In vivo topical gene therapy for recessive dystrophic epidermolysis bullosa: a phase 1 and 2 trial.

Recessive dystrophic epidermolysis bullosa (RDEB) is a lifelong genodermatosis associated with blistering, wounding, and scarring caused by mutations in COL7A1, the gene encoding the anchoring fibril component, collagen VII (C7). Here, we evaluated beremagene geperpavec (B-VEC), an engineered, non-replicating COL7A1 containing herpes simplex virus type 1 (HSV-1) vector, to treat RDEB skin. B-VEC restored C7 expression in RDEB keratinocytes, fibroblasts, RDEB mice and human RDEB xenografts. Subsequently, a randomized, placebo-controlled, phase 1 and 2 clinical trial (NCT03536143) evaluated matched wounds from nine RDEB patients receiving topical B-VEC or placebo repeatedly over 12 weeks. No grade 2 or above B-VEC-related adverse events or vector shedding or tissue-bound skin immunoreactants were noted. HSV-1 and C7 antibodies sometimes presented at baseline or increased after B-VEC treatment without an apparent impact on safety or efficacy. Primary and secondary objectives of C7 expression, anchoring fibril assembly, wound surface area reduction, duration of wound closure, and time to wound closure following B-VEC treatment were met. A patient-reported pain-severity secondary outcome was not assessed given the small proportion of wounds treated. A global assessment secondary endpoint was not pursued due to redundancy with regard to other endpoints. These studies show that B-VEC is an easily administered, safely tolerated, topical molecular corrective therapy promoting wound healing in patients with RDEB.

Cells from discarded dressings differentiate chronic from acute wounds in patients with Epidermolysis Bullosa.

Impaired wound healing complicates a wide range of diseases and represents a major cost to healthcare systems. Here we describe the use of discarded wound dressings as a novel, cost effective, accessible, and non-invasive method of isolating viable human cells present at the site of skin wounds. By analyzing 133 discarded wound dressings from 51 patients with the inherited skin-blistering disease epidermolysis bullosa (EB), we show that large numbers of cells, often in excess of 100 million per day, continually infiltrate wound dressings. We show, that the method is able to differentiate chronic from acute wounds, identifying significant increases in granulocytes in chronic wounds, and we show that patients with the junctional form of EB have significantly more cells infiltrating their wounds compared with patients with recessive dystrophic EB. Finally, we identify subsets of granulocytes and T lymphocytes present in all wounds paving the way for single cell profiling of innate and adaptive immune cells with relevance to wound pathologies. In summary, our study delineates findings in EB that have potential relevance for all chronic wounds, and presents a method of cellular isolation that has wide reaching clinical application.

Identification of Rigosertib for the Treatment of Recessive Dystrophic Epidermolysis Bullosa-Associated Squamous Cell Carcinoma.

PURPOSE: Squamous cell carcinoma (SCC) of the skin is the leading cause of death in patients with the severe generalized form of the genetic disease recessive dystrophic epidermolysis bullosa (RDEB). Although emerging data are identifying why patients suffer this fatal complication, therapies for treatment of RDEB SCC are in urgent need.Experimental Design: We previously identified polo-like kinase 1 (PLK1) as a therapeutic target in skin SCC, including RDEB SCC. Here, we undertake a screen of 6 compounds originally designated as PLK1 inhibitors, and detail the efficacy of the lead compound, the multipathway allosteric inhibitor ON-01910, for targeting RDEB SCC in vitro and in vivo. RESULTS: ON-01910 (or rigosertib) exhibited significant specificity for RDEB SCC: in culture rigosertib induced apoptosis in 10 of 10 RDEB SCC keratinocyte populations while only slowing the growth of normal primary skin cells at doses 2 orders of magnitude higher. Furthermore, rigosertib significantly inhibited the growth of two RDEB SCC in murine xenograft studies with no apparent toxicity. Mechanistically, rigosertib has been shown to inhibit multiple signaling pathways. Comparison of PLK1 siRNA with MEK inhibition, AKT inhibition, and the microtubule-disrupting agent vinblastine in RDEB SCC shows that only PLK1 reduction exhibits a similar sensitivity profile to rigosertib. CONCLUSIONS: These data support a "first in RDEB" phase II clinical trial of rigosertib to assess tumor targeting in patients with late stage, metastatic, and/or unresectable SCC.

