Delivery of Short Interfering Ribonucleic Acid-Complexed Magnetic Nanoparticles in an Oscillating Field Occurs via Caveolae-Mediated Endocytosis

Gene delivery technologies to introduce foreign genes into highly differentiated mammalian cells have improved significantly over the last few decades. Relatively new techniques such as magnetic nanoparticle-based gene transfection technology are showing great promise in terms of its high transfection efficiency and wide-ranging research applications. We have developed a novel gene delivery technique, which uses magnetic nanoparticles moving under the influence of an oscillating magnetic array. Herein we successfully introduced short interfering RNA (siRNA) against green fluorescent protein (GFP) or actin into stably-transfected GFP-HeLa cells or wild-type HeLa and rat aortic smooth muscle cells, respectively. This gene silencing technique occurred in a dose- and cell density- dependent manner, as reflected using fluorescence intensity and adhesion assays. Furthermore, using endocytosis inhibitors, we established that these magnetic nanoparticle-nucleic acid complexes, moving across the cell surface under the influence of an oscillating magnet array, enters into the cells via the caveolae-mediated endocytic pathway.     

Non-Silent Story on Synonymous Sites in Voltage-Gated Ion Channel Genes

Synonymous mutations are usually referred to as “silent”, but increasing evidence shows that they are not neutral in a wide range of organisms. We looked into the relationship between synonymous codon usage bias and residue importance of voltage-gated ion channel proteins in mice, rats, and humans. We tested whether translationally optimal codons are associated with transmembrane or channel-forming regions, i.e., the sites that are particularly likely to be involved in the closing and opening of an ion channel. Our hypothesis is that translationally optimal codons are preferred at the sites within transmembrane domains or channel-forming regions in voltage-gated ion channel genes to avoid mistranslation-induced protein misfolding or loss-of-function. Using the Mantel-Haenszel procedure, which applies to categorical data, we found that translationally optimal codons are more likely to be used at transmembrane residues and the residues involved in channel-forming. We also found that the conservation level at synonymous sites in the transmembrane region is significantly higher than that in the non-transmembrane region. This study provides evidence that synonymous sites in voltage-gated ion channel genes are not neutral. Silent mutations at channel-related sites may lead to dysfunction of the ion channel.     

Disentangling the Effects of Risk Factors and Clinical Care on Subnational Variation in Early Neonatal Mortality in the United States

Objective

Between 1990 and 2010, the U.S ranking in neonatal mortality slipped from 29^th to 45^th among countries globally. Substantial subnational variation in newborn mortality also exists. Our objective is to measure the extent to which trends and subnational variation in early neonatal mortality reflect differences in the prevalence of risk factors (gestational age and birth weight) compared to differences in clinical care.

Methods

Observational study using linked birth and death data for all births in the United States between 1996 and 2006. We examined health service area (HSA) level variation in the expected early neonatal mortality rate, based on gestational age (GA) and birth-weight (BW), and GA-BW adjusted mortality as a proxy for clinical care. We analyzed the relationship between selected health system indicators and GA-BW-adjusted mortality.

Results

The early neonatal death (ENND) rate declined 12% between 1996 and 2006 (2.39 to 2.10 per 1000 live births). This occurred despite increases in risk factor prevalence. There was significant HSA-level variation in the expected ENND rate (Rate Ratio: 0.73–1.47) and the GA-BW adjusted rate (Rate ratio: 0.63–1.68). Accounting for preterm volume (defined as <34 weeks), the number of neonatologist and NICU beds, 25.2% and 58.7% of the HSA-level variance in outcomes was explained among all births and very low birth weight babies, respectively.

Conclusion

Improvements in mortality could be realized through the expansion or reallocation of clinical neonatal resources, particularly in HSAs with a high volume of preterm births; however, prevention of preterm births and low-birth weight babies has a greater potential to improve newborn survival in the United States.     

Hwanggeumchal sorghum induces cell cycle arrest, and suppresses tumor growth and metastasis through Jak2/STAT pathways in breast cancer xenografts

Background

Cancer is one of the highly virulent diseases known to humankind with a high mortality rate. Breast cancer is the most common cancer in women worldwide. Sorghum is a principal cereal food in many parts of the world, and is critical in folk medicine of Asia and Africa. In the present study, we analyzed the effects of HSE in metastatic breast cancer.

Methodology/Principal Findings

Preliminary studies conducted on MDA-MB 231 and MCF-7 xenograft models showed tumor growth suppression by HSE. Western blotting studies conducted both in vivo and in vitro to check the effect of HSE in Jak/STAT pathways. Anti-metastatic effects of HSE were confirmed using both MDA-MB 231 and MCF-7 metastatic animal models. These studies showed that HSE can modulate Jak/STAT pathways, and it hindered the STAT5b/IGF-1R and STAT3/VEGF pathways not only by down-regulating the expression of these signal molecules and but also by preventing their phosphorylation. The expression of angiogenic factors like VEGF, VEGF-R2 and cell cycle regulators like cyclin D, cyclin E, and pRb were found down-regulated by HSE. In addition, it also targets Brk, p53, and HIF-1α for anti-cancer effects. HSE induced G1 phase arrest and migration inhibition in MDA-MB 231 cells. The metastasis of breast cancer to the lungs also found blocked by HSE in the metastatic animal model.

