Prognostic significance of metabolic tumor volume and total lesion glycolysis in patients with advanced cervical carcinoma. / Significación pronóstica del volumen metabólico tumoral y de la glucólisis tumoral total en pacientes con cáncer cervical avanzado.

AIM: 18-Fluoro-2-deoxy-d-glucose positron emission tomography (18F-FDG PET/CT) is considered to be the most accurate image method of detection of node or distant metastases in cervical cancer. Metabolic tumor volume (MTV) and total lesion glycolysis (TLG) of 18F-FDG PET/CT are volumetric measurements of tumor cells with increased 18F-FDG uptake. The prognostic value of MTV and TLG in patients with advanced cervical cancer (ACC) were evaluated. METHODS: 38 patients with ACC from one tertiary university hospital underwent 18F-FDG PET/CT between June 2009 and December 2015. Clinicopathologic factors and various PET parameters were analyzed to evaluate their relationship with recurrence-free survival (RFS) and overall survival (OS). These parameters were: maximum standardized uptake value (SUVmax), mean standardized uptake value (SUV mean), metabolic tumor volume (MTV), and total lesion glycolysis (TLG) of the primary tumor, of the pelvic nodes, of the paraaortic nodes and the metabolic volume of the metastases if any. RESULTS: A total of 38 patients with ACC fulfilled the inclusion criteria. All of them underwent a 18F-FDG PET/CT before definitive chemoradiotherapy. In the univariate analyses higher tumor size, pelvic lymph node metastasis and both MTV and TLG showed a significant association with OS and with RFS (MTV HR=1.55, p=0.011 and TLG HR=1.43, p=0.017 for RFS and MTV HR=1.82, p=0.006 and TLG HR=1.67, p=0.007 for OS). CONCLUSION: Pretreatment TLG sum and MTV sum seem to be independent prognostic factors for OS and RFS in patients with advanced cervical cancer treated with definitive chemoradiotherapy and they are better than the classic measurement of SUVmax.

Mannose-Binding Lectin-Deficient Donors Increase the Risk of Bacterial Infection and Bacterial Infection-Related Mortality After Liver Transplantation.

Mannose-binding lectin (MBL) is synthesized by the liver and binds to microbes. MBL2 gene polymorphisms produce intermediate/low/null or normal MBL serum levels (MBL-deficient or MBL-sufficient phenotypes, respectively). We aimed to evaluate the incidence and severity of infection, rejection, and survival within 1 year after liver transplantation (LT) according to donor and recipient MBL2 gene polymorphisms. A repeated-event analysis for infection episodes (negative binomial regression, Andersen-Gill model) was performed in 240 LTs. Four hundred twenty-eight infectious episodes (310 bacterial, 15 fungal, 65 cytomegalovirus [CMV]-related, and 38 viral non-CMV-related episodes) and 48 rejection episodes were recorded. The main bacterial infections were urinary (n = 82, 26%) and pneumonia (n = 69, 22%). LT recipients of MBL-deficient livers had a higher risk of bacterial infection (incidence rate ratio [IRR] 1.48 [95% confidence interval 1.04-2.09], p = 0.028), pneumonia (IRR 2.4 [95% confidence interval 1.33-4.33], p = 0.013), and septic shock (IRR 5.62 [95% confidence interval 1.92-16.4], p = 0.002) compared with recipients of MBL-deficient livers. The 1-year bacterial infection-related mortality was higher in recipients of MBL-deficient versus MBL-sufficient livers (65.8% vs. 56.1%, respectively; p = 0.0097). The incidence of rejection, viral, or fungal infection was similar in both groups. Recipient MBL2 genotype did not significantly increase the risk of bacterial infection. LT recipients of MBL-deficient livers have a higher risk of bacterial infection, pneumonia, septic shock, and 1-year bacterial infection-related mortality after LT.

High resolution melting analysis for the identification of novel mutations in DKC1 and TERT genes in patients with dyskeratosis congenita.

