Nuclear Protein Research Articles

AAV-NRIP gene therapy ameliorates motor neuron degeneration and muscle atrophy in ALS model mice

BackgroundAmyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron (MN) degeneration, leading to neuromuscular junction (NMJ) dismantling and severe muscle atrophy. The nuclear receptor interaction protein (NRIP) functions as a multifunctional protein. It directly interacts with calmodulin or α-actinin 2, serving as a calcium sensor for muscle contraction and maintaining sarcomere integrity. Additionally, NRIP binds with the acetylcholine receptor (AChR) for NMJ stabilization. Loss of NRIP in muscles results in progressive motor neuron degeneration with abnormal NMJ architecture, resembling ALS phenotypes. Therefore, we hypothesize that NRIP could be a therapeutic factor for ALS.MethodsWe used SOD1 G93A mice, expressing human SOD1 with the ALS-linked G93A mutation, as an ALS model. An adeno-associated virus vector encoding the human NRIP gene (AAV-NRIP) was generated and injected into the muscles of SOD1 G93A mice at 60 days of age, before disease onset. Pathological and behavioral changes were measured to evaluate the therapeutic effects of AAV-NRIP on the disease progression of SOD1 G93A mice.ResultsSOD1 G93A mice exhibited lower NRIP expression than wild-type mice in both the spinal cord and skeletal muscle tissues. Forced NRIP expression through AAV-NRIP intramuscular injection was observed in skeletal muscles and retrogradely transduced into the spinal cord. AAV-NRIP gene therapy enhanced movement distance and rearing frequencies in SOD1 G93A mice. Moreover, AAV-NRIP increased myofiber size and slow myosin expression, ameliorated NMJ degeneration and axon terminal denervation at NMJ, and increased the number of α-motor neurons (α-MNs) and compound muscle action potential (CMAP) in SOD1 G93A mice.ConclusionsAAV-NRIP gene therapy ameliorates muscle atrophy, motor neuron degeneration, and axon terminal denervation at NMJ, leading to increased NMJ transmission and improved motor functions in SOD1 G93A mice. Collectively, AAV-NRIP could be a potential therapeutic drug for ALS.

An electrochemical ratiometric immunosensor for the detection of NMP22 based on ZIF-8@MWCNTs@Chit@Fc@AuNPs and AuPt-MB.

Nuclear matrix protein 22 (NMP22) is one of the most important tumor markers of bladder cancer and is significantly elevated in the urine of bladder cancer patients. Therefore, in this work, a highly sensitive ratiometric electrochemical immunosensor was constructed to detect NMP22 based on ZIF-8@MWCNTs@Chit@Fc@AuNPs composites. ZIF-8 had a large surface area and good adsorption ability. Multi-Walled Carbon Nanotubes (MWCNTs) can optimize the electrical conductivity of ZIF-8, so that the electrode surface of ferrocene (Fc) obtains a stable and strong electrochemical signal. In addition, AuPt-MB provided another strong detection signal methylene blue (MB) while immobilizing the secondary antibody (Ab2) through Au-N and Pt-N bonds. A ratiometric electrochemical sensor was formed based on ZIF-8@MWCNTs@Chit@Fc@AuNPs and AuPt-MB, which showed a great linear connection between IMB/IFc and the logarithmic concentration of NMP22 with a detection limit of 3.33 fg mL-1 (S/N = 3) under optimized specifications in the concentration interval of 0.01 pg mL-1 to 1000 ng mL-1. In addition, the ratiometric immunosensor showed good selectivity and stability.

Spatiotemporal analysis of multi-scale cell structure in spheroid culture reveals hypertrophic chondrocyte differentiation.

3D cell culture has emerged as a promising approach to replicate the complex behaviors of cells within living organisms. This study aims to analyze spatiotemporal behavior of the morphological characteristics of cell structure at multiscale in 3D scaffold-free spheroids using chondrogenic progenitor ATDC5 cells. Over a 14-day culture period, it exhibited cell hypertrophy in the spheroids regarding cellular and nuclear size as well as changes in morphology. Moreover, biological analysis indicated a signification up-regulation of normal chondrocyte as well as hypertrophic chondrocyte markers, suggesting early hypertrophic chondrocyte differentiation. Cell nuclei underwent changes in volume, sphericity, and distribution in spheroid over time, indicating alterations in chromatin organization. The ratio of chromatin condensation volume to cell nuclear volume decreased as the cell nuclei enlarged, potentially signifying changes in chromatin state during hypertrophic chondrocyte differentiation. Our image analysis techniques in this present study enabled detailed morphological measurement of cell structure at multi-scale, which can be applied to various 3D culture models for in-depth investigation.

