Wild-type Research Articles

Overexpression of the RAV Transcription Factor OsAAT1 Confers Enhanced Arsenic Tolerance by Modulating Auxin Hemostasis in Rice.

Characterization of arsenic (As)-responsive genes is fundamental to solving the issue of As contamination in rice. Herein, we establish the involvement of an RAV transcription factor OsAAT1 (Arsenic Accumulation and Tolerance 1) in regulating As response in rice. The expression of OsAAT1 is significantly higher in roots and stems of rice seedlings and is clearly upregulated by higher concentrations of arsenite [As(III)]. Compared with wild-type (WT) plants, OsAAT1-overexpressed transgenic lines (OE-OsAAT1) exhibit tolerance, while OsAAT1-knockout mutants (Osaat1) are sensitive to As(III) stress. Notably, the application of exogenous 1-naphthylacetic acid (NAA) greatly enhances the As tolerance of WT and transgenic lines, with stronger effects on OE-OsAAT1. The change in OsAAT1 expression leads to the alteration of As and auxin accumulation in transgenic plants by regulating the expression of OsLsi1, OsLsi2, OsCRL4, and OsAUX1 genes. Moreover, overexpression of OsAAT1 accelerates ROS scavenging and phytochelatins (PCs) synthesis, especially with the addition of exogenous NAA. OsAAT1 localizes in the nucleus and works as a transcriptional suppressor. OsGH3-12, belonging to the auxin-responsive GH3 gene family, is the downstream target gene of OsAAT1, whose expression is extensively downregulated by As(III). These findings provide new insights into As response via auxin signaling pathway in rice.

Toxoplasma gondii infection induces the expression of the chemokine CXCL16 in macrophages to promote chemoattraction of CXCR6+ cells.

CXCL16 is a multifaceted chemokine expressed by macrophages and other immune cells in response to viral and bacterial pathogens. However, few studies have investigated its role in parasitic infections. The obligate intracellular parasite Toxoplasma gondii (T. gondii) is the causative agent of toxoplasmosis, an infection with potentially deleterious consequences in immunocompromised individuals and the developing fetus of acutely infected pregnant women. Chemokines are critical mediators of host defense and, as such, dysregulation of their expression is a subversion strategy often employed by the parasite to ensure its survival. Herein, we report that types I and II T. gondii strains upregulated the expression of both transmembrane and soluble forms of CXCL16 in infected bone marrow-derived macrophages (BMDM). Exposure to soluble T. gondii antigens (STAg) and to excreted-secreted proteins (TgESP) led to the induction of CXCL16. Cxcl16 mRNA abundance and CXCL16 protein levels increased in a time-dependent manner upon T. gondii infection. Importantly, conditioned medium (CM) collected from T. gondii-infected wild-type (WT) macrophage cultures promoted the migration of RAW264.7 cells expressing CXCR6, the cognate receptor of CXCL16, an effect that was significantly reduced by a neutralizing anti-CXCL16 antibody or use of CM from CXCL16 knockout (KO) macrophages. Lastly, T. gondii-driven CXCL16 expression appeared to modulate cytokine-induced (IL-4 + IL-13) alternative macrophage activation and M2 phenotypic marker expression. Further investigation is required to determine whether this chemokine contributes to the pathogenesis of toxoplasmosis and to elucidate the underlying molecular mechanisms.

Nicorandil treatment improves survival and spatial learning in aged granulin knockout mice.

Mutations in the human granulin (GRN) gene are associated with multiple diseases, including dementia disorders such as frontotemporal dementia (FTD) and limbic-predominant age-related TDP-43 encephalopathy (LATE). We studied a Grn knockout (Grn-KO) mouse model in order to evaluate a potential therapeutic strategy for these diseases using nicorandil, a commercially available agonist for the ABCC9/Abcc9-encoded regulatory subunit of the "K+ATP" channel that is well-tolerated in humans. Aged (13 months) Grn-KO and wild-type (WT) mice were treated as controls or with nicorandil (15 mg/kg/day) in drinking water for 7 months, then tested for neurobehavioral performance, neuropathology, and gene expression. Mortality was significantly higher for aged Grn-KO mice (particularly females), but there was a conspicuous improvement in survival for both sexes treated with nicorandil. Grn-KO mice performed worse on some cognitive tests than WT mice, but Morris Water Maze performance was improved with nicorandil treatment. Neuropathologically, Grn-KO mice had significantly increased levels of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytosis but not ionized calcium binding adaptor molecule 1 (IBA-1)-immunoreactive microgliosis, indicating cell-specific inflammation in the brain. Expression of several astrocyte-enriched genes, including Gfap, were also elevated in the Grn-KO brain. Nicorandil treatment was associated with a subtle shift in a subset of detected brain transcript levels, mostly related to attenuated inflammatory markers. Nicorandil treatment improved survival outcomes, cognition, and inflammation in aged Grn-KO mice.