Dyskeratosis congenita (DC) is a rare inherited bone-marrow failure syndrome with high clinical heterogeneity. Cells derived from DC patients present short telomeres at early ages, as a result of mutations in genes encoding components of the telomerase complex (DKC1, TERC, TERT, NHP2 and NOP10), or the shelterin complex (TINF2). However, mutations have been identified only in around 50% of the cases, indicating that other genes could be involved in the development of this disease. Indeed, mutations in TCBA1 or chromosome segment C16orf57 have been described recently. We have used HRM technology to perform genetic analysis in the above mentioned genes, in Spanish patients showing both, some clinical features of DC and short telomeres. The mutations have been identified by PCR amplification of DC genes followed by high resolution melting (HRM) and direct DNA sequencing analysis. We have identified seven new families with DC, three with X-linked DC and four with autosomal dominant DC, in which we have found two novel mutations in DKC1 (p.His68Arg and p.Lys390del) and four novel mutations in TERT gene (p.Pro530Leu, p.Arg698Trp, p.Arg971His and p.Arg698Gln). The results show that the use of HRM analysis enables a rapid and inexpensive identification of mutations in dyskeratosis congenita associated genes.

The MADS-box gene srfA is expressed in a complex pattern under the control of alternative promoters and is essential for different aspects of Dictyostelium development.

srfA displays a complex temporal and cell type-specific pattern of expression in Dictyostelium and is expressed by most of its cell types at some stage of their development. This complexity is achieved by the use of alternative promoters. The promoter activity of the proximal region was found to be restricted to a subset of prestalk cells. Little or no associated expression was observed in the lower cup and basal disc during culmination. The middle promoter region was preferentially active in prestalk cells under usual conditions of filter development. Interestingly, during slug migration, the activity of this promoter in posterior prespore cells was strongly induced. The distal region displayed a dual pattern of expression. Thus, before culmination, this region drove lacZ expression in a few cells scattered along the entire structure. However, intense lacZ staining was found in the spores by the end of culmination. We have previously reported that srfA expression is essential for spore differentiation (R. Escalante and L. Sastre, Development 125, 3801-3808). Our novel finding of the expression of the gene in prestalk cells before culmination suggested that it might play additional roles in Dictyostelium development. The study of knockout strains revealed that srfA is also required for proper slug migration. Spore differentiation and slug migration defects were rescued by reexpression of srfA in the null mutant background, under the appropriate promoter control. The expression of srfA under the activity of the distal promoter region was able to rescue spore differentiation but not slug migration. Conversely, reexpression under the control of the middle promoter rescued slug morphogenesis and migration. Our results demonstrate that the correct spatial and temporal pattern of expression of srfA is essential for the different functions that this transcription factor plays in development.

cAMP and DIF-1 repress the expression of the Dictyostelium MADS-box gene srfA at early stages of development.

The MADS-box-containing gene srfA from Dictyostelium discoideum codes for a putative transcription factor that plays multiple roles in the development of this social amoeba. We have investigated the regulation of srfA gene expression after disaggregation of the cells from developing structures. The steady-state level of srfA mRNA was strongly and transiently induced shortly after disaggregation. srfA is maximally expressed 20 min after cell disaggregation and decreases thereafter. Induction was not dependent on protein synthesis, PKA, the kinase SplA and SrfA itself. This phenomena does not occur when cells are disaggregated in a small volume of buffer, suggesting the presence of extracellular molecules that repress srfA gene expression. To test this hypothesis, several well-known extracellular signaling molecules were studied. We found that srfA mRNA induction can be efficiently repressed by addition of exogenous cAMP and DIF-1 to the buffer in which the cells were disaggregated. Addition of other extracellular compounds such as ammonia, adenosine, SDF-1, and SDF-2 had no effect. srfA promoter P2, specifically induced during slug migration, was responsible for this regulation by extracellular compounds.

Characterization of a functional serum response element in the Actin403 gene promoter from the crustacean Artemia franciscana.

The serum response factor (SRF) activates expression of several genes in response to growth factors present in serum. SRF also regulates the expression of tissue-specific genes, including those in vertebrate muscles. An SRF-binding site (CArG box) present in the Artemia franciscana Actin403 promoter was shown to be necessary for transcriptional activity in cultured cells from Drosophila melanogaster and mammals. This DNA region bound mammalian and Drosophila SRFs in vitro and mediated transcriptional activation of the Actin403 promoter in response to serum, phorbol esters and lysophosphatidic acid in transfected cultured mammalian cells. Mutations in the CArG box greatly reduced promoter activity and stimulation by extracellular compounds.

