Wild-type Controls Research Articles

Disruption of the OsWRKY71 transcription factor gene results in early rice seed germination under normal and cold stress conditions

Abstract Background Early seed germination in crops can confer a competitive advantage against weeds and reduce the time to maturation and harvest. WRKY transcription factors regulate many aspects of plant development including seed dormancy and germination. Both positive and negative regulators of seed germination have been reported in many plants such as rice and Arabidopsis. Using a transient expression system, we previously demonstrated that OsWRKY71 is a negative regulator of gibberellin (GA) signaling in aleurone cells and likely forms a “repressosome” complex with other transcriptional repressors. Hence, it has the potential to impact seed germination properties. Results In this study, we demonstrate that OsWRKY71, a Group IIa WRKY gene, appeared at the same time as seed-bearing plants. Rice mutants lacking OsWRKY71 have seeds and embryos that germinate earlier than wildtype controls. In oswrky71 aleurone layers, α-amylase activity was hypersensitive to stimulation by GA3 and hyposensitive to inhibition by abscisic acid (ABA). Early germination in oswrky71 intact seeds was also hyposensitive to ABA. Transcriptomic profiling during embryo germination and early post-germination growth demonstrates that OsWRKY71 influences the expression of 9–17% of genes in dry and imbibing embryos. Compared to wildtype embryos, the mutant transcriptomes have large temporal shifts at 4, 8 and 12 h after imbibition (HAI). Importantly, many genes involved in the ABA-dependent inhibition of seed germination were downregulated in oswrky71-1. This mutant also displayed altered expression of multiple ABA receptors (OsPYLs/RCARs) that control ABA signaling and the VP1-SDR4-DOG1L branch of ABA signaling that promotes seed dormancy. Association studies reveal an OsWRKY71-containing quantitative trait locus involved in low-temperature seed germinability, qLTG-2. Indeed, oswrky71 seeds germinated early at 15 °C. Conclusions Rice Group-IIa WRKY transcription factor OsWRKY71 is a master regulator of germination that influences the expression of 9–17% of genes in dry and imbibing embryos. It is also most likely the primary candidate of low-temperature seed germinability QTL, qLTG-2. We propose that knockouts of OsWRKY71 can generate rice varieties with improved germination properties under normal or low-temperature conditions.

Abstract B027: ISG15 modulate cancer cell mechanics and metastasis in breast cancer

Abstract Metastasis in breast cancer is a major challenge for prognosis and effective treatment, primarily because cancer cells can create an immune-suppressive microenvironment that supports their progression and spread. The physical properties of cancer cells significantly influence their metastatic potential and their interaction with the environment. Identifying targets that impact both the physical characteristics of cancer cells, and their microenvironment is crucial for tackling metastatic disease. Interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein, is often overexpressed in breast cancer tissues and is associated with poor prognosis, higher tumor grade, and increased invasiveness. Despite these associations, the precise role of ISG15 and its post-translational modification, ISGylation, in cancer cell behavior and interactions with their microenvironment remains unclear. To explore this, we interrogated the biophysical role of ISG15 by using mouse modeling and in vitro loss of function approaches. We utilized whole- body ISG15-deficient mice and conducted a series of experiments to assess the impact of ISG15 on cancer cell properties and their surrounding environment. Our preliminary results indicate that cells lacking ISG15 exhibit altered physical traits, such as increased stiffness and size, which may influence their metastatic behavior. Additionally, targeting ISG15 or disrupting ISGylation enhances the susceptibility of cancer cells to immune cell-mediated destruction. In ISG15- deficient mice, we observed significantly reduced tumor growth and metastasis compared to wild-type controls. These findings suggest that ISG15 modulates critical aspects of cancer cell behavior and their interactions with the microenvironment, contributing to immune evasion and metastatic spread. Our research highlights ISG15 as a promising therapeutic target and provides a foundation for developing new strategies to prevent and treat metastatic cancer Citation Format: Mohamed Haloul, Priya Shah, Vinay Pai, Faruk Hossen, James James Lee, Ekrem Emrah Er. ISG15 modulate cancer cell mechanics and metastasis in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B027.

Targeted TGF-βR2 Silencing in the Retrotrapezoid Nucleus Mitigates Respiratory Dysfunction and Cognitive Decline in a Mouse Model of Cerebral Amyloid Angiopathy with and without Stroke.

