Overexpression of a nitrate transporter NtNPF2.11 increases nitrogen accumulation and yield in tobacco.

Nitrogen (N) is the key essential macronutrient for crop growth and yield. Over-application of inorganic N fertilizer in fields generated serious environmental pollution and had a negative impact to human health. Therefore, improving crop N use efficiency (NUE) is helpful for sustainable agriculture. The biological functions of nitrogen transporters and regulators have been intensively studied in many crop species. However, only a few nitrogen transporters have been identified in tobacco to date. We reported the identification and functional characterization of a nitrate transporter NtNPF2.11 from tobacco (Nicotiana tabacum). qRT-PCR assay revealed that NtNPF2.11 was mainly expressed in leaf and vein. Under middle N (MN, 1.57 kg N/100 m2) and high N (HN, 2.02 kg N/100 m2) conditions, overexpression of NtNPF2.11 in tobacco greatly improved N utilization and biomass. Moreover, under middle N and high N conditions, the expression of genes for nitrate assimilation, such as NtNR1, NtNiR, NtGS and NtGOGAT, were upregulated in NtNPF2.11 overexpression plants. Compared with WT, overexpression of NtNPF2.11 increased potassium (K) accumulation under high N conditions. These results indicated that overexpression of NtNPF2.11 could increase tobacco yield, N and K accumulation under higher N conditions. Overall, these findings improve our understanding the function of NtNPF2.11 and provide useful gene for sustainable agriculture.

A novel therapeutic strategy of combined camrelizumab and apatinib for the treatment of advanced hepatocellular carcinoma.

Methods: 83 patients with hepatocellular carcinoma (HCC) admitted to the interventional oncology department were randomly divided into two groups. Apatinib and camrelizumab were administered to 42 patients in group A, whereas sorafenib was administered to 41 patients in group B for three months. The clinical efficacy was evaluated in terms of objective response rate (ORR), and disease control rate (DCR). Certain tumor markers like alpha-fetoprotein (AFP), carbohydrate antigen 199 (CA199), carcinoembryonic antigen (CEA), hypoxia-inducible factor (HIF-1), immune function T lymphocyte subsets (CD3+, CD4+, CD8+, CD4+/CD8+) were determined before and after treatment. The serum levels of vascular endothelial growth factor (VEGF), osteopontin (OPN), aspartate aminotransferase (AST), and epidermal growth factor 7 (EGF7)] were observed. The survival time between the two groups was compared, such as progression-free survival (PFS) and median survival (MS). Finally, the toxicity and side effects data were also obtained. Results: The ORR and DCR of group A were 69.05% and 88.10%, respectively, which were significantly higher (P<0.05) than group B (ORR=53.66% and DCR=70.73%). After treatment, the AFP, CA199, CEA, and HIF-1 levels of both groups decreased significantly (P<0.05), and the respective biomarker levels of group A were lower than those of group B (P<0.05). Following treatment, CD3+, CD4+, CD4+/CD8+ index in group A significantly increased (P<0.05) while CD8+ level was significantly decreased (P<0.05). Compared to group B, a significant increase was observed in group A's CD3+, CD4+, and CD4+/CD8+ index. There were no significant changes in CD3+, CD4+, CD8+, CD4+/CD8+ indexes before and after treatment in group B (P>0.05). The serum level of VEGF, OPN, EGF-7 and AST indexes of group A&B were decreased significantly (P<0.05). Compared with group B, the VEGF, OPN, EGF7 and AST indexes of group A were significantly reduced (P<0.05). PFS and MS in group A were significantly higher than in group B (P<0.05). There was no significant difference between groups A and B in terms of toxicity and adverse effects (P>0.05). Conclusion: In treating HCC, combining apatinib and camrelizumab can reduce tumor markers, enhance the immune system and curative effect, and prolong patient survival. The underline mechanism is related to the down-regulation of VEGF, OPN and HIF-1 indexes.

Evaluation in pig of an intestinal administration device for oral peptide delivery.

