Chemical conjugation of aptamer-sphingomyelin nanosystems and their potential as inhibitors of tumour cell proliferation in breast cancer cells.

Breast cancer is a complex and heterogeneous disease with a high mortality rate due to non-specific cytotoxicity, low intratumoral accumulation and drug resistance associated with the ineffectiveness of chemotherapy. In recent years, all efforts have been focused on finding new markers and therapeutic targets, protein kinase MNK1b being a promising candidate. Recently, an aptamer known as apMNK2F showed a highly specific interaction with this protein kinase, leading to a significant reduction in tumour cell proliferation, migration and colony formation. However, as aptamers are unable to penetrate the cell membrane and reach the target, these small biomolecules need to be conjugated to suitable vectors that can transport and protect them inside the cells. In this work, covalent conjugation between biocompatible and non-harmful nanoemulsions of vitamin E and sphingomyelin and the aptamer was performed to facilitate intracellular delivery of the therapeutic aptamer apMNK2F. All strategies employed were based on 2-step bioconjugation and optimized to get the simplest and most reproducible vehicle with the highest association efficiency (about 70% in all cases). The ability of the nanosystems to successfully deliver the conjugated therapeutic aptamer was demonstrated and compared to other commercial transfection agents such as Lipofectamine 2000, leading to an effective decrease of breast cancer cell proliferation in the MDA-MB-231 cell line. The proliferation inhibition of the aptamer nanoconjugates compared to the non-conjugated aptamer provides evidence that the antitumoral capacity derived from kinase interaction is improved in a dose-dependent manner. Furthermore, various experiments including cell migration and colony formation assays, along with apoptosis induction experiments, emphasize the significant antitumoral potential. Overall, the obtained results indicate that the developed formulation could be a promising therapy for the treatment of breast cancer.

Gonadotropic activity of a second relaxin-type peptide in starfish.

In starfish, a relaxin-like gonad-stimulating peptide (RGP) acts as a gonadotropin that triggers gamete maturation and spawning. In common with other relaxin/insulin superfamily peptides, RGP consists of an A- and a B-chain, with cross-linkages mediated by one intra- and two inter-chain disulfide bonds. In this study, a second relaxin-like peptide (RLP2) was identified in starfish species belonging to the orders Valvatida, Paxillosida, and Forcipulatida. Like RGP, RLP2 precursors comprise a signal peptide and a C-peptide in addition to the A- and B-chains. However, a unique cysteine motif [CC-(3X)-C-(10X)-C] is present in the A-chain of RLP2, which contrasts with the cysteine motif in other members of the relaxin/insulin superfamily [CC-(3X)-C-(8X)-C]. Importantly, in vitro pharmacological tests revealed that Patiria pectinifera RLP2 (Ppe-RLP2) and Asterias rubens RLP2 (Aru-RLP2) trigger shedding of mature eggs from ovaries of P. pectinifera and A. rubens, respectively. Furthermore, the potencies of Ppe-RLP2 and Aru-RLP2 as gonadotropic peptides were similar to those of Ppe-RGP and Aru-RGP, respectively, and the effect of RLP2 exhibited partial species-specificity. These findings indicate that two relaxin-type peptides regulate spawning in starfish and therefore we propose that RGP and RLP2 are renamed RGP1 and RGP2, respectively.

Safety and Efficacy of ApTOLL in Patients With Ischemic Stroke Undergoing Endovascular Treatment: A Phase 1/2 Randomized Clinical Trial.

