Bixin and lycopene modulation of free radical generation induced by cisplatin-DNA interaction

This work evaluated the Modulation of reactive oxygen species (ROS) produced by the cisplatin-human DNA interaction in a cell-free experimental model by the carotenoids bixin and lycopene extracted from, natural dietary Sources and purified through luminol- and Cypridina luciferin methoxy-analogue (MCLA)- enhanced chemiluminescence assays. The results showed that the ROS generation by DNA-cisplatin interaction was inhibited by both lycopene and bixin in a concentration-dependent manner. At a concentration of 100 mu M, lycopene and bixin inhibited Superoxide anion (O center dot(2)) generation at 90% and 82%, respectively, and the total ROS generation at 44% and 42%, respectively. The formation of significant amounts of isomers or degradation products of both carotenoids was not observed after ROS scavenging, as evaluated by high-performance liquid chromatography. Taken together, these results Suggest that carotenoids can be helpful to Modulate the oxidative stress found in cancer therapy with cisplatin. (c) 2008 Elsevier Ltd. All rights reserved.

Sequence analysis, chromosomal distribution and long-range organization show that rapid turnover of new and old pBuM satellite DNA repeats leads to different patterns of variation in seven species of the Drosophila buzzatii cluster

We aimed to study patterns of variation and factors influencing the evolutionary dynamics of a satellite DNA, pBuM, in all seven Drosophila species from the buzzatii cluster (repleta group). We analyzed 117 alpha pBuM-1 (monomer length 190 bp) and 119 composite alpha/beta (370 bp) pBuM-2 repeats and determined the chromosome location and long-range organization on DNA fibers of major sequence variants. Such combined methodologies in the study of satDNAs have been used in very few organisms. In most species, concerted evolution is linked to high copy number of pBuM repeats. Species presenting low-abundance and scattered distributed pBuM repeats did not undergo concerted evolution and maintained part of the ancestral inter-repeat variability. The alpha and alpha/beta repeats colocalized in heterochromatic regions and were distributed on multiple chromosomes, with notable differences between species. High-resolution FISH revealed array sizes of a few kilobases to over 0.7 Mb and mutual arrangements of alpha and alpha/beta repeats along the same DNA fibers, but with considerable changes in the amount of each variant across species. From sequence, chromosomal and phylogenetic data, we could infer that homogenization and amplification events involved both new and ancestral pBuM variants. Altogether, the data on the structure and organization of the pBuM satDNA give insights into genome evolution including mechanisms that contribute to concerted evolution and diversification.

Genetic interactions of the Ashergillus nidulans atmA(ATM) homolog with different components of the DNA damage response pathway

Ataxia telangiectasia mutated (ATM) is a phosphatidyl-3-kinase-related protein kinase that functions as a central regulator of the DNA damage response in eukaryotic cells. In humans, mutations in ATM cause the devastating neurodegenerative disease ataxia telangiectasia. Previously, we characterized the homolog of ATM (AtmA) in the filamentous fungus Aspergillus nidulans. In addition to its expected role in the DNA damage response, we found that AtmA is also required for polarized hyphal growth. Here, we extended these studies by investigating which components of the DNA damage response pathway are interacting with AtmA. The AtmA(ATM) loss of function caused synthetic lethality when combined with mutation in UvsB(ATR). Our results suggest that AtmA and UvsB are interacting and they are probably partially redundant in terms of DNA damage sensing and/or repairing and polar growth. We identified and inactivated A. nidulans chkA(CHK1) and chkB(CHK2) genes. These genes are also redundantly involved in A. nidulans DNA damage response. We constructed several combinations of double mutants for Delta atmA, Delta uvsB, Delta chkA, and Delta chkB. We observed a complex genetic relationship with these mutations during the DNA replication checkpoint and DNA damage response. Finally, we observed epistatic and synergistic interactions between AtmA, and bimE(APCI), ankA(WEE1) and the cdc2-related kinase npkA, at S-phase checkpoint and in response to DNA-damaging agents.

