A hybrid vector for ligand-directed tumor targeting and molecular imaging

Merging tumor targeting and molecular-genetic imaging into an integrated platform is limited by lack of strategies to enable systemic yet ligand-directed delivery and imaging of specific transgenes. Many eukaryotic viruses serve for transgene delivery but require elimination of native tropism for mammalian cells; in contrast, prokaryotic viruses can be adapted to bind to mammalian receptors but are otherwise poor vehicles. Here we introduce a system containing cis-elements from adeno-associated virus (AAV) and single-stranded bacteriophage. Our AAV/phage (AAVP) prototype targets an integrin. We show that AAVP provides superior tumor transduction over phage and that incorporation of inverted terminal repeats is associated with improved fate of the delivered transgene. Moreover, we show that the temporal dynamics and spatial heterogeneity of gene expression mediated by targeted AAVP can be monitored by positron emission tomography. This new class of targeted hybrid viral particles will enable a wide range of applications in biology and medicine.     

Microtubule network facilitates nuclear targeting of human cytomegalovirus capsid

We assessed the requirement of the host cytoskeleton for the intracytosolic transport of incoming human cytomegalovirus (HCMV) capsids. Treatments with microtubule (MT)-depolymerizing drugs nocodazole and colchicine led to a drastic decrease in levels of IE1 antigen, whereas cytochalasin B had no effect on the level of IE1 as determined by Western blot analyses. Sequential treatment including nocodazole washout and removal of cell surface virion revealed that HCMV entry into the cells occurred normally in the absence of the MT network. This finding was also supported by data obtained by monitoring pUL83 signals with an immunofluorescent assay (IFA). Furthermore, we demonstrated a close association of incoming HCMV capsids with MTs by IFA and ultrastructural analyses. In the absence of the MT network, the capsids which had entered the cytoplasm did not move to close proximity of the nucleus. These data suggest that HCMV capsids associate with the MT network to facilitate their own movement to the nucleus before the onset of immediate-early (IE) gene expression and that this association is required to start efficient IE gene expression.     

Preosteoblast-enriched Inc-Evf2 facilitates osteogenic differentiation by targeting Notch

Ossification of ligaments(OL)and osteoporosis(OP)are multifactorial disorders without definitive clinical biomarkers.Long non-coding RNAs(IncRNAs)are known to i...     

MicroRNA-1 facilitates hypoxia-induced injury by targeting NOTCH3

Cell proliferation, apoptosis, and autophagy have been reported to be related to myocardial ischemia injury. MicroRNAs have attracted wide attention on regulating cell proliferation, apoptosis, and autophagy. miR-1 expression has been reported to be dysregulated in cardiac tissue or cells with hypoxia, while the exact roles as well as underlying mechanism remain poorly understood. In this study, we investigated the potential roles of miR-1 in cell proliferation, apoptosis, and autophagy in hypoxia-treated cardiac injury and explored the underlying mechanism using H9c2 cells. Results showed that hypoxic stimulation inhibited cell proliferation and the expression of miR-1 but promoted cell apoptosis in H9c2 cells. Moreover, overexpression of miR-1 promoted cell apoptosis and inhibited cell proliferation and autophagy in H9c2 cells treated with hypoxia, while its knockdown played an opposite effect. In addition, bioinformatics, luciferase reporter, and RNA immunoprecipitation analyses indicated that NOTCH3 was a direct target of miR-1 and its upregulation reversed the effects of miR-1 on cell proliferation, apoptosis, and autophagy in hypoxia-treated H9c2 cells. Taken together, our data suggested that miR-1 promoted hypoxia-induced injury by targeting NOTCH3, indicating novel therapeutic targets for treatment of myocardial ischemia injury.     