Cerebral amyloid angiopathy (CAA) is characterized by the deposition of amyloid-beta peptides within cerebral blood vessels, leading to neurovascular complications. Ischemic strokes result from acute disruptions in cerebral blood flow, triggering metabolic disturbances and neurodegeneration. Both conditions often co-occur and are associated with respiratory dysfunctions. The retrotrapezoid nucleus (RTN), which is crucial for CO2 sensing and breathing regulation in the brainstem, may play a key role in breathing disorders seen in these conditions. This study aims to investigate the role of Transforming Growth Factor Beta (TGF-β) signaling in the RTN on respiratory and cognitive functions in CAA, both with and without concurrent ischemic stroke.Adult male Tg-SwDI (CAA model) mice and C57BL/6 wild-type controls underwent stereotaxic injections of lentivirus targeting TGF-βR2 in the RTN. Stroke was induced by middle cerebral artery occlusion using a monofilament. Respiratory functions were assessed using whole-body plethysmography, while cognitive functions were evaluated through the Barnes Maze and Novel Object Recognition Test (NORT). Immunohistochemical analysis was conducted to measure TGF-βR2 and GFAP expressions in the RTN.CAA mice exhibited significant respiratory dysfunctions, including reduced respiratory rates and increased apnea frequency, as well as impaired cognitive performance. TGF-βR2 silencing in the RTN improved respiratory functions and cognitive outcomes in CAA mice. In CAA mice with concurrent stroke, TGF-βR2 silencing similarly enhanced respiratory and cognitive functions. Immunohistochemistry confirmed reduced TGF-βR2 and GFAP expressions in the RTN following silencing.Our findings demonstrate that increased TGF-β signaling and gliosis in the RTN contribute to respiratory and cognitive dysfunctions in CAA and CAA with stroke. Targeting TGF-βR2 signaling in the RTN offers a promising therapeutic strategy to mitigate these impairments. This study is the first to report a causal link between brainstem gliosis and both respiratory and cognitive dysfunctions in CAA and stroke models.

Evaluation of a six-minute walk test in the DE50-MD canine model of Duchenne muscular dystrophy and its effect on blood-borne biomarkers

Background Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by mutations in the dystrophin gene resulting in cycles of muscle degeneration, inflammation and regeneration. The 6-minute walk test (6MWT) is a key functional outcome measure for DMD patient clinical trials and has been adapted for use in animal models of the disease. The DE50-MD dog model of DMD closely reflects the DMD patient phenotype prior to loss of ambulation. For pre-clinical trials using this model, functional outcome measures must be established. Methods This longitudinal study compared distance walked in a 6MWT by DE50-MD and WT control dogs and assessed the utility of the 6MWT as a functional biomarker. Dogs underwent two 6MWTs conducted approximately 48-hours apart, at 3, 6, 9, 12, 15 and 18 months of age. In addition, we evaluated the stability of selected blood-borne biomarkers in 12-month old DE50-MD and WT dogs 0, 3, 6, 24 and 48 hours following a 6MWT. Results DE50-MD dogs exhibited significantly shorter 6-minute walk distance (6MWD) than WT dogs at all timepoints (P<0.05), with no difference in 6MWD between the first and second 6MWT. C-C motif chemokine ligand 2 (CCL2), myomesin-3 (MYOM3) and myostatin (MSTN) were biomarkers of the DE50-MD phenotype that remained unchanged in DE50-MD dogs following the 6MWT, while creatine kinase (CK) activity significantly increased 3-hours following the test in DE50-MD dogs but remained unchanged in WT dogs. Conclusions The 6MWT effectively discriminates DE50-MD from WT dogs aged 3-18 months and a single 6MWT is sufficient for future studies. Serum MYOM3, CCL2 and MSTN are good biomarkers of the DE50-MD phenotype that are unaffected by this relatively low level exertion.

Double-stranded RNAs targeting Sphaerulina musiva silence DCL but not CYP51 in vitro, and fail to confer resistance to canker in transgenic poplar