Low Satellite DNA Variability in Natural Populations of Drosophila antonietae Involved in Different Evolutionary Events

Drosophila antonietae is a cactophilic species that is found in the mesophilic forest of the Parana`-Paraguay river basin and in the dunes of the South Atlantic coast of Brazil. Although the genetic structure of the Parana`-Paraguay river basin populations has already been established, the relationship between these populations and those on the Atlantic coast is controversial. In this study, we compared 33 repetitive units of pBuM-2 satellite DNA isolated from individuals from 8 populations of D. antonietae in these geographic regions, including some populations found within a contact zone with the closely related D. serido. The pBuM-2 sequences showed low interpopulational variability. This result was interpreted as a consequence of both gene flow among the populations and unequal crossing over promoting homogenization of the tandem arrays. The results presented here, together with those of previous studies, highlight the use of pBuM-2 for solving taxonomic conflicts within the D. buzzatii species cluster.

Mitochondrial DNA Variability Among Eight Tikuna Villages: Evidence for an Intratribal Genetic Heterogeneity Pattern

To study the genetic structure of the Tikuna tribe, four major Native American mitochondrial DNA (mtDNA) founder haplogroups were analyzed in 187 Amerindians from eight Tikuna villages located in the Brazilian Amazon. The central position of these villages in the continent makes them relevant for attempts to reconstruct population movements in South America. In this geographic region, there is particular concern regarding the genetic structure of the Tikuna tribe, formerly designated ""enigmatic"" due to its remarkable degree of intratribal homogeneity and the scarcity of private protein variants. In spite of its large population size and geographic distribution, the Tikuna tribe presents marked genetic and linguistic isolation. All individuals presented indigenous mtDNA haplogroups. An intratribal genetic heterogeneity pattern characterized by two highly homogeneous Tikuna groups that differ considerably from each other was observed. Such a finding was unexpected, since the Tikuna tribe is characterized by a social system that favors intratribal exogamy and patrilocality that would lead to a higher female migration rate and homogenization of the mtDNA gene pool. Demographic explosions and religious events, which significantly changed the sizes and compositions of many Tikuna villages, may be reflected in the genetic results presented here. Am J Phys Anthropol 140:526-531,2009. (C) 2009 Wiley-Liss, Inc

Physicochemical characterization of nanoparticles formed between DNA and phosphorylcholine substituted chitosans

The interactions between phosphorylcholine-substituted chitosans (PC-CH) and calf-thymus DNA (ct-DNA) were investigated focusing on the effects of the charge ratio, the pH, and phosphorylcholine content on the size and stability of the complexes using the ethidium bromide fluorescence assay, gel electrophoresis, dynamic light scattering. and fluorescence microscopy. The size and colloidal stability of deacetylated chitosan (CH/DNA) and PC-CH/DNA complexes were strongly dependent on phosphorylcholine content, charge ratios, and pH. The interaction strengths were evaluated from ethidium bromide fluorescence, and at N/P ratios higher than 5.0, no DNA release was observed in any synthesized PC-CH/DNA polyplexes by gel electrophoresis. The PC-CH/DNA polyplexes exhibited a higher resistance to aggregation compared to deacetylated chitosan (CH) at neutral pH. At low pH values highly charged chitosan and its phosphorylcholine derivatives had strong binding affinity with DNA, whereas at higher pH Values CH formed large aggregates and only C-CH derivatives were able to form small nanoparticles with hydrodynamic radii varying from 100 to 150 nm. Nanoparticles synthesized at low ionic strength with PC-CH derivatives containing moderate degrees of substitution (DS = 20% and 40%) remained stable for weeks. Photomicroscopies also confirmed that rhodamine-labeled PC(40)CH derivative nanoparticles presented higher colloidal stability than those synthesized using deacetylated chitosan. Accordingly, due to their improved physicochemical properties these phosphorylcholine-modified chitosans provide new perspectives for controlling the properties of polyplexes. (C) 2009 Elsevier Inc. All rights reserved.