Chaperone networking facilitates protein targeting to the bacterial cytoplasmic membrane

Nascent polypeptides emerging from the ribosome are assisted by a pool of molecular chaperones and targeting factors, which enable them to efficiently partition as cytosolic, integral membrane or exported proteins. Extensive genetic and biochemical analyses have significantly expanded our knowledge of chaperone tasking throughout this process. In bacteria, it is known that the folding of newly-synthesized cytosolic proteins is mainly orchestrated by three highly conserved molecular chaperones, namely Trigger Factor (TF), DnaK (HSP70) and GroEL (HSP60). Yet, it has been reported that these major chaperones are strongly involved in protein translocation pathways as well. This review describes such essential molecular chaperone functions, with emphasis on both the biogenesis of inner membrane proteins and the post-translational targeting of presecretory proteins to the Sec and the twin-arginine translocation (Tat) pathways. Critical interplay between TF, DnaK, GroEL and other molecular chaperones and targeting factors, including SecB, SecA, the signal recognition particle (SRP) and the redox enzyme maturation proteins (REMPs) is also discussed. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

超声波-微波协同盐提莲子蛋白

探索利用超声波微波协同盐提湘莲蛋白质最佳提取条件。最佳工艺条件为超声波功率50w,微波功率600W,料液比1：12（g／mE）、38℃、0．15mol／LNaCl盐溶液提取15min,最佳提取率可达88．9％。以CO2为沉淀剂,乙醇为助溶剂,研究加压CO2和乙醇对蛋白质的协同沉淀作用。在常温、5MPa加压CO2酸沉8％（质量分数）的湘莲蛋白质乙醇液（体积分数20％乙醇）0．5h时,莲子蛋白沉淀率可达81．2％。结果表明,用文中方法提取湘莲蛋白,湘莲蛋白收率为72．2％,纯度达93％.     

Computer assisted simulations and molecular graphics methods in molecular design

A survey is presented of computer-assisted statistical mechanical methods. The general theoretical background is described and special methods are discussed in detail. Practical procedures allowing for the calculation of binding energies are examined. A recent perturbation-relaxation procedure is summarized.     

Aptamer-based molecular imaging

Molecular imaging has greatly advanced basic biology and translational medicine through visualization and quantification of single/multiple molecular events temporally and spatially in a cellular context and in living organisms. Aptamers, short single-stranded nucleic acids selected in vitro to bind a broad range of target molecules avidly and specifically, are ideal molecular recognition elements for probe development in molecular imaging. This review summarizes the current state of aptamer-based biosensor development (probe design and imaging modalities) and their application in imaging small molecules, nucleic acids and proteins mostly in a cellular context with some animal studies. The article is concluded with a brief discussion on the perspective of aptamer-based molecular imaging.     

Novel targeting strategy based on multimeric ligands for drug delivery and molecular imaging: homooligomers of alpha-MSH

Homooligomers constructed with 4- and 6-amino acid fragments of melanocortin (alpha-MSH) bind with higher affinity and with apparent cooperativity to melanocortin receptor, compared to their constituent monomers. Individual ligands were tethered with various spacers of different length and rigidity and the influence of spacers on binding was studied. Binding assays were performed on cells transfected with the melanocortin receptor, hMC4R. There is a 5-7-fold decrease in the EC(50) with the addition of each subunit, going from monomer to trimer. The Hill coefficient increases from 0.76 for the monomer to 1.12 for the dimer and 1.35 for the trimer. These data show a general trend of increasing avidity with increasing number of ligands in oligomers.     

The pro-apoptotic protein Par-4 facilitates vascular contractility by cytoskeletal targeting of ZIPK

Par-4 (prostate apoptosis response 4) is a pro-apoptotic protein and tumour suppressor that was originally identified as a gene product up-regulated during apoptosis in prostate cancer cells. Here, we show, for the first time, that Par-4 is expressed and co-localizes with the actin filament bundles in vascular smooth muscle. Furthermore, we demonstrate that targeting of ZIPK to the actin filaments, as observed upon PGF-2α stimulation, is inhibited by the presence of a cell permeant Par-4 decoy peptide. The same decoy peptide also significantly inhibits PGF-2α induced contractions of smooth muscle tissue. Moreover, knockdown of Par-4 using antisense morpholino nucleotides results in significantly reduced contractility, and myosin light chain and myosin phosphatase target subunit phosphorylation. These results indicate that Par-4 facilitates contraction by targeting ZIPK to the vicinity of its substrates, myosin light chain and MYPT, which are located on the actin filaments. These results identify Par-4 as a novel regulator of myosin light chain phosphorylation in differentiated, contractile vascular smooth muscle.     

