Evaluating the efficacy and mechanisms of a ketogenic diet as adjunctive treatment for people with treatment-resistant depression: A protocol for a randomised controlled trial.

BACKGROUND: One-third of people with depression do not respond to antidepressants, and, after two adequate courses of antidepressants, are classified as having treatment-resistant depression (TRD). Some case reports suggest that ketogenic diets (KDs) may improve some mental illnesses, and preclinical data indicate that KDs can influence brain reward signalling, anhedonia, cortisol, and gut microbiome which are associated with depression. To date, no trials have examined the clinical effect of a KD on TRD. METHODS: This is a proof-of-concept randomised controlled trial to investigate the efficacy of a six-week programme of weekly dietitian counselling plus provision of KD meals, compared with an intervention involving similar dietetic contact time and promoting a healthy diet with increased vegetable consumption and reduction in saturated fat, plus food vouchers to purchase healthier items. At 12 weeks we will assess whether participants have continued to follow the assigned diet. The primary outcome is the difference between groups in the change in Patient Health Questionnaire-9 (PHQ-9) score from baseline to 6 weeks. PHQ-9 will be measured at weeks 2, 4, 6 and 12. The secondary outcomes are the differences between groups in the change in remission of depression, change in anxiety score, functioning ability, quality of life, cognitive performance, reward sensitivity, and anhedonia from baseline to 6 and 12 weeks. We will also assess whether changes in reward sensitivity, anhedonia, cortisol awakening response and gut microbiome may explain any changes in depression severity. DISCUSSION: This study will test whether a ketogenic diet is an effective intervention to reduce the severity of depression, anxiety and improve quality of life and functioning ability for people with treatment-resistant depression.

IL11-mediated stromal cell activation may not be the master regulator of pro-fibrotic signaling downstream of TGFß.

Fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF) and systemic scleroderma (SSc), are commonly associated with high morbidity and mortality, thereby representing a significant unmet medical need. Interleukin 11 (IL11)-mediated cell activation has been identified as a central mechanism for promoting fibrosis downstream of TGFß. IL11 signaling has recently been reported to promote fibroblast-to-myofibroblast transition, thus leading to various pro-fibrotic phenotypic changes. We confirmed increased mRNA expression of IL11 and IL11Rα in fibrotic diseases by OMICs approaches and in situ hybridization. However, the vital role of IL11 as a driver for fibrosis was not recapitulated. While induction of IL11 secretion was observed downstream of TGFß signaling in human lung fibroblasts and epithelial cells, the cellular responses induced by IL11 was quantitatively and qualitatively inferior to that of TGFß at the transcriptional and translational levels. IL11 blocking antibodies inhibited IL11Rα-proximal STAT3 activation but failed to block TGFß-induced profibrotic signals. In summary, our results challenge the concept of IL11 blockade as a strategy for providing transformative treatment for fibrosis.

Altered foot placement modulation with somatosensory stimulation in people with chronic stroke.

Many individuals who experience a stroke exhibit reduced modulation of their mediolateral foot placement, an important gait stabilization strategy. One factor that may contribute to this deficit is altered somatosensory processing, which can be probed by applying vibration to the involved muscles (e.g., the hip abductors). The purpose of this study was to investigate whether appropriately controlled hip abductor vibration can increase foot placement modulation among people with chronic stroke. 40 people with chronic stroke performed a series of treadmill walking trials without vibration and with vibration of either the hip abductors or lateral trunk (a control condition) that scaled with their real-time mediolateral motion. To assess participants' vibration sensitivity, we also measured vibration detection threshold and lateral sway evoked by abductor vibration during quiet standing. As a group, foot placement modulation increased significantly with either hip or trunk vibration, compared to without vibration. However, these changes were quite variable across participants, and were not predicted by either vibration detection threshold or the lateral sway evoked by hip vibration during standing. Overall, we found that somatosensory stimulation had small, positive effects on post-stroke foot placement modulation. Unexpectedly, these effects were observed with both hip abductor and lateral trunk vibration, perhaps indicating that the trunk can also provide useful somatosensory feedback during walking. Future work is needed to determine whether repeated application of such somatosensory stimulation can produce sustained effects on this important gait stabilization strategy.