Host-induced gene silencing (HIGS) is a common method for engineering plant protection against pathogens, although success requires double-stranded (dsRNA) uptake mechanisms that may not be present in all fungi. We explored HIGS in transgenic poplar to study and control Sphaerulina musiva, the cause of Septoria stem canker disease. HIGS transgenic poplars expressing dsRNA that targeted either or both S. musiva CYP51 and DCL were developed and screened for resistance to stem canker disease in two greenhouse inoculation trials. While differences in resistance between transgenic lines and wild-type controls were not detected, there was a correlation between greenhouse-expressed disease resistance and transgene expression among HIGS lines targeting S. musiva DCL. To evaluate the likelihood that HIGS or spray-induced gene silencing might be effective under some conditions, concurrent with greenhouse screening we studied: 1) S. musiva’s capacity for uptake of environmental dsRNA; 2) effects of in vitro silencing of CYP51 and DCL on fungal growth and target transcript abundance; and 3) persistence of dsRNA in culture. Uptake of fluorescently tagged dsRNA was not detected with confocal imaging. In dsRNA-treated cultures, fungal growth inhibition was not detected, and RNA was rapidly degraded. Of the five target transcripts tested after dsRNA treatment, only DCL1 had reduced expression. Knockdown of DCL1 along with the enhanced resistance among high-expressing HIGS events targeting DCL suggests some HIGS may have been observed. Further determination of the factors limiting dsRNA uptake by S. musiva are needed to determine if HIGS can be an effective technology for limiting stem canker.

Complete inhibition of liver acetyl-CoA carboxylase activity is required to exacerbate liver tumorigenesis in mice treated with diethylnitrosamine.

The metabolic pathway of de novo lipogenesis (DNL) is upregulated in fatty liver disease and liver cancer. Inhibitors of DNL are in development for the treatment of these disorders; however, our previous study showed that blocking DNL unexpectedly exacerbated liver tumorigenesis when liver acetyl-CoA carboxylase (ACC) 1 and 2 enzymes were deleted in mice treated with diethylnitrosamine (DEN) and fed high fat diet. Herein, we used 3 new approaches including ACC1 vs. ACC2 isotype-selective inhibition, delaying ACC inhibition until after carcinogen treatment, and feeding mice normal chow diet to better understand the impact of ACC inhibition on liver tumorigenesis. Six genotypes of female C57BL/6J mice with floxed ACC1 and/or ACC2 alleles were injected with DEN at 2 weeks of age followed by liver-specific knockout of ACC genes at 9 weeks. Mice were fed a normal chow diet and evaluated at 52 weeks for liver tumours. Compared to the wildtype control group, no genotype decreased tumour multiplicity or burden; however, mice completely lacking liver ACC1 and ACC2 had > 5-fold increases in liver tumour multiplicity and burden. ACC inhibition exacerbated DEN-induced liver tumorigenesis only when both ACC isotypes were completely inhibited. The pro-tumour phenotype of ACC inhibition was strongly reproducible irrespective of chow or high fat feeding, and irrespective of ACC inhibition prior to or after DEN treatment. Retaining partial ACC activity at either isotype prevented tumour exacerbation in mice at risk for developing liver tumours.

Determination of qPCR reference genes suitable for normalizing gene expression in a novel model of Duchenne muscular dystrophy, the D2-mdx mouse.

Duchenne muscular dystrophy (DMD) is a X-linked neuromuscular disorder arising from mutations in the dystrophin gene, leading to a progressive muscle wasting and disability. Currently there is no universal therapy, and there is thus a strong interest in preclinical studies for finding novel treatments. The most widely used and characterized mouse model for DMD is the C57BL/10ScSn-Dmdmdx/J (BL10-mdx), but this model exhibits mild pathology and does not replicate key features of human disease. The D2.B10-Dmdmdx/J (D2-mdx) mouse is a more recent model which seems to better mimics the complex human DMD phenotype. However, the D2-mdx mouse remains less extensively characterised than its BL10-mdx counterpart. Quantitative PCR analysis of gene expression is an important tool to monitor disease progression and evaluate therapeutic efficacy, but measurements must be normalised to stably expressed reference genes, which should ideally be determined and validated empirically. We examined gene expression in the gastrocnemius (GC), diaphragm (DIA) and heart in the D2-mdx mouse, the BL10-mdx mouse, and appropriate strain-matched wild-type controls (D2-wt and BL10-wt), from 4 to 52 weeks of age, using a large panel of candidate references (ACTB, AP3D1, CSNK2A2, GAPDH, HPRT1, PAK1IP1, RPL13A, SDHA, and in the heart, also HTATSF1 and HMBS). Data was analyzed using GeNorm, Bestkeeper, deltaCt and Normfinder algorithms to identify stable references under multiple possible scenarios. We show that CSNK2A2, AP3D1 and ACTB represent strong universal reference genes in both GC and DIA, regardless of age, muscle type, strain and genotype, while HTATSF1 and SDHA are optimal for the heart. GAPDH, HPRT1 and RPL13A were conversely revealed to be poor references, showing tissue-, age- or disease-specific changes in expression. Our results illustrate the importance of determining appropriate reference genes for specific comparative scenarios, but also reconfirm that universal panels can nevertheless be identified for normalising gene expression studies in even complex pathological states.

