Cumulative risk of future bleaching for the world's coral reefs.

Spatial and temporal patterns of future coral bleaching are uncertain, hampering global conservation efforts to protect coral reefs against climate change. Our analysis of daily projections of ocean warming establishes the severity, annual duration, and onset of severe bleaching risk for global coral reefs this century, pinpointing vital climatic refugia. We show that low-latitude coral regions are most vulnerable to thermal stress and will experience little reprieve from climate mitigation. By 2080, coral bleaching is likely to start on most reefs in spring, rather than late summer, with year-round bleaching risk anticipated to be high for some low-latitude reefs regardless of global efforts to mitigate harmful greenhouse gasses. By identifying Earth's reef regions that are at lowest risk of accelerated bleaching, our results will prioritize efforts to limit future loss of coral reef biodiversity.

Anatomical and molecular insights into the antennal gland of the giant freshwater prawn Macrobrachium rosenbergii.

In this study, the complex organization of the AnG in the giant freshwater prawn Macrobrachium rosenbergii was revealed using various techniques, including conventional histology, histochemistry, scanning electron microscopy, and X-ray tomography. The results showed the diversity of cells in the AnG and the detailed organization of the labyrinth's tubule into four radiated areas from the central to peripheral zones. The study also demonstrated the expression of some vertebrate kidney-associated homolog genes, aquaporin (AQP), solute carrier family 22 (SLC-22), nephrin, and uromodulin, in the AnG by qPCR. The result of in situ hybridization further showed the localization of SLC-22 and AQP transcript in the bladder and labyrinth's epithelium, specifically in regions 2, 3, and 4. Additionally, the study revealed neuropeptide expressions in the AnG by qPCR and in situ hybridization, i.e., crustacean hyperglycemic hormone (CHH) and molt inhibiting hormone (MIH), implying that the AnG may have a role in hormone production. Moreover, male and female prawns exhibited different levels of AQP, SLC-22, nephrin, and CHH expressions during the premolt and intermolt stages, suggesting a crucial role relevant to the molting stages. In conclusion, this study clarified the complex structure of the AnG in M. rosenbergii and demonstrated for the first time the expression of vertebrate kidney-associated genes and the possible endocrine role of the AnG. Further investigation is needed to clarify the role of these genes, particularly during ecdysis. The implications of these findings could significantly advance our understanding of the AnG in decapod crustaceans.

Double Cyclization Tandem Mass for Identification and Quantification of Phosphatidylcholines Using Isobaric Six-Plex Capillary nLC-MS/MS.

Multiplexing of phosphatidylcholine analysis is hindered by a lack of appropriate derivatization. Presented here is a tagging scheme that uses a quaternary amine tag and targets the hydroxy group of the phosphate, which switches the net charge from neutral to +2. Quantitative yields were achieved from >99% reaction completion derived by dimethoxymethyl morpholinium (DMTMM) activation. Fragmentation of phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs) releases two trimethylamines and the acyl chains through neutral loss and generates a unique double cyclization constant mass reporter. Selective incorporation of isotopes onto the tag produces a six-plex set of isobaric reagents. For equivalent six-plex-labeled samples, <14% RSD was achieved, followed by a dynamic range of 1:10 without signal compression. Quantification of PCs/LPCs in human hepatic cancer cells was conducted as six-plex using data-dependent analysis tandem MS. We report a six-plex qualitative and quantitative isobaric tagging strategy expanding the limits of analyzing PCs/LPCs.

Mechanisms for dysregulation of excitatory-inhibitory balance underlying allodynia in dorsal horn neural subcircuits.

