Hypoxia Activates the K-Ras Proto-Oncogene to Stimulate Angiogenesis and Inhibit Apoptosis in Colon Cancer Cells

The KRAS proto-oncogene plays a key role in the development of many human tumors and is commonly activated by somatic mutation or signaling through specific growth factor receptors. However, the interaction between the micro-environment and K-ras activity has not been defined. Hypoxia invariably develops as tumors outgrow their supply of oxygen. A series of well-orchestrated cellular adaptations occur that stimulate angiogenesis and enhance survival of the tumor in hypoxic conditions. Our previous studies demonstrated that mutant KRAS alleles can interact with hypoxia to induce vascular endothelial growth factor (VEGF) in colon cancer. We sought to determine whether similar hypoxic responses are also present in tumors without a KRAS mutation. Hypoxia consistently increased the levels of activated, GTP-bound K-ras in colon cancer cell lines with a wild-type KRAS gene, and this depended upon the activation of c-Src. Inhibition of c-Src by PP2 treatment or siRNA knockdown blocked the hypoxic activation of K-ras. This activation of K-ras did not depend upon EGFR and resulted in the phosphorylation of Akt and induction of VEGF expression. In addition, activation of K-ras significantly blocked apoptosis in hypoxic conditions. These studies reveal a unique adaptive mechanism in hypoxia that activates K-ras signaling in the absence of a mutant KRAS oncogene

Circulating Galectin-1 and Galectin-3 in sera from patients with systemic sclerosis: associations with clinical features and treatment

Systemic Sclerosis (SSc) is a rheumatic disease characterized by fibrosis, microvascular damage and immune dysregulation. Two major subsets, limited cutaneous systemic sclerosis (lcSSc) and diffuse cutaneous systemic sclerosis (dcSSc) can be defined, according to the extent of skin involvement. Increasing evidence indicates a role for galectins in immune and vascular programs, extracellular matrix remodeling and fibrosis, suggesting their possible involvement in SSc. Here, we determined serum levels of galectin (Gal)-1 and Gal-3 in 83 SSc patients (dcSSc n = 17; lcSSc n = 64; ssSSc n = 2), and evaluated their association with clinical manifestations of the disease. Patients with dcSSc showed lower Gal-3 levels, compared to lcSSc (p = 0.003), whereas no considerable difference in Gal-1 levels was detected between groups. Remarkably, higher concentrations of Gal-1 were associated with the presence of telangiectasias (p = 0.015), and higher concentrations Gal-3 were associated with telangiectasias (p = 0.021), diarrhea (p = 0.039) and constipation (p = 0.038). Moreover, lower Gal-3 levels were associated with the presence of tendinous retractions (p = 0.005). Patients receiving calcium blockers (p = 0.048), methotrexate (p = 0.046) or any immunosuppressive treatment (p = 0.044) presented lower concentrations of Gal-3 compared to those not receiving such treatments. The presence of telangiectasia and the type of SSc maintained their statistical association with Gal-3 (β 0.25; p = 0.022 and β 0.26; p = 0.017, respectively) in multiple linear regression models. In conclusion, serum levels of Gal-3 are associated with clinical manifestations of SSc. Among them, the presence of telangiectasias could be explained by the central role of this lectin in the vascularization programs.Fil: Sundblad, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Gomez, Ramiro A.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Stupirski, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Hockl, Pablo Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Pino, Maria S.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Laborde, Hugo. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentin

Sequential chemotherapy in nonsmall-cell lung cancer: cisplatin and gemcitabine followed by docetaxel