Triple-Gene Overexpression of the AcrA-AcrB-TolC Transporter System in Synechocystis sp. PCC 6803 Contributes to a Higher Secretion of Free Fatty Acids in Response to Nitrogen Shortage and Salt Stress

One important aspect of cyanobacterial homoeostasis is reducing the toxicity of excess free fatty acids (FFAs) generated in the cells by means of both secreting these into the medium and recycling them toward membrane lipid synthesis. In this study, the cyanobacterium Synechocystis sp. PCC 6803 served to implement the overexpression of native genes of the transportation system. Specifically, we worked with the Sll0180-Slr2131-Slr1270 homologs of Escherichia coli AcrA-AcrB-TolC, respectively, to create single- and triple-overexpressing strains of OA, OB, OC, and OABC. Remarkably, the OABC strain that triply overexpressed the sll0180_slr2131_slr1270 genes acquired a significant amount of intracellular lipids, up to 23.5% of dry cell weight, under the normal condition. Nitrogen-deficient stress undoubtedly raised extracellular FFAs and intracellular lipids in overexpressing strains, especially in the OABC strain, which exhibited 33.9% and 41.5% of dry cell weight, respectively. During the first 5 days of treatment, salt stress at 256 mM significantly increased the FFA efflux, notably for the OB strain, but had no effect on intracellular lipids. It is noteworthy that the OA and OABC strains outperformed all other strains in terms of growth throughout the 16 days of nitrogen shortage. Furthermore, in comparison to the wild-type control, all the overexpressing strains exhibited a considerable increase in carotenoid accumulation. Thus, our results point to the effective role of the sll0180_slr2131_slr1270 transportation system in facilitating FFA secretion, especially in response to environmental stressors.

MurA-catalyzed synthesis of 5-enolpyruvylshikimate-3-phosphate confers glyphosate tolerance in bryophytes

Glyphosate is a broad-spectrum herbicide that kills most vascular plant weeds but is ineffective against many bryophytes. Glyphosate competitively inhibits the enolpyruvyl transferase enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS catalyzes the production of 5-enolpyruvylshikimate-3-phosphate (EPSP)-an intermediate in the shikimate pathway-from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) substrates. Here, we show that Marchantia polymorpha mutants with loss-of-function mutations in a closely related enolpyruvyl transferase, MpMurA, were more sensitive to glyphosate than wild-type controls. Overexpression of MpMurA in M. polymorpha increased glyphosate tolerance, and heterologous expression of the M. polymorpha MurA enzyme in Arabidopsis thaliana conferred glyphosate resistance. Furthermore, we demonstrate that MpMurA catalyzes the production of EPSP from S3P and PEP substrates. These data demonstrate that MpMurA contributes to glyphosate tolerance in M. polymorpha. We speculate that the existence of two independent mechanisms for EPSP synthesis-one canonical and EPSPS-dependent, and the other MurA-dependent-may account for glyphosate tolerance in bryophytes. Alternatively, MurA in bryophytes may bind glyphosate, thereby leaving more unbound EPSPS enzymes available, allowing aromatic amino acid biosynthesis to continue.

Regulatory T cells require peripheral CCL2-CCR2 signaling to facilitate the resolution of medication overuse headache-related behavioral sensitization.