Gestational physical activity alters offspring brain APP processing in an age-specific manner.

Maternal exercise is beneficial for offspring brain development. Amyloid precursor protein (APP) influences neurogenesis and synaptic plasticity. Cleavage products of APP are implicated in the proliferation of neural progenitor cells and neuronal network development. Our study aimed to investigate differences in APP processing in active or sedentary offspring of dams who were exposed to voluntary wheel running with and without a western diet throughout gestation. Female Wistar rats (7-8 weeks old) were fed a normal chow or western diet and randomized into voluntary wheel run or sedentary conditions. Dams returned to sedentary conditions post-parturition. The pups were weaned at 6 weeks after which point half of the samples were collected, while the rest of the pups remained on a normal diet, separated into sedentary or voluntary wheel run groups, and collected 12 weeks later. In-utero exposure to maternal exercise was associated with higher neuronal nuclear protein, higher soluble APPα and lower soluble APPβ in offspring prefrontal cortex tissue at 6, but not 18 weeks of age. Neuronal nuclear protein is exclusive to mature neurons implying that offspring of mothers who exercised could have more neuron maturation potentially influenced by the higher APPα content at this early developmental stage. The voluntary wheel run offspring groups had a higher mature/pro brain derived neurotrophic factor ratio compared to the sedentary counterparts. The maternal effects were isolated to the juvenile 6-week-old pups, while the differences in the adult offspring were caused by their own exercise status.

Applying the area fraction fractionator (AFF) probe for total volume estimations of somatic, dendritic and axonal domains of the nigrostriatal dopaminergic system in a murine model

BackgroundThe Cavalieri estimator is used for volume measurement of brain and brain regions. Derived from this estimator is the Area Fraction Fractionator (AFF), used for efficient area and number estimations of small 2D elements, such as axons in cross-sectioned nerves. However, to our knowledge, the AFF has not been combined with serial sectioning analysis to measure the volume of small-size nervous structures. New methodUsing the nigrostriatal dopaminergic system as an illustrative case, we describe a protocol based on Cavalieri's principle and AFF to estimate the volume of its somatic, nuclear, dendritic, axonal and axon terminal cellular compartments in the adult mouse. The protocol consists of (1) systematic random sampling of sites within and across sections in regions of interest (substantia nigra, the nigrostriatal tract, caudate-putamen), (2) confocal image acquisition of sites, (3) marking of cellular domains using Cavalieri's 2D point-counting grids, and 4) determination of compartments’ total volume using the estimated area of each compartment, and between-sections distance. ResultsThe volume of the nigrostriatal system is ~0.38 mm3, with ~5% corresponding to perikarya and cell nuclei, ~10% to neuropil/dendrites, and ~85% to axons and varicosities. Comparison with existing methodsIn contrast to other methods to measure volume of discrete objects, such as the optical nucleator or 3D reconstructions, it stands out for its versatility and ease of use. ConclusionsThe use of a simple quantitative, unbiased approach to assess the global state of a system may allow quantification of compartment-specific changes that may accompany neurodegenerative processes.

Maintenance of germline stem cell homeostasis despite severe nuclear distortion

Stem cell loss in aging and disease is associated with nuclear deformation. Yet, how nuclear shape influences stem cell homeostasis is poorly understood. We investigated this connection using Drosophila germline stem cells, as survival of these stem cells is compromised by dysfunction of the nuclear lamina, the extensive protein network that lines the inner nuclear membrane and gives shape to the nucleus. To induce nuclear distortion in germline stem cells, we used the GAL4-UAS system to increase expression of the permanently farnesylated nuclear lamina protein, Kugelkern, a rate limiting factor for nuclear growth. We show that elevated Kugelkern levels cause severe nuclear distortion in germline stem cells, including extensive thickening and lobulation of the nuclear envelope and nuclear lamina, as well as alteration of internal nuclear compartments. Despite these changes, germline stem cell number, proliferation, and female fertility are preserved, even as females age. Collectively, these data demonstrate that disruption of nuclear architecture does not cause a failure of germline stem cell survival or homeostasis, revealing that nuclear deformation does not invariably promote stem cell loss.