Discovering a novel glycosyltransferase gene CmUGT1 enhances main metabolites production of Cordyceps militaris

Cordyceps militaris is a filamentous fungus used for both medicinal and culinary purposes. It exhibits a wide range of pharmacological activities due to its valuable contents of cordycepin, polysaccharides, carotenoids, terpenoids and other metabolites. However, C. militaris strains are highly susceptible to irreversible degradation in agricultural production, which is often manifested as a prolonged color change period and a significant decrease in the production of secondary metabolites. UDP-glycosyltransferases are an important enzyme family that participates in the synthesis of terpenoids by performing the glycosylation of key residues of enzymes or molecules. However, few studies have focused on its effect on the regulation of metabolite production in C. militaris. Therefore, in this study, we performed transcriptome analysis across four different developmental stages of C. militaris to target the putative glycosyltransferase gene CmUGT1, which plays important roles in metabolite production. We further constructed and screened a CmUGT1-overexpressing strain by Agrobacterium tumefaciens-mediated infestation of C. militaris spores. The major metabolite production of the wild-type and CmUGT1-overexpressing C. militaris strains was determined after short-term shake-flask cultivation of mycelia. The results showed that the yields of carotenoids and polysaccharides in the mycelia of the CmUGT1-overexpressing strains were 3.8 and 3.4 times greater than those in the mycelia of the wild type, respectively (p < 0.01). The levels of intracellular and extracellular cordycepin produced by the overexpression strain were 4.4 and 8.0 times greater than those produced by the wild-type strain (p < 0.01). This suggests that the overexpression of CmUGT1 in C. militaris enhances the synthesis activities of the main enzymes related to metabolite production, which provides a guide for obtaining excellent recombinant strains of C. militaris.

Development and drought escape response in Arabidopsis thaliana are regulated by AtPLC1 in response to abscisic acid.

AtPLC1 plays a critical role in plant growth, development, and response to drought stress. Phosphoinositide-specific phospholipase C (PI-PLC) hydrolyzes substrates to generate secondary messengers crucial for plant growth, development, and stress responses. Drought escape (DE) response is an adaptive strategy that plants employ under drought conditions. The expression levels of the flower meristem-specific gene APETALA 1 and flowering regulatory genes FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 were downregulated in plc1, and FLOWERING LOCUS C was upregulated. The flowering time of the plc1flc double mutant was earlier than that of the wild type. Transcriptome analysis revealed that the Gene Ontology of differentially expressed genes (DEGs) was enriched in abscisic acid (ABA) response signaling, and Kyoto Encyclopedia of Genes and Genomes analysis revealed differential gene expression annotated to plant hormone signaling pathways. Our experiments show that AtPLC1 is upregulated by ABA in Arabidopsis. Under ABA induction and water stress, wild-type plants exhibit a DE response, and the DE response in plc1 disappears. Expression levels of ABA signaling pathway transcription factors ABA-responsive element-binding factors 3 (ABF3) and ABF4 were downregulated in plc1. In conclusion, our study suggests that AtPLC1 participates in regulating plant growth and development and participates in the DE response through the regulation of ABA signaling pathway transcription factors ABF3/ABF4. The study enhances our comprehension of the role of AtPLC1 in plant development and drought stress, providing a theoretical foundation for further investigation into DE responses.

Neurosurgical application of olaparib from a thermo-responsive paste potentiates DNA damage to prolong survival in malignant glioma.

