Evaluation of radioprotective effects of propolis and its flavonoid constituents: in vitro study on human white blood cells

This in vitro study aimed to evaluate the possible radioprotective effects of the natural substances WSDP, caffeic acid, chrysin and naringin on γ‐irradiated human white blood cells. The effectiveness of tested compounds was evaluated using the alkaline comet assay, the analysis of structural chromosome aberration and the cytokinesis‐block micronucleus assay. The results obtained by the alkaline comet study indicate favourable toxicity profiles of propolis and its polyphenolic components, and confirmed the radioprotective abilities comparable to the chemical radioprotector AET. WSDP and its polyphenolic components were able to reduce the number of necrotic cells. None of tested compounds induced significant genotoxicity, but all of them offered a quite measurable protection against DNA damage. WSDP was found to be the most effective in diminishing the levels of primary and more complex cytogenetic DNA damage in white blood cells. Considering its complex composition, to undoubtedly explain the underlying mechanisms of cyto/radioprotective effects, further studies are needed. Copyright © 2009 John Wiley & Sons, Ltd.     

Computational prediction of methylation status in human genomic sequences

Epigenetic effects in mammals depend largely on heritable genomic methylation patterns. We describe a computational pattern recognition method that is used to predict the methylation landscape of human brain DNA. This method can be applied both to CpG islands and to non-CpG island regions. It computes the methylation propensity for an 800-bp region centered on a CpG dinucleotide based on specific sequence features within the region. We tested several classifiers for classification performance, including K means clustering, linear discriminant analysis, logistic regression, and support vector machine. The best performing classifier used the support vector machine approach. Our program (called hdfinder) presently has a prediction accuracy of 86%, as validated with CpG regions for which methylation status has been experimentally determined. Using hdfinder, we have depicted the entire genomic methylation patterns for all 22 human autosomes.     

Intraoperative volume restriction in esophageal cancer surgery: an exploratory randomized clinical trial

Aim

To investigate whether the fluid volume administered during esophageal cancer surgery affects pulmonary gas exchange and tissue perfusion.

Methods

An exploratory single-center randomized clinical trial was performed. Patients with esophageal cancer who underwent Lewis-Tanner procedure between June 2011 and August 2012 at the Department of Thoracic surgery “Jordanovac”, Zagreb were analyzed. Patients were randomized (1:1) to receive a restrictive volume of intraoperative fluid (≤8 mL/kg/h) or a liberal volume (>8 mL/kg/h). Changes in oxygen partial pressure (Pao₂), inspired oxygen fraction (FiO₂), creatinine, and lactate were measured during and after surgery.

Results

Overall 16 patients were randomized and they all were analyzed (restrictive group n = 8, liberal group n = 8). The baseline value Pao₂/FiO₂ ratio (restrictive) was 345.01 ± 35.31 and the value six hours after extubation was 315.51 ± 32.91; the baseline Pao₂/FiO₂ ratio (liberal) was 330.11 ± 34.71 and the value six hours after extubation was 307.11 ± 30.31. The baseline creatinine value (restrictive) was 91.91 ± 12.67 and the value six hours after extubation was 100.88 ± 18.33; the baseline creatinine value (liberal) was 90.88 ± 14.99 and the value six hours after extubation was 93.51 ± 16.37. The baseline lactate value (restrictive) was 3.93 ± 1.33 and the value six hours after extubation was 2.69 ± 0.91. The baseline lactate value (liberal) was 3.26 ± 1.25 and the value six hours after extubation was 2.40 ± 1.08. The two groups showed no significant differences in Pao₂/FiO₂ ratio (P = 0.410), creatinine (P = 0.410), or lactate (P = 0.574).

Conclusions

Restriction of intraoperative applied volume does not significantly affect pulmonary exchange function or tissue perfusion in patients undergoing surgical treatment for esophageal cancer.Trial registration number: Clinical Trials {"type":"clinical-trial","attrs":{"text":"NCT 02033213","term_id":"NCT02033213"}}NCT 02033213.Pulmonary complications remain a primary cause of morbidity after esophageal cancer surgery. Complications range from atelectasis and pneumonia to acute lung injury and acute respiratory distress syndrome; the risk of these complications is determined largely by preoperative pulmonary status and surgical approach (1). Another factor that can influence the risk of postoperative respiratory complications is the volume of fluid administered intraoperatively (2,3). Such fluid administration is a routine procedure during lung and esophageal surgery (4).The optimal type and volume of fluid are controversial issues and have not been standardized in international guidelines (5). Several studies suggest that restrictive intraoperative fluid resuscitation during open abdominal surgeries is superior to an aggressive or “liberal” fluid protocol, because it is associated with fewer postoperative complications and shorter discharge time (6-8). On the other hand, restrictive fluid management can lead to hypovolemia and impaired tissue perfusion, which can cause organ dysfunction, particularly postoperative acute kidney injury (9).In esophageal surgery, fluid management is a special concern because one-lung ventilation (OLV), which is an integral part of anesthesia, can cause postoperative pulmonary edema (10-13). When conventional ventilation is reestablished after surgery, reexpansion of the deflated lung can induce oxidative stress that leads to edema (12-15). In this way, OLV may aggravate the postoperative effects of perioperative pulmonary fluid overload (16). The aim of this exploratory trial was to compare the effects of restrictive and liberal fluid resuscitation protocol on pulmonary gas exchange and tissue perfusion.     