Chronic pain is a wide-spread condition that is debilitating and expensive to manage, costing the United States alone around $600 billion in 2010. In a common type of chronic pain called allodynia, non-painful stimuli produce painful responses with highly variable presentations across individuals. While the specific mechanisms remain unclear, allodynia is hypothesized to be caused by the dysregulation of excitatory-inhibitory (E-I) balance in pain-processing neural circuitry in the dorsal horn of the spinal cord. In this work, we analyze biophysically-motivated subcircuit structures that represent common motifs in neural circuits in layers I-II of the dorsal horn. These circuits are hypothesized to be part of the neural pathways that mediate two different types of allodynia: static and dynamic. We use neural firing rate models to describe the activity of populations of excitatory and inhibitory interneurons within each subcircuit. By accounting for experimentally-observed responses under healthy conditions, we specify model parameters defining populations of subcircuits that yield typical behavior under normal conditions. Then, we implement a sensitivity analysis approach to identify the mechanisms most likely to cause allodynia-producing dysregulation of the subcircuit's E-I signaling. We find that disruption of E-I balance generally occurs either due to downregulation of inhibitory signaling so that excitatory neurons are "released" from inhibitory control, or due to upregulation of excitatory neuron responses so that excitatory neurons "escape" their inhibitory control. Which of these mechanisms is most likely to occur, the subcircuit components involved in the mechanism, and the proportion of subcircuits exhibiting the mechanism can vary depending on the subcircuit structure. These results suggest specific hypotheses about diverse mechanisms that may be most likely responsible for allodynia, thus offering predictions for the high interindividual variability observed in allodynia and identifying targets for further experimental studies on the underlying mechanisms of this chronic pain condition.

Biophysics of Frequency-Dependent Variation in Paresthesia and Pain Relief during Spinal Cord Stimulation.

The neurophysiological effects of spinal cord stimulation (SCS) for chronic pain are poorly understood, resulting in inefficient failure-prone programming protocols and inadequate pain relief. Nonetheless, novel stimulation patterns are regularly introduced and adopted clinically. Traditionally, paresthetic sensation is considered necessary for pain relief, although novel paradigms provide analgesia without paresthesia. However, like pain relief, the neurophysiological underpinnings of SCS-induced paresthesia are unknown. Here, we paired biophysical modeling with clinical paresthesia thresholds (of both sexes) to investigate how stimulation frequency affects the neural response to SCS relevant to paresthesia and analgesia. Specifically, we modeled the dorsal column (DC) axonal response, dorsal column nucleus (DCN) synaptic transmission, conduction failure within DC fiber collaterals, and dorsal horn network output. Importantly, we found that high-frequency stimulation reduces DC fiber activation thresholds, which in turn accurately predicts clinical paresthesia perception thresholds. Furthermore, we show that high-frequency SCS produces asynchronous DC fiber spiking and ultimately asynchronous DCN output, offering a plausible biophysical basis for why high-frequency SCS is less comfortable and produces qualitatively different sensation than low-frequency stimulation. Finally, we demonstrate that the model dorsal horn network output is sensitive to SCS-inherent variations in spike timing, which could contribute to heterogeneous pain relief across patients. Importantly, we show that model DC fiber collaterals cannot reliably follow high-frequency stimulation, strongly affecting the network output and typically producing antinociceptive effects at high frequencies. Altogether, these findings clarify how SCS affects the nervous system and provide insight into the biophysics of paresthesia generation and pain relief.

Lifespan effects in male UM-HET3 mice treated with sodium thiosulfate, 16-hydroxyestriol, and late-start canagliflozin.

Genetically heterogeneous UM-HET3 mice born in 2020 were used to test possible lifespan effects of alpha-ketoglutarate (AKG), 2,4-dinitrophenol (DNP), hydralazine (HYD), nebivolol (NEBI), 16α-hydroxyestriol (OH_Est), and sodium thiosulfate (THIO), and to evaluate the effects of canagliflozin (Cana) when started at 16 months of age. OH_Est produced a 15% increase (p = 0.0001) in median lifespan in males but led to a significant (7%) decline in female lifespan. Cana, started at 16 months, also led to a significant increase (14%, p = 0.004) in males and a significant decline (6%, p = 0.03) in females. Cana given to mice at 6 months led, as in our previous study, to an increase in male lifespan without any change in female lifespan, suggesting that this agent may lead to female-specific late-life harm. We found that blood levels of Cana were approximately 20-fold higher in aged females than in young males, suggesting a possible mechanism for the sex-specific disparities in its effects. NEBI was also found to produce a female-specific decline (4%, p = 0.03) in lifespan. None of the other tested drugs provided a lifespan benefit in either sex. These data bring to 7 the list of ITP-tested drugs that induce at least a 10% lifespan increase in one or both sexes, add a fourth drug with demonstrated mid-life benefits on lifespan, and provide a testable hypothesis that might explain the sexual dimorphism in lifespan effects of the SGLT2 inhibitor Cana.

