing maize yield gaps is key to tackling food insecurity; however, closing yield gaps might imply an increased greenhouse gas (GHG) emission cost. Climate smart agriculture (CSA) seeks to minimise this cost whilst maximising productivity and resilience. One key element of CSA is resilience to extreme events, although this is rarely examined. Accordingly, we assess the climate smartness of contrasting agro-technology and climate scenarios to assess both resilience to extremes and the overall climate smartness of the scenarios. We use simulations from an existing integrated modelling framework for Malawi, Tanzania, and Zambia, centred on 2050. Four scenarios were examined, defined by combinations of high vs. low agro-technology adoption and high vs. low climate risk (RCP2.6 and RCP8.5). We calculated a climate smartness index (CSI) to the model outputs that quantify the trade-offs between greenhouse gas emissions and agricultural productivity. CSI scores showed that the increase in GHG emissions from improved agro-technology is compensated for the yield benefits. Agro-technology in SSA can therefore benefit the pillars of climate-smart agriculture, namely increased mitigation, adaptation, and productivity. Further, we show that improved maize varieties and irrigation can substantially reduce future yield shocks and enhance resilience to climate change extremes in SSA, pointing to best-bets for agro-technology adoption. Irrigation reduces mid-century yield shocks by 64% (RCP2.6) or 42% (RCP8.5). When combined with improved maize varieties, irrigation removes the majority of yield shocks (90%) in RCP8.5. We therefore conclude that: (i) irrigation has significant potential to increase resilience in SSA; and (ii) investment in strategies to improve crop varieties is critical if the benefits or irrigation are to be fully realized under an RCP8.5 future.

El Cantado Cuencano “Cuencano singing” constitutes the hallmark of Cuencano Spanish: a widely spoken Andean dialect in the Ecuadorian province of Azuay. This colloquially described “singing” makes Cuencano Spanish one of the most distinct dialects of Ecuador. The aim of the present study is to provide a preliminary analysis of intonation patterns from common utterance types in this under documented dialect. A sample of 550 utterances from 11 categories that included declarative statements, yes/no questions, exclamative statements, wh-questions, imperatives, lists, conditionals, tag-questions, interjections, negative statements, and vocatives was collected from five male and five female participants. The tokens were analyzed acoustically and labeled using the Spanish Tones and Break Indices system (Sp_ToBI). Results reveal the presence of a tritonal pitch accent (PA), labeled as L + H* + L and the extensive use of bitonal PAs (namely, L + H*). These three phenomena mark the singing quality of Cuencano Spanish and make it stand out from the dialects of Ecuadorian Spanish.

Objective: The liver is regularly exposed to changing metabolic and inflammatory environments. It must sense and adapt to metabolic need while balancing resources required to protect itself from insult. Peroxisome proliferator activated receptor gamma coactivator-1 alpha (PGC-1α) is a transcriptional coactivator expressed as multiple, alternatively spliced variants transcribed from different promoters that coordinate metabolic adaptation and protect against inflammation. It is not known how PGC-1α integrates extracellular signals to balance metabolic and anti-inflammatory outcomes. Methods: Primary mouse hepatocytes were used to evaluate the role(s) of different PGC-1α proteins in regulating hepatic metabolism and inflammatory signaling downstream of tumor necrosis factor alpha (TNFα). Gene expression and signaling analysis were combined with biochemical measurement of apoptosis using gain- and loss-of-function in vitro and in vivo. Results: Hepatocytes expressed multiple isoforms of PGC-1α, including PGC-1α4, which microarray analysis showed had common and isoform-specific functions linked to metabolism and inflammation compared with canonical PGC-1α1. Whereas PGC-1α1 primarily impacted gene programs of nutrient metabolism and mitochondrial biology, TNFα signaling showed several pathways related to innate immunity and cell death downstream of PGC-1α4. Gain- and loss-of-function models illustrated that PGC-1α4 uniquely enhanced expression of anti-apoptotic gene programs and attenuated hepatocyte apoptosis in response to TNFα or lipopolysaccharide (LPS). This was in contrast to PGC-1α1, which decreased the expression of a wide inflammatory gene network but did not prevent hepatocyte death in response to cytokines. Conclusions: PGC-1α variants have distinct, yet complementary roles in hepatic responses to metabolism and inflammation, and we identify PGC-1α4 as an important mitigator of apoptosis. Keywords: Liver, PGC-1 isoforms, Inflammation, Apoptosis, Metabolism

Maize is the most important crop grown in South Africa, but yields can be severely reduced by extreme high summer average temperatures and low precipitation, potentially adversely affecting both domestic consumption and regional food security exports. To help understand and manage climate risks to food security in Southern Africa it is essential to quantify the present-day likelihood and magnitude of climate extremes in South Africa’s maize-growing region and explore the potential for unprecedented climate conditions which would likely result in record low maize yields. We analyse a large ensemble of initialised climate model simulations, which provides almost 100 times as many plausible present-day summers as the equivalent observational dataset. We quantify the risk of unprecedented climate extremes affecting maize production in South Africa and examine the role of the El Niño-Southern Oscillation. We find that the South African maize region is at risk of experiencing record-breaking hot, cold, dry or wet events under current climatic conditions. We find that the annual chance of unprecedented high temperatures in South Africa is approximately 4%, increasing to 62% during very strong El Niño years. We also find that the chance of exceeding the present day seasonal high temperature record has increased across the 1979–2018 period, being five times more likely now than it was in 1980. These extreme events could result in a record-breaking number of days above the optimum, or even the maximum, temperature for maize production, and lead to more severe floods or droughts. Under climate change scenarios, the magnitude and frequency of climate extremes is projected to increase meaning that the unprecedented extremes studied here could become commonplace in the future. This suggests that significant investment is needed to develop adaptations that manage the climate-related risks to food systems now and build resilience to the projected impacts of climate change.