There is increased pan-cancer specific interest in repurposing the poly adenosine diphosphate-ribose polymerase-1 (PARP-1) inhibitor, olaparib, for newly diagnosed or recurrent isocitrate dehydrogenase wild type glioblastoma. We explore whether intra-cavity delivery of olaparib confers a survival benefit in a pre-clinical high-grade glioma model. Primary tumor RNA sequencing data was used to determine PARP-1 as a target in the glioblastoma infiltrative margin. We assessed radiosensitization conferred by olaparib alone and concomitant to genotoxic insults in vitro using clonal growth assays, cell cycle analysis and immunocytochemistry, and in vivo upon post-surgical delivery from a temperature-sensitive polymeric paste. RNA-sequencing confirmed PARP-1 as a viable therapy target in glioblastoma infiltrative disease. Acute exposure of glioma cells to olaparib impaired proliferation and induced late-stage apoptosis associated with DNA damage in vitro, potentiated by radiation. Using high-grade glioma orthotopic allografts, a long-term overall survival benefit was observed upon interstitial olaparib delivery concomitant with radiotherapy, compared to systemic olaparib and standard glioblastoma treatment. Combined delivery of olaparib with either temozolomide or etoposide increased long-term survival, suggestive of olaparib functioning as DNA damage sensitizer. Collectively, our data support a rationale for localized olaparib delivery concomitant with the current clinical regimen for malignant glioma treatment.

Essential role of interferon-regulatory factor 4 in regulating diabetogenic CD4+ T and innate immune cells in autoimmune diabetes in NOD mice.

Haploinsufficiency of the transcription factor interferon-regulatory factor 4 (IRF4) prevents the onset of spontaneous diabetes in NOD mice. However, the immunological mechanisms of the IRF4-mediated disease regulation remain unclear. This study aims to investigate the role of IRF4 in the pathogenesis of autoimmune diabetes by conducting adoptive transfer experiments using donor IRF4 gene-deficient CD4+ T cells from BDC2.5-transgenic (Tg) NOD mice and recipient Rag1-knockout NOD mice, respectively. Through this approach, we analyzed both clinical and immunological phenotypes of the recipient mice. Additionally, IRF4-deficient BDC2.5 CD4+ T cells were stimulated to assess their immunological and metabolic phenotypes in vitro. The findings revealed that diabetes was completely prevented in the recipients with Irf4-/- T cells and was approximately 50% lower in those with Irf4+/- T cells than in wild type (WT) controls, whereas Irf4-/- recipients with WT T cells only showed a delayed onset of diabetes. Islet-infiltrating T cells isolated from recipients with Irf4+/- T cells exhibited significantly lower proliferation and IFN-γ/IL-17 double-positive cell fraction rates compared with those in WT controls. Irf4-/- BDC2.5 CD4+ T cells stimulated in vitro showed a reduced number of cell divisions, decreased antigen-specific T-cell markers, and impairment of glycolytic capacity compared with those observed in WT controls. We concluded that IRF4 predominantly regulates the diabetogenic potential in a dose-dependent manner by mediating the proliferation and differentiation of islet-infiltrating T cells while playing an adjunctive role in the innate immune responses toward diabetes progression in NOD mice.

Characterization and ontogeny of a novel lymphoid follicle inducer cell during development of the bursa of Fabricius

The avian bursa of Fabricius (BF) is a primary lymphoid organ, where B-cell development occurs within bursal follicles of epithelial origin. During embryogenesis the epithelial anlage of the BF emerges as a diverticulum of the cloaca surrounded by undifferentiated tail bud mesenchyme. While it is believed that the epithelial-mesenchymal BF primordium provides a selective microenvironment for developing B cells, the initial events inducing lymphoid follicle formation are not fully elucidated. Using wild type and CSF1R-eGFP transgenic chick embryos, we find that separate B cell, macrophage and dendritic cell precursors enter the BF mesenchyme, migrate to the surface epithelium, and colonize the lymphoid follicle buds. Detailed immunocytochemical characterization revealed a novel EIV-E12+ blood-borne cell type, colonizing the surface epithelium of the BF rudiment before the entry of myeloid and lymphoid lineages and the appearance of this cell type coincides with the onset of follicle bud formation. Chick-duck chimeras and chick-quail tissue recombination experiments suggest that EIV-E12+ cells represent a transient lymphoid inducer cell population. They are not dendritic or B cells precursors, and they are capable of follicle bud induction in both dendritic cell- and B cell-depleted bursae.

