Green Fluorescent Protein Signal Research Articles

Bioinformatics, expression analysis, and functional verification of allene oxide synthase gene HvnAOS1 and HvnAOS2 in qingke.

Allene oxide synthase (AOS) is a key enzyme involved in the jasmonic acid (JA) synthesis pathway in plants. To explore its function on the regulatory mechanism of JA synthesis, we screened and identified two AOS genes HvnAOS1 and HvnAOS2 in qingke. Both HvnAOS1 and HvnAOS2 contained conserved heme-binding motif, which is most closely related to AtsAOS2, indicating controlled dehydration of fatty acid hydroperoxides to allene oxides. Molecular docking simulations identified the key amino acid sites that were important for heme binding and interaction with 13(S)-HPOT, respectively. The expression pattern also indicated that HvnAOS1 and HvnAOS2 were highly induced by JA, abscisic acid, and salicylic acid. Subcellular localization of HvnAOS1 and HvnAOS2 using transient expression of Agrobacterium tumefaciens showed the green fluorescent protein signal in the cell cytoplasm of the N. benthamiana leaves. Overexpression of HvnAOS1 and HvnAOS2 in Arabidopsis aos mutant restored male fertility and plant resistance to Botrytis cinerea, indicating that HvnAOS1 and HvnAOS2 can restore the functions of AOS in Arabidopsis aos mutant.

GFP-labeled Schwann cell-like cells derived from hair follicle epidermal neural crest stem cells promote the acellular nerve allografts to repair facial nerve defects in rats

BackgroundAcellular nerve allografts (ANAs) have been successfully applied to bridge facial nerve defects, and transplantation of stem cells may enhance the regenerative results. Up to now, application of hair follicle epidermal neural crest stem cell-derived Schwann cell-like cells (EPI–NCSC–SCLCs) combined with ANAs for bridging facial nerve defects has not been reported. MethodsThe effect of ANAs laden with green fluorescent protein (GFP)-labeled EPI–NCSC–SCLCs (ANA + cells) on bridging rat facial nerve trunk defects (5-mm-long) was detected by functional and morphological examination, as compared with autografts and ANAs, respectively. Results(1) EPI–NCSC–SCLCs had good compatibility with ANAs in vitro. (2) In the ANA + cells group, the GFP signals were observed by in vivo imaging system for small animals within 8 weeks, and GFP-labeled EPI–NCSC–SCLCs were detected in the tissue slices at 16 weeks postoperatively. (3) The facial symmetry at rest after surgery in the ANA + cells group was better than that in the ANA group (p < 0.05), and similar to that in the autograft group (p > 0.05). The initial recovery time of vibrissal and eyelid movement in the ANA group was 2 weeks later than that in the other two groups. (4) The myelinated fibers, myelin sheath thickness and diameter of the axons of the buccal branches in the ANA group were significantly worse than those in the other two groups (P < 0.05), and the results in the ANA + cells group were similar to those in the autograft group (p > 0.05). ConclusionsEPI–NCSC–SCLCs could promote functional and morphological recovery of rat facial nerve defects, and GFP labeling could track the transplanted EPI–NCSC–SCLCs in vivo for a certain period of time. These may provide a novel choice for clinical treatment of peripheral nerve defects.

Spatiotemporally Precise Optical Manipulation of Intracellular Molecular Activities.

Controlling chemical processes in live cells is a challenging task. The spatial heterogeneity of biochemical reactions in cells is often overlooked by conventional means of incubating cells with desired chemicals. A comprehensive understanding of spatially diverse biochemical processes requires precise control over molecular activities at the subcellular level. Herein, a closed-loop optoelectronic control system is developed that allows the manipulation of biomolecular activities in live cells at high spatiotemporal precision. Chemical-selective fluorescence signals are utilized to command lasers that trigger specific chemical processes or control the activation of photoswitchable inhibitors at desired targets. This technology is fully compatible with laser scanning confocal fluorescence microscopes. The authors demonstrate selective interactions of a 405nm laser with targeted organelles and simultaneous monitoring of cell responses by fluorescent protein signals. Notably, blue laser interaction with the endoplasmic reticulum leads to a more pronounced reduction in cytosolic green fluorescent protein signals in comparison to that with nuclei and lipid droplets. Moreover, when combined with a photoswitchable inhibitor, microtubule polymerization is selectively inhibited within the subcellular compartments. This technology enables subcellular spatiotemporal optical manipulation over chemical processes and drug activities, exclusively at desired targets, while minimizing undesired effects on non-targeted locations.