Objective: This article provides a systematic review of the team knowledge literature and guidance for further research.Background: Recent research has called attention to the need for the improved study and understanding of team knowledge. Team knowledge refers to the higher level knowledge structures that emerge from the interactions of individual team members.Method: We conducted a systematic review of the team knowledge literature, focusing on empirical work that involves the measurement of team knowledge constructs. For each study, we extracted author degree area, study design type, study setting, participant type, task type, construct type, elicitation method, aggregation method, measurement timeline, and criterion domain.Results: Our analyses demonstrate that many of the methodological characteristics of team knowledge research can be linked back to the academic training of the primary author and that there are considerable gaps in our knowledge with regard to the relationships between team knowledge constructs, the mediating mechanisms between team knowledge and performance, and relationships with criteria outside of team performance, among others. We also identify categories of team knowledge not yet examined based on an organizing framework derived from a synthesis of the literature.Conclusion: There are clear opportunities for expansion in the study of team knowledge; the science of team knowledge would benefit from a more holistic theoretical approach.Application: Human factors researchers are increasingly involved in the study of teams. This review and the resulting organizing framework provide researchers with a summary of team knowledge research over the past 10 years and directions for improving further research. [ABSTRACT FROM AUTHOR]

BACKGROUND: Oral submucous fibrosis (OSF) is a precancerous condition showing extensive fibrosis of the submucosa and affects most parts of the oral cavity, including pharynx and upper third of the oesophagus. The molecules involved in the biological pathways of the fibrotic process appeared to be either down- or upregulated at different stages of the disease. Despite the precancerous nature, malignant transformation of the epithelium in the background of fibrosis has not been studied in detail. HIF-1alpha is a known transcription factor that is induced by hypoxia. AIMS: To test the hypothesis that hypoxia plays a role in malignant transformation and progression of OSF. MATERIALS AND METHODS: We used both formalin-fixed and frozen samples of OSF and normal mucosa to investigate the relationship between HIF-1alpha and epithelial dysplasia using immunohistochemistry and RT-PCR. CONCLUSIONS: Our data indicate that HIF-1alpha is upregulated at both protein and mRNA levels in OSF and the correlation with epithelial dysplasia is statistically significant (P < 0.001). We propose that HIF-1alpha may play a role in malignant transformation of OSF. Further, over-expression of HIF-1alpha may contribute to the progression of fibrosis. It may be possible to use HIF-1alpha as a marker for malignant transformation of OSF. [ABSTRACT FROM AUTHOR]

Objective: This study measured the independent contributions of self-efficacy, outcome expectations, and dental health value in predicting an objective measure of preventive oral hygiene behavior, dental plaque. Methods: One hundred forty-eight subjects completed study measures and then had their dental plaque levels measured. Results: Structural equation modeling revealed that self-efficacy for flossing significantly predicted plaque levels but self-efficacy for brushing, outcome expectations, and dental value did not. Conclusion: Results supported Bandura's belief that self-efficacy for specific behavior can predict outcomes of that behavior, and outcome expectations and value beliefs do not play an independent role in explaining behavior. [ABSTRACT FROM AUTHOR]

A Dental Value Questionnaire (DVQ) was developed, pilot tested, and given to two samples of men and women (n = 103 and 50). Subjects reported how the loss of all teeth fared against the loss of body parts, senses, abilities, aesthetics, and having a chronic illness or disease. Principal components analysis with varimax rotation revealed three unipolar dimensions. Factor items were placed into scales called Senses and Abilities, Superficial Losses and Problems, and Body Parts, to which a Total scale was added. All scales demonstrated good internal consistency and test-retest stability. Correlations with extratest measures provided preliminary evidence for their validity. [ABSTRACT FROM AUTHOR]

Abstract Sleep restriction is associated with impaired glucose metabolism and insulin resistance, however, the underlying mechanisms leading to this impairment are unknown. This study aimed to assess whether the decrease in insulin sensitivity observed after sleep restriction is accompanied by changes in skeletal muscle PKB activity. Ten healthy young males participated in this randomized crossover study which included two conditions separated by a 3‐week washout period. Participants underwent two nights of habitual sleep (CON) and two nights of sleep which was restricted to 50% of habitual sleep duration (SR) in the home environment. Whole‐body glucose tolerance and insulin sensitivity were assessed by an oral glucose tolerance test after the second night of each condition. Skeletal muscle tissue samples were obtained from the vastus lateralis to determine PKB activity. Findings displayed no effect of trial on plasma glucose concentrations (P = 0.222). Plasma insulin area under the curve was higher after sleep restriction compared to the control (P = 0.013). Matsuda index was 18.6% lower in the sleep restriction (P = 0.010). Fold change in PKB activity from baseline tended to be lower in the sleep restriction condition at 30 min (P = 0.098) and 120 min (P = 0.087). In conclusion, we demonstrated decreased whole‐body insulin sensitivity in healthy young males following two nights of sleep restriction. Skeletal muscle insulin signaling findings are inconclusive and require further study to examine any potential changes.

Skeletal muscle is a plastic tissue capable of adapting and mal-adapting to physical activity and diet. The response of skeletal muscle to adaptive stimuli, such as exercise, can be modified by the prior nutritional status of the muscle. The influence of nutrition on skeletal muscle has the potential to substantially impact physical function and whole body metabolism. Animal and cell based models show that omega-3 fatty acids, in particular those of marine origin, can influence skeletal muscle metabolism. Furthermore, recent human studies demonstrate that omega-3 fatty acids of marine origin can influence the exercise and nutritional response of skeletal muscle. These studies show that the prior omega-3 status influences not only the metabolic response of muscle to nutrition, but also the functional response to a period of exercise training. Omega-3 fatty acids of marine origin therefore have the potential to alter the trajectory of a number of human diseases including the physical decline associated with aging. We explore the potential molecular mechanisms by which omega-3 fatty acids may act in skeletal muscle, considering the n-3/n-6 ratio, inflammation and lipidomic remodelling as possible mechanisms of action. Finally, we suggest some avenues for further research to clarify how omega-3 fatty acids may be exerting their biological action in skeletal muscle.