Importance: ApTOLL is a TLR4 antagonist with proven preclinical neuroprotective effect and a safe profile in healthy volunteers. Objective: To assess the safety and efficacy of ApTOLL in combination with endovascular treatment (EVT) for patients with ischemic stroke. Design, Setting, and Participants: This phase 1b/2a, double-blind, randomized, placebo-controlled study was conducted at 15 sites in Spain and France from 2020 to 2022. Participants included patients aged 18 to 90 years who had ischemic stroke due to large vessel occlusion and were seen within 6 hours after stroke onset; other criteria were an Alberta Stroke Program Early CT Score of 6 to 10, estimated infarct core volume on baseline computed tomography perfusion of 5 to 70 mL, and the intention to undergo EVT. During the study period, 4174 patients underwent EVT. Interventions: In phase 1b, 0.025, 0.05, 0.1, or 0.2 mg/kg of ApTOLL or placebo; in phase 2a, 0.05 or 0.2 mg/kg of ApTOLL or placebo; and in both phases, treatment with EVT and intravenous thrombolysis if indicated. Main Outcomes and Measures: The primary end point was the safety of ApTOLL based on death, symptomatic intracranial hemorrhage (sICH), malignant stroke, and recurrent stroke. Secondary efficacy end points included final infarct volume (via MRI at 72 hours), NIHSS score at 72 hours, and disability at 90 days (modified Rankin Scale [mRS] score). Results: In phase Ib, 32 patients were allocated evenly to the 4 dose groups. After phase 1b was completed with no safety concerns, 2 doses were selected for phase 2a; these 119 patients were randomized to receive ApTOLL, 0.05 mg/kg (n = 36); ApTOLL, 0.2 mg/kg (n = 36), or placebo (n = 47) in a 1:1:√2 ratio. The pooled population of 139 patients had a mean (SD) age of 70 (12) years, 81 patients (58%) were male, and 58 (42%) were female. The primary end point occurred in 16 of 55 patients (29%) receiving placebo (10 deaths [18.2%], 4 sICH [7.3%], 4 malignant strokes [7.3%], and 2 recurrent strokes [3.6%]); in 15 of 42 patients (36%) receiving ApTOLL, 0.05 mg/kg (11 deaths [26.2%], 3 sICH [7.2%], 2 malignant strokes [4.8%], and 2 recurrent strokes [4.8%]); and in 6 of 42 patients (14%) receiving ApTOLL, 0.2 mg/kg (2 deaths [4.8%], 2 sICH [4.8%], and 3 recurrent strokes [7.1%]). ApTOLL, 0.2 mg/kg, was associated with lower NIHSS score at 72 hours (mean difference log-transformed vs placebo, -45%; 95% CI, -67% to -10%), smaller final infarct volume (mean difference log-transformed vs placebo, -42%; 95% CI, -66% to 1%), and lower degrees of disability at 90 days (common odds ratio for a better outcome vs placebo, 2.44; 95% CI, 1.76 to 5.00). Conclusions and Relevance: In acute ischemic stroke, 0.2 mg/kg of ApTOLL administered within 6 hours of onset in combination with EVT was safe and associated with a potential meaningful clinical effect, reducing mortality and disability at 90 days compared with placebo. These preliminary findings await confirmation from larger pivotal trials. Trial Registration: ClinicalTrials.gov Identifier: NCT04734548.

An Aptamer against MNK1 for Non-Small Cell Lung Cancer Treatment.

Lung cancer is the leading cause of cancer-related death worldwide. Its late diagnosis and consequently poor survival make necessary the search for new therapeutic targets. The mitogen-activated protein kinase (MAPK)-interacting kinase 1 (MNK1) is overexpressed in lung cancer and correlates with poor overall survival in non-small cell lung cancer (NSCLC) patients. The previously identified and optimized aptamer from our laboratory against MNK1, apMNKQ2, showed promising results as an antitumor drug in breast cancer in vitro and in vivo. Thus, the present study shows the antitumor potential of apMNKQ2 in another type of cancer where MNK1 plays a significant role, such as NSCLC. The effect of apMNKQ2 in lung cancer was studied with viability, toxicity, clonogenic, migration, invasion, and in vivo efficacy assays. Our results show that apMNKQ2 arrests the cell cycle and reduces viability, colony formation, migration, invasion, and epithelial-mesenchymal transition (EMT) processes in NSCLC cells. In addition, apMNKQ2 reduces tumor growth in an A549-cell line NSCLC xenograft model. In summary, targeting MNK1 with a specific aptamer may provide an innovative strategy for lung cancer treatment.

Potential Therapeutic Use of Aptamers against HAT1 in Lung Cancer.