Medication overuse headache (MOH) is the most common secondary headache disorder, resulting from chronic and excessive use of medication to treat headaches, for example, sumatriptan. In a recent study, we have shown that the peripheral C-C motif ligand 2 (CCL2), C-C motif chemokine receptor 2 (CCR2) and calcitonin-gene-related peptide (CGRP) signaling pathways interact with each other and play critical roles in the development of chronic migraine-related behavioral and cellular sensitization. In the present study, we investigated whether CCL2-CCR2 and CGRP signaling pathways play a role in the development of sumatriptan overuse-induced sensitization, and whether they are involved in its resolution by the low-dose interleukin-2 (LD-IL-2) treatment. Mice received daily sumatriptan administration for 12 days. MOH-related behavioral sensitization was assessed by measuring changes of periorbital mechanical thresholds for 3 weeks. CCL2-CCR2 and CGRP signaling pathways were inhibited by targeted gene deletion or with an anti-CCL2 antibody. Ca2+-imaging was used to examine whether repetitive sumatriptan treatment enhances CGRP and pituitary adenylate cyclase-activating polypeptide (PACAP) signaling in trigeminal ganglion (TG) neurons. LD-IL-2 treatment was initiated after the establishment of sumatriptan-induced sensitization. Immunohistochemistry and flow cytometry analyses were used to examine whether CCL2-CCR2 signaling controls regulatory T (Treg) cell proliferation and/or trafficking. CCL2, CCR2 and CGRPα global KO mice exhibited robust sumatriptan-induced behavioral sensitization comparable to wild-type controls. Antibody neutralization of peripheral CCL2 did not affect sumatriptan-induced behaviors either. Repeated sumatriptan administration did not enhance the strength of CGRP or PACAP signaling in TG neurons. Nevertheless, LD-IL-2 treatment, which facilitated the resolution of sumatriptan-induced sensitization in wild-type and CGRPα KO mice, was completely ineffective in mice with compromised CCL2-CCR2 signaling. In CCL2 KO mice, we observed normal LD-IL-2-induced Treg expansion in peripheral blood, but the increase of Treg cells in dura and TG tissues was significantly reduced in LD-IL-2-treated CCL2 KO mice relative to wild-type controls. These results indicate that the endogenous CCL2-CCR2 and CGRP signaling pathways are not involved in sumatriptan-induced behavioral sensitization, suggesting that distinct molecular mechanisms underlie chronic migraine and MOH. On the other hand, peripheral CCL2-CCR2 signaling is required for LD-IL-2 to reverse chronic headache-related sensitization.

Alterations in Zinc, Copper, and Iron Levels in the Retina and Brain of Alzheimer's Disease Patients and the APP/PS1 Mouse Model.

Transition metals like copper, iron, and zinc are vital for normal central nervous system function and are also linked to neurodegeneration, particularly in the onset and progression of Alzheimer's disease (AD). Their alterations in AD, identified prior to amyloid plaque aggregation, offer a unique target for staging pre-amyloid AD. However, analysing their levels in the brain is extremely challenging, necessitating the development of alternative approaches. Here, we utilised laser ablation-inductively coupled plasma-mass spectrometry and solution nebulisation-inductively coupled plasma-mass spectrometry to quantitatively measure Cu, Fe, and Zn concentrations in the retina and hippocampus samples obtained from human donors (i.e., AD and healthy controls), and in the APP/PS1 mouse model of AD, and Wild Type controls, aged 9 and 18 months. Our findings revealed significantly elevated Cu, Fe, and Zn levels in the retina (*p < 0.05, **p < 0.01, ***p < 0.001) and hippocampus (*p < 0.05, *p < 0.05, *p < 0.05) of human AD samples compared to healthy controls. Conversely, APP/PS1 mouse models exhibited notably lower metal levels in the same regions compared to WT mice, Cu, Fe, and Zn levels in the retina (**p < 0.01, *p < 0.05, *p < 0.05) and hippocampus (**p < 0.01, **p < 0.01, *p < 0.05). The contrasting metal profiles in human and mouse samples, yet similar patterns within each species' retina and brain, suggest the retina mirrors cerebral metal dyshomeostasis in AD. Our findings lay the groundwork for staging pre-AD pathophysiology through assessment of transition metal levels in the retina.

Klrb1 Loss Promotes Chronic Hepatic Inflammation and Metabolic Dysregulation

Background/Objectives: CD161, encoded by the KLRB1 gene, is an inhibitory receptor expresses on various immune cell and has gained attention in immune checkpoint research. In recent studies, KLRB1 has been found to be one of the potential markers of liver diseases such as cirrhosis. Therefore, it will be important to understand what process KLRB1 involved in the liver for the prevention of liver diseases. Methods: We compared KO mice with wild-type controls by routine blood analysis and RNA-seq, and additionally performed H&amp;E staining and qPCR to validate the differentially expressed genes (DEGs). Results:KO mice had fewer lymphocytes compared to the wild-type mice. A transcriptomic analysis showed that Klrb1 loss causes the upregulation of immune-related genes and pathways like NOD-like receptor and p53 signaling, while causing the downregulation of lipid metabolism-related genes. A protein interaction analysis indicated a potential cancer risk under chronic inflammation. Histological examination with H&amp;E staining reveals an inflammatory response around the central venous vessels in the liver tissue of the KO mice. Conclusions: We conclude that Klrb1 knockout disrupts the immune and metabolic functions in the liver, which may possibly lead to chronic inflammation and malignancy risks. These findings highlight the role of Klrb1 in hepatic health.

Enhanced Gαq Signaling in TSC2-deficient Cells Is Required for Their Neoplastic Behavior.