Optimisation of virtual monoenergetic reconstructions for the diagnosis of pulmonary embolism using photon-counting detector computed tomography angiography.

Purpose: Computed tomography (CT) pulmonary angiography is considered the gold standard for pulmonary embolism (PE) diagnosis, relying on the discrimination between contrast and embolus. Photon-counting detector CT (PCD-CT) generates monoenergetic reconstructions through energy-resolved detection. Virtual monoenergetic images (VMI) at low keV can be used to improve pulmonary artery opacification. While studies have assessed VMI for PE diagnosis on dual-energy CT (DECT), there is a lack of literature on optimal settings for PCD-CT-PE reconstructions, warranting further investigation. Material and methods: Twenty-five sequential patients who underwent PCD-CT pulmonary angiography for suspicion of acute PE were retrospectively included in this study. Quantitative metrics including signal-to-noise ratio (SNR) and contrast-to-noise (CNR) ratio were calculated for 4 VMI values (40, 60, 80, and 100 keV). Qualitative measures of diagnostic quality were obtained for proximal to distal pulmonary artery branches by 2 cardiothoracic radiologists using a 5-point modified Likert scale. Results: SNR and CNR were highest for the 40 keV VMI (49.3 ± 22.2 and 48.2 ± 22.1, respectively) and were inversely related to monoenergetic keV. Qualitatively, 40 and 60 keV both exhibited excellent diagnostic quality (mean main pulmonary artery: 5.0 ± 0 and 5.0 ± 0; subsegmental pulmonary arteries 4.9 ± 0.1 and 4.9 ± 0.1, respectively) while distal segments at high (80-100) keVs had worse quality. Conclusions: 40 keV was the best individual VMI for the detection of pulmonary embolism by quantitative metrics. Qualitatively, 40-60 keV reconstructions may be used without a significant decrease in subjective quality. VMIs at higher keV lead to reduced opacification of the distal pulmonary arteries, resulting in decreased image quality.

Music, occupational, physical, and speech therapy interventions for patients in disorders of consciousness: An umbrella review.

BACKGROUND: Current clinical guidelines recommend that a multidisciplinary team inclusive of allied healthcare practitioners deliver assessment and intervention for disorders of consciousness. Allied health professionals include music, occupational, physical, and speech therapists. These allied health clinicians are challenged to select interventions due to a lack of evidence-based recommendations regarding rehabilitation interventions that support recovery of consciousness. This umbrella review synthesizes available systematic reviews (SRs) that describe occupational, speech and language, physical and/or musical therapeutic interventions for people with disorders of consciousness. OBJECTIVES: Identify and summarize evidence from systematic reviews (SRs) that examine allied healthcare interventions for patients with disorders of consciousness. Additionally, this umbrella review aims to evaluate the impact of allied health interventions on recovery of consciousness, methodological quality and risk of bias for the included systematic reviews. METHODS: An umbrella review was completed. The review was reported according to the Preferred Reporting Items for Overview of Reviews (PRIOR) guidance. Five academic databases (PubMed, CINAHL, PsycInfo, Web of Science, and the Cochrane Library) were searched for SRs and/or meta-analyses of allied health (i.e., music, occupational, physical, and speech therapy) interventions for disorders of consciousness. For included studies, data were extracted and quality of the SRs appraised using the A Measurement Tool to Assess Systematic Reviews (AMSTAR) 2 checklist. Data extracted from each SR identified the authors and years of primary studies, interventions, comparators, and outcomes related to recovery of consciousness (i.e., neurobehavioral/cognitive), functional status, physiological response pain, and adverse events. Rehabilitation interventions were categorized and described. RESULTS: Fifteen SRs were included and three of these reviews conducted meta-analyses. Identified rehabilitation interventions included: 1) sensory stimulation, 2) median nerve stimulation, 3) communication/environmental control through assistive technology, 4) mobilization, and 5) music-based therapy. SRs were published between 2002 and 2022 and included 2286 participants. Using the AMSTAR 2, the quality of reviews was critically low (k = 6), low (k = 3), moderate (k = 4), and high (k = 2). SRs within this umbrella review demonstrated significant heterogeneity in research methods and use of outcome measures to evaluate the recovery of consciousness within the primary studies. These factors influenced the ability to conduct meta-analyses. CONCLUSIONS: Sensory stimulation, median nerve stimulation, music therapy and mobilization are all interventions that demonstrate some level of benefit, but current SRs fail to prove benefit through high-level quality evidence. There is an indisputable need for continued rehabilitation research to expand options for treatment modalities and to ensure that the interventions being applied to DoC rehabilitation are evidence-based to improve consciousness and recovery.