Reflex sympathetic activation to inspiratory muscle loading is attenuated in females relative to males.

Intense inspiratory muscle work can evoke a metabolite-stimulated pressor reflex, commonly referred to as the respiratory muscle metaboreflex. When completing similar relative and absolute levels of inspiratory work, females have an attenuated blood pressure response. We sought to test the hypothesis that the lower blood pressure response to the respiratory muscle metaboreflex in females is associated with a reduced sympathetic response. Healthy young (26 ± 4 yr) males (n = 9) and females (n = 7) completed two experimental days. On day 1, participants completed pulmonary function testing and became familiarized with an inspiratory pressure-threshold loading (PTL) task. On the second day, balloon-tipped catheters were placed in the esophagus and stomach to measure pleural and gastric pressures, and transdiaphragmatic pressure was calculated. A microelectrode was inserted into the fibular nerve to quantify muscle sympathetic nerve activity (MSNA), and participants then completed isocapnic PTL to task failure. There was a significant sex-by-time interaction in the mean arterial pressure (MAP, P = 0.015) and burst frequency (P = 0.039) response to PTL. Males had a greater rise in MAP (Δ21 ± 9 mmHg) than females (Δ13 ± 5 mmHg, P = 0.026). Males also demonstrated a greater rise in MSNA burst frequency (Δ18 ± 7 bursts/min) than females (Δ10 ± 5 bursts/min, P = 0.015). The effect of sex was observed despite females and males completing the same magnitude of diaphragm work throughout the task (P = 0.755). Our findings provide novel evidence that the lower blood pressure response to similar relative and absolute inspiratory muscle work in females is associated with lower sympathetic activation.NEW & NOTEWORTHY The blood pressure response to high levels of inspiratory muscle work is lower in females and occurs alongside a reduced sympathetic response. The reduced blood pressure and sympathetic response occur despite males and females performing similar levels of absolute inspiratory work. Our findings provide evidence that sex differences in the respiratory muscle metaboreflex are, in part, sympathetically mediated.

Real-world patterns and sequences of targeted therapy use in chronic lymphocytic leukemia and small lymphocytic lymphoma in the United States: a longitudinal study.

With increasing focus on novel targeted therapies for chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), this longitudinal claims-based study evaluated real-world CLL/SLL treatment sequences, particularly sequential targeted therapy. Among patients with first-line (1 L) treatment in 2014-2017 (N = 2,612; median follow-up = 3 years), the most common 1 L treatment was chemoimmunotherapy (CIT; 44.6%), followed by CD20 (25.2%) and Bruton's tyrosine kinase inhibitors (BTKi; 21.7%). Among those with 1 L in 2018-2021 (N = 4,534; median follow-up = 1 year), these were BTKi (45.5%), CD20 (20.4%), CIT (17.5%), and B-cell lymphoma 2 inhibitor (8.3%). In 2014-2017, the proportion of patients receiving sequential targeted therapy in the first 2 LOTs was 11.2% (80.2% was BTKi→BTKi); in 2018-2021, this proportion was 34.3% (66.4% was BTKi→BTKi). Over time, there was a substantial increase in targeted therapy use in 1 L and sequential targeted therapy, particularly with BTKi→BTKi. Future studies should assess clinical outcomes to determine optimal sequences for CLL/SLL and reasons for restarting BTKi.

Quantifying Diaphragm Blood Flow With Contrast-Enhanced Ultrasound in Humans.

