Identification of cell culture contamination by an unusual species of Mycoplasma related to the M. mycoides cluster.

Mycoplasma contamination is a significant problem in cell culture replication and maintenance. From more than 200 known species, a limited number of Mycoplasma species have been detected in cell cultures, representing new species or variants that can escape detection systems. A qPCR commercial kit was used for Mycoplasma detection in cell cultures. Furthermore, an amplified Mycoplasma species was sequenced and summited for sequence assembly, clustering, and evolutionary analysis study. Our work has identified a new and unusual variant or species of Mycoplasma that possesses a high degree of homology with species related with M. mycoides cluster. This variant is usually associated with cattle but has been detected contaminating a cell culture. Mycoplasma testing (even for unusual species) in cell cultures is essential to ensure the validity and reproducibility of research that uses cell cultures and to ensure the quality of cell line deposits in biobanks. For this reason, it is necessary to perform continuous checks for the absence of Mycoplasma in cell cultures and engage in the continuous adaptation of relevant detection systems.

Evaluation of RNA purification methods by using different blood stabilization tubes: identification of key features for epidemiological studies.

OBJECTIVE: Peripheral blood is the most promising source of RNA biomarkers for diagnostic and epidemiological studies, because the presence of disease and prognostic information is reflected in the gene expression pattern. Quality RNA is used by a number of different downstream applications, so the selection of the most appropriate RNA stabilization and purification method is important. We have analyzed the RNA purified from 300 blood samples from 25 donors processed by two technicians using three methodologies with Tempus and PaxGene tubes. RESULTS: The best quality sample results were obtained with the Tempus Spin RNA Isolation Kit and the PaxGene Blood miRNA Kit, although larger amounts of RNA were obtained with the Tempus Spin RNA Isolation Kit. Lower Cq values were observed for RNA and miRNA genes in samples that were tested with PaxGene Blood miRNA Kit and Tempus Spin RNA Isolation Kit respectively. We identify the Tempus Spin RNA Isolation Kit as the most robust methodology, whilst the MagMax for Stabilized Blood Tubes RNA Isolation Kit showed the most instability. For biobanks, which process a large cohort and conduct epidemiological studies, the Tempus Spin RNA Isolation Kit is the most appropriate methodology. The study demonstrates the robustness of real-life procedures.

A clinically oriented computer model for radiofrequency ablation of hepatic tissue with internally cooled wet electrode.

PURPOSE: To improve the computer modelling of radiofrequency ablation (RFA) by internally cooled wet (ICW) electrodes with added clinically oriented features. METHODS: An improved RFA computer model by ICW electrode included: (1) a realistic spatial distribution of the infused saline, and (2) different domains to distinguish between healthy tissue, saline-infused tumour, and non-infused tumour, under the assumption that infused saline is retained within the tumour boundary. A realistic saline spatial distribution was obtained from an in vivo pig liver study. The computer results were analysed in terms of impedance evolution and coagulation zone (CZ) size, and were compared to the results of clinical trials conducted on 17 patients with the same ICW electrode. RESULTS: The new features added to the model provided computer results that matched well with the clinical results. No roll-offs occurred during the 4-min ablation. CZ transversal diameter (4.10 ± 0.19 cm) was similar to the computed diameter (4.16 cm). Including the tumour and saline infusion in the model involved (1) a reduction of the initial impedance by 10 - 20 Ω, (2) a delay in roll-off of 20 s and 70 - 100 s, respectively, and (3) 18 - 31% and 22 - 36% larger CZ size, respectively. The saline spatial distribution geometry was also seen to affect roll-off delay and CZ size. CONCLUSIONS: Using a three-compartment model and a realistic saline spatial distribution notably improves the match with the outcome of the clinical trials.

The impact of radiofrequency-assisted transection on local hepatic recurrence after resection of colorectal liver metastases.

