Chitosan-Gelatin-EGCG Nanoparticle-Meditated LncRNA TMEM44-AS1 Silencing to Activate the P53 Signaling Pathway for the Synergistic Reversal of 5-FU Resistance in Gastric Cancer.

Chemoresistance is one of the leading causes of therapeutic failure in gastric cancer (GC) treatment. Recent studies have shown lncRNAs play pivotal roles in regulating GC chemoresistance. Nanocarriers delivery of small interfering RNAs (siRNAs) to silence cancer-related genes has become a novel approach to cancer treatment research. However, finding target genes and developing nanosystems capable of selectively delivering siRNAs for disease treatment remains a challenge. In this study, a novel lncRNA TMEM44-AS1 that is related to 5-FU resistance is identified. TMEM44-AS1 has the ability to bind to and sponge miR-2355-5p, resulting in the upregulated PPP1R13L expression and P53 pathway inhibition. Next, a new nanocarrier called chitosan-gelatin-EGCG (CGE) is developed, which has a higher gene silencing efficiency than lipo2000, to aid in the delivery of a si-TMEM44-AS1 can efficiently silence TMEM44-AS1 expression to synergistically reverse 5-FU resistance in GC, leading to a markedly enhanced 5-FU therapeutic effect in a xenograft mouse model of GC. These findings indicate that TMEM44-AS1 may estimate 5-FU therapy outcome among GC cases, and that systemic si-TMEM44-AS1 delivery combined with 5-FU therapy is significant in the treatment of patients with recurrent GC.

Mitochondrial bioenergetics boost macrophage activation, promoting liver regeneration in metabolically compromised animals.

BACKGROUND AND AIMS: Hepatic ischemia-reperfusion injury (IRI) is the leading cause of early posttransplantation organ failure as mitochondrial respiration and ATP production are affected. A shortage of donors has extended liver donor criteria, including aged or steatotic livers, which are more susceptible to IRI. Given the lack of an effective treatment and the extensive transplantation waitlist, we aimed at characterizing the effects of an accelerated mitochondrial activity by silencing methylation-controlled J protein (MCJ) in three preclinical models of IRI and liver regeneration, focusing on metabolically compromised animal models. APPROACH AND RESULTS: Wild-type (WT), MCJ knockout (KO), and Mcj silenced WT mice were subjected to 70% partial hepatectomy (Phx), prolonged IRI, and 70% Phx with IRI. Old and young mice with metabolic syndrome were also subjected to these procedures. Expression of MCJ, an endogenous negative regulator of mitochondrial respiration, increases in preclinical models of Phx with or without vascular occlusion and in donor livers. Mice lacking MCJ initiate liver regeneration 12 h faster than WT and show reduced ischemic injury and increased survival. MCJ knockdown enables a mitochondrial adaptation that restores the bioenergetic supply for enhanced regeneration and prevents cell death after IRI. Mechanistically, increased ATP secretion facilitates the early activation of Kupffer cells and production of TNF, IL-6, and heparin-binding EGF, accelerating the priming phase and the progression through G1 /S transition during liver regeneration. Therapeutic silencing of MCJ in 15-month-old mice and in mice fed a high-fat/high-fructose diet for 12 weeks improves mitochondrial respiration, reduces steatosis, and overcomes regenerative limitations. CONCLUSIONS: Boosting mitochondrial activity by silencing MCJ could pave the way for a protective approach after major liver resection or IRI, especially in metabolically compromised, IRI-susceptible organs.

Minimal Physiologically Based Pharmacokinetic-Pharmacodynamic (mPBPK-PD) Model of N-Acetylgalactosamine-Conjugated Small Interfering RNA Disposition and Gene Silencing in Preclinical Species and Humans.