A computational model for single cell Lamin-A structural organization after microfluidic compression.

In recent years, nuclear mechanobiology gained a lot of attention for the study of cell responses to external cues like adhesive forces, applied compression, and/or shear-stresses. In details, the Lamin-A protein-as major constituent of the cell nucleus structure-plays a crucial role in the overall nucleus mechanobiological response. However, modeling and analysis of Lamin-A protein organization upon rapid compression conditions in microfluidics are still difficult to be performed. Here, we introduce the possibility to control an applied microfluidic compression on single cells, deforming them up to the nucleus level. In a wide range of stresses (~1-102 kPa) applied on healthy and cancer cells, we report increasing Lamin-A intensities which scale as a power law with the applied compression. Then, an increase up to two times of the nuclear viscosity is measured in healthy cells, due to the modified Lamin-A organization. This is ascribable to the increasing assembly of Lamin-A filament-like branches which increment both in number and elongation (up to branches four-timelonger). Moreover, the solution of a computational model of differential equations is presented as a powerful tool for a single cell prediction of the Lamin-A assembly as a function of the applied compression.

The Role of Long Non-Coding RNF144A-AS1 in Cancer Progression.

RNAs transcribing more than 200 nucleotides without encoding proteins are termed long non-coding RNAs (LncRNAs). LncRNAs can be used as decoy molecules, signal molecules, scaffolds, and guide molecules. Long non-coding RNAs can interact with DNA, chromatin-modifying complexes, and transcriptional regulatory proteins, regulating gene expression in the cell nucleus. It is distributed in cytoplasm; they also participate in mRNA degradation and translational regulation via miRNAs, other transcription products, and proteins. They play a significant role in the development of various diseases, including tumors. Cancer seriously threatens human life and health. Regretfully, a great deal of newly diagnosed cancer patients found to have metastasized. RNF144A-AS1, also referred to as GRASLND, was initially recognized for its regulation of chondrogenic differentiation in MSCs. Focusing on RNF144A-AS1, this review summarizes and discusses the latest progress of RNF144A-AS1 in bladder cancer, glioblastoma, papillary renal cell carcinoma, gastric cancer, osteosarcoma, head and neck squamous cell carcinoma, and ovarian cancer. RNF144A-AS1 has good potential in tumor treatment and diagnosis.

AKT-dependent nuclear localization of EPRS1 activates PARP1 in breast cancer cells

Glutamyl-prolyl-tRNA synthetase (EPRS1) is a bifunctional aminoacyl-tRNA-synthetase (aaRS) essential for decoding the genetic code. EPRS1 resides, with seven other aaRSs and three noncatalytic proteins, in the cytoplasmic multi-tRNA synthetase complex (MSC). Multiple MSC-resident aaRSs, including EPRS1, exhibit stimulus-dependent release from the MSC to perform noncanonical activities distinct from their primary function in protein synthesis. Here, we show EPRS1 is present in both cytoplasm and nucleus of breast cancer cells with constitutively low phosphatase and tensin homolog (PTEN) expression. EPRS1 is primarily cytosolic in PTEN-expressing cells, but chemical or genetic inhibition of PTEN, or chemical or stress-mediated activation of its target, AKT, induces EPRS1 nuclear localization. Likewise, preferential nuclear localization of EPRS1 was observed in invasive ductal carcinoma that were also P-Ser473-AKT+. EPRS1 nuclear transport requires a nuclear localization signal (NLS) within the linker region that joins the catalytic glutamyl-tRNA synthetase and prolyl-tRNA synthetase domains. Nuclear EPRS1 interacts with poly(ADP-ribose) polymerase 1 (PARP1), a DNA-damage sensor that directs poly(ADP-ribosyl)ation (PARylation) of proteins. EPRS1 is a critical regulator of PARP1 activity as shown by markedly reduced ADP-ribosylation in EPRS1 knockdown cells. Moreover, EPRS1 and PARP1 knockdown comparably alter the expression of multiple tumor-related genes, inhibit DNA-damage repair, reduce tumor cell survival, and diminish tumor sphere formation by breast cancer cells. EPRS1-mediated regulation of PARP1 activity provides a mechanistic link between PTEN loss in breast cancer cells, PARP1 activation, and cell survival and tumor growth. Targeting the noncanonical activity of EPRS1, without inhibiting canonical tRNA ligase activity, provides a therapeutic approach potentially supplementing existing PARP1 inhibitors.