Abstract Background Neonatal cerebellar cortical degeneration is a neurodegenerative disease described in several canine breeds including the Beagle. Affected Beagles are unable to ambulate normally from the onset of walking and the main pathological findings include Purkinje cell loss with swollen dendritic processes. Previous reports suggest an autosomal recessive mode of inheritance. The development of massively parallel sequencing techniques has presented the opportunity to investigate individual clinical cases using genome-wide sequencing approaches. We used genome-wide mRNA sequencing (mRNA-seq) of cerebellum tissue from a single Beagle with neonatal cerebellar cortical degeneration as a method of candidate gene sequencing, with the aim of identifying the causal mutation. Results A four-week old Beagle dog presented with progressive signs of cerebellar ataxia and the owner elected euthanasia. Histopathology revealed findings consistent with cerebellar cortical degeneration. Genome-wide mRNA sequencing (mRNA-seq) of RNA from cerebellum tissue was used as a method of candidate gene sequencing. After analysis of the canine orthologues of human spinocerebellar ataxia associated genes, we identified a homozygous 8 bp deletion in the β-III spectrin gene, SPTBN2, associated with spinocerebellar type 5 in humans. Genotype analysis of the sire, dam, ten clinically unaffected siblings, and an affected sibling from a previous litter, showed the mutation to fully segregate with the disorder. Previous studies have shown that β-III spectrin is critical for Purkinje cell development, and the absence of this protein can lead to cell damage through excitotoxicity, consistent with the observed Purkinje cell loss, degeneration of dendritic processes and associated neurological dysfunction in this Beagle. Conclusions An 8 bp deletion in the SPTBN2 gene encoding β-III spectrin is associated with neonatal cerebellar cortical degeneration in Beagle dogs. This study shows that mRNA-seq is a feasible method of screening candidate genes for mutations associated with rare diseases when a suitable tissue resource is available.

Abstract Background Malaria is the third most prevalent cause of infectious disease in the world. Resistance of the parasite to classical drugs makes the discovery of new and effective drugs more urgent. The oxidized derivative of hydroxy-cis terpenone (OHCT) is a synthetic molecule that is not toxic to cultured human liver cells at concentrations as high as 60 μM and inhibits activity of cytochrome P450s that metabolize many drugs. Methods OHCT activity against chloroquine-sensitive and -resistant strains of Plasmodium falciparum, and a P. falciparum clone that is partially resistant to artemisinin was assayed in vitro. Results OHCT at nanomolar concentrations was effective against all intraerythrocytic stages of P. falciparum and exhibited activity in vitro against both chloroquine-sensitive and -resistant strains of P. falciparum as well as a P. falciparum clone that is partially resistant to artemisinin. Moreover, OHCT exhibited potent activity against gametocytes, the form that is transmitted by mosquitoes and essential for the spread of malaria. Conclusion OHCT displays strong growth inhibitory activity against all stages of P. falciparum and no evidence of toxicity to human cells in culture. It is easily synthesized and has the potential for inhibiting metabolism of drugs used in combination therapies.

Abstract Background Biologists are becoming increasingly aware that the interaction of animals, including humans, with their coevolved bacterial partners is essential for health. This growing awareness has been a driving force for the development of models for the study of beneficial animal-bacterial interactions. In the squid-vibrio model, symbiotic Vibrio fischeri induce dramatic developmental changes in the light organ of host Euprymna scolopes over the first hours to days of their partnership. We report here the creation of a juvenile light-organ specific EST database. Results We generated eleven cDNA libraries from the light organ of E. scolopes at developmentally significant time points with and without colonization by V. fischeri. Single pass 3' sequencing efforts generated 42,564 expressed sequence tags (ESTs) of which 35,421 passed our quality criteria and were then clustered via the UIcluster program into 13,962 nonredundant sequences. The cDNA clones representing these nonredundant sequences were sequenced from the 5' end of the vector and 58% of these resulting sequences overlapped significantly with the associated 3' sequence to generate 8,067 contigs with an average sequence length of 1,065 bp. All sequences were annotated with BLASTX (E-value < -03) and Gene Ontology (GO). Conclusion Both the number of ESTs generated from each library and GO categorizations are reflective of the activity state of the light organ during these early stages of symbiosis. Future analyses of the sequences identified in these libraries promise to provide valuable information not only about pathways involved in colonization and early development of the squid light organ, but also about pathways conserved in response to bacterial colonization across the animal kingdom.

Abstract Background Invasion is an important early step of cancer metastasis that is not well understood. Developing therapeutics to limit metastasis requires the identification and validation of candidate proteins necessary for invasion and migration. Methods We developed a functional proteomic screen to identify mediators of tumor cell invasion. This screen couples Fluorophore Assisted Light Inactivation (FALI) to a scFv antibody library to systematically inactivate surface proteins expressed by human fibrosarcoma cells followed by a high-throughput assessment of transwell invasion. Results Using this screen, we have identified CD155 (the poliovirus receptor) as a mediator of tumor cell invasion through its role in migration. Knockdown of CD155 by FALI or by RNAi resulted in a significant decrease in transwell migration of HT1080 fibrosarcoma cells towards a serum chemoattractant. CD155 was found to be highly expressed in multiple cancer cell lines and primary tumors including glioblastoma (GBM). Knockdown of CD155 also decreased migration of U87MG GBM cells. CD155 is recruited to the leading edge of migrating cells where it colocalizes with actin and αv-integrin, known mediators of motility and adhesion. Knockdown of CD155 also altered cellular morphology, resulting in cells that were larger and more elongated than controls when plated on a Matrigel substrate. Conclusion These results implicate a role for CD155 in mediating tumor cell invasion and migration and suggest that CD155 may contribute to tumorigenesis.