Relationships between mediolateral step modulation and clinical balance measures in people with chronic stroke.

BACKGROUND: Many people with chronic stroke (PwCS) exhibit walking balance deficits linked to increased fall risk and decreased balance confidence. One potential contributor to these balance deficits is a decreased ability to modulate mediolateral stepping behavior based on pelvis motion. This behavior, hereby termed mediolateral step modulation, is thought to be an important balance strategy but can be disrupted in PwCS. RESEARCH QUESTION: Are biomechanical metrics of mediolateral step modulation related to common clinical balance measures among PwCS? METHODS: In this cross-sectional study, 93 PwCS walked on a treadmill at their self-selected speed for 3-minutes. We quantified mediolateral step modulation for both paretic and non-paretic steps by calculating partial correlations between mediolateral pelvis displacement at the start of each step and step width (ρSW), mediolateral foot placement relative to the pelvis (ρFP), and final mediolateral location of the pelvis (ρPD) at the end of the step. We also assessed several common clinical balance measures (Functional Gait Assessment [FGA], Activities-specific Balance Confidence scale [ABC], self-reported fear of falling and fall history). We performed Spearman correlations to relate each biomechanical metric of step modulation to FGA and ABC scores. We performed Wilcoxon rank sum tests to compare each biomechanical metric between individuals with and without a fear of falling and a history of falls. RESULTS: Only ρFP for paretic steps was significantly related to all four clinical balance measures; higher paretic ρFP values tended to be observed in participants with higher FGA scores, with higher ABC scores, without a fear of falling and without a history of falls. However, the strength of each of these relationships was only weak to moderate. SIGNIFICANCE: While the present results do not provide insight into causality, they justify future work investigating whether interventions designed to increase ρFP can improve clinical measures of post-stroke balance in parallel.

Modulation of neuronal activity in cortical organoids with bioelectronic delivery of ions and neurotransmitters.

Precise modulation of brain activity is fundamental for the proper establishment and maturation of the cerebral cortex. To this end, cortical organoids are promising tools to study circuit formation and the underpinnings of neurodevelopmental disease. However, the ability to manipulate neuronal activity with high temporal resolution in brain organoids remains limited. To overcome this challenge, we introduce a bioelectronic approach to control cortical organoid activity with the selective delivery of ions and neurotransmitters. Using this approach, we sequentially increased and decreased neuronal activity in brain organoids with the bioelectronic delivery of potassium ions (K+) and γ-aminobutyric acid (GABA), respectively, while simultaneously monitoring network activity. This works highlights bioelectronic ion pumps as tools for high-resolution temporal control of brain organoid activity toward precise pharmacological studies that can improve our understanding of neuronal function.

Enhancing diagnostic precision for acute chest syndrome in sickle cell disease: insights from dual-energy CT lung perfusion mapping.