BACKGROUND: Despite the known interplay between blood flow and function, there is currently no minimally invasive method to monitor diaphragm hemodynamics. We used contrast-enhanced ultrasound (CEUS) to quantify relative diaphragm blood flow (Q˙DIA) in humans and assessed the technique's efficacy and reliability during graded inspiratory pressure threshold loading. We hypothesized that: (1) Q˙DIA would linearly increase with pressure generation: and (2) that there would be good test-retest reliability and interanalyzer reproducibility. RESEARCH QUESTION: Can the first minimally invasive method to measure relative diaphragm blood flow be validated in humans? STUDY DESIGN AND METHODS: Quantitative contrast-enhanced ultrasound of the costal diaphragm was performed in healthy participants (10 male subjects, 6 female subjects; mean age 28 ± 5 years; BMI 22.8 ± 2.0 kg/m) during unloaded breathing and three stages of loaded breathing on two separate days. Gastric and esophageal balloon catheters measured diaphragmatic pressure. Ultrasonography was performed during a constant-rate IV infusion of lipid-stabilized microbubbles following each stage. Ultrasound images were acquired after a destruction-replenishment sequence and diaphragm specific time-intensity data were used to determine Q˙DIA by two individuals. RESULTS: Transdiaphragmatic pressure for unloaded and each loading stage were 15.2 ± 0.8, 26.1 ± 0.8, 34.6 ± 0.8, and 40.0 ± 0.8 percentage of the maximum, respectively. Q˙DIA increased with each stage of loading (3.1 ± 3.1, 6.9 ± 3.6, 11.0 ± 4.9, and 13.5 ± 5.4 AU/s; P < .0001). The linear relationship between diaphragmatic flow and pressure was reproducible from day to day. Q˙DIA had good to excellent test-retest reliability (0.86 [0.77, 0.92]; P < .0001) and excellent interanalyzer reproducibility (0.93 [0.90, 0.95]; P < .0001) with minimal bias. INTERPRETATION: Relative Q˙DIA measurements have valid physiological underpinnings, are reliable day to day, and reproducible analyzer-to-analyzer. Contrast-enhanced ultrasound is a viable, minimally invasive method for assessing costal Q˙DIA in humans and may provide a tool to monitor diaphragm hemodynamics in clinical settings.

Serum Vitamin D and Chronic Musculoskeletal Pain: A Cross-Sectional Study of 349,221 Adults in the UK.

Insufficient and deficient vitamin D may be associated with chronic musculoskeletal pain, but study findings are conflicting, and few account for important confounding factors. This cross-sectional study explored the association between serum vitamin D status and chronic musculoskeletal pain in various body sites, adjusting for a wide range and a number of potential confounding factors. Data collected at the baseline assessments of 349,221 UK Biobank participants between 2006 and 2010 were analyzed. Serum 25-hydroxyvitamin D was measured and categorized as <25.0 nmol/L (severe deficiency), 25.0 to 49.9 nmol/L (deficiency), 50.0 to 74.9 nmol/L (insufficiency), and ≥75.0 nmol/L (sufficiency). The outcome was self-reported chronic musculoskeletal pain at any site, neck/shoulder, back, hip, knee, or widespread pain that interfered with usual activities. Potential confounders were identified using directed acyclic graphs and included sociodemographic, lifestyle, psychological factors, and medical comorbidities. Simple models adjusted for age and sex showed significant associations between suboptimal vitamin D status and chronic pain across all sites (odds ratios [ORs] ranged 1.07-2.85). These associations were weakened or became insignificant after accounting for all confounding factors (ORs ≤ 1.01) for chronic regional musculoskeletal pain. Severe vitamin D deficiency remained a significant and positive association with chronic widespread pain after adjusting for all confounding factors (OR [95% confidence interval]: 1.26 [1.07, 1.49]). This study suggests that, while vitamin D status is not a key independent determinant of chronic regional musculoskeletal pain, severe vitamin D deficiency may be associated with chronic widespread pain. PERSPECTIVES: After accounting for various confounders, vitamin D deficiency was not associated with regional musculoskeletal pain. However, the relationship between chronic widespread pain severe vitamin D deficiency remained after confounder adjustment. Use of vitamin D supplements in individuals with chronic widespread pain and severe vitamin D deficiency warrants further exploration.

Flavin-containing monooxygenase (FMO): Beyond xenobiotics.