Conjugation of small interfering RNA (siRNA) to tris N-acetylgalactosamine [(GalNAc)3] can enable highly selective, potent, and durable knockdown of targeted proteins in the liver. However, potential knowledge gaps between in vitro experiments, preclinical species, and clinical scenarios remain. A minimal physiologically based pharmacokinetic-pharmacodynamic model for GalNAc-conjugated siRNA (GalNAc-siRNA) was developed using published data for fitusiran (ALN-AT3), an investigational compound targeting liver antithrombin (AT), to delineate putative determinants governing the whole-body-to-cellular pharmacokinetic (PK) and pharmacodynamic (PD) properties of GalNAc-siRNA and facilitate preclinical-to-clinical translation. The model mathematically linked relevant mechanisms: 1) hepatic biodistribution, 2) tris-GalNAc binding to asialoglycoprotein receptors (ASGPRs) on hepatocytes, 3) ASGPR endocytosis and recycling, 4) endosomal transport and escape of siRNA, 5) cytoplasmic RNA-induced silencing complex (RISC) loading, 6) degradation of target mRNA by bound RISC, and 7) knockdown of protein. Physiologic values for 36 out of 48 model parameters were obtained from the literature. Kinetic parameters governing (GalNAc)3-ASGPR binding and internalization were derived from published studies of uptake in hepatocytes. The proposed model well characterized reported pharmacokinetics, RISC dynamics, and knockdown of AT mRNA and protein by ALN-AT3 in mice. The model bridged multiple PK-PD data sets in preclinical species (mice, rat, monkey) and successfully captured reported plasma pharmacokinetics and AT knockdown in a phase I ascending-dose study. Estimates of in vivo potency were similar (∼2-fold) across species. Subcutaneous absorption and serum AT degradation rate constants scaled across species by body weight with allometric exponents of -0.29 and -0.22. The proposed mechanistic modeling framework characterizes the unique PK-PD properties of GalNAc-siRNA. SIGNIFICANCE STATEMENT: Tris N-acetylgalactosamine (GalNAc)3-conjugated small interfering RNA (siRNA) therapeutics enable liver-targeted gene therapy and precision medicine. Using a translational and systems-based minimal physiologically based pharmacokinetic-pharmacodynamic (mPBPK-PD) modeling approach, putative determinants influencing GalNAc-conjugated siRNA (GalNAc-siRNA) functionality in three preclinical species and humans were investigated. The developed model successfully integrated and characterized relevant published in vitro-derived biomeasures, mechanistic PK-PD profiles in animals, and observed clinical PK-PD responses for an investigational GalNAc-siRNA (fitusiran). This modeling effort delineates the disposition and liver-targeted pharmacodynamics of GalNAc-siRNA.

Prolonged cultured human hepatocytes as an in vitro experimental system for the evaluation of potency and duration of activity of RNA therapeutics: Demonstration of prolonged duration of gene silencing effects of a GalNAc-conjugated human hypoxanthine phosphoribosyl transferase (HPRT1) siRNA.

We report here the evaluation of a novel in vitro experimental model, prolonged cultured human hepatocytes (PCHC), as an experimental system to evaluate the potency and duration of effects of oligonucleotide therapeutics. A novel observation was made on the redifferentiation of PCHC upon prolonged culturing based on mRNA profiling of characteristic hepatic differentiation marker genes albumin, transferrin, and transthyretin. Consistent with the known de-differentiation of cultured human hepatocytes, decreases in marker gene expression were observed upon culturing of the hepatocytes for 2 days. A novel observation of re-differentiation was observed on day 7 as demonstrated by an increase in expression of the marker genes to levels similar to that observed on the first day of culture. The expression of the differentiation marker genes was highest on day 7, followed by a gradual decrease but remained higher than that on day 2 for up to the longest culture duration evaluated of 41 days. The redifferentiation phenomenon suggests that PCHC may be useful for the evaluation of the duration of effects of oligonucleotide therapeutics on gene expression in human hepatocytes. A proof of concept study was thereby conducted with PCHC with a GalNAc-conjugated siRNA targeting human hypoxanthine phosphoribosyl transferase1 (HPRT1). HPRT1 mRNA expression in siRNA-treated cultures decreased to 21% of that in untreated hepatocytes on day 1, <10% from days 2 to 12, <20% from days 16 to 33, and eventually recovered to 64% by day 41. Our results suggest that PCHC represent a clinically-relevant cost- and time-efficient experimental tool to aid in the evaluation of GalNAc-siRNA silencing activity, providing information on both efficacy and duration of efficacy. PCHC may be applicable in the drug development setting as a species- and cell type-relevant experimental tool to aid the development of oligonucleotide therapeutics.