Background: Improving results in nonsmall-cell lung cancer (NSCLC) will require the development of new drugs and strategies to combine available agents. On the basis of data indicating the activity of docetaxel as second-line therapy, a Phase II study was conducted to evaluate the efficacy and toxicity of the sequential combination of chemotherapy consisting of cisplatin (P) and gemcitabine (G) followed by docetaxel (DOC) in patients with advanced NSCLC. Methods: Patients with 1997 TNM stage IIIB (pleural effusion)/stage IV NSCLC, performance status (PS) of 0-1, and normal organ function were eligible. Therapy consisted of P at 75 mg/m(2) on Day 1 and G 1200 mg/m(2) on Days 1 and 8 every 3 weeks for 3 cycles followed, in nonprogressive patients, by DOC 30 mg/m(2) every week for 6 consecutive weeks every 8 weeks for 2 cycles. Results: Fifty-two eligible patients were enrolled (M/F, 39/13; stage IIIB/IV, 8/44; PS 0, 73%, PS 1, 27%; median age, 58 years; range, 36-73). The overall response rate was 36.5% (95% confidence interval [CI]: 23-49). The median overall survival was 11 months (95% CI: 9-13); the median progression-free survival was 6 months (95% CI: 5-7); and the 1- and 2-year survivals were 48% and 25%, respectively. One- and 2-year progression-free survivals were 12% and 8%, respectively. Both phases of the treatment protocol were well tolerated. Conclusions: P/G followed by weekly DOC is well tolerated and active as first-line therapy for NSCLC patients and provides a feasible chemotherapeutic option in this clinical setting

Land use legacies drive higher growth, lower wood density and enhanced climatic sensitivity in recently established forests

Europe is undergoing significant forest expansion due to the abandonment of rural areas driven by economic and demographic changes. Recently established forests provide key ecosystem services such as habitat provision and increased carbon stocks. However, we lack understanding of whether past land use might alter their resilience to climate change compared with long-established forests. Forests established in former agricultural areas may benefit from land use legacies resulting in higher fertility, yet such a benefit might turn into a disadvantage if it involves changes in functional attributes that lower their ability to cope with negative climatic events (e.g. droughts). Here we examined whether recently (post 1956) and long-established (pre 1956) beech forests in Catalonia (NE Spain) differ in their growth patterns, wood density, sensitivity to climate and response to extreme climatic events. Our results indicate higher growth (32%) and lower wood density (3%) in trees from recently established forests, even when controlling for tree age and competition. In addition, recently established forests showed a higher sensitivity to Standardised Precipitation-Evapotranspiration Index (SPEI)SPEI, precipitation and temperature and to extreme negative and positive climatic events. In particularly wet years, recently established forests show twice the number of positive pointer years than long-established forests. Compensatory growth during positive years in recently established forests, may be driving the similar or even higher recovery and resilience detected after drought episodes. Nevertheless, the higher climatic sensitivity of the recently established forests, together with their greater growth and lower wood density indicates that they may be particularly vulnerable to future droughts. Such enhanced vulnerability might question their ability to contribute to carbon sequestration in the long term and emphasises the need to account for land use legacies to better predict future forest function as climate changes

Smart biofilms produced from fish filleting wastes

The myofibrillar proteins utilization from fish processing wastes to produce films has an importance to decrease the environmental impact due to usual inappropriate disposal of these wastes. The aim of this work was to make and characterize biodegradable films developed from myofibrillar proteins of gó whitefish (Macrodon ancylodon) wastes, with anthocyanin addition as a pH change indicator. The material proteic extracted was chacterized presenting high concentration of total proteins (95.01%), important for the development of filmogenic solution. According to the results, there was a significant difference (p < 0.05) between the control and the added with anthocyanin biofilms. The film with lower concentration of anthocyanin presented better barrier property, with water vapor permeability of 3.8 × 1011 g.m1.s1.Pa1. There is a great potential for fish processing wastes utilization to obtain biodegradable films even as the anthocyanin pigment powder is promising to act as pH indicator.This work was financially supported by Project POCI-01–0145-FEDER-007457, funded by FEDER funds through “COMPETE 2020 – Programa Operacional Competitividade e Internacionalização” (POCI) and by national funds through “FCT—Fundação para a Ciência e a Tecnologia”. J.R. Carneiro would also like to thank “FCT” for the research grant SFRH/ BPD/88730/ 2012 (grant supported by POPH/POCH/FSE funding).info:eu-repo/semantics/publishedVersio

Detection of RNA from a Novel West Nile-like Virus and High Prevalence of an Insect-specific Flavivirus in Mosquitoes in the Yucatan Peninsula of Mexico