Inherited or sporadic loss of the TSC2 gene can lead to pulmonary lymphangioleiomyomatosis (LAM), a rare cystic lung disease caused by protease-secreting interstitial tumor nodules. The nodules arise by metastasis of cells that exhibit features of neural crest and smooth muscle lineage ('LAM cells'). Their aberrant growth is attributed to increased activity of 'mechanistic target of rapamycin complex 1' (mTORC1), an anabolic protein kinase that is normally suppressed by the TSC1-TSC2 protein complex. The mTORC1 inhibitor rapamycin slows the progression of LAM, but fails to eradicate disease, indicating a role for mTORC1-independent mechanisms in LAM pathogenesis. Our previous studies revealed G-protein coupled urotensin-II receptor (UT) signaling as a candidate mechanism, but how it promotes oncogenic signaling in TSC2-deficient cells remained unknown. Using a human pluripotent stem cell-derived in vitro model of LAM, we now show hyperactivation of UT, which was required for their enhanced migration and pro-neoplastic signaling in a rapamycin-insensitive mechanism that required heterotrimeric Gαq/11 (Gαq). Bioluminescence resonance energy transfer assays in HEK 293T cells lacking TSC2 demonstrated selective and enhanced activation of Gαq and its RhoA-associated effectors compared to wild-type control cells. By immunoprecipitation, recombinant UT was physically associated with Gαq and TSC2. The augmented Gαq signaling in TSC2-deleted cells was independent of mTOR activity, and associated with increased endosomal targeting of p63RhoGEF, a known RhoA-activating effector of Gαq. These studies identify potential mTORC1-independent pro-neoplastic mechanisms that can be targeted for prevention or eradication of pulmonary and extrapulmonary LAM tumors.

Seasonal Variation in ATP-Induced Retinal Damage in the Cone-Dominant 13-Lined Ground Squirrel.

To examine whether time of year (relative to hibernation emergence) influences the retinal degenerative effects of intravitreal injection of adenosine triphosphate (ATP) in the 13-lined ground squirrel (13-LGS). Eighteen (9 male, 9 female) 13-LGS in three experimental cohorts (early season, mid-season, late season) (n = 6 each) underwent baseline imaging using scanning light ophthalmoscopy (SLO) and optical coherence tomography (OCT). Animals then received a 10-µL intravitreal injection of 0.723 M ATP, followed by OCT and SLO imaging at 3, 10, and 21 days. Adaptive optics SLO (AOSLO) was performed in animals without retinal damage after the 21-day follow-up. Retinal thickness, choroidal thickness, and cone density measures were compared to values from wild-type controls (n = 12). Five animals (four early season, one late season) showed retinal damage post-ATP injection (Fisher's exact test, P = 0.065). Animals with retinal damage displayed areas of disrupted retinal lamination on OCT. Any changes in OCT thickness were generally present on initial follow-up and resolved at later time points. Follow-up imaging with AOSLO on animals without retinal damage showed no significant differences in the cone mosaic topography from control eyes. Axial length was increased in mid-/late-season cohorts relative to early season (P = 0.0025 and P = 0.0007). In this pilot study, the 13-LGS appears more susceptible to ATP-induced retinal damage during the early season. Future studies adjusting dose based on ocular biometry may help elucidate the impact of time of year on chemical response. Consideration of ocular biometry in this and other animal models is merited when using intravitreal methods of chemical administration.

Identifying the bioimaging features of Alzheimer’s disease based on pupillary light response-driven brain-wide fMRI in awake mice

Pupil dynamics has emerged as a critical non-invasive indicator of brain state changes. In particular, pupillary-light-responses (PLR) in Alzheimer’s disease (AD) patients show potential as biomarkers for brain degeneration. To investigate AD-specific PLR and its underlying neuromodulatory sources, we combine high-resolution awake mouse fMRI with real-time pupillometry to map brain-wide event-related correlation patterns based on illumination-driven pupil constriction (Pc\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${P}_{c}$$\\end{document}) and post-illumination pupil dilation recovery (amplitude, Pd\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${P}_{d}$$\\end{document}, and time, T). The Pc\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${P}_{c}$$\\end{document}-driven differential analysis reveals altered visual signal processing and reduced thalamocortical activation in AD mice in comparison with wild-type (WT) control mice. In contrast, the post-illumination pupil dilation recovery-based fMRI highlights multiple brain areas associated with AD brain degeneration, including the cingulate cortex, hippocampus, septal area of the basal forebrain, medial raphe nucleus, and pontine reticular nuclei (PRN). Additionally, the brain-wide functional connectivity analysis highlights the most significant changes in PRN of AD mice, which serves as the major subcortical relay nuclei underlying oculomotor function. This work integrates non-invasive pupil-fMRI measurements in preclinical models to identify pupillary biomarkers based on brain-wide functional changes, including neuromodulatory dysfunction coupled with AD brain degeneration.