PURPOSE: Acute chest syndrome (ACS) is secondary to occlusion of the pulmonary vasculature and a potentially life-threatening complication of sickle cell disease (SCD). Dual-energy CT (DECT) iodine perfusion map reconstructions can provide a method to visualize and quantify the extent of pulmonary microthrombi. METHODS: A total of 102 patients with sickle cell disease who underwent DECT CTPA with perfusion were retrospectively identified. The presence or absence of airspace opacities, segmental perfusion defects, and acute or chronic pulmonary emboli was noted. The number of segmental perfusion defects between patients with and without acute chest syndrome was compared. Sub-analyses were performed to investigate robustness. RESULTS: Of the 102 patients, 68 were clinically determined to not have ACS and 34 were determined to have ACS by clinical criteria. Of the patients with ACS, 82.4% were found to have perfusion defects with a median of 2 perfusion defects per patient. The presence of any or new perfusion defects was significantly associated with the diagnosis of ACS (P = 0.005 and < 0.001, respectively). Excluding patients with pulmonary embolism, 79% of patients with ACS had old or new perfusion defects, and the specificity for new perfusion defects was 87%, higher than consolidation/ground glass opacities (80%). CONCLUSION: DECT iodine map has the capability to depict microthrombi as perfusion defects. The presence of segmental perfusion defects on dual-energy CT maps was found to be associated with ACS with potential for improved specificity and reclassification.

Low-temperature and circadian signals are integrated by the sigma factor SIG5.

Chloroplasts are a common feature of plant cells and aspects of their metabolism, including photosynthesis, are influenced by low-temperature conditions. Chloroplasts contain a small circular genome that encodes essential components of the photosynthetic apparatus and chloroplast transcription/translation machinery. Here, we show that in Arabidopsis, a nuclear-encoded sigma factor that controls chloroplast transcription (SIGMA FACTOR5) contributes to adaptation to low-temperature conditions. This process involves the regulation of SIGMA FACTOR5 expression in response to cold by the bZIP transcription factors ELONGATED HYPOCOTYL5 and ELONGATED HYPOCOTYL5 HOMOLOG. The response of this pathway to cold is gated by the circadian clock, and it enhances photosynthetic efficiency during long-term cold and freezing exposure. We identify a process that integrates low-temperature and circadian signals, and modulates the response of chloroplasts to low-temperature conditions.

Cell wall fucosylation in Arabidopsis influences control of leaf water loss and alters stomatal development and mechanical properties.

The Arabidopsis sensitive-to-freezing8 (sfr8) mutant exhibits reduced cell wall (CW) fucose levels and compromised freezing tolerance. To examine whether CW fucosylation also affects the response to desiccation, we tested the effect of leaf excision in sfr8 and the allelic mutant mur1-1. Leaf water loss was strikingly higher than in the wild type in these, but not other, fucosylation mutants. We hypothesized that reduced fucosylation in guard cell (GC) walls might limit stomatal closure through altering mechanical properties. Multifrequency atomic force microscopy (AFM) measurements revealed a reduced elastic modulus (E'), representing reduced stiffness, in sfr8 GC walls. Interestingly, however, we discovered a compensatory mechanism whereby a concomitant reduction in the storage modulus (E'') maintained a wild-type viscoelastic time response (tau) in sfr8. Stomata in intact leaf discs of sfr8 responded normally to a closure stimulus, abscisic acid, suggesting that the time response may relate more to closure properties than stiffness does. sfr8 stomatal pore complexes were larger than those of the wild type, and GCs lacked a fully developed cuticular ledge, both potential contributors to the greater leaf water loss in sfr8. We present data that indicate that fucosylation-dependent dimerization of the CW pectic domain rhamnogalacturonan-II may be essential for normal cuticular ledge development and leaf water retention.

Imaging the Alternatively Spliced D Domain of Tenascin C in a Preclinical Model of Inflammatory Bowel Disease.

PURPOSE: To image colon-expressed alternatively spliced D domain of tenascin C in preclinical colitis models using near infrared (NIR)-labeled targeted molecular imaging agents. PROCEDURES: A human IgG1 with nanomolar binding affinity specific to the alternatively spliced D domain of tenascin C was generated. Immunohistochemistry identified disease-specific expression of this extracellular matrix protein in the colon of mice given dextran sulfate sodium in the drinking water. The antibody reagent was labeled with the NIR fluorophore IRDye 800CW via amine chemistry and intravenously dosed to evaluate in vivo targeting specificity. Increasing doses of imaging agent were given to estimate the saturating dose. RESULTS: The NIR-labeled proteins successfully targeted colonic lesions in a murine model of colitis. Co-administration of a molar excess competing unlabeled dose reduced normalized uptake in diseased colon by > 70%. Near infrared ex vivo images of colon resected from diseased animals showed saturation at doses exceeding 1 nmol and was confirmed with additional quantitative ex vivo biodistribution. Cellular-level specificity and protein stability were assessed via microscopy. CONCLUSIONS: Our imaging data suggest the alternatively spliced D domain of tenascin C is a promising target for delivery-based applications in inflammatory bowel diseases.

