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Alternative splicing (AS) plays a crucial role in plant growth, development and response to various environmental changes. However, whether alternative splicing of MADS-box transcription factors contributes to the flower bud dormancy process in fruit trees still remains unknown. In this work, the AS profile of genes in the dormant flower buds of 'Dangshansu' pear tree were examined. A total number of 3661 alternatively spliced genes were identified, and three mRNA isoforms of the dormancy associated MADS box (DAM) gene, PpDAM1, derived by alternative splicing, designated as PpDAM1.1, PpDAM1.2 and PpDAM1.3, were characterized. Bimolecular fluorescence complementation (BiFC) analysis indicated that AS of PpDAM1 didn't affect the nucleus localization and homo-/heterodimerization of PpDAM1.1, PpDAM1.2 and PpDAM1.3 proteins, but disturbed the translocation of PpDAM1.1/PpDAM1.1, PpDAM1.3/PpDAM1.3, PpDAM1.1/PpDAM1.3, and PpDAM1.2/PpDAM1.3 dimers to the nucleus. Constitutive expression of PpDAM1.2, but not PpDAM1.1 and PpDAM1.3, in Arabidopsis retarded the growth and development of transgenic plants. Further comparative expression analyses of PpDAM1.1, PpDAM1.2 and PpDAM1.3 in the flower buds of 'Dangshansu' and a less dormant pear cultivar, 'Cuiguan', exhibited that the expression of all the three isoforms in 'Dangshansu' were significantly higher than in 'Cuiguan', especially PpDAM1.2, which showed a predominantly higher expression than PpDAM1.1 and PpDAM1.3 in both cultivars. Our results suggest that alternative splicing of PpDAM1 could play a crucial role in pear flower bud dormancy process.

Objective : The invisibility of domestic oocyte nucleus in bright field currently forces operators to blindly aspirate nucleus out in oocyte enucleation, usually causing large cytoplasm losses and poor developmental competences of cloned embryos. Although fluorescent labeling of nucleus allows for nucleus localization, the involved photobleaching problems and barriers to the execution of enucleation process limit its online-application in oocyte enucleation. This paper reports a novel label-free oocyte enucleation method for precise removal of the nucleus with less cytoplasm loss. Methods : The relative positions between the injection pipette and nucleus for complete removal of nucleus with less cytoplasm loss were determined through a finite element modeling of nucleus aspiration. To position injection pipette to the above positions relative to nucleus, the appropriate oocyte orientation and trajectory of injection pipette inside oocyte were derived according to the offline-calibrated 3-D distribution of nucleus and the simulated dynamic drift of nucleus that occurs as injection pipette is maneuvered inside oocyte. Finally, a robotic label-free precise enucleation procedure was established. Results : The experimental results on more than 1000 porcine oocytes proved that this system is capable of reducing cytoplasm loss by 60% at the same level of enucleation success rate and almost doubling the cleavage rate of clone embryos in comparison to blind aspiration method. Conclusions : The results prove that our method significantly improves the developmental competence of cloned embryos in comparison to manual enucleation method. Significance : Our method is expected to improve the extremely low success rate of animal cloning in the future.

Kindlin-2 is known to play important roles in the development of mesoderm-derived tissues including myocardium, smooth muscle, cartilage and blood vessels. However, nothing is known for the role of Kindlin-2 in mesoderm-derived reproductive organs. Here, we report that loss of Kindlin-2 in Sertoli cells caused severe testis hypoplasia, abnormal germ cell development and complete infertility in male mice. Functionally, loss of Kindlin-2 inhibits proliferation, increases apoptosis, impairs phagocytosis in Sertoli cells and destroyed the integration of blood-testis barrier structure in testes. Mechanistically, Kindlin-2 interacts with LATS1 and YAP, the key components of Hippo pathway. Kindlin-2 impedes LATS1 interaction with YAP, and depletion of Kindlin-2 enhances LATS1 interaction with YAP, increases YAP phosphorylation and decreases its nuclear translocation. For clinical relevance, lower Kindlin-2 expression and decreased nucleus localization of YAP was found in SCOS patients. Collectively, we demonstrated that Kindlin-2 in Sertoli cells is essential for sperm development and male reproduction.

Background Esophageal squamous cell carcinoma (ESCC) is an aggressive and lethal cancer with a low 5 year survival rate. Identification of new therapeutic targets and its inhibitors remain essential for ESCC prevention and treatment. Methods TYK2 protein levels were checked by immunohistochemistry. The function of TYK2 in cell proliferation was investigated by MTT [(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and anchorage-independent cell growth. Computer docking, pull-down assay, surface plasmon resonance, and kinase assay were used to confirm the binding and inhibition of TYK2 by cirsiliol. Cell proliferation, western blot and patient-derived xenograft tumor model were used to determine the inhibitory effects and mechanism of cirsiliol in ESCC. Results TYK2 was overexpressed and served as an oncogene in ESCC. Cirsiliol could bind with TYK2 and inhibit its activity, thereby decreasing dimer formation and nucleus localization of signal transducer and activator of transcription 3 (STAT3). Cirsiliol could inhibit ESCC growth in vitro and in vivo. Conclusions TYK2 is a potential target in ESCC, and cirsiliol could inhibit ESCC by suppression of TYK2.

Long non-coding RNAs (lncRNAs) have been potentially identified as new diagnostic markers, prognostic factors and therapeutic targets in cancer. The acquisition of a mesenchymal (MES) phenotype in glioblastomas (GBMs) results into therapeutic resistance and poor clinical outcomes. The correlation between lncRNAs and MES differentiation remains elusive. Here, we report that LINC01057 as a lncRNA is overexpressed in GBMs, especially in MES subtype. LINC01057 knockdown suppresses proliferation, invasion and radioresistance of GBM cells in vitro, and tumor growth in vivo. LINC01057 knockdown leads to loss of MES signature in MES subpopulation of GBM cells, but LINC01057 overexpression promotes MES differentiation in proneural (PN) subpopulation. LINC01057 interacts with IKKα and maintains IKKα nucleus localization, leading to effective chromatin accessibility at NF-κB responsive promoters via histone modification and final NF-κB activation. IKKα knockdown disrupts the effect of LINC01057 overexpression on PN to MES transition (PMT). LINC01057 level is negatively correlated with patient prognosis in MES-subtype GBM. Collectively, our findings uncover LINC01057 as a regulator of NF-κB signaling to promote MES differentiation and a potential target for therapeutic intervention for MES-subtype GBM.

