Comparison of DreamFect Gold, Lipofectamine 2000 and DreamFect Gold + CombiMag when transfection cmRNAs in MSC

Chemically modifed RNA induces osteogenesis of stem cells and human tissue explants as well as accelerates bone healing in rats

Biomaterials fev 2016

Elizabeth R. Balmayor et al.

Abstract
Limitations associated to the use of growth factors represent a major hurdle to musculoskeletal regeneration. On the one hand, they are needed to induce neo-tissue formation for the substitution of a necrotic or missing tissue. On the other hand, these factors are used in supraphysiological concentrations, are short lived and expensive and result in many side effects. Here we develop a gene transfer strategy based on the use of chemically modified mRNA (cmRNA) coding for human bone morphogenetic protein 2 (hBMP-2) that is non-immunogenic and highly stable when compared to unmodified mRNA. Transfected stem cells secrete hBMP-2, show elevated alkaline phosphatase levels and upregulated expression of RunX2, ALP, Osterix, Osteocalcin, Osteopontin and Collagen Type I genes. Mineralization was induced as seen by positive Alizarin red staining. hBMP-2 cmRNA transfected human fat tissue also yielded an osteogenic response in vitro as indicated by expression of hBMP-2, RunX2, ALP and Collagen Type I. Delivering hBMP-2 cmRNA to a femur defect in a rat model results in new bone tissue formation as early as 2 weeks after application of very low doses. Overall, our studies demonstrate the feasibility and therapeutic potential of a new cmRNA-based gene therapy strategy that is safe and efficient. When applied clinically, this approach could overcome BMP-2 growth factor associated limitations in bone regeneration.

Lentiviral Transduction of Tumor Organoids for Tumor Transplantation with ViroMag, a magnetic transduction enhancer

Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters

PNAS Dec 2015, doi: 10.1073/pnas.1508541113
Kevin J. Cheung et al

ViroMag is used to enhance Lentiviral Transduction of Tumor Organoids for Tumor Transplantation.

Significance

Conventional models of cancer progression propose that single cells leave the primary tumor, enter the circulation, and seed clonal metastases. However, metastases can contain multiple clones, raising the question: How do polyclonal metastases form? We demonstrate that cancer cells seed distant organs as cohesive clusters, composed of two molecularly distinct subpopulations, whose proportions vary systematically during metastasis. We establish that collective dissemination is a frequent mechanism for metastasis and identify a molecular program in the most invasive, keratin 14⁺ (K14⁺) cancer cells, regulating cell–cell adhesion, cell–matrix adhesion, and immune evasion. We demonstrate that this metastatic phenotype is dependent upon K14 expression. Understanding the molecular basis of collective dissemination may therefore enable novel prognostics and therapies to improve patient outcomes.

Abstract

Recent genomic studies challenge the conventional model that each metastasis must arise from a single tumor cell and instead reveal that metastases can be composed of multiple genetically distinct clones. These intriguing observations raise the question: How do polyclonal metastases emerge from the primary tumor? In this study, we used multicolor lineage tracing to demonstrate that polyclonal seeding by cell clusters is a frequent mechanism in a common mouse model of breast cancer, accounting for >90% of metastases. We directly observed multicolored tumor cell clusters across major stages of metastasis, including collective invasion, local dissemination, intravascular emboli, circulating tumor cell clusters, and micrometastases. Experimentally aggregating tumor cells into clusters induced a >15-fold increase in colony formation ex vivo and a >100-fold increase in metastasis formation in vivo. Intriguingly, locally disseminated clusters, circulating tumor cell clusters, and lung micrometastases frequently expressed the epithelial cytoskeletal protein, keratin 14 (K14). RNA-seq analysis revealed that K14⁺ cells were enriched for desmosome and hemidesmosome adhesion complex genes, and were depleted for MHC class II genes. Depletion of K14 expression abrogated distant metastases and disrupted expression of multiple metastasis effectors, including Tenascin C (Tnc), Jagged1 (Jag1), and Epiregulin (Ereg). Taken together, our findings reveal K14 as a key regulator of metastasis and establish the concept that K14⁺ epithelial tumor cell clusters disseminate collectively to colonize distant organs.  

Increase protein production yield at a considerably reduced cost with HYPE-5 transfection reagent

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization.