HIP is involved in NaCl and endoplasmic reticulum stress resistance in Arabidopsis

Heat shock protein 70 (HSP70)-interacting proteins (HIPs) have been studied in animals. HIPs perform diverse cellular functions, ranging from alleviating stress to suppressing the formation of insoluble protein, but how their orthologs function in plants is less known. This study shows that Arabidopsis HIP is a cytosolic and nuclear protein associated with HSP70. The hip mutants showed compromised tolerance to NaCl and endoplasmic reticulum (ER) stress, although they grew normally under standard growth conditions. Furthermore, hip mutants presented a decreased HSP70 level compared with the wild type under NaCl and ER stress conditions. These findings suggest the involvement of HIP in NaCl and ER stress tolerance.

Vascular inflammaging: Endothelial CEACAM1 expression is upregulated by TNF-α via independent activation of NF-κB and β-catenin signaling.

Chronic inflammation with progressive age, called inflammaging, contributes to the pathogenesis of cardiovascular diseases. Previously, we have shown increased vascular expression of the Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in aged mice and humans, presumably via mutual upregulation with the pro-inflammatory cytokine TNF-α. CEACAM1 is critical for aging-associated vascular alterations like endothelial dysfunction, fibrosis, oxidative stress, and sustained inflammation and can be regarded as a main contributor to vascular inflammaging. This study was conducted to elucidate the mechanisms underlying endothelial CEACAM1 upregulation by TNF-α in detail. Using wildtype (WT) and TNF-α knockout (Tnf-/-) mice, we confirmed that the aging-related upregulation of endothelial CEACAM1 critically depends on TNF-α. The underlying mechanisms were analyzed in an endothelial cell culture model. TNF-α time-dependently upregulated CEACAM1 invitro. In pharmacological experiments, we identified an early NF-κB- and a delayed β-catenin-mediated response. Involvement of β-catenin was further substantiated by siRNA-mediated knockdown of the β-catenin-targeted transcription factor TCF4. Both signaling pathways acted independent from each other. Elucidating the delayed response, co-immunoprecipitation analysis revealed release of β-catenin from adherens junctions by TNF-α. Finally, TNF-α activated Akt kinase by increasing its Ser473 phosphorylation. Consequently, Akt kinase facilitated β-catenin signaling by inhibiting its degradation via phosphorylation of GSK3β at Ser9 and by increased phosphorylation of β-catenin at Ser552 that augments its transcriptional activity. Taken together, our study provides novel mechanistic insights into the aging-related, inflammation-mediated endothelial upregulation of CEACAM1. Beyond the pathogenesis of cardiovascular diseases, these findings may be significant to all fields of inflammaging.

NeoI represents a group of transcriptional repressors regulating the biosynthesis of multiple aminoglycosides.

In general, the initiation or closure of antibiotic biosynthesis is determined by regulatory proteins, but most of their mechanisms of action remain unknown. The 2-deoxystreptamine-containing aminoglycosides (2-DOS AGs) form a unique category among antibiotics. Genomic analysis revealed that a group of hypothetical regulatory genes represented by neoI are widely distributed in the biosynthetic gene clusters (BGCs) of natural products from Streptomyces species, including several 2-DOS AGs. Only limited knowledge is available for the roles of NeoI-type regulators although neomycin and some of the related AGs have been developed as therapeutic drugs for decades. This study focuses on the functional determination of neoI and its homologues situated in the BGCs of six AGs. We found that the yield of neomycin in neoI disruption mutant (ΔneoI) increased by 50% compared to the wild-type (WT) strain ((420.6±44.1) mg L-1), while it was partially restored by the complementation of neoI, demonstrating that NeoI acted as a repressor in neomycin biosynthesis. Further electrophoretic mobility shift assays (EMSAs) and DNase I footprinting assays indicated that NeoI could specifically bind to the promoter region between neoE and neoI with conserved nucleotides (5'-CVHYMRCHDKAGYGGACR-3'), as determined by site-directed mutagenesis. Interestingly, cross-bindings of the NeoI homologues from the six different BGCs to their corresponding DNA targets were manifested, and the five exogenous NeoI homologues could complement NeoI function of repressing neomycin biosynthesis. Our results suggested that NeoI-type regulators represent widespread and conservative regulatory characteristics in the biosynthesis of 2-DOS AGs, which would be significant for optimizing the biosynthetic pathways of valuable commercialized aminoglycoside antibiotics.