Lung cancer is one of the leading causes of death worldwide and the most common of all cancer types. Histone acetyltransferase 1 (HAT1) has attracted increasing interest as a potential therapeutic target due to its involvement in multiple pathologies, including cancer. Aptamers are single-stranded RNA or DNA molecules whose three-dimensional structure allows them to bind to a target molecule with high specificity and affinity, thus making them exceptional candidates for use as diagnostic or therapeutic tools. In this work, aptamers against HAT1 were obtained, subsequently characterized, and optimized, showing high affinity and specificity for HAT1 and the ability to inhibit acetyltransferase activity in vitro. Of those tested, the apHAT610 aptamer reduced cell viability, induced apoptosis and cell cycle arrest, and inhibited colony formation in lung cancer cell lines. All these results indicate that the apHAT610 aptamer is a potential drug for the treatment of lung cancer.

DNA Aptamers against Vaccinia-Related Kinase (VRK) 1 Block Proliferation in MCF7 Breast Cancer Cells.

Vaccinia-related kinase (VRK) 1 is a serin/threonine kinase that plays an important role in DNA damage response (DDR), phosphorylating some proteins involved in this process such as 53BP1, NBS1 or H2AX, and in the cell cycle progression. In addition, VRK1 is overexpressed in many cancer types and its correlation with poor prognosis has been determined, showing VRK1 as a new therapeutic target in oncology. Using in vitro selection, high-affinity DNA aptamers to VRK1 were selected from a library of ssDNA. Selection was monitored using the enzyme-linked oligonucleotide assay (ELONA), and the selected aptamer population was cloned and sequenced. Three aptamers were selected and characterized. These aptamers recognized the protein kinase VRK1 with an affinity in the nanomolar range and showed a high sensibility. Moreover, the treatment of the MCF7 breast cell line with these aptamers resulted in a decrease in cyclin D1 levels, and an inhibition of cell cycle progression by G1 phase arrest, which induced apoptosis in cells. These results suggest that these aptamers are specific inhibitors of VRK1 that might be developed as potential drugs for the treatment of cancer.

The Relevance of the SH2 Domain for c-Src Functionality in Triple-Negative Breast Cancer Cells.

The role of Src family kinases (SFKs) in human tumors has been always associated with tyrosine kinase activity and much less attention has been given to the SH2 and SH3 adapter domains. Here, we studied the role of the c-Src-SH2 domain in triple-negative breast cancer (TNBC). To this end, SUM159PT and MDA-MB-231 human cell lines were employed as model systems. These cells conditionally expressed, under tetracycline control (Tet-On system), a c-Src variant with point-inactivating mutation of the SH2 adapter domain (R175L). The expression of this mutant reduced the self-renewal capability of the enriched population of breast cancer stem cells (BCSCs), demonstrating the importance of the SH2 adapter domain of c-Src in the mammary gland carcinogenesis. In addition, the analysis of anchorage-independent growth, proliferation, migration, and invasiveness, all processes associated with tumorigenesis, showed that the SH2 domain of c-Src plays a very relevant role in their regulation. Furthermore, the transfection of two different aptamers directed to SH2-c-Src in both SUM159PT and MDA-MB-231 cells induced inhibition of their proliferation, migration, and invasiveness, strengthening the hypothesis that this domain is highly involved in TNBC tumorigenesis. Therefore, the SH2 domain of c-Src could be a promising therapeutic target and combined treatments with inhibitors of c-Src kinase enzymatic activity may represent a new therapeutic strategy for patients with TNBC, whose prognosis is currently very negative.

Deeping in the Role of the MAP-Kinases Interacting Kinases (MNKs) in Cancer.

The mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs) are involved in oncogenic transformation and can promote metastasis and tumor progression. In human cells, there are four MNKs isoforms (MNK1a/b and MNK2a/b), derived from two genes by alternative splicing. These kinases play an important role controlling the expression of specific proteins involved in cell cycle, cell survival and cell motility via eukaryotic initiation factor 4E (eIF4E) regulation, but also through other substrates such as heterogeneous nuclear ribonucleoprotein A1, polypyrimidine tract-binding protein-associated splicing factor and Sprouty 2. In this review, we provide an overview of the role of MNK in human cancers, describing the studies conducted to date to elucidate the mechanism involved in the action of MNKs, as well as the development of MNK inhibitors in different hematological cancers and solid tumors.

MAFG is a potential therapeutic target to restore chemosensitivity in cisplatin-resistant cancer cells by increasing reactive oxygen species.