J Vis Exp. 2015 Dec 23;(106). doi: 10.3791/53445.

Kober DL¹, Yurtsever Z², Brett TJ³.

This article demonstrates the high efficiency of HYPE-5 for protein production.

Abstract

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

"Optimal cell viability and transfection conditions are the most crucial for efficient protein production. For better cell viability, low-passage cell cultures are boosted with cell culture supplements and the antibiotic concentration in the growth media is reduced. The modified PEITMC-25 has suitable transfection efficiency with most proteins; however, there are other options available at moderate price that offer increased transfection efficiency and reduced cytotoxicity. Hype 5 (Oz Bioscience) increases yields compared to PEI or other reagents, at a considerably reduced cost compared to more expensive transfection reagents, such as 293fectin. The reagent type and DNA: reagent ratios can be optimized for individual expressions and the needs of the desired experiment."

 

Cervical epithelial carcinoma and Embryonic kidney cells were transiently transfected with siRNA using Magnetofectamine

CUL2-mediated clearance of misfolded TDP-43 is paradoxically affected by VHL in oligodendrocytes in ALS.

Sci Rep. 2016 Jan 11;6:19118. doi: 10.1038/srep19118.

Yin PT, Shah S, Pasquale NJ, Garbuzenko OB, Minko T, Lee KB.

This article demonstrates the high efficiency of Magnetofectamine from OZ Biosciences to transfect primary cells

Abstract:

Stem cell-based gene therapies, wherein stem cells are genetically engineered to express therapeutic molecules, have shown tremendous potential for cancer applications owing to their innate ability to home to tumors. However, traditional stem cell-based gene therapies are hampered by our current inability to control when the therapeutic genes are actually turned on, thereby resulting in detrimental side effects. Here, we report the novel application of magnetic core-shell nanoparticles for the dual purpose of delivering and activating a heat-inducible gene vector that encodes TNF-related apoptosis-inducing ligand (TRAIL) in adipose-derived mesenchymal stem cells (AD-MSCs). By combining the tumor tropism of the AD-MSCs with the spatiotemporal MCNP-based delivery and activation of TRAIL expression, this platform provides an attractive means with which to enhance our control over the activation of stem cell-based gene therapies. In particular, we found that these engineered AD-MSCs retained their innate ability to proliferate, differentiate, and, most importantly, home to tumors, making them ideal cellular carriers. Moreover, exposure of the engineered AD-MSCS to mild magnetic hyperthermia resulted in the selective expression of TRAIL from the engineered AD-MSCs and, as a result, induced significant ovarian cancer cell death in vitro and in vivo.  

Magnetofectamine™ is the association of Lipofectamine™ 2000 from Life Technologies Corporation with the CombiMag Transfection Reagent from OZ Biosciences. Ideal to transfect primary and hard-to-transfect cells, the alliance of these two reagents leads to increased transfection efficiency, minimized toxicity and enhanced gene expression.

MC3T3 Pre-osteoblasts were transiently transfected with plasmid DNA using LipoMag kit

Osteoclast TGF-b Receptor Signaling Induces Wnt1 Secretion and Couples Bone Resorption to Bone Formation

This article demonstrates the high efficiency of LipoMag from OZ Biosciences for DNA transfection into pre-osteoblasts

J Bone Miner Res. 2016 Jan;31(1):76-85. doi: 10.1002/jbmr.2586. Epub 2015 Aug 6.

Weivoda MM, Ruan M, Pederson L, Hachfeld C, Davey RA, Zajac J, Westendorf JJ, Khosla S, Oursler MJ.

Abstract:

Osteoblast-mediated bone formation is coupled to osteoclast-mediated bone resorption. These processes become uncoupled with age, leading to increased risk for debilitating fractures. Therefore, understanding how osteoblasts are recruited to sites of resorptionis vital to treating age-related bone loss. Osteoclasts release and activate TGF-b from the bone matrix. Here we show that osteoclastspecific inhibition of TGF-b receptor signaling in mice results in osteopenia due to reduced osteoblast numbers with no significant impact on osteoclast numbers or activity. TGF-b induced osteoclast expression of Wnt1, a protein crucial to normal bone formation,and this response was blocked by impaired TGF-b receptor signaling. Osteoclasts in aged murine bones had lower TGF-b signaling and Wnt1 expression in vivo. Ex vivo stimulation of osteoclasts derived from young or old mouse bone marrow macrophages showed no difference in TGF-b–induced Wnt1 expression. However, young osteoclasts expressed reduced Wnt1 when cultured on aged mouse bone chips compared to young mouse bone chips, consistent with decreased skeletal TGF-b availability with age.