APOBEC mutation drives early-onset squamous cell carcinomas in recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare inherited skin and mucous membrane fragility disorder complicated by early-onset, highly malignant cutaneous squamous cell carcinomas (SCCs). The molecular etiology of RDEB SCC, which arises at sites of sustained tissue damage, is unknown. We performed detailed molecular analysis using whole-exome, whole-genome, and RNA sequencing of 27 RDEB SCC tumors, including multiple tumors from the same patient and multiple regions from five individual tumors. We report that driver mutations were shared with spontaneous, ultraviolet (UV) light-induced cutaneous SCC (UV SCC) and head and neck SCC (HNSCC) and did not explain the early presentation or aggressive nature of RDEB SCC. Instead, endogenous mutation processes associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) deaminases dominated RDEB SCC. APOBEC mutation signatures were enhanced throughout RDEB SCC tumor evolution, relative to spontaneous UV SCC and HNSCC mutation profiles. Sixty-seven percent of RDEB SCC driver mutations was found to emerge as a result of APOBEC and other endogenous mutational processes previously associated with age, potentially explaining a >1000-fold increased incidence and the early onset of these SCCs. Human papillomavirus-negative basal and mesenchymal subtypes of HNSCC harbored enhanced APOBEC mutational signatures and transcriptomes similar to those of RDEB SCC, suggesting that APOBEC deaminases drive other subtypes of SCC. Collectively, these data establish specific mutagenic mechanisms associated with chronic tissue damage. Our findings reveal a cause for cancers arising at sites of persistent inflammation and identify potential therapeutic avenues to treat RDEB SCC.

Stromal cyclin D1 promotes heterotypic immune signaling and breast cancer growth.

The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that drives cell autonomous cell cycle progression and proliferation. Herein we show cyclin D1 abundance is increased >30-fold in the stromal fibroblasts of patients with invasive breast cancer, associated with poor outcome. Cyclin D1 transformed hTERT human fibroblast to a cancer-associated fibroblast phenotype. Stromal fibroblast expression of cyclin D1 (cyclin D1Stroma) in vivo, enhanced breast epithelial cancer tumor growth, restrained apoptosis, and increased autophagy. Cyclin D1Stroma had profound effects on the breast tumor microenvironment increasing the recruitment of F4/80+ and CD11b+ macrophages and increasing angiogenesis. Cyclin D1Stroma induced secretion of factors that promoted expansion of stem cells (breast stem-like cells, embryonic stem cells and bone marrow derived stem cells). Cyclin D1Stroma resulted in increased secretion of proinflammatory cytokines (CCL2, CCL7, CCL11, CXCL1, CXCL5, CXCL9, CXCL12), CSF (CSF1, GM-CSF1) and osteopontin (OPN) (30-fold). OPN was induced by cyclin D1 in fibroblasts, breast epithelial cells and in the murine transgenic mammary gland and OPN was sufficient to induce stem cell expansion. These results demonstrate that cyclin D1Stroma drives tumor microenvironment heterocellular signaling, promoting several key hallmarks of cancer.

Amlexanox Enhances Premature Termination Codon Read-Through in COL7A1 and Expression of Full Length Type VII Collagen: Potential Therapy for Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare monogenic blistering disorder caused by the lack of functional type VII collagen, leading to skin fragility and subsequent trauma-induced separation of the epidermis from the underlying dermis. A total of 46% of patients with RDEB harbor at least one premature termination codon (PTC) mutation in COL7A1, and previous studies have shown that aminoglycosides are able to overcome RDEB PTC mutations by inducing "read-through" and incorporation of an amino acid at the PTC site. However, aminoglycoside toxicity will likely prevent widespread clinical application. Here the FDA-approved drug amlexanox was tested for its ability to read-through PTC mutations in cells derived from patients with RDEB. Eight of 12 different PTC alleles responded to treatment and produced full length protein, in some cases more than 50% relative to normal controls. Read-through type VII collagen was readily detectable in cell culture media and also localized to the dermal-epidermal junction in organotypic skin culture. Amlexanox increased COL7A1 transcript and the phosphorylation of UPF-1, an RNA helicase associated with nonsense-mediated mRNA decay, suggesting that amlexanox inhibits nonsense-mediated mRNA decay in cells from patients with RDEB that respond to read-through treatment. This preclinical study demonstrates the potential of repurposing amlexanox for the treatment of patients with RDEB harboring PTC mutation in COL7A1.