Structure and Energetics of PET-Hydrolyzing Enzyme Complexes: A Systematic Comparison from Molecular Dynamics Simulations.

Discovered in 2016, the enzyme PETase, secreted by bacterial Ideonella Sakaiensis 201-F6, has an excellent hydrolytic activity toward poly(ethylene terephthalate) (PET) at room temperature, while it decreases at higher temperatures due to the low thermostability. Many variants have been engineered to overcome this limitation, which hinders industrial application. In this work, we systematically compare PETase wild-type (WT) and four mutants (DuraPETase, ThermoPETase, FastPETase, and HotPETase) using standard molecular dynamics (MD) simulations and unbinding free energy calculations. In particular, we analyze the enzymes' structural characteristics and binding to a tetrameric PET chain (PET4) under two temperature conditions: T1─300 K and T2─350 K. Our results indicate that (i) PET4 forms stable complexes with the five enzymes at room temperature (∼300 K) and (ii) most of the interactions are localized close to the active site of the protein, where the W185 and Y87 residues interact with the aromatic rings of the substrate. Specifically, (iii) the W185 side-chain explores different conformations in each variant (a phenomenon known in the literature as "W185 wobbling"). This suggests that the binding pocket retains structural plasticity and flexibility among the variants, facilitating substrate recognition and localization events at moderate temperatures. Moreover, (iv) PET4 establishes aromatic interactions with the catalytic H237 residue, stabilizing the catalytic triad composed of residues S160-H237-D206, and helping the system achieve an effective configuration for the hydrolysis reaction. Conversely, (v) the binding affinity decreases at a higher temperature (∼350 K), retaining moderate interactions only for HotPETase. Finally, (vi) MD simulations of complexes formed with poly(ethylene-2,5-furan dicarboxylate) (PEF) show no persistent interactions, suggesting that these enzymes are not yet optimized for binding this alternative semiaromatic plastic polymer. Our study offers valuable insights into the structural stability of these enzymes and the molecular determinants driving PET binding onto their surfaces, sheds light on the mechanistic steps that precede the onset of hydrolysis, and provides a foundation for future enzyme optimization.

AtZAT10/STZ1 improves drought tolerance and increases fiber yield in cotton

Drought poses a significant challenge to global crop productivity, necessitating innovative approaches to bolster plant resilience. Leveraging transgenic technology to bolster drought tolerance in crops emerges as a promising strategy for addressing the demands of a rapidly growing global populace. AtZAT10/STZ1, a C2H2-type zinc finger protein transcription factor has shown to significantly improve Arabidopsis’ tolerance to various abiotic stresses. In this study, we reports that AtSTZ1 confers notable drought resistance in upland cotton (Gossypium hirsutum), amplifying cotton fiber yield under varying conditions, including irrigated and water-limited environments, in field trials. Notably, AtSTZ1-overexpressing transgenic cotton showcases enhanced drought resilience across critical growth stages, including seed germination, seedling establishment, and reproductive phases. Morphological analysis reveals an expanded root system characterized by an elongated taproot system, increased lateral roots, augmented root biomass, and enlarged cell dimensions from transgenic cotton plants. Additionally, higher contents of proline, chlorophyll, soluble sugars, and enhanced ROS-scavenging enzyme activities are observed in leaves of transgenic plants subjected to drought, underscoring improved physiological adaptations. Furthermore, transgenic lines exhibit heightened photosynthetic rate, increased water use efficiency, and larger stomatal and epidermal cell sizes, coupled with a decline in leaf stomatal conductance and density, as well as diminished transpiration rates compared to the wild type counterparts. Transcriptome profiling unveils 106 differentially expressed genes in transgenic cotton leaves post-drought treatment, including protein kinases, transcription factors, aquaporins, and heat shock proteins, indicative of an orchestrated stress response. Collectively, these findings underscore the capacity of AtSTZ1 to augment the expression of abiotic stress-related genes in cotton following drought conditions, thus presenting a compelling candidate for genetic manipulation aimed at enhancing crop resilience.