Polyethylene glycol–mediated genetic transformation and fluorescent labelling of Colletotrichum sublineola for studying sorghum anthracnose pathogenicity

AbstractAnthracnose, a disease primarily caused by the fungus Colletotrichum sublineola, affects sorghum production in China. However, the infection mechanisms and complex host interactions of C. sublineola remain to be elucidated. Thus, this study aimed to establish and optimize a protoplast transformation system of C. sublineola so as to lay a foundation for further research on the infection mechanisms and complex host interactions of C. sublineola. A genetic transformation system was established and optimized for C. sublineola strain HX‐5‐1 and used to introduce fluorescent reporter genes, namely, green fluorescent protein (GFP) and mCherry. Results showed that the growth of HX‐5‐1 was completely inhibited on a regenerating medium containing 300 μg mL−1 hygromycin B, indicating that hygromycin B acted as a transformant‐selective marker. HX‐5‐1 produced the highest yield of protoplasts when the mycelia were cultured for 60 h and dissolved in a 0.1% lysozyme solution (0.7 M NaCl) for 3 h. Subsequently, a 106 mL−1 protoplast solution transformed using 5 μg of plasmid DNA exhibited the highest transformation efficiency. Laser confocal microscopy showed that GFP and mCherry fluorescent signals were successfully expressed in the mycelia and spores of HX‐5‐1, indicating that they were stably inherited in the transformants and did not affect colony morphology, growth rate, or pathogenicity. In conclusion, we successfully established a genetic transformation system for C. sublineola and employed it to obtain stable fluorescent protein expression. This study may serve as a foundation for understanding the infection process of C. sublineola.

Endometrial organoids: a reservoir of functional mitochondria for uterine repair.

Background: Asherman's syndrome (AS) is a dreadful gynecological disorder of the uterus characterized by intrauterine adhesion with severe fibrotic lesions, resulting in a damaged basalis layer with infertility. Despite extensive research on overcoming AS, evidence-based effective and reproducible treatments to improve the structural and functional morphology of the AS endometrium have not been established. Methods: Endometrial organoids generated from human or mouse endometrial tissues were transplanted into the uterine cavity of a murine model of AS to evaluate their transplantable feasibility to improve the AS uterine environment. The successful engraftment of organoid was confirmed by detection of human mitochondria and cytosol (for human endometrial organoid) or enhanced green fluorescent protein signals (for mouse endometrial organoid) in the recipient endometrium. The therapeutic effects mediated by organoid transplantation were examined by the measurements of fibrotic lesions, endometrial receptivity and angiogenesis, and fertility assessment by recording the number of implantation sites and weighing the fetuses and placenta. To explore the cellular and molecular mechanisms underlying the recovery of AS endometrium, we evaluated the status of mitochondrial movement and biogenetics in organoid transplanted endometrium. Results: Successfully engrafted endometrial organoids with similar morphological and molecular features to the parental tissues dramatically repaired the AS-induced damaged endometrium, significantly reducing fibrotic lesions and increasing fertility outcomes in mice. Moreover, dysfunctional mitochondria in damaged tissues, which we propose might be a key cellular feature of the AS endometrium, was fully recovered by functional mitochondria transferred from engrafted endometrial organoids. Endometrial organoid-originating mitochondria restored excessive collagen accumulation in fibrotic lesions and shifted uterine metabolic environment to levels observed in the normal endometrium. Conclusions: Our findings suggest that endometrial organoid-originating mitochondria might be key players to mediate uterine repair resulting in fertility enhancement by recovering abrogated metabolic circumstance of the endometrium with AS. Further studies addressing the clinical applicability of endometrial organoids may aid in identifying new therapeutic strategies for infertility in patients with AS.