Adjuvant chemotherapy for solid tumors based on platinum-derived compounds such as cisplatin is the treatment of choice in most cases. Cisplatin triggers signaling pathways that lead to cell death, but it also induces changes in tumor cells that modify the therapeutic response, thereby leading to cisplatin resistance. We have recently reported that microRNA-7 is silenced by DNA methylation and is involved in the resistance to platinum in cancer cells through the action of the musculoaponeurotic fibrosarcoma oncogene family, protein G (MAFG). In the present study, we first confirm the miR-7 epigenetic regulation of MAFG in 44 normal- and/or tumor-paired samples in non-small-cell lung cancer (NSCLC). We also provide translational evidence of the role of MAFG and the clinical outcome in NSCLC by the interrogation of two extensive in silico databases of 2019 patients. Moreover, we propose that MAFG-mediated resistance could be conferred due to lower reactive oxygen species production after cisplatin exposure. We developed specifically selected aptamers against MAFG, with high sensitivity to detect the protein at a nuclear level probed by aptacytochemistry and histochemistry analyses. The inhibition of MAFG activity through the action of the specific aptamer apMAFG6F increased the levels of reactive oxygen species production and the sensitivity to cisplatin. We report first the specific nuclear identification of MAFG as a novel detection method for diagnosis in NSCLC, and then we report that MAFG modulates the redox response and confers cell protection against free radicals generated after platinum administration, thus also being a promising therapeutic target.

Increased expression of MNK1b, the spliced isoform of MNK1, predicts poor prognosis and is associated with triple-negative breast cancer.

MAP kinase interacting kinases (MNKs) modulate the function of oncogene eukaryotic initiation factor 4E (eIF4E) through phosphorylation, which is necessary for oncogenic transformation. MNK1 gives rise to two mRNAs and thus two MNK1 isoforms, named MNK1a and MNK1b. MNK1b, the splice variant of human MNK1a, is constitutively active and independent of upstream MAP kinases. In this study, we have analyzed the expression of both MNK1 isoforms in 69 breast tumor samples and its association with clinicopathologic/prognostic characteristics of breast cancer. MNK1a and MNK1b expression was significantly increased in tumors relative to the corresponding adjacent normal tissue (p < 0.001). In addition, MNK1b overexpression was found in most of the triple-negative tumors and was associated with a shorter overall and disease-free survival time. Overexpression of MNK1b in MDA-MB-231 cells induced an increase in the expression of the MCL1 antiapoptotic protein and promoted proliferation, invasion and colony formation. In conclusion, a high expression level of MNK1b protein could be used as a marker of poor prognosis in breast cancer patients and it could be a therapeutic target in triple-negative tumors.

Characterization of MNK1b DNA Aptamers That Inhibit Proliferation in MDA-MB231 Breast Cancer Cells.

Elevated expression levels of eukaryotic initiation factor 4E (eIF4E) promote cancer development and progression. MAP kinase interacting kinases (MNKs) modulate the function of eIF4E through the phosphorylation that is necessary for oncogenic transformation. Therefore, pharmacologic MNK inhibitors may provide a nontoxic and effective anticancer strategy. MNK1b is a truncated isoform of MNK1a that is active in the absence of stimuli. Using in vitro selection, high-affinity DNA aptamers to MNK1b were selected from a library of ssDNA. Selection was monitored using the enzyme-linked oligonucleotide assay (ELONA), and the selected aptamer population was cloned and sequenced. Four groups of aptamers were identified, and the affinities of one representative for rMNK1b were determined using ELONA and quantitative polymerase chain reaction. Two aptamers, named apMNK2F and apMNK3R, had a lower Kd in the nmol/l range. The secondary structure of the selected aptamers was predicted using mFold, and the QGRS Mapper indicated the presence of potential G-quadruplex structures in both aptamers. The selected aptamers were highly specific against MNK1, showing higher affinity to MNK1b than to MNK1a. Interestingly, both aptamers were able to produce significant translation inhibition and prevent tumor cell proliferation and migration and colony formation in breast cancer cells. These results indicate that MNK1 aptamers have an attractive therapeutic potential.

Apoptosis-related proteins are potential markers of neonatal hypoxic-ischemic encephalopathy (HIE) injury.