How Service Robots Can Improve Workplace Experience: Camaraderie, Customization, and Humans-in-the-Loop.

This paper presents the results of a three-week in-the-wild deployment of a wizarded service robot in a shared campus workplace. The study introduces robot-centric ethnography, a concept in which a wizarded robot acts as a mediated anthropologist, used in this case, to further our understandings of how service robots impact and integrate into everyday workplace experiences. Our research site included participants familiar with robots, recruited from 90+ students and faculty working in a shared lab space. Our wizarding team visited these participants each workday they were there for three weeks, navigating open office and lab spaces to remind participants to be aware of their mental, physical, and nutritional health needs. Using a semi-structured format, the wizards adapted the standard interaction flow to the situation. This interaction sequence was guided via pre-populated buttons on our health coach interface, with human wizards triggering the timing and adding extra responses as felt natural. Our ethnography-informed approach used the social knowledge of both participants and wizards, blending the robot into the cultural environment in which it was operating. Our data supports the positive impact of fluent service robot experience on participant mood and overall workplace experience. This suggests that effectively designed service robots can benefit workplace environments above and beyond their intended functions.

Fibronectin extra domain A as a drug delivery targeting epitope for rheumatoid arthritis.

OBJECTIVES: To assess the ability of monoclonal antibodies (mAbs) specific for fibronectin extra-domain A (FnEDA) to target diseased tissues of mouse collagen induced arthritis (mCIA) models. To explore the parameters of the targeting exhibited by anti-FnEDA mAbs including timing and location. METHODS: Targeting capabilities of anti-FnEDA mAbs were demonstrated by biodistribution study where i.v. injected antibodies were detected by conjugated near-infrared (NIR) fluorophore, 125I label and immunohistochemistry (IHC) of the injected antibody. Location of FnEDA expression in both mCIA and human RA tissue were mapped by IHC. Quantification of anti-FnEDA mAbs targeted to disease tissue was measured by whole-body autoradiography (WBA). Timing of the targeting was interrogated with fluorescent and confocal microscopy using anti-FnEDA mAbs labeled with different fluorophores and injected at different times. RESULTS: Anti-FnEDA mAbs show specific targeting to diseased paws of mCIA animal. The targeting was focused on inflamed synovium which is consistent with FnEDA expression profile in both mCIA and human RA tissues. Anti-FnEDA mAbs accumulated in diseased tissue at pharmacologically relevant concentrations, the targeting was sustained for up to 14 days and FnEDA was able to support targeting of multiple doses of anti-FnEDA mAbs given 5 days apart. CONCLUSION: FnEDA is specifically upregulated in the inflamed tissues of mCIA. Antibodies specific for FnEDA can be useful as molecular delivery vehicles for disease specific targeting of payloads to inflamed joint tissue.

Fibronectin extra domain A as a drug delivery targeting epitope for rheumatoid arthritis