Resection is the gold standard in the treatment of liver metastases from colorectal cancer. An internal cooled radiofrequency electrode was described to achieve tissue coagulation to a greater margin width. The aim of this study is to determinate if a RF-assisted transection device (RFAT) has any effect on local hepatic recurrence (LHER) compared to conventional technologies. A study population of 103 patients who had undergone a hepatic surgical resection was retrospectively analysed. Patients were classified into two groups according to the device used: a RF-assisted device (RFAT group; n = 45) and standard conventional devices (control group; n = 58). LHER was defined as any growing or enhancing tumour in the margin of hepatic resection during follow-up. Cox proportional models were constructed and variables were eliminated only if p > 0.20 to protect against residual confounding. To assess the stability of Cox's regression model and its internal validity, a bootstrap investigation was also performed. Baseline and operative characteristics were similar in both groups. With a mean follow-up of 28.5 months (range 2-106), in patients with positive margins, we demonstrated 0% of LHER in RFAT vs. 27% in control group (p = 0.032). In the multivariate analysis five factors demonstrated significant influence on the final model of LHER: RFAT group, size of the largest metastases, number of resected metastases, positive margin and usage of Pringle-manoeuvre. This study suggests that parenchymal transection using a RFAT able to create deep thermal lesions may reduce LHER especially in case of margin invasion during transection.

Cell lines authentication and mycoplasma detection as minimun quality control of cell lines in biobanking.

Establishment of continuous cell lines from human normal and tumor tissues is an extended and useful methodology for molecular characterization of cancer pathophysiology and drug development in research laboratories. The exchange of these cell lines between different labs is a common practice that can compromise assays reliability due to contamination with microorganism such as mycoplasma or cells from different flasks that compromise experiment reproducibility and reliability. Great proportions of cell lines are contaminated with mycoplasma and/or are replaced by cells derived for a different origin during processing or distribution process. The scientific community has underestimated this problem and thousand of research experiment has been done with cell lines that are incorrectly identified and wrong scientific conclusions have been published. Regular contamination and authentication tests are necessary in order to avoid negative consequences of widespread misidentified and contaminated cell lines. Cell banks generate, store and distribute cell lines for research, being mandatory a consistent and continuous quality program. Methods implementation for guaranteeing both, the absence of mycoplasma and authentication in the supplied cell lines, has been performed in the Andalusian Health System Biobank. Specifically, precise results were obtained using real time PCR detection for mycoplasma and 10 STRs identification by capillary electrophoresis for cell line authentication. Advantages and disadvantages of these protocols are discussed.

Long-term effectiveness of irreversible electroporation in a murine model of colorectal liver metastasis.

Irreversible electroporation (IRE) has recently gained in popularity as an ablative technique, however little is known about its oncological long-term outcomes. To determine the long-time survival of animals treated with a high dose of IRE and which histological changes it induces in tumoral tissue, IRE ablation was performed in forty-six athymic-nude mice with KM12C tumors implanted in the liver by applying electric current with different voltages (2000 V/cm, 1000 V/cm). The tumors were allowed to continue to grow until the animals reached the end-point criteria. Histology was harvested and the extent of tumor necrosis was semi-quantitatively assessed. IRE treatment with the 2000 V/cm protocol significantly prolonged median mouse survival from 74.3 ± 6.9 days in the sham group to 112.5 ± 15.2 days in the 2000 V/cm group. No differences were observed between the mean survival of the 1000 V/cm and the sham group (83.2 ± 16.4 days, p = 0.62). Histology revealed 63.05% ± 23.12 of tumor necrosis in animals of the 2000 V/cm group as compared to 17.50% ± 2.50 in the 1000 V/cm group and 25.6% ± 22.1 in the Sham group (p = 0.001). IRE prolonged the survival of animals treated with the highest electric field (2000 V/cm). The animals in this group showed significantly higher rate of tumoral necrosis.

Laparoscopic partial splenectomy for giant cyst using a radiofrequency-assisted device: a case report.

BACKGROUND: Although radiofrequency-assisted devices have sometimes been used in partial splenectomy, this is not a common technique. This report describes the first case of laparoscopic partial splenectomy using an RF-assisted device (Coolinside) which allows both coagulation and transection of the parenchyma and eventually the protective coagulation of the remnant side. CASE PRESENTATION: A 27-year-old woman was found to have a giant hydatic cyst measuring 12.0 × 14.0 × 16.6 cm that mainly occupied the lower pole of the spleen and retroperitoneal space. The patient underwent a laparoscopic partial splenectomy using an RF-based device designed to accomplish both the coagulation and dissection of the splenic tissue. The estimated blood loss was less than 200 mL. CONCLUSIONS: Even though RF ablation has traditionally been used for hepatic parenchymal transection, it seems equally suited to partial splenectomy. This device seems to provide good results, minimizing blood loss during partial splenectomy; however, randomized trials will be necessary to see if the results are superior to those of other techniques.