Flavin-containing monooxygenases (FMOs), traditionally known for detoxifying xenobiotics, are now recognized for their involvement in endogenous metabolism. We recently discovered that an isoform of FMO, fmo-2 in Caenorhabditis elegans, alters endogenous metabolism to impact longevity and stress tolerance. Increased expression of fmo-2 in C. elegans modifies the flux through the key pathway known as One Carbon Metabolism (OCM). This modified flux results in a decrease in the ratio of S-adenosyl-methionine (SAM) to S-adenosyl-homocysteine (SAH), consequently diminishing methylation capacity. Here we discuss how FMO-2-mediated formate production during tryptophan metabolism may serve as a trigger for changing the flux in OCM. We suggest formate bridges tryptophan and OCM, altering metabolic flux away from methylation during fmo-2 overexpression. Additionally, we highlight how these metabolic results intersect with the mTOR and AMPK pathways, in addition to mitochondrial metabolism. In conclusion, the goal of this essay is to bring attention to the central role of FMO enzymes but lack of understanding of their mechanisms. We justify a call for a deeper understanding of FMO enzyme's role in metabolic rewiring through tryptophan/formate or other yet unidentified substrates. Additionally, we emphasize the identification of novel drugs and microbes to induce FMO activity and extend lifespan.

Prospective memory performance in veterans with and without histories of mild traumatic brain injury: effect of the apolipoprotein E (APOE) ε4 genotype.

OBJECTIVE: Identifying factors that moderate cognitive outcomes following mild traumatic brain injury (mTBI) is crucial. Prospective memory (PM) is a cognitive domain of interest in mTBI recovery as it may be especially sensitive to TBI-related changes. Since studies show that genetic status - particularly possession of the apolipoprotein E (APOE) ε4 allele - can modify PM performance, we investigated associations between mTBI status and APOE-ε4 genotype on PM performance in a well-characterized sample of Veterans with neurotrauma histories. METHODS: 59 Veterans (mTBI = 33, Military Controls [MCs] = 26; age range: 24-50; average years post-injury = 10.41) underwent a structured clinical interview, neuropsychological assessment, and genotyping. The Memory for Intentions Test (MIST) measured PM across multiple subscales. ANCOVAs, adjusting for age and posttraumatic stress symptoms, tested the effects of mTBI status (mTBI vs. MC) and ε4 status (ε4+ vs. ε4-) on MIST scores. RESULTS: Veterans with mTBI history performed more poorly compared to MCs on the MIST 15-min delay (p=.002, ηp2 =.160), Time Cue (p = .003, ηp2 =.157), and PM Total (p = .016, ηp2 =.102). Those with at least one copy of the ε4 allele performed more poorly compared to ε4- Veterans on the MIST 15-min delay (p = .011, ηp2 =.113) and PM Total (p = .048, ηp2 = .071). No significant interactions were observed between mTBI and APOE-ε4 status on MIST outcomes (ps>.25). Within the mTBI group, APOE-ε4+ Veterans performed worse than APOE-ε4- Veterans on the MIST 15-min delay subscale (p = .031, ηp2 = .150). CONCLUSIONS: mTBI history and APOE-ε4 genotype status were independently associated with worse PM performance compared to those without head injury histories or possession of the APOE-e4 genotype. Performance on the MIST 15-min delay was worse in Veterans with both risk factors (mTBI history and APOE-ε4 positivity). Findings suggest that genetic status may modify outcomes even in relatively young Veterans with mTBI histories. Future research examining longitudinal associations and links to neuroimaging and biomarker data are needed.

Reprint of: Prelude to molecularization: The double gradient model of Sulo Toivonen and Lauri Saxén.

The present molecular investigations of Organizer phenomena show a remarkable connection to the earlier classical embryological studies that used transplantation as a method for making mechanistic models of induction. One of the most prominent of these connections is the dual gradient model for anterior-posterior and dorsal-ventral polarity. This paper will discuss some of the history of how transplantation experiments provided data that could be interpreted in terms of two gradients of biologically active materials. It will highlight how the attempts to discover the elusive Induktionsstoffen gave rise to the double gradient model of Sulo Toivonen and Lauri Saxén in the 1950s and 1960s. This paper will also document how this research into the identity of these molecules gave rise to the developmental genetics that eventually would find the molecules responsible for primary embryonic induction.

Sea anemone-anemonefish symbiosis: Behavior and mucous protein profiling.