The bioavailability of peptides co-delivered with permeation enhancers following oral administration remains low and highly variable. Two factors that may contribute to this are the dilution of the permeation enhancer in the intestinal fluid, as well as spreading of the released permeation enhancer and peptide in the lumen by intestinal motility. In this work we evaluated an Intestinal Administration Device (IAD) designed to reduce the luminal dilution of drug and permeation enhancer, and to minimize movement of the dosage form in the intestinal lumen. To achieve this, the IAD utilizes an expanding design that holds immediate release mini tablets and places these in contact with the intestinal epithelium, where unidirectional drug release can occur. The expanding conformation limits movement of the IAD in the intestinal tract, thereby enabling drug release at a single focal point in the intestine. A pig model was selected to study the ability of the IAD to promote intestinal absorption of the peptide MEDI7219 formulated together with the permeation enhancer sodium caprate. We compared the IAD to intestinally administered enteric coated capsules and an intestinally administered solution. The IAD restricted movement of the immediate release tablets in the small intestine and histological evaluation of the mucosa indicated that high concentrations of sodium caprate were achieved. Despite significant effect of the permeation enhancer on the integrity of the intestinal epithelium, the bioavailability of MEDI7219 was of the same order of magnitude as that achieved with the solution and enteric coated capsule formulations (2.5-3.8%). The variability in plasma concentrations of MEDI7219 were however lower when delivered using the IAD as compared to the solution and enteric coated capsule formulations. This suggests that dosage forms that can limit intestinal dilution and control the position of drug release can be a way to reduce the absorptive variability of peptides delivered with permeation enhancers but do not offer significant benefits in terms of increasing bioavailability.

Pancreatic Cancer: Nucleic Acid Drug Discovery and Targeted Therapy.

Pancreatic cancer (PC) is one of the most lethal cancers with an almost 10% 5-year survival rate. Because PC is implicated in high heterogeneity, desmoplastic tumor-microenvironment, and inefficient drug-penetration, the chemotherapeutic strategy currently recommended for the treatment of PC has limited clinical benefit. Nucleic acid-based targeting therapies have become strong competitors in the realm of drug discovery and targeted therapy. A vast evidence has demonstrated that antibody-based or alternatively aptamer-based strategy largely contributed to the elevated drug accumulation in tumors with reduced systematic cytotoxicity. This review describes the advanced progress of antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), messenger RNA (mRNAs), and aptamer-drug conjugates (ApDCs) in the treatment of PC, revealing the bright application and development direction in PC therapy.

Poly (ADP-ribose) polymerase 1 (PARP1) inhibition promotes pulmonary metastasis of osteosarcoma by boosting ezrin phosphorylation.

Due to the large proportion of BRCA deficiency and chromosomal instability in OS patients, poly (ADP-ribose) polymerase inhibitors (PARPi) could be an effective strategy for anti-OS therapy. In two orthotopic OS mouse models, we discovered that although PARPi had inhibitory effect on the growth of the orthotopic OS tumors regardless of BRCA deficiency, the treatment of PARPi essentially aggravated the pulmonary metastasis of OS in both models. A protein playing a crucial role in OS metastasis, ezrin, was identified as an interactive protein for PARP1. The phosphorylation of ezrin was significantly promoted during PARP inhibition. Besides the traditional function of phosphorylated ezrin at plasma membrane, we newly identified its nuclear speckle localization and its function with mRNA export. Ezrin knockdown or phosphorylation inhibition could partially rescue PARPi induced metastasis. Collectively, we unveiled a new mechanism for PARP-involved OS metastasis, which proposed a novel combinational therapy strategy using PARP and ezrin inhibitors for future OS treatment.

A nucleocytoplasmic-localized E3 ligase affects the NLR receptor stability.