Here, we report a strategy to deliver drug nanoparticles into cells with nucleus-targeting ability under a spatiotemporal control. The nanoparticles were constructed through self-assembly of photoresponsive prodrugs and free drugs. By incorporating a nucleus localization sequence in the system, drug nanoparticles could be delivered into nuclei upon visible light irradiation. The drug nanoparticles showed high drug loading capacity and specific nucleus-targeting ability, which efficiently killed cancer cells. This self-assembly strategy could be applied to other hydrophobic drugs and targeting ligands for photo-controlled organelle-targeted drug delivery.

We here investigated molecular basis of notch receptor GLP-1 in controlling simulated microgravity stress in Caenorhabditis elegans. glp-1 expression was decreased by simulated microgravity. Meanwhile, glp-1 mutation caused resistance to toxicity of simulated microgravity. GLP-1 acted in germline cells to control toxicity of simulated microgravity. In germline cells, RNAi knockdown of glp-1 increased daf-16 expression. RNAi knockdown of daf-16 suppressed resistance to toxicity of simulated microgravity in glp-1 mutant. In simulated microgravity treated worms, germline RNAi knockdown of glp-1 decreased expressions of daf-28, ins-39, and ins-8 encoding insulin peptides, and resistance to simulated microgravity toxicity could be detected in daf-28(RNAi), ins-39(RNAi), and ins-8(RNAi) worms. In simulated microgravity treated worms, RNAi knockdown of daf-28, ins-39, or ins-8 in germline cells further increased expression and nucleus localization of transcriptional factor DAF-16 in intestinal cells. Therefore, the GLP-1-activated germline-intestine communication of insulin signaling is required for control of simulated microgravity toxicity in C. elegans.

The enhanced permeability efficiencies still remain a big challenge in crossing the blood-brain barrier (BBB). Herein, a BBB-targeting delivery system based on transferrin (Tf)-poly(ethylene glycol) (PEG) PEGylated-cationic liposome was prepared for delivering the protamine labeled nerve growth factor (NGF) gene. The nanoparticle (TLDP) could preferentially accumulate into the BBB by receptor-mediated transcytosis via the Tf receptor present on cerebral endothelial cells. The polyplex showed good encapsulation of the NGF gene as well as triggered corresponding protein release in the BBB. Surface modification of liposomes with PEG imparts a steric barrier to the NPs that decreases their recognition and clearance by the reticuloendothelial system for increasing the circulation time, and cationic liposomes with protamine are indicated with nuclear localization function to improve the efficiency of nucleus localization and gene expression. The polyplex at a DOTAP/DNA ratio of 3 showed an appropriate diameter, desired serum stability, and much higher encapsulation efficiency. The polyplex had no cytotoxicity against cells. The cell uptake of the TLDP was stronger than other groups without transferrin, which suggested that the TLDP could successfully deliver the NGF gene to the BBB cell and enhanced the expression and secretion of the NGF protein in the brain. In vivo imaging further verified that the TLDP exhibited a higher brain distribution than other groups. Consequently, these findings showed that BBB cells as the "transit station" is a promising method to overcome the BBB and increase the concentration of drug in the brain.

BES1/BZR1 is a kind of transcription factors in brassinosteroid signal pathway, which play a vital role in plant growth and development, the responses to stresses in many plants, like Arabidopsis and rice. However, there are few reports for BES1 in potato, which is an important crop in China. Here, we identified nine potato BES1 genes by genome-wide analysis, and amplified all these CDS sequences which were localized on eight chromosomes. All StBES1 proteins had conserved domains including bHLH domains, BIN2 phosphorylation sites and nucleus localization sites, and shared similar motif compositions. The gene expression displayed differential in different organs and under different hormone and stress treatments. Subcellular localization showed that all StBES1 proteins localized in nucleus and cytoplasm, and transactivation assay indicated these nine StBES1 proteins played roles as transcription factors. All these results provide a theoretic basis for further studying StBES1 functions in potato.

Development of human mesenchymal stem cell (hMSC)-based therapies is hindered by the limited availability of adult stem cells. Long-term retention of multipotency and stem-like phenotypes remains a...

Objective: Nucleus recognition is a critical yet challenging step in histopathology image analysis, for example, in Ki67 immunohistochemistry stained images. Although many automated methods have been proposed, most use a multi-stage processing pipeline to categorize nuclei, leading to cumbersome, low-throughput, and error-prone assessments. To address this issue, we propose a novel deep fully convolutional network for single-stage nucleus recognition. Methods: Instead of conducting direct pixel-wise classification, we formulate nucleus identification as a deep structured regression model. For each input image, it produces multiple proximity maps, each of which corresponds to one nucleus category and exhibits strong responses in central regions of the nuclei. In addition, by taking into consideration the nucleus distribution in histopathology images, we further introduce an auxiliary task, region of interest (ROI) extraction, to assist and boost the nucleus quantification with weak ROI annotation. The proposed network can be learned in an end-to-end, pixel-to-pixel manner for simultaneous nucleus detection and classification. Results: We have evaluated this network on a pancreatic neuroendocrine tumor Ki67 image dataset, and the experiments demonstrate that our method outperforms recent state-of-the-art approaches. Conclusion: We present a new, pixel-to-pixel deep neural network with two sibling branches for effective nucleus recognition and observe that learning with another relevant task, ROI extraction, can further boost individual nucleus localization and classification. Significance: Our method provides a clean, single-stage nucleus recognition pipeline for histopathology image analysis, especially a new perspective for Ki67 image quantification, which would potentially benefit individual object quantification in whole-slide images.

Initiation of the innate immune response requires recognition of pathogen-associated molecular patterns by pathogen recognition receptors such as Toll-like receptors (TLRs). MyD88 adaptor-like (Mal) is an adaptor that responds to TLR activation and acts as a bridging adaptor for MyD88. In the present study, the open reading frame of Mal was identified in orange-spotted grouper (Epinephelus coioides), and named EcMal. It contained 831 bp encoding 276 aa, and was encoded by a 1299 bp DNA sequence with three exons and two introns. EcMal and the Mal sequence of other species shared different degrees of sequence identity, and clustered into the same group. EcMal was distributed in all tissues tested in healthy grouper, with the highest expression level in the head kidney. After infection with Cryptocaryon irritans, the expression level of EcMal was up-regulated in the gill and spleen. In addition, EcMal exhibited global cytosolic and nucleus localization, and could significantly activate NF-κB activity in grouper spleen cells.

