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Effects of the Incredible Years parenting program on sibling conduct problems: A latent transition analysis
Background: Behavioral parenting programs are a primary strategy used to reduce children's conduct problems. Although behavior problems in siblings may co-occur, behavioral parenting program trials typically report outcomes for one child per family (the index child), with potential program effects on any non-targeted sibling largely neglected. This study examined co-occurring patterns of index child and non-targeted sibling conduct problems, and how parental participation in the Incredible Years (IY) program changes these patterns. Methods: We used individual participant data pooled across three randomized trials of the IY parenting program in England, Wales, and Ireland, with data for the index child and one non-targeted sibling (N = 240 families, 480 children; index child: M age = 4.73, SD = 1.44, range 2–9 years, 62% male; non-targeted sibling: M age = 5.94 years, SD = 3.15, range 6 months−15 years, 49% male). We used latent transition analysis to identify latent classes at both baseline and posttest based on families' combinations of index child and non-targeted sibling conduct problems. Results: We identified two classes with distinct patterns of co-occurring sibling dyad conduct problems: one with moderate clinical levels of index child conduct problems and non-clinical levels for the non-targeted sibling; and one with severe clinical levels for both children. In terms of the effects of IY, most intervention families maintained their patterns of sibling dyad conduct problems, but with lower levels across classes. Most intervention families reported improvements predominantly for the index child. However, a minority of families with severe baseline levels of conduct problems in both children moved to a class with non-clinical levels for both children. Conclusions: For most families, IY had limited effects on non-targeted sibling disruptive behavior. However, IY may reduce co-occurring sibling conduct problems for a small number of families with initially severe levels in both children.
Thermal constraints on Middle Pleistocene hominin brain evolution and cognition
High latitude habitats are subject to thermally-driven energetic constraints that make their occupation challenging. This is likely to have had a particularly significant impact on energy-expensive tissue like the brain, especially during periods of lower global temperatures during the Mid-Pleistocene Ice Ages. I analyse data on endocranial volumes for archaic humans (Homo heidelbergensis, H. neanderthalensis and allies) to show (1) that cranial volumes were typically smaller at high latitudes than in the tropics and (2) that they declined during cold phases and increased during warm phases of the Middle Pleistocene Ice Ages. Within this broad pattern, there is a significant uplift in cranial volumes after 400 ka that seems to coincide with widespread presence of hearths at high latitudes, suggesting that hominin populations might have gained at least partial release from this constraint through cultural control over fire. While this might pinpoint the time at which hominins first began to cook on a regular basis, fire offers other important benefits (notably warmth and extending the length of the working day) that might have played an equally important role in buffering populations against thermal stresses. The larger brain sizes that this made possible have implications for social cognitive capacities like mentalising, that in turn have implications for language skills, cultural behaviour and social group size.
Multimodal population study reveals the neurobiological underpinnings of chronotype.
The rapid shifts in society have altered human behavioural patterns, with increased evening activities, increased screen time and changed sleep schedules. As an explicit manifestation of circadian rhythms, chronotype is closely intertwined with physical and mental health. Night owls often exhibit unhealthier lifestyle habits, are more susceptible to mood disorders and have poorer physical fitness compared with early risers. Although individual differences in chronotype yield varying consequences, their neurobiological underpinnings remain elusive. Here we conducted a pattern-learning analysis with three brain-imaging modalities (grey matter volume, white-matter integrity and functional connectivity) and capitalized on 976 phenotypes in 27,030 UK Biobank participants. The resulting multilevel analysis reveals convergence on the basal ganglia, limbic system, hippocampus and cerebellum. The pattern derived from modelling actigraphy wearables data of daily movement further highlighted these key brain features. Overall, our population-level study comprehensively investigates chronotype, emphasizing its close connections with habit formation, reward processing and emotional regulation.
Collaborative care for pregnant women with eye conditions.
The management of ophthalmic conditions in pregnancy presents unique challenges that demand a nuanced approach. Significant knowledge gaps and practice variations persist, likely as a result of the infrequent nature of these issues in pregnancy, as well as the lack of crossover in the specialty training curriculum for both specialties. This commentary explores how multidisciplinary team (MDT) working can address these uncertainties to support shared decision-making and potentially improve outcomes in this vulnerable patient population. We highlight the need to involve ophthalmologists in obstetric MDTs, where appropriate, and the importance of establishing clear communication channels and referral pathways between both specialties and across hospitals in the region. We also share our experience of establishing these pathways locally, the feedback we have received from interdisciplinary educational initiatives to improve knowledge sharing, and possible future directions for this collaborative approach to help fill the evidence gap.
Structures of the human adult muscle-type nicotinic receptor in resting and desensitized states.