Heterogeneity in killing efficacy of individual effector CD8+ T cells against Plasmodium liver stages.

Vaccination strategies in mice inducing high numbers of memory CD8+ T cells specific to a single epitope are able to provide sterilizing protection against infection with Plasmodium sporozoites. We have recently found that Plasmodium-specific CD8+ T cells cluster around sporozoite-infected hepatocytes but whether such clusters are important in elimination of the parasite remains incompletely understood. Here, we used our previously generated data in which we employed intravital microscopy to longitudinally image 32 green fluorescent protein (GFP)-expressing Plasmodium yoelii parasites in livers of mice that had received activated Plasmodium-specific CD8+ T cells after sporozoite infection. We found significant heterogeneity in the dynamics of the normalized GFP signal from the parasites (termed 'vitality index' or VI) that was weakly correlated with the number of T cells near the parasite. We also found that a simple model assuming mass-action, additive killing by T cells well describes the VI dynamics for most parasites and predicts a highly variable killing efficacy by individual T cells. Given our estimated median per capita kill rate of k = 0.031/h we predict that a single T cell is typically incapable of killing a parasite within the 48 h lifespan of the liver stage in mice. Stochastic simulations of T cell clustering and killing of the liver stage also suggested that: (i) three or more T cells per infected hepatocyte are required to ensure sterilizing protection; (ii) both variability in killing efficacy of individual T cells and resistance to killing by individual parasites may contribute to the observed variability in VI decline, and (iii) the stable VI of some clustered parasites cannot be explained by measurement noise. Taken together, our analysis for the first time provides estimates of efficiency at which individual CD8+ T cells eliminate intracellular parasitic infection in vivo.

Suppression of pinoid mutant phenotypes by mutations in PIN-FORMED 1 and PIN1-GFP fusion.

Disruption of either the auxin transporter PIN-FORMED 1 (PIN1) or the protein kinase PINOID (PID) leads to the development of pin-like inflorescences. Previous studies have shown that phosphoregulation of PIN1 by AGC kinases including PID directs auxin flux to drive organ initiation. Here, we report unexpected findings on the genetic interactions between these two genes. We deleted the first 2/3 of the PIN1 coding sequence using CRISPR/Cas9, and the resulting pin1 mutant (pin1-27) was a strong allele. Surprisingly, heterozygous pin1-27 suppressed two independent pid null mutants, whereas homozygous pin1-27 enhanced the phenotypes of the pid mutants during embryogenesis. Furthermore, we show that deletion of either the hydrophilic loop or the second half of PIN1 also abolished PIN1 function, yet those heterozygous pin1 mutants were also capable of rescuing pid nulls. Moreover, we inserted green fluorescent protein (GFP) into the hydrophilic loop of PIN1 through CRISPR-mediated homology-directed repair (HDR). The GFP signal and pattern in the PIN1-GFPHDR line are similar to those in the previously reported PIN1-GFP transgenic lines. Interestingly, the PIN1-GFPHDR line also rescued various pid null mutant alleles in a semidominant fashion. We conclude that decreasing the number of functional PIN1 copies is sufficient to suppress the pid mutant phenotype, suggesting that PIN1 is likely part of a larger protein complex required for organogenesis.

Optimised Agrobacterium-Mediated Transformation and Application of Developmental Regulators Improve Regeneration Efficiency in Melons.

Melon (Cucumis melo L.) is a protected crop in China with high economic value. Agrobacterium-mediated genetic transformation is a powerful tool to improve agronomic traits and obtain elite germplasm. However, current transformation protocols in melons are inefficient and highly genotype-dependent. To improve transformation in melon, we tested different infiltration methods for Agrobacterium-mediated transformation. Among these methods, micro-brushing and sonication for 20 s, followed by vacuum infiltration at -1.0 kPa for 90 s, resulted in the strongest green fluorescent protein signal and increased the proportion of infected explants. We transformed melon with developmental regulatory genes AtGRF5, AtPLT5, AtBBM, AtWUS, AtWOX5, and AtWIND1 from Arabidopsis and estimated regeneration frequencies as the number of regenerating shoots/total number of inoculated explants in the selection medium. The overexpression of AtGRF5 and AtPLT5 in melon resulted in transformation efficiencies of 42.3% and 33% in ZHF and 45.6% and 32.9% in Z12, respectively, which were significantly higher than those of the control. AtGRF5 and AtPLT5 expression cassettes were added to CRISPR/Cas9 genome-editing vectors to obtain transgenic phytoene desaturase CmPDS knockout mutants. Using AtGRF5 or AtPLT5, multi-allelic mutations were observed at CmPDS target sites in recalcitrant melon genotypes. This strategy enables genotype-flexible transformation and promotes precise genome modification technologies in melons.