Optimizing sparse sequencing of single cells for highly multiplex copy number profiling

Genome-wide analysis at the level of single cells has recently emerged as a powerful tool to dissect genome heterogeneity in cancer, neurobiology, and development. To be truly transformative, single-cell approaches must affordably accommodate large numbers of single cells. This is feasible in the case of copy number variation (CNV), because CNV determination requires only sparse sequence coverage. We have used a combination of bioinformatic and molecular approaches to optimize single-cell DNA amplification and library preparation for highly multiplexed sequencing, yielding a method that can produce genome-wide CNV profiles of up to a hundred individual cells on a single lane of an Illumina HiSeq instrument. We apply the method to human cancer cell lines and biopsied cancer tissue, thereby illustrating its efficiency, reproducibility, and power to reveal underlying genetic heterogeneity and clonal phylogeny. The capacity of the method to facilitate the rapid profiling of hundreds to thousands of single-cell genomes represents a key step in making single-cell profiling an easily accessible tool for studying cell lineage.Tumor cells evolve via the acquisition of somatic genetic lesions that bestow the capacity to proliferate and survive (Vogelstein et al. 2013). Consequently, genetically distinct subpopulations are likely to evolve and dynamically interact with each other (Marusyk et al. 2012; Yates and Campbell 2012; Burrell et al. 2013). The presence of tumor genome heterogeneity has long been acknowledged (Nowell 1976), and recent investigations have tied it to disease progression and metastasis, as well as therapeutic resistance (Turke et al. 2010; Walter et al. 2012; Wu et al. 2012). Unfortunately, our knowledge of cancer genome heterogeneity is still lacking, due primarily to the lack of sensitive approaches that explore genetic heterogeneity at a genome-wide scale. New technologies are needed to facilitate the dissection of intra-tumoral heterogeneity.Recently, with the advent of next-generation sequencing (NGS) technologies and whole-genome amplification (WGA) approaches, single-cell genomic investigations have emerged as a powerful approach to analyze cancer genetic heterogeneity (Navin et al. 2011; Baslan et al. 2012). Genome-wide single-cell sequencing investigations have begun to illuminate valuable and novel aspects of cancer biology and promise to deliver more (Ni et al. 2013; Dago et al. 2014; Francis et al. 2014; Lohr et al. 2014). To realize the potential of single-cell sequencing in understanding the biology of heterogeneity, methods are needed that allow the investigation of hundreds of single-cell genomes at a reasonable cost in time, effort, and reagents. Sequencing hundreds of single cells to the nucleotide level is simply not affordable even with the remarkable NGS platforms that are available. Fortunately, copy number analysis requires only sparse sequence coverage, yet it can distinguish subpopulations and provides deep insights into genetic heterogeneity. Thus, in theory, coupling sparse sequencing with molecular barcoding approaches offers a means to profile many cells together.Indeed, we and others have recently demonstrated the feasibility of this approach by combining up to eight barcoded single cells on a single sequencing lane (McConnell et al. 2013; Dago et al. 2014), but the potential for higher level multiplexing has not been explored at either the bioinformatic or operational levels. To accomplish this, informatic analysis aimed at identifying minimal sequence read requirements for robust copy number identification is required. Furthermore, while technically feasible, amplifying and creating barcoded sequencing libraries from many single cells using traditional library preparation protocols involving sonication, end repair, A-tailing, and adaptor ligation is time-consuming and expensive. We have therefore set out to create an optimized multiplexing process by determining the minimum number of reads that can be used to determine genome-wide copy number profiles at specific levels of resolution and then to develop a simplified preparative method that is faster and cheaper and yet maximizes the amount of information that can be extracted from each sequencing read from a single sequencing lane of the Illumina HiSeq machine.Here, we describe a robust and affordable, high-throughput method that employs a modified version of degenerate oligonucleotide priming-PCR (DOP-PCR) amplification, simplified library preparation, and multiplex sequencing that facilitates the retrieval of the genome-wide copy number landscape of hundreds of individual cancer cells. Our method drastically lowers the cost of profiling single-cell genomes (down to ∼$30 per single cell), significantly cuts sequence library preparation time, and maximizes the amount of information extracted from each single-cell sequencing data set. We apply our approach to human cancer cell lines and clinical cancer biopsies to demonstrate its power to reveal population heterogeneity.     

Nesidiodysplasia and Nesidioblastosis of Infancy: Structural and Functional Correlations with the Syndrome of Hyperinsulinemic Hypoglycemia

Subtotal pancreatectomy specimens of seven infants with persistent hyperinsulinemic hypoglycemia were studied; all showed the characteristic light microscopic picture of nesidioblastosis. Specimens were studied by electron and conventional light microscopy and by light microscopic immunohistochemistry for insulin, glucagon, somatostatin, and HPP (human pancreatic polypeptide); double staining and quantitative methods were also used. Findings were compared with those in age-matched controls.