Background and objective Bone remodeling during orthodontic treatment is achieved by the osteogenesis of human periodontal ligament cells (PDLCs) subjected to mechanical loadings. Transcriptional co-activator with PDZ-binding motif (TAZ) mediates bone remodeling in response to extracellular mechanical signals. This study aims to investigate the role of TAZ in osteogenesis of PDLCs under tensile strain. Materials and methods A uniaxial cyclic tensile stress (CTS) at 12% elongation and 6 cycles/min (5 s on and 5 s off) was applied to PDLCs. The osteogenic differentiation was determined by the protein and gene expressions of osteogenic markers using qRT-PCR and Western blot, respectively, and further by alkaline phosphatase (ALP) activity and Alizarin Red S staining. The interaction of TAZ with core-binding factor α1 (Cbfα1) was examined by co-immunoprecipitation. The immunofluorescence histochemistry was used to examine the nucleus aggregation of TAZ and the reorganization of actin filaments. Moreover, small interfering RNA-targeting TAZ (TAZsiRNA) was used for TAZ inhibition and Y-27632 was employed for Ras homologue-associated coiled-coil protein kinase (ROCK) signaling blockage. Results CTS clearly stimulated the nucleus accumulation of TAZ and its interaction with Cbfα1. CTS-induced osteogenesis in PDLCs was significantly abrogated by the infection with TAZsiRNA, as shown by the decreased stained nodules and protein expressions of Cbfα1, collagen type I, osterix, and osteocalcin, along with the inhibition of β-catenin signaling. Moreover, ROCK inhibition by Y-27632 hindered TAZ nucleus aggregation and its binding with Cbfα1, which subsequently lead to the decreased osteoblastic differentiation of PDLCs. Conclusions Taken together, we propose that TAZ nucleus localization and its interaction with Cbfα1 are essential for the CTS-induced osteogenic differentiation in PDLCs. And such TAZ activation by CTS could be mediated by ROCK signaling, indicating the pivot role of ROCK-TAZ pathway for PDLCs differentiation.

Aim: The aim of this study was to report a novel POU Class 3 Homeobox 4 (POU3F4) variant and to provide further guidance on genetic counseling for incomplete partition (IP) type III families in the Korean population by showing two new contrasting cases in terms of genotypes and inheritance. Materials and Methods: Two consecutively recruited hearing-impaired probands with seemingly nonsyndromic features and their biological mothers were included in this study. Sanger sequencing and quantitative polymerase chain reaction (PCR) assays were performed for POU3F4. Results: A novel frameshift variant of POU3F4, c.852delC (p.Ile285Serfs*3), was identified in one of the patients. This mutation is predicted to truncate the protein within the POU homeodomain, resulting in the complete loss of the last nucleus localization signal. The proband's biological mother was also shown to be a carrier of this c.852delC (p.Ile285Serfs*3) mutant allele. A de novo genomic deletion on chromosome Xq21.2 was confirmed in another subject via quantitative PCR. This subject's biological mother, however, was not a carrier of this deletion. This indicates that the large upstream deletion of POU3F4 in the second proband occurred de novo. This finding is compatible with the previously proposed tendency for a high de novo rate of large genomic deletions involving the X-linked deafness-2 (DFNX2) locus. Conclusion: This study adds a novel, probably pathogenic POU3F4 truncation variant to the literature and provides guidance toward effective genetic counseling for IP III subjects based on more frequent de novo occurrence of POU3F4 deletions than POU3F4 point variants.

The aim of the present study was to investigate the effects of phosphorylatable nucleus localization signal linked nucleic kinase substrate short peptide (NNSp)-conjugated chitosan (NNSp-CS) mediated miR-140 and IGF-1 in both rabbit chondrocytes and cartilage defects model. NNSp-CS was combined with pBudCE4.1-IGF-1, pBudCE4.1-miR-140, and negative control pBudCE4.1 to form pDNA/NNSp-CS complexes. Then these complexes were transfected into chondrocytes or injected intra-articularly into the knee joints. High levels of IGF-1 and miR-140 expression were detected both in vitro and in vivo. Compared with pBudCE4.1 group, in vitro, the transgenic groups significantly promoted chondrocyte proliferation, increased glycosaminoglycan (GAG) synthesis, and ACAN, COL2A1, and TIMP-1 levels, and reduced the levels of nitric oxide (NO), MMP-13, and ADAMTS-5. In vivo, the exogenous genes enhanced COL2A1, ACAN, and TIMP-1 expression in cartilage and reduced cartilage Mankin score and the contents of NO, IL-1β, TNF-α, and GAG contents in synovial fluid of rabbits, MMP-13, ADAMTS-5, COL1A2, and COL10A1 levels in cartilage. Double gene combination showed better results than single gene. This study indicate that NNSp-CS is a better gene delivery vehicle in gene therapy for cartilage defects and that miR-140 combination IGF-1 transfection has better biologic effects on cartilage defects.

A series of physiological barriers have impeded nanoparticle-based drug formulations (NDFs) from reaching their targeted sites and achieving therapeutic outcomes. In this study, we develop size-controllable stealth doxorubicin-loaded nanodrug coated with CD47 peptides (DOX/sNDF-CD47) based on supramolecular chemistry to overcome multiple biological barriers. The smart DOX/sNDF-CD47 can efficiently decrease sequestration by macrophages and disassemble into poly(amidoamine) dendrimers with nuclear localization sequences (DOX/PAMAM-NLS) in the presence of matrix metalloproteinase-2 (MMP-2). Such structure transformation endows DOX/sNDF-CD47 with the ability of deep penetration in multicellular tumor spheroid, lysosomal escape, and nucleus localization, resulting in excellent cytotoxicity and drug resistance combating. In vivo experiments further confirmed that DOX/sNDF-CD47 has good tumor-targeting ability and can significantly improve therapeutic efficacy of DOX on xenograft tumor model. The ability to overcome multiple biological barriers makes sNDF-CD47 a promising NDFs to treat cancer expressing MMP-2 and combating drug resistance.

The presence of cancer stem cells (CSC), which possess the ability of self-renewal and cancer initiation, is correlated with poor prognosis and drug resistance of breast cancer patients. But the molecular regulatory networks for maintenance of CSC function still remain unclear. Here, we identified that an estrogen-inducible gene FXYD3, whose expression is significantly upregulated in ER+ breast CSCs, is a critical player for regulating ER+ breast CSC function. FXYD3 amplification is crucial in mediating tamoxifen resistance in ER+ breast cancer cells. Interestingly, we also find that stem cell–related transcription factor SOX9 directly promotes FXYD3 expression, and FXYD3 is indispensable for SOX9 nucleus localization, thus forming a positive regulatory feedback loop for FXYD3 amplification and function. In terms of mechanism, FXYD3 interacts with Src and ERα to form an activated complex and triggers Src to transduce nongenomic estrogen signaling for facilitating ER+ breast CSCs. Collectively, these results establish a critical role for SOX9/FXYD3/Src axis in boosting nongenomic estrogen signaling and SOX9 nucleus entry, which is required for maintenance of ER+ breast CSCs and endocrine resistance. Targeting FXYD3-mediated pathway might be a promising therapeutic strategy for hormone therapy–refractory ER+ breast cancer. Implications: SOX9/FXYD3/Src axis is critical for promoting CSC function and tamoxifen resistance in ER+ breast cancer.