Novel Treatment Immediately after Myelomeningocele Repair Applying Low-Level Laser Therapy in Newborns: A Pilot Study

Background/Aims: The use of low-level laser therapy (LLLT) in neurosurgery is still hardly disseminated and there are situations in which the effects of this therapeutic tool would be extremely relevant in this medical field. The aim of the present study is to analyze the effect of LLLT on tissue repair after the corrective surgical incision in neonates with myelomeningocele, in an attempt to diminish the incidence of postoperative dehiscences following surgical repair performed immediately after birth. Materials and Methods: Prospective pilot study with 13 patients submitted to surgery at birth who received adjuvant treatment with LLLT (group A). A diode laser CW, lambda = 685 nm, p = 21 mW, was applied punctually along the surgical incision, with 0.19 J delivered per point, accounting for a total of 4-10 J delivered energy per patient, according to the surgical wound area and then compared with the results obtained in 23 patients who underwent surgery without laser therapy (group B). Results: This pilot study disclosed a significant decline in dehiscences of the surgical wounds in neonates who were submitted to LLLT (7.69 vs. 17.39%). Conclusion: This new adjuvant therapeutic modality with LLLT aided the healing of surgical wounds, preventing morbidities, as well as shortening the period of hospital stay, which implies a reduction of costs for patients and for the institution. Copyright (C) 2010 S. Karger AG, Basel

Immune response to vaccination with DNA-hsp65 in a phase I clinical trial with head and neck cancer patients

DNA-hsp65, a DNA vaccine encoding the 65-kDa heat-shock protein of Mycobacterium leprae (Hsp65) is capable of inducing the reduction of established tumors in mouse models. We conducted a phase I clinical trial of DNA-hsp65 in patients with advanced head and neck carcinoma. In this article, we report on the vaccine`s potential to induce immune responses to Hsp65 and to its human homologue, Hsp60, in these patients. Twenty-one patients with unresectable squamous cell carcinoma of the head and neck received three doses of 150, 400 or 600 mu g naked DNA-hsp65 plasmid by ultrasound-guided intratumoral injection. Vaccination did not increase levels of circulating anti-hsp65 IgG or IgM antibody, or lead to detectable Hsp65-specific cell proliferation or interferon-gamma (IFN-gamma) production by blood mononuclear cells. Frequency of antigen-induced IL-10-producing cells increased after vaccination in 4 of 13 patients analyzed. Five patients showed disease stability or regression following immunization; however, we were unable to detect significant differences between these patients and those with disease progression using these parameters. There was also no increase in antibody or IFN-gamma responses to human Hsp60 in these patients. Our results suggest that although DNA-hsp65 was able to induce some degree of immunostimulation with no evidence of pathological autoimmunity, we were unable to differentiate between patients with different clinical outcomes based on the parameters measured. Future studies should focus on characterizing more reliable correlations between immune response parameters and clinical outcome that may be used as predictors of vaccine success in immunosuppressed individuals. Cancer Gene Therapy (2009) 16, 598-608; doi:10.1038/cgt.2009.9; published online 6 February 2009

Phase I trial of DNA-hsp65 immunotherapy for advanced squamous cell carcinoma of the head and neck

Considering that mycobacterial heat-shock protein 65 (hsp65) gene transfer can elicit a profound antitumoral effect, this study aimed to establish the safety, maximum-tolerated dose (MTD) and preliminary efficacy of DNA-hsp65 immunotherapy in patients with advanced head and neck squamous cell carcinoma (HNSCC). For this purpose, 21 patients with unresectable and recurrent HNSCC were studied. Each patient received three ultrasound-guided injections at 21-day intervals of: 150, 600 or 400 mu g of DNA-hsp65. Toxicity was graded according to CTCAE directions. Tumor volume was measured before and after treatment using computed tomography scan. The evaluation included tumor mass variation, delayed-type hypersensitivity response and spontaneous peripheral blood mononuclear cell proliferation before and after treatment. The MTD was 400 mg per dose. DNA-hsp65 immunotherapy was well tolerated with moderate pain, edema and infections as the most frequent adverse effects. None of the patients showed clinical or laboratory alterations compatible with autoimmune reactions. Partial response was observed in 4 out of 14 patients who completed treatment, 2 of which are still alive more than 3 years after the completion of the trial. Therefore, DNA-hsp65 immunotherapy is a feasible and safe approach at the dose of 400 mg per injection in patients with HNSCC refractory to standard treatment. Further studies in a larger number of patients are needed to confirm the efficacy of this novel strategy.

