4-Deoxyphorbol inhibits HIV-1 infection in synergism with antiretroviral drugs and reactivates viral reservoirs through PKC/MEK activation synergizing with vorinostat.

Latent HIV reservoirs are the main obstacle to eradicate HIV infection. One strategy proposes to eliminate these viral reservoirs by pharmacologically reactivating the latently infected T cells. We show here that a 4-deoxyphorbol ester derivative isolated from Euphorbia amygdaloides ssp. semiperfoliata, 4ß-dPE A, reactivates HIV-1 from latency and could potentially contribute to decrease the viral reservoir. 4ß-dPE A shows two effects in the HIV replication cycle, infection inhibition and HIV transactivation, similarly to other phorboids PKC agonists such PMA and prostratin and to other diterpene esters such SJ23B. Our data suggest 4ß-dPE A is non-tumorigenic, unlike the related compound PMA. As the compounds are highly similar, the lack of tumorigenicity by 4ß-dPE A could be due to the lack of a long side lipophilic chain that is present in PMA. 4ß-dPE activates HIV transcription at nanomolar concentrations, lower than the concentration needed by other latency reversing agents (LRAs) such as prostratin and similar to bryostatin. PKCθ/MEK activation is required for the transcriptional activity, and thus, anti-latency activity of 4ß-dPE A. However, CD4, CXCR4 and CCR5 receptors down-regulation effect seems to be independent of PCK/MEK, suggesting the existence of at least two different targets for 4ß-dPE A. Furthermore, NF-κb transcription factor is involved in 4ß-dPE HIV reactivation, as previously shown for other PKCs agonists. We also studied the effects of 4ß-dPE A in combination with other LRAs. When 4ß-dPE A was combined with another PKC agonists such as prostratin an antagonic effect was achieved, while, when combined with an HDAC inhibitor such as vorinostat, a strong synergistic effect was obtained. Interestingly, the latency reversing effect of the combination was synergistically diminishing the EC50 value but also increasing the efficacy showed by the drugs alone. In addition, combinations of 4ß-dPE A with antiretroviral drugs as CCR5 antagonist, NRTIs, NNRTIs and PIs, showed a consistent synergistic effect, suggesting that the combination would not interefer with antiretroviral therapy (ART). Finally, 4ß-dPE A induced latent HIV reactivation in CD4 + T cells of infected patients under ART at similar levels than the tumorigenic phorbol derivative PMA, showing a clear reactivation effect. In summary, we describe here the mechanism of action of a new potent deoxyphorbol derivative as a latency reversing agent candidate to decrease the size of HIV reservoirs.

Disponibilidad de técnicas moleculares para niños con tumores de difícil caracterización. Un problema en la Oncopatología pediátrica. Aplicación y difusión de técnicas de diagnóstico complementarias en el ámbito público / Availability of molecular techniques for children with difficult-to-characterize tumors. A problem in pediatric oncopathology. Application and diffusion of complementary diagnostic techniques in the public setting

La revolución de la biología molecular y el desarrollo de la investigación biomédica básica para el diagnóstico y posterior manejo del cáncer infantil han llevado a la necesidad de organización de grupos interdisciplinarios de profesionales, los cuales se encargan de afrontar los nuevos desafíos diagnósticos y terapéuticos. Los sarcomas indiferenciados pediátricos constituyen un grupo heterogéneo de neoplasias malignas de aspecto primitivo y polifenotípico. La categorización de gran parte de este tipo de tumores es posible gracias a la aplicación de técnicas moleculares complementarias al estudio histopatológico. El objetivo del presente estudio fue recategorizar sarcomas indiferenciados mediante la implementación de una nueva metodología diagnóstica. Se efectuaron técnicas de inmunohistoquimica (IHQ), FISH de interfase y RT-PCR a partir de tejido fijado en formol e incluido en parafina en 144 casos de sarcomas indiferenciados. Se logró la recategorización del 95.1% de los casos, arribando a 24 diagnósticos diferentes. Sólo un 4.9% permanece aún como sarcoma indiferenciado o inclasificable. Los resultados alcanzados por este estudio demuestran la importancia de contar con nuevas herramientas diagnósticas a nivel molecular y recursos humanos especializados que posibiliten su correcta implementación para el diagnóstico de neoplasias de difícil caracterización (AU)

