Ehf controls mammary alveolar lineage differentiation and is a putative suppressor of breast tumorigenesis.

The transcription factor EHF is highly expressed in the lactating mammary gland, but its role in mammary development and tumorigenesis is not fully understood. Utilizing a mouse model of Ehf deletion, herein, we demonstrate that loss of Ehf impairs mammary lobuloalveolar differentiation at late pregnancy, indicated by significantly reduced levels of milk genes and milk lipids, fewer differentiated alveolar cells, and an accumulation of alveolar progenitor cells. Further, deletion of Ehf increased proliferative capacity and attenuated prolactin-induced alveolar differentiation in mammary organoids. Ehf deletion also increased tumor incidence in the MMTV-PyMT mammary tumor model and increased the proliferative capacity of mammary tumor organoids, while low EHF expression was associated with higher tumor grade and poorer outcome in luminal A and basal human breast cancers. Collectively, these findings establish EHF as a non-redundant regulator of mammary alveolar differentiation and a putative suppressor of mammary tumorigenesis.

BCL-XL inhibitors enhance the apoptotic efficacy of BRAF inhibitors in BRAFV600E colorectal cancer.

Metastatic BRAFV600E colorectal cancer (CRC) carries an extremely poor prognosis and is in urgent need of effective new treatments. While the BRAFV600E inhibitor encorafenib in combination with the EGFR inhibitor cetuximab (Enc+Cet) was recently approved for this indication, overall survival is only increased by 3.6 months and objective responses are observed in only 20% of patients. We have found that a limitation of Enc+Cet treatment is the failure to efficiently induce apoptosis in BRAFV600E CRCs, despite inducing expression of the pro-apoptotic protein BIM and repressing expression of the pro-survival protein MCL-1. Here, we show that BRAFV600E CRCs express high basal levels of the pro-survival proteins MCL-1 and BCL-XL, and that combining encorafenib with a BCL-XL inhibitor significantly enhances apoptosis in BRAFV600E CRC cell lines. This effect was partially dependent on the induction of BIM, as BIM deletion markedly attenuated BRAF plus BCL-XL inhibitor-induced apoptosis. As thrombocytopenia is an established on-target toxicity of BCL-XL inhibition, we also examined the effect of combining encorafenib with the BCL-XL -targeting PROTAC DT2216, and the novel BCL-2/BCL-XL inhibitor dendrimer conjugate AZD0466. Combining encorafenib with DT2216 significantly increased apoptosis induction in vitro, while combining encorafenib with AZD0466 was well tolerated in mice and further reduced growth of BRAFV600E CRC xenografts compared to either agent alone. Collectively, these findings demonstrate that combined BRAF and BCL-XL inhibition significantly enhances apoptosis in pre-clinical models of BRAFV600E CRC and is a combination regimen worthy of clinical investigation to improve outcomes for these patients.

Structure-Activity Relationship Studies and Optimization of 4-Hydroxypyridones as GPR84 Agonists.

GPR84 is a putative medium-chain fatty acid receptor that is implicated in regulation of inflammation and fibrogenesis. Studies have indicated that GPR84 agonists may have therapeutic potential in diseases such as Alzheimer's disease, atherosclerosis, and cancer, but there is a lack of quality tool compounds to explore this potential. The fatty acid analogue LY237 (4a) is the most potent GPR84 agonist disclosed to date but has unfavorable physicochemical properties. We here present a SAR study of 4a. Several highly potent agonists were identified with EC50 down to 28 pM, and with SAR generally in excellent agreement with structure-based modeling. Proper incorporation of rings and polar groups resulted in the identification of TUG-2099 (4s) and TUG-2208 (42a), both highly potent GPR84 agonists with lowered lipophilicity and good to excellent solubility, in vitro permeability, and microsomal stability, which will be valuable tools for exploring the pharmacology and therapeutic prospects of GPR84.

BECLIN1 is essential for intestinal homeostasis involving autophagy-independent mechanisms through its function in endocytic trafficking.