Impact of mild acute kidney injury (AKI) on outcome after open repair of aortic aneurysms

Recently, mild AKI has been considered as a risk factor for mortality in different scenarios. We conducted a retrospective analysis of the risk factors for two distinct definitions of AKI after elective repair of aortic aneurysms. Logistic regression was carried out to identify independent risk factors for AKI ( defined as >= 25% or >= 50% increase in baseline SCr within 48 h after surgery, AKI 25% and AKI 50%, respectively) and for mortality. Of 77 patients studied ( mean age 68 +/- 10, 83% male), 57% developed AKI 25% and 33.7% AKI 50%. There were no differences between AKI and control groups regarding comorbidities and diameter of aneurysms. However, AKI patients needed a supra-renal aortic cross-clamping more frequently and were more severely ill. Overall in-hospital mortality was 27.3%, which was markedly higher in those requiring a supra-renal aortic cross-clamping. The risk factors for AKI 25% were suprarenal aortic cross-clamping ( odds ratio 5.51, 95% CI 1.05-36.12, p = 0.04) and duration of operation for AKI 25% ( OR 6.67, 95% CI 2.23-19.9, p < 0.001). For AKI 50%, in addition to those factors, post-operative use of vasoactive drugs remained as an independent factor ( OR 6.13, 95% CI 1.64-22.8, p = 0.005). The risk factors associated with mortality were need of supra-renal aortic cross-clamping ( OR 9.6, 95% CI 1.37-67.88, p = 0.02), development of AKI 50% ( OR 8.84, 95% CI 1.31-59.39, p = 0.02), baseline GFR lower than 49 mL/min ( OR 17.07, 95% CI 2.00 145.23, p = 0.009), and serum glucose > 118 mg/dL in the post-operative period ( OR 19.99, 95% CI 2.32-172.28, p = 0.006). An increase of at least 50% in baseline SCr is a common event after surgical repair of aortic aneurysms, particularly when a supra-renal aortic cross-clamping is needed. Along with baseline moderate chronic renal failure, AKI is an independent factor contributing to the high mortality found in this scenario.

Evaluation of Primary Cleft Nose Repair: Severity of the Cleft Versus Final Position of the Nose

Background: Since 1957, when the concept of rotation-advancement repair was introduced by Millard, this technique has become the procedure of choice for unilateral cleft lip worldwide. More recently, modifications described by Noordhoof, Mohler, Skoog, and McComb started being jointly performed so that better results could be obtained. In this study, the nasal position was evaluated and related to the size of the cleft. The primary unilateral cleft lip repair was performed through a modified technique. Methods: Forty-five patients with unilateral cleft lip underwent primary surgical repair through this technique. To analyze aesthetic results, a severity classification of deformities and a scoring system for evaluation of the results were established based on nasal alar lateralization, dome position, alignment of bone segments, and deviation of the columella. Results: By means of the established system, 26.6% of mild forms, 13.4% of moderate forms, and 60% of severe forms were observed. Among aesthetic results, 17.8% were found to be good, and 82.2% were considered excellent. Among aspects considered negative, late deformity of the lower lateral cartilage prevailed. Conclusions: Through the presented evaluation, the authors observed that there was no relation between severity of the cleft and final position of the nose. Among the 27 patients considered to have had severe forms of cleft deformity, 22 were classified as excellent results (81.5%). To obtain better results along time, technical refinements and the critical analysis of results must be performed on a routinely basis.