In the hyperinsulinemic hypoglycemic infants, an increase in total endocrine cell volume was found; however, the typical features of nesidioblastosis were also found in the controls. In both groups, immunohistochemistry and electron microscopy suggested that some endocrine cells are capable of producing synchronously more than one hormone. Amphicrine (”composite” or “intermediate”) cells with exocrine and endocrine differentiation were found in three hypoglycemic infants.

Observations are discussed in relation to current concepts of embryo genesis of the gastroenteropancreative endocrine system. We conclude that nesidioblastosis, as defined anatomically cannot be considered as the morphologic basis of hyperinsulinemic hypoglycemia. The term “nesidiodysplasia” is suggested and includes increased, maldistributed, and malregulated or malprogrammed endocrine and amphicrine cells when associated with endocrine abnormality.     

Significance of crown-down root canal preparation technique in endodontic therapy by using the hydroxylapatite sealer

AIM: To evaluate the crown-down preparation technique, and the use of hydroxylapatite based material for the definitive root canal obturation. METHODS: The investigation included 20 single-canal roots with chronic periapical inflammatory lesion. Biomechanical medicamentous canal preparation was done using the double-flared technique with balanced force, and the obturation was performed with hydroxylapatite sealer (unicone technique). Clinical and radiographic check-up performed 12 months after the treatment, used the following parameters: pain, swelling, percussion and palpation sensitivity, and the presence of fistula. RESULTS: The obtained results showed a successful treatment in 18 cases, while in the 2 cases the treatment failed. CONCLUSION: These findings suggested that the crown-down preparation technique efficiently cleaned and shaped the root canal, and that the hydroxylapatite-based material created the homogenous and hermetic root canal obturation, so this methodology could be recommended for the endodontic therapy.     

Flow cytometric detection of DNA cell cycle alterations in hemocytes of mussels (Mytilus galloprovincialis) off the Adriatic coast,Croatia

Studies were carried out to determine the alteration in DNA cell cycle characteristics of hemocytes of the mussel Mytilus galloprovincialis collected at 17 different locations (146 individuals) along the Adriatic coast, Croatia. In order to connect possible genomic manifestation to urban and/or industrial waste flow cytometry was used. We studied incidence of altered DNA profile reflective of chromosomal fragmentation phenomena or aneuploid mosaicism, coefficient of variation (CV) in DNA fluorescence as a measure of intraindividual genome size variability and DNA index (DI) as a measure of ploidy. The different classes of DNA cell cycle alterations found in this study mirror either acute or cumulative genotoxic effects of the surrounding environment on mussel hemocyte DNA. These are intraindividual genome size variability (CV>8, seven individuals from four sites), aneuploidy (altered DNA profile and DI<0.9, 45 individuals from 14 sites) and accidental apoptotic processes (altered DNA profile and presence of apoptotic cells, two individuals from two sites). Normal cell cycle DNA profiles were obtained for 89 (60.9%) individuals from all 17 sites and for 146 examined samples polyploids were absent. Flow cytometry proved to be a powerful technique for the determination of alterations in cell cycle characteristics in mussel hemocyte DNA. Therefore, it may be used in pollution control measurements to distinguish affected or vulnerable populations from healthy populations living in the presence of a wide variety of marine environmental contaminants.     

Thrombendvenectomy for Organized Portal Vein Thrombosis at the Time of Liver Transplantation

OBJECTIVE: To determine the efficacy of portal thrombendvenectomy in cases of portal vein thrombosis at the time of orthotopic liver transplantation. SUMMARY BACKGROUND DATA: Portal vein thrombosis (PVT) has been reported to have an incidence of 2% to 39% in end-stage liver disease. Multiple techniques have been suggested to treat this finding. Several reports have suggested suboptimal results after liver transplantation in recipients with PVT. METHODS: The authors prospectively collected data on 1,546 patients who underwent an initial orthotopic liver transplant at the authors' institution between December 1984 and October 1999. There were 820 male patients and 726 female patients. All recipients received either cyclosporine or tacrolimus immunosuppression. Intraoperative flows of the portal vein and hepatic artery were routinely measured. Duplex sonography was routinely performed on the first postoperative day and routinely 1, 2, 5, and 10 years after transplantation. Eighty-five patients underwent thrombendvenectomy for organized thrombus partially or completely occluding the portal vein. Postoperative treatment included low-molecular-weight dextran for 48 hours and daily aspirin for 3 months. There were 53 male patients and 32 female patients. The PVT group was compared with a control group consisting of transplant recipients without PVT. RESULTS: When compared with the control group, PVT patients were older at the time of transplantation and had a higher incidence of liver disease secondary to cryptogenic cirrhosis and Laennec's cirrhosis. There were no significant differences among both groups for 1-, 3-, and 6-year patient and graft survival rates. CONCLUSIONS: Thrombendvenectomy provides a rapid resolution of an otherwise complex problem. It is the authors' procedure of choice in cases of organized PVT at the time of transplantation. Operative time and length of stay in the intensive care unit are not prolonged, and patient and graft survival rates are not compromised.