Exosome-mediated transfer from the tumor microenvironment increases TGFß signaling in squamous cell carcinoma.

Transforming growth factor-beta (TGFß) signaling in cancer is context dependent and acts either as a tumor suppressor or a tumor promoter. Loss of function mutation in TGFß type II receptor (TßRII) is a frequent event in oral cavity squamous cell carcinoma (SCC). Recently, heterogeneity of TGFß response has been described at the leading edge of SCC and this heterogeneity has been shown to influence stem cell renewal and drug resistance. Because exosome transfer from stromal to breast cancer cells regulates therapy resistance pathways we investigated whether exosomes contain components of the TGFß signaling pathway and whether exosome transfer between stromal fibroblasts and tumor cells can influence TGFß signaling in SCC. We demonstrate that exosomes purified from stromal fibroblasts isolated from patients with oral SCC contains TßRII. We also demonstrate that transfer of fibroblast exosomes increases TGFß signaling in SCC keratinocytes devoid of TßRII which remain non-responsive to TGFß ligand in the absence of exosome transfer. Overall our data show that stromal communication with tumor cells can direct TGFß signaling in SCC.

Outcomes of Chopart Amputation in a Tertiary Referral Diabetic Foot Clinic: Data From a Consecutive Series of 83 Hospitalized Patients.

The purpose of the present retrospective study was to evaluate the outcomes (ie, ulcer recurrence, major amputation, death) in diabetic patients undergoing Chopart amputation because of deep infection or gangrene extending to the midfoot. From 2009 to 2011, 83 patients, aged 71.4 ± 9.3 years, underwent a midtarsal amputation and were followed up until December 31, 2012 (mean follow-up 2.8 ± 0.8 years). Of the 83 patients, 26 were female, 61 required insulin, 47 had renal insufficiency, 19 underwent hemodialysis, 65 had hypertension, 34 had a history of cardiac disease, and 4 had a history of stroke. Chopart amputation was performed in 38 patients (45.8%) with gangrene, 31 (37.4%) with abscess, and 14 (16.9%) with osteomyelitis. Urgent surgery was performed in 56 patients (67.5%). Effective revascularization was performed in 64 patients (77.1%) patients. Of the 83 patients, 47 had healed at a mean period of 164.7 (range 11 to 698) days. Ulcer recurrence developed in 15 patients (31.9%). A major amputation was necessary in 23 patients (27.7%), with an annual incidence of 13.0%. None of the included variables on logistic regression analysis was significantly associated with proximal amputation. Of the 83 patients, 38 (45.8%) died, with an annual incidence of 25.8%. On logistic regression analysis, age (odds ratio [OR] 1.11, 95% confidence interval [CI] 1.01 to 1.16), history of stroke (OR 9.94, 95% CI 3.16 to 31.24), and urgent surgery (OR 2.60, 95% CI 1.14 to 5.93) were associated with mortality. Chopart amputation represents the last chance to avoid major amputation for diabetic patients with serious foot complications. Our success rate was great enough to consider Chopart amputation a viable option for limb salvage in this high-risk population.

Lysyl Hydroxylase 3 Localizes to Epidermal Basement Membrane and Is Reduced in Patients with Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention.

Severity of demographic and clinical characteristics, revascularization feasibility, major amputation, and mortality rate in diabetic patients admitted to a tertiary diabetic foot center for critical limb ischemia: comparison of 2 cohorts recruited at a 10-year distance.

BACKGROUND: To compare demographic and clinical characteristics, revascularization, major amputation, and mortality among patients admitted to a diabetic foot center because of critical limb ischemia (CLI) during 1999-2003 (cohort 1) and 2009 (cohort 2). METHODS: During 1999-2003, 564 diabetic patients with CLI (cohort 1) were admitted to our center, and 344 patients (360 affected limbs) were admitted during 2009 (cohort 2). Data on demographic and clinical characteristics, revascularization by peripheral angioplasty (PTA) or bypass graft (BPG), major amputation, and mortality were recorded. RESULTS: Patients belonging to cohort 2 were older than patients of cohort 1 (P = 0.001). In cohort 2, there were more subjects requiring insulin (P = 0.008) and duration of diabetes was longer (P = 0.001); moreover, there were more patients requiring dialysis (P = 0.001), patients with history of stroke (P = 0.004), or foot ulcer (P = 0.001). No significant difference between the 2 groups was found concerning gender, metabolic control, hypertension, lipid values, neuropathy, and retinopathy. Occlusion was more frequent than stenosis in the posterior tibial (P < 0.001) and peroneal (P = 0.016) arteries. However, the revascularization rate did not differ (P = 0.318) between the 2 groups. Restenosis after PTA was not significantly different (P = 0.627), whereas BPG failure was significantly more frequent (P = 0.010) in cohort 2 (2009). Major amputation (P = 0.222) and mortality rate (P = 0.727) did not differ between the 2 groups. CONCLUSIONS: The severity of either foot lesions or patients comorbidities should be concomitantly assessed and taken into proper consideration when evaluating changes in the amputation rate among different studies or in different temporal settings.