Autophagy-related genes have been closely associated with intestinal homeostasis. BECLIN1 is a component of Class III phosphatidylinositol 3-kinase complexes that orchestrate autophagy initiation and endocytic trafficking. Here we show intestinal epithelium-specific BECLIN1 deletion in adult mice leads to rapid fatal enteritis with compromised gut barrier integrity, highlighting its intrinsic critical role in gut maintenance. BECLIN1-deficient intestinal epithelial cells exhibit extensive apoptosis, impaired autophagy, and stressed endoplasmic reticulum and mitochondria. Remaining absorptive enterocytes and secretory cells display morphological abnormalities. Deletion of the autophagy regulator, ATG7, fails to elicit similar effects, suggesting additional novel autophagy-independent functions of BECLIN1 distinct from ATG7. Indeed, organoids derived from BECLIN1 KO mice show E-CADHERIN mislocalisation associated with abnormalities in the endocytic trafficking pathway. This provides a mechanism linking endocytic trafficking mediated by BECLIN1 and loss of intestinal barrier integrity. Our findings establish an indispensable role of BECLIN1 in maintaining mammalian intestinal homeostasis and uncover its involvement in endocytic trafficking in this process. Hence, this study has important implications for our understanding of intestinal pathophysiology.

Brief cognitive behavioural therapy for eating disorders symptomatology among a mixed sample of adolescents and young adults in primary care: A non-randomised feasibility and pilot study.

OBJECTIVE: Brief and accessible therapies for people with an eating disorder is an important health target. Ten-session cognitive behavioural therapy (CBT-T) is a brief treatment evaluated in people with a non-underweight eating disorder. This study aimed to evaluate the feasibility and preliminary effectiveness of CBT-T for young people in primary care. METHOD: This cohort pilot study used group (adolescents vs. young adults) by time (over four time points) Generalised Linear Mixed Model analysis. Participants included 13-25-year-olds attending an early intervention mental health service, receiving 10 sessions of CBT-T. Feasibility was assessed using recruitment, retention and satisfaction. Eating and other pathology measures were administered at baseline, weeks four and 10, and 12-week follow-up. RESULTS: Of the 63 commencing treatment, 38 completed 10 CBT-T sessions (60%). Most (94%) reported high treatment satisfaction. Significant reductions in eating pathology, depression and stress were found. Age group did not yield differences in CBT-T outcome, with large to very large effect sizes across outcome variables. Anxiety was associated with attrition. CONCLUSION: This study provides preliminary support for the use of CBT-T in primary care, across adolescence and early adulthood. Findings require replication in other clinical settings and comparison to other clinical approaches and control populations.

A genomic region associated with iteroparous spawning phenology is linked with age-at-maturity in female steelhead trout.

Age-at-maturity and iteroparity are two life history variations of steelhead trout (Oncorhynchus mykiss) that are believed to increase population resilience and stability. While repeat-spawning individuals are thought to have historically made up a substantial portion of the reproductive population in the Columbia River and the majority of females still attempt outmigration as kelts, return rates of repeat-spawner are low throughout the basin and below 1% for the furthest migrating stocks. Notably, outmigrating adults exhibit variation in rematuration phenology, displaying either "consecutive" (reproduce immediately the following season) or "skip" (delay spawning for future seasons) spawning patterns. Here, we use low coverage whole genome sequencing of consecutive versus skip spawning female Columbia River steelhead from two populations to test for genomic differences between these two iteroparous phenotypes. We identified genomic regions on several chromosomes which were associated with the phenology of iteroparity, including a region on chromosome 25 containing two genes, estradiol receptor beta (ERß) and glycoprotein hormone beta-5 (GPHB5), which, in mammals, are estrogen-sensitive and expressed in reproductive tissues. Allele frequencies in this ERß/GPHB5 region differed among female steelhead of different age at maturity, but not males. These genes also shared an island of linkage disequilibrium with the SIX6 gene, 600Kbp away on the same chromosome, a region of known association with age-at-maturity. These observations contribute to growing evidence that age-at-maturity and the phenology of iteroparity are determined by overlapping physiological processes and genetic pathways.

Structural basis for the ligand recognition and signaling of free fatty acid receptors.