Irreversible electroporation of the liver: is there a safe limit to the ablation volume?

Irreversible electroporation is a fast-growing liver ablation technique. Although safety has been well documented in small ablations, our aim is to assess its safety and feasibility when a large portion of liver is ablated. Eighty-seven mice were subjected to high voltage pulses directly delivered across parallel plate electrodes comprising around 40% of mouse liver. One group consisted in 55 athymic-nude, in which a tumor from the KM12C cell line was grown and the other thirty-two C57-Bl6 non-tumoral mice. Both groups were subsequently divided into subsets according to the delivered field strength (1000 V/cm, 2000 V/cm) and whether or not they received anti-hyperkalemia therapy. Early mortality (less than 24 hours post-IRE) in the 2000 V/cm group was observed and revealed considerably higher mean potassium levels. In contrast, the animals subjected to a 2000 V/cm field treated with the anti-hyperkalemia therapy had higher survival rates (OR = 0.1, 95%CI = 0.02-0.32, p < 0.001). Early mortality also depended on the electric field magnitude of the IRE protocol, as mice given 1000 V/cm survived longer than those given 2000 V/cm (OR = 4.7, 95%CI = 1.8-11.8, p = 0.001). Our findings suggest that ionic disturbances, mainly due to potassium alterations, should be warned and envisioned when large volume ablations are performed by IRE.

Deep-subwavelength negative-index waveguiding enabled by coupled conformal surface plasmons.

In this Letter we introduce a novel route for achieving negative-group-velocity waveguiding at deep-subwavelength scales. Our scheme is based on the strong electromagnetic coupling between two conformal surface plasmon structures. Using symmetry arguments and detailed numerical simulations, we show that the coupled system can be geometrically tailored to yield negative-index dispersion. A high degree of subwavelength modal confinement, of λ/10 in the transversal dimensions, is also demonstrated. These results can assist in the development of ultrathin surface circuitry for the low-frequency region (microwave and terahertz regimes) of the electromagnetic spectrum.

Interaction between PARP-1 and HIF-2α in the hypoxic response.

Hypoxia-inducible factors (HIFs) mediate the transcriptional adaptation of hypoxic cells. The extensive transcriptional programm regulated by HIFs involves the induction of genes controlling angiogenesis, cellular metabolism, cell growth, metastasis, apoptosis, extracellular matrix remodeling and others. HIF is a heterodimer of HIF-α and HIF-ß subunits. In addition to HIF-1α, HIF-2α has evolved as an isoform that contributes differently to the hypoxic adaptation by performing non-redundant functions. Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear protein involved in the control of DNA repair and gene transcription by modulating chromatin structure and acting as part of gene-specific enhancer/promoter-binding complexes. Previous results have shown that PARP-1 regulates HIF-1 activity. In this study, we focused on the cross-talk between HIF-2α and PARP-1. By using different approaches to suppress PARP-1, we show that HIF-2α mRNA expression, protein levels and HIF-2-dependent gene expression, such as ANGPTL4 and erythropoietin (EPO), are regulated by PARP-1. This regulation occurs at both the transcriptional and post-trancriptional level. We also show a complex formation between HIF-2α with PARP-1. This complex is sensitive to PARP inhibition and seems to protect against the von Hippel-Lindau-dependent HIF-2α degradation. Finally, we show that parp-1(-/-) mice display a significant reduction in the circulating hypoxia-induced EPO levels, number of red cells and hemoglobin concentration. Altogether, these results reveal a complex functional interaction between PARP-1 and the HIF system and suggest that PARP-1 is involved in the fine tuning of the HIF-mediated hypoxic response in vivo.

Identification of dual mTORC1 and mTORC2 inhibitors in melanoma cells: prodigiosin vs. obatoclax.