As part of our ongoing surveillance efforts for West Nile virus (WNV) in the Yucatan Peninsula of Mexico, 96,687 mosquitoes collected from January through December 2007 were assayed by virus isolation in mammalian cells. Three mosquito pools caused cytopathic effect. Two isolates were orthobunyaviruses (Cache Valley virus and Kairi virus) and the identity of the third infectious agent was not determined. A subset of mosquitoes was also tested by reverse transcription-polymerase chain reaction (RT-PCR) using WNV-, flavivirus-, alphavirus-, and orthobunyavirus-specific primers. A total of 7,009 Culex quinquefasciatus in 210 pools were analyzed. Flavivirus RNA was detected in 146 (70%) pools, and all PCR products were sequenced. The nucleotide sequence of one PCR product was most closely related (71-73% identity) with homologous regions of several other flaviviruses, including WNV, St. Louis encephalitis virus, and Ilheus virus. These data suggest that a novel flavivirus (tentatively named T\u27Ho virus) is present in Mexico. The other 145 PCR products correspond to Culex flavivirus, an insect-specific flavivirus first isolated in Japan in 2003. Culex flavivirus was isolated in mosquito cells from approximately one in four homogenates tested. The genomic sequence of one isolate was determined. Surprisingly, heterogeneous sequences were identified at the distal end of the 5\u27 untranslated region

Smart biofilms produced from fish filleting wastes

The myofibrillar proteins utilization from fish processing wastes to produce films has an importance to decrease the environmental impact due to usual inappropriate disposal of these wastes. The aim of this work was to make and characterize biodegradable films developed from myofibrillar proteins of gó whitefish (Macrodon ancylodon) wastes, with anthocyanin addition as a pH change indicator. The material proteic extracted was chacterized presenting high concentration of total proteins (95.01%), important for the development of filmogenic solution. According to the results, there was a significant difference (p < 0.05) between the control and the added with anthocyanin biofilms. The film with lower concentration of anthocyanin presented better barrier property, with water vapor permeability of 3.8 × 1011 g.m1.s1.Pa1. There is a great potential for fish processing wastes utilization to obtain biodegradable films even as the anthocyanin pigment powder is promising to act as pH indicator.This work was financially supported by Project POCI-01–0145-FEDER-007457, funded by FEDER funds through “COMPETE 2020 – Programa Operacional Competitividade e Internacionalização” (POCI) and by national funds through “FCT—Fundação para a Ciência e a Tecnologia”. J.R. Carneiro would also like to thank “FCT” for the research grant SFRH/ BPD/88730/ 2012 (grant supported by POPH/POCH/FSE funding).info:eu-repo/semantics/publishedVersio

Universal primers for the amplification and sequence analysis pf actin-1 from diverse mosquito species

We report the development of universal primers for the reverse-transcription polymerase chain reaction (RT-PCR) amplification and nucleotide sequence analysis of actin cDNAs from taxonomically diverse mosquito species. Primers specific to conserved regions of the invertebrate actin-1 gene were designed after actin cDNA sequences of Anopheles gambiae, Bombyx mori, Drosophila melanogaster, and Caenorhabditis elegans. The efficacy of these primers was determined by RT-PCR with the use of total RNA from mosquitoes belonging to 30 species and 8 genera (Aedes, Anopheles, Culex, Deinocerites, Mansonia, Psorophora, Toxorhynchites, and Wyeomyia). The RT-PCR products were sequenced, and sequence data were used to design additional primers. One primer pair, denoted as Act-2F (5′-ATGGTCGGYATGGGNCAGAAGGACTC-3′) and Act-8R (5′-GATTCCATACCCAGGAAG-GADGG-3′), successfully amplified an RT-PCR product of the expected size (683-nt) in all mosquito spp. tested. We propose that this primer pair can be used as an internal control to test the quality of RNA from mosquitoes collected in vector surveillance studies. These primers can also be used in molecular experiments in which the detection, amplification or silencing of a ubiquitously expressed mosquito housekeeping gene is necessary. Sequence and phylogenetic data are also presented in this report

Connecting nutritional deprivation and pubertal inhibition via GRK2-mediated repression of kisspeptin actions in GnRH neurons