Signatures of Structural Disorder in the Developing Drosophila Germband Epithelium

Epithelial cells generate functional tissues in developing embryos through collective movements and shape changes. In some morphogenetic events, a tissue dramatically reorganizes its internal structure—often generating high degrees of structural disorder—to accomplish changes in tissue shape. However, the origins of structural disorder in epithelia and what roles it might play in morphogenesis are poorly understood. We study this question in the germband epithelium, which undergoes dramatic changes in internal structure as cell rearrangements drive elongation of the embryo body axis. Using two order parameters that quantify volumetric and shear disorder, we show that structural disorder increases during body axis elongation and is strongly linked with specific developmental processes. Both disorder metrics begin to increase around the onset of axis elongation, but then plateau at values that are maintained throughout the process. Notably, the disorder plateau values for volumetric disorder are similar to those for random cell packings, suggesting this may reflect a limit on tissue behavior. In mutant embryos with disrupted external stresses from the ventral furrow, both disorder metrics reach wild-type maximum disorder values with a delay, correlating with delays in cell rearrangements. In contrast, in mutants with disrupted internal stresses and cell rearrangements, volumetric disorder is reduced compared to wild type, and shear disorder depends on specific external stress patterns. Together, these findings demonstrate that internal and external stresses both contribute to epithelial tissue disorder and suggest that the maximum values of disorder in a developing tissue reflect physical or biological limits on morphogenesis. Published by the American Physical Society 2024

Gpr55 deficiency crucially alters cardiomyocyte homeostasis and counteracts angiotensin II induced maladaption in female mice.

Cannabis stimulates several G-protein-coupled-receptors and causes bradycardia and hypotension upon sustained consumption. Moreover, in vitro studies suggest an interference of cannabinoid-signalling with cardiomyocyte contractility and hypertrophy. We aimed at revealing a functional contribution of the cannabinoid-sensitive receptor GPR55 to cardiomyocyte homeostasis and neurohumorally induced hypertrophy in vivo. Gpr55-/- and wild-type (WT) mice were characterized after 28-day angiotensin II (AngII; 1·μg·kg-1min-1) or vehicle infusion. In isolated adult Gpr55-/- and WT cardiomyocytes, mitochondrial function was assessed under naïve conditions, while cytosolic Ca2+ handling was additionally determined following application of the selective GPR55 antagonist CID16020046. Gpr55 deficiency did not affect angiotensin II (AngII) mediated hypertrophic growth, yet, especially in females, it alleviated maladaptive pro-hypertrophic and -inflammatory gene expression and improved inotropy and adrenergic responsiveness compared to WT. In-depth analyses implied increased cytosolic Ca2+ concentrations and transient amplitudes, and accelerated sarcomere contraction kinetics in Gpr55-/- myocytes, which could be mimicked by GPR55 blockade with CID16020046 in female WT cells. Moreover, Gpr55 deficiency up-regulated factors involved in glucose and fatty acid transport independent of the AngII challenge, accelerated basal mitochondrial respiration and reduced basal protein kinase (PK) A, G and C activity and phospholemman (PLM) phosphorylation. Our study suggests GPR55 as crucial regulator of cardiomyocyte hypertrophy and homeostasis presumably by regulating PKC/PKA-PLM and PKG signalling, and identifies the receptor as potential target to counteract maladaptation, adrenergic desensitization and metabolic shifts as unfavourable features of the hypertrophied heart in females.