Cloning, subcellular localization and expression of phosphate transporter gene HvPT6 of hulless barley.

Deficiency of phosphate (Pi) is one of the main growth-limiting factors for crops. Generally, phosphate transporters play a key role in the uptake of P in the crops. However, current knowledge regarding the molecular mechanism underlying Pi transport is still limited. In this study, a phosphate transporter (PT) gene, designated HvPT6, was isolated from a cDNA library constructed from hulless barley "Kunlun 14." The promoter of HvPT6 showed a large number of elements related to plant hormones. The expression pattern also indicated that HvPT6 was highly induced by low phosphorus, drought, abscisic acid, methyl jasmonate and gibberellin. Phylogenetic tree analysis revealed that HvPT6 belongs to the same subfamily of the major facilitator superfamily as OsPT6 from Oryza sativa. Subcellular localization of HvPT6:GFP using transient expression of Agrobacterium tumefaciens showed the green fluorescent protein signal in the membrane and nucleus of the Nicotiana benthamiana leaves. Overexpressing HvPT6 led to a longer and higher lateral root length and dry matter yield in the transgenic Arabidopsis lines under low Pi conditions, indicating that HvPT6 improves plant tolerance under Pi-deficient conditions. This study will lay a molecular basis for phosphate absorption mechanism in barley and breeding barley with high-efficient phosphate uptake.

Systemic Colonization of Potato Plants by Verticillium dahliae Leads to Infection of Tubers and Sprouting Buds.

A green fluorescent protein (GFP)-tagged isolate of Verticillium dahliae was used to study its colonization in potato plants and tubers. Three-week-old potato plants of the highly susceptible cultivar 'Shepody' were inoculated with a conidial suspension of a GFP-tagged isolate of V. dahliae using a wound inoculation method. Colonization was studied using confocal microscopy combined with tissue sections. Conidia germinated and hyphae grew along the root hairs, elongation zones, and root caps between 24 and 96 h postinoculation (HPI). At 7 days postinoculation (DPI), the pathogen advanced to cortical tissues and grew into the root vascular bundles. At 8 weeks postinoculation (WPI), the stem epidermal cells, cortical tissues, vascular elements, and petioles were fully colonized by the mycelium of V. dahliae. At 11 WPI, the pathogen was detected in the stolon and progeny tubers, as confirmed by both GFP signals in tissues and reisolation of the pathogen on the semiselective NP-10 medium. Progeny potato tubers were harvested from the inoculated potato plants, and the GFP-signal was observed in the epidermal cells and vascular elements of sprouting buds that emerged from the harvested tubers. The infection rate of progeny tubers detected on semiselective NP-10 medium ranged from 34.55 to 55.56%, with an average of 45.31%. In conclusion, we report, for the first time, the entire progression of colonization by V. dahliae in potato plant tissues, progeny tubers, as well as of the sprouting buds that emerged from progeny tubers.