Background: Perturbations in the timing of puberty, with potential adverse consequences in later health, are increasingly common. The underlying neurohormonal mechanisms are unfolded, but nutritional alterations are key contributors. Efforts to unveil the basis of normal puberty and its metabolic control have focused on mechanisms controlling expression of Kiss1, the gene encoding the puberty-activating neuropeptide, kisspeptin. However, other regulatory phenomena remain ill-defined. Here, we address the putative role of the G protein-coupled-receptor kinase-2, GRK2, in GnRH neurons, as modulator of pubertal timing via repression of the actions of kisspeptin, in normal maturation and conditions of nutritional deficiency. Methods: Hypothalamic RNA and protein expression analyses were conducted in maturing female rats. Pharmacological studies involved central administration of GRK2 inhibitor, βARK1-I, and assessment of gonadotropin responses to kisspeptin or phenotypic and hormonal markers of puberty, under normal nutrition or early subnutrition in female rats. In addition, a mouse line with selective ablation of GRK2 in GnRH neurons, aka G-GRKO, was generated, in which hormonal responses to kisspeptin and puberty onset were monitored, in normal conditions and after nutritional deprivation. Results: Hypothalamic GRK2 expression increased along postnatal maturation in female rats, especially in the preoptic area, where most GnRH neurons reside, but decreased during the juvenile-to-pubertal transition. Blockade of GRK2 activity enhanced Ca responses to kisspeptin in vitro, while central inhibition of GRK2 in vivo augmented gonadotropin responses to kisspeptin and advanced puberty onset. Postnatal undernutrition increased hypothalamic GRK2 expression and delayed puberty onset, the latter being partially reversed by central GRK2 inhibition. Conditional ablation of GRK2 in GnRH neurons enhanced gonadotropin responses to kisspeptin, accelerated puberty onset, and increased LH pulse frequency, while partially prevented the negative impact of subnutrition on pubertal timing and LH pulsatility in mice. Conclusions: Our data disclose a novel pathway whereby GRK2 negatively regulates kisspeptin actions in GnRH neurons, as major regulatory mechanism for tuning pubertal timing in nutritionally-compromised conditions.This work was supported by grants BFU2017-83934-P, PID2020-118660GB-I00 (to MTS) and SAF2017-84125-R (to FM), Agencia Estatal de Investigación, Spain; co-funded with EU funds from FEDER Program; Project PIE-00005 (to MTS) and CB16/11/00278 (to FM) - Instituto de Salud Carlos III, Ministerio de Sanidad, Spain; Project P12-FQM-01943 (to MTS) and Project PI-0370-2016 (to MSA- Junta de Andalucía, Spain); and Project REP-655232 (ReprObesity; to MTS – European Union). CIBER Fisiopatología de la Obesidad y Nutrición and CIBER Enfermedades Cardiovasculares are initiatives of Instituto de Salud Carlos III, Spai

Frequency of Fabry disease in male and female haemodialysis patients in Spain

<p>Abstract</p> <p>Background</p> <p>Fabry disease (FD), an X-linked lysosomal storage disorder, is caused by a reduced activity of the lysosomal enzyme α-galactosidase A. The disorder ultimately leads to organ damage (including renal failure) in males and females. However, heterozygous females usually present a milder phenotype with a later onset and a slower progression.</p> <p>Methods</p> <p>A combined enzymatic and genetic strategy was used, measuring the activity of α-galactosidase A and genotyping the α-galactosidase A gene (<it>GLA</it>) in dried blood samples (DBS) of 911 patients undergoing haemodialysis in centers across Spain.</p> <p>Results</p> <p><it>GLA </it>alterations were found in seven unrelated patients (4 males and 3 females). Two novel mutations (p.Gly346AlafsX347 and p.Val199GlyfsX203) were identified as well as a previously described mutation, R118C. The R118C mutation was present in 60% of unrelated patients with <it>GLA </it>causal mutations. The D313Y alteration, considered by some authors as a pseudo-deficiency allele, was also found in two out of seven patients.</p> <p>Conclusions</p> <p>Excluding the controversial D313Y alteration, FD presents a frequency of one in 182 individuals (0.55%) within this population of males and females undergoing haemodialysis. Moreover, our findings suggest that a number of patients with unexplained and atypical symptoms of renal disease may have FD. Screening programmes for FD in populations of individuals presenting severe kidney dysfunction, cardiac alterations or cerebrovascular disease may lead to the diagnosis of FD in those patients, the study of their families and eventually the implementation of a specific therapy.</p