Therefore, osteoclast responses to TGF-b are essential for coupling bone resorption to bone formation, and modulating this pathway may provide opportunities to treat age-related bone loss. © 2015 American Society for Bone and Mineral Research.

LipoMag (OZBiosciences USA, San Diego, CA, USA) was used to transfect MC3T3 preosteoblasts with a Wnt reporter plasmid containing three copies of the TCF/Lef binding site upstream of the firefly luciferase gene (TOPFlash)(31) or control DNA.

LipoMag Transfection Kit associates our most efficient lipid-based transfection reagent, DreamFect Gold with CombiMag magnetic nanoparticles, the most versatile Magnetofection™ reagent. By taking advantage of the Lipofection and Magnetofection transfection techniques, LipoMag Kit reaches even higher transfection efficiency especially in primary cells while reducing toxicity. It is suitable for all type of nucleic acids, including: plasmid DNA,linearized DNA, siRNA, oligonucleotides, double stranded RNA, mRNA, shRNA.

Genome Editing: the CRISPR/Cas9 system

Genome Editing

Genetic modification is the intended modulation of gene expression in specific cells or organisms to treat pathological disorders. The introduction of exogenous nucleic acids such as DNA, messenger RNA (mRNA), small interfering RNA (siRNA), micro RNA (miRNA) or antisense nucleotides settles the basis of gene-based therapy. Given the plurality of size and charge of these macromolecules, OZ Biosciences has developed numerous versatile transfection reagents to mediate their delivery. 

A very recent technology named “Genome editing” or “genome engineering” gives now the investigators ability to precisely and efficiently introduces a variety of genetic alterations into mammalian cells (deletion, insertion…). During the past decade, zinc finger nucleases (ZFNs) and transcription activator-like effector nuclease (TALENs) illustrate the rapid development and innovation in genome-editing technologies. ZFNs and TALENs defined a new powerful class of tools that redefined the limits of biological research until the very recent discovery of the clustered regularly interspaced short palindromic repeat (CRISPR) arrays and their CRISPR associated (Cas) proteins.

With more than 10 years of expertise in the development of transfection reagents, OZ Biosciences offers tailored transfection solutions for CRISPR/Cas9 technology.

 

How does CRISPR/Cas9 work?

CRISPR/Cas9 system is originated from bacteria in which it provides acquired immunity against invading foreign DNA via RNA-guided cleavage [1]. The bacteria collect “protospacers”, short segments of foreign DNA (e.g. from bacteriophages) and integrate them into their genome. Sequences from CRISPR genomic loci are then transcribed into short CRISPR RNA (crRNA) that anneal to transactivating crRNA (tracrRNAs) to destroy any DNA sequence matching the protospacers. After transcription and processing, crRNA first complexes with Cas9 and tracrRNA and then bind target sequence onto DNA. An R-loop forms and both strands of DNA are cut. crRNA is used as a guide while Cas9 acts as an endonuclease to cleave the DNA (figure 1).

Figure 1. The CRISPR-Cas9 nuclease programmed with sgRNA. Upon binding the sgRNA guide (tracrRNA-crRNA) specifically targets a short DNA sequence-tag (PAM) and unzips DNA complementary to the sgRNA. sgRNA–target DNA heteroduplex, triggering R-loop formation results in a further structural rearrangement: Recognition (REC) and Nuclease lobes (NUC) undergo rotation to fully enclose the DNA target sequence. Two nuclease domains (RuvC, HNH) each nicking one DNA strand, generate a double-strand break. Structurally, REC domain interacts with the sgRNA, while NUC lobe drives interaction with the PAM and target DNA.

