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.

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.

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.

Complementary DNA cloning of a protein highly homologous to mammalian sarcoplasmic reticulum Ca-ATPase from the crustacean Artemia.

Complementary DNA clones coding for an Artemia ATPase have been isolated using an oligonucleotide probe for a region highly conserved between P-type ATPases. The nucleotide sequence of three overlapping clones, 3309 base-pairs, has been established. This sequence includes 78 nucleotides of 5' untranslated sequence, an open reading frame of 3009 nucleotides and 222 nucleotides of 3' untranslated sequences. The amino acid sequence predicted for the coding region is 71% similar to that of slow and fast twitch rabbit muscle sarcoplasmic reticulum Ca-ATPases. The homology is specially high in some regions of the protein that include the previously described regions that are similar between all known P-type ATPases, as well as transmembrane domains and intra- and extracellular domains adjacent to the membrane that are not conserved in P-type ATPases but have been proposed to be involved in calcium binding and transport in rabbit sarcoplasmic reticulum Ca-ATPases. Probes of this likely sarcoplasmic reticulum Ca-ATPase hybridize to two mRNAs of 5200 and 4500 bases. Although both mRNAs are already present in cryptobiotic embryos, the levels of the 5200 base mRNA decrease after development is reassumed, being undetectable after hatching of the nauplii. The levels of the 4500 base mRNA increase during development; maximal levels are reached by ten hours and are maintained at later stages of development.

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.

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).

Isolation of cDNA clones coding for mitochondrial 16S ribosomal RNA from the crustacean Artemia.

cDNA clones coding for Artemia mitochondrial 16S ribosomal RNA (rRNA) have been isolated. The clones cover from nucleotide 650 of the RNA molecule to its 3' end. The comparison of Artemia sequence with both vertebrate and invertebrate mitochondrial 16S rRNA sequences has shown the existence of regions of high similarity between them. A model for the secondary structure of the 3' half of Artemia mitochondrial 16S rRNA is proposed. The size of the rRNA molecule has been estimated at 1.35 kb. Despite the similarity of the Artemia gene to insect rRNA in size, sequence and secondary structure, the G + C content of the Artemia gene (42%) is closer to that of mammals than to the insect genes. The number of mitochondria in Artemia has been estimated at 1500 per diploid genome in the cyst and 4000 in the nauplius. In contrast, the amount of mt 16S rRNA is constant at all stages of Artemia development.

Cloning of a cDNA encoding an Artemia franciscana Na/K ATPase alpha-subunit.

Clones of cDNA that code for an isoform of the Artemia franciscana Na/K ATPase alpha subunit (NaKA alpha) have been isolated. The sequence of the longest of these clones (pArATNa136) is 3595 nucleotides; it codes for a 1004-amino acid protein whose sequence is identical to that of two previously sequenced Artemia NaKA alpha peptides. The encoded protein is over 73% identical to Drosophila melanogaster and vertebrate NaKA alpha s, and 73.8% identical to another Artemia NaKA alpha isoform previously described (named alpha 2850 in this article). The two Artemia cDNA clones code for mRNAs of different size; the clone pArATNa136 codes for a 4.5-kb mRNA while the alpha 2850 clone codes for a 3.6-kb mRNA. The degree of homology and the different size of the mRNAs encoded by both cDNAs suggest that they code for two different isoforms of the protein.

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.

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.

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.

Developmental changes in poly(A) polymerase activity in Artemia.

The levels of poly(A) polymerase activity have been determined during Artemia early development. Poly(A) polymerase activity increases steadily during postgastrular embryonic development reaching a maximum shortly after hatching. The rise of poly(A) polymerase is concomitant with an increase in poly(A) content and with a change in the subcellular distribution of the enzyme activity, the major increase corresponding to the nuclear fraction. Only one isoenzyme of poly(A) polymerase has been identified in Artemia embryos and nauplii despite changes in enzyme levels and subcellular changes during early development. Poly(A) polymerase is not associated with the cytoplasmic poly(A)-containing ribonucleoprotein particles stored in Artemia dormant embryos.

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.

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.

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.

Similar alternative splicing events generate two sarcoplasmic or endoplasmic reticulum Ca-ATPase isoforms in the crustacean Artemia franciscana and in vertebrates.

A second isoform of the Artemia franciscana sarcoplasmic or endoplasmic reticulum Ca-ATPase has been identified through the isolation of cDNA clones. This isoform differs from the previously identified one only at the C-terminal end of the protein. The last 6 amino acids of the former isoform change to 30 hydrophobic amino acids in the newly identified isoform that have the potentiality of being an additional transmembrane domain. The two A. franciscana isoforms are highly homologous to the two isoforms coded by the mammalian and bird SERCA2 gene, except that the C-terminal extensions share their hydrophobic character but have no significant amino acid homology. The isolation of genomic clones coding for this region of the gene shows that both isoforms arise from the same gene by alternative splicing. The donor splicing site of the penultimate exon can either be recognized and fused to the last exon, giving rise to the mRNA coding for the shortest protein, or remain unrecognized, in which case a polyadenylation site is recognized before the last exon of the gene and the mRNA coding for the largest protein is originated. The alternative splicing used to originate the two isoforms is similar in Artemia and vertebrates. The conservation of the alternative splicing between species so distant in evolution suggests an important physiological role for the existence of the two isoforms of the protein.