Controlling tissue patterning by translational regulation of signaling transcripts through the core translation factor eIF3c.

Although gene expression is tightly regulated during embryonic development, the impact of translational control has received less experimental attention. Here, we find that eukaryotic translation initiation factor-3 (eIF3) is required for Shh-mediated tissue patterning. Analysis of loss-of-function eIF3 subunit c (Eif3c) mice reveal a unique sensitivity to the Shh receptor patched 1 (Ptch1) dosage. Genome-wide in vivo enhanced cross-linking immunoprecipitation sequence (eCLIP-seq) shows unexpected specificity for eIF3 binding to a pyrimidine-rich motif present in subsets of 5'-UTRs and a corresponding change in the translation of these transcripts by ribosome profiling in Eif3c loss-of-function embryos. We further find a transcript specific effect in Eif3c loss-of-function embryos whereby translation of Ptch1 through this pyrimidine-rich motif is specifically sensitive to eIF3 amount. Altogether, this work uncovers hidden specificity of housekeeping translation initiation machinery for the translation of key developmental signaling transcripts.

Molecular Design and Production of AAV Viral Vectors for Gene Therapy.

Adeno-associated virus (AAV) is a helper-dependent single-stranded DNA parvovirus. Over the years, AAV has become the vector of choice in the gene therapy field due to its safety profile and low immunogenicity. With a carrying capacity of 4.2 kbp, these vectors have demonstrated their clinical value, especially in the field of ophthalmology. Herein we describe methods for the molecular design and packaging of AAV viral vectors. These methods apply to the design of single-stranded or self-complementary AAV vectors.

Changing how I feel about the food: experimentally manipulated affective associations with fruits change fruit choice behaviors.

Fewer than half of Americans meet current recommendations for fruit and vegetable intake. The behavioral affective associations model posits that feelings and emotions associated with a behavior are a proximal influence on decision making. Cross-sectional evidence supports the model and suggests that affective associations predict fruit and vegetable consumption. The purpose of this study was to test whether a causal relation exists between affective associations about fruits and future fruit consumption behavior, as measured by a snack selection task. Following a baseline assessment of cognitive and affective variables, participants' (N = 161) affective associations about fruits were experimentally manipulated with an implicit priming paradigm. Images of fruits were repeatedly paired with positive, negative, or neutral affective stimuli. The key outcome measure was a behavioral choice task in which participants chose between fruit and a granola bar. Participants in the positive prime condition were three times more likely than those in the negative condition to select a piece of fruit over the granola bar alternative in the snack selection task. They were also twice as likely as those in the neutral condition to select fruit. There were no changes in self-reported affective associations or cognitive beliefs. These findings provide further evidence of the implicit and direct influence of affective associations on behavior, suggesting the need to both incorporate the role of affect in health decision making models, as well as the potential utility of intervention strategies targeting affective associations with health-related behaviors.

The relationship of positive work environments and workplace injury: evidence from the National Nursing Assistant Survey.

BACKGROUND: With estimates of a 51% growth in the number of nursing assistants needed by 2016, there is a critical need to examine workplace factors that negatively contribute to the recruitment and retention of nursing assistants. Studies have shown that high demands, physical stress, and chronic workforce shortages contribute to a working environment that fosters one of the highest workforce injury rates in the United States. PURPOSES: The aim of this study was to explore the relationship between nursing assistant injury rates and key outcomes, such as job satisfaction and turnover intent, while exploring workplace environment factors, such as injury prevention training, supervisor support, and employee engagement, that can decrease the rates of workplace injury. METHODOLOGY/APPROACH: Data from the 2004 National Nursing Assistant Survey were used to examine the negative effects of workplace injury on nursing assistants and the workplace environment factors that are related to the rate of worker injury. FINDINGS: Nursing assistants who experience job-related injuries have lower levels of job satisfaction, increased turnover intentions, and are less likely to recommend their facility as a place to work or seek care services. It was also found that nursing assistant injury rates are related to employee ratings of injury prevention training, supervisor support, and employee engagement. NAs with multiple injuries (>2) were 1.3-1.6 times more likely to report being injured at work than NAs who had not been injured when supervisor support, employee engagement, and training ratings were low. PRACTICE IMPLICATIONS: Evidence that health care organizations can use to better understand how workplace injuries occur and insight into ways to reduce the current staggering rate of on-the-job injuries occurring in health care workplaces were offered in this study. The findings also offer empirical support for an extension of the National Institute for Occupational Health and Safety/National Occupational Research Agenda Work Organization Framework for Occupational Illness and Injury.