 

Genome Editing with CRISPR/Cas9

In 2013, four groups demonstrated that CRISPR/Cas9 associated with guide RNA can be utilized for gene editing [2-5]. Based on the type II CRISPR/Cas9 mechanism, researchers created a single guide RNA (sgRNA), a chimeric form of crRNA and tracrRNA which is able to bind to a specific dsDNA sequence. This resulted in double strand breaks (DSB) at target site with: (1) a 20-bp sequence matching the protospacer of the guide RNA and (2) a protospacer-adjacent motif (PAM) 3 bp downstream NGG sequence. CRISPR/Cas9-mediated genome editing thus depends on the generation of DSB and subsequent cellular DNA repair process. The presence of DSB in the DNA generated by CRISPR/Cas9 leads to activation of cellular DNA repair processes, including non-homologous end-joining (NHEJ)-mediated error prone DNA repair and homology-directed repair (HDR)-mediated error-free DNA repair. Insertions and deletion mutations at target site generated by NHEJ and HDR allow disrupting or abolishing the function of a target gene. Moreover, modifications in this system can also be used to silence gene, insert new exogenous DNA or to block RNA transcription. 

Various Cas9-based applications:

• Indel mutations,
• Specific sequence insertion or replacement
• Large deletion or genomic rearrangement (inversions or translocation)
• Fusion to an activation domain :

o   Gene Activation

o   Other modifications (histone modification, DNA methylation, fluorescent protein)

o   Imaging location of genomic locus.

 

CRISPR/Cas9 advantages over ZFNs and TALENs

CRISPR/Cas9 can be easily adapted to virtually any genomic sequence by changing the 20-bp protospacer of the guide RNA; the Cas9 protein component remaining unchanged. This ease of use presents a main advantage over ZFNs and TALENs in generating genome-wide libraries or multiplexing guide RNA into the same cells.

• ZFNs and TALENs are built on protein-guided DNA cleavage that needs complex protein engineering.
• CRISPR/Cas9 only needs a short guide RNA for DNA targeting.
• CRISPR/Cas9 allows using several gRNA with different target sites: simultaneously genomic modifications at multiple independent sites (Cong et al, 2013).
• Accelerates the generation of transgenic animals with multiple gene mutations [6].

CRISPR/Cas9 system presents a versatile and reliable genome editing tool to facilitate a large variety of targeting genome applications. CRISPR/Cas9 components comprise endonuclease and sgRNA that can be delivered into cells under several forms. For each form we can propose a solution. 

 

OZBIOSCIENCES’ Tools for CRISPR/Cas9 system

For generation of cellular models, Cas9 and the appropriately designed sgRNA (a chimeric RNA containing all essential crRNA and tracRNA components) can be easily introduced into the target cells. The type II CRISPR/Cas system only needs a single Cas protein that can be expressed into target cells by: (1) plasmid transfection^**, (2) direct delivery of the active Cas9 endonuclease, (3) transfection of mRNA enconding for Cas9 or (4) by viral vectors transduction.

OZ Biosciences offers solutions transfecting any kind of cell with every form of Cas9 (figure 2).

 ^**most CRISPR/Cas9 system also expresses the guide RNA from a plasmid using a RNA Polymerase III promoter such as the U6 promoter.

Figure 2. For each CRISPR/Cas9 application a specific transfection reagent is provided by OZBiosciences.

Bibliographic references

1. Wiedenheft B, et al. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331-338.

2. Cong L, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339 (6121):819-823.

3. Mali P, et al. RNA-guided genome engineering via Cas9. Science. 2013;339 (6121):823-826.

4. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337 (6096):816-821.

5. Cho SW, Kim S, Kim JM, Kim JS.  Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol. 2013;31(3):230-232.

6. Wang H, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153 (4):910-8.

HeLa cells were transiently transfected with plasmid DNA using DreamFect

DNA encoding the protein of interest was transiently transfected into HeLa cells using DreamFect transfection reagent.

This article demonstrates the high efficiency of DreamFect from OZ Biosciences for DNA transfection into cell lines.

article reference: J Neurosci. 2014 Nov 26;34(48):15851-60.

Gap Junctional Coupling is Essential for Epithelial Repair in the Avian Cochlea.

Jagger DJ, Nickel R, Forge A.