Ketones and lactate increase cancer cell "stemness," driving recurrence, metastasis and poor clinical outcome in breast cancer: achieving personalized medicine via Metabolo-Genomics.

Previously, we showed that high-energy metabolites (lactate and ketones) "fuel" tumor growth and experimental metastasis in an in vivo xenograft model, most likely by driving oxidative mitochondrial metabolism in breast cancer cells. To mechanistically understand how these metabolites affect tumor cell behavior, here we used genome-wide transcriptional profiling. Briefly, human breast cancer cells (MCF7) were cultured with lactate or ketones, and then subjected to transcriptional analysis (exon-array). Interestingly, our results show that treatment with these high-energy metabolites increases the transcriptional expression of gene profiles normally associated with "stemness," including genes upregulated in embryonic stem (ES) cells. Similarly, we observe that lactate and ketones promote the growth of bonafide ES cells, providing functional validation. The lactate- and ketone-induced "gene signatures" were able to predict poor clinical outcome (including recurrence and metastasis) in a cohort of human breast cancer patients. Taken together, our results are consistent with the idea that lactate and ketone utilization in cancer cells promotes the "cancer stem cell" phenotype, resulting in significant decreases in patient survival. One possible mechanism by which these high-energy metabolites might induce stemness is by increasing the pool of Acetyl-CoA, leading to increased histone acetylation, and elevated gene expression. Thus, our results mechanistically imply that clinical outcome in breast cancer could simply be determined by epigenetics and energy metabolism, rather than by the accumulation of specific "classical" gene mutations. We also suggest that high-risk cancer patients (identified by the lactate/ketone gene signatures) could be treated with new therapeutics that target oxidative mitochondrial metabolism, such as the anti-oxidant and "mitochondrial poison" metformin. Finally, we propose that this new approach to personalized cancer medicine be termed "Metabolo-Genomics," which incorporates features of both 1) cell metabolism and 2) gene transcriptional profiling. Importantly, this powerful new approach directly links cancer cell metabolism with clinical outcome, and new therapeutic strategies for inhibiting the TCA cycle and mitochondrial oxidative phosphorylation in cancer cells.

Expression of the transcriptional repressor Gfi-1 is regulated by C/EBP{alpha} and is involved in its proliferation and colony formation-inhibitory effects in p210BCR/ABL-expressing cells.

Ectopic expression of CAAT/enhancer binding protein α (C/EBPα) in p210BCR/ABL-expressing cells induces granulocytic differentiation, inhibits proliferation, and suppresses leukemogenesis. To dissect the molecular mechanisms underlying these biological effects, C/EBPα-regulated genes were identified by microarray analysis in 32D-p210BCR/ABL cells. One of the genes whose expression was activated by C/EBPα in a DNA binding-dependent manner in BCR/ABL-expressing cells is the transcriptional repressor Gfi-1. We show here that C/EBPα interacts with a functional C/EBP binding site in the Gfi-1 5'-flanking region and enhances the promoter activity of Gfi-1. Moreover, in K562 cells, RNA interference-mediated downregulation of Gfi-1 expression partially rescued the proliferation-inhibitory but not the differentiation-inducing effect of C/EBPα. Ectopic expression of wild-type Gfi-1, but not of a transcriptional repressor mutant (Gfi-1P2A), inhibited proliferation and markedly suppressed colony formation but did not induce granulocytic differentiation of BCR/ABL-expressing cells. By contrast, Gfi-1 short hairpin RNA-tranduced CD34(+) chronic myeloid leukemia cells were markedly more clonogenic than the scramble-transduced counterpart. Together, these studies indicate that Gfi-1 is a direct target of C/EBPα required for its proliferation and survival-inhibitory effects in BCR/ABL-expressing cells.