Abstract Objectives: To assess the ability of monoclonal antibodies (mAbs) specific for fibronectin extra-domain A (FnEDA) to target diseased tissues of mouse collagen induced arthritis (mCIA) models. To explore the parameters of the targeting exhibited by anti-FnEDA mAbs including timing and location. Methods: Targeting capabilities of anti-FnEDA mAbs were demonstrated by biodistribution study where i.v. injected antibodies were detected by conjugated near-infrared (NIR) fluorophore, 125I label and immunohistochemistry (IHC) of the injected antibody. Location of FnEDA expression in both mCIA and human RA tissue were mapped by IHC. Quantification of anti-FnEDA mAbs targeted to disease tissue was measured by whole-body autoradiography (WBA). Timing of the targeting was interrogated with fluorescent and confocal microscopy using anti-FnEDA mAbs labeled with different fluorophores and injected at different times. Results: Anti-FnEDA mAbs show specific targeting to diseased paws of mCIA animal. The targeting was focused on inflamed synovium which is consistent with FnEDA expression profile in both mCIA and human RA tissues. Anti-FnEDA mAbs accumulated in diseased tissue at pharmacologically relevant concentrations, the targeting was sustained for up to 14 days and FnEDA was able to support targeting of multiple doses of anti-FnEDA mAbs given 5 days apart. Conclusion: FnEDA is specifically upregulated in the inflamed tissues of mCIA. Antibodies specific for FnEDA can be useful as molecular delivery vehicles for disease specific targeting of payloads to inflamed joint tissue.

Mediator Subunits MED16, MED14, and MED2 Are Required for Activation of ABRE-Dependent Transcription in Arabidopsis.

The Mediator complex controls transcription of most eukaryotic genes with individual subunits required for the control of particular gene regulons in response to various perturbations. In this study, we reveal the roles of the plant Mediator subunits MED16, MED14, and MED2 in regulating transcription in response to the phytohormone abscisic acid (ABA) and we determine which cis elements are under their control. Using synthetic promoter reporters we established an effective system for testing relationships between subunits and specific cis-acting motifs in protoplasts. Our results demonstrate that MED16, MED14, and MED2 are required for the full transcriptional activation by ABA of promoters containing both the ABRE (ABA-responsive element) and DRE (drought-responsive element). Using synthetic promoter motif concatamers, we showed that ABA-responsive activation of the ABRE but not the DRE motif was dependent on these three Mediator subunits. Furthermore, the three subunits were required for the control of water loss from leaves but played no role in ABA-dependent growth inhibition, highlighting specificity in their functions. Our results identify new roles for three Mediator subunits, provide a direct demonstration of their function and highlight that our experimental approach can be utilized to identify the function of subunits of plant transcriptional regulators.

Mechanisms and regulation of IL-22-mediated intestinal epithelial homeostasis and repair.

AIMS: Ulcerative colitis and Crohn's disease, collectively known as inflammatory bowel disease (IBD), are chronic inflammatory disorders of the intestine for which key elements in disease initiation and perpetuation are defects in epithelial barrier integrity. Achieving mucosal healing is essential to ameliorate disease outcome and so new therapies leading to epithelial homeostasis and repair are under investigation. This study was designed to determine the mechanisms by which IL-22 regulates intestinal epithelial cell function. MAIN METHODS: Human intestinal organoids and resections, as well as mice were used to evaluate the effect of IL-22 on stem cell expansion, proliferation and expression of mucus components. IL-22 effect on barrier function was assessed in polarized T-84 cell monolayers. Butyrate co-treatments and organoid co-cultures with immune cells were performed to monitor the impact of microbial-derived metabolites and inflammatory environments on IL-22 responses. KEY FINDINGS: IL-22 led to epithelial stem cell expansion, proliferation, barrier dysfunction and anti-microbial peptide production in human and mouse models evaluated. IL-22 also altered the mucus layer by inducing an increase in membrane mucus but a decrease in secreted mucus and goblet cell content. IL-22 had the same effect on anti-microbial peptides and membrane mucus in both healthy and IBD human samples. In contrast, this IL-22-associated epithelial phenotype was different when treatments were performed in presence of butyrate and organoids co-cultured with immune cells. SIGNIFICANCE: Our data indicate that IL-22 promotes epithelial regeneration, innate defense and membrane mucus production, strongly supporting the potential clinical utility of IL-22 as a mucosal healing therapy in IBD.

The calcium transporter ANNEXIN1 mediates cold-induced calcium signaling and freezing tolerance in plants.