Abstract
The loss of auditory hair cells triggers repair responses within the population of nonsensory supporting cells. When hair cells are irreversibly lost from the mammalian cochlea, supporting cells expand to fill the resulting lesions in the sensory epithelium, an initial repair process that is dependent on gap junctional intercellular communication (GJIC). In the chicken cochlea (the basilar papilla or BP), dying hair cells are extruded from the epithelium and supporting cells expand to fill the lesions and then replace hair cells via mitotic and/or conversion mechanisms. Here, we investigated the involvement of GJIC in the initial epithelial repair process in the aminoglycoside-damaged BP. Gentamicin-induced hair cell loss was associated with a decrease of chicken connexin43 (cCx43) immunofluorescence, yet cCx30-labeled gap junction plaques remained. Fluorescence recovery after photobleaching experiments confirmed that the GJIC remained robust in gentamicin-damaged explants, but regionally asymmetric coupling was no longer evident. Dye injections in slice preparations from undamaged BP explants identified cell types with characteristic morphologies along the neural-abneural axis, but these were electrophysiologically indistinct. In gentamicin-damaged BP, supporting cells expanded to fill space formerly occupied by hair cells and displayed more variable electrophysiological phenotypes. When GJIC was inhibited during the aminoglycoside damage paradigm, the epithelial repair response halted. Dying hair cells were retained within the sensory epithelium and supporting cells remained unexpanded. These observations suggest that repair of the auditory epithelium shares common mechanisms across vertebrate species and emphasize the importance of functional gap junctions in maintaining a homeostatic environment permissive for subsequent hair cell regeneration.

DreamFect from OZ Biosciences allows transfecting all types of nucleic acids with a very high efficiency; it is fully biodegradable and does not interfere with cellular mechanisms

HeLa transfection with plasmid DNA using DreamFect reagent

3x10^6 HeLa cells seeded in 15 cm diameter dishes were transfected with 5 µg plasmid DNA using 40 µL DreamFect transfection reagent.

This article demonstrates the high efficiency of DreamFect from OZ Biosciences for DNA transfection into HeLa cells.

article reference: Bio-protocol 4(7): Vol 4, Iss 7, 4/5/2014

UPF1 RNA Immunoprecipitation from Mini-μ Construct–expressing Cells

David Zünd and Oliver Mühlemann
 
Abstract
UPF1, an RNA helicase and a core factor of nonsense-mediated mRNA decay (NMD), interacts with RNA independently of the sequence context. To investigate the influence of translation on the association of UPF1 with specific reporter transcripts, UPF1 RNA immunoprecipitations (RIPs) are performed from Hela cells that either express a normally translated immunoglobulin-µ (Ig-µ) reporter (mini µ) or a version with a stable stem loop in the 5' UTR (SL mini µ) that efficiently inhibit translation initiation (Zund et al., 2013). Both the cloning of the SL mini µ reporter construct and the UPF1 RIP experiment are described in detail.

DreamFect from OZ Biosciences allows transfecting all types of nucleic acids with a very high efficiency; it is fully biodegradable and does not interfere with cellular mechanisms.

Gene Silencing in ovarian cancer cell line SKOV3 with siRNA using DreamFect Gold

Transient knockdown was performed using siRNA and DreamFect Gold transfection reagent.

This article demonstrates the high efficiency of DreamFect Gold from OZ Biosciences for gene silencing using siRNA in various cell lines.

article reference: Epigenomics, 2014, DOI:10.4161/15592294.2014.971608 

TGF-β induces global changes in DNA methylation during the epithelial-to-mesenchymal transition in ovarian cancer cells

Horacio Cardenas, Edyta Vieth, Jiyoon Lee, Mathew Segar, Yunlong Liu, Kenneth P. Nephew & Daniela Matei