Ubiquitination is a pivotal post-translational modification that regulates turnover of nucleotide-binding site and leucine-rich repeat receptors (NLRs). As a RING-type E3 ligase, BOI (Botrytis susceptible1 interactor) has been reported to interact with different proteins, and function in the nucleus. New studies have identified that BOI can interact and ubiquitinate L5 (AT1G12290), a CC-NBS-LRR protein in vitro, and mediate the proteasomal degradation of L5 in Nicotiana benthamiana and Arabidopsis thaliana. However, there still remains an unanswered question about where the degradation occurs at the subcellular level. In this study, the ubiquitination of L5 by BOI was determined in N. benthamiana. Meanwhile, we discovered that BOI exhibited nucleocytoplasmic localization and mediated the degradation of the plasma membrane localized L5 outside the nucleus. BOI and its homologs BRG1 and BRG3 function redundantly in negatively regulate the protein level of L5. Overall, this report reveals BOI and its homologs have multiple targets and function at different subcellular locations.

The RING-type protein BOI negatively regulates the protein level of a CC-NBS-LRR in Arabidopsis.

Nucleotide-binding site and leucine-rich repeat receptors (NLRs) play pivotal roles in plant immunity. The regulation of NLR stability is essential to ensure effective immunity, whereas the exact mechanism is largely unclear. The Arabidopsis CC-NBS-LRR protein L5 (At1g12290) can induce cell death in Nicotiana benthamiana, but not in Arabidopsis thaliana. We screened the interactors of L5 by yeast two-hybrid, and found that the BOI can interact with the CC domain of L5. Transiently expressed BOI reduced the protein level of L5, and suppressed the auoactivity of L5 in N. benthamiana. BOI can interact and ubiquitinate L5 in vitro, and mediate the proteasomal degradation of L5 in N. benthamiana and Arabidopsis. The Lys425 in the NBS domain of L5 is the critical unbiquitin site for the degradation. In conclusion, our results reveal a mechanism for the control of the stability of L5 protein and for the suppressed of L5-triggered cell death by a RING-type E3 ligase through the ubiquitin proteasome system.

Mechanism of Nucleic Acid Sensing in Retinal Pigment Epithelium (RPE): RIG-I Mediates Type I Interferon Response in Human RPE.

Age-related macular degeneration (AMD), a degenerative disease of the outer retina, is the leading cause of blindness among the elderly. A hallmark of geographic atrophy (GA), an advanced type of nonneovascular AMD (dry AMD), is photoreceptor and retinal pigment epithelium (RPE) cell death. Currently, there are no FDA-approved therapies for GA due to a lack of understanding of the disease-causing mechanisms. Increasing evidence suggests that chronic inflammation plays a predominant role in the pathogenesis of dry AMD. Dead or stressed cells release danger signals and inflammatory factors, which causes further damage to neighboring cells. It has been reported that type I interferon (IFN) response is activated in RPE cells in patients with AMD. However, how RPE cells sense stress to initiate IFN response and cause further damage to the retina are still unknown. Although it has been reported that RPE can respond to extracellularly added dsRNA, it is unknown whether and how RPE detects and senses internally generated or internalized nucleic acids. Here, we elucidated the molecular mechanism by which RPE cells sense intracellular nucleic acids. Our data demonstrate that RPE cells can respond to intracellular RNA and induce type I IFN responses via the RIG-I (DExD/H-box helicase 58, DDX58) RNA helicase. In contrast, we showed that RPE cells were unable to directly sense and respond to DNA through the cGAS-STING pathway. We demonstrated that this was due to the absence of the cyclic GMP-AMP synthase (cGAS) DNA sensor in these cells. The activation of IFN response via RIG-I induced expression of cell death effectors and caused barrier function loss in RPE cells. These data suggested that RPE-intrinsic pathways of nucleic acid sensing are biased toward RNA sensing.

A Novel Graphene Quantum Dot-Based mRNA Delivery Platform.

During the last decades, there has been growing interest in using therapeutic messager RNA (mRNA) together with drug delivery systems. Naked, unformulated mRNA is, however, unable to cross the cell membrane and is susceptible to degradation. Here we use graphene quantum dots (GQDs) functionalized with polyethyleneimine (PEI) as a novel mRNA delivery system. Our results show that these modified GQDs can be used to deliver intact and functional mRNA to Huh-7 hepatocarcinoma cells at low doses and, that the GQDs are not toxic, although cellular toxicity is a problem for these first-generation modified particles. Functionalized GQDs represent a potentially interesting delivery system that is easy to manufacture, stable and effective.