A serum response factor homologue is expressed in ectodermal tissues during development of the crustacean Artemia franciscana.

Complementary DNA clones have been isolated from the crustacean Artemia franciscana coding for a serum response factor (SRF)-homologue that is more than 96% identical to human and Drosophila melanogaster SRFs in their MADS boxes. The SRF homologue is expressed in ectodermal tissues, as determined by in situ hybridization experiments. A SRF-binding site has been identified in the promoter region of the Actin403 gene that is also expressed in ectodermal tissues, in accordance with its transcriptional regulation by the SRF homologue. The mRNA coding for A. franciscana SRF is present at similar levels in cryptobiotic encysted embryos and in developing nauplii. However, there is a significant increase in CArG-binding activity at the later developmental stage, indicating a postranscriptional regulation of SRF during A. franciscana embryonic development.

High DNA sequence variability at the alpha 1 Na/K-ATPase locus of Artemia franciscana (brine shrimp): polymorphism in a gene for salt-resistance in a salt-resistant organism.

We previously reported that the Na/K-ATPase alpha 1 subunit coding gene showed signs of being a very polymorphic locus in Artemia franciscana. This species is adapted to highly saline waters, and the Na/K-ATPase alpha 1 isoform presumably plays a key role in this adaptation. Therefore, we were interested in further study of the alpha 1 Na/K-ATPase polymorphisms to examine whether they might be due to an adaptation to salt resistance driven by natural selection. Using coding sequences from 10 genomic clones and 3 cDNAs, we observed that most substitutions are in synonymous positions (88.8%). The 12 nonsynonymous substitutions code for conservative amino acid replacements with an apparent scattered distribution across functional domains of the protein. Interspecific comparison between these sequences and two genomic clones from Artemia parthenogenetica containing 1,122 bp of the alpha 1 Na/K-ATPase locus coding sequence showed independence of the synonymous/nonsynonymous ratio in the comparison within A. franciscana and between A. franciscana and A. parthenogenetica, which fits the neutral model of evolution. Since there were no previous studies on DNA polymorphism for other A. franciscana genes, we also studied variability at the Actin 302 locus for comparison. Both loci were amplified by reverse transcription-polymerase chain reaction, and 20 sequences were obtained for each. This study shows that the amplified region of the alpha 1 Na/K-ATPase gene is 3.5 times as polymorphic as the Actin 302 gene and 2.9 times as heterozygotic. Interestingly, under a model of neutral evolution, the data observed would be expected with a probability of approximately 0.05, suggesting an excess of intraspecific variation of alpha 1 Na/K-ATPase with respect to Actin 302. Restriction fragment length polymorphism studies show similar patterns of polymorphism along the approximately 41-kb span of the alpha 1 Na/K-ATPase locus. Most of the nucleotide differences are linked in a few haplotypes, although recombination events are also inferred from the data. We propose a possible explanation for the high polymorphic levels at the alpha 1 Na/K-ATPase locus which invokes positive selection acting tightly to the locus in transiently isolated or semi-isolated subpopulations.

Isolation and characterization of the gene coding for Artemia franciscana TATA-binding protein: expression in cryptobiotic and developing embryos.

Genomic and cDNA clones coding for the Artemia franciscana homolog of the TATA box-binding protein (TBP) were isolated. The C-terminal region of the predicted protein displays up to 92% sequence identity with the conserved C-terminal regions of TBPs from other species. The gene is divided in seven exons that expand over a region of 33 kb. The position of the four introns located in the conserved C-terminal region has been compared with those of other species. Two of these introns have been generally conserved during evolution, another is an arthropod specific intron, present in Drosophila melanogaster and A. franciscana, and the other is only conserved between vertebrates and A. franciscana. Primer extension experiments detected several transcription initiation sites. Northern blot analyses showed the presence of four mRNAs of estimated sizes of 6.8, 2.6, 1.6 and 1.1 kb. Except for the low expression of the 6.8 and 2. 6 kb RNAs in encysted embryos, steady-state levels showed little variation during the activation of the encysted embryo and the first steps of embryonic and larval development. The amount of TBP protein expressed in encysted embryos and developing larvae has been analyzed by Western blot. Cryptobiotic embryos contain significant amounts of TBP although the level of expression increased almost twice during the first 20 h of development. The presence of TBP protein in cryptobiotic embryos suggests that TBP does not play, by itself, a critical role in the arrest of transcription characteristic of these resistance forms.

