RUNX3 Regulates Intercellular Adhesion Molecule 3 (ICAM-3) Expression during Macrophage Differentiation and Monocyte Extravasation

The adhesion molecule ICAM-3 belongs to the immunoglobulin gene superfamily and functions as a ligand for the β2 integrins LFA-1, Mac-1 and αdβ2. The expression of ICAM-3 is restricted to cells of the hematopoietic lineage. We present evidences that the ICAM-3 gene promoter exhibits a leukocyte-specific activity, as its activity is significantly higher in ICAM-3+ hematopoietic cell lines. The activity of the ICAM-3 gene promoter is dependent on the occupancy of RUNX cognate sequences both in vitro and in vivo, and whose integrity is required for RUNX responsiveness and for the cooperative actions of RUNX with transcription factors of the Ets and C/EBP families. Protein analysis revealed that ICAM-3 levels diminish upon monocyte-derived macrophage differentiation, monocyte transendothelial migration and dendritic cell maturation, changes that correlate with an increase in RUNX3. Importantly, disruption of RUNX-binding sites led to enhanced promoter activity, and small interfering RNA-mediated reduction of RUNX3 expression resulted in increased ICAM-3 mRNA levels. Altogether these results indicate that the ICAM-3 gene promoter is negatively regulated by RUNX transcription factors, which contribute to the leukocyte-restricted and the regulated expression of ICAM-3 during monocyte-to-macrophage differentiation and monocyte extravasation

Diet-Induced Muscle Insulin Resistance Is Associated With Extracellular Matrix Remodeling and Interaction With Integrin α2β1 in Mice

OBJECTIVE: The hypothesis that high-fat (HF) feeding causes skeletal muscle extracellular matrix (ECM) remodeling in C57BL/6J mice and that this remodeling contributes to diet-induced muscle insulin resistance (IR) through the collagen receptor integrin α(2)β(1) was tested. RESEARCH DESIGN AND METHODS: The association between IR and ECM remodeling was studied in mice fed chow or HF diet. Specific genetic and pharmacological murine models were used to study effects of HF feeding on ECM in the absence of IR. The role of ECM-integrin interaction in IR was studied using hyperinsulinemic-euglycemic clamps on integrin α(2)β(1)-null (itga2(-/-)), integrin α(1)β(1)-null (itga1(-/-)), and wild-type littermate mice fed chow or HF. Integrin α(2)β(1) and integrin α(1)β(1) signaling pathways have opposing actions. RESULTS: HF-fed mice had IR and increased muscle collagen (Col) III and ColIV protein; the former was associated with increased transcript, whereas the latter was associated with reduced matrix metalloproteinase 9 activity. Rescue of muscle IR by genetic muscle-specific mitochondria-targeted catalase overexpression or by the phosphodiesterase 5a inhibitor, sildenafil, reversed HF feeding effects on ECM remodeling and increased muscle vascularity. Collagen remained elevated in HF-fed itga2(-/-) mice. Nevertheless, muscle insulin action and vascularity were increased. Muscle IR in HF-fed itga1(-/-) mice was unchanged. Insulin sensitivity in chow-fed itga1(-/-) and itga2(-/-) mice was not different from wild-type littermates. CONCLUSIONS: ECM collagen expansion is tightly associated with muscle IR. Studies with itga2(-/-) mice provide mechanistic insight for this association by showing that the link between muscle IR and increased collagen can be uncoupled by the absence of collagen-integrin α(2)β(1) interaction

Hybrid inorganic-organic capsules for efficient intracellular delivery of novel siRNAs against influenza A (H1N1) virus infection

This work was supported by ARUK project grant 21210 ‘Sustained and Controllable Local Delivery of Anti-inflammatory Therapeutics with Nanoengineered Microcapsules’. The work was also supported in part by Russian Foundation of Basic Research grants No. 16-33-50153 mol_nr, No. 16-33-00966 mol_a, Russian Science Foundation grant No. 15-15-00170 and Russian Governmental Program ‘‘Nauka’’, No. 1.1658.2016, 4002

Immunostimulatory Motifs Enhance Antiviral siRNAs Targeting Highly Pathogenic Avian Influenza H5N1

Highly pathogenic avian influenza (HPAI) H5N1 virus is endemic in many regions around the world and remains a significant pandemic threat. To date H5N1 has claimed almost 300 human lives worldwide, with a mortality rate of 60% and has caused the death or culling of hundreds of millions of poultry since its initial outbreak in 1997. We have designed multi-functional RNA interference (RNAi)-based therapeutics targeting H5N1 that degrade viral mRNA via the RNAi pathway while at the same time augmenting the host antiviral response by inducing host type I interferon (IFN) production. Moreover, we have identified two factors critical for maximising the immunostimulatory properties of short interfering (si)RNAs in chicken cells (i) mode of synthesis and (ii) nucleoside sequence to augment the response to virus. The 5-bp nucleoside sequence 5′-UGUGU-3′ is a key determinant in inducing high levels of expression of IFN -α, -β, -λ and interleukin 1- β in chicken cells. Positioning of this 5′-UGUGU-3′ motif at the 5′- end of the sense strand of siRNAs, but not the 3′- end, resulted in a rapid and enhanced induction of type I IFN. An anti-H5N1 avian influenza siRNA directed against the PB1 gene (PB1-2257) tagged with 5′-UGUGU-3′ induced type I IFN earlier and to a greater extent compared to a non-tagged PB1-2257. Tested against H5N1 in vitro, the tagged PB1-2257 was more effective than non-tagged PB1-2257. These data demonstrate the ability of an immunostimulatory motif to improve the performance of an RNAi-based antiviral, a finding that may influence the design of future RNAi-based anti-influenza therapeutics