The resistance associated protein RIN4 promotes the extracellular transport of AtEXO70E2.

RIN4 is an important immunomodulator in Arabidopsis, which is targeted by multiple pathogenic effectors, and consequently guarded by different immune receptors. Although RIN4 plays a significant role in plant immunity, its molecular function is not fully understood. We found that RIN4 interacts with the exocyst subunit EXO70E2. Transiently expressed RIN4 can recruits EXO70E2 vesicles to the plasma membrane, and promote the transport of the vesicles to the extracellular matrix. RIN4 also can decrease the protein level of EXO70E2. Base on the fact that EXO70 proteins positively mediates plant immunity, the function of RIN4 is to promote the extracellular export of defense related vesicles. Pathogens will secret effectors to modify or cleavage it to interfere this exocytosis.

The Roles of Sclerostin in Immune System and the Applications of Aptamers in Immune-Related Research.

Wnt signaling is one of the fundamental pathways that play a major role in almost every aspect of biological systems. In addition to the well-known influence of Wnt signaling on bone formation, its essential role in the immune system also attracted increasing attention. Sclerostin, a confirmed Wnt antagonist, is also proven to modulate the development and differentiation of normal immune cells, particularly B cells. Aptamers, single-stranded (ss) oligonucleotides, are capable of specifically binding to a variety of target molecules by virtue of their unique three-dimensional structures. With in-depth study of those functional nucleic acids, they have been gradually applied to diagnostic and therapeutic area in immune diseases due to their various advantages over antibodies. In this review, we focus on several issues including the roles of Wnt signaling and Wnt antagonist sclerostin in the immune system. For the sake of understanding, current examples of aptamers applications for the immune diseases are also discussed. At the end of this review, we propose our ideas for the future research directions.

Structure and function analysis of a CC-NBS-LRR protein AT1G12290.

Nucleotide-binding site (NBS) and leucine-rich repeat (LRR) receptors (NLRs) play important roles in plant immunity. The genome of Arabidopsis thaliana contains about 150 genes encoding NLR proteins, but few of them have been studied. We transiently expressed a series of NBS-LRR proteins in the leaves of Nicotiana benthamiana, and found that the CC-NBS-LRR protein (AT1G12290) was able to trigger cell death, a characterized function for the activation of an NLR protein. We observed that the YFP-tagged AT1G12290 was localized on the plasma membrane (PM), and the predicted myristoylation site Gly2 is required for the localization and function of the protein. Further structure dissection revealed that the CC domain was enough to activate cell death, and the N-terminal 1-100 amino acid fragment was the minimal region to induce cell death and self-association. Our research provides important clues to elucidate the activation mechanism of AT1G12290.

Recent Progress in Aptamer Discoveries and Modifications for Therapeutic Applications.

Aptamers are oligonucleotide sequences with a length of about 25-80 bases which have abilities to bind to specific target molecules that rival those of monoclonal antibodies. They are attracting great attention in diverse clinical translations on account of their various advantages, including prolonged storage life, little batch-to-batch differences, very low immunogenicity, and feasibility of chemical modifications for enhancing stability, prolonging the half-life in serum, and targeted delivery. In this Review, we demonstrate the emerging aptamer discovery technologies in developing advanced techniques for producing aptamers with high performance consistently and efficiently as well as requiring less cost and resources but offering a great chance of success. Further, the diverse modifications of aptamers for therapeutic applications including therapeutic agents, aptamer-drug conjugates, and targeted delivery materials are comprehensively summarized.

A PD-L1 Aptamer Selected by Loss-Gain Cell-SELEX Conjugated with Paclitaxel for Treating Triple-Negative Breast Cancer.