Abstract Purpose To validate the treatment-specific Treatment Satisfaction Survey for Intraocular Pressure (TSS-IOP). Methods Item content was developed by 4 heterogeneous patient focus groups (n = 32). Instrument validation involved 250 patients on ocular hypotensive medications recruited from ophthalmology practices in the Southern USA. Participants responded to demographic and test questions during a clinic visit. Standard psychometric analyses were performed on the resulting data. Sample Of the 412 patients screened, 253 consented to participate, and 250 provided complete datasets. The sample included 44% male (n = 109), 44% Black (n = 109) and 57% brown eyed (n = 142) participants, with a mean age of 64.6 years (SD 13.1) and a history of elevated IOP for an average of 8.4 yrs (SD 7.8). A majority was receiving monotherapy (60%, n = 151). Results A PC Factor analysis (w/ varimax rotation) of the 31 items yielded 5 factors (Eigenvalues > 1.0) explaining 70% of the total variance. Weaker and conceptually redundant items were removed and the remaining 15 items reanalyzed. The satisfaction factors were; Eye Irritation (EI; 4 items), Convenience of Use (CofU; 3 items), Ease of Use (EofU; 3 items), Hyperemia (HYP; 3 items), and Medication Effectiveness (EFF; 2 items). Chronbach's Alphas ranged from .80 to .86. Greater distributional skew was found for less common experiences (i.e., HYP & EI with 65% & 48.4% ceilings) than for more common experiences (i.e., EofU, CofU, EFF with 10.8%, 20.8% & 15.9% ceilings). TSS-IOP scales converged with conceptually related scales on a previously validated measure of treatment satisfaction, the TSQM (r = .36 to .77). Evidence of concurrent criterion-related validity was found. Patients' symptomatic ratings of eye irritation, hyperemia and difficulties using the medication correlated with satisfaction on these dimensions (r = .30-.56, all p < .001). Clinicians' ratings of IOP control, severity of side effects and problematic medication use correlated with patients' satisfaction scores on these dimensions (r = .13-.26, all p < .01). Conclusions This study provides initial evidence that the TSS-IOP is a reliable and valid measure, assessing patients' satisfaction with ocular hypotensive medications.

Abstract Background Many RNA viruses do not have a single, representative genome but instead form a set of related variants that has been called a quasispecies. The sequence variability of such viruses presents a significant bioinformatics challenge. In order for the sequence information to be understood, the complete mutational spectrum needs to be distilled to a biologically relevant and analyzable representation. Results Here, we develop a "selection mapping" algorithm--QUASI--that identifies the positively selected variants of viral proteins. The key to the selection mapping algorithm is the identification of particular replacement mutations that are overabundant relative to silent mutations at each codon (e.g., threonine at hemagglutinin position 262). Selection mapping identifies such replacement mutations as positively selected. Conversely, selection mapping recognizes negatively selected variants as mutational "noise" (e.g., serine at hemagglutinin position 262). Conclusion Selection mapping is a fundamental improvement over earlier methods (e.g., dN/dS) that identify positive selection at codons but do not identify which amino acids at these codons confer selective advantage. Using QUASI's selection maps, we characterize the selected mutational landscapes of influenza A H3 hemagglutinin, HIV-1 reverse transcriptase, and HIV-1 gp120.

Fires in coniferous forests throughout the northern United States alter ecosystem processes and ecological communities, including the diversity and composition of microbial communities living in the soil. In addition to its influence on ecosystem processes and functions, the soil microbiome can interact with soilborne pathogens to facilitate or suppress plant disease development. Altering the microbiome composition to promote taxa that inhibit pathogenic activity has been suggested as a management strategy for forest diseases, including Armillaria root disease caused by Armillaria solidipes , which causes growth loss and mortality of conifers. These forest ecosystems are experiencing increased wildfire burn severity that could influence A. solidipes activity and interactions of the soil microbiome with Armillaria root disease. In this research, we examine changes to the soil microbiome following three levels of burn severity in a coniferous forest in northern Idaho, United States, where Armillaria root disease is prevalent. We further determine how these changes correspond to the soil microbiomes associated with the pathogen A. solidipes, and a putatively beneficial species, A. altimontana . At 15-months post-fire, we found significant differences in richness and diversity between bacterial communities associated with unburned and burned areas, yet no significant changes to these metrics were found in fungal communities following fire. However, both bacterial and fungal communities showed compositional changes associated with burn severity, including microbial taxa with altered relative abundance. Further, significant differences in the relative abundance of certain microbial taxa in communities associated with the three burn severity levels overlapped with taxa associated with various Armillaria spp. Following severe burn, we observed a decreased relative abundance of beneficial ectomycorrhizal fungi associated with the microbial communities of A. altimontana, which may contribute to the antagonistic activity of this soil microbial community. Additionally, A. solidipes and associated microbial taxa were found to dominate following high-severity burns, suggesting that severe fires provide suitable environmental conditions for these species. Overall, our results suggest that shifts in the soil microbiome and an associated increase in the activity of A. solidipes following high-severity burns in similar conifer forests may result in priority areas for monitoring and proactive management of Armillaria root disease., Importance: With its influence on ecosystem processes and functions, the soil microbiome can interact with soilborne pathogens to facilitate or suppress plant disease development. These forest ecosystems are experiencing increased wildfire frequency and burn severity that could influence the fungal root pathogen, Armillaria solidipes , and interactions with the soil microbiome. We examined changes to the soil microbiome following three levels of burn severity, and examined how these changes correspond with A. solidipes , and a putatively beneficial species, A. altimontana . Following severe burn, there was a decreased relative abundance of ectomycorrhizal fungi associated A. altimontana . A. solidipes and associated microbial taxa dominated following high-severity burns, suggesting that severe fires provide suitable environmental conditions for these species. Our results suggest that shifts in the soil microbiome and an associated increase in the activity of A. solidipes following high-severity burns in conifer forests may result in priority areas for monitoring and proactive management of Armillaria root disease., Competing Interests: The authors declare no conflict of interest.