The PI3K/AKT/mTOR signaling pathway regulates cell proliferation, survival and angiogenesis. The mammalian target of rapamycin (mTOR) is a protein kinase ubiquitously expressed within cells that regulates cell growth and survival by integrating nutrient and hormonal signals. mTOR exists in two complexes, mTORC1 and mTORC2. Hyperactivation of the mTOR protein has been linked to development of cancer, raising mTOR as an attractive target for cancer therapy. Prodigiosin (PG) and obatoclax (OBX), two members of the prodiginines family, are small molecules with anticancer properties which are currently under clinical trials. In the present paper, we demonstrate that mTOR is a molecular target of both prodiginines in melanoma, a highly drug-resistant cancer model. The inhibition of mTORC1 and mTORC2 complexes by PG or OBX resulted in a loss of AKT phosphorylation at S473, preventing its full activation, with no significant effect on T308. The strongest activity inhibition (89%) was induced by PG on mTORC2. Binding assays using Surface Plasmon Resonance (SPR) provide kinetic and affinity data of the interaction of these small molecules with mTOR. In addition, in silico modeling produced a detailed atomic description of the binding modes. These results provide new data to understand the mechanism of action of these molecules, and provide new structural data that will allow the development of more specific mTOR inhibitors for cancer treatment.

Production of haemolytic factor by clinical isolates of Candida tropicalis.

Although haemolytic factor is known to be a putative virulence factor contributing to pathogenicity in Candida species, its production by Candida tropicalis is poorly understood. In this study, we analysed the culture conditions under which C. tropicalis can display haemolytic factor on plate assay and the secretion of haemolytic factor in liquid medium by clinical isolates obtained from different specimens. All the tested isolates exhibited an internal translucent ring, resembling beta-haemolysis, surrounding by a peripheral greenish-grey halo on sheep blood agar medium. Similar haemolytic pattern was observed on human blood enriched medium. Furthermore, incubation either under normal atmosphere or under increased CO(2) had no effect on haemolysis. Overall, no differences were observed on beta-haemolytic activities (P > 0.05) among tested isolates of C. tropicalis. In glucose-limited medium (RPMI 1640 with 0.2% glucose), none of the isolates induced haemolysis on red blood cells. Similarly to found on plate assays, there were no significant differences (P > 0.05) in the activity of secreted haemolytic factor in liquid medium among C. tropicalis isolates. However, after growth, the number of yeast cells varied among isolates revealing different efficiencies of haemolytic factor production. Haemolytic activity was neither inhibited by heat treatment (100 °C) nor by the addition of pepstatin A. The obtained results extend our knowledge about haemolytic factor production by Candida species.

Haemolytic and proteinase activities in clinical isolates of Candida parapsilosis and Candida tropicalis with reference to the isolation anatomic site.

The aim of this study was to determine in vitro haemolytic and protease activities of Candida parapsilosis and Candida tropicalis isolates, obtained from anatomically distinct sites. Analysis of haemolytic activity of C. parapsilosis and C. tropicalis isolates obtained from the same anatomic site revealed that C. tropicalis isolates from blood had statistically higher activity (P < 0.05) than C. parapsilosis. On comparison of haemolytic activities of Candida isolates obtained from different anatomic sites, C. parapsilosis isolates from tracheal secretion were found to have higher activity than blood isolates. Protease activity was detected in the majority of the isolates analysed. Analysis of proteinase activity of C. parapsilosis and C. tropicalis isolates obtained from the same anatomic site revealed that C. parapsilosis isolates from tracheal secretion had statistically higher activity than C. tropicalis isolates. On comparison of proteinase activities of Candida isolates obtained from different anatomic sites, C. parapsilosis isolates from tracheal secretion were found to have higher activity than blood and superficial lesions isolates. Furthermore, C. tropicalis isolates from superficial lesions had higher activity than tracheal secretion isolates. Our results show the potential of C. parapsilosis and C. tropicalis isolates, obtained from distinct anatomic sites, to produce haemolytic factor and proteinases. Anatomic sites of isolation seem to be correlated with these activities, particularly for C. parapsilosis isolates.