Conditional inactivation of Npy1r gene in mice induces sex-related differences of metabolic and behavioral functions.

Sex hormone-driven differences in gene expression have been identified in experimental animals, highlighting brain neuronal populations implicated in dimorphism of metabolic and behavioral functions. Neuropeptide Y-Y1 receptor (NPY-Y1R) system is sexually dimorphic and sensitive to gonadal steroids. In the present study we compared the phenotype of male and female conditional knockout mice (Npy1rrfb mice), carrying the inactivation of Npy1r gene in excitatory neurons of the brain limbic system. Compared to their male control (Npy1r2lox) littermates, male Npy1rrfb mice exhibited hyperactivation of the hypothalamic-pituitary-adrenal (HPA) axis that is associated with anxiety and executive dysfunction, reduced body weight growth, after-fasting refeeding, white adipose tissue (WAT) mass and plasma leptin levels. Conversely, female Npy1rrfb mice displayed an anxious-like behavior but no differences in HPA axis activity, executive function and body weight, compared to control females. Moreover, conditional inactivation of Npy1r gene induced an increase of subcutaneous and gonadal WAT weight and plasma leptin levels and a compensatory decrease of Agouti-related protein immunoreactivity in the hypothalamic arcuate (ARC) nucleus in females, compared to their respective control littermates. Interestingly, Npy1r mRNA expression was reduced in the ARC and in the paraventricular hypothalamic nuclei of female, but not male mice. These results demonstrated that female mice are resilient to hormonal and metabolic effects of limbic Npy1r gene inactivation, suggesting the existence of an estrogen-dependent relay necessary to ensure the maintenance of the homeostasis, that can be mediated by hypothalamic Y1R.

Virus-Induced Gene Silencing in Diploid and Tetraploid Potato Species.

Potato is the world's fourth largest food crop and a vegetatively propagated model polyploid plant. To facilitate genomic studies in potato, here we describe detailed protocols to silence genes in both diploid potato Solanum bulbocastanum and tetraploid potato cultivars such as Maris Bard, Arran Pilot, Ancilla, and Serrana using tobacco rattle virus (TRV)- or potato virus X (PVX)-induced gene silencing (VIGS) system, respectively. The established VIGS system represents an efficient and powerful approach for functional analysis of genes involved in growth, development, metabolism, and responses to biotic and abiotic stresses in potato.

The ethylene responsive factor CdERF1 from bermudagrass (Cynodon dactylon) positively regulates cold tolerance.

Cold stress is one of the major environmental factors that limit growth and utilization of bermudagrass [Cynodon dactylon (L.) Pers], a prominent warm-season turfgrass. However, the molecular mechanism of cold response in bermudagrass remains largely unknown. In this study, we characterized a cold-responsive ERF (ethylene responsive factor) transcription factor, CdERF1, from bermudagrass. CdERF1 expression was induced by cold, drought and salinity stresses. The CdERF1 protein was nucleus-localized and encompassed transcriptional activation activity. Transgenic Arabidopsis plants overexpressing CdERF1 showed enhanced cold tolerance, whereas CdERF1-underexpressing bermudagrass plants via virus induced gene silencing (VIGS) method exhibited reduced cold resistance compared with control, respectively. Under cold stress, electrolyte leakage (EL), malondialdehyde (MDA), H2O2 and O2- contents were reduced, while the activities of SOD and POD were elevated in transgenic Arabidopsis. By contrast, these above physiological indicators in CdERF1-underexpressing bermudagrass exhibited the opposite trend. To further explore the possible molecular mechanism of bermudagrass cold stress response, the RNA-Seq analyses were performed. The result indicated that overexpression of CdERF1 activated a subset of stress-related genes in transgenic Arabidopsis, such as CBF2, pEARLI1 (lipid transfer protein), PER71 (peroxidase) and LTP (lipid transfer protein). Interestingly, under-expression of CdERF1 suppressed the transcription of many genes in CdERF1-underexpressing bermudagrass, also including pEARLI1 (lipid transfer protein) and PER70 (peroxidase). All these results revealed that CdERF1 positively regulates plant cold response probably by activating stress-related genes, PODs, CBF2 and LTPs. This study also suggests that CdERF1 may be an ideal candidate in the effort to improve cold tolerance of bermudagrass in the further molecular breeding.