Brown root rot disease (BRRD) is a highly destructive tree disease. Early diagnosis of BRRD has been challenging because the first symptoms and signs are often observed after extensive tissue colonization. Existing molecular detection methods, all based on the internal transcribed spacer (ITS) region, were developed without testing against global Phellinus noxius isolates, other wood-decay fungi, or host plant tissues. This study aimed to develop SYBR Green real-time quantitative PCR (qPCR) assays for P. noxius . The primer pair Pn&#95;ITS&#95;F/Pn&#95;ITS&#95;R targets the ITS, and the primer pair Pn&#95;NLR&#95;F/Pn&#95;NLR&#95;R targets a P. noxius -unique group of homologous genes identified through a comparative genomics analysis. The homologous genes belong to the nucleotide-binding-oligomerization-domain-like receptor (NLR) superfamily. The new primer pairs and a previous primer pair G1F/G1R were optimized for qPCR conditions and tested for specificity using 61 global P. noxius isolates, 5 other Phellinus species, and 22 non- Phellinus wood-decay fungal species. Although all three primer pairs could detect as little as 100 fg (approximately 2.99 copies) of P. noxius genomic DNA, G1F/G1R had the highest specificity and Pn&#95;NLR&#95;F/Pn&#95;NLR&#95;R had the highest efficiency. To avoid false positives, the cutoff quantification cycle values were determined as 34 for G1F/G1R, 29 for Pn&#95;ITS&#95;F/Pn&#95;ITS&#95;R, and 32 for Pn&#95;NLR&#95;F/Pn&#95;NLR&#95;R. We further validated these qPCR assays using Ficus benjamina seedlings artificially inoculated with P. noxius , six tree species naturally infected by P. noxius , rhizosphere soil, and bulk soil. The newly developed qPCR assays provide sensitive detection and quantification of P. noxius , which is useful for long-term monitoring of BRRD status., Competing Interests: The author(s) declare no conflict of interest.

Leptomeningeal dissemination (LMD) is the primary cause of treatment failure in children with group 3 medulloblastoma (MB). Building on our previous work on protein phosphatase 2A (PP2A) activation in MB, here we present preclinical and molecular data on the effects of two novel classes of PP2A activators on disease processes of LMD in group 3 MB. The PP2A activators used in this study are ATUX-6156 and ATUX-6954 (diarylmethylcycloamine sulfonylureas), and ATUX-1215 and ATUX-5800 (diarylmethyl-4-aminotetrahydropyran-sulfonamides). Treatment with these compounds led to suppression of the endogenous PP2A inhibitor, cancerous inhibitor of PP2A (CIP2A), enhanced phosphatase activity (10-60%), and reduced MB viability, migration, and invasion, prerequisites for MB cells to access the cerebrospinal fluid, affecting the initiation stage of LMD. PP2A activator treatment of MB cells led to apoptosis mediated via caspase 9/PARP signaling due to decreased phosphorylation of Bad, impeding the dispersal stage of LMD. Cell proliferation and LMD-driving cellular traits and molecules pertinent to the third stage, colonization, were also affected. Treatment with ATUX-1215 or ATUX-5800 prevented LMD in an intraventricular murine model of MB, possibly mediated by disruption of the CCL2-CCR2 axis by altered NF-kB phosphorylation via disrupted AKT signaling. The present investigation offers proof-of-principle data for PP2A-based reactivation therapy for Group 3 MB and provides the first indications that PP2A reactivation may challenge the current paradigm in targeting the 3-stage process of MB LMD. Further investigations of PP2A activators are warranted as these compounds may prove beneficial as therapeutics for MB., Competing Interests: Conflict of interest Patent applications WO2023/023594 and WO2021/188949, inventor MO, are assigned to Atux Iskay LLC. The other authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Here, we describe patterns of reproduction and flight phenology of putative Phloeosinus punctatus in giant sequoia groves and compare morphology and genotypes of beetles from sympatric giant sequoia (Sequoiadendron giganteum) and California incense-cedar (Calocedrus decurrens). Surveys conducted in 2022 revealed that numerous branches fall from giant sequoia crowns (on average ~30 branches/tree), with 20%-50% of trees per site shedding branches, depositing breeding material for beetles on the forest floor that subsequently becomes colonized. When noninfested branches cut from mature giant sequoias were placed at the ground surface, they were colonized by P. punctatus and produced an average of 28 beetles/kg branch. Climbing and examination of sequoia crowns in 2023 showed that 75% of mature trees across 11 groves showed evidence of adult beetle entrance holes in their crowns. In 2021, tests with sticky traps showed that beetles alighted on fallen branches from 20th May to 20th August (peak landing: 2nd July); a logistic model developed from emergence data in 2021 and 2022 predicts the emergence of F1 offspring from branches between 10th July and 1st September (peak emergence: 8th August). Beetles emerging from giant sequoia preferred to settle on giant sequoia, did not reproduce in incense-cedar, and diverged morphologically from beetles emerging from incense-cedar. However, phylogenetic analysis of three genes (28S, CAD, and COI) revealed no clear pattern of sequence divergence, suggesting a single species (P. punctatus) that colonizes both hosts, though cryptic speciation may not be detectable with standard barcoding genes. Ecological and potential management implications are discussed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America.)