The G9a histone methyltransferase represses osteoclastogenesis and bone resorption by regulating NFATc1 function.

Epigenetic modifications affect cell differentiation via transcriptional regulation. G9a/EHMT2 is an important epigenetic modifier that catalyzes the methylation of histone 3 lysine 9 (H3K9) and interacts with various nuclear proteins. In this study, we investigated the role of G9a in osteoclast differentiation. When we deleted G9a by infection of Cre-expressing adenovirus into bone marrow macrophages (BMMs) from G9afl/fl (Ehmt2fl/fl) and induced osteoclastic differentiation by the addition of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), the number of TRAP-positive multinucleated osteoclasts significantly increased compared with control. Furthermore, the mRNA expression of osteoclast markers, TRAP, and cathepsin K, and to a lesser extent, NFATc1, a critical transcription factor, increased in G9a KO cells. Infection of wild-type (WT) G9a-expressing adenovirus in G9a KO cells restored the number of TRAP-positive multinucleated cells. In G9a KO cells, increased nuclear accumulation of NFATc1 protein and decreased H3K9me2 accumulation were observed. Furthermore, ChIP experiments revealed that NFATc1 binding to its target, Ctsk promoter, was enhanced by G9a deletion. For invivo experiments, we created G9a conditional knock-out (cKO) mice by crossing G9afl/fl mice with Rank Cre/+ (Tnfrsf11aCre/+) mice, in which G9a is deleted in osteoclast lineage cells. The trabecular bone volume was significantly reduced in female G9a cKO mice. The serum concentration of the C-terminal telopeptide of type I collagen (CTX), a bone-resorbing indicator, was higher in G9a cKO mice. In addition, osteoclasts differentiated from G9a cKO BMMs exhibited greater bone-resorbing activity. Our findings suggest that G9a plays a repressive role in osteoclastogenesis by modulating NFATc1 function.

The dual role of FSP1 in programmed cell death: resisting ferroptosis in the cell membrane and promoting necroptosis in the nucleus of THP-1 cells

BackgroundAcute monocytic leukemia-M5 (AML-M5) remains a challenging disease due to its high morbidity and poor prognosis. In addition to the evidence mentioned earlier, several studies have shown that programmed cell death (PCD) serves a critical function in treatment of AML-M5. However, the role and relationship between ferroptosis and necroptosis in AML-M5 remains unclear.MethodsTHP-1 cells were mainly treated with Erastin and IMP-366. The changes of ferroptosis and necroptosis levels were detected by CCK-8, western blot, quantitative real-time PCR, and electron microscopy. Flow cytometry was applied to detect the ROS and lipid ROS levels. MDA, 4-HNE, GSH and GSSG were assessed by ELISA kits. Intracellular distribution of FSP1 was studied by immunofluorescent staining and western blot.ResultsThe addition of the myristoylation inhibitor IMP-366 to erastin-treated acute monocytic leukemia cell line THP-1 cell not only resulted in greater susceptibility to ferroptosis characterized by lipid peroxidation, glutathione (GSH) depletion and mitochondrial shrinkage, as the FSP1 position on membrane was inhibited, but also increased p-RIPK1 and p-MLKL protein expression, as well as a decrease in caspase-8 expression, and triggered the characteristic necroptosis phenomena, including cytoplasmic translucency, mitochondrial swelling, membranous fractures by FSP1 migration into the nucleus via binding importin α2. It is interesting to note that ferroptosis inhibitor fer-1 reversed necroptosis.ConclusionWe demonstrated that inhibition of myristoylation by IMP-366 is capable of switching ferroptosis and ferroptosis-dependent necroptosis in THP-1 cells. In these findings, FSP1-mediated ferroptosis and necroptosis are described as alternative mechanisms of PCD of THP-1 cells, providing potential therapeutic strategies and targets for AML-M5.