Free fatty acid receptors 1 to 4 (FFA1 to FFA4) are class A G protein-coupled receptors (GPCRs). FFA1 to FFA3 share substantial sequence similarity, whereas FFA4 is unrelated. However, FFA1 and FFA4 are activated by long-chain fatty acids, while FFA2 and FFA3 respond to short-chain fatty acids generated by intestinal microbiota. FFA1, FFA2, and FFA4 are potential drug targets for metabolic and inflammatory conditions. Here, we determined the active structures of FFA1 and FFA4 bound to docosahexaenoic acid, FFA4 bound to the synthetic agonist TUG-891, and butyrate-bound FFA2, each complexed with an engineered heterotrimeric Gq protein (miniGq), by cryo-electron microscopy. Together with computational simulations and mutagenesis studies, we elucidated the similarities and differences in the binding modes of fatty acid ligands to their respective GPCRs. Our findings unveiled distinct mechanisms of receptor activation and G protein coupling. We anticipate that these outcomes will facilitate structure-based drug development and underpin future research on this group of GPCRs.

Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies.

Neurodevelopmental proteasomopathies represent a distinctive category of neurodevelopmental disorders (NDD) characterized by genetic variations within the 26S proteasome, a protein complex governing eukaryotic cellular protein homeostasis. In our comprehensive study, we identified 23 unique variants in PSMC5 , which encodes the AAA-ATPase proteasome subunit PSMC5/Rpt6, causing syndromic NDD in 38 unrelated individuals. Overexpression of PSMC5 variants altered human hippocampal neuron morphology, while PSMC5 knockdown led to impaired reversal learning in flies and loss of excitatory synapses in rat hippocampal neurons. PSMC5 loss-of-function resulted in abnormal protein aggregation, profoundly impacting innate immune signaling, mitophagy rates, and lipid metabolism in affected individuals. Importantly, targeting key components of the integrated stress response, such as PKR and GCN2 kinases, ameliorated immune dysregulations in cells from affected individuals. These findings significantly advance our understanding of the molecular mechanisms underlying neurodevelopmental proteasomopathies, provide links to research in neurodegenerative diseases, and open up potential therapeutic avenues.

Inviting the Patient to Talk About a Conversation They Had with Another Healthcare Practitioner: A Way of Promoting Discussion About Disease Progression and End of Life in Palliative Care Interactions.

Discussing disease progression is a core task in palliative care. This is especially important when there are indications that a patient considers their death as less imminent than the clinical team does. This article examines a communicative action that palliative medicine doctors use to address such discrepancies in knowledge and understanding of the patient's prognosis: inviting the patient to talk about the contents of a conversation they had with another healthcare practitioner. The study used conversation analysis to examine five consultations in which this action was identified. These were part of a larger data set of 37 consultations recorded in a large UK hospice and involving patients with palliative care needs, sometimes accompanied by family or friends, and palliative medicine doctors. Findings are that the action of inviting the patient to talk about a previous conversation creates an opportunity for patients to articulate what they know and understand about their disease progression - but without requiring them to do so. Discussing such sensitive matters is thus made a matter of 'opting in' (rather than 'opting out'). Doctors thereby avoid being interactionally accountable for directly initiating a potentially distressing topic. The article shows how the task of discussing disease progression and end of life is intertwined with the delicate management of patients' displayed states of awareness regarding their disease progression. The study thus has practical implications by documenting ways in which clinicians can help patients realign their expectations about such delicate matters.

"Are you otherwise fit and well?": Past medical history questions in UK paediatric consultations.

Accurate diagnosis and treatment depend upon detailed knowledge of both the child's presenting symptoms and their past medical history. However, the process of soliciting past medical history has never been subject to systematic scrutiny in actual clinical practice. OBJECTIVE: To examine the function of the question "are you otherwise fit and well?" to elicit a child's general medical history in UK paediatric allergy outpatient consultations. METHODS: Examination of 30 video-recorded UK paediatric outpatient consultations involving children (2-10 years), caregivers, and one doctor. We identified, transcribed, and interrogated 13 examples, deploying the systematic and rigorous method of conversation analysis to elucidate the question's micro-design elements and their consequences for the consultation's trajectory. RESULTS: Asking "Are you otherwise fit and well?" is built to efficiently solicit a problem-free report of good health. Nonetheless patients can and do raise other relevant matters. In practice, the question initiates several interactional matters simultaneously: establishing/resolving (mis)understandings of "fitness" and "wellness"; negotiating opportunities for children's participation; and importantly, a shift towards discussing more general wellbeing. CONCLUSION: Past medical history questions unavoidably generate broader interactional matters which are skilfully resolved in real-time between clinicians, caregivers, and children. PRACTICE IMPLICATIONS: Clinical training could be greatly enhanced by integrating insights into the interactional consequences of asking questions, particularly in the complex multiparty environment of paediatrics. While the question 'Are you otherwise fit and well' clearly serves an important function, clinicians should be alert to the possible problems it might raise, especially when directed towards younger children.