Neonatal hypoxic-ischemic encephalopathy (HIE) causes high mortality and long-term morbidity rates. The magnitude of the neuronal damage depends on the duration and severity of the initial insult combined with the deleterious effects of reperfusion and apoptosis. Currently, a diagnosis of HIE is based largely on the neurological and histological findings. Therefore, the aim of this study was to identify apoptosis-related proteins that might serve as potential markers of HIE injury. As an initial step toward reaching this objective, we analyzed changes in protein levels in an in vitro model of hypoxia using antibody arrays, and we have identified changes in the expression level of two proteins involved in apoptosis, Smac-DIABLO and cathepsin D. We obtained brain sections from eight neonatal HIE patients and performed histological staining, TUNEL assays and Smac-DIABLO and cathepsin D immunolocalization. Our results revealed a high number of TUNEL-positive cells, including neurons, astrocytes and ependymal cells, in the various regions that were analyzed. Interestingly, many of the areas that were positive for TUNEL staining did not appear to be damaged in the histological evaluation. In addition, using immunostaining, we found that Smac-DIABLO and cathepsin D had the same regional distribution pattern. Taken together, these findings indicate that these two proteins could serve as markers to identify injured regions that might not to be detectable using histological observations alone.

Selection and characterization of single stranded DNA aptamers for the hormone abscisic Acid.

The hormone abscisic acid (ABA) is a small molecule involved in pivotal physiological functions in higher plants. Recently, ABA has been also identified as an endogenous hormone in mammals, regulating different cell functions including inflammatory processes, stem cell expansion, insulin release, and glucose uptake. Aptamers are short, single-stranded (ss) oligonucleotidesable to recognize target molecules with high affinity. The small size of the ABA molecule represented a challenge for aptamer development and the aim of this study was to develop specific anti-ABA DNA aptamers. Biotinylated abscisic acid (bio-ABA) was immobilized on streptavidin-coated magnetic beads. DNA aptamers against bio-ABA were selected with 7 iterative rounds of the systematic evolution of ligands by exponential enrichment method (SELEX), each round comprising incubation of the ABA-binding beads with the ssDNA sequences, DNA elution, electrophoresis, and polymerase chain reaction (PCR) amplification. The PCR product was cloned and sequenced. The binding affinity of several clones was determined using bio-ABA immobilized on streptavidin-coated plates. Aptamer 2 and aptamer 9 showed the highest binding affinity, with dissociation constants values of 0.98 ± 0.14 µM and 0.80 ± 0.07 µM, respectively. Aptamers 2 and 9 were also able to bind free, unmodified ABA and to discriminate between different ABA enantiomers and isomers. Our findings indicate that ssDNA aptamers can selectively bind ABA and could be used for the development of ABA quantitation assays.

eIF4F complex disruption causes protein synthesis inhibition during hypoxia in nerve growth factor (NGF)-differentiated PC12 cells.

Poor oxygenation (hypoxia) influences important physiological and pathological situations, including development, ischemia, stroke and cancer. Hypoxia induces protein synthesis inhibition that is primarily regulated at the level of initiation step. This regulation generally takes place at two stages, the phosphorylation of the subunit α of the eukaryotic initiation factor (eIF) 2 and the inhibition of the eIF4F complex availability by dephosphorylation of the inhibitory protein 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1). The contribution of each of them is mainly dependent of the extent of the oxygen deprivation. We have evaluated the regulation of hypoxia-induced translation inhibition in nerve growth factor (NGF)-differentiated PC12 cells subjected to a low oxygen concentration (0.1%) at several times. Our findings indicate that protein synthesis inhibition occurs primarily by the disruption of eIF4F complex through 4E-BP1 dephosphorylation, which is produced by the inhibition of the mammalian target of rapamycin (mTOR) activity via the activation of REDD1 (regulated in development and DNA damage 1) protein in a hypoxia-inducible factor 1 (HIF1)-dependent manner, as well as the translocation of eIF4E to the nucleus. In addition, this mechanism is reinforced by the increase in 4E-BP1 levels, mainly at prolonged times of hypoxia.

Analysis of the protein expression changes during taxol-induced apoptosis under translation inhibition conditions.