Cell-type-specific markers are valuable tools to reveal developmental processes underlying cell differentiation during plant reproduction. Here we report the pollen vegetative cell marker gene VCS1 (Vegetative Cell Specific 1) of rice (Oryza sativa japonica). VCS1 was expressed specifically in late pollen and was predicted to encode a small FAF domain-containing protein of 205 amino acid residues (aa). The expression of reporter fusion proteins showed that VCS1 was exclusively targeted to the vegetative nucleus of pollen. Upon pollen germination, VCS1 lost vegetative nucleus localization, and appeared diffused in the vegetative cytoplasm of pollen grain but not in the pollen tube. T-DNA insertional mutation which disrupted the carboxyl-terminal 21 aa of VCS1 did not affect plant vegetative growth and pollen development, while destruction of VCS1 by CRISPR/Cas9 only moderately affect pollen viability. VCS1 is evolutionally conserved in monocots but appeared absent in dicotyledons. This study reveals a molecular tool for visualizing the vegetative cell of rice and possible other monocots, which has potential values in the genetic engineering of male-sterile lines.

The Myc proto-oncogene family consists of three members, C-MYC, MYCN, and MYCL, which encodes the transcription factor c-Myc (hereafter Myc), N-Myc, and L-Myc, respectively. Myc protein orchestrates diverse physiological processes, including cell proliferation, differentiation, survival, and apoptosis. Myc modulates about 15% of the global transcriptome, and its deregulation rewires the cellular signaling modules inside tumor cells, thereby acquiring selective advantages. The deregulation of Myc occurs in >70% of human cancers, and is related to poor prognosis; hence, hyperactivated Myc oncoprotein has been proposed as an ideal drug target for decades. Nevertheless, no specific drug is currently available to directly target Myc, mainly because of its “undruggable” properties: lack of enzymatic pocket for conventional small molecules to bind; inaccessibility for antibody due to the predominant nucleus localization of Myc. Although the topic of targeting Myc has actively been reviewed in the past decades, exciting new progresses in this field keep emerging. In this review, after a comprehensive summarization of valuable sources for potential druggable targets of Myc-driven cancer, we also peer into the promising future of utilizing macropinocytosis to deliver peptides like Omomyc or antibody agents to intracellular compartment for cancer treatment.

Membrane localized transcription factors play essential roles in various plant developmental processes. The XVP/NAC003 protein is a NAC domain transcription factor associated with the plasma membrane and involved in the TDIF-PXY signaling during vascular development. We report here the mechanisms of XVP membrane localization and its nuclear translocation. Using a transient transformation approach, we found that XVP is associated with the plasma membrane through positively charged KR-rich regions. Mutagenesis studies found that the threonine amino acid at position 354 (T354) is critical for XVP translocation to the nucleus. In particular, the threonine to alanine mutation (T354A) resulted in a partial nucleus localization, while threonine to aspartic acid (T354D) mutation showed no effect on protein localization, indicating that dephosphorylation at T354 may serve as a nucleus translocation signal. This research sheds new light on the nucleus partitioning of plasma membrane-associated transcription factors.

Protein Kinase A (PKA) exists as a tetrameric holoenzyme which activates with increase of cAMP and plays an important role in many physiological processes including cardiac physiology, neuronal development, and adipocyte function. Although this kinase has been the subject of numerous biosensor designs, a single-fluorophore reporter that performs comparably to Förster resonance energy transfer (FRET) has not yet been reported. Here, we have used basic observations of electrostatic interactions in PKA substrate recognition mechanism and nucleus localization sequence motif to design a phosphorylation switch that shuttles between the cytosol and the nucleus, a strategy that should be generalizable to all basophilic kinases. The resulting reporter yielded comparable kinetics and dynamic range to the PKA FRET reporter, AKAR3EV. We also performed basic characterization and demonstrated its potential use in monitoring multiple signaling molecules inside cells using basic fluorescence microscopy. Due to the single-fluorophore nature of this reporter, we envision that this could find broad applications in studies involving single cell analysis of PKA activity.

TGF-&beta, is a critical cytokine to regulate multiple pathophysiological functions. For tumor development and progression, TGF-&beta, was reported to play dual functions as a tumor suppressor and epithelial-mesenchymal transition (EMT) inducer. The mechanism of the TGF-&beta, signaling pathway is essential for TGF-&beta, /Smad-targeted therapy in clinic. Here, ATBF1 was demonstrated to participate in dual functions of TGF-&beta, via different ways. On one hand, ATBF1 expression level was associated with EMT and migration induced by TGF-&beta, After TGF-&beta, treatment, ATBF1 expression was reduced in a dose- and time-dependent manner, along with the alteration of cell morphology and EMT marker expression. Knockdown of ATBF1 by siRNA further promoted EMT progression and cell migration. On the other hand, ATBF1 localization was associated with cell proliferation inhibited by TGF-&beta, The number of cells with nucleus localization of ATBF1 in TGF-&beta, activation group was much higher than that in control group. After that, knockdown of ATBF1 by siRNA rescued the inhibition of cell proliferation affected by TGF-&beta, These data revealed that ATBF1 is a key gene for the dual roles of TGF-&beta, which may contribute to future therapy.

Pap smear method which is based on the morphological properties of cell nuclei is used to detect pre-cancerous cells in the uterine cervix. An automated and accurate segmentation of nuclei is essential in detection. In this paper, we propose an Interacting Convolution with Pyramid Structure Network (ICPN), which consists of a sufficient aggregating path that focus on more nucleus contexts and a selecting path that enable nucleus localization. The two paths are built on Interacting Convolutional Modules (ICM) and Internal Pyramid Resolution Complementing Modules (IPRCM) respectively. ICM reciprocally aggregates different details of contexts from two sizes of kernels for capturing distinguishing features of diverse sizes and shapes of nuclei. Meanwhile, IPRCM hierachically complements kinds of resolution features to prevent information loss in encoding precedure. The proposed method shows a Zijdenbos similarity index (ZSI) of 0.972±0.04 on Herlev dataset compared to the state-of-the-art approach.