'Nano-in-nano' hybrid liposomes increase target specificity and gene silencing efficiency in breast cancer induced SCID mice.

Gene silencing has immense potential in the treatment of cancer. However, enhancement of its efficiency requires the development of specifically targeted and safe carrier systems. Cationic carriers are generally limited by their immunogenicity. Hence, in this study, we report hybrid liposomes encapsulating Poly (L-lysine)-siRNA complex to silence epithelial cell adhesion molecule (EpCAM), highly expressed in epithelial cancers. The hybrid liposomes LL1 (Egg PC:DSPE-PEG, 10:0) and hybrid immunoliposomes LL2 (Egg PC:DSPE-PEG, 8:2) linked with EpCAM antibody as the targeting ligand showed an encapsulation efficiency of 70% and 86%, respectively. LL2 liposomes with a zeta potential of -26mV exhibited good colloidal stability in phosphate buffered saline containing bovine serum albumin and fetal bovine serum at 37°C. Cell uptake studies showed increased uptake of the LL2 when compared to LL1 liposomes. Finally, the hybrid immunoliposomes were evaluated for their efficacy in regressing the tumor volume in SCID mice. Eight doses each of 0.15mg/kg, which is among the lowest reported siRNA concentrations, were administered to the animals. About 45% reduction in tumor volume was achieved after 28days in the mice treated with LL2 when compared with the positive control and LL1 treated groups. Thus, our results demonstrate that the 'nano-in-nano' concept of encapsulating poly (l-Lysine) complexed EpCAM siRNA in immunoliposomes may be a promising strategy to treat EpCAM-positive epithelial cancers, especially as an adjuvant therapy.

TNFAIP3 downregulation mediated by histone modification contributes to T-cell dysfunction in systemic lupus erythematosus.

Objective: TNF-α-induced protein 3 ( TNFAIP3 ) is one of the major SLE susceptibility genes involved in the regulation of inflammatory responses through modulation of the nuclear factor-κB (NF-κB) pathway. We aim to assess TNFAIP3 expression in CD4 + T cells and the molecular mechanism underlying TNFAIP3 regulation in the pathogenesis of SLE. Methods: The expression and epigenetic regulation of TNFAIP3 in CD4 + T cells from SLE patients and normal controls (NCs) were investigated by RT-quantitative PCR, western blot and chromatin immunoprecipitation. The functional effect of TNFAIP3 was further evaluated by knockdown or overproduction of TNFAIP3 in CD4 + T cells from SLE patients and NCs. Results: TNFAIP3 mRNA was significantly downregulated in the CD4 + T cells of SLE patients compared with NCs. The reduced expression of TNFAIP3 was associated with the reduction of H3K4me3 in the gene promoter region. Functional blockage of TNFAIP3 in normal CD4 + T cells using small interfering RNA increased the expression of IFN-γ and IL-17, but not IL-2, IL-4 and IL-5. Nevertheless, overexpression of TNFAIP3 in CD4 + T cells from SLE patients resulted in the suppression of IFN-γ and IL-17 production. Conclusion: The downregulation of TNFAIP3 in CD4 + T cells of SLE was potentially regulated by demethylation of histone H3K4, which led to a decreased amount of H3K4me3 in the promoter of the TNFAIP3 gene. The dysregulation of TNFAIP3 in CD4 + T cells may contribute to the pathogenesis of SLE by overproduction of inflammatory cytokine IFN-γ and IL-17. TNFAIP3 may serve as a promising target for the treatment of SLE in clinical practice.

Silencing of the potato StNAC103 gene enhances the accumulation of suberin polyester and associated wax in tuber skin.