Validation of endophytic bacteria colonisation in tissue culture-derived oil palm plantlets via green fluorescent visualization

This study aimed to validate the colonisation capability of endophytic bacteria (EB) isolates, Bacillus cereus EB2 and Pseudomonas aeruginosa EB35, that previously exhibited their potentials as biological control agents (BCAs) against the Ganoderma spp., a pathogen for Ganoderma disease in oil palm. Here, we demonstrated a rapid method to determine the colonisation capacity of the selected EB using oil palm tissue culture plantlets and a green fluorescent protein (GFP) visual marker. Wounded plantlet roots were inoculated with GFP-tagged B. cereus EB2 and P. aeruginosa EB35 while the plantlets without EB inoculation served as controls. The GFP signals appeared as bright green spots or lines in the inoculated GFP-tagged EB cells in root and leaf plantlet tissues, respectively, under the confocal laser scanning microscopy (CLSM) 5 days post-inoculation. In contrast, there was no intense GFP spots in neither the control root nor leaf tissues. The cracks in the roots by wounding facilitated the entry of the GFP-tagged EB cells into root tissues, allowing for endophytically colonisation of the root and above-ground tissues. Subsequent result of polymerase chain reaction (PCR)-GFP analysis further displayed the endophytic nature and early chronological colonisation of the tested EB. This is a preliminary report on root colonisation by a Gram-positive endophyte, B. cereus EB2 and leaf tissues colonisation by both EB isolates as internal colonisers, demonstrating their potential as BCAs to protect oil palm against Ganoderma spp. for a sustainable disease management.

Endoplasmic Reticulum Bodies in the Lateral Root Cap Are Involved in the Direct Transport of Beta-Glucosidase to Vacuoles.

Programmed cell death (PCD) in lateral root caps (LRCs) is crucial for maintaining root cap functionality. Endoplasmic reticulum (ER) bodies play important roles in plant immunity and PCD. However, the distribution of ER bodies and their communication with vacuoles in the LRC remain elusive. In this study, we investigated the ultrastructure of LRC cells of wild-type and transgenic Arabidopsis lines using an auto-acquisition transmission electron microscope (TEM) system and high-pressure freezing. Gigapixel-scale high-resolution TEM imaging of the transverse and longitudinal sections of roots followed by three-dimensional imaging identified sausage-shaped structures budding from the ER. These were subsequently identified as ER bodies using GFPh transgenic lines expressing green fluorescent protein (GFP) fused with an ER retention signal (HDEL). Immunogold labeling using an anti-GFP antibody detected GFP signals in the ER bodies and vacuoles. The fusion of ER bodies with vacuoles in LRC cells was identified using correlative light and electron microscopy. Imaging of the root tips of a GFPh transgenic line with a PYK10 promoter revealed the localization of PYK10, a member of the β-glucosidase family with an ER retention signal, in the ER bodies in the inner layer along with a fusion of ER bodies with vacuoles in the middle layer and collapse of vacuoles in the outer layer of the LRC. These findings suggest that ER bodies in LRC directly transport β-glucosidases to the vacuoles, and that a subsequent vacuolar collapse triggered by an unknown mechanism releases protective substances to the growing root tip to protect it from the invaders.

Binding immunoglobulin 2 functions as a proviral factor for potyvirus infections in Nicotiana benthamiana.

Infection of viruses from the genera Bromovirus, Potyvirus, and Potexvirus in Nicotiana benthamiana induces significant up-regulation of the genes that encode the HSP70 family, including binding immunoglobulin protein 2 (BiP2). Three up-regulated genes were knocked down and infection assays with these knockdown lines demonstrated the importance of the BiP2 gene for potyvirus infection but not for infection by the other tested viruses. Distinct symptoms of cucumber mosaic virus (CMV) and potato virus X (PVX) were observed in the BiP2 knockdown line at 10 days postagroinfiltration. Interestingly, following inoculation with either soybean mosaic virus (SMV) or pepper mottle virus (PepMoV) co-expressing green fluorescent protein (GFP), neither crinkle symptoms nor GFP signals were observed in the BiP2 knockdown line. Subsequent reverse transcription-quantitative PCR analysis demonstrated that knockdown of BiP2 resulted in a significant decrease of SMV and PepMoV RNA accumulation but not PVX or CMV RNA accumulation. Further yeast two-hybrid and co-immunoprecipitation analyses validated the interaction between BiP2 and nuclear inclusion protein b (NIb) of SMV. Together, our findings suggest the crucial role of BiP2 as a proviral host factor necessary for potyvirus infection. The interaction between BiP2 and NIb may be the critical factor determining susceptibility in N.benthamiana, but further studies are needed to elucidate the underlying mechanism.