Background: Neuroblastoma is the most common extracranial solid tumor in children and accounts for 15% of pediatric cancer related deaths. Targeting neuroblastoma with immunotherapies has proven challenging due to a paucity of immune cells in the tumor microenvironment and the release of immunosuppressive cytokines by neuroblastoma tumor cells. We hypothesized that combining an oncolytic Herpes Simplex Virus (oHSV) with natural killer (NK) cells might overcome these barriers and incite tumor cell death., Methods: We utilized MYCN amplified and non-amplified neuroblastoma cell lines, the IL-12 expressing oHSV, M002, and the human NK cell line, NK-92 MI. We assessed the cytotoxicity of NK cells against neuroblastoma with and without M002 infection, the effects of M002 on NK cell priming, and the impact of M002 and priming on the migratory capacity and CD107a expression of NK cells. To test clinical applicability, we then investigated the effects of M002 and NK cells on neuroblastoma in vivo., Results: NK cells were more attracted to neuroblastoma cells that were infected with M002. There was an increase in neuroblastoma cell death with the combination treatment of M002 and NK cells both in vitro and in vivo. Priming the NK cells enhanced their cytotoxicity, migratory capacity and CD107a expression., Conclusions: To the best of our knowledge, these investigations are the first to demonstrate the effects of an oncolytic virus combined with self-maintaining NK cells in neuroblastoma and the priming effect of neuroblastoma on NK cells. The current studies provide a deeper understanding of the relation between NK cells and neuroblastoma and these data suggest that oHSV increases NK cell cytotoxicity towards neuroblastoma., (© 2024. The Author(s).)

Background: Pediatric gastroenteropancreatic neuroendocrine tumors are exceedingly rare, resulting in most pediatric treatment recommendations being based on data derived from adults. Trametinib is a kinase inhibitor that targets MEK1/2 and has been employed in the treatment of cancers harboring mutations in the Ras pathway., Methods: We utilized an established human pediatric gastroenteropancreatic neuroendocrine-like tumor patient-derived xenograft (PDX) with a known NRAS mutation to study the effects of MEK inhibition. We evaluated the effects of trametinib on proliferation, motility, and tumor growth in vivo. We created an intraperitoneal metastatic model of this PDX, characterized both the phenotype and the genotype of the metastatic PDX and again, investigated the effects of MEK inhibition., Results: We found target engagement with decreased ERK1/2 phosphorylation with trametinib treatment. Trametinib led to decreased in vitro cell growth and motility, and decreased tumor growth and increased animal survival in a murine flank tumor model. Finally, we demonstrated that trametinib was able to significantly decrease gastroenteropancreatic neuroendocrine intraperitoneal tumor metastasis., Conclusions: The results of these studies support the further investigation of MEK inhibition in pediatric NRAS mutated solid tumors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

This study aimed to elucidate the effects of storage temperature on various fruit quality attributes, physiological disorders, and associated metabolites in the 0.5, 3, or 10 °C stored hardy kiwifruit. Peel pitting, which was highest in the 0.5 °C stored fruit, was identified as a chilling injury symptom of hardy kiwifruit. Proline and branched-chain amino acid contents showed higher values at 0.5 °C stored fruit as chilling responses. On the other hand, fruit shriveling and decay were highest in the 10 °C after 5 weeks of storage. The 10 °C storage induced fruit ripening during 3 weeks, but fruit shriveling and decay were severe after 5 weeks of storage. Therefore, storing the 'Autumn Sense' hardy kiwifruit at proper temperatures would be more beneficial, as it alters targeted metabolites and helps reduce the incidence of physiological disorders during cold storage., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Introduction: Soil-borne pathogens cause considerable crop losses and food insecurity in smallholder systems of sub-Saharan Africa. Soil and crop testing is critical for estimating pathogen inoculum levels and potential for disease development, understanding pathogen interactions with soil nutrient and water limitations, as well as for developing informed soil health and disease management decisions. However, formal laboratory analyses and diagnostic services for pathogens are often out of reach for smallholder farmers due to the high cost of testing and a lack of local laboratories., Methods: To address this challenge, we assessed the performance of a suite of simplified soil bioassays to screen for plant parasitic nematodes (e.g., Meloidogyne , Pratylenchus ) and other key soil-borne pathogens ( Pythium and Fusarium ). We sampled soils from on-farm trials in western Kenya examining the impact of distinct nutrient inputs (organic vs. synthetic) on bean production. Key soil health parameters and common soil-borne pathogens were evaluated using both simple bioassays and formal laboratory methods across eleven farms, each with three nutrient input treatments (66 samples in total)., Results and Discussion: The soil bioassays, which involved counting galls on lettuce roots and lesions on soybean were well correlated with the abundance of gall forming ( Meloidogyne ) and root lesion nematodes (e.g., Pratylenchus ) recovered in standard laboratory-based extractions. Effectiveness of a Fusarium bioassay, involving the counting of lesions on buried bean stems, was verified via sequencing and a pathogenicity test of cultured Fusarium strains. Finally, a Pythium soil bioassay using selective media clearly distinguished pathogen infestation of soils and infected seeds. When examining management impact on nematode communities, soils amended with manure had fewer plant parasites and considerably more bacterivore and fungivore nematodes compared to soils amended with synthetic N and P. Similarly, Pythium presence was 35% lower in soils amended with manure, while the Fusarium assays indicated 23% higher Fusarium infection in plots with amended manure. Our findings suggest that relatively simple bioassays can be used to help farmers assess soil-borne pathogens in a timely manner, with minimal costs, thus enabling them to make informed decisions on soil health and pathogen management., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Mutai, Stewart, Medvecky, Dobbs, Vanek, Ojiem, Chege and Fonte.)