Suberin and wax deposited in the cork (phellem) layer of the periderm form the lipophilic barrier that protects mature plant organs. Periderm lipids have been widely studied for their protective function with regards to dehydration and for how they respond to environmental stresses and wounding. However, despite advances in the biosynthetic pathways of suberin and associated wax, little is known about the regulation of their deposition. Here, we report on a potato NAC transcription factor gene, StNAC103, induced in the tuber phellem (skin). The StNAC103 promoter is active in cells undergoing suberization such as in the basal layer of the phellem, but also in the root apical meristem. Gene silencing in potato periderm correlates with an increase in the suberin and wax load, and specifically in alkanes, ω-hydroxyacids, diacids, ferulic acid, and primary alcohols. Concomitantly, silenced lines also showed up-regulation of key genes related to the biosynthesis and transport of suberin and wax in the tuber periderm. Taken together, our results suggest that StNAC103 has a role in the tight regulation of the formation of apoplastic barriers and is, to the best of our knowledge, the first candidate gene to be identified as being involved in the repression of suberin and wax deposition.

Efficient Virus-Induced Gene Silencing in Solanum rostratum.

Solanum rostratum is a "super weed" that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum.

DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing.

Antisense oligonucleotides (ASOs) are recognized therapeutic agents for the modulation of specific genes at the post-transcriptional level. Similar to any medical drugs, there are opportunities to improve their efficacy and safety. Here we develop a short DNA/RNA heteroduplex oligonucleotide (HDO) with a structure different from double-stranded RNA used for short interfering RNA and single-stranded DNA used for ASO. A DNA/locked nucleotide acid gapmer duplex with an α-tocopherol-conjugated complementary RNA (Toc-HDO) is significantly more potent at reducing the expression of the targeted mRNA in liver compared with the parent single-stranded gapmer ASO. Toc-HDO also improves the phenotype in disease models more effectively. In addition, the high potency of Toc-HDO results in a reduction of liver dysfunction observed in the parent ASO at a similar silencing effect. HDO technology offers a novel concept of therapeutic oligonucleotides, and the development of this molecular design opens a new therapeutic field.

Vacuolar invertase gene silencing in potato (Solanum tuberosum L.) improves processing quality by decreasing the frequency of sugar-end defects.

Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form dark-colored Maillard reaction products during frying. Acrylamide is another Maillard reaction product formed from reducing sugars and acrylamide consumption has raised health concerns worldwide. Vacuolar invertase gene (VInv) expression was suppressed in cultivars Russet Burbank and Ranger Russet using RNA interference to determine if this approach could control sugar-end defect formation. Acid invertase activity and reducing sugar content decreased at both ends of tubers. Sugar-end defects and acrylamide in fried potato strips were strongly reduced in multiple transgenic potato lines. Thus vacuolar invertase silencing can minimize a long-standing French fry quality problem while providing consumers with attractive products that reduce health concerns related to dietary acrylamide.

Prognostic value of PTEN loss in men with conservatively managed localised prostate cancer.

BACKGROUND: The natural history of prostate cancer is highly variable and difficult to predict. We report on the prognostic value of phosphatase and tensin homologue (PTEN) loss in a cohort of 675 men with conservatively managed prostate cancer diagnosed by transurethral resection of the prostate. METHODS: The PTEN status was assayed by immunohistochemistry (PTEN IHC) and fluorescent in situ hybridisation (PTEN FISH). The primary end point was death from prostate cancer. RESULTS: The PTEN IHC loss was observed in 18% cases. This was significantly associated with prostate cancer death in univariate analysis (hazard ratio (HR)=3.51; 95% CI 2.60-4.73; P=3.1 × 10(-14)). It was highly predictive of prostate cancer death in the 50% of patients with a low risk score based on Gleason score, PSA, Ki-67 and extent of disease (HR=7.4; 95% CI 2.2-24.6; P=0.012) ), but had no prognostic value in the higher risk patients. The PTEN FISH loss was only weakly associated with PTEN IHC loss (κ=0.5). Both PTEN FISH loss and amplification were univariately predictive of death from prostate cancer, but this was not maintained in the multivariate analyses. CONCLUSION: In low-risk patients, PTEN IHC loss adds prognostic value to Gleason score, PSA, Ki-67 and extent of disease.

A novel target of microRNA-29, Ring1 and YY1-binding protein (Rybp), negatively regulates skeletal myogenesis.