A Serum Response Factor homolog is required for spore differentiation in Dictyostelium.

A homolog of the Serum Response Factor (SRF) has been isolated from Dictyostelium discoideum and its function studied by analyzing the consequences of its gene disruption. The MADS-box region of Dictyostelium SRF (DdSRF) is highly conserved with those of the human, Drosophila and yeast homologs. srfA is a developmentally regulated gene expressed in prespore and spore cells. This gene plays an essential role in sporulation as its disruption leads to abnormal spore morphology and loss of viability. The mutant spores were round and cellulose deposition seemed to be partially affected. Initial prestalk and prespore cell differentiation did not seem to be compromised in the mutant since the expression of several cell-type-specific markers were found to be unaffected. However, the mRNA level of the spore marker spiA was greatly reduced. Activation of the cAMP-dependent protein kinase (PKA) by 8-Br-cAMP was not able to fully bypass the morphological defects of srfA- mutant spores, although this treatment induced spiA mRNA expression. Our results suggest that DdSRF is required for full maturation of spores and participates in the regulation of the expression of the spore-coat marker spiA and probably other maturation genes necessary for proper spore cell differentiation.

Polymorphism and structure of the gene coding for the alpha 1 subunit of the Artemia franciscana Na/K-ATPase.

Genomic clones coding for one of the two identified Artemia franciscana Na/K-ATPase alpha subunits, the alpha 1 subunit, have been isolated. Several overlapping clones were obtained, although their restriction maps showed a large heterogeneity. Sequencing of their exons showed that they differ in up to 3.46% of their nucleotides in translated regions and 8.18% in untranslated regions. Southern blot analysis of DNA purified from different lots of A. franciscana cysts and from isolated individuals suggests that the variation is due to the existence of multiple Na/K-ATPase alpha 1 subunit alleles in A. franciscana. The Na/K-ATPase alpha 1 subunit gene is divided into 15 exons. Ten of the 14 introns are located in identical positions in this gene as in the human Na/K-ATPase alpha 3 subunit gene. Analysis of the 5' flanking region of the gene has allowed identification of the transcription-initiation sites. The adjacent upstream region has been shown to have functional promoter activity in cultured mammalian cells, suggesting the evolutionary conservation of some of the promoter regulatory sequences.

Actin gene structure in two Artemia species, A. franciscana and A. parthenogenetica.

Genomic clones coding for actin have been isolated from two species of the crustacean Artemia, A. parthenogenetica and A. franciscana. The Act211 isoform gene was isolated from A. parthenogenetica, and the two other isoform genes, Act302 and Act403, were isolated from A. franciscana. The comparison of the nucleotide sequence of genomic and cDNA clones showed an interspecific divergence of 4% in translated and 6.1% in untranslated regions. However, the establishment of the partial structure of the Act211 gene in A. franciscana and of the Act302 gene in A. parthenogenetica suggests their similarity in the two species. The Act211 gene is divided into four exons, the Act302 gene into six exons, and the Act403 gene into seven exons. The three genes have introns in the 5' untranslated region and between codons 41 and 42. The Act211 and 403 genes have one common intron in codon 168. The Act302 and 403 genes have common introns between codons 121-122, 246-247, and within codon 301. While introns in the 5' untranslated region and between codons 41-42 and 121-122 are present in many organisms, the introns in positions 168 and 246-247 had only been found previously in actin genes from the nematode Onchocerca volvulus and the green alga Volvox carterii, respectively. The intron in position 301 had not been reported before. The transcription initiation sites of these three genes as well as the nucleotide sequences of the promoter regions have been also determined.

Tissue-specific expression of two Artemia franciscana sarco/endoplasmic reticulum Ca-ATPase isoforms.

The sarco/endoplasmic reticulum Ca-ATPase (SERCA) gene from Artemia franciscana is transcribed into two mRNAs that code for two different enzyme isoforms. We investigated the tissue-specific expression of each mRNA by in situ hybridization of larval tissue sections. One of the isoforms is expressed in the muscle fibers of the appendages. The other isoform is generally expressed throughout all tissues of the larvae. The tissue distribution of these two isoforms is very similar to the one described for the two homologous isoforms generated from the vertebrate SERCA 2 gene, and shows the evolutionarily conserved nature of their tissue-specific expression.