Mitonuclear compatibility is maintained despite relaxed selection on male mitochondrial DNA in bivalves with doubly uniparental inheritance.

Mitonuclear coevolution is common in eukaryotes, but bivalve lineages that have doubly uniparental inheritance (DUI) of mitochondria may be an interesting example. In this system, females transmit mtDNA (F mtDNA) to all offspring, while males transmit a different mtDNA (M mtDNA) solely to their sons. Molecular evolution and functional data suggest oxidative phosphorylation (OXPHOS) genes encoded in M mtDNA evolve under relaxed selection due to their function being limited to sperm only (vs. all other tissues for F mtDNA). This has led to the hypothesis that mitonuclear coevolution is less important for M mtDNA. Here, we use comparative phylogenetics, transcriptomics, and proteomics to understand mitonuclear interactions in DUI bivalves. We found nuclear OXPHOS proteins coevolve and maintain compatibility similarly with both F and M mtDNA OXPHOS proteins. Mitochondrial recombination did not influence mitonuclear compatibility and nuclear-encoded OXPHOS genes were not upregulated in tissues with M mtDNA to offset dysfunction. Our results support that selection maintains mitonuclear compatibility with F and M mtDNA despite relaxed selection on M mtDNA. Strict sperm transmission, lower effective population size, and higher mutation rates may explain the evolution of M mtDNA. Our study highlights that mitonuclear coevolution and compatibility may be broad features of eukaryotes.

Differential expression of various isoforms of superoxide dismutase in the cells of the human exocrine pancreas.

Superoxide dismutase (SOD) is an enzyme that plays acrucial role in protecting cells from oxidative damage. Our study aims to address the lack of papers simultaneously analyzing the immunoreactivity of all three distinct isoforms of SOD in human exocrine pancreas cells. Superoxide dismutases (SODs) facilitate the conversion of superoxide radicals into less harmful substances. By neutralizing superoxide radicals, SODs help prevent the formation of highly reactive and destructive species that can adversely affect manifold cellular components. The study analyzed immunoreactivity of SODs in samples of six healthy adult human pancreases, while using the indirect immunohistochemical method under alight microscope. SOD1 was predominantly found in centroacinar cells and epithelial cells of the duct system while SOD2 was mainly detected in the epithelial cells of interlobular ducts. Both enzymes were prominently present in the basal region of acinar cells near the cell nucleus. The expression of SOD3 was observed to be rare. Understanding the intracellular metabolism of SODs in healthy exocrine pancreas cells serves as abasis for determining the precise role of oxidative damage and SOD signaling in the pathogenesis of various pancreatic diseases, including chronic pancreatitis and pancreatic cancer (Fig. 6, Ref. 24). Text in PDF www.elis.sk Keywords: antioxidants, histology, immunohistochemistry, pancreas, superoxide dismutase.

Molecular cloning and functional study of a Rel homologous gene in sea urchin Strongylocentrotus intermedius

NF-κB (Nuclear factor-kappa B) family proteins are versatile transcription factors that play crucial regulatory roles in cell development, growth, apoptosis, inflammation, and immune response. However, there is limited research on the function of these key genes in echinoderms. In this study, an NF-κB family gene (SiRel) was identified in sea urchin Strongylocentrotus intermedius. The gene has an open reading frame length of 1809 bp and encodes for 602 amino acids. Domain prediction results revealed that the N-terminal of SiRel protein encodes a conserved Rel homology domain (RHD), including the RHD-DNA binding domain and the RHD-dimerization domain. Multiple sequence comparison results showed that the protein sequences of these two domains were conserved. Phylogenetic analysis indicated that SiRel clustered with Strongylocentrotus purpuratus p65 protein and Rel protein of other echinoderms. Results from quantitative real-time PCR demonstrated detectable SiRel mRNA expression in all tested sea urchin tissues, with the highest expression level found in the gills. And SiRel mRNA expression levels were significantly induced after LPS (Lipopolysaccharide) and poly(I:C) (Polyinosinic:polycytidylic acid) stimulation. In addition, SiRel protein expression can be found in cytoplasm and nucleus of HEK293T cells. Co-immunoprecipitation results showed that SiRel could interact with sea urchin IκB (Inhibitor of NF-κB) protein. Western blotting and dual-luciferase reporter gene assay results indicated that overexpression of SiRel in HEK293T cells could impact the phosphorylation levels of JNK (c-Jun N-terminal kinase) and Erk1/2 (Extracellular signal-regulated kinases1/2) and activate interleukin-6 (IL-6), activating protein 1 (AP-1), interferon (IFN)α/β/γ, and signal transducer and activator of transcription 3 (STAT3) reporter genes in HEK293T cells. In conclusion, this study reveals that SiRel plays an important role in the innate immune response of sea urchins and enriches our understanding of comparative immunology theory.