Abstract

A key step in the process of metastasis is the epithelial-to-mesenchymal transition (EMT). We hypothesized that epigenetic mechanisms play a key role in EMT and to test this hypothesis we analyzed global and gene-specific changes in DNA methylation during TGF-β-induced EMT in ovarian cancer cells. Epigenetic profiling using the Infinium HumanMethylation450 BeadChip (HM450) revealed extensive (P < 0.01) methylation changes after TGF-β stimulation (468 and 390 CpG sites altered at 48 and 120 hour post cytokine treatment, respectively). The majority of gene-specific TGF-β-induced methylation changes occurred in CpG islands located in or near promoters (193 and 494 genes hypermethylated at 48 and 120 hours after TGF-β stimulation, respectively). Furthermore, methylation changes were sustained for the duration of TGF-β treatment and reversible after the cytokine removal. Pathway analysis of the hypermethylated loci identified functional networks strongly associated with EMT and cancer progression, including cellular movement, cell cycle, organ morphology, cellular development, and cell death and survival. Altered methylation and corresponding expression of specific genes during TGF-β-induced EMT included CDH1 (E-cadherin) and COL1A1 (collagen 1A1). Furthermore, TGF-β induced both expression and activity of DNA methyltransferases (DNMT) -1, -3A, and -3B, and treatment with the DNMT inhibitor SGI-110 prevented TGF-β-induced EMT. These results demonstrate that dynamic changes in the DNA methylome are implicated in TGF-β-induced EMT and metastasis. We suggest that targeting DNMTs may inhibit this process by reversing the EMT genes silenced by DNA methylation in cancer.

DreamFect Gold allows transfecting all types of nucleic acids with a very high efficiency. Due to its formulation, DreamFect™Gold delivers a large quantity of nucleic acids leading to higher protein expression compared to other transfection reagents. It is fully biodegradable and does not interfere with cellular mechanisms.

Gene Silencing in Human Coronary Artery Endothelial Cells (HCAEC) with siRNA using SilenceMag (Magnetofection)

siRNA (10 nM) were incubated with SilenceMag according to the recommendation protocol and added onto HCAEC seeded into 6 well plates. 

This article highlights the capacity of Magnetofection technology to efficiently silence gene expression into primary endothelial cells with siRNA using SilenceMag from OZ Biosciences.

article reference: Cardiovasc Diabetol. 2014 Nov 13;13(1):152.

Activation of AMP-activated protein kinase by metformin protects human coronary artery endothelial cells against diabetic lipoapoptosis.

Eriksson L, Nyström T.


abstract
BackgroundThe prevalence of type 2 diabetes (T2D) among adults worldwide is rapidly increasing, and in patients with diabetes the major cause of death is macrovascular disease. Endothelial cells play an important role in maintaining vascular homeostasis. Free fatty acids, which are elevated in T2D, have previously been shown to induce endothelial dysfunction and apoptosis of endothelial cells, which is considered as an important and early factor in the onset of atherosclerosis and cardiovascular disease. Metformin, which is used as first line treatment of T2D patients, is believed to exert its pharmacological effects through activation of AMP-activated protein kinase, which has emerged as a new potential target in reversing endothelial dysfunction.MethodsHere we studied the protective effect of metformin against free fatty acid-induced apoptosis of human coronary artery endothelial cells (HCAECs) by assessing DNA fragmentation and cleaved caspase 3 levels. We also attempted to elucidate the underlying mechanisms by investigating the involvement of AMP-activated protein kinase, p38 MAPK and eNOS. Generation of reactive oxygen species by free fatty acid exposure was also examined.ResultsOur results suggest that metformin protects HCAECs from lipoapoptosis, an effect that involves eNOS and p38 MAPK, downstream of AMPK signaling, but not as previously suggested through suppression of reactive oxygen species.ConclusionThe protective effect of metformin against free fatty acid induced apoptosis is potentially clinically relevant as metformin is first line treatment for patients with T2D, a patient group which is rapidly increasing and carries a high burden of cardiovascular disease.

SilenceMag from OZ Biosciences is a very efficient siRNA delivery reagent based on Magnetofection™ technology.

Gene Silencing in primary myometrial cells with siRNA using SilenceMag (Magnetofection)

Primary myometrial cells were transfected at 50% confluence with 200 nM siRNA for 48 H using SilenceMag transfection reagent as previsouly described in many papers (for more info refer to Lim Ratana and/or Martha Lappas).

This article highlights the capacity of Magnetofection technology to efficiently silence gene expression into primary mammary epithelial cells with siRNA using SilenceMag from OZ Biosciences.

article reference: J Reprod Immunol. 2014 Nov 11. 

Human cathelicidin antimicrobial protein 18 (hCAP18/LL-37) is increased in foetal membranes and myometrium after spontaneous labour and delivery.

Lim R, Barker G, Lappas M.