Biosensors and molecular imaging

The increase in the understanding of the physical and functional properties of the biological material, from the cellular level down to single molecules, owes its success to the development of suitable high-sensitivity platforms to image the biomaterial and analyze its response to specific stimuli. Imaging has indeed reached molecular capabilities, thanks to optical or magnetic markers [1], to the atomic force microscopy (AFM) in surface reconstruction [2], and is nearing success in three-dimensional (3-D) reconstruction thanks to X-ray holography [3].     

CircRNA circ_0043533 facilitates cell growth in polycystic ovary syndrome by targeting miR-1179

Increasing evidence indicates that circular RNAs (CircRNAs) have an important role in human diseases, including polycystic ovary syndrome (PCOS). Recently, circ_0043533, a novel circRNA, was proposed to be involved in the progression of PCOS. However, its role in PCOS has not been explored. In this study, the expression levels of circ_0043533 and miR-1179 in ovarian granulosa cells (OGCs) were examined by qRT-PCR analysis. Moreover, knockdown of circ_0043533 in OGC lines COV434 and KGN, respectively, the cell viability, proliferation, apoptosis, and cycle-related markers of insulin-triggered OGCs were examined by CCK-8, EdU staining, flow cytometry, and western blot assays, respectively. The interaction between circ_0043533 and miR-1179 was examined by bioinformatics, dual-luciferase assay, and RNA immunoprecipitation. Besides, effects of the miR-1179 inhibitor on cell viability and apoptosis in OGC lines with circ_0043533 knockdown were also evaluated. OGCs and insulin-treated OGCs exhibited higher circ_0043533 levels in comparison to the IOSE80 cells. Additionally, knockdown of circ_0043533 remarkably inhibited the cell viability and proliferation and promoted the apoptosis of insulin-treated COV434 and KGN cells, respectively. Meanwhile, circ_0043533 knockdown could down-regulate the Bcl-2, CDK2, and Cyclin D1 expressions, and up-regulate the Bax levels. Furthermore, we demonstrated that circ_0043533 acted as a sponge to absorb miR-1179. Interestingly, miR-1179 inhibition remarkably attenuated the effect of circ_0043533 silence on cell proliferation and apoptosis in insulin-treated COV434 and KGN cells. Taken together, this study revealed that circ_0043533 knockdown restrained the malignant progression of PCOS via targeting miR-1179. Our data suggested that circ_0043533 would serve as a novel therapeutic target for PCOS.     

The somatodendritic endosomal regulator NEEP21 facilitates axonal targeting of L1/NgCAM

Correct targeting of proteins to axons and dendrites is crucial for neuronal function. We showed previously that axonal accumulation of the cell adhesion molecule L1/neuron-glia cell adhesion molecule (NgCAM) depends on endocytosis (Wisco, D., E.D. Anderson, M.C. Chang, C. Norden, T. Boiko, H. Folsch, and B. Winckler. 2003. J. Cell Biol. 162:1317-1328). Two endocytosis-dependent pathways to the axon have been proposed: transcytosis and selective retrieval/retention. We show here that axonal accumulation of L1/NgCAM occurs via nondegradative somatodendritic endosomes and subsequent anterograde axonal transport, which is consistent with transcytosis. Additionally, we identify the neuronal-specific endosomal protein NEEP21 (neuron-enriched endosomal protein of 21 kD) as a regulator of L1/NgCAM sorting in somatodendritic endosomes. Down-regulation of NEEP21 leads to missorting of L1/NgCAM to the somatodendritic surface as well as to lysosomes. Importantly, the axonal accumulation of endogenous L1 in young neurons is also sensitive to NEEP21 depletion. We propose that small endosomal carriers derived from somatodendritic recycling endosomes can serve to redistribute a distinct set of membrane proteins from dendrites to axons.     