Synaptic Vesicle-Related Proteins and Ubiquilin 2 in Cortical Synaptosomes Mediate Cognitive Impairment in Vascular Dementia Rats.

Synaptic dysfunction is considered the best neuropathological correlate of cognitive decline in vascular dementia (VaD). However, the alterations of synaptic proteins at the synaptosomal level in VaD remain unclear. In this study, a VaD model was established in male rats using bilateral common carotid artery occlusion (2VO). We performed a novel object recognition task to evaluate cognitive impairment. Immunohistochemistry was used to assess the expression of neuron-specific nuclear binding protein (NeuN). Brain synaptosomes were isolated and subjected to label-free proteomic analysis to quantify and identify the synaptic features of differentially expressed proteins (DEPs). Synaptic and hub protein expression was detected in synaptosomes using western blotting. We found that male rats with VaD presented impaired memory and decreased NeuN protein expression in the cortex. Synaptosome proteomic analysis revealed 604 DEPs, with 493 and 111 markedly downregulated and upregulated proteins, respectively. KEGG analysis and SynGO annotation revealed that the synaptic vesicle (SV) cycle may be a key signaling pathway in VaD. Hub protein analysis of the main nodes in the protein network identified UBQLN2 and SV-related proteins, including CLTC, SNAP91, AP2S1, CLTA, VAMP2, EPN1, UBQLN2, AP2B1, AP2A2, and AP2M1. Western blotting showed that the levels of SV2A, CLTC, AP2S1, and VAMP2 decreased in the synaptosomes of 2VO rats, while UBQLN2 expression significantly increased. Our results suggest that the disruption in the presynaptic SV cycle is a key event in male rats with VaD, which could be characterized by the aberrant SV2A expression. SV-related proteins and UBQLN2 may be essential in synaptopathy. Thus, targeting the specific molecular markers in synaptosomes may be critical for the development of mechanism-directed therapies against VaD.

Nucleus-targeting Oxaplatin(IV) prodrug Amphiphile for enhanced chemotherapy and immunotherapy

Platinum(II)-based drugs (PtII), which hinder DNA replication, are the most widely used chemotherapeutics. However, current PtII drugs often miss their DNA targets, leading to severe side effects and drug resistance. To overcome this challenge, we developed a novel oxaliplatin-based platinum(IV) (PtIV) prodrug amphiphile (C16-OPtIV-R8K), integrating a long-chain hydrophobic lipid and a nucleus-targeting hydrophilic peptide (R8K). This design allows the prodrug to self-assemble into highly uniform lipid nanoparticles (NTPtIV) for enhanced targeting chemotherapy and immunotherapy. Subsequently, NTPtIV's bioactivity and effects were examined at diverse levels, encompassing cancer cells, 3D tumor spheres, and in vivo. Our in vitro studies show a 74% cancer cell nucleus localization of platinum drugs-3.6 times higher than that of oxaliplatin, achieving more than a ten-fold increase in eliminating drug-resistant cancer cells. In vivo, NTPtIV shows efficient tumor accumulation, leading to suppressed tumor growth of murine breast cancer. Moreover, NTPtIV recruited more CD4+ and CD8+ T cells and reduced CD4+ Foxp3+ Tregs to synergistically enhance targeted chemotherapy and immunotherapy. Overall, this strategy presents a promising advancement in nucleus-targeted cancer therapy, synergistically boosting the efficacy of chemotherapy and immunotherapy.

The transcription factor ERF110 promotes cold tolerance by directly regulating sugar and sterol biosynthesis in citrus.