The revolution of molecular biology and the development of basic medical research for the diagnosis and subsequent management of childhood cancer have led to a need to organize interdisciplinary groups of professionals in charge of facing new diagnostic and treatment challenges. Childhood undifferentiated sarcomas are a heterogeneous group of malignant neoplasms that are primitive in appearance and have polyphenotypic features. Categorization of a large part of this type of tumor has become possible with molecular techniques as a complement to histopathological studies. The aim of this study was to categorize undifferentiated sarcomas using new diagnostic tools. Immunohistochemistry (IHC), interfase FISH, and RT-PCR techniques were used on formalin-fixed and paraffin-embedded tissues of 144 cases of undifferentiated sarcomas. Overall, 95.1% of the cases could be recategorized resulting in 24 different diagnoses. In only 4.9% the diagnosis of undifferentiated or unclassifiable sarcoma was maintained. These results emphasize the importance of the availability of new diagnostic tools at the molecular level and specialized human resources enabling adequate implementation for the diagnosis of difficult-to-characterize neoplasms (AU)

Long-term fiscal implications of funding assisted reproduction: a generational accounting model for Spain.

The aim of this study was to assess the lifetime economic benefits of assisted reproduction in Spain by calculating the return on this investment. We developed a generational accounting model that simulates the flow of taxes paid by the individual, minus direct government transfers received over the individual's lifetime. The difference between discounted transfers and taxes minus the cost of either IVF or artificial insemination (AI) equals the net fiscal contribution (NFC) of a child conceived through assisted reproduction. We conducted sensitivity analysis to test the robustness of our results under various macroeconomic scenarios. A child conceived through assisted reproduction would contribute €370,482 in net taxes to the Spanish Treasury and would receive €275,972 in transfers over their lifetime. Taking into account that only 75% of assisted reproduction pregnancies are successful, the NFC was estimated at €66,709 for IVF-conceived children and €67,253 for AI-conceived children. The return on investment for each euro invested was €15.98 for IVF and €18.53 for AI. The long-term NFC of a child conceived through assisted reproduction could range from €466,379 to €-9,529 (IVF) and from €466,923 to €-8,985 (AI). The return on investment would vary between €-2.28 and €111.75 (IVF), and €-2.48 and €128.66 (AI) for each euro invested. The break-even point at which the financial position would begin to favour the Spanish Treasury ranges between 29 and 41 years of age. Investment in assisted reproductive techniques may lead to positive discounted future fiscal revenue, notwithstanding its beneficial psychological effect for infertile couples in Spain.

Reflux patterns and risk factors of primary varicose veins' clinical severity.

OBJECTIVES: Primarily, to determine the association between the clinical severity of primary varicose veins and different reflux patterns in an anatomic and haemodynamic clinical study using duplex ultrasonography (DU). Secondly, to analyse the association of clinical severity with other aspects, such as risk factors for chronic venous insufficiency (CVI) and other concurrent diseases. METHOD: A total of 2036 limbs were evaluated using DU. Clinical status was characterized by the CEAP (clinical, aetiological, anatomical and pathological elements) classification. The degree of clinical severity was grouped into two categories, mild to moderate CVI (C1­C3)and severe CVI, characterized by the presence of skin changes (C4­C6). We analysed the association of the different reflux patterns with CEAP status. RESULTS: Saphenofemoral junction (SFJ) reflux of the great saphenous vein (GSV) was associated with the most severe form of the disease (odds ratio [OR] » 2.96; confidence interval [CI] 95%: 2.2­3.8), whereas competent SFJ of the GSV with reflux from proximal veins (OR » 2; CI 95%: 1.4­2.7) and the pure non-saphenous reflux (OR » 4.1; CI 95%:1.8­9.0) were associated with mild to moderate CVI. Obesity increased the frequency of severe CVI 2.7 times (OR » 2.7; CI 95%: 1.6­4.6); being a woman also increased the frequency of more severe disease 1.3 times (OR » 1.3; CI 95%: 1.0­1.7). CONCLUSION: Anatomical and haemodynamic studies by DU are postulated as a useful diagnostic tool that allow, by identifying the pattern of venous reflux of varicose pathology, characterization of the probable association to CVI clinical severity.

Immune response to racotumomab in a child with relapsed neuroblastoma.