Muscle-type nicotinic acetylcholine receptor (AChR) is the key signaling molecule in neuromuscular junctions. Here, we present the structures of full-length human adult receptors in complex with Fab35 in α-bungarotoxin (αBuTx)-bound resting states and ACh-bound desensitized states. In addition to identifying the conformational changes during recovery from desensitization, we also used electrophysiology to probe the effects of eight previously unstudied AChR genetic variants found in patients with congenital myasthenic syndrome (CMS), revealing they cause either slow- or fast-channel CMS characterized by prolonged or abbreviated ion channel bursts. The combined kinetic and structural data offer a better understanding of both the AChR state transition and the pathogenic mechanisms of disease variants.
Neuroimaging-based data-driven subtypes of spatiotemporal atrophy due to Parkinson's disease.
Parkinson's disease is the second most common neurodegenerative disease. Despite this, there are no robust biomarkers to predict progression, and understanding of disease mechanisms is limited. We used the Subtype and Stage Inference algorithm to characterize Parkinson's disease heterogeneity in terms of spatiotemporal subtypes of macroscopic atrophy detectable on T1-weighted MRI-a successful approach used in other neurodegenerative diseases. We trained the model on covariate-adjusted cortical thicknesses and subcortical volumes from the largest known T1-weighted MRI dataset in Parkinson's disease, Enhancing Neuroimaging through Meta-Analysis consortium Parkinson's Disease dataset (n = 1100 cases). We tested the model by analyzing clinical progression over up to 9 years in openly-available data from people with Parkinson's disease from the Parkinson's Progression Markers Initiative (n = 584 cases). Under cross-validation, our analysis supported three spatiotemporal atrophy subtypes, named for the location of the earliest affected regions as: 'Subcortical' (n = 359, 33%), 'Limbic' (n = 237, 22%) and 'Cortical' (n = 187, 17%). A fourth subgroup having sub-threshold/no atrophy was named 'Sub-threshold atrophy' (n = 317, 29%). Statistical differences in clinical scores existed between the no-atrophy subgroup and the atrophy subtypes, but not among the atrophy subtypes. This suggests that the prime T1-weighted MRI delineator of clinical differences in Parkinson's disease is atrophy severity, rather than atrophy location. Future work on unravelling the biological and clinical heterogeneity of Parkinson's disease should leverage more sensitive neuroimaging modalities and multimodal data.
Discovery of active mouse, plant and fungal cytochrome P450s in endogenous proteomes and upon expression in planta.
Eukaryotes produce a large number of cytochrome P450s that mediate the synthesis and degradation of diverse endogenous and exogenous metabolites. Yet, most of these P450s are uncharacterized and global tools to study these challenging, membrane-resident enzymes remain to be exploited. Here, we applied activity profiling of plant, mouse and fungal P450s with chemical probes that become reactive when oxidized by P450 enzymes. Identification by mass spectrometry revealed labeling of a wide range of active P450s, including six plant P450s, 40 mouse P450s and 13 P450s of the fungal wheat pathogen Zymoseptoria tritici. We next used transient expression of GFP-tagged P450s by agroinfiltration to show ER-targeting and NADPH-dependent, activity-based labeling of plant, mouse and fungal P450s. Both global profiling and transient expression can be used to detect a broad range of active P450s to study e.g. their regulation and discover selective inhibitors.
The root pathogen Aphanomyces euteiches secretes modular proteases in pea apoplast during host infection.
To successfully colonize the host, phytopathogens have developed a large repertoire of components to both combat the host plant defense mechanisms and to survive in adverse environmental conditions. Microbial proteases are predicted to be crucial components of these systems. In the present work, we aimed to identify active secreted proteases from the oomycete Aphanomyces euteiches, which causes root rot diseases on legumes. Genome mining and expression analysis highlighted an overrepresentation of microbial tandemly repeated proteases, which are upregulated during host infection. Activity Based Protein Profiling and mass spectrometry (ABPP-MS) on apoplastic fluids isolated from pea roots infected by the pathogen led to the identification of 35 active extracellular microbial proteases, which represents around 30% of the genes expressed encoding serine and cysteine proteases during infection. Notably, eight of the detected active secreted proteases carry an additional C-terminal domain. This study reveals novel active modular extracellular eukaryotic proteases as potential pathogenicity factors in Aphanomyces genus.
Chemoproteomics Reveals the Pan-HER Kinase Inhibitor Neratinib To Target an Arabidopsis Epoxide Hydrolase Related to Phytohormone Signaling.