Phosphorylation bar-coding of free fatty acid receptor 2 is generated in a tissue-specific manner.

Free fatty acid receptor 2 (FFAR2) is activated by short-chain fatty acids and expressed widely, including in white adipocytes and various immune and enteroendocrine cells. Using both wild-type human FFAR2 and a designer receptor exclusively activated by designer drug (DREADD) variant we explored the activation and phosphorylation profile of the receptor, both in heterologous cell lines and in tissues from transgenic knock-in mouse lines expressing either human FFAR2 or the FFAR2-DREADD. FFAR2 phospho-site-specific antisera targeting either pSer296/pSer297 or pThr306/pThr310 provided sensitive biomarkers of both constitutive and agonist-mediated phosphorylation as well as an effective means to visualise agonist-activated receptors in situ. In white adipose tissue, phosphorylation of residues Ser296/Ser297 was enhanced upon agonist activation whilst Thr306/Thr310 did not become phosphorylated. By contrast, in immune cells from Peyer's patches Thr306/Thr310 become phosphorylated in a strictly agonist-dependent fashion whilst in enteroendocrine cells of the colon both Ser296/Ser297 and Thr306/Thr310 were poorly phosphorylated. The concept of phosphorylation bar-coding has centred to date on the potential for different agonists to promote distinct receptor phosphorylation patterns. Here, we demonstrate that this occurs for the same agonist-receptor pairing in different patho-physiologically relevant target tissues. This may underpin why a single G protein-coupled receptor can generate different functional outcomes in a tissue-specific manner.

Intestinal-specific Hdac3 deletion increases susceptibility to colitis and small intestinal tumor development in mice fed a high-fat diet.

High-fat (HF) diets (HFDs) and inflammation are risk factors for colon cancer; however, the underlying mechanisms remain to be fully elucidated. The transcriptional corepressor HDAC3 has recently emerged as a key regulator of intestinal epithelial responses to diet and inflammation with intestinal-specific Hdac3 deletion (Hdac3IKO) in mice increasing fatty acid oxidation genes and the rate of fatty acid oxidation in enterocytes. Hdac3IKO mice are also predisposed to experimentally induced colitis; however, whether this is driven by the intestinal metabolic reprogramming and whether this predisposes these mice to intestinal tumorigenesis is unknown. Herein, we examined the effects of intestinal-specific Hdac3 deletion on colitis-associated intestinal tumorigenesis in mice fed a standard (STD) or HFD. Hdac3IKO mice were highly prone to experimentally induced colitis, which was further enhanced by an HFD. Hdac3 deletion also accelerated intestinal tumor development, specifically when fed an HFD and most notably in the small intestine where lipid absorption is maximal. Expression of proteins involved in fatty acid metabolism and oxidation (SCD1, EHHADH) were elevated in the small intestine of Hdac3IKO mice fed an HFD, and these mice displayed increased levels of lipid peroxidation, DNA damage, and apoptosis in their villi, as well as extensive expansion of the stem cell and progenitor cell compartment. These findings reveal a novel role for Hdac3 in suppressing colitis and intestinal tumorigenesis, particularly in the context of consumption of an HFD, and reveal a potential mechanism by which HFDs may increase intestinal tumorigenesis by increasing fatty acid oxidation, DNA damage, and intestinal epithelial cell turnover.NEW & NOTEWORTHY We reveal a novel role for the transcriptional corepressor Hdac3 in suppressing colitis and intestinal tumorigenesis, particularly in the context of consumption of an HFD, and reveal a potential mechanism by which HFDs may increase intestinal tumorigenesis by increasing fatty acid oxidation, DNA damage, and intestinal epithelial cell turnover. We also identify a unique mouse model for investigating the complex interplay between diet, metabolic reprogramming, and tumor predisposition in the intestinal epithelium.

G protein-receptor kinases 5/6 are the key regulators of G protein-coupled receptor 35-arrestin interactions.