Disulfide-Based Poly(amido amine)s for siRNA Delivery: Effects of Structure on siRNA Complexation, Cellular Uptake, Gene Silencing and Toxicity

Purpose\ud \ud RNA interference (RNAi) is a process by which small interfering RNAs (siRNA) induce sequence-specific gene silencing. Therefore, siRNA is an emerging promise as a novel therapeutic. In order to realize the high expectations for therapeutic applications, efficient delivery systems for siRNA are necessary.\ud \ud Methods\ud \ud In this study, a new series of biodegradable poly(amido amine)s with disulfide linkages in the backbone was synthesized out of N,N′-cystaminebisacrylamide (CBA), 4-amino-1-butanol (ABOL) and ethylene diamine (EDA). Effects of different percentages of butanolic side chains and protonatable fragments in the main chain on siRNA complexation, cellular uptake, gene silencing and toxicity were investigated.\ud \ud Results\ud \ud Incorporation of EDA in the polymer resulted in increased siRNA condensation. Efficient siRNA condensation was shown to be necessary for cellular uptake; however, excess of polymer decreased siRNA uptake for polymers with high amounts of EDA. Silencing efficiency did not correlate with uptake, since silencing increased with increasing w/w ratio for all polymers. More than 80% knockdown was found for polyplexes formed with polymers containing 25% or 50% EDA, which was combined with low cytotoxicity.\ud \ud Conclusions\ud \ud Poly(amido amine)s with minor fractions of protonatable fragments in the main chain are promising carriers for delivery of siRNA\u

Human BCAS3 Expression in Embryonic Stem Cells and Vascular Precursors Suggests a Role in Human Embryogenesis and Tumor Angiogenesis

Cancer is often associated with multiple and progressive genetic alterations in genes that are important for normal development. BCAS3 (Breast Cancer Amplified Sequence 3) is a gene of unknown function on human chromosome 17q23, a region associated with breakpoints of several neoplasms. The normal expression pattern of BCAS3 has not been studied, though it is implicated in breast cancer progression. Rudhira, a murine WD40 domain protein that is 98% identical to BCAS3 is expressed in embryonic stem (ES) cells, erythropoiesis and angiogenesis. This suggests that BCAS3 expression also may not be restricted to mammary tissue and may have important roles in other normal as well as malignant tissues. We show that BCAS3 is also expressed in human ES cells and during their differentiation into blood vascular precursors. We find that BCAS3 is aberrantly expressed in malignant human brain lesions. In glioblastoma, hemangiopericytoma and brain abscess we note high levels of BCAS3 expression in tumor cells and some blood vessels. BCAS3 may be associated with multiple cancerous and rapidly proliferating cells and hence the expression, function and regulation of this gene merits further investigation. We suggest that BCAS3 is mis-expressed in brain tumors and could serve as a human ES cell and tumor marker

A Microcosm of the Biomedical Research Experience for Upper-level Undergraduates

The skill set required of biomedical researchers continues to grow and evolve as biology matures as a natural science. Science necessitates creative yet critical thinking, persuasive communication skills, purposeful use of time, and adeptness at the laboratory bench. Teaching these skills can be effectively accomplished in an inquiry-based, active-learning environment at a primarily undergraduate institution. Cell Biology Techniques, an upper-level cell biology laboratory course at St. John Fisher College, features two independent projects that take advantage of the biology of the nematode Caenorhabditis elegans, a premier yet simple model organism. First, students perform a miniature epigenetic screen for novel phenotypes using RNA interference. The results of this screen combined with literature research direct students toward a singe gene that they attempt to subclone in the second project. The biology of the chosen gene/protein also becomes an individualized focal point with respect to the content of the laboratory. Progress toward course goals is evaluated using written, oral, and group-produced assignments, including a concept map. Pre- and postassessment indicates a significant increase in the understanding of broad concepts in cell biological research

siRNA-Like Double-Stranded RNAs Are Specifically Protected Against Degradation in Human Cell Extract

RNA interference (RNAi) is a set of intracellular pathways in eukaryotes that controls both exogenous and endogenous gene expression. The power of RNAi to knock down (silence) any gene of interest by the introduction of synthetic small-interfering (si)RNAs has afforded powerful insight into biological function through reverse genetic approaches and has borne a new field of gene therapeutics. A number of questions are outstanding concerning the potency of siRNAs, necessitating an understanding of how short double-stranded RNAs are processed by the cell. Recent work suggests unmodified siRNAs are protected in the intracellular environment, although the mechanism of protection still remains unclear. We have developed a set of doubly-fluorophore labeled RNAs (more precisely, RNA/DNA chimeras) to probe in real-time the stability of siRNAs and related molecules by fluorescence resonance energy transfer (FRET). We find that these RNA probes are substrates for relevant cellular degradative processes, including the RNase H1 mediated degradation of an DNA/RNA hybrid and Dicer-mediated cleavage of a 24-nucleotide (per strand) double-stranded RNA. In addition, we find that 21- and 24-nucleotide double-stranded RNAs are relatively protected in human cytosolic cell extract, but less so in blood serum, whereas an 18-nucleotide double-stranded RNA is less protected in both fluids. These results suggest that RNAi effector RNAs are specifically protected in the cellular environment and may provide an explanation for recent results showing that unmodified siRNAs in cells persist intact for extended periods of time