Lack of AMP-activated protein kinase-α1 reduces nitric oxide synthesis in thoracic aorta perivascular adipose tissue

ObjectivePerivascular adipose tissue (PVAT) releases anti-contractile bioactive molecules including NO. PVAT anti-contractile activity is attenuated in mice lacking AMPKα1 (AMP-activated protein kinase-α1). As AMPK regulates endothelial NO synthase (eNOS) activity in cultured cells, NO synthesis was examined in PVAT from AMPKα1 knockout (KO) mice. Methods and resultsEndothelium-denuded thoracic or abdominal aortic rings were isolated from wild type (WT) and KO mice. NOS inhibition enhanced vasoconstriction in PVAT-intact thoracic aortic rings from mice of either genotype yet had no effect on abdominal rings as assessed by wire myography. Thoracic aorta PVAT exhibited increased NO production, NOS activity and levels of the brown adipose tissue marker uncoupling protein-1 (UCP1) compared to abdominal PVAT. In KO mice, NO production was significantly reduced in thoracic but not abdominal PVAT. Reduced NO production in KO thoracic PVAT was not due to altered levels or phosphorylation of eNOS but was associated with increased caveolin-1:eNOS association and caveolin-1 Tyr14 phosphorylation. A peptide that disrupts eNOS:caveolin-1 association increased NO synthesis and reduced vasoconstriction of PVAT-intact thoracic but not abdominal aortic rings. KO thoracic PVAT also exhibited reduced UCP1 levels. ConclusionsMurine thoracic aorta PVAT exhibits higher NO synthesis and UCP1 levels than abdominal aortic PVAT. Downregulation of AMPK suppresses NO synthesis which may contribute to the reduced anticontractile activity and reduced brown adipose tissue phenotype of KO thoracic PVAT. The mechanism underlying the effect of AMPK downregulation likely results from increased caveolin-1:eNOS association associated with caveolin-1 Tyr14 phosphorylation.

OsNAL11 and OsGASR9 Regulate the Low-Temperature Germination of Rice Seeds by Affecting GA Content

Low temperatures cause serious threat to rice seed emergence, which has become one of the main limiting factors in the production of direct seeding rice. It is of great importance to study the genes controlling low-temperature tolerance during seed germination and to mine the possible regulatory mechanism for developing new rice varieties with immense low-temperature germination ability. In the current research study, two types of mutants of nal11 and gasr9, derived from the WT (wild type) ZH11, were used for the analysis of low-temperature germinability. The results showed that the nal11 and gasr9 mutants displayed no significant difference in germination rate with ZH11 at room temperature, but the mutants showed significantly lower germination rates, germination potential and germination index, and slowed seedling growth in the simulated direct seeding experiments at low temperatures compared to ZH11. Additionally, the activity of POD, SOD, CAT, and anti-superoxide anion radial activity were significantly reduced, but the levels of MDA and H2O2 were significantly higher in the nal11 and gasr9 mutant seeds that were germinated at low temperatures compared to ZH11. Further analysis revealed that the levels of total active GA, especially GA4 and GA7, were significantly lower in the nal11 and gasr9 mutants than that in ZH11 during low-temperature germination. Based on qRT-PCR analysis, the expression levels of some GA synthesis-related genes were higher, whereas some were lower in the nal11 and gasr9 mutants than those in ZH11, however, the GA metabolism-related genes OsGA2ox8 and OsGA2ox10 and the GA signaling negative regulator gene SLR1 were significantly up-regulated in both nal11 and gasr9 mutants at several time points during low-temperature germination. This may explain the lower GA levels in the nal11 and gasr9 mutants. Furthermore, the interaction between the OsNAL11 and OsGASR9 proteins was confirmed by Y2H, LUC, and Co-IP assays. This study provides preliminary insights into the regulatory mechanism of the OsNAL11 and OsGASR9 genes, which control the low-temperature germination of rice seeds by affecting the GA pathway. Our study will provide the basis for further mining the molecular mechanisms of low-temperature germination in rice and valuable theoretical reference for breeding varieties with strong low-temperature germinability.