Human G protein-coupled receptor 35 is regulated by agonist-mediated phosphorylation of a set of five phospho-acceptor amino acids within its C-terminal tail. Alteration of both Ser300 and Ser303 to alanine in the GPR35a isoform greatly reduces the ability of receptor agonists to promote interactions with arrestin adapter proteins. Here, we have integrated the use of cell lines genome edited to lack expression of combinations of G protein receptor kinases (GRKs), selective small molecule inhibitors of subsets of these kinases, and antisera able to specifically identify either human GPR35a or mouse GPR35 only when Ser300 and Ser303 (orce; the equivalent residues in mouse GPR35) have become phosphorylated to demonstrate that GRK5 and GRK6 cause agonist-dependent phosphorylation of these residues. Extensions of these studies demonstrated the importance of the GRK5/6-mediated phosphorylation of these amino acids for agonist-induced internalization of the receptor. Homology and predictive modeling of the interaction of human GPR35 with GRKs showed that the N terminus of GRK5 is likely to dock in the same methionine pocket on the intracellular face of GPR35 as the C terminus of the α5 helix of Gα13 and, that while this is also the case for GRK6, GRK2 and GRK3 are unable to do so effectively. These studies provide unique and wide-ranging insights into modes of regulation of GPR35, a receptor that is currently attracting considerable interest as a novel therapeutic target in diseases including ulcerative colitis.

Pro-phagocytic function and structural basis of GPR84 signaling.

GPR84 is a unique orphan G protein-coupled receptor (GPCR) that can be activated by endogenous medium-chain fatty acids (MCFAs). The signaling of GPR84 is largely pro-inflammatory, which can augment inflammatory response, and GPR84 also functions as a pro-phagocytic receptor to enhance phagocytic activities of macrophages. In this study, we show that the activation of GPR84 by the synthetic agonist 6-OAU can synergize with the blockade of CD47 on cancer cells to induce phagocytosis of cancer cells by macrophages. We also determine a high-resolution structure of the GPR84-Gi signaling complex with 6-OAU. This structure reveals an occluded binding pocket for 6-OAU, the molecular basis of receptor activation involving non-conserved structural motifs of GPR84, and an unusual Gi-coupling interface. Together with computational docking and simulations studies, this structure also suggests a mechanism for the high selectivity of GPR84 for MCFAs and a potential routes of ligand binding and dissociation. These results provide a framework for understanding GPR84 signaling and developing new drugs targeting GPR84.

Structural basis for the ligand recognition and signaling of free fatty acid receptors.

Free fatty acid receptors 1-4 (FFA1-4) are class A G protein-coupled receptors (GPCRs). FFA1-3 share substantial sequence similarity whereas FFA4 is unrelated. Despite this FFA1 and FFA4 are activated by the same range of long chain fatty acids (LCFAs) whilst FFA2 and FFA3 are instead activated by short chain fatty acids (SCFAs) generated by the intestinal microbiota. Each of FFA1, 2 and 4 are promising targets for novel drug development in metabolic and inflammatory conditions. To gain insights into the basis of ligand interactions with, and molecular mechanisms underlying activation of, FFAs by LCFAs and SCFAs, we determined the active structures of FFA1 and FFA4 bound to the polyunsaturated LCFA docosahexaenoic acid (DHA), FFA4 bound to the synthetic agonist TUG-891, as well as SCFA butyrate-bound FFA2, each complexed with an engineered heterotrimeric Gq protein (miniGq), by cryo-electron microscopy. Together with computational simulations and mutagenesis studies, we elucidated the similarities and differences in the binding modes of fatty acid ligands with varying chain lengths to their respective GPCRs. Our findings unveil distinct mechanisms of receptor activation and G protein coupling. We anticipate that these outcomes will facilitate structure-based drug development and underpin future research to understand allosteric modulation and biased signaling of this group of GPCRs.

Effects of post-spawning ration restriction on reproductive development and the growth hormone/insulin-like growth factor-1 axis in female rainbow trout (Oncorhynchus mykiss).