BACKGROUND The clinical challenges of triple-negative breast cancer (TNBC) includes the lack of targeted therapy and chemoresistance. TNBC has relatively high PD-L1 expression, and PD-L1 antibody in combination with nab-paclitaxel has been approved by FDA for TNBC treatment. Aptamers, also termed chemical antibody, are widely used in targeted drug delivery. The present study aimed to select a DNA aptamer that could specifically bind and deliver drugs to TNBC cells. MATERIAL AND METHODS An innovative loss-gain cell-SELEX strategy was used to select DNA aptamer for PD-L1 protein. Construction of PD-L1 knock-out and over-expression MDA-MB-231 cell lines were conducted through transfection and confirmed by western blot and flow cytometry. Confocal microscopy and flow cytometry were used to analyze the binding ability of aptamer with TNBC cells. The cytotoxicity of aptamer-paclitaxel complex against TNBC cells was evaluated by Cell Counting Kit-8 assay. The reactivation of the T cell function by aptamer was measured by IL-2 enzyme-linked immunosorbent assay after T cells co-cultured with tumor cells. RESULTS In this work, using an innovative loss-gain cell-SELEX strategy, we screened a PD-L1-targeting aptamer. PD-L1 aptamer selectively bound to PD-L1 over-expressed TNBC cells with a dissociation constant in the nanomolar range. PD-L1 aptamer could also inhibit PD-1/PD-L1 interaction and restore the function of T cells. Moreover, we developed a PD-L1 aptamer-paclitaxel conjugate which showed improved cellular uptake and anti-proliferation efficacy in PD-L1 over-expressed TNBC cells. CONCLUSIONS In summary, these findings suggest that the selected PD-L1 aptamer might have potential implication in immune modulation and targeted therapy against TNBC.

RANKL/RANK System-Based Mechanism for Breast Cancer Bone Metastasis and Related Therapeutic Strategies.

Breast cancer remains one of the most life-threatening tumors affecting women. Most patients with advanced breast cancer eventually develop metastatic diseases, which cause significant morbidity and mortality. Approximately two-thirds of patients with advanced breast cancer exhibit osteolytic-type bone metastasis, which seriously reduce the quality of life. Therefore, development of novel therapeutic strategies for treating breast cancer patients with bone metastasis is urgently required. The "seed and soil" theory, which describes the interaction between the circulating breast cancer cells (seeds) and bone microenvironment (soil), is widely accepted as the mechanism underlying metastasis. Disruption of any step in this cycle might have promising anti-metastasis implications. The interaction of receptor activator of nuclear factor-κB ligand (RANKL) and its receptor RANK is fundamental in this vicious cycle and has been shown to be a novel effective therapeutic target. A series of therapeutic strategies have been developed to intervene in this cross-talk. Therefore, in this review, we have systematically introduced the functions of the RANKL/RANK signaling system in breast cancer and discussed related therapeutic strategies.

Elucidation and Structural Modeling of CD71 as a Molecular Target for Cell-Specific Aptamer Binding.

Pancreatic cancer is a highly lethal malignancy associated with tissues of the pancreas. Early diagnosis and effective treatment are crucial to improving the survival rate of patients with pancreatic cancer. In a previous study, we employed the cell-SELEX strategy to obtain an ssDNA aptamer termed XQ-2d with high binding affinity for pancreatic cancer. Here, we first identify CD71 as the XQ-2d-binding target. We found that knockdown of CD71 abolished the binding of XQ-2d and that the binding affinity of XQ-2d is associated with membrane-bound CD71, rather than total CD71 levels. Competitive analysis revealed that XQ-2d shares the same binding site on CD71 with transferrin (Tf), but not anti-CD71 antibody. We then used a surface energy transfer (SET) nanoruler to measure the distance between the binding sites of XQ-2d and anti-CD71 antibody, and it was about 15 nm. Furthermore, we did molecular dynamics simulation to clarify that the spatial structure of XQ-2d and Tf competitively binding to CD71. We also engineered XQ-2d-mediated targeted therapy for pancreatic cancer, using an XQ-2d-based complex for loading doxorubicin (Dox). Because CD71 is overexpressed not only in pancreatic cancer but also in a variety of tumors, our work provides a systematic novel way of studying a potential biomarker and also promising tools for cancer diagnosis and therapy, opening new doors for effective cancer theranostics.