ERFs (ethylene-responsive factors) are known to play a key role in orchestrating cold stress signal transduction. However, the regulatory mechanisms and target genes of most ERFs are far from being well deciphered. In this study, we identified a cold-induced ERF, designated as PtrERF110, from trifoliate orange (Poncirus trifoliata L. Raf., also known as Citrus trifoliata L.), an elite cold-hardy plant. PtrERF110 is a nuclear protein with transcriptional activation activity. Overexpression of PtrERF110 remarkably enhanced cold tolerance in lemon (Citrus limon) and tobacco (Nicotiana tabacum), whereas VIGS (virus-induced gene silencing)-mediated knockdown of PtrERF110 drastically impaired the cold tolerance. RNA sequence analysis revealed that PtrERF110 overexpression resulted in global transcriptional reprogramming of a range of stress-responsive genes. Three of the genes, including PtrERD6L16 (early responsive dehydration 6-like transporters), PtrSPS4 (sucrose phosphate synthase 4), and PtrUGT80B1 (UDP-glucose: sterol glycosyltransferases 80B1), were confirmed as direct targets of PtrERF110. Consistently, PtrERF110-overexpressing plants exhibited higher levels of sugars and sterols compared to their wild type counterparts, whereas the VIGS plants had an opposite trend. Exogenous supply of sucrose restored the cold tolerance of PtrERF110-silencing plants. In addition, knockdown of PtrSPS4, PtrERD6L16, and PtrUGT80B1 substantially impaired the cold tolerance of P. trifoliata. Taken together, our findings indicate that PtrERF110 positively modulates cold tolerance by directly regulating sugar and sterol synthesis through transcriptionally activating PtrERD6L16, PtrSPS4, and PtrUGT80B1. The regulatory modules (ERF110-ERD6L16/SPS4/UGT80B1) unraveled in this study advance our understanding of the molecular mechanisms underlying sugar and sterol accumulation in plants subjected to cold stress.

The transcription factor ERF110 promotes cold tolerance by directly regulating sugar and sterol biosynthesis in citrus.

ERFs (ethylene-responsive factors) are known to play a key role in orchestrating cold stress signal transduction. However, the regulatory mechanisms and target genes of most ERFs are far from being well deciphered. In this study, we identified a cold-induced ERF, designated as PtrERF110, from trifoliate orange (Poncirus trifoliata L. Raf., also known as Citrus trifoliata L.), an elite cold-hardy plant. PtrERF110 is a nuclear protein with transcriptional activation activity. Overexpression of PtrERF110 remarkably enhanced cold tolerance in lemon (Citrus limon) and tobacco (Nicotiana tabacum), whereas VIGS (virus-induced gene silencing)-mediated knockdown of PtrERF110 drastically impaired the cold tolerance. RNA sequence analysis revealed that PtrERF110 overexpression resulted in global transcriptional reprogramming of a range of stress-responsive genes. Three of the genes, including PtrERD6L16 (early responsive dehydration 6-like transporters), PtrSPS4 (sucrose phosphate synthase 4), and PtrUGT80B1 (UDP-glucose: sterol glycosyltransferases 80B1), were confirmed as direct targets of PtrERF110. Consistently, PtrERF110-overexpressing plants exhibited higher levels of sugars and sterols compared to their wild type counterparts, whereas the VIGS plants had an opposite trend. Exogenous supply of sucrose restored the cold tolerance of PtrERF110-silencing plants. In addition, knockdown of PtrSPS4, PtrERD6L16, and PtrUGT80B1 substantially impaired the cold tolerance of P. trifoliata. Taken together, our findings indicate that PtrERF110 positively modulates cold tolerance by directly regulating sugar and sterol synthesis through transcriptionally activating PtrERD6L16, PtrSPS4, and PtrUGT80B1. The regulatory modules (ERF110-ERD6L16/SPS4/UGT80B1) unraveled in this study advance our understanding of the molecular mechanisms underlying sugar and sterol accumulation in plants subjected to cold stress.