Establishment of potential reference measurement procedure and reference materials for EML4-ALK fusion variants measurement

Echinoderm microtubule-associated protein 4 (EML4) - anaplastic lymphoma kinase (ALK) fusion gene detection is of great significance in personalized tumor treatment. With the development of EML4-ALK fusion variants detection, it is necessary to establish traceability to ensure the consistency and comparability of its detection results in clinical practice. The establishment of traceability relies on SI traceable reference materials (RMs) and potential reference measurement procedures (RMPs). In this study, a potential RMP for the quantitative detection of V1 and V3 fusion mutations and the reference type (ALK-ref, including wild type, V1 and V3 mutant type) based on reverse transcription dPCR (RT-dPCR) and EML4-ALK fusion gene RMs were established. The proposed potential RMP has high specificity, good inter-laboratory reproducibility (CV < 7.3%) and good linear relationship (0.92 < slope < 1.06, R2 ≧ 0.99). The limit of detection (LoD) of V1, V3, and ALK-ref are 2 copies/reaction, 2 copies/reaction, and 1 copy/reaction, respectively. Interlaboratory studies using the EML4-ALK RMs and potential RMP showed that participating laboratories can produce consistent copy concentrations of fusion variant and ALK-ref as well as the ratio of EML4-ALK/ALK-ref. The established potential RMP with high specificity and accuracy can be used to characterize the EML4-ALK RMs, and the potential RMP and RM are useful to establish the traceability of EML4-ALK fusion measurement to improve the comparability and consistency in clinical tests.

Increased expression of mesencephalic astrocyte-derived neurotrophic factor (MANF) contributes to synapse loss in Alzheimer’s disease

BackgroundThe activation of endoplasmic reticulum (ER) stress is an early pathological hallmark of Alzheimer’s disease (AD) brain, but how ER stress contributes to the onset and development of AD remains poorly characterized. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a non-canonical neurotrophic factor and an ER stress inducible protein. Previous studies reported that MANF is increased in the brains of both pre-symptomatic and symptomatic AD patients, but the consequence of the early rise in MANF protein is unknown.MethodsWe examined the expression of MANF in the brain of AD mouse models at different pathological stages. Through behavioral, electrophysiological, and neuropathological analyses, we assessed the level of synaptic dysfunctions in the MANF transgenic mouse model which overexpresses MANF in the brain and in wild type (WT) mice with MANF overexpression in the hippocampus. Using proteomic and transcriptomic screening, we identified and validated the molecular mechanism underlying the effects of MANF on synaptic function.ResultsWe found that increased expression of MANF correlates with synapse loss in the hippocampus of AD mice. The ectopic expression of MANF in mice via transgenic or viral approaches causes synapse loss and defects in learning and memory. We also identified that MANF interacts with ELAV like RNA-binding protein 2 (ELAVL2) and affects its binding to RNA transcripts that are involved in synaptic functions. Increasing or decreasing MANF expression in the hippocampus of AD mice exacerbates or ameliorates the behavioral deficits and synaptic pathology, respectively.ConclusionsOur study established MANF as a mechanistic link between ER stress and synapse loss in AD and hinted at MANF as a therapeutic target in AD treatment.

Exploring Functional Gene XsPDAT1’s Involvement in Xanthoceras sorbifolium Oil Synthesis and Its Acclimation to Cold Stress

Phospholipid: diacylglycerol acyltransferase (PDAT) is crucial in triacylglycerol (TAG) synthesis as it represents the final rate-limiting step of the acyl-CoA-independent acylation reaction. PDAT not only regulates lipid synthesis in plants, but also plays an important function in improving stress tolerance. In this study, the full-length coding sequence (CDS) of XsPDAT1, totaling 2022 base pairs and encoding 673 amino acids, was cloned from Xanthoceras sorbifolium. The relative expression of XsPDAT1 was significantly and positively correlated with oil accumulation during seed kernel development; there were some differences in the expression patterns under different abiotic stresses. Transgenic Arabidopsis thaliana plants overexpressing XsPDAT1 were obtained using the Agrobacterium-mediated method. Under low-temperature stress, the transgenic plants exhibited a smaller decrease in chlorophyll content, a smaller increase in relative conductivity, and a larger increase in POD enzyme activity and proline content in the leaves compared with the wild type. Additionally, lipid composition analysis revealed a significant increase in unsaturated fatty acids, such as oleic (C18:1) and linoleic (C18:2), in the seeds of transgenic plants compared to the wild type. These results suggest that XsPDAT1 plays a dual role in regulating the ratio of fatty acid composition and low-temperature stress in plants.