It is critical to understand the risk factors responsible for the tumorigenesis and progression of nasopharyngeal carcinoma (NPC). Bisphenol A (BPA) can regulate the estrogenic signals to modulate cancer progression, while its roles in NC were not investigated. Our present study revealed that the BPA can increase proliferation and migration of NPC cells while decrease the chemosensitivity to doxorubicin (Dox). The inhibitor of GSK-3β/β-catenin (LiCl) can restore BPA-induced cell proliferation of NPC cells, which is due to that BPA can decrease phosphorylation while increase expression and nucleus localization of β-catenin. Mechanistically, BPA can increase the mRNA stability of β-catenin (encoded by CTNNB1) via suppressing the expression of miR-214-3p, which can direct target the 3'UTR of β-catenin mRNA. Further, BPA can decrease phosphorylation of β-catenin via repressing the expression of CK1α. Collectively, our data showed that BPA can trigger the proliferation and malignancy of NPC cells via activation of Wnt/β-catenin pathway. It indicated that body accumulation and inhalation exposure of BPA might be a risk factor for NPC development.

The human nuclear pore complex is a 120 nm structure composed of nearly 1000 protein molecules (multiple copies of nearly 30 different proteins) with a mass of 110–120 MDa. It spans the double membrane of the nuclear envelope and selectively transports both proteins, nucleic acids, and small signaling molecules bidirectionally. The diameter of the channel in the nuclear pore complex is approximately 5 nm in diameter and 45 nm in depth. Selective transport through the nuclear pore complex is mediated by nuclear transport receptors that bind to the cargo to be transported. Importins mediate transport of cargo molecules into the nucleus, whereas exportins facilitate the selective transport of cargo out of the nucleus. Cargoes with a nucleus localization signal are efficiently transported into the nucleus through the nuclear pore complex. The import and export cycles require GTP hydrolysis, and thus the transport process through the nuclear pore complex is energy-dependent. Since the nuclear pore complex is the gateway to the genome, the number of nuclear pore complexes varies during the different stages of the cell cycle. For example, between G1 and G2 phase of the cell cycle, the number of nuclear pore complexes at the nuclear envelope increase to accommodate greater transcriptional demand. Assembly of the nuclear pore complex like other cellular nanomachines is little understood.

Burkitt lymphoma is one of the most common lymphatic system cancers with poor outcome in adult patients. p53-induced apoptosis is a critical signaling for preventing tumor development. Cyclin B/cyclin-dependent kinase 1 (CDK1) phosphorylates inhibitor of apoptosis stimulating protein of P53 (iASPP) to promote iASPP nucleus localization and its inhibitory effect on p53. However, p53 is frequently mutated in Burkitt lymphoma, which gains novel oncogenic properties. Recently, the p53 family member, p63, became an attractive gene for the therapeutic strategies for patients with cancer. Therefore, we investigated the role of iASPP in the transactivation domain p63 (TAp63)-dependent cell proliferation inhibition in Burkitt lymphoma. We verified that the oncogenic effect of iASPP on Burkitt lymphoma is TAp63 dependent rather than p53 and confirmed that the interaction between CDK1 and iASPP enhanced the inhibitory effect of iASPP on p53 and TAp63. An online tool predicated that miR-129 might bind to 3'-untranslated region of iASPP and CDK1. We revealed that miR-129 acted as a tumor suppressor by inhibiting cancer cell proliferation and inhibiting CDK1 and iASPP via direct binding. An miR-129 inhibitor increased nucleus iASPP and decreased nucleus p53 and TAp63 levels, which could be reversed by the CDK1 knockdown, indicating that miR-129 might target CDK1 to inhibit iASPP phosphorylation, thus hindering iASPP nucleus localization and its inhibitory effect on p53 and TAp63 protein levels. Taken together, miR-129 could targetedly inhibit the expression of CDK1 and iASPP. CDK1 knockdown inhibits iASPP S84/S113 phosphorylation, thus blocking iASPP nucleus localization, suppressing the inhibitory effect of iASPP on p53 and TAp63, and restoring TAp63-induced proliferation inhibition in Burkitt lymphoma cells.

Pyropia tenera is a marine red alga that grows in the intertidal zone and may lose more than 90% of its water during twice-daily low tides. Based on the differentially expressed genes (DEGs) identified from the desiccation transcriptome of P. tenera gametophyte, a complementary DNA was isolated that upregulated strongly under desiccation stress and encoded a polypeptide of 591-amino acid residues showing a sequence homology with α-1,2-fucosyltransferase, named PtFUT. Fucosyltransferases transfer an l-fucose residue to various acceptor molecules, but information of their activity in the red algae is limited. PtFUT showed upregulation in transcription in gametophytes of P. tenera under desiccation and osmotic stress conditions induced by mannitol. Transcription of the PtFUT gene was also increased by abscisic acid as well as H2O2 treatment. The presence of a signal peptide for nucleus localization and fluorescence of the PtFUT-GFP fusion proteins in tobacco protoplasts indicated that PtFUT is located in the nucleus. When PtFUT was overexpressed in Chlamydomonas, the PtFUT improved the growth of transgenic cells under osmotic stress. These results suggest that PtFUT may play a role in tolerance to desiccation stress and provide insights into the molecular functions of the fucosyltransferases and understanding the desiccation tolerance mechanisms in marine red algae growing in intertidal zones.

Aim To develop a new method to produce recombinant reprogramming proteins, cMyc, Klf4, Oct4, and Sox2, in soluble format with low cost for the generation of induced pluripotent stem cells (iPSCs). Methods A short polypeptide sequence derived from the HIV trans-activator of transcription protein (TAT) and the nucleus localization signal (NLS) polypeptide were fused to the N terminus of the reprogramming proteins and they were constructed into pCold-SUMO vector which can extremely improve the solubility of recombinant proteins. Then these vector plasmids were transformed into E. coli BL21 (DE3) Chaperone competent cells for amplification. The solubility of these recombinant proteins was determined by SDS-PAGE and Coomassie brilliant blue staining. The recombinant proteins were purified by Ni-NTA resin and identified by Western blot. The transduction of these proteins into HEK 293T cells were evaluated by immunofluorescence staining. Results These four reprogramming proteins could be produced in soluble format in pCold-SUMO expression vector system with the assistance of chaperone proteins in bacteria. The proteins were purified successfully with a purity of over 70% with a relative high transduction rate into 293 cells. Conclusion The results in the present study indicate the four important reprogramming proteins, cMyc, Klf4, Oct4, and Sox2, can be produced in soluble format in bacteria with low cost. Our new method thus might be expected to greatly contribute to the future study of iPSCs.