Transcriptional repression of c-Myb and GATA-2 is involved in the biologic effects of C/EBPalpha in p210BCR/ABL-expressing cells.

Ectopic C/EBPalpha expression in p210(BCR/ABL)-expressing hematopoietic cells induces granulocytic differentiation, inhibits proliferation, and suppresses leukemogenesis. To assess the underlying mechanisms, C/EBPalpha targets were identified by microarray analyses. Upon C/EBPalpha activation, expression of c-Myb and GATA-2 was repressed in 32D-BCR/ABL, K562, and chronic myelogenous leukemia (CML) blast crisis (BC) primary cells but only c-Myb levels decreased slightly in CD34(+) normal progenitors. The role of these 2 genes for the effects of C/EBPalpha was assessed by perturbing their expression in K562 cells. Ectopic c-Myb expression blocked the proliferation inhibition- and differentiation-inducing effects of C/EBPalpha, whereas c-Myb siRNA treatment enhanced C/EBPalpha-mediated proliferation inhibition and induced changes in gene expression indicative of monocytic differentiation. Ectopic GATA-2 expression suppressed the proliferation inhibitory effect of C/EBPalpha but blocked in part the effect on differentiation; GATA-2 siRNA treatment had no effects on C/EBPalpha induction of differentiation but inhibited proliferation of K562 cells, alone or upon C/EBPalpha activation. In summary, the effects of C/EBPalpha in p210(BCR/ABL)-expressing cells depend, in part, on transcriptional repression of c-Myb and GATA-2. Since perturbation of c-Myb and GATA-2 expression has nonidentical consequences for proliferation and differentiation of K562 cells, the effects of C/EBPalpha appear to involve dif-ferent transcription-regulated targets.

Micro RNA 145 targets the insulin receptor substrate-1 and inhibits the growth of colon cancer cells.

The insulin receptor substrate-1 (IRS-1), a docking protein for both the type 1 insulin-like growth factor receptor (IGF-IR) and the insulin receptor, is known to send a mitogenic, anti-apoptotic, and anti-differentiation signal. Several micro RNAs (miRs) are suggested by the data base as possible candidates for targeting IRS-1. We show here that one of the miRs predicted by the data base, miR145, whether transfected as a synthetic oligonucleotide or expressed from a plasmid, causes down-regulation of IRS-1 in human colon cancer cells. IRS-1 mRNA is not decreased by miR145, while it is down-regulated by an siRNA targeting IRS-1. Targeting of the IRS-1 3'-untranslated region (UTR) by miR145 was confirmed using a reporter gene (luciferase) expressing the miR145 binding sites of the IRS-1 3'-UTR. In agreement with the role of IRS-1 in cell proliferation, we show that treatment of human colon cancer cells with miR145 causes growth arrest comparable to the use of an siRNA against IRS-1. Taken together, these results identify miR145 as a micro RNA that down-regulates the IRS-1 protein, and inhibits the growth of human cancer cells.

Insulin receptor substrate (IRS)-1 regulates murine embryonic stem (mES) cells self-renewal.

Mouse embryonic stem (mES) cells are pluripotent cells that can be propagated in vitro with leukemia inhibitory factor (LIF) and serum. Intracellular signaling by LIF is principally mediated by activation of STAT-3, although additional pathways for self-renewal have been described. Here, we identified a novel role for Insulin receptor substrate-1 (IRS-1) as a critical factor in mES cells self-renewal and differentiation. IRS-1 is expressed and tyrosyl phosphorylated during mES cells self-renewal. Differentiation of mES cells, by LIF withdrawal, is associated with a marked reduction in IRS-1 expression. Targeting of IRS-1 by si-IRS-1 results in a severe reduction of Oct-4 protein expression and alkaline phosphatase activity, markers of undifferentiated mES cells. IRS-1 targeting does not interfere with LIF-induced STAT-3 phosphorylation, but negatively affects protein kinase B (PKB/AKT) and glycogen synthase kinase-3 (GSK-3beta) phosphorylation, which are downstream effectors of the LIF-mediated PI3K signaling cascade. Targeting of IRS-1 also results in a marked down regulation of Id-1 and Id-2 proteins expression, which are important components for self-renewal of ES cells. Conversely, over expression of IRS-1 inhibits mES cell differentiation. Taken together, these results suggest that expression and activity of IRS-1 are critical to the maintenance of the self-renewal program in mES cells.