Importance of interaction between nerve growth factor and α9ß1 integrin in glial tumor angiogenesis.

NGF is a growth factor for which the role in the promotion of angiogenesis is still not completely understood. We found that NGF promotes the pathological neovascularization process in glioma through a direct interaction with α9ß1 integrin, which is up-regulated on microvascular endothelial cells in cancer tissue. We propagated gHMVEC primary cells using a new method of immune-selection, and these cells demonstrated α9ß1 integrin-dependent binding of NGF in a cell adhesion assay. Moreover, NGF induced gHMVEC proliferation and chemotaxis inhibited by specific blockers of α9ß1 integrin, such as MLD-disintegrins and monoclonal antibody Y9A2. A Matrigel tube formation assay revealed that NGF significantly increased capillary-like growth from gHMVEC to a level comparable to treatment with VEGF. The snake venom disintegrin, VLO5, inhibited the agonistic effect of both growth factors, whereas the effect of Y9A2 was not statistically significant. Angiogenesis exogenously induced by NGF  was also α9ß1-integrin dependent in an embryonic quail CAM system. However, angiogenesis pathologically induced by developing glioma in this system was only sensitive for inhibition with MLD-disintegrin, suggesting a more complex effect of cancer cells on the neovascularization process. The anti-angiogenic effect of MLD-disintegrins is probably related to their pro-apoptotic ability induced in activated tumoral endothelial cells. Therefore, the molecular basis of these disintegrins may be useful for developing new angiostatic pharmaceuticals for application in cancer therapy.

Non-RGD-containing snake venom disintegrins, functional and structural relations.

Snake venom disintegrins are present in a variety of species and are functionally divided into three families: RGD, MLD and R/KTS. The RGD family of disintegrins, which bind and inhibit the physiological functions of RGD-dependent integrins, constitute the largest and most investigated family. This review will be focused on characterization of two relatively new families of snake venom disintegrins, expressing in their active site MLD and R/KTS motifs. The MLD motif, present only in heterodimeric disintegrins, mediates binding of these disintegrins to α4ß1, α4ß7 and α9ß1 integrins, whereas the presence of a KTS or RTS sequence in the active site selectively directs activity of disintegrins to the collagen receptor α1ß1 integrin. Structurally, KTS-disintegrins are short, monomeric molecules containing 41 amino acids in its polypeptide chain. Biological activities of MLD and KTS-disintegrins were investigated in many systems in vitro and in vivo. Purified disintegrins are non-toxic in therapeutic doses in rodent and avian models. Their modulatory properties were observed in investigations of cancer angiogenesis and metastasis, immunosuppression of IDDM (insulin-dependent diabetes mellitus) and asthma, as well as in neurodegenerative assays and cell apoptosis.

alpha(9)beta(1) integrin engagement inhibits neutrophil spontaneous apoptosis: involvement of Bcl-2 family members.

Integrin signaling is comprised of well-characterized pathways generally involved in cell survival. alpha(9)beta(1) integrin has recently become a target of study and has been shown to present pro-survival effects on neutrophils. However, there are no detailed studies on how alpha(9)beta(1) integrin-coupled signaling pathways interact and how they converge to finally modulate spontaneous apoptosis in neutrophils. In this regard we sought to investigate the main signaling events triggered by alpha(9)beta(1) integrin engagement and how these signaling pathways modulate the apoptotic program of human neutrophils. Using VLO5, a snake venom disintegrin shown to bind to alpha(9)beta(1) integrin in neutrophils, we demonstrate that alpha(9)beta(1) integrin engagement leads to the activation of integrin signaling pathways and potently reduces neutrophil spontaneous apoptosis. These effects are dependent on the activation of PI3K and MAPK pathways, since both LY294002 (PI3K inhibitor) or PD95059 (MEK inhibitor) reverted the effects of VLO5/alpha(9)beta(1) interaction. Moreover we show that VLO5/alpha(9)beta(1) engagement induces NF-kappaB nuclear translocation and increases the ratio between anti- and pro-apoptotic proteins by inducing the degradation of pro-apoptotic protein Bad and increasing the expression of anti-apoptotic protein Bcl-x(L). VLO5 also inhibited the early steps of neutrophil spontaneous apoptosis by preventing Bax translocation to the outer mitochondrial membrane and consequent cytochrome c release. In conclusion, as the mechanistic details of alpha(9)beta(1) integrin signaling pathways in human neutrophils becomes clearer, it should become possible to develop new therapeutic agents for human diseases where neutrophils play a prominent role.