Conclusions/Significance

Usage of HS as a dietary supplement is an inexpensive natural cancer therapy, without any side effects. We strongly recommend the use of HS as an edible therapeutic agent as it possesses tumor suppression, migration inhibition, and anti-metastatic effects on breast cancer.     

Inhibition of TDP-43 Accumulation by Bis(thiosemicarbazonato)-Copper Complexes

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, motor neuron disease with no effective long-term treatment options. Recently, TDP-43 has been identified as a key protein in the pathogenesis of some cases of ALS. Although the role of TDP-43 in motor neuron degeneration is not yet known, TDP-43 has been shown to accumulate in RNA stress granules (SGs) in cell models and in spinal cord tissue from ALS patients. The SG association may be an early pathological change to TDP-43 metabolism and as such a potential target for therapeutic intervention. Accumulation of TDP-43 in SGs induced by inhibition of mitochondrial activity can be inhibited by modulation of cellular kinase activity. We have also found that treatment of cells and animal models of neurodegeneration, including an ALS model, with bioavailable bis(thiosemicarbazonato)copper(II) complexes (Cu(II)(btsc)s) can modulate kinase activity and induce neuroprotective effects. In this study we examined the effect of diacetylbis(-methylthiosemicarbazonato)copper(II) (Cu(II)(atsm)) and glyoxalbis(-methylthiosemicarbazonato)copper(II) (Cu(II)(gtsm)) on TDP-43-positive SGs induced in SH-SY5Y cells in culture. We found that the Cu(II)(btsc)s blocked formation of TDP-43-and human antigen R (HuR)-positive SGs induced by paraquat. The Cu(II)(btsc)s protected neurons from paraquat-mediated cell death. These effects were associated with inhibition of ERK phosphorylation. Co-treatment of cultures with either Cu(II)(atsm) or an ERK inhibitor, PD98059 both prevented ERK activation and blocked formation of TDP-43-and HuR-positive SGs. Cu(II)(atsm) treatment or ERK inhibition also prevented abnormal ubiquitin accumulation in paraquat-treated cells suggesting a link between prolonged ERK activation and abnormal ubiquitin metabolism in paraquat stress and inhibition by Cu. Moreover, Cu(II)(atsm) reduced accumulation of C-terminal (219-414) TDP-43 in transfected SH-SY5Y cells. These results demonstrate that Cu(II)(btsc) complexes could potentially be developed as a neuroprotective agent to modulate neuronal kinase function and inhibit TDP-43 aggregation. Further studies in TDP-43 animal models are warranted.     

TRAF6 mediates IL-1β/LPS-induced suppression of TGF-β signaling through its interaction with the type III TGF-β receptor

Transforming growth factor-β1 (TGF-β1) is an important anti-inflammatory cytokine that modulates and resolves inflammatory responses. Recent studies have demonstrated that inflammation enhances neoplastic risk and potentiates tumor progression. In the evolution of cancer, pro-inflammatory cytokines such as IL-1β must overcome the anti-inflammatory effects of TGF-β to boost pro-inflammatory responses in epithelial cells. Here we show that IL-1β or Lipopolysaccharide (LPS) suppresses TGF-β-induced anti-inflammatory signaling in a NF-κB-independent manner. TRAF6, a key molecule in IL-1β signaling, mediates this suppressive effect through interaction with the type III TGF-β receptor (TβRIII), which is TGF-β-dependent and requires type I TGF-β receptor (TβRI) kinase activity. TβRI phosphorylates TβRIII at residue S829, which promotes the TRAF6/TβRIII interaction and consequent sequestration of TβRIII from the TβRII/TβRI complex. Our data indicate that IL-1β enhances the pro-inflammatory response by suppressing TGF-β signaling through TRAF6-mediated sequestration of TβRIII, which may be an important contributor to the early stages of tumor progression.     

A Key Role for the Endothelium in NOD1 Mediated Vascular Inflammation: Comparison to TLR4 Responses

Understanding the mechanisms by which pathogens induce vascular inflammation and dysfunction may reveal novel therapeutic targets in sepsis and related conditions. The intracellular receptor NOD1 recognises peptidoglycan which features in the cell wall of Gram negative and some Gram positive bacteria. NOD1 engagement generates an inflammatory response via activation of NFκB and MAPK pathways. We have previously shown that stimulation of NOD1 directly activates blood vessels and causes experimental shock in vivo. In this study we have used an ex vivo vessel-organ culture model to characterise the relative contribution of the endothelium in the response of blood vessels to NOD1 agonists. In addition we present the novel finding that NOD1 directly activates human blood vessels. Using human cultured cells we confirm that endothelial cells respond more avidly to NOD1 agonists than vascular smooth muscle cells. Accordingly we have sought to pharmacologically differentiate NOD1 and TLR4 mediated signalling pathways in human endothelial cells, focussing on TAK1, NFκB and p38 MAPK. In addition we profile novel inhibitors of RIP2 and NOD1 itself, which specifically inhibit NOD1 ligand induced inflammatory signalling in the vasculature. This paper is the first to demonstrate activation of whole human artery by NOD1 stimulation and the relative importance of the endothelium in the sensing of NOD1 ligands by vessels. This data supports the potential utility of NOD1 and RIP2 as therapeutic targets in human disease where vascular inflammation is a clinical feature, such as in sepsis and septic shock.