Background: Chemoresistance contributes to relapse in high-risk neuroblastoma. Cancer cells acquire resistance through multiple mechanisms, including drug efflux pumps. In neuroblastoma, multidrug resistance-associated protein-1 (MRP1/ABCC1) efflux pump expression correlates with worse outcomes. These pumps are regulated by PIM kinases, a family of serine-threonine kinases, overexpressed in neuroblastoma. We hypothesized PIM kinase inhibition would sensitize neuroblastoma cells by modulating MRP1., Methods: Kocak database query evaluated ABCC1, PIM1, PIM2, and PIM3 expression in neuroblastoma patients. SK-N-AS and SK-N-BE(2) cells were treated with doxorubicin or the pan-PIM kinase inhibitor, AZD1208. Flow cytometry assessed intracellular doxorubicin accumulation. AlamarBlue assay measured viability. The lethal dose 50% (LD 50 ) of each drug and combination indices (CI) were calculated and isobolograms constructed to determine synergy., Results: Kocak database query demonstrated positive correlation between PIM genes and ABCC1. PIM kinase inhibition increased intracellular doxorubicin accumulation in both cell lines, suggesting PIM kinase regulation of MRP1. Isobolograms showed synergy between AZD1208 and doxorubicin., Conclusions: The correlation between PIM and ABCC1 gene expression suggests PIM kinases may contribute to neuroblastoma chemotherapeutic resistance. PIM kinase inhibition increased intracellular doxorubicin accumulation. Combination treatment with AZD1208 and doxorubicin decreased neuroblastoma cell viability in a synergistic fashion. These findings support further investigations of PIM kinase inhibition in neuroblastoma., Type of Study: Basic Science Research., Level of Evidence: NA., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Passalora sequoiae is a foliar pathogen to conifer tree species. In this study, we conducted whole-genome and transcriptome analyses on isolates of P. sequoiae collected from symptomatic Leyland cypress leaves from a Christmas tree farm in Mississippi. The objectives for this research were to elucidate the pathogenicity mechanisms of P. sequoiae by characterizing the genome and transcriptome and possibly identify unique and shared predicted genes in comparison with non-conifer/canker and foliar pathogens in the family Mycosphaerellaceae . P. sequoiae was found to be similar to other foliar Mycosphaerellaceae pathogens and likely represents a hemibiotrophic lifestyle based on comparisons across pathogens. The genome and in planta transcriptome highlighted some unique features of P. sequoiae : the significant presence of chitin synthases and fructose-degrading carbohydrate-degrading enzymes, trans-AT PKS genes, and antibiotic gene clusters that were unique to P. sequoiae compared with the other Mycosphaerellaceae species genomes. Several transcripts that were highly expressed in planta were identified as effectors, yet the functions were not characterized. These targets provide ample resources to continue to characterize pathogen-conifer host interactions in conifer foliar pathogens. Furthermore, this research helps build genomic resources for an important plant pathogen on Leyland cypress that will further our ability to develop novel management practices that could begin with breeding for resistance., Competing Interests: The author(s) declare no conflict of interest.

Cardiac mitochondrial dysfunction is a critical contributor to the pathogenesis of aging and many age-related conditions. As such, complete control of mitochondrial function is critical to maintain cardiac efficiency in the aged heart. Lysine acetylation is a reversible post-translational modification shown to regulate several mitochondrial metabolic and biochemical processes. In the present study, we investigated how mitochondrial lysine acetylation regulates fatty acid oxidation (FAO) and cardiac function in the aged heart. We found a significant increase in mitochondrial protein acetylation in the aged heart which correlated with increased level of mitochondrial acetyltransferase-related protein GCN5L1. We showed that acetylation status of several fatty acid and glucose oxidation enzymes (long-chain acyl-coenzyme A dehydrogenase, hydroxyacyl-coA dehydrogenase, and pyruvate dehydrogenase) were significantly up-regulated in aged heart which correlated with decreased enzymatic activities. Using a cardiac-specific GCN5L1 knockout (KO) animal model, we showed that overall acetylation of mitochondrial proteins was decreased in aged KO animals, including FAO proteins which led to improved FAO activity and attenuated cardiac diastolic dysfunction observed in the aged heart. Together, these findings indicate that lysine acetylation regulates FAO in the aged heart which results in improved cardiac diastolic function and this is in part regulated by GCN5L1., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background: Sarcomas are a heterogenous collection of bone and soft tissue tumors. The heterogeneity of these tumors makes it difficult to standardize treatment. CDK 4/6 inhibitors are a family of targeted agents which limit cell cycle progression and have been shown to be upregulated in sarcomas. In the current preclinical study, we evaluated the effects of lerociclib, a CDK4/6 inhibitor, on pediatric sarcomas in vitro and in 3D bioprinted tumors., Methods: The effects of lerociclib on viability, proliferation, cell cycle, motility, and stemness were assessed in established sarcoma cell lines, U-2 OS and MG-63, as well as sarcoma patient-derived xenografts (PDXs). 3D printed biotumors of each of the U-2 OS, MG-63, and COA79 cells were utilized to study the effects of lerociclib on tumor growth ex vivo., Results: CDK 4/6, as well as the intermediaries retinoblastoma protein (Rb) and phosphorylated Rb were identified as targets in the four sarcoma cell lines. Lerociclib treatment induced cell cycle arrest, decreased proliferation, motility, and stemness of sarcoma cells. Treatment with lerociclib decreased sarcoma cell viability in both traditional 2D culture as well as 3D bioprinted microtumors., Conclusions: Inhibition of CDK 4/6 activity with lerociclib was efficacious in traditional 2D sarcoma cell culture as well as in 3D bioprints. Lerociclib holds promise and warrants further investigation as a novel therapeutic strategy for management of these heterogenous groups of tumors., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The Neonatal Intensive Care Unit (NICU) has a language and culture that is its own. For professionals, it is a place of intense and constant attention to microdetails and cautious optimism. For parents, it is a foreign place with a new and unique language and culture. It is also the setting in which they are introduced to their child and parenthood for this child. This combination has been referred to as an emotional cauldron. The neonatal ethics literature mainly examines complex ethical dilemmas about withholding/drawing life sustaining interventions for fragile children. Rarely are everyday ethics or mundane ethics discussed. Microethics describe the mundane, discrete moments that occur between patients/families and clinicians. A key piece of these microethics is the language used to discuss patient care. Perception of prognoses, particularly around long-term neurodevelopmental outcome, is shaped with the language used. Despite this, clinicians in the NICU often have no specific training in the long-term neurodevelopment outcomes that they discuss. This paper focuses on the microethics of language used to discuss long-term neurodevelopmental outcomes, the developmental neuroscience behind language processing, and offers recommendations for more accurate and improved communication around long-term outcomes with families with critically ill neonates.