The Rules and Functions of Nucleocytoplasmic Shuttling Proteins.

Biological macromolecules are the basis of life activities. There is a separation of spatial dimension between DNA replication and RNA biogenesis, and protein synthesis, which is an interesting phenomenon. The former occurs in the cell nucleus, while the latter in the cytoplasm. The separation requires protein to transport across the nuclear envelope to realize a variety of biological functions. Nucleocytoplasmic transport of protein including import to the nucleus and export to the cytoplasm is a complicated process that requires involvement and interaction of many proteins. In recent years, many studies have found that proteins constantly shuttle between the cytoplasm and the nucleus. These shuttling proteins play a crucial role as transport carriers and signal transduction regulators within cells. In this review, we describe the mechanism of nucleocytoplasmic transport of shuttling proteins and summarize some important diseases related shuttling proteins.

PARP1 in Carcinomas and PARP1 Inhibitors as Antineoplastic Drugs.

Poly (ADP-ribose) polymerase 1 (PARP1), the best-studied isoform of the nuclear enzyme PARP family, plays a pivotal role in cellular biological processes, such as DNA repair, gene transcription, and so on. PARP1 has been found to be overexpressed in various carcinomas. These all indicate the clinical potential of PARP1 as a therapeutic target of human malignancies. Additionally, multiple preclinical research studies and clinical trials demonstrate that inhibition of PARP1 can repress tumor growth and metastasis. Up until now, PARP1 inhibitors are clinically used not only for monotherapy to suppress various tumors, but also for adjuvant therapy, to maintain or enhance therapeutic effects of mature antineoplastic drugs, as well as protect patients from chemotherapy and surgery-induced injury. To supply a framework for understanding recent research progress of PARP1 in carcinomas, we review the structure, expression, functions, and mechanisms of PARP1, and summarize the clinically mature PARP1-related anticancer agents, to provide some ideas for the development of other promising PARP1 inhibitors in antineoplastic therapy.

Potential Diagnostic and Therapeutic Applications of Oligonucleotide Aptamers in Breast Cancer.

Breast cancer is one of the most common causes of cancer related deaths in women. Currently, with the development of early detection, increased social awareness and kinds of treatment options, survival rate has improved in nearly every type of breast cancer patients. However, about one third patients still have increased chances of recurrence within five years and the five-year relative survival rate in patients with metastasis is less than 30%. Breast cancer contains multiple subtypes. Each subtype could cause distinct clinical outcomes and systemic interventions. Thereby, new targeted therapies are of particular importance to solve this major clinical problem. Aptamers, often termed "chemical antibodies", are functionally similar to antibodies and have demonstrated their superiority of recognizing target with high selectivity, affinity and stability. With these intrinsic properties, aptamers have been widely studied in cancer biology and some are in clinical trials. In this review, we will firstly discuss about the global impacts and mechanisms of breast cancer, then briefly highlight applications of aptamers that have been developed for breast cancer and finally summarize various challenges in clinical translation of aptamers.

Functional analysis of plant NB-LRR gene L3 by using E. coli.

Plant NB-LRR genes mediate plant innate immunity and cause the programmed cell death of plant cells. Very little, however, is known about these processes. Taken advantage of easy manipulation of bacteria, genetic analysis was made to understand the mechanism of lethality of NB-LRR proteins to bacteria and correlate the information back to how NB-LRR proteins cause cell death in plants. It was found that only L3 encoded by NB-LRR gene L3 (At1g15890) specifically caused significant death of BL21(DE3), while other NBS-LRR proteins did not, and 760-851, the truncated form of L3, was essential to the lethality of L3. Gene yedZ (EG14048) and nupG (EG10664) were identified by genome re-sequencing from E. coli, both of which mediate the toxicity of L3 in E. coli. Furthermore, NupG can affect the activity of peroxidase and significantly suppress plant cell death, which is induced by NB-LRR protein RPM1(D505V) encoded by RPM1 (At3g07040) in N. benthamiana. These findings provide evidence that functional analysis of plant NB-LRR genes in microorganisms might be a potential and rapid method.