Remotely and locally triggered release of therapeutic species by X-ray irradiation is highly desired to enhance the efficacy of radiotherapy. However, the development of such X-ray responsive nanosystems remains a challenge, especially in response to high energy clinically relevant X-ray radiation. Herein, we report novel nitroimidazole ligated gold nanoparticles (AuNPs) that synergistically function to release nitrite, an important precursor for nitric oxide and reactive nitrogen species that sensitize cancer cells, upon radiation with clinically used 6 MeV X-rays, while no release was detected without radiation. These functional AuNPs were prepared with surface-grafted nitroimidazole as the nitrite-releasing agent, cell-penetrating peptide (CPP) to induce nucleus localization, and poly(ethylene glycol) for water solubility. In vitro radiotherapy using such nanoparticles showed enhanced sensitivity of hypoxic cancer cells to X-ray radiation, presumably due to the generation of both reactive oxygen and nitrogen species. The dose modifying factor (DMF) was found to be 0.71 for the dual-functionalized nanoparticle, which indicates that significant lower X-ray doses are required to achieve the same therapeutic effects. Thus, X-ray triggered nitrite release from gold-nitroimidazole nanosystems offers a novel strategy to sensitize cancer cells for improved radiotherapy.

Protein Kinase A (PKA) exists as a tetrameric holoenzyme which activates with increase of cAMP and plays an important role in many physiological processes including cardiac physiology, neuronal development, and adipocyte function. Although this kinase has been the subject of numerous biosensor designs, a single-fluorophore reporter that performs comparably to Förster resonance energy transfer (FRET) has not yet been reported. Here, we have used basic observations of electrostatic interactions in PKA substrate recognition mechanism and nucleus localization sequence motif to design a phosphorylation switch that shuttles between the cytosol and the nucleus, a strategy that should be generalizable to all basophilic kinases. The resulting reporter yielded comparable kinetics and dynamic range to the PKA FRET reporter, AKAR3EV. We also performed basic characterization and demonstrated its potential use in monitoring multiple signaling molecules inside cells using basic fluorescence microscopy. Due to the single-fluorophore nature of this reporter, we envision that this could find broad applications in studies involving single cell analysis of PKA activity.

As a family of negatively feedback regulating factors, the suppressor of cytokine signaling (SOCS) can depress cytokine signal transduction, and eventually modulate growth, development, differentiation, and immune response. In the present study, a SOCS homologue (designated as CgSOCS6) was identified from oyster Crassostrea gigas. The open reading frame of CgSOCS6 cDNA was of 1167 bp encoding a peptide of 388 amino acid residues with a central Src homology 2 (SH2) domain, a conserved C-terminal SOCS box, and a nucleus localization sequence (NLS) in its N-terminus. The deduced amino acid sequence of CgSOCS6 shared 37.9-45.5% similarity with other SOCS6/7 family members. In the unrooted phylogenetic tree, CgSOCS6 was clustered with EsSOCS6 from Chinese mitten crab Eriocheir sinensis and assigned into the SOCS6/7 group. The mRNA transcripts of CgSOCS6 were constitutively distributed in all the tested tissues, with the highest level in hemocytes. After lipopolysaccharide (LPS) stimulation, the mRNA expression of CgSOCS6 in hemocytes was significantly up-regulated to the highest level at 6 h (8.48-fold compared to the control group, p 0.01), and then kept at a relatively higher level from 12 h to 72 h. CgSOCS6 protein could be translocated into the hemocyte nucleus after LPS stimulation. The mRNA expressions of interleukin 17-4 (CgIL17-4), CgIL17-5, and defensin (CgDefh1) in the hemocytes of CgSOCS6-knockdown oysters increased significantly (2.55-fold, 2.68-fold, 4.68-fold of that in EGFP-RNAi oysters, p 0.05, p 0.05, p 0.001, respectively) after LPS stimulation. These findings suggested that CgSOCS6 was involved in the oyster immune response by regulating the expressions of CgIL17-4, CgIL17-5, and CgDefh1.

We have previously identified a Foxo1 binding protein, FCoR, which suppresses Foxo1 activity by increasing acetylation. FCoR is expressed in pancreatic α- and β-cells. FCoR expression is decreased in high-fat fed mice and in Leprdb/db mice, and FcorKO mice exhibited decreased insulin secretion, increased α-cell mass and β to α-cell conversion, suggesting FCoR involvement in the pathophysiology of diabetes. In FcorKO islets, nucleus localization of Foxo1 was increased, and Foxo1 acetylation was significantly decreased compared to WT. These data suggest that FCoR regulates acetylation and activity of Foxo1 in islets. The qPCR in isolated islets from FcorKO showed increased Arx, Mafb and Gcg expression compared to WT, supporting the phenotype of increased α-cell mass. Interestingly, expression level of Mafa and Neurod1, which are the β-cell markers and target genes of Foxo1, were also significantly increased, confirming activation of Foxo1 in the absence of FCoR. FCoR suppresses the expression of Aristaless related homeobox (Arx), which is essential for α-cell differentiation, via increased methylation of the CG rich promoter region of Arx. One of the epigenetic modifications of histone, the tri-methylation at H3K4 (H3K4me3), is an important marker of Arx expression in α-cells. Chromatin immunoprecipitation assay revealed that H3K4me3 was significantly decreased in αTC cells transduced with Flag-FCoR compared to LacZ-transduced-cells, suggesting that FCoR suppresses Arx expression through epigenetic regulation involving both DNA and histone methylation. Our data show that FCoR regulates Foxo1 activity in islets, and that FCoR regulates pancreatic α- and β-cell identity by epigenetic regulation of DNA and histone methylation. Disclosure N. Kodani: Research Support; Self; Lilly Diabetes, Novartis Pharmaceuticals Corporation. M. Kobayashi: None. O. Kikuchi: None. T. Kitamura: None. H. Itoh: None. J. Nakae: None.

Chromosomal maintenance 1 (CRM1) inhibitors display antihypertrophic effects and control protein trafficking between the nucleus and the cytoplasm. PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1alpha) is a type of transcriptional coactivator that predominantly resides in the nucleus and is downregulated during heart failure. NT-PGC-1α is an alternative splicing variant of PGC-1α that is primarily distributed in the cytoplasm. We hypothesized that the use of a CRM1 inhibitor could shuttle NT-PGC-1α into the nucleus and activate PGC-1α target genes to potentially improve cardiac function in a mouse model of myocardial infarction (MI). We showed that PGC-1α and NT-PGC-1α were decreased in MI-induced heart failure mice. Phenylephrine and angiotensin II were applied to induce hypertrophy in neonatal rat ventricular myocytes (NRVMs). The antihypertrophic effects of the CRM1-inhibitor Selinexor was verified through profiling the expression of β-MHC and through visualizing the cell cross-sectional area. NRVMs were transfected with adenovirus-NT-PGC-1α or adenovirus-NLS (nucleus localization sequence)-NT-PGC-1α and then exposed to Selinexor. Confocal microscopy was then used to observe the shuttling of NT-PGC-1α. After NT-PGC-1α was shuttled into the nucleus, there was increased expression of its related genes, including PPAR-α, Tfam, ERR-γ, CPT1b, PDK4, and Nrf2. The effects of Selinexor on post-MI C57BL/6j mice were determined by echocardiography and qPCR. We found that Selinexor showed antihypertrophic effects but did not influence the ejection fraction of MI-mice. Interestingly, the antihypertrophic effects of Selinexor might be independent of NT-PGC-1α transportation.