Plant phytohormone pathways are regulated by an intricate network of signaling components and modulators, many of which still remain unknown. Here, we report a forward chemical genetics approach for the identification of functional SA agonists in Arabidopsis thaliana that revealed Neratinib (Ner), a covalent pan-HER kinase inhibitor drug in humans, as a modulator of SA signaling. Instead of a protein kinase, chemoproteomics unveiled that Ner covalently modifies a surface-exposed cysteine residue of Arabidopsis epoxide hydrolase isoform 7 (AtEH7), thereby triggering its allosteric inhibition. Physiologically, the Ner application induces jasmonate metabolism in an AtEH7-dependent manner as an early response. In addition, it modulates PATHOGENESIS RELATED 1 (PR1) expression as a hallmark of SA signaling activation as a later effect. AtEH7, however, is not the exclusive target for this physiological readout induced by Ner. Although the underlying molecular mechanisms of AtEH7-dependent modulation of jasmonate signaling and Ner-induced PR1-dependent activation of SA signaling and thus defense response regulation remain unknown, our present work illustrates the powerful combination of forward chemical genetics and chemical proteomics for identifying novel phytohormone signaling modulatory factors. It also suggests that marginally explored metabolic enzymes such as epoxide hydrolases may have further physiological roles in modulating signaling.
Cysteine Reactivity Profiling to Unveil Redox Regulation in Phytopathogens.
Reactivity-based chemical proteomics is a powerful technology based on the use of tagged chemicals that covalently react with surface-exposed residues on proteins in native proteomes. Reactivity profiling involves the purification, identification, and quantification of labeled peptides by LC-MS/MS. Here, we have detailed a protocol for reactivity profiling of Cys residues using iodoacetamide probes, displaying >1000 reactive Cys residues in the proteome of phytopathogen Pseudomonas syringae pv. tomato DC3000 (PtoDC3000). Comparative reactivity profiling of PtoDC3000 treated with or without hydrogen peroxide (H2O2) identified ~200 H2O2-sensitive Cys residues in antioxidant enzymes, metabolic enzymes, and transcription regulators. Interestingly, half of these H2O2-sensitive Cys residues are more reactive in response to H2O2 and several proteins have multiple Cys residues with opposite reactivities in response to H2O2 exposure.
Purification of His-Tagged Proteases from the Apoplast of Agroinfiltrated N. benthamiana.
Protein expression in plants by agroinfiltration and subsequent purification is increasingly used for the biochemical characterization of plant proteins. In this chapter we describe the purification of secreted, His-tagged proteases from the apoplast of agroinfiltrated Nicotiana benthamiana using immobilized metal affinity chromatography (IMAC). We show quality checks for the purified protease and discuss potential problems and ways to circumvent them. As a proof of concept, we produce and purify tomato immune protease Pip1 and demonstrate that the protein is active after purification.
Activity-based probes trap early active intermediates during metacaspase activation.
Metacaspases are essential cysteine proteases present in plants, fungi, and protists that are regulated by calcium binding and proteolytic maturation through mechanisms not yet understood. Here, we developed and validated activity-based probes for the three main metacaspase types, and used them to study calcium-mediated activation of metacaspases from their precursors in vitro. By combining substrate-inspired tetrapeptide probes containing an acyloxymethylketone (AOMK) reactive group, with purified representatives of type-I, type-II, and type-III metacaspases, we were able to demonstrate that labeling of mature metacaspases is strictly dependent on calcium. The probe with the highest affinity for all metacaspases also labels higher molecular weight proteoforms of all three metacaspases only in the presence of calcium, displaying the active, unprocessed metacaspase intermediates. Our data suggest that metacaspase activation proceeds through previously unknown active intermediates that are formed upon calcium binding, before precursor processing.
Broad-range metalloprotease profiling in plants uncovers immunity provided by defence-related metalloenzyme.
Plants encode > 100 metalloproteases representing > 19 different protein families. Tools to study this large and diverse class of proteases have not yet been introduced into plant research. We describe the use of hydroxamate-based photoaffinity probes to explore plant proteomes for metalloproteases. We detected labelling of 23 metalloproteases in leaf extracts of the model plant Arabidopsis thaliana that belong to nine different metalloprotease families and localize to different subcellular compartments. The probes identified several chloroplastic FtsH proteases, vacuolar aspartyl aminopeptidase DAP1, peroxisomal metalloprotease PMX16, extracellular matrix metalloproteases and many cytosolic metalloproteases. We also identified nonproteolytic metallohydrolases involved in the release of auxin and in the urea cycle. Studies on tobacco plants (Nicotiana benthamiana) infected with the bacterial plant pathogen Pseudomonas syringae uncovered the induced labelling of PRp27, a secreted protein with implicated metalloprotease activity. PRp27 overexpression increases resistance, and PRp27 mutants lacking metal binding site are no longer labelled, but still show increased immunity. Collectively, these studies reveal the power of broad-range metalloprotease profiling in plants using hydroxamate-based probes.
Leaf Apoplast of Field-Grown Potato Analyzed by Quantitative Proteomics and Activity-Based Protein Profiling.