Expression profiles of micro RNA in proliferating and differentiating 32D murine myeloid cells.

32D cells are murine myeloid cells that grow indefinitely in Interleukin-3 (IL-3). In these cells, the type 1 insulin-like growth factor (IGF-I) and granulocytic-colony stimulating factor (G-CSF) induce differentiation to granulocytes. 32D cells do not express insulin receptor substrate-1 (IRS-1) or IRS-2, docking proteins of the IGF-I receptor. Ectopic expression of IRS-1 in these cells inhibits differentiation, the cells become IL-3 independent and IGF-1 dependent and can form tumors in mice. 32D and 32D-derived cells offer a good model in which to study the expression profiles of Micro Rna (miR) related to sustained proliferation or differentiation. We present here the data obtained with miR micro-arrays and identify the miR that are regulated by IGF-1 or G-CSF and are associated with either differentiation or indefinite cell proliferation of 32D murine myeloid cells.

24p3 in differentiation of myeloid cells.

24p3 is a secreted lipocalin that has been variously related to apoptosis, proliferation, and the neutrophil lineage of blood cells. We have investigated the expression of 24p3 mRNA and protein in myeloid cell lines induced to differentiate by insulin-like growth factor 1 (IGF-1) and the granulocytic-colony simulating factor (G-CSF). Both these growth factors, which cause myeloid cells to differentiate into granulocytes, induced a marked increase in the expression of both 24p3 protein and mRNA. The mRNA especially appeared early after the cells were induced with either IGF-1 or G-CSF, at a time when the cells were still proliferating and are morphologically undifferentiated. 24p3 can be considered an early marker of granulocytic differentiation.

Regulation of upstream binding factor 1 activity by insulin-like growth factor I receptor signaling.

The upstream binding factor 1 (UBF1) is one of the proteins in a complex that regulates the activity of RNA polymerase I, which controls the rate of ribosomal RNA (rRNA) synthesis. We have shown previously that insulin receptor substrate-1 (IRS-1) can translocate to the nuclei and nucleoli of cells and bind UBF1. We report here that activation of the type I insulin-like growth factor receptor (IGF-IR) by IGF-I increases transcription from the ribosomal DNA (rDNA) promoter in both myeloid cells and mouse fibroblasts. The increased activity of the rDNA promoter is accompanied by increased phosphorylation of UBF1, a requirement for UBF1 activation. Phosphorylation occurs on a number of UBF1 peptides, most prominently on the highly acidic, serine-rich C terminus. In myeloid cells (but not in mouse embryo fibroblasts) IRS-1 signaling stabilizes the levels of UBF1 protein. These findings demonstrate that IGF-IR signaling can increase the activity of UBF1 and transcription from the rDNA promoter, providing one explanation for the reported effects of the IGF/IRS-1 axis on cell and body size in animals and cells in culture.

A modified tandem affinity purification tag technique for the purification of protein complexes in mammalian cells.

The tandem affinity purification (TAP) tag technique has been used with success to identify under nondenaturing conditions protein complexes in yeast. The technique can be used in mammalian cells, but we found that the original technique does not yield enough recovery for the identification of proteins when mammalian cells growing in monolayer have to be used. We present here a modified TAP tag technique that allows sufficient recovery of proteins from mouse fibroblasts growing in monolayer cultures. The recovery allows protein identification by mass spectrometry.

Uso de cianocrilato en la síntesis de las fimosectomías / Cianocrilato use in the synthesis of the phymosectomy

El objetivo de este trabajo es investigar la utilidad del uso de Cianocrilato en la síntesis de heridas de fimosectomías realizadas en el Hospital "Miguel Pérez Carreño" durante el período de enero a diciembre de 2003. Se realizó estudio prospectivo, procediéndose a la síntesis de heridas de fimosectomías con Cianocrilato en 10 pacientes y comparándose en 10 pacientes en quienes la síntesis se realizó con Catgut Crómico, tomando en cuenta el tiempo utilizado en la síntesis, dolor, complicaciones y el tiempo para reinicio de relaciones sexules. Con el uso de Cianocrilato no se evidenciaron cicatrices hipertróficas. El tiempo utilizado en la síntesis con Cianocrilato fue significativamente menor que con la sutura (P<0,0001), al igual que el dolor a las dos semanas de postoperatorio (p<0,000092) y el tiempo de reinicio de relaciones sexuales (P<0,00033) con un valor de 4.87 y 11.03 días más corto