Interspecific hybridization plays a key role in the evolution of novel fungal pathogens, and when it occurs between native and invasive species, can lead to potentially serious consequences. In this study, we examined the temporal and spatial distribution of a recently detected hybrid (Cronartium x flexili) of two tree pathogens, invasive to North America Cronartium ribicola and native Cronartium comandrae. In total, 726 and 1452 aecia from 178 Pinus contorta ssp. latifolia and 357 Pinus flexilis trees were collected from 26 sites in four national forests in 2019-2021. Using morphological and molecular analyses, 71 aecia collected from 25 P. flexilis trees had intermediate morphology and contained heterozygous SNPs in two genomic regions. Population analyses revealed the presence of multiple hybrid genotypes randomly distributed among sites and years. No aecia from P. contorta ssp. latifolia were identified as hybrids suggesting unidirectional gene flow from native C. comandrae to invasive C. ribicola. Aeciospores from 2 hybrid aecia produced urediniospores on Ribes nigrum. Overall, these results suggest that, even though low in frequency, C. x flexili is persistent in the region and has pathogenic potential. Hybrid expansion into the large range of susceptible pines could have cascading impacts on forest health., Competing Interests: Declaration of competing interest All authors have edited the manuscript and agreed to publishing the research findings in Fungal Biology. This research and it's findings have not been previously published and there is no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hepatoblastoma is the most common primary pediatric liver tumor. Children with pulmonary metastases at diagnosis experience survival rates as low as 25%. We have shown PIM kinases play a role in hepatoblastoma tumorigenesis. In this study, we assessed the role of PIM kinases in metastatic hepatoblastoma. We employed the metastatic hepatoblastoma cell line, HLM&#95;2. PIM kinase inhibition was attained using PIM3 siRNA and the pan-PIM inhibitor, AZD1208. Effects of PIM inhibition on proliferation were evaluated via growth curve. Flow cytometry determined changes in cell cycle. AlamarBlue assay assessed effects of PIM kinase inhibition and cisplatin treatment on viability. The lethal dose 50% (LD 50 ) of each drug and combination indices (CI) were calculated and isobolograms constructed to determine synergy. PIM kinase inhibition resulted in decreased HLM&#95;2 proliferation, likely through cell cycle arrest mediated by p21. Combination therapy with AZD1208 and cisplatin resulted in synergy, potentially through downregulation of the ataxia-telangiectasia mutated (ATM) kinase DNA damage response pathway. When assessing the combined effects of pharmacologic PIM kinase inhibition with cisplatin on HLM&#95;2 cells, we found the agents to be synergistic, potentially through inhibition of the ATM pathway. These findings support further exploration of PIM kinase inhibition as a therapeutic strategy for metastatic hepatoblastoma.

Leptographium wageneri is a native fungal pathogen in western North America that causes black stain root disease (BSRD) of conifers. Three host-specialized varieties of this pathogen were previously described: L. wageneri var. wageneri on pinyon pines ( Pinus monophylla and P . edulis ); L. wageneri var. ponderosum , primarily on hard pines (e.g., P . ponderosa , P . jeffreyi ); and L. wageneri var. pseudotsugae on Douglas-fir ( Pseudotsuga menziesii ). Morphological, physiological, and ecological differences among the three pathogen varieties have been previously determined; however, DNA-based characterization and analyses are needed to determine the genetic relationships among these varieties. The objective of this study was to use DNA sequences of 10 gene regions to assess phylogenetic relationships among L. wageneri isolates collected from different hosts. The multigene phylogenetic analyses, based on maximum likelihood and Bayesian inference, strongly supported species-level separation of the three L. wageneri varieties. These results, in conjunction with previously established phenotypic differences, support the elevation of L. wageneri var. ponderosum and L. wageneri var. pseudotsugae to the species level as L . ponderosum comb. nov. and L . pseudotsugae comb. nov., respectively, while maintaining L. wageneri var. wageneri as Leptographium wageneri . Characterization of the three Leptographium species, each with distinct host ranges, provides a baseline to further understand the ecological interactions and evolutionary relationships of these forest pathogens, which informs management of black stain root disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Choi, Harrington, Shaw, Stewart, Klopfenstein, Kroese and Kim.)