Fbxo45 facilitates pancreatic carcinoma progression by targeting USP49 for ubiquitination and degradation

Fbxo45, a conserved F-box protein, comprises of an atypical SKP1, CUL1, F-box protein (SCF) ubiquitin ligase complex that promotes tumorigenesis and development. However, the biological function and molecular mechanisms of Fbxo45 involved in pancreatic carcinogenesis are ambiguous. We conducted several approaches, including transfection, coIP, real-time polymerase chain reaction (RT-PCR), Western blotting, ubiquitin assays, and animal studies, to explore the role of Fbxo45 in pancreatic cancer. Here, we report that USP49 stability is governed by Fbxo45-mediated ubiquitination and is enhanced by the absence of Fbxo45. Moreover, Fbxo45 binds to a short consensus sequence of USP49 through its SPRY domain. Furthermore, Fbxo45-mediated USP49 ubiquitination and degradation are enhanced by NEK6 kinase. Functionally, Fbxo45 increases cell viability and motility capacity by targeting USP49 in pancreatic cancer cells. Xenograft mouse experiments demonstrated that ectopic expression of Fbxo45 enhanced tumor growth in mice and that USP49 overexpression inhibited tumor growth in vivo. Notably, Fbxo45 expression was negatively associated with USP49 expression in pancreatic cancer tissues. Fbxo45 serves as an oncoprotein to facilitate pancreatic oncogenesis by regulating the stability of the tumor suppressor USP49 in pancreatic cancer.Subject terms: Pancreatic cancer, Pancreatic cancer     

The RNA-binding protein PCBP2 facilitates gastric carcinoma growth by targeting miR-34a

Gastric carcinoma is the fourth most common cancer worldwide, with a high rate of death and low 5-year survival rate. However, the mechanism underling gastric cancer is still not fully understood. Here in the present study, we identify the RNA-binding protein PCBP2 as an oncogenic protein in human gastric carcinoma. Our results show that PCBP2 is up-regulated in human gastric cancer tissues compared to adjacent normal tissues, and that high level of PCBP2 predicts poor overall and disease-free survival. Knockdown of PCBP2 in gastric cancer cells inhibits cell proliferation and colony formation in vitro, whereas opposing results are obtained when PCBP2 is overexpressed. Our in vivo subcutaneous xenograft results also show that PCBP2 can critically regulate gastric cancer cell growth. In addition, we find that PCBP2-depletion induces apoptosis in gastric cancer cells via up-regulating expression of pro-apoptotic proteins and down-regulating anti-apoptotic proteins. Mechanically, we identify that miR-34a as a target of PCBP2, and that miR-34a is critically essential for the function of PCBP2. In summary, PCBP2 promotes gastric carcinoma development by regulating the level of miR-34a.     

Bispecific affibody molecule targeting HPV16 and HPV18E7 oncoproteins for enhanced molecular imaging of cervical cancer

Applied Microbiology and Biotechnology - High-risk human papillomavirus (HPV16 and HPV18) are now widely recognized as responsible for cervical cancer, which remains to be the most common...     

Protein scaffold-based molecular probes for cancer molecular imaging

Protein scaffold molecules are powerful reagents for targeting various cell signal receptors, enzymes, cytokines and other cancer-related molecules. They belong to the peptide and small protein platform with distinct properties. For the purpose of development of new generation molecular probes, various protein scaffold molecules have been labeled with imaging moieties and evaluated both in vitro and in vivo. Among the evaluated probes Affibody molecules and analogs, cystine knot peptides, and nanobodies have shown especially good characteristics as protein scaffold platforms for development of in vivo molecular probes. Quantitative data obtained from positron emission tomography, single photon emission computed tomography/CT, and optical imaging together with biodistribution studies have shown high tumor uptakes and high tumor-to-blood ratios for these probes. High tumor contrast imaging has been obtained within 1 h after injection. The success of those molecular probes demonstrates the adequacy of protein scaffold strategy as a general approach in molecular probe development.