Biphasic hydrogenation over PVP stabilized Rh nanoparticles in hydroxyl functionalized ionic liquids.

Polyvinyl pyrrolidone stabilized rhodium nanoparticles are highly soluble in hydroxyl-functionalized ionic liquids, providing an effective and highly stable catalytic system. In hydrogenation reactions, excellent results were obtained, and transmission electron microscopy, solubility determinations, and leaching experiments were employed to quantify the advantages of this catalytic system.

[Toxic epidermal necrolysis induced by phenytoin and whole brain radiotherapy]. / Necrolisis epidérmica tóxica inducida por fenitoína y radioterapia holocraneal.

Toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) have been associated with some drugs, particularly anticonvulsants such as phenytoin. Some authors have pointed out an increased risk of TEN/SJS when phenytoin is associated with whole brain radiotherapy. We report a patient diagnosed with breast adenocarcinoma and brain metastases that was on treatment with phenytoin and, shortly after receiving whole brain radiotherapy, developed toxic epidermal necrolysis.

Modulation of transcription by PARP-1: consequences in carcinogenesis and inflammation.

Post-translational modification of proteins by poly(ADP-ribosyl)ation is involved in the regulation of a number of biological functions. While an 18 member superfamily of poly(ADP-ribose) polymerases (PARP)s has been described PARP-1 accounts for more than 90% of the poly(ADP-ribosyl)ating capacity of the cells. PARP-1 act as a DNA nick sensor and is activated by DNA breaks to cleave NAD(+) into nicotinamide and ADP-ribose to synthesize long branching poly(ADP-ribose) polymers (PAR) covalently attached to nuclear acceptor proteins. Whereas activation of PARP-1 by mild genotoxic stimuli facilitate DNA repair and cell survival, severe DNA damage triggers different pathways of cell death including PARP-mediated cell death through the translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus. PAR and PARP-1 have also been described as having a function in transcriptional regulation through their ability to modify chromatin-associated proteins and as a cofactor of different transcription factors, most notably NF-kappaB and AP-1. Pharmacological inhibition or genetic ablation of PARP-1 not only provided remarkable protection from tissue injury in various oxidative stress-related disease models but it result in a clear benefit in the treatment of cancer by different mechanisms including selective killing of homologous recombination-deficient tumor cells, down regulation of tumor-related gene expression and decrease in the apoptotic threshold in the co-treatment with chemo and radiotherapy. We will summarize in this review the current findings and concepts for the role of PARP-1 and poly(ADP-ribosyl)ation in the regulation of transcription, oxidative stress and carcinogenesis.

Aspectos evolutivos de la hipertensión portal en pacientes cirróticos / Evolutive aspects of portal hypertension in cirrhotic

El diagnóstico de hipertensión portal en pacientes cirróticos conlleva importantes consecuencias pronósticas y terapéuticas. La complicación más importante es el sangrado por rotura de várices esófagogástricas. El objetivo del presente estudio fue evaluar aspectos clínicos y endoscópicos de la historia natural de la hipertensión portal en pacientes cirróticos. La prevalencia de várices esfágicas en el grupo de pacientes estudiados fue de 60 por ciento. Una vez que las várices se han desarrollado éstas aumentaron de tamaño en el 38 por ciento durante los siguientes dos años de seguimiento. La hemorragia variceal fue el motivo de ingreso en un 20 por ciento. Esta complicación se relacionó con una pobre función hepática (score Child-Pugh > 10) y un recuento plaquetario < de 140.000. El riesgo de sangrado por varices no se relacionó con el tamaño variceal en nuestro grupo. La mortalidad precoz fue de un 6 por ciento y se presentó en los primeros 3 días de hospitalización. Los pacientes que sobrevivieron a un episodio de hemorragia variceal aguda, tuvieron un alto riesgo de resangrar. A pesar del tratamiento farmacológico con beta-bloqueadores, frecuencia de resangrado fue de 57 por ciento, similar a la frecuencia observada en estudios previos para los grupos placebo o sin terapia activa. Ya que el riesgo de resangrar puede disminuir significativamente con caídas marcadas de la presión portal, es que podría considerarse la medición de ésta, cada vez que el tratamiento farmacológico sea considerado como opción terapéutica