Mechanistic Studies of Intracellular Delivery of Proteins by Cell-Penetrating Peptides in Cyanobacteria

Background: The plasma membrane plays an essential role in selective permeability, compartmentalization, osmotic balance, and cellular uptake. The characteristics and functions of cyanobacterial membranes have been extensively investigated in recent years. Cell-penetrating peptides (CPPs) are special nanocarriers that can overcome the plasma membrane barrier and enter cells directly, either alone or with associated cargoes. However, the cellular entry mechanisms of CPPs in cyanobacteria have not been studied. Results: In the present study, we determine CPP-mediated transduction efficiency and internalization mechanisms in cyanobacteria using a combination of biological and biophysical methods. We demonstrate that both Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 strains of cyanobacteria possess red autofluorescence. Green fluorescent protein (GFP), either alone or noncovalently associated with a CPP comprised of nine arginine residues (R9/GFP complexes), entered cyanobacteria. The ATP-depleting inhibitor of classical endocytosis, N-ethylmaleimide (NEM), could block the spontaneous internalization of GFP, but not the transduction of R9/GFP complexes. Three specific inhibitors of macropinocytosis, cytochalasin D (CytD), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and wortmannin, reduced the efficiency of R9/GFP complex transduction, indicating that entry of R9/GFP complexes involves macropinocytosis. Both the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) and membrane leakage analyses confirmed that R9/GFP complexes were not toxic to the cyanobacteria, nor were the endocytic and macropinocytic inhibitors used in these studies. Conclusions: In summary, we have demonstrated that cyanobacteria use classical endocytosis and macropinocytosis to internalize exogenous GFP and CPP/GFP proteins, respectively. Moreover, the CPP-mediated delivery system is not toxic to cyanobacteria, and can be used to investigate biological processes at the cellular level in this species. These results suggest that both endocytic and macropinocytic pathways can be used for efficient internalization of regular protein and CPP-mediated protein delivery in cyanobacteria, respectively

Hypotoxic Fluorescent Nanoparticles Delivery by Cell-Penetrating Peptides in Multiple Organisms: From Prokaryotes to Mammalians Cells

Nanotechnology is the study of materials in the nanoscale. By its nature, nanotechnology is interdisciplinary. Nanotechnology has made a significant stride in recent two decades in various industries. Numerous nanomaterials are devised for biomedical applications which include intracellular tracking and labeling, gene detection and hybridization, tumor or tissue targeting, pharmaceutical therapies, pathogenic inhibiting, and medical instrument coating for disinfections. High photostability and quantum yield of fluorescent nanoparticles are ideal for long-term monitoring of molecular events in living organisms. Here, we discuss delivery of three fluorescent nanoparticles in A549 cells, rotifers, Gram-negative bacteria, Gram-positive bacteria, and archaea. As these nanoparticles cannot enter cells, arginine-rich cell-penetrating peptides (CPPs) were used to enhance their internalization at the cellular or organismal level. The 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay and sulforhodamine B (SRB) assay demonstrated that CPP complexed fluorescent nanoparticles did not produce lethal effect in all organisms tested. The discussion of these nanomaterials in this chapter intends to broaden our understanding of their biocompatibility in organisms of various hierarchical levels

Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells

Cell-penetrating peptides (CPPs) have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy

CR3 and Dectin-1 Collaborate in Macrophage Cytokine Response through Association on Lipid Rafts and Activation of Syk-JNK-AP-1 Pathway

Copyright: © 2015 Huang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Acknowledgments We are grateful to the Second Core Laboratory of Research Core Facility at the National Taiwan University Hospital for confocal microscopy service and providing ultracentrifuge. We thank Dr. William E. Goldman (University of North Carolina, Chapel Hill, NC) for kindly providing WT and ags1-null mutant of H. capsulatum G186A. Funding: This work is supported by research grants 101-2320-B-002-030-MY3 from the Ministry of Science and Technology (http://www.most.gov.tw) and AS-101-TP-B06-3 from Academia Sinica (http://www.sinica.edu.tw) to BAWH. GDB is funded by research grant 102705 from Welcome Trust (http://www.wellcome.ac.uk). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Olf1/EBF associated zinc finger protein interfered with antinuclear antibody production in patients with systemic lupus erythematosus