Immunotherapy targeting ganglioside antigens is a powerful tool for the treatment of high risk neuroblastoma. However, only treatment with anti-GD2 antibodies has been used in clinical practice and other options may be pursued. We report the use of racotumomab, an anti-idiotype vaccine against N-glycolyl neuraminic acid (NeuGc)- containing gangliosides, eliciting an immune response in a child with relapsed neuroblastoma expressing the NeuGcGM3 ganglioside.

Engineering platforms for directed evolution of Laccase from Pycnoporus cinnabarinus.

While the Pycnoporus cinnabarinus laccase (PcL) is one of the most promising high-redox-potential enzymes for environmental biocatalysis, its practical use has to date remained limited due to the lack of directed evolution platforms with which to improve its features. Here, we describe the construction of a PcL fusion gene and the optimization of conditions to induce its functional expression in Saccharomyces cerevisiae, facilitating its directed evolution and semirational engineering. The native PcL signal peptide was replaced by the α-factor preproleader, and this construct was subjected to six rounds of evolution coupled to a multiscreening assay based on the oxidation of natural and synthetic redox mediators at more neutral pHs. The laccase total activity was enhanced 8,000-fold: the evolved α-factor preproleader improved secretion levels 40-fold, and several mutations in mature laccase provided a 13.7-fold increase in k(cat). While the pH activity profile was shifted to more neutral values, the thermostability and the broad substrate specificity of PcL were retained. Evolved variants were highly secreted by Aspergillus niger (∼23 mg/liter), which addresses the potential use of this combined-expression system for protein engineering. The mapping of mutations onto the PcL crystal structure shed new light on the oxidation of phenolic and nonphenolic substrates. Furthermore, some mutations arising in the evolved preproleader highlighted its potential for heterologous expression of fungal laccases in yeast (S. cerevisiae).

Effect of pH on the stability of Pleurotus eryngii versatile peroxidase during heterologous production in Emericella nidulans.

Complementary DNA (cDNA) encoding the new versatile peroxidase from the ligninolytic basidiomycete Pleurotus eryngii has been expressed in the ascomycete Emericella nidulans. In recombinant E. nidulans cultures, the pH reached values as high as 8.3, correlating with a sharp decrease in peroxidase activity. Peroxidase was rapidly inactivated at alkaline pH, but was comparatively stable at acidic pH. The peroxidase inactivation in alkaline buffer could be reversed by adding Ca(2+) and lowering the pH. However, reactivation did not result after incubating the enzyme in non-buffered E. nidulans cultures that reached pH 7.5. To optimize recombinant peroxidase production, the effect of controlling the pH in E. nidulans bioreactor cultures was studied. An extended growth period, and a significant increase in the recombinant peroxidase level (5.3-fold higher activity than in the bioreactor without pH control) was obtained when the pH was maintained at 6.8, showing that culture pH is an important parameter for recombinant peroxidase production.

Pycnoporus cinnabarinus laccases: an interesting tool for food or non-food applications.

The effects of the addition of ferulic acid and ethanol in P. cinnabarinus ss3 culture medium in fermentor were compared in 15-L fermentor. In the presence of 30 g l(-1) ethanol, laccase activity (270,000 U/L1) was 3-fold higher as compared with ferulic acid-induced cultures, and 150-fold higher as compared with non-induced cultures, respectively. High-quality flax pulp was bleached in a totally-chlorine free (TCF) sequence using a laccase-mediator system constituted by laccase from Pycnoporus cinnabarinus and 1-hydroxybenzotriazole (HBT) as mediator. Up to 90% delignification and strong brightness increase were attained after the laccase-mediator treatment followed by H2O2 bleaching. This TCF sequence was further improved by applying H2O2 under pressurized O2. In this way, up to 82% ISO brightness was obtained (compared with 37% in the initial pulp and 60% in the peroxide-bleached control) as well as very low kappa number. A positive evaluation of the laccase has been also performed in a food application. The colour of a tea-based beverage was significantly improved by incubating an infusion of green tea with the Pycnoporus laccase.

A new versatile peroxidase from Pleurotus.