Effect of VP12 and viperistatin on inhibition of collagen-receptor-dependent melanoma metastasis.

Viperistatin and VP12 isolated from Vipera paleastinae venom showed a potent inhibitory activity against collagen receptors, alpha1beta1 and alpha2beta1 integrins, respectively. Structurally, viperistatin belongs to the disintegrin family of proteins, whereas VP12 is composed of two subunits VP12A and VP12B displaying amino acid sequence homology with heterodimeric C-lectin type proteins. Viperistatin and VP12 used separately and simultaneously inhibited pro-metastatic activities of melanoma cells lines. The level of inhibition of MV3 and HS.939T human cell lines in cell adhesion and migration assays by both compounds was correlated with expression of alpha1beta1 and alpha2beta1 integrins on the cell surface. MV3 cells express collagen receptors to much higher extent than HS.939T and required the application of higher concentrations of inhibitors to block their adhesion to collagen types I and IV. A melanoma cell transmigration assay through a dHMVEC layer revealed that alpha1beta1 integrin plays a significant role in invasion of HS.939T cells, while alpha2beta1 integrin appears to be more important for MV3 cells. In an animal model of hematogenous metastasis of the mouse B16F10 cell line, the inhibitory effect of viperistatin and VP12 was only partial. These data suggest that collagen receptors may be an interesting target for development of new anti-metastatic therapies.

Integrin alpha9 beta1 is a receptor for nerve growth factor and other neurotrophins.

The integrin alpha9beta1 is a multifunctional receptor that interacts with a variety of ligands including vascular cell adhesion molecule 1, tenascin C and osteopontin. We found that this integrin is a receptor for nerve growth factor (NGF) and two other neurotrophins, brain-derived neurotrophic factor and NT3, using a cell adhesion assay with the alpha9SW480 cell line. Interaction of alpha9beta1 with NGF was confirmed in an ELISA assay by direct binding to purified integrin. alpha9beta1 integrin binds to neurotrophins in a manner similar to another common neurotrophin receptor, p75(NTR) (NGFR), although alpha9beta1 activity is correlated with induction of pro-survival and pro-proliferative signaling cascades. This property of alpha9beta1 resembles the interaction of NGF with a high affinity receptor, TrkA, however, this integrin shows a low affinity for NGF. NGF induces chemotaxis of cells expressing alpha9beta1 and their proliferation. Moreover, alpha9beta1 integrin is a signaling receptor for NGF, which activates the MAPK (Erk1/2) pathway. The alpha9beta1-dependent chemotactic ability of NGF appears to result from the activation of paxillin.

VEGF-related protein isolated from Vipera palestinae venom, promotes angiogenesis.

Therapeutic angiogenesis is one of the major approaches in designing new therapies for cardiovascular diseases. vpVEGF was purified from Vipera palestinae venom using two steps of reverse-phase HPLC. Structurally, vpVEGF belongs to the VEGF-F1 family of snake venom proteins, and potently stimulated dHMVEC proliferation in a VEGFR-2 dependent manner. This growth factor appeared to be a chemoattractant for migration of these cells and stimulated their radial migration in a collagen gel. The stimulatory effect on dHMVEC was correlated with activation of the MAPK Erk1/2 signaling pathway. In vivo vpVEGF induced angiogenesis in a Japanese quail assay and in a Matrigel plug assay in mice. Although in the quail assay vpVEGF showed lower activity than hrVEGF-A165 in mammalian-related systems there were no significant differences. The experiments with dHMVEC, as well as angiogenesis in vivo suggest that the pro-angiogenic effect of vpVEGF is related to its interaction with VEGFR-2 (flk-1).