Abstract Introduction The aim of the study was to determine whether Olf1/EBF associated zinc finger protein (OAZ), a transcription factor encoded by a positional systemic lupus erythematosus (SLE) candidate gene, plays a functional role in the pathogenesis in SLE. Methods Gene expression levels in peripheral blood cells (PBLs) measured using quantitative real-time polymerase chain reaction (qPCR) were assessed for association with disease activity and the presence of specific autoantibodies. Peripheral blood mononuclear cells (PBMCs) were incubated with specific siRNAs for three days, then cells were harvested for measuring mRNA levels using qPCR, and supernatants for levels of total immunoglobulin (Ig)G and IgM as well as secreted cytokines, chemokine and antinuclear antibodies (ANA) using ELISA. Indirect immunofluorescence was also applied for ANA detection. Results OAZ gene expressions in PBLs from 40 ANA-positive SLE patients were significantly increased than those from 30 normal controls (P < 0.0001) and 18 patients with rheumatoid arthritis (P < 0.01). In SLE patients, OAZ transcripts were positively correlated with SLE disease activity index (SLEDAI) score (r = 0.72, P < 0.0001) and higher in those positive for anti-dsDNA or anti-Sm antibodies (both P < 0.05). Co-culturing with OAZ siRNAs reduced mRNA levels of OAZ by 74.6 ± 6.4% as compared to those co-cultured with non-targeting siRNA and OAZ silencing resulted in reduced total IgG, ANA, interferon (IFN)-γ, interleukin (IL)-10, IL-12 and IL-21, but elevated CCL2 levels in culture supernatants (P < 0.05). The declined ANA levels correlated with inhibited OAZ expression (r = 0.88, P = 0.05), reduced IL-21 levels (r = 0.99, P < 0.01), and elevated chemokine (C-C motif) ligand 2 levels (r = -0.98, P < 0.01). Expressions of ID1-3 were significantly down-regulated by 68.7%, 70.2% and 67.7% respectively after OAZ silence, while ID3 was also highly expressed in SLE PBLs (P < 0.0001) and associated with disease activity (r = 0.76, P < 0.0001) as well as anti-dsDNA or anti-Sm antibodies (both P < 0.05). Conclusions Elevated expression of OAZ transcripts in SLE PBLs were strongly correlated with disease activity. Suppression of OAZ expression inhibited downstream ID levels, and secretion of ANA and IL-21, implicating a role of OAZ pathway in the pathogenesis of SLE

Distribution of Nitrobacter and Nitrospira Communities in an Aerobic Activated Sludge Bioreactor and their Contributions to Nitrite Oxidation

ABSTRACT An analysis of nitrite-oxidizing bacteria in the activated sludge process of a full-scale partially nitrifying wastewater treatment plant revealed Nitrospira and Nitrobacter averaged 10 13 cells·L -1 and 10 12 cells·L -1 , respectively. Correlation coefficients linking shifts in NOB community to operational or environmental variables illustrated Nitrospira were negatively correlated to nitrite (r = -0.45, P<0.01), while Nitrobacter showed no significant relationship to nitrite (P=0.1017). Nitrospira was negative correlation to DO (r = -0.46, P<0.01) and positively correlated to temperature (r = 0.59, P<0.0001). However, Nitrobacter was positively correlated to DO (r = 0.38, P<0.01) and HRT (R = 0.33, P<0.05), as well as negatively correlated to temperature (r = -0.49, P<0.001) suggesting niche adaptations within the NOB community. The positive association between Nitrobacter and DO supports a selective advantage over Nitrospira in completely nitrifying plants. Given the operational schematic at this WWTP, Nitrospira contributed more to nitrification than Nitrobacter in this WWTP

The Rho-GEF Kalirin regulates bone mass and the function of osteoblasts and osteoclasts