Tissue-specific alternative promoters regulate the expression of the two sarco/endoplasmic reticulum Ca-ATPase isoforms from Artemia franciscana.

The sarco/endoplasmic reticulum Ca-ATPase gene from Artemia franciscana is transcribed into two mRNAs of 4.5 and 5.2 kb that code for protein isoforms differing at their carboxyl terminus. Northern blot assays and anchored polymerase chain reaction (PCR) experiments have shown that these two mRNAs also differ at the initial part of their 5' untranslated region. The 5.2-kb mRNA-specific 5' untranslated region is present as an independent exon whose transcription is regulated by a promoter different from the one previously described that regulates the expression of the 4.5-kb mRNA. The nucleotide sequence of the 5.2-kb mRNA promoter and the transcription initiation site have been determined. These results suggest that the expression of the two protein isoforms is regulated in A. franciscana at the transcription initiation step, in contrast with the vertebrates sarco/endoplasmic reticulum Ca-ATPase genes 1 and 2 which have unique promoters for transcription of the two isoforms encoded by each gene.

In situ hybridization analyses of Na, K-ATPase alpha-subunit expression during early larval development of Artemia franciscana.

The spatial pattern of expression of the mRNA encoded by the Na,K-ATPase alpha-subunit cDNA clone pArATNa136 was determined by in situ hybridization of first, second, and third instar Artemia franciscana larvae. This mRNA was expressed at high levels in the salt gland, the antennal gland, and the end of the midgut, which are the three main osmoregulatory organs in Artemia at these stages of development. The pattern of expression was similar at the three stages of development analyzed, although the level of expression increased during development, especially in the salt and antennal glands. The expression of the mRNA coding for another Na, K-ATPase alpha-subunit isoform, the proposed alpha 2-isoform, was also determined and was shown to be limited to the salt gland. These results suggest that the clone pArATNa136 codes for the biochemically defined alpha 1-isoform of the Na,K-ATPase alpha-subunit and reinforce the importance of this isoform in osmoregulation at the three larval stages studied. The alpha 2-isoform may also be involved in osmoregulation during the first stages of larval development.

Identification of an Artemia franciscana retropseudogene containing part of the last exons of the sarco/endoplasmic reticulum Ca-ATPase-encoding SERCA gene.

A genomic clone has been isolated which contains sequences highly homologous to part of exon 14 and exons 15, 16 and 17 of the Artemia franciscana sarco-endoplasmic reticulum Ca-ATPase(SERCA)-encoding gene, but none of the introns. The homologous region extends to the 3' end of the mRNA, although the poly(A) tail is not present. The structure of this clone suggests that it represents a 5'-end-truncated retropseudogene (r psi).

Structure of Artemia franciscana sarco/endoplasmic reticulum Ca-ATPase gene.

Genomic clones coding for the Artemia franciscana sarco(endo)plasmic reticulum Ca-ATPase have been isolated. The restriction map of the overlapping clones covers a region of 65 kilobases of DNA. Nucleotide sequence of mRNA coding regions shows that the gene is divided into 18 exons separated by 17 introns. Compared with the structure of the rabbit sarco(endo)plasmic reticulum Ca-ATPase 1 gene, 12 of the introns are in the same position, 8 introns present in the rabbit gene are absent from A. franciscana, 4 introns present in A. franciscana are not found in rabbit, and the position of 1 intron is shifted one base between both genes. Southern blot analysis strongly suggests that this is the only sarco(endo)plasmic reticulum Ca-ATPase gene present in A. franciscana. Primer extension and nuclease S1 protection experiments have shown the existence of two main regions of transcription initiation separated by 30 nucleotides. Transcription is initiated in both regions at two or three consecutive bases. A hexanucleotide that includes the initiation sites is repeated in both transcription initiation regions. The nucleotide sequence of the promoter region shows the existence of several putative regulatory sites, including some that are muscle-specific such as one CArG box, 3 MEF-2, and 8 putative binding sites for muscle transcription factors of the MyoD family.