In iteroparous female salmonids, the growth and reproductive endocrine axes interact during the period after spawning. Energy depletion due to pre-spawn fasting, migration, and ovarian development must be restored, and the next reproductive cycle is initiated in consecutively maturing fish. In the natural environment, food availability is often limited during the post-spawn period. To investigate the growth and reproductive endocrinology of the post-spawn period, we sampled female rainbow trout over the 30 weeks following their first spawning. Fish were fasted for 2 months prior to spawning, then fed a standard or a restricted ration. Analysis was confined to reproductive fish. Plasma estradiol-17ß decreased during the 8 weeks following spawning and then began increasing in both ration groups and was lower in feed-restricted versus standard ration fish from 8 weeks onward. Plasma insulin-like growth factor-1 increased over the same period and then remained constant in both ration groups and was lower in feed-restricted versus standard ration fish from week 8 to week 30. Plasma growth hormone decreased following spawning in standard ration fish and became elevated in feed-restricted versus standard ration fish at 20- and 30-weeks post-spawn. Growth rates, condition factor, and muscle lipid levels were higher in standard ration versus feed-restricted fish within 2-4 weeks after spawning. These results suggest that two phases occurred during the post-spawn period: recovery from spawning and restoration of energy reserves over weeks 0 to 8, followed by adjustment of the growth and reproductive endocrine axes to ration level over weeks 8 to 30.

Discovery of Potent Tetrazole Free Fatty Acid Receptor 2 Antagonists.

The free fatty acid receptor 2 (FFA2), also known as GPR43, mediates effects of short-chain fatty acids and has attracted interest as a potential target for treatment of various metabolic and inflammatory diseases. Herein, we report the results from bioisosteric replacement of the carboxylic acid group of the established FFA2 antagonist CATPB and SAR investigations around these compounds, leading to the discovery of the first high-potency FFA2 antagonists, with the preferred compound TUG-2304 (16l) featuring IC50 values of 3-4 nM in both cAMP and GTPγS assays, favorable physicochemical and pharmacokinetic properties, and the ability to completely inhibit propionate-induced neutrophil migration and respiratory burst.

Pro-phagocytic function and structural basis of GPR84 signaling.

GPR84 is a unique orphan G protein-coupled receptor (GPCR) that can be activated by endogenous medium-chain fatty acids (MCFAs). The signaling of GPR84 is largely pro-inflammatory, which can augment inflammatory response, and GPR84 also functions as a pro-phagocytic receptor to enhance the phagocytic activities of macrophages. In this study, we first showed that the activation of GPR84 by the synthetic agonist 6-OAU could synergize with the blockade of CD47 on cancer cells to induce phagocytosis of cancer cells by macrophages. Then, we determined a high-resolution structure of the GPR84-Gi signaling complex with 6-OAU. This structure revealed a completely occluded binding pocket for 6-OAU, the molecular basis of receptor activation involving non-conserved structural motifs of GPR84, and an unusual Gi-coupling interface. Together with computational docking and simulations studies, our structure also suggested the mechanism for the high selectivity of GPR84 for MCFAs and the potential routes of ligand binding and dissociation. Our results provide a framework for understanding GPR84 signaling and developing new drugs targeting GPR84.

Feeding after spawning and energy balance at spawning are associated with repeat spawning interval in steelhead trout.

Consecutive and skip repeat spawning (1- or ≥2-year spawning interval) life histories commonly occur in seasonally breeding iteroparous fishes. Spawning interval variation is driven by energetic status and impacts fisheries management. In salmonids, energetic status (either absolute level of energy reserves or the rate of change of energy reserves, i.e., energy balance) is thought to determine reproductive trajectory during a critical period ∼1 year prior to initial spawning. However, information on repeat spawners is lacking. To examine the timing and the aspects of energetic status that regulate repeat spawning interval, female steelhead trout (Oncorhynchus mykiss) were fasted for 10 weeks after spawning and then fed ad libitum and compared to ad libitum fed controls. Plasma growth hormone (GH) and insulin-like growth factor-I (IGF-I) levels were measured to assess long-term energy balance. Plasma estradiol levels showed that some fish in both groups initiated a consecutive spawning cycle. In fasted fish, GH was lower at spawning in consecutive versus skip spawners. In consecutive spawners, GH was higher at spawning in fed versus fasted fish. These results suggest that fish with a less negative energy balance at spawning initiated reproductive development in the absence of feeding, but that feeding during the post-spawning period enabled initiation of reproduction in some fish with a more negative energy balance at spawning. Thus, both energy balance at spawning and feeding after spawning regulated reproductive schedules. These results show that the critical period model of salmonid maturation applies to regulation of repeat spawning, and that the reproductive decision window extends into the first 10 weeks after spawning.