Bone homeostasis is maintained by the balance between bone resorption by osteoclasts and bone formation by osteoblasts. Dysregulation in the activity of the bone cells can lead to osteoporosis, a disease characterized by low bone mass and an increase in bone fragility and risk of fracture. Kalirin is a novel GTP-exchange factor protein that has been shown to play a role in cytoskeletal remodeling and dendritic spine formation in neurons. We examined Kalirin expression in skeletal tissue and found that it was expressed in osteoclasts and osteoblasts. Furthermore, micro-CT analyses of the distal femur of global Kalirin knockout (Kal-KO) mice revealed significantly reduced trabecular and cortical bone parameters in Kal-KO mice, compared to WT mice, with significantly reduced bone mass in 8, 14 and 36 week-old female Kal-KO mice. Male mice also exhibited a decrease in bone parameters but not to the level seen in female mice. Histomorphometric analyses also revealed decreased bone formation rate in 14 week-old female Kal-KO mice, as well as decreased osteoblast number/bone surface and increased osteoclast surface/bone surface. Consistent with our in vivo findings, the bone resorbing activity and differentiation of Kal-KO osteoclasts was increased in vitro. Although alkaline phosphatase activity by Kal-KO osteoblasts was increased in vitro, Kal-KO osteoblasts showed decreased mineralizing activity, as well as decreased secretion of OPG, which was inversely correlated with ERK activity. Taken together, our findings suggest that deletion of Kalirin directly affects osteoclast and osteoblast activity, leading to decreased OPG secretion by osteoblasts which is likely to alter the RANKL/OPG ratio and promote osteoclastogenesis. Therefore, Kalirin may play a role in paracrine and/or endocrine signaling events that control skeletal bone remodeling and the maintenance of bone mass

Positive Association of Metabolic Syndrome with a Single Nucleotide Polymorphism of Syndecan-3 (rs2282440) in the Taiwanese Population

Background/Purpose. Metabolic syndrome (MetS) poses a major public health burden on the general population worldwide. Syndecan-3 (SDC3), a heparin sulfate proteoglycan, had been found by previous studies to be linked with energy balance and obesity, but its association with MetS is not known. The objective of this study is to investigate whether SDC3 polymorphism (rs2282440) is associated with MetS in the Taiwanese population. Methods. Genotypes of SDC3 polymorphism (rs2282440) were analyzed in 545 Taiwanese adult subjects, of which 154 subjects had MetS. Results. Subjects with SDC3 rs2282440 TT homozygote had higher frequency of MetS than those with CC or CT genotype (p=0.0217). SDC3 rs2282440 TT homozygote had a 1.96-fold risk of being obese and 1.8-fold risk of having MetS (with CC genotype as reference). As for the individual components of MetS, subjects with SDC3 rs2282440 TT homozygote were more likely to have large waist circumference and low high-density lipoprotein cholesterol (OR = 1.75 and OR = 1.84, resp.). Conclusion. SDC3 rs2282440 polymorphism is positively associated with MetS in the Taiwanese population. Further investigation is needed to see if this association is mediated by mere adiposity or SDC3 polymorphism is also linked with other components of MetS such as lipid metabolism

Nona-Arginine Facilitates Delivery of Quantum Dots into Cells via Multiple Pathways

Semiconductor quantum dots (QDs) have recently been used to deliver and monitor biomolecules, such as drugs and proteins. However, QDs alone have a low efficiency of transport across the plasma membrane. In order to increase the efficiency, we used synthetic nona-arginine (SR9), a cell-penetrating peptide, to facilitate uptake. We found that SR9 increased the cellular uptake of QDs in a noncovalent binding manner between QDs and SR9. Further, we investigated mechanisms of QD/SR9 cellular internalization. Low temperature and metabolic inhibitors markedly inhibited the uptake of QD/SR9, indicating that internalization is an energy-dependent process. Results from both the pathway inhibitors and the RNA interference (RNAi) technique suggest that cellular uptake of QD/SR9 is predominantly a lipid raft-dependent process mediated by macropinocytosis. However, involvement of clathrin and caveolin-1 proteins in transducing QD/SR9 across the membrane cannot be completely ruled out