Suppressing neutrophil itaconate production attenuates Mycoplasma pneumoniae pneumonia.

Mycoplasma pneumoniae is a common cause of community-acquired pneumonia in which neutrophils play a critical role. Immune-responsive gene 1 (IRG1), responsible for itaconate production, has emerged as an important regulator of inflammation and infection, but its role during M. pneumoniae infection remains unknown. Here, we reveal that itaconate is an endogenous pro-inflammatory metabolite during M. pneumoniae infection. Irg1 knockout (KO) mice had lower levels of bacterial burden, lactate dehydrogenase (LDH), and pro-inflammatory cytokines compared with wild-type (WT) controls after M. pneumoniae infection. Neutrophils were the major cells producing itaconate during M. pneumoniae infection in mice. Neutrophil counts were positively correlated with itaconate concentrations in bronchoalveolar lavage fluid (BALF) of patients with severe M. pneumoniae pneumonia. Adoptive transfer of Irg1 KO neutrophils, or administration of β-glucan (an inhibitor of Irg1 expression), significantly attenuated M. pneumoniae pneumonia in mice. Mechanistically, itaconate impaired neutrophil bacterial killing and suppressed neutrophil apoptosis via inhibiting mitochondrial ROS. Moreover, M. pneumoniae induced Irg1 expression by activating NF-κB and STAT1 pathways involving TLR2. Our data thus identify Irg1/itaconate pathway as a potential therapeutic target for the treatment of M. pneumoniae pneumonia.

A seed-specific DNA-binding with One Finger transcription factor, RPBF, positively regulates galactinol synthase to maintain seed vigour in rice.

Seed vigour and longevity are intricate yet indispensable physiological traits for agricultural crops, as they play a crucial role in facilitating the successful emergence of seedlings and exert a substantial influence on crop productivity. Transcriptional regulation plays an important role in seed development, maturation, and desiccation tolerance, which are important attributes for seed vigour and longevity. Here, we have investigated the regulatory role of the seed-specific DNA binding with the One Finger (DOF) transcription factor and the RPBF (Rice P-box Binding Factor) in seed vigour. RPBF modulates the transcription of galactinol synthase and improves seed vigour. The promoter region of Galactinol synthase (GolS)-encoding genes from different species was enriched with DOF binding sites, and the expression levels of both RPBF and OsGolS were found to enhance during seed development. Furthermore, direct interaction of RPBF with OsGolS promoter has been demonstrated through multiple approaches: yeast one-hybrid (Y1H) assays, in planta promoter-GUS assays, dual luciferase assay, and in silico molecular docking. To assess functionality, Agrobacterium-mediated genetic transformation of rice was performed to generate the RNAi lines with reduced RPBF expression. In these RNAi lines, a reduction in both galactinol and raffinose content was observed. Since galactinol and raffinose are known contributors to seed vigour, the T2-transgenic lines were assessed for vigour and viability. For this, RNAi seeds were subjected to accelerated ageing by exposing them to high relative humidity and temperature, followed by scoring the germination and viability potential. Tetrazolium and seed germination assay revealed that the RNAi seeds were more sensitive to ageing compared to their wild-type and vector control counterparts. Collectively, this is the first report demonstrating that the DOF transcription factor RPBF controls the seed vigour through transcriptional regulation of galactinol synthase.

Overexpression of tae-miR9670 enhances cadmium tolerance in wheat by targeting mTERFs without yield penalty

Cadmium (Cd) is a widely distributed heavy metal that poses significant hazards to both crop productivity and human health. MicroRNAs (miRNAs) play pivotal roles in plant growth, development and responses to environmental stresses, yet little is known about their roles in regulating Cd tolerance in wheat. In this study, we identified tae-miR9670, a Triticeae-specific miRNA, as responsive to Cd exposure in wheat through miRNAome analysis. Tae-miR9670 can target genes that encode mitochondrial transcription termination factors (mTERFs), mediating their mRNA cleavage and suppressing their expression. Overexpression of tae-miR9670 significantly enhanced Cd tolerance in wheat seedlings, as demonstrated by increased biomass and reduced levels of malondialdehyde (MDA), H2O2, and Cd content. Consequently, multiple downstream genes involved in ROS scavenging, detoxification and heavy metal transport were upregulated in tae-miR9670 overexpression plants. Moreover, the grain Cd content in mature plants overexpressing tae-miR9670 was reduced by over 60% compared to wild-type controls. Our results also indicated that overexpressing tae-miR9670 in wheat preserved yield-related traits, thereby overcoming the trade-off between stress resistance and grain yield. Overall, our findings provide new insights into the role of tae-miR9670 in Cd tolerance in wheat and its potential application in breeding low-Cd cultivars.