Abstract
Infection and/or inflammation are most commonly associated with preterm birth. Studies have shown that antimicrobial peptides can modulate the inflammatory response in non-gestational tissues; the human cathelicidin hCAP18 (and its active component LL-37) has such anti-microbial and immunomodulatory properties. The aim of this study was to determine the effect of human labour on hCAP18 expression in foetal membranes and myometrium, and to determine the effect of the synthetic LL-37 peptide on pro-inflammatory and pro-labour mediators in foetal membranes and myometrium. The localisation and expression of hCAP18 in non-labouring and labouring tissues was determined by immunohistochemistry and Western blot, respectively. Tissue explants were used to determine the effect of LL-37 on pro-labour mediators. hCAP18 was localised to the amnion epithelium, cytotrophoblasts and decidua in the foetal membranes, and in the longitudinal and transverse muscle fibres of the myometrium. Additional hCAP18 staining was present in leukocytes. In foetal membranes and myometrium, human labour was associated with significantly higher hCAP18 protein expression. Treatment of foetal membranes and myometrium with LL-37 significantly induced the expression and secretion of the pro-inflammatory cytokines IL-6 and TNF-α, and the chemokines IL-8 and MCP-1. LL-37 also induced expression of MMP-9 mRNA and pro MMP-9 expression in foetal membranes. Co-treatment with BAY 11-7082 was associated with a decrease in LL-37-induced pro-inflammatory cytokine expression. Moreover, inhibition of MyD88 in myometrial cells decreased LL-37-induced pro-inflammatory cytokine expression and release. LL-37 also significantly increased NF-κB transcriptional activity. In conclusion, hCAP18/LL-37 induces pro-inflammatory and pro-labour mediators, via the MyD88/NF-κB pathway.

SilenceMag from OZ Biosciences is a very efficient siRNA delivery reagent based on Magnetofection™ technology.

D-Luciferin from OZ Biosciences to monitor Luciferase activity of B16 melanoma cells in vivo

B16-luciferase melanoma cells were injected into tail vein. 3 mg/mouse of D-Luciferin from OZ Biosciences was injected intraperitoneally to monitor luciferase expression by in vivo imaging.

This article demonstrates the capacity of the D-Luciferin from OZ Biosciences  to efficiently monitor luciferase expression in vivo.

article reference: Nat Commun. 2014 Oct 30;5:5108.

SHP-1-mediated inhibitory signals promote responsiveness and anti-tumour functions of natural killer cells.

Viant C, Fenis A, Chicanne G, Payrastre B, Ugolini S, Vivier E.

abstract 

Natural killer (NK) cells are cytotoxic innate lymphoid cells that are involved in immune defense. NK cell reactivity is controlled in part by MHC class I recognition by inhibitory receptors, but the underlying molecular mechanisms remain undefined. Using a mouse model of conditional deletion in NK cells, we show here that the protein tyrosine phosphatase SHP-1 is essential for the inhibitory function of NK cell MHC class I receptors. In the absence of SHP-1, NK cells are hyporesponsive to tumour cells in vitro and their early Ca(2+) signals are compromised. Mice without SHP-1 in NK cells are unable to reject MHC class I-deficient transplants and to control tumours in vivo. Thus, the inhibitory activity of SHP-1 is needed for setting the threshold of NK cell reactivity.  

D-Luciferin K+ and Na+ salts are dedicated to in vitro and in vivo bioluminescent assays. The quality and purity of the D-Luciferin are essential to obtain good and reproducible results. OZ Biosciences is offering high quality of Endotoxin-Free D-Luciferin K+ and Na+ salts.

Gene Silencing into Primary Rat and Mouse L cells with siRNA using Magnetofection and SilenceMag

L Cells were cultured for 3 days before transfection: SilenceMag was mixed with siRNA (final concentration 20 to 50 nmol/L and incubated for 20 min at 37°C and complexes were added onto L Cells for 15 min on a magnetic plate.