The transient elevation of cytosolic free calcium concentration ([Ca2+ ]cyt ) induced by cold stress is a well-established phenomenon; however, the underlying mechanism remains elusive. Here, we report that the Ca2+ -permeable transporter ANNEXIN1 (AtANN1) mediates cold-triggered Ca2+ influx and freezing tolerance in Arabidopsis thaliana. The loss of function of AtANN1 substantially impaired freezing tolerance, reducing the cold-induced [Ca2+ ]cyt increase and upregulation of the cold-responsive CBF and COR genes. Further analysis showed that the OST1/SnRK2.6 kinase interacted with and phosphorylated AtANN1, which consequently enhanced its Ca2+ transport activity, thereby potentiating Ca2+ signaling. Consistent with these results and freezing sensitivity of ost1 mutants, the cold-induced [Ca2+ ]cyt elevation in the ost1-3 mutant was reduced. Genetic analysis indicated that AtANN1 acts downstream of OST1 in responses to cold stress. Our data thus uncover a cascade linking OST1-AtANN1 to cold-induced Ca2+ signal generation, which activates the cold response and consequently enhances freezing tolerance in Arabidopsis.

Optimized Culture Conditions for Improved Growth and Functional Differentiation of Mouse and Human Colon Organoids.

Background & Aims: Diligent side-by-side comparisons of how different methodologies affect growth efficiency and quality of intestinal colonoids have not been performed leaving a gap in our current knowledge. Here, we summarize our efforts to optimize culture conditions for improved growth and functional differentiation of mouse and human colon organoids. Methods: Mouse and human colon organoids were grown in four different media. Media-dependent long-term growth was measured by quantifying surviving organoids via imaging and a cell viability readout over five passages. The impact of diverse media on differentiation was assessed by quantifying the number of epithelial cell types using markers for enterocytes, stem cells, Goblet cells, and enteroendocrine cells by qPCR and histology upon removal of growth factors. Results: In contrast to Wnt3a-conditioned media, media supplemented with recombinant Wnt3a alone did not support long-term survival of human or mouse colon organoids. Mechanistically, this observation can be attributed to the fact that recombinant Wnt3a did not support stem cell survival or proliferation as demonstrated by decreased LGR5 and Ki67 expression. When monitoring expression of markers for epithelial cell types, the highest level of organoid differentiation was observed after combined removal of Wnt3a, Noggin, and R-spondin from Wnta3a-conditioned media cultures. Conclusion: Our study defined Wnt3a-containing conditioned media as optimal for growth and survival of human and mouse organoids. Furthermore, we established that the combined removal of Wnt3a, Noggin, and R-spondin results in optimal differentiation. This study provides a step forward in optimizing conditions for intestinal organoid growth to improve standardization and reproducibility of this model platform.

MUR1-mediated cell-wall fucosylation is required for freezing tolerance in Arabidopsis thaliana.

Forward genetic screens play a key role in the identification of genes contributing to plant stress tolerance. Using a screen for freezing sensitivity, we have identified a novel freezing tolerance gene, SENSITIVE-TO-FREEZING8, in Arabidopsis thaliana. We identified SFR8 using recombination-based mapping and whole-genome sequencing. As SFR8 was predicted to have an effect on cell wall composition, we used GC-MS and polyacrylamide gel electrophoresis to measure cell-wall fucose and boron (B)-dependent dimerization of the cell-wall pectic domain rhamnogalacturonan II (RGII) in planta. After treatments to promote borate-bridging of RGII, we assessed freeze-induced damage in wild-type and sfr8 plants by measuring electrolyte leakage from freeze-thawed leaf discs. We mapped the sfr8 mutation to MUR1, a gene encoding the fucose biosynthetic enzyme GDP-d-mannose-4,6-dehydratase. sfr8 cell walls exhibited low cell-wall fucose levels and reduced RGII bridging. Freezing sensitivity of sfr8 mutants was ameliorated by B supplementation, which can restore RGII dimerization. B transport mutants with reduced RGII dimerization were also freezing-sensitive. Our research identifies a role for the structure and composition of the plant primary cell wall in determining basal plant freezing tolerance and highlights the specific importance of fucosylation, most likely through its effect on the ability of RGII pectin to dimerize.