Fish species of the genus Amphiprion (Perciformes: Pomacentridae) seek protection from predators among the tentacles of sea anemones as their natural habitat, where they live essentially unharmed from stinging by the host's nematocysts. The skin mucus of these anemonefish has been suggested as a protective mechanism that prevents the discharge of the nematocysts upon contact. Whereas some anemonefish species seem to produce their own protective mucous coating, others may acquire mucus (or biomolecules within) from the sea anemone during an acclimation period. In controlled experiments, we show that Amphiprion ocellaris acclimated successfully to their natural host anemone species Stichodactyla gigantea, and also to Stichodactyla haddoni, and in some cases Heteractis crispa, neither of which are natural host species. No symbiosis was observed for three other anemone species tested, Entacmaea quadricolor, Macrodactyla doreensis, and Heteractis malu. We explored the skin mucous protein profile from naive and experienced A. ocellaris during their acclimation to natural and unnatural host anemones. We confidently report the presence of metabolic and structural proteins in the skin mucus of all samples, likely involved in immunological defense, molecular transport, stress response, and signal transduction. For those anemonefish that established symbiosis, there was a clear increase in ribosomal-type proteins. We additionally provide evidence for the presence of anemone proteins only in the skin mucus of individuals that established symbiosis. Our results support previous speculation of the role of skin mucous-associated proteins in anemonefish-anemone symbiosis. Further exploration of these mucosal proteins could reveal the mechanism of anemonefish acclimation to host anemones.

Fmo induction as a tool to screen for pro-longevity drugs.

Dietary restriction (DR) and hypoxia (low oxygen) extend lifespan in Caenorhabditis elegans through the induction of a convergent downstream longevity gene, fmo-2. Flavin-containing monooxygenases (FMOs) are highly conserved xenobiotic-metabolizing enzymes with a clear role in promoting longevity in nematodes and a plausible similar role in mammals. This makes them an attractive potential target of small molecule drugs to stimulate the health-promoting effects of longevity pathways. Here, we utilize an fmo-2 fluorescent transcriptional reporter in C. elegans to screen a set of 80 compounds previously shown to improve stress resistance in mouse fibroblasts. Our data show that 19 compounds significantly induce fmo-2, and 10 of the compounds induce fmo-2 more than twofold. Interestingly, 9 of the 10 high fmo-2 inducers also extend lifespan in C. elegans. Two of these drugs, mitochondrial respiration chain complex inhibitors, interact with the hypoxia pathway to induce fmo-2, whereas two dopamine receptor type 2 (DRD2) antagonists interact with the DR pathway to induce fmo-2, indicating that dopamine signaling is involved in DR-mediated fmo-2 induction. Together, our data identify nine drugs that each (1) increase stress resistance in mouse fibroblasts, (2) induce fmo-2 in C. elegans, and (3) extend nematode lifespan, some through known longevity pathways. These results define fmo-2 induction as a viable approach to identifying and understanding mechanisms of putative longevity compounds.

DengueSeq: a pan-serotype whole genome amplicon sequencing protocol for dengue virus.

BACKGROUND: The increasing burden of dengue virus on public health due to more explosive and frequent outbreaks highlights the need for improved surveillance and control. Genomic surveillance of dengue virus not only provides important insights into the emergence and spread of genetically diverse serotypes and genotypes, but it is also critical to monitor the effectiveness of newly implemented control strategies. Here, we present DengueSeq, an amplicon sequencing protocol, which enables whole-genome sequencing of all four dengue virus serotypes. RESULTS: We developed primer schemes for the four dengue virus serotypes, which can be combined into a pan-serotype approach. We validated both approaches using genetically diverse virus stocks and clinical specimens that contained a range of virus copies. High genome coverage (>95%) was achieved for all genotypes, except DENV2 (genotype VI) and DENV 4 (genotype IV) sylvatics, with similar performance of the serotype-specific and pan-serotype approaches. The limit of detection to reach 70% coverage was 10-100 RNA copies/µL for all four serotypes, which is similar to other commonly used primer schemes. DengueSeq facilitates the sequencing of samples without known serotypes, allows the detection of multiple serotypes in the same sample, and can be used with a variety of library prep kits and sequencing instruments. CONCLUSIONS: DengueSeq was systematically evaluated with virus stocks and clinical specimens spanning the genetic diversity within each of the four dengue virus serotypes. The primer schemes can be plugged into existing amplicon sequencing workflows to facilitate the global need for expanded dengue virus genomic surveillance.