Immune checkpoint blockade (ICB) is an attractive option in cancer therapy, but its efficacy is still less than expected due to the transient and incomplete blocking and the low responsiveness. Herein, an unprecedented programmable unlocking nano‐matryoshka‐CRISPR system (PUN) targeting programmed cell death ligand 1 (PD‐L1) and protein tyrosine phosphatase N2 (PTPN2) is fabricated for permanent and complete and highly responsive immunotherapy. While PUN is inert at normal physiological conditions, enzyme‐abundant tumor microenvironment and preternatural intracellular oxidative stress sequentially trigger programmable unlocking of PUN to realize a nano‐matryoshka‐like release of CRISPR/Cas9. The successful nucleus localization of CRISPR/Cas9 ensures the highly efficient disruption of PD‐L1 and PTPN2 to unleash cascade amplified adaptive immune response via revoking the immune checkpoint effect. PD‐L1 downregulation in tumor cells not only disrupts PD‐1/PD‐L1 interaction to attenuate the immunosurveillance evasion but also spurs potent immune T cell responses to enhance adaptive immunity. Synchronously, inhibition of JAK/STAT pathway is relieved by deleting PTPN2, which promotes tumor susceptibility to CD8+ T cells depending on IFN‐γ, thus further amplifying adaptive immune responses. Combining these advances together, PUN exhibits optimal antitumor efficiency and long‐term immune memory with negligible toxicity, which provides a promising alternative to current ICB therapy., Programmable unlocking nano‐matryoshka‐CRISPR (PUN) possesses multistage sensitive properties and exhibits excellent performances, including prolonged blood circulation, precise tumor recognition, deep tumor penetration, robust lysosomal escape, and effective transfection. With the precise control of CRISPR/Cas9 activation, PUN realizes complete and thorough intracellular disruption of PD‐L1 and PTPN2, thus eliciting cascade amplified adaptive immune response to boost antitumor immune effects.

Cervical cancer occurs as a result of the persistent infection of high-risk human papillomavirus (HPV). HPV16 oncoproteins E6 and E7 exert different and concerted pro-tumor actions in cell transformation and malignance maintenance in various m echanisms. Nanobody expressed as “intracellular antibodies” (intrabodies) can target intracellular antigens to hamper their function efficaciously and specifically. In this work, phage-display approach was employed to select the high affinity HPV16 E6-specific nanobody, nanobody Nb9 against HPV16 E6 was selected. Nb9 has high affinity (Kaff =6.3 × 108 M−) and can specifically bind endogenous HPV16 E6 protein in HPV16 positive CaSki and SiHa cells. In Nb9 overexpressed SiHa and CaSki cells, nucleus localization of HPV16 E6 was inhibited, p53 inactivation was prevented and increased apoptosis was observed. Moreover, tumor growth was inhibited in mouse xenograft model. Taken together, our results suggested that nanobody Nb9 could be a useful inhibitor for HPV16 E6 function and particularly appropriate for the treatment of HPV-associated disease.

A novel cold responsive HbICE1 gene from Hevea brasiliensis was identified, and its function as a transcriptional factor in cold stress responses was further analyzed. The inducers of CBF expression (ICEs) play a crucial role in the response of plants to cold stress. To date, information is limited about the ICE genes from rubber trees (Hevea brasiliensis Muell. Arg.), a tropical woody plant. In this study, an ICE homologuous gene, referred to as HbICE1, was isolated and characterized by a combination of molecular and physiological techniques and bioinformatics analyses. The full-length cDNA of HbICE1 was 1806-bp in length and contained a 1617 bp open reading frame (ORF) encoding a putative protein of 538 amino acids. The predicted HbICE1 harbored S-rich, bHLH and ACT-like domains that were conserved in ICE1 orthologs from Arabidopsis and other plant species. Phylogenetic analysis demonstrated that HbICE1 was closely related to MeICE1 and RcICE1 as well as PeICE1, MdICE1 and PuICE1. It harbored transcriptional activity and nucleus localization. qRT-PCR analysis showed that HbICE1 was mainly expressed in leaves and stems under natural conditions. Its expression was significantly up-regulated in the leaves of the cold-resistant rubber tree clone ‘93-114’ in response to cold stress by comparison with the cold-sensitive rubber tree clone ‘Reken501’. Functional analysis in yeast revealed that HbICE1 conferred cold stress resistance. Collectively, these results suggest that HbICE1 acts as a transcriptional factor and indicatively mediates the cold resistance in rubber trees.

The HIV-1 nucleocapsid (NC) is an essential viral protein containing two highly conserved retroviral-type zinc finger (ZF) motifs, which functions in multiple stages of the HIV-1 life cycle. Although a number of functions for NC either in its mature form or as a domain of Gag have been revealed, little is known about the intracellular localization of NC and, moreover, its role in Gag protein trafficking. Here, we have investigated various forms of HIV-1 NC protein for its cellular localization and found that the NC has a strong nuclear and nucleolar localization activity. The linker region, composed of a stretch of basic amino acids between the two ZF motifs, was necessary and sufficient for the activity.

Phosphorylated Ser473-Akt (p-Ser473-Akt) is extensively studied as a correlate for the activity of Akt, which plays an important role in mouse oogenesis and preimplantation embryogenesis. However, little progress has been made about its effect on the mouse zygotic genome activation (ZGA) of 2-cell stage in mouse preimplantation embryos. In this study, we confirmed its localization in the pronuclei of 1-cell embryos and found that p-Ser473-Akt acquired prominent nucleus localization in 2-cell embryos physiologically. Akt specific inhibitors API-2 and MK2206 could inhibit the development of mouse preimplantation embryos in vitro, and induce 2-cell arrest at certain concentrations. 2-cell embryos exposed to 2.0 μmol/L API-2 or 30 μmol/L MK2206 displayed attenuated immunofluorescence intensity of p-Ser473-Akt in the nucleus. Simultaneously, qRT-PCR results revealed that 2.0 μmol/L API-2 treatment significantly downregulated the mRNA pattern of MuERV-L and eIF-1A, two marker genes of ZGA, suggesting a defect in ZGA compared with that of control group. Collectively, our work demonstrated the nuclear localization of p-Ser473-Akt during major ZGA, and Akt specific inhibitors API-2 and MK2206 which led to 2-cell arrest inhibited p-Ser473-Akt from translocating into the nucleus of 2-cell embryos with defective ZGA as well, implying p-Ser473-Akt may be a potential player in the major ZGA of 2-cell mouse embryos.