Lignin peroxidase (LiP) and manganese peroxidase (MnP) have been investigated in Phanerochaete chrysosporium. A third ligninolytic peroxidase has been described in Pleurotus and Bjerkandera. Two of these versatile peroxidases (VPs) have been cloned, sequenced and characterized. They have high affinity for Mn(2+), hydroquinones and dyes, and also oxidize veratryl alcohol, dimethoxybenzene and lignin dimers. The deduced sequences show higher identity with Ph. chrysosporium LiP than MnP, but the molecular models obtained include a Mn(2+)-binding site. Concerning aromatic substrate oxidation, Pl. eryngii VP shows a putative long-range electron transfer pathway from an exposed trytophan to haem. Mutagenesis and chemical modification of this tryptophan and the acidic residues forming the Mn(2+)-binding site confirmed their role in catalysis. The existence of several substrate oxidation sites is supported further by biochemical evidence. Residue conservation in other fungal peroxidases is discussed.

The cloning of a new peroxidase found in lignocellulose cultures of Pleurotus eryngii and sequence comparison with other fungal peroxidases.

We report cloning and sequencing of gene ps1 encoding a versatile peroxidase combining catalytic properties of lignin peroxidase (LiP) and manganese peroxidase (MnP) isolated from lignocellulose cultures of the white-rot fungus Pleurotus eryngii. The gene contains 15 putative introns, and the deduced amino acid sequence consists of a 339-residue mature protein with a 31-residue signal peptide. Several putative response elements were identified in the promoter region. Amino acid residues involved in oxidation of Mn(2+) and aromatic substrates by direct electron transfer to heme and long-range electron transfer from superficial residues as predicted by analogy with Phanerochaete chrysosporium MnP and LiP, respectively. A dendrogram is presented illustrating sequence relationships between 29 fungal peroxidases.

Regulation of peroxidase transcript levels in liquid cultures of the ligninolytic fungus Pleurotus eryngii.

A versatile peroxidase able to oxidize Mn(2+) as well as phenolic and nonphenolic aromatic compounds is produced in peptone-containing liquid cultures of Pleurotus eryngii encoded by the gene mnpl. The regulation of its transcript levels was investigated by Northern blotting of total RNA. High-peroxidase transcripts and activity were found in cultures grown in glucose-peptone medium, whereas only basal levels were detected in glucose-ammonium medium. The addition of more than 25 microM Mn(2+) to the former medium did not result in detectable peroxidase transcripts or activity. Potential regulators were also added to isolated mycelium. In this way, it was shown that high transcript levels (in peroxidase-expressing mycelium) were maintained on peptone, whereas expression was not induced in short-term incubation experiments. Similar results were obtained with Mn(2+) ions. Strong induction of mnpl expression was caused by exogenous H(2)O(2) or by continuous H(2)O(2) generation during redox cycling of menadione. By the use of the latter system in the presence of Fe(3+), which catalyzes the reduction of H(2)O(2) to hydroxyl radical, it was shown for the first time that the presence of this strong oxidant causes a rapid increase of the transcripts of a ligninolytic peroxidase. In conclusion, peptone and Mn(2+) affect the levels of transcripts of this versatile peroxidase in culture, and reduced oxygen species induce short-term expression in isolated mycelium, probably via a stress response mechanism.

Description of a versatile peroxidase involved in the natural degradation of lignin that has both manganese peroxidase and lignin peroxidase substrate interaction sites.

Two major peroxidases are secreted by the fungus Pleurotus eryngii in lignocellulose cultures. One is similar to Phanerochaete chrysosporium manganese-dependent peroxidase. The second protein (PS1), although catalyzing the oxidation of Mn2+ to Mn3+ by H2O2, differs from the above enzymes by its manganese-independent activity enabling it to oxidize substituted phenols and synthetic dyes, as well as the lignin peroxidase (LiP) substrate veratryl alcohol. This is by a mechanism similar to that reported for LiP, as evidenced by p-dimethoxybenzene oxidation yielding benzoquinone. The apparent kinetic constants showed high activity on Mn2+, but methoxyhydroquinone was the natural substrate with the highest enzyme affinity (this and other phenolic substrates are not efficiently oxidized by the P. chrysosporium peroxidases). A three-dimensional model was built using crystal models from four fungal peroxidase as templates. The model suggests high structural affinity of this versatile peroxidase with LiP but shows a putative Mn2+ binding site near the internal heme propionate, involving Glu36, Glu40, and Asp181. A specific substrate interaction site for Mn2+ is supported by kinetic data showing noncompetitive inhibition with other peroxidase substrates. Moreover, residues reported as involved in LiP interaction with veratryl alcohol and other aromatic substrates are present in peroxidase PS1 such as His82 at the heme-channel opening, which is remarkably similar to that of P. chrysosporium LiP, and Trp170 at the protein surface. These residues could be involved in two different hypothetical long range electron transfer pathways from substrate (His82-Ala83-Asn84-His47-heme and Trp170-Leu171-heme) similar to those postulated for LiP.