Brain injury triggers cell-type-specific and time-dependent endoplasmic reticulum stress responses.

The unfolded protein response (UPR) is a signal transduction network that responds to endoplasmic reticulum (ER) stress by coordinating protein homeostasis to maintain cell viability. The UPR can also trigger cell death when adaptive responses fail to improve protein homeostasis. Despite accumulating evidence suggesting that the UPR plays a role in neurodegenerative diseases and brain insults, our understanding of how ER stress is induced under neuropathological conditions is limited. Here, we investigated the cell- and time-specific patterns of the ER stress response after brain injury using ER stress-activated indicator (ERAI) mice, which enable monitoring of the UPR in vivo via increased fluorescence of a spliced XBP-1 protein fused with the green fluorescent protein (GFP) variant Venus. Following cortical stab injury of ERAI mice, the GFP signal and number of GFP+ cells increased in the ipsilateral cortex throughout the observation period (6h to 7 days post-injury), confirming the induction of the UPR. GFP signals were observed in injured neurons early (from 6h) after brain injury. However, non-neuronal cells, mainly endothelial cells followed by astrocytes, accounted for the majority of GFP+ cells after brain injury. Similar results were obtained in a mouse model of focal cerebral ischemia. These findings suggest that activation of the UPR in both neuronal and non-neuronal cells, especially endothelial cells and astrocytes, may play an important role in and could be a potential therapeutic target for acute brain injuries.

Reliable online measurement of population dynamics for filamentous co‐cultures

Understanding population dynamics is a key factor for optimizing co-culture processes to produce valuable compounds. However, the measurement of independent population dynamics is difficult, especially for filamentous organisms and in presence of insoluble substrates like cellulose. We propose a workflow for fluorescence-based online monitoring of individual population dynamics of two filamentous microorganisms. The fluorescent tagged target co-culture is composed of the cellulolytic fungus Trichoderma reesei RUT-C30-mCherry and the pigment-producing bacterium Streptomyces coelicolor A3(2)-mNeonGreen (mNG) growing on insoluble cellulose as a substrate. To validate the system, the fluorescence-to-biomass and fluorescence-to-scattered-light correlation of the two strains was characterized in depth under various conditions. Thereby, especially for complex filamentous microorganisms, microbial morphologies have to be considered. Another bias can arise from autofluorescence or pigments that can spectrally interfere with the fluorescence measurement. Green autofluorescence of both strains was uncoupled from different green fluorescent protein signals through a spectral unmixing approach, resulting in a specific signal only linked to the abundance of S. coelicolor A3(2)-mNG. As proof of principle, the population dynamics of the target co-culture were measured at varying inoculation ratios in presence of insoluble cellulose particles. Thereby, the respective fluorescence signals reliably described the abundance of each partner, according to the variations in the inocula. With this method, conditions can be fine-tuned for optimal growth of both partners along with natural product formation by the bacterium.

Novel role of UHRF1 in the epigenetic repression of the latent HIV-1.