The biggest challenge in the effective management of breast cancer is the limited duration of therapeutic benefit that is achieved with the current treatment options. Patients diagnosed with triple-negative breast cancer (TNBC), a type of breast tumor with worst prognoses, are currently treated with surgery, radiation and chemotherapy. Unfortunately, despite initial responses cancer cells become resistant to drugs and tumors relapse. We previously identified the cell surface protein GPNMB as a key mediator of breast cancer metastasis. Recently, an antibody-drug conjugate targeting GPNMB (Cdx-011) was developed that has been tested as single agent for the treatment of metastatic TNBC in a clinical trial. We have recently uncovered a novel response of TNBCs to therapeutic treatments: GPNMB expression is greatly elevated following treatment with mTOR pathway inhibitors (i.e., rapamycin, INK-128, AZD2014 and Torin-1) as well as conventional chemotherapy agents (doxorubicin, paclitaxel or cisplatin) and experimental treatments, i.e., MEK pathway inhibitors. Pharmacologically induced enhancement of GPNMB was also observed in ER+ cancers treated with antiestrogens (i.e., Fulvestrant). Interestingly, tumor cells that developed resistance to chemotherapy or antiestrogens exhibit higher levels of GPNMB. We found that therapy-induced GPNMB upregulation is mediated by the mobilization of the stress-induced MiTF/TFE family of transcription factors. In TNBC cells, mTOR inhibition promotes TFE3 nuclear translocation and GPNMB transcription. In ER+ cells, antiestrogens inactivate mTOR pathway causing TFE3 nucleus localization and GPNMB expression. We are currently conducting preclinical studies to investigate whether the upregulation of GPNMB caused by treatment with mTOR inhibitors could sensitize breast cancer cells to the activity of an antibody-drug conjugate targeting GPNMB (Cdx-011). Citation Format: Marco Biondini, April Rose, Matthew Annis, Cristiano Ferrario, Rosa Puertollano, Mark Basik, Peter Siegel. mTOR pathway inhibition induces GPNMB expression and sensitizes breast cancer cells to an antibody-drug conjugate targeting GPNMB [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B11.

Opal shale, as a naturally occurring and noncrystalline silica material with porous structure, has the potential to be a drug delivery carrier. In this study, we obtained opal shale nanoparticles (OS NPs) through the techniques of ultrasonic emulsion and differential centrifugation. The OS NPs exhibited markedly lower cytotoxicity than crystalline mesoporous silica nanoparticles. The highly porous structure and the strong adsorbability endowed OS NPs with the ability of loading and sustained release of doxorubicin (DOX). DOX-loaded OS NPs improved tumor cellular uptake and antiproliferation compared with free drug. Interestingly, OS NPs possessed strong binding with the nuclear envelope, which can be beneficial to the nucleus localization and apoptosis inducing of loaded DOX. We further demonstrated the tumor passive targeting ability, prolonged blood circulation, and enhanced antitumor effect with limited in vivo toxicity. Our results suggest that OS NPs can be applied for tumor targeting drug delivery, which may have a significant influence on the development of silica-based drug delivery system.

ERF proteins are plant-specific transcription factors that play significant roles in plant defense against various stresses. However, only little information regarding stress-related ERF genes is available in tomato (Solanum lycopersicum, Sl). In this study, a tomato ERF gene, SlERF84, was cloned and functionally characterized. The nucleus localization of SlERF84-sGFP was confirmed through a transient expression assay. Transactivation assays in yeast demonstrated that SlERF84 functions as a transcriptional activator. Real-time PCR analysis revealed that SlERF84 could be markedly induced by drought, salt and by several phytohormones (ABA, MeJA and ACC). Overexpression of SlERF84 in Arabidopsis endows transgenic plants with ABA hypersensitivity and enhanced tolerance to drought and salt stress. Histochemical staining assay showed that SlERF84 renders transgenic plants better ROS-scavenging capability. Pathogen inoculation assay revealed that SlERF84 might negatively modulate plant defense response to Pseudomonas syringae pv. tomato DC3000. Moreover, the transcript levels of pathogenesis-related genes AtPR1 and AtPR3 were compromised in transgenic Arabidopsis, as compared to that in Col-0 plants when inoculated with Pseudomonas syringae pv. tomato DC3000. These results suggest that SlERF84 functions as a stress-responsive transcription factor in differentially modulation of abiotic and biotic stress tolerance, and may have applications in the engineering of economically important crops.

We review our new imaging technique for quantitatively measuring the integral (thickness-average) refractive index of the nuclei of live biological cells during flow, combining quantitative phase microscopy with simultaneous 2-D fluorescence microscopy for nucleus localization.

Relish is an NF-kB transcription factor involved in immune-deficiency (IMD) signal pathway. In this study, a Relish gene (MrRelish) was identified from Macrobrachium rosenbergii. The full length of MrRelish comprises 5072 bp, including a 3510 bp open reading frame encoding a 1169 bp amino acid protein. MrRelish contains a Rel homology domain (RHD), a nucleus localization signal, an IκB-like domain (6 ankyrin repeats), and a death domain. Phylogenetic analysis showed that MrRelish and other Relish from crustaceans belong to one group. MrRelish was expressed in all detected tissues, with the highest expression level in hemocytes and intestines. MrRelish was also upregulated in hepatopancreas at 6 h after Vibrio anguillarum challenge. The over-expression of MrRelish could induce the expression of antimicrobial peptides (AMPs), such as Drosophila Metchnikowin (Mtk), Attacin (Atta), Drosomycin (Drs), and Cecropin (CecA) and shrimp Penaeidin (Pen4). The RNAi of MrRelish in gills showed that the expression of crustin (cru) 2, Cru5, Cru8, lysozyme (Lyso) 1, and Lyso2 was inhibited. However, the expression of anti-lipopolysaccharide factor (ALF) 1 and ALF3 did not change when MrRelish was knocked down. These results indicate that MrRelish may play an important role in innate immune defense against V. anguillarum in M. rosenbergii.