Role of nuclear protein Akirin in the modulation of female reproduction in Nilaparvata lugens (Hemiptera: Delphacidae)

Introduction: Akirin as a highly conserved transcription factor, exerts a profound influence on the growth, development, immune response, and reproductive processes in animals. The brown planthopper (BPH), Nilaparvata lugens, a major pest in rice production in Asia, possesses high reproductive capacity, a critical factor contributing to reduced rice yields. The aims of this study were to demonstrate the regulatory role of Akirin in the reproduction of BPH.Methods: In this study, quantitative PCR (qPCR) was used to detect the mRNA expression of genes. RNA interference (RNAi) was used to downregulate the expression of Akirin gene, and RNA sequencing (RNA-seq) was used to screen for differentially expressed genes caused by Akirin downregulation. Hormone contents were measured with the enzyme linked immunosorbent assay (ELISA), and protein content was evaluated with the bicinchoninic acid (BCA) method.Results: Using BPH genome data, we screened for an Akirin gene (NlAkirin). An analysis of tissue-specific expressions showed that NlAkirin was expressed in all tissues tested in female BPH, but its expression level was highest in the ovary. After inhibiting the mRNA expression of NlAkirin in BPH females, the number of eggs laid, hatching rate, and number of ovarioles decreased. Transcriptome sequencing was performed, following a NlAkirin double-stranded RNA treatment. Compared with the genes of the control, which was injected with GFP double-stranded RNA, there were 438 upregulated genes and 1012 downregulated genes; the expression of vitellogenin (Vg) and vitellogenin receptor (VgR) genes as well as the mRNA expression of genes related to the target of rapamycin (TOR), juvenile hormone (JH), and insulin pathways involved in Vg synthesis was significantly downregulated. As a result of NlAkirin knockdown, the titers of JH III and Ecdysone (Ecd) were downregulated in unmated females but returned to normal levels in mated females. The ovarian protein contents in both unmated and mated females were downregulated.Discussion and conclusion: Our results suggest that NlAkirin affects female BPH reproduction by regulating the mRNA expression of genes related to the Vg, VgR, TOR, JH, and insulin signaling pathways, in addition to the titers of JH III and Ecd. The findings of this research provide novel insights into the regulatory role of Akirin in insect reproductive capacity.

Role of nuclear protein Akirin in the modulation of female reproduction in Nilaparvata lugens (Hemiptera: Delphacidae).

Introduction: Akirin as a highly conserved transcription factor, exerts a profound influence on the growth, development, immune response, and reproductive processes in animals. The brown planthopper (BPH), Nilaparvata lugens, a major pest in rice production in Asia, possesses high reproductive capacity, a critical factor contributing to reduced rice yields. The aims of this study were to demonstrate the regulatory role of Akirin in the reproduction of BPH. Methods: In this study, quantitative PCR (qPCR) was used to detect the mRNA expression of genes. RNA interference (RNAi) was used to downregulate the expression of Akirin gene, and RNA sequencing (RNA-seq) was used to screen for differentially expressed genes caused by Akirin downregulation. Hormone contents were measured with the enzyme linked immunosorbent assay (ELISA), and protein content was evaluated with the bicinchoninic acid (BCA) method. Results: Using BPH genome data, we screened for an Akirin gene (NlAkirin). An analysis of tissue-specific expressions showed that NlAkirin was expressed in all tissues tested in female BPH, but its expression level was highest in the ovary. After inhibiting the mRNA expression of NlAkirin in BPH females, the number of eggs laid, hatching rate, and number of ovarioles decreased. Transcriptome sequencing was performed, following a NlAkirin double-stranded RNA treatment. Compared with the genes of the control, which was injected with GFP double-stranded RNA, there were 438 upregulated genes and 1012 downregulated genes; the expression of vitellogenin (Vg) and vitellogenin receptor (VgR) genes as well as the mRNA expression of genes related to the target of rapamycin (TOR), juvenile hormone (JH), and insulin pathways involved in Vg synthesis was significantly downregulated. As a result of NlAkirin knockdown, the titers of JH III and Ecdysone (Ecd) were downregulated in unmated females but returned to normal levels in mated females. The ovarian protein contents in both unmated and mated females were downregulated. Discussion and conclusion: Our results suggest that NlAkirin affects female BPH reproduction by regulating the mRNA expression of genes related to the Vg, VgR, TOR, JH, and insulin signaling pathways, in addition to the titers of JH III and Ecd. The findings of this research provide novel insights into the regulatory role of Akirin in insect reproductive capacity.