The methyltransferase MLL4 promotes non-alcoholic steatohepatitis by enhancing NF-κB signaling.

Non-alcoholic fatty liver disease (NAFLD) is a growing health problem worldwide, ranging from non-alcoholic fatty liver (NAFL) to the more severe metabolic non-alcoholic steatohepatitis (NASH). Although many studies have elucidated the pathogenesis of NAFLD, the epigenetic regulatory mechanism from NAFL to NASH remains incompletely understood. The histone H3 lysine 4 methyltransferase, MLL4 (also called KMT2D), is a critical epigenetic transcriptional coactivator that mediates overnutrition-induced steatosis in mice, but its potential role in the progression of NASH remains largely unknown. Here, we show that mice lacking the one allele of the Mll4 gene are resistant to hepatic steatosis, inflammation, and fibrosis in NASH conditions compared to wild-type controls. Transcriptome analysis of the livers of control and Mll4+/- mice identified pro-inflammatory genes regulated by the nuclear factor kappa B (NF-κB) signaling pathway as major target genes of MLL4. We show that MLL4 binds to p65 and that MLL4 is required for NF-κB transactivation. Myeloid-specific Mll4 knockout mice showed an almost complete block of NASH, while hepatocyte-specific Mll4 knockout mice showed mild inhibition of steatosis. Pro-inflammatory M1 polarization is decreased and anti-inflammatory M2 polarization is increased in liver macrophages from myeloid-specific Mll4 knockout mice. Importantly, we show that histone H3-lysine 4 methylation mediated by the MLL4-complex plays a critical role in promoting the expression of Ccl2 in hepatocytes and M1 marker genes in macrophages. Our results demonstrate that MLL4, through the NF-κB-MLL4 regulatory axis, exacerbates steatohepatitis in the context of an inflammatory response and represents a potential therapeutic target for NASH.

Rhynchophylline Alleviates Hyperactivity and Cognitive Flexibility Impairment Associated With Inhibition of Inflammatory Responses in Mice That Partly Lack the Dopamine Transporter Protein.

Rhynchophylline (RHY) can alleviate some cognitive flexibility impairment and stereotyped behavior for attention-deficit hyperactivity disorder (ADHD) and Tourette syndrome (TS) patients as one of a keyextract and an active ingredient in Ningdong granule (NDG), which is a Traditional Chinese medicine (TCM) preparation widely used in the treatment of ADHD and TS children in China; however, the underlying mechanism is not well understood. Therefore, this study aimed to evaluate how RHY alleviates hyperactivity and cognitive flexibility impairment while inhibiting inflammatory responses in mice that partly lack dopamine transporter protein (DAT- mice). Male DAT- mice were randomly divided into the RHY group (n = 8) and administered RHY (30mg/kg) in the DAT- group (n = 8) and administered saline (i.p., 10mL/kg) in wild-type (WT) mice as the WT control group (n = 8). Hyperactivity and cognitive flexibility impairment were evaluated by the open field test (OFT) and the Morris water maze (MWM) test. The levels of the inflammatory factors of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in cortical homogenates were tested by enzyme-linked immunosorbent assays (ELISA) after 8 weeks of treatment with RHY. In vitro, primary microglia and astrocytes extracted from the cortices of DAT- neonatal mice and WT neonatal mice were treated with lipopolysaccharide (LPS) (1mg/mL) to induce neuroinflammatory responses and with RHY (20mM) for 48h. The levels of the inflammatory factors TNF-α, IL-1β, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX2) in the culture medium were measured at 6h, 24h, and 48h after treatment with LPS and RHY. RHY ameliorated hyperactivity and cognitive flexibility impairment in DAT- mice and inhibited the expression of the inflammatory factors TNF-α, IL-1β, iNOS, and COX-2 in microglia and astrocytes in vitro, and also inhibited the expression of TNF-α and IL-1β in cortical homogenates after 8 weeks of treatment. RHY improved hyperactivity and cognitive flexibility impairment through inhibiting inflammatory responses in DAT- mice.