Total Chemical Synthesis of Aggregation-Prone Disulfide-Rich Starfish Peptides.

A relaxin-like gonad-stimulating peptide (RGP), Aso-RGP, featuring six cysteine residues, was identified in the Crown-of-Thorns Starfish (COTS, Acanthaster cf. solaris) and initially produced through recombinant yeast expression. This method yielded a single-chain peptide with an uncleaved C-peptide (His Tag) and suboptimal purity. Our objective was to chemically synthesize Aso-RGP in its mature form, comprising two chains (A and B) and three disulfide bridges, omitting the C-peptide. Furthermore, we aimed to synthesize a newly identified relaxin-like peptide, Aso-RLP2, from COTS, which had not been previously synthesized. This paper reports the first total chemical synthesis of Aso-RGP and Aso-RLP2. Aso-RGP synthesis proceeded without major issues, whereas the A-chain of Aso-RLP2, in its reduced and unfolded state with two free thiols, presented considerable challenges. These were initially marked by "messy" RP-HPLC profiles, typically indicative of synthesis failure. Surprisingly, oxidizing the A-chain significantly improved the RP-HPLC profile, revealing the main issue was not synthesis failure but the peptide's aggregation tendency, which initially obscured analysis. This discovery highlights the critical need to account for aggregation in peptide synthesis and analysis. Ultimately, our efforts led to the successful synthesis of both peptides with purities exceeding 95 %.

Fmo5 plays a sex-specific role in goblet cell maturation and mucus barrier formation.

The intestine plays a key role in metabolism, nutrient and water absorption, and provides both physical and immunological defense against dietary and luminal antigens. The protective mucus lining in the intestine is a critical component of intestinal barrier function that when compromised, can lead to dysfunctional intestinal barriers that are a defining characteristic of inflammatory bowel disease (IBD), among other intestinal diseases. Here, we define a new role for the flavin-containing monooxygenase family of enzymes in maintaining a healthy intestinal epithelium. In nematodes, we find that Cefmo-2 is necessary and sufficient for proper intestinal barrier function, intestinal actin expression, and is induced by intestinal damage. In mice, we utilize an intestine-specific, inducible knockout model of the prevalent gut Fmo (Fmo5) and find striking phenotypes within two weeks of knockout. These phenotypes include sex-dependent changes in colon epithelial histology, goblet cell localization and maturation factors, and mucus barrier formation. Each of these changes are significantly more severe in female mice, plausibly mirroring differences observed in some types of IBD in humans. Looking further at these phenotypes, we find increased protein folding stress in Fmo5 knockout animals and successfully rescue the severe female phenotype with addition of a chemical ER chaperone. Together, our results identify a new role for Fmo5 in the mammalian intestine and support a key role for Fmo5 in maintenance of ER/protein homeostasis and proper mucus barrier formation.

A diet of oxidative stress-adapted bacteria improves stress resistance and lifespan in C. elegans via p38-MAPK.

Organisms across taxa face stresses including variable temperature, redox imbalance, and xenobiotics. Successfully responding to stress and restoring homeostasis are crucial for survival. Aging is associated with a decreased stress response and alterations in the microbiome, which contribute to disease development. Animals and their microbiota share their environment; however, microbes have short generation time and can rapidly evolve and potentially affect host physiology during stress. Here, we leverage Caenorhabditis elegans and its simplified bacterial diet to demonstrate how microbial adaptation to oxidative stress affects the host's lifespan and stress response. We find that worms fed stress-evolved bacteria exhibit enhanced stress resistance and an extended lifespan. Through comprehensive genetic and metabolic analysis, we find that iron in stress-evolved bacteria enhances worm stress resistance and lifespan via activation of the mitogen-activated protein kinase pathway. In conclusion, our study provides evidence that understanding microbial stress-mediated adaptations could be used to slow aging and alleviate age-related health decline.