Skeletal muscle cell differentiation (myogenesis) is a process orchestrated by a complex network involving transcription factors, epigenetic regulators, and microRNAs. Previous studies identified miR-29 as a pro-myogenic factor that interacts with components of Polycomb repressive complex, YY1 and Ezh2. In a genome-wide survey of miR-29-mediated transcriptome changes in C2C12 myoblasts, many epigenetic factors were found to be down-regulated by miR-29. Among them, Rybp was shown to be a direct target of miR-29 through binding to its 3' UTR. Functional studies demonstrated that Rybp is down-regulated during myogenesis and acts as a negative regulator of skeletal myogenesis both in vitro during C2C12 differentiation and in vivo in injury-induced muscle regeneration. Furthermore, we found that Rybp and YY1 co-occupy several myogenic loci, including miR-29 itself, to silence their expression, thus forming a Rybp-miR-29 feedback loop. Rybp overexpression was found to enhance the enrichment of Ezh2 and trimethylation of H3K27 at target loci, suggesting it may facilitate the recruitment or stabilization of the Polycomb repressive complex. Collectively, our results identify Rybp as a novel regulator of myogenesis that co-acts with YY1 to silence miR-29 and other myogenic loci.

Applying gene silencing technology to contraception.

Population control of feral animals is often difficult, as it can be dangerous for the animals, labour intensive and expensive. Therefore, a useful tool for control of animal populations would be a non-surgical method to induce sterility. Our laboratories utilize methods aimed at targeting brain cells in vivo with vehicles that deliver a payload of either inhibitory RNAs or genes intended to correct cellular dysfunction. A useful framework for design of a new approach will be the combination of these methods with the intended goal to produce a technique that can be used to non-invasively sterilize cats and dogs. For this approach to succeed, it has to meet several conditions: the target gene must be essential for fertility; the method must include a mechanism to effectively and specifically silence the gene of interest; the method of delivering the silencing agent must be minimally invasive, and finally, the silencing effect must be sustained for the lifespan of the target species, so that expansion of the population can be effectively prevented. In this article, we discuss our work to develop gene silencing technology to induce sterility; we will use examples of our previous studies demonstrating that this approach is viable. These studies include (i) the use of viral vectors able to disrupt reproductive cyclicity when delivered to the regions of the brain involved in the control of reproduction and (ii) experiments with viral vectors that are able to ameliorate neuronal disease when delivered systemically using a novel approach of gene therapy.

Negative transcriptional regulation of the human periostin gene by YingYang-1 transcription factor.

Periostin (POSTN), an osteoblast-specific secreted protein known to be associated with cell adhesion activity for bone formation and development by the epithelial cell-derived tumors, leads to a significant enhancement in angiogenesis and tumorigenesis. At present, little is known about the mechanisms underlying its transcriptional control either in physiological or neoplastic conditions. In this study we demonstrate that the ability of the human POSTN promoter to drive transcription mostly depends on the activity of YingYang-1 (YY1) zinc finger transcription factor. YY1, whose regulatory role in biology includes, besides transcriptional control, also chromatin remodeling, DNA damage repair and tumorigenesis, acts as a strong negative modulator of the POSTN expression. We retain that the identification of the functional role of YY1 in the transcriptional control of the human POSTN gene adds new insights in the studies focused on gene expression in normal and transformed cells.

Inactivation of TNF-α ameliorates diabetic neuropathy in mice.