SummaryBackgroundThe multiplicity, heterogeneity, and dynamic nature of human immunodeficiency virus type-1 (HIV-1) latency mechanisms are reflected in the current lack of functional cure for HIV-1. Accordingly, all classes of latency-reversing agents (LRAs) have been reported to present variable ex vivo potencies. Here, we investigated the molecular mechanisms underlying the potency variability of one LRA: the DNA methylation inhibitor 5-aza-2’-deoxycytidine (5-AzadC).MethodsWe employed epigenetic interrogation methods (electrophoretic mobility shift assays, chromatin immunoprecipitation, Infinium array) in complementary HIV-1 infection models (latently-infected T-cell line models, primary CD4+ T-cell models and ex vivo cultures of PBMCs from HIV+ individuals). Extracellular staining of cell surface receptors and intracellular metabolic activity were measured in drug-treated cells. HIV-1 expression in reactivation studies was explored by combining the measures of capsid p24Gag protein, green fluorescence protein signal, intracellular and extracellular viral RNA and viral DNA.FindingsWe uncovered specific demethylation CpG signatures induced by 5-AzadC in the HIV-1 promoter. By analyzing the binding modalities to these CpG, we revealed the recruitment of the epigenetic integrator Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) to the HIV-1 promoter. We showed that UHRF1 redundantly binds to the HIV-1 promoter with different binding modalities where DNA methylation was either non-essential, essential or enhancing UHRF1 binding. We further demonstrated the role of UHRF1 in the epigenetic repression of the latent viral promoter by a concerted control of DNA and histone methylations.InterpretationA better understanding of the molecular mechanisms of HIV-1 latency allows for the development of innovative antiviral strategies. As a proof-of-concept, we showed that pharmacological inhibition of UHRF1 in ex vivo HIV+ patient cell cultures resulted in potent viral reactivation from latency. Together, we identify UHRF1 as a novel actor in HIV-1 epigenetic silencing and highlight that it constitutes a new molecular target for HIV-1 cure strategies.FundingFunding was provided by the Belgian National Fund for Scientific Research (F.R.S.-FNRS, Belgium), the « Fondation Roi Baudouin », the NEAT (European AIDS Treatment Network) program, the Internationale Brachet Stiftung, ViiV Healthcare, the Télévie, the Walloon Region (« Fonds de Maturation »), « Les Amis des Instituts Pasteur à Bruxelles, asbl », the University of Brussels (Action de Recherche Concertée ULB grant), the Marie Skodowska Curie COFUND action, the European Union's Horizon 2020 research and innovation program under grant agreement No 691119-EU4HIVCURE-H2020-MSCA-RISE-2015, the French Agency for Research on AIDS and Viral Hepatitis (ANRS), the Sidaction and the “Alsace contre le Cancer” Foundation. This work is supported by 1UM1AI164562-01, co-funded by National Heart, Lung and Blood Institute, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke, National Institute on Drug Abuse and the National Institute of Allergy and Infectious Diseases.

MicroRNA828 negatively regulates lignin biosynthesis in stem of Populus tomentosa through MYB targets.

Lignin biosynthesis in the sclerenchyma cells is strictly controlled by a complex network of genetic and environmental signals. In the last decades, the transcriptional regulation of lignin synthesis in woody species has been established. However, the role of microRNA-mediated post-transcriptional modulation in secondary cell wall biosynthesis remains poorly understood. Here, we identified a microRNA, miR828, involved in the regulation specific to lignin biosynthesis during stem development in Populus tomentosa Carr. miR828 is preferentially expressed in the secondary vascular tissues during stem development. Two MYB genes (MYB171 and MYB011) were validated as direct targets of miR828 by degradome analysis and green fluorescent protein signal detection. Overexpression of miR828 in poplar downregulated genes for lignin biosynthesis, resulting in reduced lignin content in cell walls. Conversely, suppression of miR828 in plants by the short tandem target mimics elevated the expression of lignin biosynthetic genes and increased lignin deposition. We further revealed that poplar MYB171, as the most abundant miR828 target in the stem, is a positive regulator for lignin biosynthesis. Transient expression assays showed that both MYB171 and MYB011 activated PAL1 and CCR2 transcription, whereas the introduction of miR828 significantly suppressed their expression that was induced by MYB171 or MYB011. Collectively, our results demonstrate that the miR828-MYBs module precisely regulates lignin biosynthesis during the stem development in P. tomentosa through transcriptional and post-transcriptional manners.

ACE2 internalization induced by a SARS-CoV-2 recombinant protein is modulated by angiotensin II type 1 and bradykinin 2 receptors