This article highlights the capacity of Magnetofection technology to efficiently silence gene expression into primary epithelial cells with siRNA using SilenceMag from OZ Biosciences.

article reference: Am J Pathol, 2014, doi:10.1016/j.ajpath.2014.09.010

Tumor Necrosis Factor α Decreases Glucagon-Like Peptide-2 Expression by Up-Regulating G-Protein–Coupled Receptor 120 in Crohn Disease 

Takuya Tsukahara, Kenji Watanabe, Toshio Watanabe, Hirokazu Yamagami, Mitsue Sogawa, Tetsuya Tanigawa, Masatsugu Shiba, Kazunari Tominaga, Yasuhiro Fujiwara, Kiyoshi Maeda, Kosei Hirakawa, Tetsuo Arakawa,

Abstract

Glucagon-like peptide (GLP)-2, secreted by L cells in the small intestine, has anti-inflammatory effects in the gastrointestinal tract. A GLP-2 analogue has been an effective treatment for Crohn disease (CD). G-protein–coupled receptor (GPR) 40 and GPR120 are probably involved in GLP-2 production, the mechanisms of which remain unclear. In our experiments, normal ileal mucosa expressed GPR40, but rarely expressed GPR120. However, both GPRs were overexpressed in the L cells of the inflamed ileal mucosa of CD patients. Mucosal inflammation induced the overexpression of GPR40, GPR120, and several inflammatory cytokines, with correlations between ileal concentrations of tumor necrosis factor (TNF)-α and GPR expression levels; however, inflammation did not induce the expression of proglucagon, a precursor of GLP-2 in CD patients. In rat L cells and GLUTag cells, TNF-α treatment increased GPR120 mRNA expression without affecting GPR40 mRNA expression. Dual agonists of GPR40 and GPR120, GW9508 and linoleic acid, respectively, increased GLP-2 production from L cells, but these agonists decreased it in the presence of TNF-α. The GPR40 antagonist, GW1100, inhibited the GW9508-induced increase in GLP-2 production, and silencing GPR120 resulted in further elevation of GLP-2 production. Thus, GPR120-dependent signaling inhibited the stimulatory effects of GPR40 on GLP-2 expression, and TNF-α treatment decreased GLP-2 expression by up-regulating GPR120 expression in L cells.

SilenceMag from OZ Biosciences is a very efficient siRNA delivery reagent based on Magnetofection™ technology.

Gene Silencing in LNCaP and CWR22 cells with siRNA using DreamFect Gold transfection reagent; assay after 48H or 7 days follow-up.

LNCaP or CWR22 cells were transiently transfected with siRNA using DreamFect Gold transfection reagent. Assays were performed 48 H or 7 days after transfection.

This article demonstrates the high efficiency of DreamFect Gold from OZ Biosciences for gene silencing using siRNA in various cell lines.

article reference: Mol Cancer Ther. 2014 Oct 24.

Elevated LIM kinase 1 in non-metastatic prostate cancer reflects its role in facilitating androgen receptor nuclear translocation.

Mardilovich K, Gabrielsen M, McGarry L, Orange C, Patel R, Shanks E, Edwards J, Olson MF.
Abstract
Prostate cancer (PC) affects a large proportion of the male population, and is primarily driven by androgen receptor (AR) activity. First-line treatment typically consists of reducing AR signaling by hormone-depletion, but resistance inevitably develops over time. One way to overcome this issue is to block AR function via alternative means, preferably by inhibiting protein targets that are more active in tumors than in normal tissue. By staining PC tumor sections, elevated LIM kinase 1 (LIMK1) expression and increased phosphorylation of its substrate cofilin were found to be associated with poor outcome and reduced survival in patients with non-metastatic PC. A LIMK selective small molecule inhibitor (LIMKi) was used to determine whether targeted LIMK inhibition was a potential PC therapy. LIMKi reduced PC cell motility, as well as inhibiting proliferation and increasing apoptosis in androgen-dependent PC cells more effectively than in androgen-independent PC cells. LIMK inhibition blocked ligand-induced AR nuclear translocation, reduced AR protein stability and transcriptional activity, consistent with its effects on proliferation and survival acting via inhibition of AR activity. Furthermore, inhibition of LIMK activity increased alphaTubulin acetylation and decreased AR interactions with alphaTubulin, indicating that the role of LIMK in regulating microtubule dynamics contributes to AR function. These results indicate that LIMK inhibitors could be beneficial for the treatment of PC both by reducing nuclear AR translocation, leading to reduced proliferation and survival, and by inhibiting PC cell dissemination.

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