Tumor necrosis factor (TNF)-α is a potent proinflammatory cytokine involved in the pathogenesis of diabetic neuropathy. We inactivated TNF-α to determine if it is a valid therapeutic target for the treatment of diabetic neuropathy. We effected the inactivation in diabetic neuropathy using two approaches: by genetic inactivation of TNF-α (TNF-α(-/-) mice) or by neutralization of TNF-α protein using the monoclonal antibody infliximab. We induced diabetes using streptozotocin in wild-type and TNF-α(-/-) mice. We measured serum TNF-α concentration and the level of TNF-α mRNA in the dorsal root ganglion (DRG) and evaluated nerve function by a combination of motor (MNCV) and sensory (SNCV) nerve conduction velocities and tail flick test, as well as cytological analysis of intraepidermal nerve fiber density (IENFD) and immunostaining of DRG for NF-κB p65 serine-276 phosphorylated and cleaved caspase-3. Compared with nondiabetic mice, TNF-α(+/+) diabetic mice displayed significant impairments of MNCV, SNCV, tail flick test, and IENFD as well as increased expression of NF-κB p65 and cleaved caspase-3 in their DRG. In contrast, although nondiabetic TNF-α(-/-) mice showed mild abnormalities of IENFD under basal conditions, diabetic TNF-α(-/-) mice showed no evidence of abnormal nerve function tests compared with nondiabetic mice. A single injection of infliximab in diabetic TNF-α(+/+) mice led to suppression of the increased serum TNF-α and amelioration of the electrophysiological and biochemical deficits for at least 4 wk. Moreover, the increased TNF-α mRNA expression in diabetic DRG was also attenuated by infliximab, suggesting infliximab's effects may involve the local suppression of TNF-α. Infliximab, an agent currently in clinical use, is effective in targeting TNF-α action and expression and amelioration of diabetic neuropathy in mice.

Silencing of RhoA nucleotide exchange factor, ARHGEF3, reveals its unexpected role in iron uptake.

Genomewide association meta-analysis studies have identified > 100 independent genetic loci associated with blood cell indices, including volume and count of platelets and erythrocytes. Although several of these loci encode known regulators of hematopoiesis, the mechanism by which most sequence variants exert their effect on blood cell formation remains elusive. An example is the Rho guanine nucleotide exchange factor, ARHGEF3, which was previously implicated by genomewide association meta-analysis studies in bone cell biology. Here, we report on the unexpected role of ARHGEF3 in regulation of iron uptake and erythroid cell maturation. Although early erythroid differentiation progressed normally, silencing of arhgef3 in Danio rerio resulted in microcytic and hypochromic anemia. This was rescued by intracellular supplementation of iron, showing that arhgef3-depleted erythroid cells are fully capable of hemoglobinization. Disruption of the arhgef3 target, RhoA, also produced severe anemia, which was, again, corrected by iron injection. Moreover, silencing of ARHGEF3 in erythromyeloblastoid cells K562 showed that the uptake of transferrin was severely impaired. Taken together, this is the first study to provide evidence for ARHGEF3 being a regulator of transferrin uptake in erythroid cells, through activation of RHOA.

A signaling cascade of nuclear calcium-CREB-ATF3 activated by synaptic NMDA receptors defines a gene repression module that protects against extrasynaptic NMDA receptor-induced neuronal cell death and ischemic brain damage.

Synapse-to-nucleus signaling triggered by synaptic NMDA receptors can lead to the buildup of a neuroprotective shield. Nuclear calcium activating the cAMP response element binding protein (CREB) plays a key role in neuroprotection acquired by synaptic activity. Here we show that in mouse hippocampal neurons, the transcription factor Atf3 (activating transcription factor 3) is a direct target of CREB. Induction of ATF3 expression by CREB in hippocampal neurons was initiated by calcium entry through synaptic NMDA receptors and required nuclear calcium transients and calcium/calmodulin-dependent protein kinase IV activity. Acting as a transcriptional repressor, ATF3 protects cultured hippocampal neurons from apoptosis and extrasynaptic NMDA receptor-induced cell death triggered by bath application of NMDA or oxygen-glucose deprivation. Expression of ATF3 in vivo using stereotaxic delivery of recombinant adeno-associated virus reduces brain damage following a cerebral ischemic insult in mice. Conversion of ATF3 to a transcriptional activator transforms ATF3 into a potent prodeath protein that kills neurons in cell culture and, when expressed in vivo in the hippocampus, ablates the neuronal cell layer. These results link nuclear calcium-CREB signaling to an ATF3-mediated neuroprotective gene repression program, indicating that activity-dependent shutoff of genes is an important process for survival. ATF3 supplementation may counteract age- and disease-related neuronal cell loss caused by a reduction in synaptic activity, malfunctioning of calcium signaling toward and within the nucleus ("nuclear calciopathy"), or increases in death signaling by extrasynaptic NMDA receptors.