AimsAngiotensin-converting enzyme 2 (ACE2) is a key regulator of the renin-angiotensin system (RAS) recently identified as the membrane receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we aim to study whether two receptors from RAS, the angiotensin receptor type 1 (AT1R) and the bradykinin 2 receptor (B2R) modulate ACE2 internalization induced by a recombinant receptor binding domain (RBD) of SARS-CoV-2 spike protein. Also, we investigated the impact of ACE2 coexpression on AT1R and B2R functionality. Materials and methodsTo study ACE2 internalization, we assessed the distribution of green fluorescent protein (GFP) signal in HEK293T cells coexpressing GFP-tagged ACE2 and AT1R, or B2R, or AT1R plus B2R in presence of RBD alone or in combination with AT1R or B2R ligands. To estimate ACE2 internalization, we classified GFP signal distribution as plasma membrane uniform GFP (PMU-GFP), plasma membrane clustered GFP (PMC-GFP) or internalized GFP and calculated its relative frequency. Additionally, we investigated the effect of ACE2 coexpression on AT1R and B2R inhibitory action on voltage-gated calcium channels (CaV2.2) currents by patch-clamp technique. Key findingsRBD induced ACE2-GFP internalization in a time-dependent manner. RBD-induced ACE2-GFP internalization was increased by angiotensin II and reduced by telmisartan in cells coexpressing AT1R. RBD-induced ACE2-GFP internalization was strongly inhibited by B2R co-expression. This effect was mildly modified by bradykinin and rescued by angiotensin II in presence of AT1R. ACE2 coexpression impacted on B2R- and AT1R-mediated inhibition of CaV2.2 currents. SignificanceOur work contributes to understand the role of RAS modulators in the susceptibility to SARS-CoV-2 infection and severity of COVID-19.

TWIK-1 BAC-GFP Transgenic Mice, an Animal Model for TWIK-1 Expression.

TWIK-1 is the first identified member of the two-pore domain potassium (K2P) channels that are involved in neuronal excitability and astrocytic passive conductance in the brain. Despite the physiological roles of TWIK-1, there is still a lack of information on the basic expression patterns of TWIK-1 proteins in the brain. Here, using a modified bacterial artificial chromosome (BAC), we generated a transgenic mouse (Tg mouse) line expressing green fluorescent protein (GFP) under the control of the TWIK-1 promoter (TWIK-1 BAC-GFP Tg mice). We confirmed that nearly all GFP-producing cells co-expressed endogenous TWIK-1 in the brain of TWIK-1 BAC-GFP Tg mice. GFP signals were highly expressed in various brain areas, including the dentate gyrus (DG), lateral entorhinal cortex (LEC), and cerebellum (Cb). In addition, we found that GFP signals were highly expressed in immature granule cells in the DG. Finally, our TWIK-1 BAC-GFP Tg mice mimic the upregulation of TWIK-1 mRNA expression in the hippocampus following the injection of kainic acid (KA). Our data clearly showed that TWIK-1 BAC-GFP Tg mice are a useful animal model for studying the mechanisms regulating TWIK-1 gene expression and the physiological roles of TWIK-1 channels in the brain.

Generation of the Y-chromosome linked red fluorescent protein transgenic mouse model and sexing at the preimplantation stage.

In mammals, sexual fate is determined by the chromosomes of the male and female gametes during fertilization. Males (XY) or females (XX) are produced when a sperm containing a Y or X-chromosome respectively fertilizes an X-chromosome-containing unfertilized egg. However, sexing of preimplantation stage embryos cannot be conducted visually. To address this, transgenic male mouse models with the ubiquitously expressed green fluorescent protein (GFP) transgene on X- (X-GFP) or Y-chromosomes (Y-GFP) have been established. However, when crossed with wild-type females, sexing of the preimplantation stage embryos by observing the GFP signal is problematic in some cases due to X-inactivation, loss of Y-chromosome (LOY), or loss of transgene fluorescence. In this study, a mouse model with the ubiquitously expressed red fluorescent protein (RFP) transgene on the Y-chromosome was generated since RFP is easily distinguishable from GFP signals. Unfortunately, the ubiquitously expressed tdTomato RFP transgene on the Y-chromosome (Y-RFP) mouse showed the lethal phenotype after birth. No lethal phenotypes were observed when the mitochondrial locating signal N-terminal of tdTomato (mtRFP) was included in the transgene construct. Almost half of the collected fertilized eggs from Y-mtRFP male mice crossed with wild-type females had an RFP signal at the preimplantation stage (E1.5). Therefore, XY eggs were recognized as RFP-positive embryos at the preimplantation stage. Furthermore, 100% sexing was observed at the preimplantation stage using the X-linked GFP/Y-linked RFP male mouse. The established Y-mtRFP mouse models may be used to study sex chromosome related research.