Taxol is currently used in chemotherapeutic treatments of different types of cancers. In this article, we demonstrate that taxol induces apoptosis and translation down-regulation in human embryonic kidney (HEK293T) cells. Antibody arrays are a promising new tool for the analysis of protein levels changes in cells responding to different stimuli. Using this approach, we have identified changes in the expression of 38 proteins (20 down-regulated and 18 up-regulated), implicated in several cellular processes mainly in apoptosis, cell cycle and signal transduction pathways, and also cytoskeleton proteins. Among them, we have confirmed a considerable decrease in the expression of p14(ARF) and a significant increase in the levels of dystrophin and c-Myc. It is known that c-Myc mRNA has an internal ribosome entry segment (IRES) element in its 5'UTR that could regulate its expression under global protein synthesis inhibition conditions. We demonstrate that after taxol treatment, the c-Myc IRES activity is maintained meanwhile cap-dependent activity is inhibited. In addition, an increase in c-Myc mRNA was also observed after taxol treatment. We conclude that taxol-induced c-Myc expression is regulated at both transcriptional and translational levels, the last of them by a mechanism mediated by IRES.

Translation regulation after taxol treatment in NIH3T3 cells involves the elongation factor (eEF)2.

Changes to the translational machinery that occur during apoptosis have been described in the last few years. The two principal ways in which translational factors are modified during apoptosis are: (i) changes in protein phosphorylation and (ii) specific proteolytic cleavages. Taxol, a member of a new class of anti-tubulin drugs, is currently used in chemotherapeutic treatments of different types of cancers. We have previously demonstrated that taxol induces calpain-mediated apoptosis in NIH3T3 cells [Piñeiro et al., Exp. Cell Res., 2007, 313:369-379]. In this study we found that translation was significantly inhibited during taxol-induced apoptosis in these cells. We have studied the phosphorylation status and expression levels of eIF2a, eIF4E, eIF4G and the regulatory protein 4E-BP1, all of which are implicated in translation regulation. We found that taxol treatment did not induce changes in eIF2alpha phosphorylation, but strongly decreased eIF4G, eIF4E and 4E-BP1 expression levels. MDL28170, a specific inhibitor of calpain, prevented reduction of eIF4G, but not of eIF4E or 4E-BP1 levels. Moreover, the calpain inhibitor did not block taxol-induced translation inhibition. All together these findings demonstrated that none of these factors are responsible for the taxol-induced protein synthesis inhibition. On the contrary, taxol treatment increased elongation factor eEF2 phosphorylation in a calpain-independent manner, supporting a role for eEF2 in taxol-induced translation inhibition.

Calpain inhibition stimulates caspase-dependent apoptosis induced by taxol in NIH3T3 cells.

Taxol is an anticancer drug that triggers apoptosis in a wide spectrum of cancers such as ovarian, breast, lung, head and neck, and bladder carcinoma by both caspase-dependent and -independent apoptosis mechanisms. However, the exact signaling pathways involved in taxol-induced apoptosis strongly depend on the cellular background and they are not completely established yet. In this study we demonstrate that taxol induces caspase-3-independent apoptosis in NIH3T3 cells by a calpain-mediated mechanism. Taxol treatment produced changes in the mitochondrial membrane potential (Delta Psi m) which could be responsible of Ca(2+) release from the mitochondria and the consequent calpain activation. Interestingly, we show that calpain produced proteolysis of caspase-3 and demonstrate that, accordingly, calpain inhibition increased taxol-induced apoptosis. In addition, we reveal that poly (ADP-ribose) polymerase (PARP) was processed by calpain in taxol-treated cells and by caspase-3 after calpain inhibition. In conclusion, these results demonstrate for the first time that calpain could play an important role modulating taxol-induced apoptosis. Further studies are needed to address the potentiality of inducing apoptosis by a combined use of taxol and calpain inhibitors in cells with increased calpain activity.

The genome project of Taenia solium.