Manganese-Mediated Lignin Degradation by Pleurotus pulmonarius.

Pleurotus pulmonarius produced the strongest degradation of lignin during solid-state fermentation of [(sup14)C]lignin wheat straw with different fungi. A manganese-oxidizing peroxidase seemed to be involved in lignin attack, since the addition of Mn(sup2+) to the culture increased lignin mineralization by ca. 125%. This enzyme was purified and characterized from both solid-state fermentation and liquid cultures.

Preferential degradation of phenolic lignin units by two white rot fungi.

The differential biodegradation of phenolic and nonphenolic (C-4-etherified) lignin units in wheat straw treated with the white rot fungi Pleurotus eryngii and Phanerochaete chrysosporium was investigated under solid-state fermentation conditions. Two analytical techniques applied to permethylated straw were used for this purpose, i.e., alkaline CuO degradation and analytical pyrolysis (both followed by gas chromatography-mass spectrometry for product identification). Despite differences in the enzymatic machinery produced, both ligninolytic fungi caused a significant decrease in the relative amount of phenolic lignin units during the degradation process. Nevertheless, no differences in the biodegradation rates of phenolic and etherified cinnamic acids were observed. Changes in lignin composition and cinnamic acid content were also analyzed in the phenolic and nonphenolic lignin moieties. The results obtained are discussed in the context of the enzymatic mechanisms of lignin biodegradation.

[The surgical treatment of an aneurysm of the celiac trunk and hepatic artery]. / Tratamiento quirúrgico de un aneurisma del tronco celíaco y arteria hepática.

We report a case of splenic aneurysms with double location, at the celiac trunks and at the common hepatic artery, in the same patient that was treated by a surgical procedure. We also review literature about the hepatic aneurysms at the celiac trunks, with special attention on the aspects of etiology, natural evolutions and different surgical procedures available. It is remarkable on this kind of pathology that special attention should be paid in order to avoid the simple ligature-exclusion of the aneurysm, it involves realizing an endoaneurysmorrhaphy followed by an aorto-hepatic by-pass.

[Acute arterial thrombosis of the extremity in pseudoxanthoma elasticum]. / Trombosis arterial aguda extremitaria en el pseudoxantoma elástico.

We report a case of a patient with an elastic pseudoxanthoma (PXE) who presented an acute ischaemia at the left lower limb. The cause of such ischaemia was a thrombosis into the iliac and femoropopliteal arteries. Patient underwent a surgical procedure. The arteriopathy associated with a PXE rarely cause an arterial major occlusion. We did not found a case of acute arterial thrombotic ischaemia and PXE, treated with direct arterial revascularization in the reviewed literature.

[Carotid endarterectomy with Javid bypass and supraorbital photoplethysmography]. / Endarterectomía carotídea con cortocircuito de Javid y fotopletismografía supraorbitaria.

Bypass patency was monitorized by photoplethismography of supraorbital artery during Javid's bypass clamping in the course of carotid endarterectomy in 12 patients. This technique allows a rapid and easy detection of partial or total bypass occlusion which is manifested by a reduction in the plethysmographic wave amplitude in the ipsilateral supraorbital artery with respect to data obtained before operation or with the plethysmographic curve recorded at the contralateral artery. Among the twelve patients only five (41.6%) showed no evidence of bypass malfunction. Two patients (16.6%) suffered one episode of bypass insufficiency, four (33.3%) patients presented three episodes and one patient (8.33%) complained four episodes of bypass dysfunction. None of bypass dysfunctions were detected by the surgical team. Bypass insufficiency was due to excessive traction that caused leaning of the vessel or to malposition of the end of the bypass. Both were promptly corrected. We conclude that besides implantation of the bypass it is worthwhile monitoring its function by recording the photoplethysmographic response of the supraorbital artery which, in turn, is a safe and easy technique.