Multiple biotic and abiotic stresses challenge plants growing in agricultural fields. Most molecular studies have aimed to understand plant responses to challenges under controlled conditions. However, studies on field-grown plants are scarce, limiting application of the findings in agricultural conditions. In this study, we investigated the composition of apoplastic proteomes of potato cultivar Bintje grown under field conditions, i.e., two field sites in June-August across two years and fungicide treated and untreated, using quantitative proteomics, as well as its activity using activity-based protein profiling (ABPP). Samples were clustered and some proteins showed significant intensity and activity differences, based on their field site and sampling time (June-August), indicating differential regulation of certain proteins in response to environmental or developmental factors. Peroxidases, class II chitinases, pectinesterases, and osmotins were among the proteins more abundant later in the growing season (July-August) as compared to early in the season (June). We did not detect significant differences between fungicide Shirlan treated and untreated field samples in two growing seasons. Using ABPP, we showed differential activity of serine hydrolases and β-glycosidases under greenhouse and field conditions and across a growing season. Furthermore, the activity of serine hydrolases and β-glycosidases, including proteins related to biotic stress tolerance, decreased as the season progressed. The generated proteomics data would facilitate further studies aiming at understanding mechanisms of molecular plant physiology in agricultural fields and help applying effective strategies to mitigate biotic and abiotic stresses.
The chemical compound 'Heatin' stimulates hypocotyl elongation and interferes with the Arabidopsis NIT1-subfamily of nitrilases.
Temperature passively affects biological processes involved in plant growth. Therefore, it is challenging to study the dedicated temperature signalling pathways that orchestrate thermomorphogenesis, a suite of elongation growth-based adaptations that enhance leaf-cooling capacity. We screened a chemical library for compounds that restored hypocotyl elongation in the pif4-2-deficient mutant background at warm temperature conditions in Arabidopsis thaliana to identify modulators of thermomorphogenesis. The small aromatic compound 'Heatin', containing 1-iminomethyl-2-naphthol as a pharmacophore, was selected as an enhancer of elongation growth. We show that ARABIDOPSIS ALDEHYDE OXIDASES redundantly contribute to Heatin-mediated hypocotyl elongation. Following a chemical proteomics approach, the members of the NITRILASE1-subfamily of auxin biosynthesis enzymes were identified among the molecular targets of Heatin. Our data reveal that nitrilases are involved in promotion of hypocotyl elongation in response to high temperature and Heatin-mediated hypocotyl elongation requires the NITRILASE1-subfamily members, NIT1 and NIT2. Heatin inhibits NIT1-subfamily enzymatic activity in vitro and the application of Heatin accordingly results in the accumulation of NIT1-subfamily substrate indole-3-acetonitrile in vivo. However, levels of the NIT1-subfamily product, bioactive auxin (indole-3-acetic acid), were also significantly increased. It is likely that the stimulation of hypocotyl elongation by Heatin might be independent of its observed interaction with NITRILASE1-subfamily members. However, nitrilases may contribute to the Heatin response by stimulating indole-3-acetic acid biosynthesis in an indirect way. Heatin and its functional analogues present novel chemical entities for studying auxin biology.
Cleavage of a pathogen apoplastic protein by plant subtilases activates host immunity.
The plant apoplast is a harsh environment in which hydrolytic enzymes, especially proteases, accumulate during pathogen infection. However, the defense functions of most apoplastic proteases remain largely elusive. We show here that a newly identified small cysteine-rich secreted protein PC2 from the potato late blight pathogen Phytophthora infestans induces immunity in Solanum plants only after cleavage by plant apoplastic subtilisin-like proteases, such as tomato P69B. A minimal 61 amino acid core peptide carrying two key cysteines, conserved widely in most oomycete species, is sufficient for PC2-induced cell death. Furthermore, we showed that Kazal-like protease inhibitors, such as EPI1, produced by P. infestans prevent PC2 cleavage and dampen PC2 elicited host immunity. This study reveals that cleavage of pathogen proteins to release immunogenic peptides is an important function of plant apoplastic proteases.
How to build an effective research network: lessons from two decades of the GARNet plant science community.
Successful collaborative research is dependent on excellent ideas and innovative experimental approaches, as well as the provision of appropriate support networks. Collaboration requires venues, infrastructures, training facilities, and, perhaps most importantly, a sustained commitment to work together as a community. These activities do not occur without significant effort, yet can be facilitated and overseen by the leadership of a research network that has a clearly defined role to help build resources for their community. Over the past 20 years, this is a role that the UKRI-BBSRC-funded GARNet network has played in the support of the UK curiosity-driven, discovery-led plant science research community. This article reviews the lessons learnt by GARNet in the hope that they can inform the practical implementation of current and future research networks.