We have constituted a consortium of key laboratories at the National Autonomous University of Mexico to carry out a genomic project for Taenia solium. This project will provide powerful resources for the study of taeniasis/cysticercosis, and, in conjunction with the Echinococcus granulosus and Echinococcus multilocularis genome project of expressed sequence tags (ESTs), will mark the advent of genomics for cestode parasites. Our project is planned in two consecutive stages. The first stage is being carried out to determine some basic parameters of the T. solium genome. Afterwards, we will evaluate the best strategy for the second stage, a full blown genome project. We have estimated the T. solium genome size by two different approaches: cytofluorometry on isolated cyton nuclei, as well as a probabilistic calculation based on approximately 2000 sequenced genomic clones, approximately 3000 ESTs, resulting in size estimates of 270 and 251 Mb, respectively. In terms of sequencing, our goal for the first stage is to characterize several thousand EST's (from adult worm and cysticerci cDNA libraries) and genomic clones. Results obtained so far from about 16,000 sequenced ESTs from the adult stage, show that only about 40% of the T. solium coding sequences have a previously sequenced homologue. Many of the best hits are found with mammalian genes, especially with humans. However, 1.5% of the hits lack homologues in humans, making these genes immediate candidates for investigation on pharmaco-therapy, diagnostics and vaccination. Most T. solium ESTs are related to gene regulation, and signal transduction. Other important functions are housekeeping, metabolism, cell division, cytoskeleton, proteases, vacuolar transport, hormone response, and extracellular matrix activities. Preliminary results also suggest that the genome of T. solium is not highly repetitive.

[Results of nephrectomy in patients with autosomal dominant polycystic kidney disease]. / Resultados de la nefrectomía abierta en pacientes con enfermedad renal poliquística autosómica dominante.

BACKGROUND: Nephrectomy in patients with polycystic kidney disease (PKD) is indicated in cases of hematuria, pain, hypertension, infections or before a renal transplant. The purpose of this study is to report our results of this procedure during a contemporary period of time in patients with PKD. MATERIALS AND METHODS: The study consists on a retrospective of files from patients with PKD, including all cases with unilateral or bilateral nephrectomy. We analyzed general data and compared the results from the surgical procedure between bilateral nephrectomy, unilateral nephrectomy and 2 staged bilateral nephrectomy. RESULTS: A total of 14 PKD patients treated with nephrectomy where gathered. Mean patient age was 46 years; 78.5% has chronic renal insufficiency treated with dialysis. The decision of surgery was based predominantly on the presence of two or more symptoms. A total of 24 procedures where done; 7 patients with simultaneous bilateral nephrectomy, 3 with bilateral nephrectomy done in 2 different stages and 4 patients with unilateral nephrectomy. Good operative results where observed with minimal complications. Bilateral simultaneous nephrectomy was completed in a longer time interval than unilateral procedure (255 vs. 195 min, p = 0.008) and with a slight more bleeding (775 vs. 400cc, p = 0.008). CONCLUSIONS: Open nephrectomy remains as the standard procedure for patients with polycystic kidney disease (PKD). Although minimal operative differences where seen between unilateral or bilateral 2 stage nephrectomy and bilateral simultaneous nephrectomy, the overall morbidity was similar between procedures.

Enfermedad cerebrovascular: factores de riesgo en un área de salud

Se realiza un estudio de casos controles pareado 1-1 sobre factores de riesgo de la enfermedad cerebrovascular en el Policlínico "Carlos J. Finlay de Camagüey. Se señala que la muestra fue diseñada a partir de la dispensarización en las historias clínicas familiares, y los datos se completaron con una encuesta diseñada al efecto. Se informa que el tamaño muestral y la selección se realizaron mediante el programa EPIDAT, y la muestra fue de 97 casos. Se analiza que el antecedente de madre con enfermedad vasculocerebral fue el factor de riesgo de mayor intensidad con odds ratio de 15,6. Se comprueba que el hábito de fumar, la obesidad, la dislipidemia, la hipertensión arterial, la diabetes mellitus y la cardiopatía isquémica, fueron también factores de riesgo en la serie estudiada, y que el sexo femenino se comportó como un factor protector.

A 1-1 matched case-control study on risk factors for cerebrovascular diseases at "Carlos J. Finlay" polyclinc in Camagüey province was performed. It is suggested that the sample was designed from the classification of the family medical records and data were completed from a survey prepared to this and. The sample included 97 cases and sample selection and size were determined by an EPIDAT program. It is analyzed that having a mother affected by cerebrovascular disorders was the highest risk factor (odd ratio 15.6). It was proved that smoking, obesity, dislipidosis, blood hypertension, diabetes mellitus and ischemic cardiopathy were also risk factors in the study and that being female worked as a protective factor.