The surgical repair of half-nose

Absence of half-nose is an extremely rare congenital malformation, which has a devastating impact on the patient and the family. A review of indexed English-language literature found 91 cases of half-nose, including 50 patients with proboscis lateralis. Pathogenesis is not clear, and the reported cases have sporadically occurred. Many aspects must be considered when reconstructing a congenital half-nose, such as timing of surgery, type of tissue to be used and the need to reconstruct nasal airway. The aim of this article is to present personal experience in seven cases of half-nose reconstruction, in order to review the literature regarding to this rare entity, highlighting aspects of incidence, pathogenesis and surgical treatment. Nasal reconstruction was performed at ages of 5-7 years to minimise psychological trauma. Forehead skin demonstrated to be an excellent donor site to re-surface the nose. For the inner lining, contralateral cutaneous nasal flap was our preference. Concerning the nasal framework reconstruction, alar contour was restored using a cartilage graft from the lower portion of ear tragus and concha. (C) 2008 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

Challenges in Using Stem Cells for Cardiac Repair

Of the many diseases discussed in the context of stem cell therapy, those concerning the heart account for almost one-third of the publications in the field. However, the long-term clinical outcomes have been disappointing, in part because of preclinical studies failing to optimize the timing, number, type, and method of cell delivery and to account for shape changes that the heart undergoes during failure. In situations in which cardiomyocytes have been used in cell therapy, their alignment and integration with host tissue have not been realized. Here we review the present status of direct delivery of stem cells or their derivative cardiomyocytes to the heart and the particular challenges each cell type brings, and consider where we should go from here.

SUITABILITY OF A RAPID DNA ISOLATION AND AMPLIFICATION FOR DETECTION OF TRYPANOSOMA CRUZI IN TRIATOMA INFESTANS DRY FECAL SPOTS COLLECTED ON FILTER PAPER

A rapid DNA extraction was used for T. cruzi detection in triatomines dry fecal spots collected on filter paper and analyzed by PCR. Fifty T infestans were fed on experimentally infected Balb/C mice with high T. cruzi parasitemia and divided into five groups of len triatomines, and 100 triatomines were infected with lower parasitemia and divided into five groups of 20 triatomines, One dry fecal spot was analyzed per group on days 1, 2, 3, 4 and 5 post feeding. Amplification targeted T. cruzi TCZ sequence and resulted positive from day 4 after bugs feeding in the two models (high and lower parasitemia). The rapid DNA isolation and PCR proposed are suitable for detection of T. cruzi DNA in in filter paper and should be considered in field research.

Usefulness of qualitative polymerase chain reaction for Trypanosoma cruzi DNA in endomyocardial biopsy specimens of chagasic heart transplant patients

BACKGROUND: Chagas` disease reactivation (CDR) after heart transplantation is characterized by relapse of the infectious disease, with direct detection of Trypanosoma cruzi parasites in blood, cerebrospinal fluid, or tissues. CDR affecting the myocardium induces lymphocytic myocarditis and should be distinguished from acute cellular rejection in endomyocardial biopsy (EMB) specimens. METHODS: We performed retrospectively qualitative polymerase chain reaction for T cruzi DNA using 2 sets of primers targeting nuclear DNA (nDNA) or kinetoplast DNA (kDNA) in 61 EMB specimens of 11 chagasic heart transplant recipients who presented with CDR. Thirty-five EMB specimens were obtained up to 6 months before (pre-CDR group) and 26 up to 2 years after the diagnosis of CDR. The control group consisted of 6 chagasic heart transplant recipients with 18 EMB specimens who never experienced CDR. RESULTS: Amplification of kDNA occurred in 8 of 35 (22.9%) EMB specimens of the pre-CDR group, in 5 of 18(27.8%) of the control group, and in 17 of 26(65.4%) EMB specimens obtained after the successful treatment of CDR. Amplification of nDNA occurred in 3 of 35 (8.6%) EMB specimens of the pre-CDR group, 0 of 18 (0%) of the control group, and 6 of 26 (23.1%) EMB specimens obtained after the successful treatment of CDR. CONCLUSIONS: Amplification of kDNA in EMB specimens is not specific for the diagnosis of CDR, occurring also in patients with no evidence of CDR (control group). However, amplification of nDNA occurred in a few EMB specimens obtained before CDR, but in none of the control group specimens. Qualitative PCR for T cruzi DNA in EMB specimens should not be used as a criterion for cure of CDR because it can persist positive despite favorable clinical evolution of the patients. J Heart Lung Transplant 2011;30:799-804 (C) 2011 International Society for Heart and Lung Transplantation. All rights reserved.